"Management of Fruit Genetic Resources" Held On

Virtual Training Course on Management of Fruit Genetic Resources

February 1-2, 2021

Training Director

Dr Kuldeep Singh Director ICAR - National Bureau of Plant Genetic Resources (NBPGR), New Delhi

Training Coordinator Training Convenor

Dr Prakash Patil Dr Anuradha Agrawal Project Coordinator (Fruits) Officer-In-Charge, ICAR-AICRP on Fruits, Tissue Culture and Cryopreservation Unit, ICAR-IIHR, Bengaluru ICAR-NBPGR, New Delhi

Training Co-Convenors

Dr Vartika Srivastava Dr Sridhar Gutam Scientist Sr. Scientist, AICRP on Fruits, ICAR-NBPGR, New Delhi ICAR-IIHR, Bengaluru

Dr Era Vaidya Malhotra Dr K.S. Hooda Scientist HRD Nodal Officer ICAR-NBPGR, New Delhi ICAR-NBPGR, New Delhi Contents

1 Preface 5 2 Training Schedule 6 3 List of Faculty 8 4 List of Trainees 9 5 PGR Management System in India 16 Kuldeep Singh, S. Rajkumar and Kavita Gupta 6 Exploration and Collecting Fruit Genetic Resources for Food and Agriculture 27 S. P. Ahlawat 7 Plant Exploration and Germplasm Collecting in Fruit Crops – 41 Practical Considerations Joseph John K. and Suma A. 8 Access to Germplasm: Policies, Guidelines and Procedures 47 Pratibha Brahmi and Pragya 9 National Plant Quarantine System 67 S. C. Dubey and Kavita Gupta 10 PGR Informatics and its Utility in Conservation and Utilization of 77 Genetic Resources of Fruits and Vegetables Sunil Archak and Rajeev Gambhir 11 Principles of Germplasm Characterization and Evaluation 85 Ashok Kumar and Kuldeep Tripathi 12 Characterization and Evaluation of Fruit Crops – Role of Descriptors 92 and Other Practical Considerations Narender Negi, Rahul Chandora, Badal Singh, Dayal Singh, Ram Chander and Mohar Singh 13 Germplasm Core collections - Principles and Methodologies 199 Rakesh Singh and D.R. Chaudhury 14 Registration of Trait Specific Germplasm at ICAR-NBPGR 110 Anjali Kak Koul and Veena Gupta 15 Holistic Approach for Germplasm Conservation of Fruit Crops 115 Anuradha Agrawal, Vartika Srivastava, Era Vaidya Malhotra, Sandhya Gupta and Sangita Bansal 16 DNA Fingerprinting and Use of Genomics in Plant Genetic 132 Resources Management Mukesh Kumar Rana and Amit Kumar Singh 17 Organizing Committee 139 Contents

1 Preface 5 2 Training Schedule 6 ©2021 3 List of Faculty 8 The Director, ICAR-ICAR-National Bureau of Plant Genetic Resources (NBPGR), 4 List of Trainees 9 New Delhi-110012, India 5 PGR Management System in India 16 Kuldeep Singh, S. Rajkumar and Kavita Gupta 6 Exploration and Collecting Fruit Genetic Resources for Food and Agriculture 27 Citation S. P. Ahlawat Agrawal A., V. Srivastava, E. V. Malhotra, P. Patil and K. Singh (eds) (2021) Training Manual for 7 Plant Exploration and Germplasm Collecting in Fruit Crops – 41 Virtual Training Course on Management of Fruit Genetic Resources. ICAR-National Bureau of Practical Considerations Plant Genetic Resources, New Delhi, ICAR-All India Coordinated Research Project on Fruits, Joseph John K. and Suma A. ICAR-Indian Institute of Horticultural Research, Bengaluru, Feb. 1-2, 2021, p. 140 8 Access to Germplasm: Policies, Guidelines and Procedures 47 Pratibha Brahmi and Pragya 9 National Plant Quarantine System 67 S. C. Dubey and Kavita Gupta 10 PGR Informatics and its Utility in Conservation and Utilization of 77 Genetic Resources of Fruits and Vegetables Sunil Archak and Rajeev Gambhir 11 Principles of Germplasm Characterization and Evaluation 85 Ashok Kumar and Kuldeep Tripathi 12 Characterization and Evaluation of Fruit Crops – Role of Descriptors 92 and Other Practical Considerations Narender Negi, Rahul Chandora, Badal Singh, Dayal Singh, Ram Chander and Mohar Singh 13 Germplasm Core collections - Principles and Methodologies 199 Rakesh Singh and D.R. Chaudhury 14 Registration of Trait Specific Germplasm at ICAR-NBPGR 110 Anjali Kak Koul and Veena Gupta 15 Holistic Approach for Germplasm Conservation of Fruit Crops 115 Anuradha Agrawal, Vartika Srivastava, Era Vaidya Malhotra, Sandhya Gupta and Sangita Bansal The contents of this manual are collated for the academic purpose of the 'Virtual Training Program on Management of Fruit Genetic Resources', held online and organized by ICAR- 16 DNA Fingerprinting and Use of Genomics in Plant Genetic 132 NBPGR, New Delhi, AICRP on Fruits and ICAR-IIHR, Bengaluru, India, from Feb 1-2, 2021. The Resources Management information in the manual consists of unpublished as well as published data. Some contents of Mukesh Kumar Rana and Amit Kumar Singh chapters have been sourced by the authors from their prior publications. 17 Organizing Committee 139 Preface

his training manual is a compilation of lectures delivered during the two-day. Training Course on TManagement of Fruit Genetic Resources, held during Feb. 1-2, 2021. The training was organized by ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, in collaboration with ICAR-All India Coordinated Research Project (AICRP) on Fruits and ICAR-Indian Institute of Horticultural Research (IIHR), Bengaluru. The genesis of this training was a recommendation emanating from the Seventh Group Discussion of ICAR-AICRP on Fruits, held at Punjab Agricultural University (PAU), Ludhiana. Due to the online mode of the training, a large number (72) of researchers (Scientists and University Faculty) belonging to 9 institutes of the Indian Council of Agricultural Research (ICAR) and departments of 32 State Agricultural Universities (SAUs) participated in the program. Participants predominantly specialized in Horticulture (86.3%) and others belonged to disciplines like Agril Entomology (9.6%), Plant Pathology (2.7%) and Soil Chemistry (1.4%). The faculty belonged to ICAR-NBPGR (headquarters and regional stations) and the Project Coordinating Unit of ICAR-AICRP on Fruits. Information in the manual has been provided to enable the participants for easy access to basic overview on principles of plant genetic resources (PGR) management.

Organization of this training course has been possible due to support of several individuals. At the outset, sincere gratitude is accorded to Dr Anand Kumar Singh, Deputy Director General (Horticultural Science), ICAR, for motivating and facilitating this activity. Dr M.R. Dinesh, Director, ICAR-IIHR and Dr B. K. Pandey, ADG (HS-II), are thanked for their support in organizing in the course. Dr Kuldeep Singh, Director, ICAR- NBPGR & Course Director, provided excellent guidance and institutional support in planning and execution of the event. Dr Prakash Patil, Project Coordinator, AICRP on Fruits, ICAR-IIHR & Course Coordinator, has been the guiding force and extended immense support for preparation of the training course. He and his entire team are thanked profusely.

The Training Co-Convenors are sincerely thanked for carrying out all the 'behind-the-scene' work very diligently and quietly. Dr Sridhar Gutam, Sr. Scientist, AICRP on Fruits, Bengaluru, has provided excellent support, especially in conducting the quiz and providing all the communication support. Dr Vartika Srivastava and Dr Era Vaidya Malhotra, Scientists, ICAR-NBPGR, New Delhi, have extended all the logistic support in invitations, lecture arrangement, manual preparation and convening the technical sessions. Dr K.S. Hooda, HRD Nodal Officer, ICAR-NBPGR, has helped in the administrative matters for conducting the training. Mr Vijay Mandal, Technical Assistant, ICAR-NBPGR, is thanked for technical support in online management (hosting) of the meeting.

Sincere gratitude is extended to all the Resource Persons for agreeing to be faculty of the training course, and sparing their valuable time to share their rich experience with the trainees. All trainees are thanked for agreeing to join the course, even at very short notice. Grateful thanks are accorded to all the respective organizations for nominating and supporting their participation.

(Anuradha Agrawal) Course Convenor & Officer-In-Charge, Tissue Culture and Cryopreservation Unit ICAR-NBPGR, New Delhi, India

05 Preface

The Training Co-Convenors are sincerely thanked for carrying out all the 'behind-the-scene' work very diligently and quietly. Dr Sridhar Gutam, Sr. Scientist, AICRP on Fruits, Bengaluru, has provided excellent support, especially in conducting the quiz and providing all the communication support. Dr Vartika Srivastava and Dr Era Vaidya Malhotra, Scientists, ICAR-NBPGR, New Delhi, have extended all the logistic support in invitations, lecture arrangement, manual preparation and convening the technical sessions. Dr K.S. Hooda, HRD Nodal Officer, ICAR-NBPGR, has helped in the administrative matters for conducting the training. Mr Vijay Kumar Mandal, Senior Technical Assistant, ICAR-NBPGR, is thanked for technical support in online management (hosting) of the meeting.

(Anuradha Agrawal) Course Convenor & Officer-In-Charge, Tissue Culture and Cryopreservation Unit ICAR-NBPGR, New Delhi, India

05 Training Schedule Time Topic/Activity Speaker 11.15-12.00 PM Lecture 10: Germplasm Core Collection Rakesh Singh, – Principle and Methodologies ICAR-NBPGR, New Delhi Time Topic/Activity Speaker 12.00-12.45 PM Lecture 11: Registration of PGR Anjali Kak, Day 1 – Feb. 1 (Monday) ICAR-NBPGR, New Delhi 9.30-10.00 AM INAUGURAL SESSION Co-Chairs: Kuldeep Singh, 12.45-1.30 PM Lunch Director, ICAR-NBPGR and Prakash Patil, PC, AICRP (Fruits) TECHNICAL SESSION -III Convenor: Era Vaidya Malhotra Convenor: Anuradha Agrawal 1.30-2.15 PM Lecture 12: Holistic Approach for Anuradha Agrawal, Germplasm Conservation of Fruit Crops ICAR-NBPGR, New Delhi 10.30-11.15 AM Lecture 1: PGR Management System Kuldeep Singh, in India ICAR-NBPGR, New Delhi 2.15-3.00 PM Lecture 13: DNA Fingerprinting and Mukesh Rana, Use of Genomics in PGR Management ICAR-NBPGR, New Delhi 11.15-11.30 AM Break 3.00-3.15 AM Break TECHNICAL SESSION -1 Convenor: Vartika Srivastava 3.15-4.00 PM INTERACTIVE SESSION Convenors: Prakash Patil and 11.15-12.00 PM Lecture 2: Principles of Plant S. P. Ahlawat, Sridhar Gutam, Exploration and Germplasm Collection ICAR-NBPGR, New Delhi AICRP (Fruits), Bengaluru 12.00-12.45 PM Lecture 3: Plant Exploration and K. Joseph John, Invited Speaker: S. K. Malik, Germplasm Collecting in Fruit Crops – ICAR-NBPGR, Thrissur ICAR, New Delhi Practical Considerations 4.00-5.10 PM VALEDICTORY SESSION 12.45-1.30 PM Lunch 04.00 PM ICAR Song TECHNICAL SESSION -II Convenor: Era Vaidya Malhotra 04.05 - 04.10 PM Training Report Dr. Anuradha Agrawal 1.30-2.15 PM Lecture 4: Germplasm Access and Pratibha Brahmi, (Training Convenor) Related Policy Issues ICAR-NBPGR, New Delhi ICAR-NBPGR, New Delhi 2.15-3.00 PM Lecture 5: Procedures for Germplasm Pragya Ranjan, 04.10– 04.15 PM Trainees Feedback From Trainees Exchange ICAR-NBPGR, New Delhi 04.15 –04.20 PM Remarks by Project Coordinator Dr. Prakash Patil 3.00-3.15 PM Break (Training Coordinator) 3.15-4.00 PM Lecture 6: National Plant Quarantine S. C. Dubey, Project Coordinator (Fruits) System ICAR-NBPGR, New Delhi ICAR-IIHR, Bengaluru 4.00-4.45 PM Lecture 7: PGR Informatics and Data Sunil Archak, 04.20–04.25 PM Remarks by Director Dr. Kuldeep Singh Management ICAR-NBPGR, New Delhi (Training Director) Director, ICAR-NBPGR, New Delhi Day 2, Feb. 2 (Tuesday) 04.25 – 04.35 PM Remarks by Guests of Honour Dr. M. R. Dinesh TECHNICAL SESSION -III Convenor: Vartika Srivastava Director, ICAR-IIHR, Bengaluru 9.30-10.15 AM Lecture 8: Principles of Germplasm Kuldeep Tripathi, 04.35 – 04.45 PM Remarks by Guests of Honour Dr. B. K. Pandey Characterization and Evaluation ICAR-NBPGR, New Delhi Assistant Director General (Hort. Sci.- II) 10.15-11.00 AM Lecture 9: Germplasm Characterization Narendra Negi, ICAR, New Delhi and Evaluation of Fruit Crops – Use of ICAR-NBPGR, Shimla 04.45 – 05.05 PM Remarks by Chief Guest Dr. Anand Kumar Singh Descriptors and Other Practical Deputy Director General (Hort. Sci.) Considerations ICAR, New Delhi 11.00-11.15 AM Break 05.05 – 05.10 PM Vote of Thanks D. K.S. Hooda HRD Nodal Officer, ICAR-NBPGR ICAR-NBPGR, New Delhi

06 07 Training Schedule Time Topic/Activity Speaker 11.15-12.00 PM Lecture 10: Germplasm Core Collection Rakesh Singh, – Principle and Methodologies ICAR-NBPGR, New Delhi Time Topic/Activity Speaker 12.00-12.45 PM Lecture 11: Registration of PGR Anjali Kak, Day 1 – Feb. 1 (Monday) ICAR-NBPGR, New Delhi 9.30-10.00 AM INAUGURAL SESSION Co-Chairs: Kuldeep Singh, 12.45-1.30 PM Lunch Director, ICAR-NBPGR and Prakash Patil, PC, AICRP (Fruits) TECHNICAL SESSION -III Convenor: Era Vaidya Malhotra Convenor: Anuradha Agrawal 1.30-2.15 PM Lecture 12: Holistic Approach for Anuradha Agrawal, Germplasm Conservation of Fruit Crops ICAR-NBPGR, New Delhi 10.30-11.15 AM Lecture 1: PGR Management System Kuldeep Singh, in India ICAR-NBPGR, New Delhi 2.15-3.00 PM Lecture 13: DNA Fingerprinting and Mukesh Rana, Use of Genomics in PGR Management ICAR-NBPGR, New Delhi 11.15-11.30 AM Break 3.00-3.15 AM Break TECHNICAL SESSION -1 Convenor: Vartika Srivastava 3.15-4.00 PM INTERACTIVE SESSION Convenors: Prakash Patil and 11.15-12.00 PM Lecture 2: Principles of Plant S. P. Ahlawat, Sridhar Gutam, Exploration and Germplasm Collection ICAR-NBPGR, New Delhi AICRP (Fruits), Bengaluru 12.00-12.45 PM Lecture 3: Plant Exploration and K. Joseph John, Invited Speaker: S. K. Malik, Germplasm Collecting in Fruit Crops – ICAR-NBPGR, Thrissur ICAR, New Delhi Practical Considerations 4.00-5.10 PM VALEDICTORY SESSION 12.45-1.30 PM Lunch 04.00 PM ICAR Song TECHNICAL SESSION -II Convenor: Era Vaidya Malhotra 04.05 - 04.10 PM Training Report Dr. Anuradha Agrawal 1.30-2.15 PM Lecture 4: Germplasm Access and Pratibha Brahmi, (Training Convenor) Related Policy Issues ICAR-NBPGR, New Delhi ICAR-NBPGR, New Delhi 2.15-3.00 PM Lecture 5: Procedures for Germplasm Pragya Ranjan, 04.10– 04.15 PM Trainees Feedback From Trainees Exchange ICAR-NBPGR, New Delhi 04.15 –04.20 PM Remarks by Project Coordinator Dr. Prakash Patil 3.00-3.15 PM Break (Training Coordinator) 3.15-4.00 PM Lecture 6: National Plant Quarantine S. C. Dubey, Project Coordinator (Fruits) System ICAR-NBPGR, New Delhi ICAR-IIHR, Bengaluru 4.00-4.45 PM Lecture 7: PGR Informatics and Data Sunil Archak, 04.20–04.25 PM Remarks by Director Dr. Kuldeep Singh Management ICAR-NBPGR, New Delhi (Training Director) Director, ICAR-NBPGR, New Delhi Day 2, Feb. 2 (Tuesday) 04.25 – 04.35 PM Remarks by Guests of Honour Dr. M. R. Dinesh TECHNICAL SESSION -III Convenor: Vartika Srivastava Director, ICAR-IIHR, Bengaluru 9.30-10.15 AM Lecture 8: Principles of Germplasm Kuldeep Tripathi, 04.35 – 04.45 PM Remarks by Guests of Honour Dr. B. K. Pandey Characterization and Evaluation ICAR-NBPGR, New Delhi Assistant Director General (Hort. Sci.- II) 10.15-11.00 AM Lecture 9: Germplasm Characterization Narendra Negi, ICAR, New Delhi and Evaluation of Fruit Crops – Use of ICAR-NBPGR, Shimla 04.45 – 05.05 PM Remarks by Chief Guest Dr. Anand Kumar Singh Descriptors and Other Practical Deputy Director General (Hort. Sci.) Considerations ICAR, New Delhi 11.00-11.15 AM Break 05.05 – 05.10 PM Vote of Thanks D. K.S. Hooda HRD Nodal Officer, ICAR-NBPGR ICAR-NBPGR, New Delhi

06 07 List of Faculty List of Trainees

Dr Kuldeep Singh Dr Sridhar Gutam S.No. Details Mobile No. Email Director, Senior Scientist 1. Ashok Kumar Singh ICAR - National Bureau of Plant Genetic ICAR-AICRP on Fruits, Horticulturist, Department of Horticulture, Resources, New Delhi ICAR- Indian Institute of Horticultural Research, College of Agriculture G.B. Pant University Bengaluru of Agriculture and Technology, Pantnagar, Drashokkumar71 Dr Prakash Patil Uttarakhand 09411324825 @gmail.com Project Coordinator (Fruits) Dr Joseph John K. ICAR-AICRP on Fruits Principal Scientist 2. Anjali R. Mendhe ICAR - Indian Institute of Horticultural Research, ICAR- National Bureau of Plant Genetic Jr. Soil Chemist, Banana Research Station Mahatma Phule Krishi Vidyapeeth, Jalgaon, Bengaluru Resources, Regional Station, Thrissur, Kerala Maharashtra 09421149695 [email protected] Dr Anuradha Agrawal Dr Anjali Kak 3. A. Snehalatha Rani Principal Scientist and Officer-In-Charge (OIC) Principal Scientist Jr. Plant Pathologist, Horticultural Research Tissue Culture and Cryopreservation Unit Division of Germplasm Conservation Station, Kovvur, Dr Y.S.R. Horticultural ICAR - National Bureau of Plant Genetic ICAR - National Bureau of Plant Genetic University, West Godavari, Andhra Pradesh 09491880596 [email protected] Resources, New Delhi Resources, New Delhi 4. Ajay Y. Munj Jr. Entomologist, Regional Fruit Research Dr S.P. Ahlawat Dr S.K. Malik Station, Vengurle, Dr Balasaheb Sawant Konkan Head (Acting) and Principal Scientist Scientific Officer to DG Krishi Vidyapeeth, Sindhudurg, Maharashtra 09421916947 [email protected] Division of Plant Exploration and Germplasm ICAR, New Delhi 5. Ankit Bhandari Collection Jr. Horticulturist, Agriculture Experimental ICAR - National Bureau of Plant Genetic Dr Narender Negi Station, Navsari Agricultural University, Paria, Resources, New Delhi Scientist Gujarat 09033171336 [email protected] ICAR- National Bureau of Plant Genetic Dr Sunil Archak Resources, Regional Station, Shimla, H. P. 6. Ankur P. Patel ICAR-National Fellow and Principal Scientist Horticulturist, Fruit Research Station Agriculture Knowledge Management Unit Dr Mukesh Rana Navsari Agricultural University, Gandevi, ICAR - National Bureau of Plant Genetic Principal Scientist Gujarat 09427112947 [email protected] Resources, New Delhi Division of Genomic Resources 7. Ashok Yadav ICAR - National Bureau of Plant Genetic Scientist, ICAR-Central Institute for Subtropical Dr Pratibha Brahmi Resources, New Delhi Horticulture Regional Research Station, Principal Scientist Malda, West Bengal 07042360966 [email protected] Germplasm Exchange & Policy Unit Dr Rakesh Singh 8. Asish Kumar Panda ICAR - National Bureau of Plant Genetic Principal Scientist Assistant Professor (Fruit Science), Department Resources, New Delhi Division of Genomic Resources of Horticulture, Dr. Rajendra Prasad Central ICAR - National Bureau of Plant Genetic Agricultural University, Pusa, Samastipur, Bihar 09468356627 [email protected] Dr S.C. Dubey Resources, New Delhi ADG (Plant Protection) ICAR and Ex-Head 9. Awachare Chandrakant Madhav Division of Plant Quarantine Dr Kuldeep Tripathi Scientist, Central Horticultural Experiment Station-IIHR, Chettalli, Kodagu, Karnataka 08073541570 [email protected] ICAR - National Bureau of Plant Genetic Scientist Resources, New Delhi Division of Germplasm Evaluation 10. Bhabesh Deka ICAR - National Bureau of Plant Genetic Horticulturist, Department of Horticulture Dr Kavita Gupta Resources, New Delhi Assam Agricultural University, Jorhat, Assam 07086462046 [email protected] Principal Scientist and OIC, PME 11. Chinnasamy Kavitha Division of Plant Quarantine Jr. Horticulturist, Department of Fruit Science, ICAR - National Bureau of Plant Genetic Horticultural College and Research Institute Resources, New Delhi Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 08870733444 [email protected]

08 09 List of Faculty List of Trainees

08 09 List of Trainees

12. C. S. Maiti 24. Harinder Singh Rattanpal Professor & Horticulturist, Department of Principal Horticulturist, Department of Fruit Horticulture, School of Agricultural Sciences Science, Punjab Agricultural University, and Rural Development, Nagaland University, Ludhiana, Punjab 07999777773 [email protected] Medziphema, Nagaland 08132030283 [email protected] 25. Inee Gogoi 13. Chanda Kushwaha Jr. Scientist (Entomology), Department of Jr. Plant Pathologist, Department of Plant Horticulture, Assam Agricultural University, Pathology, Bihar Agricultural University, chanda.kushwaha Jorhat, Assam 09678084720 [email protected] Sabour, Bhagalpur, Bihar 09532982363 @gmail.com 26. Jyoti P. Dutta 14. Chirag R. Patel Horticulturist, Citrus Research Station Horticulturist, Agriculture Experimental Station Assam Agricultural University, Tinsukia, Assam 09435387295 [email protected] Navsari Agricultural University, Paria, Gujarat 09428160937 [email protected] 27. Julius Uchoi 15. Dinesh H.Paithankar Scientist, ICAR-National Bureau of Plant Jr. Horticulturist, Dr Panjabrao, Deshmukh dineshpaithankar1972@ Genetic Resources, Regional Station-Shillong, Krishi Vidyapeeth, Akola Maharashtra 07038037642 gmail.com Umaim, Meghalaya [email protected] 16. Dilip K. Misra 28. K. Abirami Jr. Plant Pathologist, Directorate of Research, Senior Scientist, Division of Horticulture and Bidhan Chandra Krishi Viswavidyalaya Forestry, ICAR-Central Island Agricultural Kalyani, Nadia, West Bengal 09433743506 [email protected] Research Institute, Port Blair, Andaman and Nicobar Islands 09476012040 [email protected] 17. Debalina Majhi Jr. Horticulturist, Directorate of Research, 29. K. D. Bisane Bidhan Chandra Krishi Viswavidyalaya drdebalina.bckv.fruits@ Jr. Entomologist, Fruit Research Station Kalyani, Nadia, West Bengal 09007902376 gmail.com Navsari Agricultural University, Gandevi, Gujarat 09426603068 [email protected] 18. H. Lembisana Devi Scientist, ICAR Research Complex for North 30. Krishna Madhav Rai Eastern Hill Region, Tripura Centre, Scientist, ICAR-National Bureau of Plant Lembucherra, West Tripura 08415917083 [email protected] Genetic Resources, Regional Station-Bhowali, Nainital, Uttarakhand [email protected] 19. Evening Stone Marboh Scientist, ICAR-National Research Centre for 31. Katakam Mounika Litchi, Mushahari, Muzaffarpur, Bihar 07070993308 [email protected] Jr. Plant Pathologist, Fruit Research Station, Sangareddy, Sri Konda Laxman Telangana State mounikakatakam 20. Fatik Kumar Bauri Horticultural University, Medak, Telangana 08790688931 @gmail.com Horticulturist, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya 09674605720 32. KantharajuVenkatappa Kalyani, Nadia, West Bengal 09433678461 [email protected] Jr. Plant Pathologist, Kittur Rani Channama College of Horticulture, Arabhavi, University 21. Ganeshi Lal Sharma of Horticultural Sciences, Belagavi, Karnataka 09448584749 [email protected] Associate Professor, Department of Fruit Science, College of Agriculture, Indira Gandhi 33. L. Mukunda Lakshmi Krishi Vishwavidyalaya, Raipur, Chhattisgarh 09425575505 [email protected] Jr. Horticulturist, Citrus Research Station Dr Y.S.R. Horticultural University, Tirupati, 22. Gunna Ramanandam Andhra Pradesh 09347115175 [email protected] Horticulturist, Horticultural Research Station Kovvur, West Godavari, Andhra Pradesh 07382633659 [email protected] 34. Lobsang Wangchu Associate Professor, Department of Fruit 23. Ganesh Deshmukh Science, College of Horticulture and Forestry Plant Pathologist, Banana Research Station Central Agricultural University, Pasighat, Mahatma Phule Krishi Vidyapeeth, Jalgaon, Arunachal Pradesh 09436053001 [email protected] Maharashtra 09422021016 [email protected]

10 11 List of Trainees

10 11 35. Madhukar H. Shete 46. Piloo Ngangbam Jr. Plant Pathologist, Department of Horticulture Associate Professor, Dept. of Horticulture, Mahatma Phule Krishi Vidyapeeth, Rahuri, mhshete123 College of Agriculture, Central Agricultural Rahuri, Ahmednagar, Maharashtra 09421951972 @rediffmail.com University, Imphal, Manipur 09436838579 [email protected] 36. M. S. Aneesa Rani 47. Poonam Srivastava Sr. Horticulturist, Department of Fruit Science, Sr. Entomologist, Department of Entomology, Horticultural College and Research Institute College of Agriculture, G.B. Pant University Tamil Nadu Agricultural University, of Agriculture And Technology, Pantnagar, Coimbatore, Tamil Nadu 09788923254 [email protected] Uttarakhand 09411159448 [email protected] 37. Mahendra Gawankar 48. Popy Bora Horticulturist, Regional Fruit Research Station, Jr. Scientist (Plant Pathology), Department of Vengurle, Dr BalasahebSawant Konkan Krishi Horticulture, Assam Agricultural University, Vidyapeeth, Sindhudurg, Maharashtra 09420110596 [email protected] Jorhat, Assam 08638493087 [email protected] 38. M. S. Saraswathi 49. Pravin Modi Principal Scientist (Horticulture), Crop Jr. Horticulturist, Fruit Research Station Improvement Division, ICAR-National Navsari Agricultural University, Gandevi, Research Centre of Banana, Tiruchirappalli, Gujarat 07228012765 [email protected] Tamil Nadu 09443590188 [email protected] 50. S. Priya Devi 39. Mahesh Kumar Dhakar Principal Scientist, ICAR-Indian Institute of Scientist, ICAR Research Complex for Horticultural Research, Bengaluru, Karnataka 08806367111 [email protected] Eastern Region, Research Centre, Rajaulathu, 51. Ramajayam Devarajan Plandu, Ranchi, Jharkhand 08603819863 [email protected] Principal Scientist (Horticulture), Crop 40. Manju P.R. Improvement Division, ICAR-National Research Jr. Horticulturist,Banana Research Station Centre for Banana, Tiruchirapalli, Tamil Nadu 08333027598 [email protected] Kerala Agricultural University, Kannara, 52. Ravindra Kumar Thrissur, Kerala 09847102430 [email protected] Jr. Horticulturist, Department of Horticulture 41. Nagalakshmi Thota Bihar Agricultural University, Sabour, kravindra70 Jr. Plant Pathologist, Horticultural Research Bhagalpur, Bihar 09431860187 @rediffmail.com Station, Anantharajupet, Dr Y.S.R. Horticultural 53. Roshni Samarth University, Kadapa, Andhra Pradesh 09110521374 [email protected] Senior Scientist, ICAR-National Research 42. Nitin Soni Centre for Grapes, Pune, Maharashtra 09970111722 [email protected] Jr. Horticulturist, College of Horticulture 54. Shri Kishan Bairwa Mandsaur, Madhya Pradesh 07223997907 [email protected] Jr. Pathologist, Agricultural Research Station 43. Polamuri Ashok Swami Keshwanand Rajasthan Agricultural Jr. Horticulturist, Horticultural Research Station, University, Sriganganagar, Rajasthan 09414269869 [email protected] Kovvur, Dr Y.S.R. Horticultural University, 55. S. Muthuramalingam West Godavari, Andhra Pradesh 09440834898 [email protected] Jr. Horticulturist, Department of Fruit Crops & 44. P. B. Pushpalatha PHT, Horticultural College and Research Horticulturist, Banana Research Station Institute, Tamil Nadu Agricultural University, muthuramhort Kerala Agricultural University, Kannara, pushpavellanikkara@ Periyakulam, Tamil Nadu 09790058011 @yahoo.co.in Thrissur, Kerala 09496245127 gmail.com 56. Shreedhar Singh Lakhawat 45. Pandiyan Indiragandhi Associate Professor, Department of Horticulture, Jr. Entomologist, Department of Fruit Crops, Rajasthan College of Agriculture, Maharana Horticultural College and Research Institute Pratap University of Agriculture & Technology, Tamil Nadu Agricultural University, Periyakulam, Udaipur, Rajasthan 09784034655 [email protected] Tamil Nadu 09655867995 [email protected]

12 13 35. Madhukar H. Shete 46. Piloo Ngangbam Jr. Plant Pathologist, Department of Horticulture Associate Professor, Dept. of Horticulture, Mahatma Phule Krishi Vidyapeeth, Rahuri, mhshete123 College of Agriculture, Central Agricultural Rahuri, Ahmednagar, Maharashtra 09421951972 @rediffmail.com University, Imphal, Manipur 09436838579 [email protected] 36. M. S. Aneesa Rani 47. Poonam Srivastava Sr. Horticulturist, Department of Fruit Science, Sr. Entomologist, Department of Entomology, Horticultural College and Research Institute College of Agriculture, G.B. Pant University Tamil Nadu Agricultural University, of Agriculture And Technology, Pantnagar, Coimbatore, Tamil Nadu 09788923254 [email protected] Uttarakhand 09411159448 [email protected] 37. Mahendra Gawankar 48. Popy Bora Horticulturist, Regional Fruit Research Station, Jr. Scientist (Plant Pathology), Department of Vengurle, Dr BalasahebSawant Konkan Krishi Horticulture, Assam Agricultural University, Vidyapeeth, Sindhudurg, Maharashtra 09420110596 [email protected] Jorhat, Assam 08638493087 [email protected] 38. M. S. Saraswathi 49. Pravin Modi Principal Scientist (Horticulture), Crop Jr. Horticulturist, Fruit Research Station Improvement Division, ICAR-National Navsari Agricultural University, Gandevi, Research Centre of Banana, Tiruchirappalli, Gujarat 07228012765 [email protected] Tamil Nadu 09443590188 [email protected] 50. S. Priya Devi 39. Mahesh Kumar Dhakar Principal Scientist, ICAR-Indian Institute of Scientist, ICAR Research Complex for Horticultural Research, Bengaluru, Karnataka 08806367111 [email protected] Eastern Region, Research Centre, Rajaulathu, 51. Ramajayam Devarajan Plandu, Ranchi, Jharkhand 08603819863 [email protected] Principal Scientist (Horticulture), Crop 40. Manju P.R. Improvement Division, ICAR-National Research Jr. Horticulturist,Banana Research Station Centre for Banana, Tiruchirapalli, Tamil Nadu 08333027598 [email protected] Kerala Agricultural University, Kannara, 52. Ravindra Kumar Thrissur, Kerala 09847102430 [email protected] Jr. Horticulturist, Department of Horticulture 41. Nagalakshmi Thota Bihar Agricultural University, Sabour, kravindra70 Jr. Plant Pathologist, Horticultural Research Bhagalpur, Bihar 09431860187 @rediffmail.com Station, Anantharajupet, Dr Y.S.R. Horticultural 53. Roshni Samarth University, Kadapa, Andhra Pradesh 09110521374 [email protected] Senior Scientist, ICAR-National Research 42. Nitin Soni Centre for Grapes, Pune, Maharashtra 09970111722 [email protected] Jr. Horticulturist, College of Horticulture 54. Shri Kishan Bairwa Mandsaur, Madhya Pradesh 07223997907 [email protected] Jr. Pathologist, Agricultural Research Station 43. Polamuri Ashok Swami Keshwanand Rajasthan Agricultural Jr. Horticulturist, Horticultural Research Station, University, Sriganganagar, Rajasthan 09414269869 [email protected] Kovvur, Dr Y.S.R. Horticultural University, 55. S. Muthuramalingam West Godavari, Andhra Pradesh 09440834898 [email protected] Jr. Horticulturist, Department of Fruit Crops & 44. P. B. Pushpalatha PHT, Horticultural College and Research Horticulturist, Banana Research Station Institute, Tamil Nadu Agricultural University, muthuramhort Kerala Agricultural University, Kannara, pushpavellanikkara@ Periyakulam, Tamil Nadu 09790058011 @yahoo.co.in Thrissur, Kerala 09496245127 gmail.com 56. Shreedhar Singh Lakhawat 45. Pandiyan Indiragandhi Associate Professor, Department of Horticulture, Jr. Entomologist, Department of Fruit Crops, Rajasthan College of Agriculture, Maharana Horticultural College and Research Institute Pratap University of Agriculture & Technology, Tamil Nadu Agricultural University, Periyakulam, Udaipur, Rajasthan 09784034655 [email protected] Tamil Nadu 09655867995 [email protected]

12 13 57. Samik Sengupta 68. Vishnu K. Garande Jr. Horticulturist, Department of Horticulture Horticulturist, Department of Horticulture Bihar Agricultural University, Sabour, samik.sengupta@ Mahatma Phule Krishi Vidyapeeth, Bhagalpur, Bihar 09608922431 rediffmail.com Ahmednagar, Maharashtra 09850028986 [email protected] 58. Sanjit Debnath 69. Vijay Singh Meena Jr. Horticulturist, Directorate of Research Scientist, ICAR-National Bureau of Plant Bidhan Chandra Krishi Viswavidyalaya, Genetic Resources, Regional Station-Jodhpur, Kalyani, West Bengal 08420117115 [email protected] Rajasthan [email protected] 59. Sarad Gurung 70. Vikas Kumar Sharma Associate Professor, Regional Research Station, Assistant Professor, Department of Fruit Science, Hill Zone, Kalimpong, Uttar Banga Krishi College of Horticulture & Forestry, Viswavidyalaya, Darjeeling, West Bengal Dr. Y.S. Parmar University of Horticulture and Regional Research Station (UBKV), Hill Zone, sgurung_hort Forestry, Hamirpur, Himachal Pradesh 07018601976 [email protected] Kalimpong-734301, West Bengal 09434429066 @rediffmail.com 71. Y. Sharath Kumar Reddy 60. Satish B. Jadhav Jr. Horticulturist, Horticultural Research Station, Horticulturis, Department of Horticulture, Anantharajupet, Dr Y.S.R. Horticultural Rahuri, Mahatma Phule Krishi Vidyapeeth, satishjadhav2010@ University, Kadapa, Andhra Pradesh 09966642541 [email protected] Ahmednagar, Maharashtra 09404683709 rediffmail.com 72. Yogesh Ingle 61. Satish Chand Jr. Plant Pathologist, AICRP on Fruits Jr. Horticulturist, Department of Horticulture, Dr Panjabrao Deshmukh Krishi Vidyapeeth College of Agriculture, G.B. Pant University Akola, Maharashtra 09422766437 [email protected] of Agriculture and Technology, Pantnagar, Uttarakhand 09412120885 [email protected] 62. Saudamini Swain Jr. Horticulturist, College of Agriculture Odisha University of Agriculture & Technology, Bhubaneswar, Odisha 09437133261 [email protected] 63. Sikha Deka Jr. Entomologist, Citrus Research Station Assam Agricultural University, Tinsukia, Assam 09435593421 [email protected] 64. Sumanjit Kaur Fruit Scientist, M.S. Randhawa Fruit Research Station, Gangian, Punjab Agricultural University, Hoshiarpur, Punjab 08872428885 [email protected] 65. T. K. Singh Jr. Horticulturist, Fruit Research Station, Kuthuliya, Jawaharlal Nehru Krishi Vishwavidyalaya, Rewa, Madhya Pradesh 09752505265 [email protected] 66. Thallapally Baby Rani Horticulturist, Grape Research Station, Rajendranagar, Sri Konda Laxman Telangana State Horticultural University, Hyderabad Telangana 09959986597 [email protected] 67. U.S. Bose Horticulturist, Fruit Research Station, Kuthuliya Jawaharlal Nehru Krishi Vishwavidyalaya, Rewa, Madhya Pradesh 09424908404 [email protected]

14 15 57. Samik Sengupta 68. Vishnu K. Garande Jr. Horticulturist, Department of Horticulture Horticulturist, Department of Horticulture Bihar Agricultural University, Sabour, samik.sengupta@ Mahatma Phule Krishi Vidyapeeth, Bhagalpur, Bihar 09608922431 rediffmail.com Ahmednagar, Maharashtra 09850028986 [email protected] 58. Sanjit Debnath 69. Vijay Singh Meena Jr. Horticulturist, Directorate of Research Scientist, ICAR-National Bureau of Plant Bidhan Chandra Krishi Viswavidyalaya, Genetic Resources, Regional Station-Jodhpur, Kalyani, West Bengal 08420117115 [email protected] Rajasthan [email protected] 59. Sarad Gurung 70. Vikas Kumar Sharma Associate Professor, Regional Research Station, Assistant Professor, Department of Fruit Science, Hill Zone, Kalimpong, Uttar Banga Krishi College of Horticulture & Forestry, Viswavidyalaya, Darjeeling, West Bengal Dr. Y.S. Parmar University of Horticulture and Regional Research Station (UBKV), Hill Zone, sgurung_hort Forestry, Hamirpur, Himachal Pradesh 07018601976 [email protected] Kalimpong-734301, West Bengal 09434429066 @rediffmail.com 71. Y. Sharath Kumar Reddy 60. Satish B. Jadhav Jr. Horticulturist, Horticultural Research Station, Horticulturis, Department of Horticulture, Anantharajupet, Dr Y.S.R. Horticultural Rahuri, Mahatma Phule Krishi Vidyapeeth, satishjadhav2010@ University, Kadapa, Andhra Pradesh 09966642541 [email protected] Ahmednagar, Maharashtra 09404683709 rediffmail.com 72. Yogesh Ingle 61. Satish Chand Jr. Plant Pathologist, AICRP on Fruits Jr. Horticulturist, Department of Horticulture, Dr Panjabrao Deshmukh Krishi Vidyapeeth College of Agriculture, G.B. Pant University Akola, Maharashtra 09422766437 [email protected] of Agriculture and Technology, Pantnagar, Uttarakhand 09412120885 [email protected] 62. Saudamini Swain Jr. Horticulturist, College of Agriculture Odisha University of Agriculture & Technology, Bhubaneswar, Odisha 09437133261 [email protected] 63. Sikha Deka Jr. Entomologist, Citrus Research Station Assam Agricultural University, Tinsukia, Assam 09435593421 [email protected] 64. Sumanjit Kaur Fruit Scientist, M.S. Randhawa Fruit Research Station, Gangian, Punjab Agricultural University, Hoshiarpur, Punjab 08872428885 [email protected] 65. T. K. Singh Jr. Horticulturist, Fruit Research Station, Kuthuliya, Jawaharlal Nehru Krishi Vishwavidyalaya, Rewa, Madhya Pradesh 09752505265 [email protected] 66. Thallapally Baby Rani Horticulturist, Grape Research Station, Rajendranagar, Sri Konda Laxman Telangana State Horticultural University, Hyderabad Telangana 09959986597 [email protected] 67. U.S. Bose Horticulturist, Fruit Research Station, Kuthuliya Jawaharlal Nehru Krishi Vishwavidyalaya, Rewa, Madhya Pradesh 09424908404 [email protected]

14 15 • Ranchi (Jharkhand): Established in 1988 for germplasm of tropical fruits and other field crops of Bihar, PGR Management System in India eastern Uttar Pradesh, Jharkhand and West Bengal. Kuldeep Singh*, S. Rajkumar and Kavita Gupta • Srinagar (Jammu & Kashmir): Established in 1988 for agri-horticultural germplasm of temperate crops. ICAR- National Bureau of Plant Genetic Resources, New Delhi PGR are conserved in the form of seeds, *Email: [email protected] vegetative propagules, tissue/ cell cultures, embryos, pollen etc. in the Genebank. The National Genebank Introduction facility commissioned in 1997 has 12 The ICAR-National Bureau of Plant Genetic Resources (NBPGR) was established by the Indian Council of long-term storage modules, each with a Agricultural Research (ICAR) in 1976 with its headquarters at New Delhi. The chronology of events leading to storage capacity of 50,000 to 76,000 the present day ICAR-NBPGR dates back to 1905 when Botany Division was established under the then samples depending upon the size of Imperial Agricultural Research Institute. ICAR-NBPGR has been given the mandate to act as a nodal institute seeds. Its cryopreservation facility at the national level for acquisition and management of indigenous and exotic plant genetic resources (PGR) contains six liquid nitrogen storage for agriculture, and to carry out related research and human resources development for sustainable growth tanks (cryo-tanks), each having a of agriculture. The Bureau is also vested with the authority to issue Import Permit and Phytosanitary capacity to hold 1,000 litres of liquid Certificate and conduct quarantine checks in seed material and vegetative propagules (including transgenic nitrogen. These six cryo-tanks have a material) introduced from abroad or exported for research purposes. Besides having a 40 ha experimental total capacity to store 0.25 million farm at Issapur village (about 45 km west of Pusa Campus), the Bureau has a strong national network samples. Thus, the National Genebank comprising Regional Stations/ Base Centres and ICAR Institutes/ SAUs that provide access to representative has a total capacity to store 0.85 to 1.25 agro-ecological situations in the country (Fig. 1). million samples. This is one of the most modern Genebanks of the world. Mandate The Bureau not only conserves PGR safely to The mandate includes management and sustainable use of plant genetic and genomic resources of agri- meet the needs of future generations, but horticultural crops and carry out related research; coordination of capacity building in PGR management also provides these to the nation's crop and policy issues governing access and benefit sharing of their use, and molecular profiling of varieties of improvement programmes to sustain Fig. 1: NBPGR network of regional stations and base centres agri-horticultural crops and GM detection technology research. continued advances in agricultural ICAR-NBPGR has its headquarters in New Delhi that hosts the second largest genebank in the world. The productivity and stabilize production. The Bureau works in close collaboration with several international operations are administered by Divisions of Plant Exploration and Germplasm Collection, Germplasm institutes/ organizations through memoranda/work-plans developed under bilateral/multilateral Evaluation, Germplasm Conservation, Genomic Resources and Plant Quarantine in addition to the Units of agreements. It exchanges plant germplasm with over 100 countries. The Bureau is gradually developing and Germplasm Exchange and Tissue Culture and Cryopreservation. ICAR-NBPGR has the network of 10 strengthening the national plant genetic resources system by linking up the National Base Collection (kept Regional Stations covering different agro-climatic zones to carry out PGR activities including collection, under long-term storage at ICAR-NBPGR) with National Active Germplasm Sites (NAGS) responsible for characterization, evaluation and maintenance of various crops as mentioned below: different crops where germplasm collections are evaluated and multiplied under field conditions backed by medium-term storage facilities. The various activities and achievement of the Bureau are presented here, • Shimla (Himachal Pradesh): Established in 1960 for temperate crops. briefly. • Jodhpur (Rajasthan): Established in 1965 for agri-horticultural crops germplasm of arid and semi-arid PGR Exploration and Collection zones. • To develop new varieties in various agri-horticultural crops for farmers suitable to different agro- • Thrissur (Kerala): Established in 1977 for agri-horticultural crops germplasm of southern peninsular climatic conditions, new germplasm/parent material with desired traits or genes is a continuous region with particular emphasis on spices and plantation crops. requirement of the plant breeders. Therefore, periodically such germplasm is collected by ICAR-NBPGR • Akola (Maharashtra): Established in 1977 for agri-horticultural crops germplasm of central India and Scientists in collaboration with crop-based institutes of ICAR. Also, the trait-specific germplasm of Deccan Plateau. various crops has been collected from diversity-rich spots (including the difficult unreached areas in different parts of the country). • Shillong (Meghalaya): Established in 1978 for agri-horticultural crops germplasm of north-eastern region including Sikkim and parts of north Bengal. • The institute has so far undertaken 2,762 explorations and collected about 2.85 lakh accessions of crop species and their wild relatives. • Bhowali (Uttarakhand): Established in 1985 for agri-horticultural crops germplasm of sub-temperate region. • Focus on North-East and rescue missions: In total, 165 explorations undertaken and a total of 9,698 accessions (cultivated-6,622, wild-3,076) collected. 45 exploration trips to North-Eastern Hill Region • Cuttack (Odisha): Established in 1985 for agri-horticultural crops germplasm of eastern peninsular and five rescue missions to natural calamity affected areas of Uttarakhand were undertaken. Systematic region with main emphasis on rice germplasm. explorations have been conducted in the remotest parts of the country including Mon district of • Hyderabad (Telangana): Established in 1985 for quarantine clearance of agri-horticultural crops Nagaland; Anjaw, Changlang and Tirap districts of Arunachal Pradesh; and Great Nicobar. germplasm of Telangana, Andhra Pradesh and adjoining areas. Disturbed/insurgency-prone areas such as Bastar region in Chhatttisgarh; Gadchiroli in Maharashtra;

16 17 • Ranchi (Jharkhand): Established in 1988 for germplasm of tropical fruits and other field crops of Bihar, PGR Management System in India eastern Uttar Pradesh, Jharkhand and West Bengal. Kuldeep Singh*, S. Rajkumar and Kavita Gupta • Srinagar (Jammu & Kashmir): Established in 1988 for agri-horticultural germplasm of temperate crops. ICAR- National Bureau of Plant Genetic Resources, New Delhi PGR are conserved in the form of seeds, *Email: [email protected] vegetative propagules, tissue/ cell cultures, embryos, pollen etc. in the Genebank. The National Genebank Introduction facility commissioned in 1997 has 12 The ICAR-National Bureau of Plant Genetic Resources (NBPGR) was established by the Indian Council of long-term storage modules, each with a Agricultural Research (ICAR) in 1976 with its headquarters at New Delhi. The chronology of events leading to storage capacity of 50,000 to 76,000 the present day ICAR-NBPGR dates back to 1905 when Botany Division was established under the then samples depending upon the size of Imperial Agricultural Research Institute. ICAR-NBPGR has been given the mandate to act as a nodal institute seeds. Its cryopreservation facility at the national level for acquisition and management of indigenous and exotic plant genetic resources (PGR) contains six liquid nitrogen storage for agriculture, and to carry out related research and human resources development for sustainable growth tanks (cryo-tanks), each having a of agriculture. The Bureau is also vested with the authority to issue Import Permit and Phytosanitary capacity to hold 1,000 litres of liquid Certificate and conduct quarantine checks in seed material and vegetative propagules (including transgenic nitrogen. These six cryo-tanks have a material) introduced from abroad or exported for research purposes. Besides having a 40 ha experimental total capacity to store 0.25 million farm at Issapur village (about 45 km west of Pusa Campus), the Bureau has a strong national network samples. Thus, the National Genebank comprising Regional Stations/ Base Centres and ICAR Institutes/ SAUs that provide access to representative has a total capacity to store 0.85 to 1.25 agro-ecological situations in the country (Fig. 1). million samples. This is one of the most modern Genebanks of the world. Mandate The Bureau not only conserves PGR safely to The mandate includes management and sustainable use of plant genetic and genomic resources of agri- meet the needs of future generations, but horticultural crops and carry out related research; coordination of capacity building in PGR management also provides these to the nation's crop and policy issues governing access and benefit sharing of their use, and molecular profiling of varieties of improvement programmes to sustain Fig. 1: NBPGR network of regional stations and base centres agri-horticultural crops and GM detection technology research. continued advances in agricultural ICAR-NBPGR has its headquarters in New Delhi that hosts the second largest genebank in the world. The productivity and stabilize production. The Bureau works in close collaboration with several international operations are administered by Divisions of Plant Exploration and Germplasm Collection, Germplasm institutes/ organizations through memoranda/work-plans developed under bilateral/multilateral Evaluation, Germplasm Conservation, Genomic Resources and Plant Quarantine in addition to the Units of agreements. It exchanges plant germplasm with over 100 countries. The Bureau is gradually developing and Germplasm Exchange and Tissue Culture and Cryopreservation. ICAR-NBPGR has the network of 10 strengthening the national plant genetic resources system by linking up the National Base Collection (kept Regional Stations covering different agro-climatic zones to carry out PGR activities including collection, under long-term storage at ICAR-NBPGR) with National Active Germplasm Sites (NAGS) responsible for characterization, evaluation and maintenance of various crops as mentioned below: different crops where germplasm collections are evaluated and multiplied under field conditions backed by medium-term storage facilities. The various activities and achievement of the Bureau are presented here, • Shimla (Himachal Pradesh): Established in 1960 for temperate crops. briefly. • Jodhpur (Rajasthan): Established in 1965 for agri-horticultural crops germplasm of arid and semi-arid PGR Exploration and Collection zones. • To develop new varieties in various agri-horticultural crops for farmers suitable to different agro- • Thrissur (Kerala): Established in 1977 for agri-horticultural crops germplasm of southern peninsular climatic conditions, new germplasm/parent material with desired traits or genes is a continuous region with particular emphasis on spices and plantation crops. requirement of the plant breeders. Therefore, periodically such germplasm is collected by ICAR-NBPGR • Akola (Maharashtra): Established in 1977 for agri-horticultural crops germplasm of central India and Scientists in collaboration with crop-based institutes of ICAR. Also, the trait-specific germplasm of Deccan Plateau. various crops has been collected from diversity-rich spots (including the difficult unreached areas in different parts of the country). • Shillong (Meghalaya): Established in 1978 for agri-horticultural crops germplasm of north-eastern region including Sikkim and parts of north Bengal. • The institute has so far undertaken 2,762 explorations and collected about 2.85 lakh accessions of crop species and their wild relatives. • Bhowali (Uttarakhand): Established in 1985 for agri-horticultural crops germplasm of sub-temperate region. • Focus on North-East and rescue missions: In total, 165 explorations undertaken and a total of 9,698 accessions (cultivated-6,622, wild-3,076) collected. 45 exploration trips to North-Eastern Hill Region • Cuttack (Odisha): Established in 1985 for agri-horticultural crops germplasm of eastern peninsular and five rescue missions to natural calamity affected areas of Uttarakhand were undertaken. Systematic region with main emphasis on rice germplasm. explorations have been conducted in the remotest parts of the country including Mon district of • Hyderabad (Telangana): Established in 1985 for quarantine clearance of agri-horticultural crops Nagaland; Anjaw, Changlang and Tirap districts of Arunachal Pradesh; and Great Nicobar. germplasm of Telangana, Andhra Pradesh and adjoining areas. Disturbed/insurgency-prone areas such as Bastar region in Chhatttisgarh; Gadchiroli in Maharashtra;

16 17 Crop Variety/Hybrid Exotic Source Chilli Co3 CA 856 (Srilanka) Pea Uttra EC109185 / HFP4 Carrot Pusa Yamdagni EC 9981 / Nantes Sweet potato Pusa Sunehari Porto Blanco / Wanrap) //Australian Cannanes (USA) Squash Pusa Alankar (EC 27050 / Selection No. PL.8) Cauliflower Pusa Snowball-1 EC 12012 / EC12013 Cowpea Aseem Pusa Phalguni / EC 21622 (Philippines) Rituraj Pusa Dofasali / EC 26410 (Mexico) Fig 2: Increased focus on CWR in germplasm collecting Pusa Dofasali Pusa Phalguni / EC 21622 (Philippines) and West Medinipur in West Bengal resulting in collection of 858 landraces of different agri- Cluster bean RGS 936 EC 248 / RGC 401 horticultural crops. Gap analysis, geo-referencing and diversity distribution mapping completed in nine crops. Not only the exotic germplasm introduced from other countries has helped in growth of Indian agriculture but several germplasm lines of Indian origin have helped in saving crops in other countries. Some of the • Priority was given to the crop wild relatives (CWR) which resulted in collection of 576 unique classical examples of use of germplasm of Indian origin include: accessions which resulted in a significant increase in the share of wild species (32%) in the total collection. • The entire rice crop of Indonesia was threatened some decades ago by a growth-stunting virus. A gene transferred from Oryza nivara from Odhisa saved rice crop against the virus. PGR Exchange • It was single gene from India for downy mildew resistance that saved the muskmelon crop in the United • ICAR-NBPGR is the nodal agency for import and export of all PGR for research purpose, adhering to States. guidelines of National Biodiversity Act, 2002. • Another gene from India that provided American sorghum resistance to greenbug insect had resulted in • ICAR-NBPGR is instrumental in introduction of several new crops in India such as soybean, sunflower, millions of dollars of annual benefit to American farmers. kiwi, tree tomato, oil palm, jojoba, guayule, hops etc. and aromatic plants like rose geranium which are • Dr. William Saunders of Canada used wheat variety Hard Red Calcutta and released new series of wheat getting popular in Himalayan states, Uttarakhand and HP. later called Marquis A and B which were early and resistant to rusts. • More than 10,000 accessions of indigenous and introduced germplasm are supplied annually to the • Recently in rice, Sub1A (from FR13A) and PSTOL1 (from Kasalath) are being used globally to save rice researchers throughout the country. from losses due to flooding and improving P use efficiency. • Exchange carried out with >100 countries and CGIAR institutes under bi-or multi-lateral agreements. Plant Quarantine Annually, ~25,000 accessions of PGR and ~75,000 samples of trial material (primarily from CG Institutes) are introduced into India for use in crop improvement programmes. Till date, about 6.5 lakh germplasm • Introduction of planting material, including transgenics from other countries carries risk of entry of the accessions of various crops including the transgenic planting material have been introduced/ imported associated pests (fungi, bacteria, viruses, insects, nematodes and weeds etc.). Hence, all genetic into the country. This is now facilitated through online application for import permit resources acquired from foreign countries are tested using plant quarantine measures (legislative (http://www.nbpgr.ernet.in/gep/). These introductions have been used both for direct release as measures) to prevent the entry of exotic pests and to avoid their spread to the fields. varieties and in crossing programme as parents. Hundreds of such examples are available and some • ICAR-NBPGR has been empowered under the Plant Quarantine (Regulation of Import into India) Order classical examples of use of exotic germplasm in varietal development is presented in Table 1. 2003 of the Government of India to carry out quarantine checks on the germplasm being exchanged Table 1. Use of introduced exotic germplasm in development of new varieties meant for research purposes, including transgenics. It undertakes quarantine processing of germplasm meant for export and issues the Phytosanitary Certificate for the material meant for export. Crop Variety/Hybrid Exotic Source • The quarantine has resulted in the interception of several pests of high economic significance including Rice Annada MTU-15 / Yaikaku Nantoku (China) (>70) those not yet reported from the country. Such interception signify the success of quarantine as Barley Clipper, Alfa 93, Rekha Introduction from Australia otherwise these pests could have entered the country and played havoc with the plant biodiversity and Chickpea C104, L550 Rabat (Morocco) agriculture. BG261, BG244 P9847 (Russia) • So far > 35 lakh samples of various crops have been processed for quarantine clearance. BG267 USA 613 (USA) • Infestation/ infection/ contamination detected in > 2 lakh samples of which 99.95% were salvaged and Tomato Kashi Hemant Sel 18 / Flora Dade 75 exotic pests intercepted. Punjab Kesari EC 55055 / Punjab Tropics Inadvertent introduction of any pest not present in the country could lead to serious economic losses to Punjab Chhuhara EC 55055 / Punjab Tropics farmers and the country. Estimated losses that could occur, if any of the diseases/weeds was introduced in Bottle gourd Kashi Ganga IC92465 / DvBG-151 the country are given in Table 2.

18 19 Crop Variety/Hybrid Exotic Source Chilli Co3 CA 856 (Srilanka) Pea Uttra EC109185 / HFP4 Carrot Pusa Yamdagni EC 9981 / Nantes Sweet potato Pusa Sunehari Porto Blanco / Wanrap) //Australian Cannanes (USA) Squash Pusa Alankar (EC 27050 / Selection No. PL.8) Cauliflower Pusa Snowball-1 EC 12012 / EC12013 Cowpea Aseem Pusa Phalguni / EC 21622 (Philippines) Rituraj Pusa Dofasali / EC 26410 (Mexico) Fig 2: Increased focus on CWR in germplasm collecting Pusa Dofasali Pusa Phalguni / EC 21622 (Philippines) and West Medinipur in West Bengal resulting in collection of 858 landraces of different agri- Cluster bean RGS 936 EC 248 / RGC 401 horticultural crops. Gap analysis, geo-referencing and diversity distribution mapping completed in nine crops. Not only the exotic germplasm introduced from other countries has helped in growth of Indian agriculture but several germplasm lines of Indian origin have helped in saving crops in other countries. Some of the • Priority was given to the crop wild relatives (CWR) which resulted in collection of 576 unique classical examples of use of germplasm of Indian origin include: accessions which resulted in a significant increase in the share of wild species (32%) in the total collection. • The entire rice crop of Indonesia was threatened some decades ago by a growth-stunting virus. A gene transferred from Oryza nivara from Odhisa saved rice crop against the virus. PGR Exchange • It was single gene from India for downy mildew resistance that saved the muskmelon crop in the United • ICAR-NBPGR is the nodal agency for import and export of all PGR for research purpose, adhering to States. guidelines of National Biodiversity Act, 2002. • Another gene from India that provided American sorghum resistance to greenbug insect had resulted in • ICAR-NBPGR is instrumental in introduction of several new crops in India such as soybean, sunflower, millions of dollars of annual benefit to American farmers. kiwi, tree tomato, oil palm, jojoba, guayule, hops etc. and aromatic plants like rose geranium which are • Dr. William Saunders of Canada used wheat variety Hard Red Calcutta and released new series of wheat getting popular in Himalayan states, Uttarakhand and HP. later called Marquis A and B which were early and resistant to rusts. • More than 10,000 accessions of indigenous and introduced germplasm are supplied annually to the • Recently in rice, Sub1A (from FR13A) and PSTOL1 (from Kasalath) are being used globally to save rice researchers throughout the country. from losses due to flooding and improving P use efficiency. • Exchange carried out with >100 countries and CGIAR institutes under bi-or multi-lateral agreements. Plant Quarantine Annually, ~25,000 accessions of PGR and ~75,000 samples of trial material (primarily from CG Institutes) are introduced into India for use in crop improvement programmes. Till date, about 6.5 lakh germplasm • Introduction of planting material, including transgenics from other countries carries risk of entry of the accessions of various crops including the transgenic planting material have been introduced/ imported associated pests (fungi, bacteria, viruses, insects, nematodes and weeds etc.). Hence, all genetic into the country. This is now facilitated through online application for import permit resources acquired from foreign countries are tested using plant quarantine measures (legislative (http://www.nbpgr.ernet.in/gep/). These introductions have been used both for direct release as measures) to prevent the entry of exotic pests and to avoid their spread to the fields. varieties and in crossing programme as parents. Hundreds of such examples are available and some • ICAR-NBPGR has been empowered under the Plant Quarantine (Regulation of Import into India) Order classical examples of use of exotic germplasm in varietal development is presented in Table 1. 2003 of the Government of India to carry out quarantine checks on the germplasm being exchanged Table 1. Use of introduced exotic germplasm in development of new varieties meant for research purposes, including transgenics. It undertakes quarantine processing of germplasm meant for export and issues the Phytosanitary Certificate for the material meant for export. Crop Variety/Hybrid Exotic Source • The quarantine has resulted in the interception of several pests of high economic significance including Rice Annada MTU-15 / Yaikaku Nantoku (China) (>70) those not yet reported from the country. Such interception signify the success of quarantine as Barley Clipper, Alfa 93, Rekha Introduction from Australia otherwise these pests could have entered the country and played havoc with the plant biodiversity and Chickpea C104, L550 Rabat (Morocco) agriculture. BG261, BG244 P9847 (Russia) • So far > 35 lakh samples of various crops have been processed for quarantine clearance. BG267 USA 613 (USA) • Infestation/ infection/ contamination detected in > 2 lakh samples of which 99.95% were salvaged and Tomato Kashi Hemant Sel 18 / Flora Dade 75 exotic pests intercepted. Punjab Kesari EC 55055 / Punjab Tropics Inadvertent introduction of any pest not present in the country could lead to serious economic losses to Punjab Chhuhara EC 55055 / Punjab Tropics farmers and the country. Estimated losses that could occur, if any of the diseases/weeds was introduced in Bottle gourd Kashi Ganga IC92465 / DvBG-151 the country are given in Table 2.

18 19 Table 2: Probable annual losses due to various exotic pests, if introduced into India Table 4: Germplasm in the in vitro and cryopreservation facility

Crop Pest Yield loss in exporting *Probable annual loss Crop / Crop Group No. of Accessions country (%) in India (INR in million) In vitro bank Wheat Monographella nivalis (fungus) 3.0 to 52.4 (USSR) 1967.16 1. Tropical fruits 443 Barley stripe mosaic virus Up to 30.0 (USA) 2. Temperate and minor tropical fruits 360 Bromus secalinus (weed) 28.0 to 48.0 (USA) 3. Tuber crops 520 Soybean Bean pod mottle virus Up to 52.0 (USA) 511.61 4. Bulbous crops 171 Peronospora manshurica (fungus) Up to 80.0 (USA) 5. Medicinal & aromatic plants 181 Cotton Anthonomus grandis (insect) Up to 51.0 (USA) 256.48 6. Spices and industrial crops 227 Maize Maize chlorotic mottle virus 90.0 (USA) 479.25 TOTAL 1, 902 High plains virus Up to 100.0 (USA) Cryobank

*Losses based on 0.1% yield loss due to appearance of pests. The total yield for 2018-2019 and minimum support price (MSP) for 1. Recalcitrant 0 2019-2020 have been taken. Yield (million tonnes) for wheat -102.19, soybean-13.79, cotton-28.71 (million bales with 170 kg each 2. Intermediate 6,948 bale) and maize-27.23) and MSP (INR/quintal) for wheat-1925, soybean-3710, cotton-5255 and maize-1760. Sources: Directorate of Economics and Statistics, Ministry of Agriculture Farmers and Welfare, Government of India. 3. Orthodox 3,911 4. Dormant bud 389 PGR Conservation 5. Pollen (Mango) 591 The Indian National Genebank (NGB) was established at ICAR-NBPGR to conserve the PGR for posterity in TOTAL 11,839 the form of seeds, vegetative propagules, in vitro cultures, budwoods, embryos/embryonic axes, genomic (Source: http:// www.nbpgr.ernet.in accessed on 27.01.2021) resources and pollen. The NGB has four kinds of facilities, namely, Seed Genebank (-18°C), Cryogenebank (-170°C to -196°C), In Vitro Genebank (25°C), and Field Genebank, to cater to long- PGR Characterization and Evaluation term as well as medium-term conservation. The utilization of PGR in crop improvement programs rests on identification of promising accessions. The The NGB with a capacity to conserve about one million germplasm in the form of seeds is currently collected or introduced germplasm is characterized and evaluated to assess its potential, by recording data conserving about 0.44 million accessions (Table 3) belonging to nearly 1,800 species. Over 12,000 samples on agronomic traits such as yield, quality, and tolerance to biotic and abiotic stresses. The germplasm is also of seed, dormant buds, and pollen are cryopreserved (Table 4) and about 1,900 accessions are conserved in evaluated for new traits using molecular tools to identify the genes to develop new varieties as per the in vitro genebank (Table 4). The NGB is supported by active partnership of other intuitions designated as requirement of the farmers. Salient achievements are as: the NAGS. The NAGS are responsible for maintaining, evaluating and distributing germplasm from their • Approximately 10,000 accessions are characterized/ evaluated every year at ICAR-NBPGR and its active collections to NGB and other user scientists. regional stations. So far, more than 2.35 lakhs accessions of different agri-horticultural crops have been characterized and evaluated and passport data is available. Table 3: Status of Base Collection National Genebank (-18°C) • Core sets have been developed in four crops viz., okra, mungbean, sesame brinjal and wheat to facilitate Crop / Crop No. of accessions Crop / Crop No. of accessions the enhanced utilization of germplasm. Group conserved Group conserved Cereals 1,66,218 Medicinal & Aromatic • Genetic diversity in large collection has been determined using morphological and DNA fingerprinting Plants & Narcotics 8,392 markers in crops like rice, mungbean, banana, cashew, mango, oilseeds, brassicas, tomato, sesame, Millets 59,722 cucumber and cotton. Forages 7,319 Ornamental 670 Spices & Condiments 3,265 • Mega programme on characterization and evaluation under the National Initiative for climate Resilient Pseudo-Cereals 7,833 Agriculture (NICRA) executed in collaboration with SAUs for 21,822 accessions of wheat and 18,775 of Agroforestry 1,654 Legumes 67,279 chickpea. Oilseeds 60,907 Safety Duplicates (Lentil, Pigeonpea) 10,235 DNA Fingerprinting and development of Genomic Resources Fibre 16,194 Trail Material • About 2,300 varieties in more than 35 crops have been fingerprinted so far (Table 5). Also, the new Vegetables 27,462 (Wheat, Barley) 10,771 varieties are being DNA fingerprinted to avoid any biopiracy by any unauthorized person or country. Fruits & Nuts 293 Total 4,48,214* • Established the National Genomic Resources Repository to collect, generate, conserve and distribute * The figure includes 5,034 Released Varieties and 4,316 Genetic Stocks; No. of Crop Species conserved: 1,762. genomic resources for agricultural research in the country. The aim is to promote deposition, sharing Source: http:// www.nbpgr.ernet.in accessed on 27.1.2021 and utilization of enormous amount of genomic resources generated in the country and elsewhere. • All forms of genomic resources including clones, gene constructs, large DNA fragment libraries as well as genomic sequence information in soft copy form can be deposited in this repository. 20 21 • All depositions or requests are to be made along with material transfer agreements in order to protect been validated in three different crops (jute, cotton, linseed) representing five different species the interest of the depositor and the sovereignty of the Nation over the genetic resources. The IP rights (Corchorus capsularis, C. olitorius, Gossypium hirsutum, Gossypium arboratum) and linseed (Linum (if any) shall remain with the depositor. usitatissimum). • Newly identified genes (952) are also conserved in the form of DNA libraries, etc. • A draft genome assembly of 480 Mb of black pepper (Piper nigrum) genome has been generated at NBPGR under the ICAR funded Consortium Research Platform on Genomics. A large number of genomic Table 5: Details of the crop varieties fingerprinted at ICAR-NBPGR resources in the form of genomic and genic SSRs have been generated. S.No. Crop No. of Varieties S.No. Crop No. of Varieties • A rice core consisting of 701 accessions has been developed from 6,984 accessions of North-Eastern 1 Rice 729 16 Safflower 26 region of India. This core was further characterized with 50K SNP chip of Rice for development of mini- core. Cluster analysis based on 50K SNP markers grouped 192 accessions of core into seven clusters. This 2 Wheat 108 17 Saffron 13 analysis shows that the core developed from NE rice collection is very diverse and has captured 3 Barley 54 18 Chickpea 77 maximum diversity present. 4 Maize 140 19 Jute 31 • DNA Barcoding loci rbcL, matK, trnH-psbA and ITS region alone and/combination of two loci identified 5 Finger Millet 11 20 Oats 9 21 genomic species in Oryza and were used for establishing correct genetic identity of mis-labeled 6 Sorghum 57 21 Pea 43 species. Two combined loci DNA barcodes (rbcL + ITS) gave better species delineation and proper barcode gaps for species identification in genus Luffa. 7 Pearl Millet 53 22 Lentil 25 8 Soybean 69 23 Mustard 42 • DNA profiling services were rendered to various public and private sector organizations. A total of 558 varieties belonging to 33 crops have been profiled in the last four years itself. 9 Pigeonpea 49 24 Linseed 46 10 Mungbean 78 25 Cotton 116 • Discovery of non-Kranz C4 photosynthesis in two cell layers (cross- and tube-cells) of pericarp in developing wheat grains. Named it as “Bose anatomy” in honour of his earliest works on C4 in Hydrilla 11 Urdbean 76 26 Sunflower 7 reported in 1924 when C3 itself was not known. 12 Ricebean 4 27 Bittergourd 38 • Technology has been developed for Identification of SRAP markers linked to the Single Dominant 13 Mothbean 2 28 Mango 23 Resistance Gene against Tomato Leaf Curl New Delhi Virus in Luffa cylindrica Roem: Two sequence- 14 Cowpea 11 29 Cashew 105 related amplified polymorphism (SRAP) markers closely-linked to the ToLCNDV-susceptible gene in the 15 Sesame 52 30 Tomato 30 susceptible parent and in a susceptible bulk population; and two SRAP markers closely-linked to the resistance gene in the resistant parent and in a resistant bulk population were found. These can be used Total: 2,195 for large-scale screening of genotypes of L. cylindrica for resistance against ToLCNDV at the seedling DNA fingerprinting (Status) for protection of over 6440 released varieties of 52 species and native landraces to stage, and to accelerate the breeding of high yielding, ToLCNDV resistant varieties and hybrids. prevent unauthorized commercial exploitation. • Qualitative and quantitative PCR and real-time PCR assays have been developed/validated for detection of more than 50 events of 14 GM crops (brinjal, cabbage, cauliflower, cotton, maize, mustard, oilseed ICAR-NBPGR also has the mandate to carry out molecular profiling of varieties of agri-horticultural crops. rape, okra, papaya, potato, rice, soybean, tomato, wheat). This involves generation and utilization of molecular markers for molecular characterization of these crops. • Rapid and cost-efficient assays have been developed for screening of GM crops employing a) visual and Although a lot of dominant multilocus marker systems such as RAPD (Random Amplified Polymorphic DNA), Real-time Loop-mediated Isothermal Amplification (LAMP) for rapid on-site detection; b) GMO AFLP (Amplified Fragment Length Polymorphism) etc. have been used, the preferred markers for molecular Screening Matrix as decision support system, and c) ready-to-use TaqMan® Real-time PCR based Multi- characterization are SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers target system. owing to their codominant nature. Whereas a lot of molecular markers are available in crops like rice, wheat, maize, potato, cotton, soybean etc. there are others which despite being important contributors to the food • GM-free Conservation of Germplasm in National Genebank: Bt cotton has been commercially cultivated basket are less worked at the genomics front. in India since 2002 and other GM events of cotton, brinjal, okra and maize were under field trials. Hence, to ensure GM-free conservation of germplasm in the National Genebank, the adventitious presence of Generation, validation and utilization of genomic resources is one of the major objective of ICAR-NBPGR. transgenes was monitored in ex situ collection including cotton (200 accns.), brinjal (150 accns.), okra These resources are utilized for value addition to the plant germplasm resources harboured in the genebank (50 accns,), maize (100 accns) using PCR/real-time PCR-based markers. None of the accessions screened and for generating molecular profiles varieties of agri-horticultural crops. The advent of next generation so far showed adventitious presence of transgenes based on tests conducted. sequencing with improved chemistries and lower input costs have resulted high throughput data that can be mined for generating SSR and SNP markers. Germplasm Utilization • Genomic SSRs have been generated and validated at NBPGR in crops like okra, snake gourd and moth The Bureau has supplied germplasm, collected indigenously or from exotic sources, to the breeders and bean using NGS technologies in house. SSRs from transcriptome sequencing have been mined and other researchers in the country. The germplasm supplied by ICAR-NBPGR to various breeders have been validated in finger millet, kodo millet, little millet and sponge gourd. used in varietal development. Several indigenously supplied germplasm accessions have helped to develop • A novel gene targeted marker technique CBDP (CAAT Box-Derived Polymorphism) has been developed improved varieties in various national programmes. These include rice variety (Maruteru sannalu), sorghum that can be used for various genotyping applications in plants. The technique exploits conserved CCAAT variety (Parbhani Moti), red okra (Aruna), Chinese potato (Nidhi), coriander variety (Sudha), and yam variety motif in the CAAT box region of promoter region of plant genes to generate markers. The concept has (Indu) a few to name.

22 23 • All depositions or requests are to be made along with material transfer agreements in order to protect been validated in three different crops (jute, cotton, linseed) representing five different species the interest of the depositor and the sovereignty of the Nation over the genetic resources. The IP rights (Corchorus capsularis, C. olitorius, Gossypium hirsutum, Gossypium arboratum) and linseed (Linum (if any) shall remain with the depositor. usitatissimum). • Newly identified genes (952) are also conserved in the form of DNA libraries, etc. • A draft genome assembly of 480 Mb of black pepper (Piper nigrum) genome has been generated at NBPGR under the ICAR funded Consortium Research Platform on Genomics. A large number of genomic Table 5: Details of the crop varieties fingerprinted at ICAR-NBPGR resources in the form of genomic and genic SSRs have been generated. S.No. Crop No. of Varieties S.No. Crop No. of Varieties • A rice core consisting of 701 accessions has been developed from 6,984 accessions of North-Eastern 1 Rice 729 16 Safflower 26 region of India. This core was further characterized with 50K SNP chip of Rice for development of mini- core. Cluster analysis based on 50K SNP markers grouped 192 accessions of core into seven clusters. This 2 Wheat 108 17 Saffron 13 analysis shows that the core developed from NE rice collection is very diverse and has captured 3 Barley 54 18 Chickpea 77 maximum diversity present. 4 Maize 140 19 Jute 31 • DNA Barcoding loci rbcL, matK, trnH-psbA and ITS region alone and/combination of two loci identified 5 Finger Millet 11 20 Oats 9 21 genomic species in Oryza and were used for establishing correct genetic identity of mis-labeled 6 Sorghum 57 21 Pea 43 species. Two combined loci DNA barcodes (rbcL + ITS) gave better species delineation and proper barcode gaps for species identification in genus Luffa. 7 Pearl Millet 53 22 Lentil 25 8 Soybean 69 23 Mustard 42 • DNA profiling services were rendered to various public and private sector organizations. A total of 558 varieties belonging to 33 crops have been profiled in the last four years itself. 9 Pigeonpea 49 24 Linseed 46 10 Mungbean 78 25 Cotton 116 • Discovery of non-Kranz C4 photosynthesis in two cell layers (cross- and tube-cells) of pericarp in developing wheat grains. Named it as “Bose anatomy” in honour of his earliest works on C4 in Hydrilla 11 Urdbean 76 26 Sunflower 7 reported in 1924 when C3 itself was not known. 12 Ricebean 4 27 Bittergourd 38 • Technology has been developed for Identification of SRAP markers linked to the Single Dominant 13 Mothbean 2 28 Mango 23 Resistance Gene against Tomato Leaf Curl New Delhi Virus in Luffa cylindrica Roem: Two sequence- 14 Cowpea 11 29 Cashew 105 related amplified polymorphism (SRAP) markers closely-linked to the ToLCNDV-susceptible gene in the 15 Sesame 52 30 Tomato 30 susceptible parent and in a susceptible bulk population; and two SRAP markers closely-linked to the resistance gene in the resistant parent and in a resistant bulk population were found. These can be used Total: 2,195 for large-scale screening of genotypes of L. cylindrica for resistance against ToLCNDV at the seedling DNA fingerprinting (Status) for protection of over 6440 released varieties of 52 species and native landraces to stage, and to accelerate the breeding of high yielding, ToLCNDV resistant varieties and hybrids. prevent unauthorized commercial exploitation. • Qualitative and quantitative PCR and real-time PCR assays have been developed/validated for detection of more than 50 events of 14 GM crops (brinjal, cabbage, cauliflower, cotton, maize, mustard, oilseed ICAR-NBPGR also has the mandate to carry out molecular profiling of varieties of agri-horticultural crops. rape, okra, papaya, potato, rice, soybean, tomato, wheat). This involves generation and utilization of molecular markers for molecular characterization of these crops. • Rapid and cost-efficient assays have been developed for screening of GM crops employing a) visual and Although a lot of dominant multilocus marker systems such as RAPD (Random Amplified Polymorphic DNA), Real-time Loop-mediated Isothermal Amplification (LAMP) for rapid on-site detection; b) GMO AFLP (Amplified Fragment Length Polymorphism) etc. have been used, the preferred markers for molecular Screening Matrix as decision support system, and c) ready-to-use TaqMan® Real-time PCR based Multi- characterization are SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers target system. owing to their codominant nature. Whereas a lot of molecular markers are available in crops like rice, wheat, maize, potato, cotton, soybean etc. there are others which despite being important contributors to the food • GM-free Conservation of Germplasm in National Genebank: Bt cotton has been commercially cultivated basket are less worked at the genomics front. in India since 2002 and other GM events of cotton, brinjal, okra and maize were under field trials. Hence, to ensure GM-free conservation of germplasm in the National Genebank, the adventitious presence of Generation, validation and utilization of genomic resources is one of the major objective of ICAR-NBPGR. transgenes was monitored in ex situ collection including cotton (200 accns.), brinjal (150 accns.), okra These resources are utilized for value addition to the plant germplasm resources harboured in the genebank (50 accns,), maize (100 accns) using PCR/real-time PCR-based markers. None of the accessions screened and for generating molecular profiles varieties of agri-horticultural crops. The advent of next generation so far showed adventitious presence of transgenes based on tests conducted. sequencing with improved chemistries and lower input costs have resulted high throughput data that can be mined for generating SSR and SNP markers. Germplasm Utilization • Genomic SSRs have been generated and validated at NBPGR in crops like okra, snake gourd and moth The Bureau has supplied germplasm, collected indigenously or from exotic sources, to the breeders and bean using NGS technologies in house. SSRs from transcriptome sequencing have been mined and other researchers in the country. The germplasm supplied by ICAR-NBPGR to various breeders have been validated in finger millet, kodo millet, little millet and sponge gourd. used in varietal development. Several indigenously supplied germplasm accessions have helped to develop • A novel gene targeted marker technique CBDP (CAAT Box-Derived Polymorphism) has been developed improved varieties in various national programmes. These include rice variety (Maruteru sannalu), sorghum that can be used for various genotyping applications in plants. The technique exploits conserved CCAAT variety (Parbhani Moti), red okra (Aruna), Chinese potato (Nidhi), coriander variety (Sudha), and yam variety motif in the CAAT box region of promoter region of plant genes to generate markers. The concept has (Indu) a few to name.

22 23 NBPGR is involved in the release of about 100 varieties in the past in different agri-horticultural crops either Human Resources Development through direct introduction or by selection from the introduced germplasm and popularized several such introductions for commercial cultivation. Also many temperate fruits including kiwi, hops and several • NBPGR faculty conducts M.Sc and Ph.D. courses in PGR under the Post-Graduate School of Indian medicinal and aromatic plants like rose geranium are getting increasingly popular in Himalayan states, Agriculture Research Institute (IARI), New Delhi. Uttarakhand and HP. • National and international training program conducted routinely on various aspects of PGR management. PGR Documentation • NBPGR is designated as the Centre of Excellence since 2006 by ICAR and Bioversity International to A PGR Portal has been hosted on NBPGR website, which is a gateway to information on plant genetic impart training on in vitro conservation and cryopreservation of PGR and more than eight trainings resources conserved. The Portal contains information on about 0.4 million accessions belonging to about conducted. So far, more than 120 researchers from 40 countries have benefitted from this training 1800 species. PGR documentation is done in various forms including printing of books, crop catalogues, course. inventories, research papers, popular articles, pamphlets etc. In addition, NBPGR has developed mobile apps Genebank and PGR map in PGR Informatics which can accessed through NBPGR web pages, National and International Linkages genebank.nbpgr.ernet.in and http://pgrinformatics.nbpgr.ernet.in/pgrmap/ • Close collaborations with Bioversity International, ICARDA, IRRI, CIMMYT and other countries on • Two mobile apps “Genebank” and “PGR Map” have been developed to enhance access to PGR genetic resources management and utilization. information with an easy user interface. The apps have been hosted on Google Play and App Store. • Collaborations with all ICAR institute, State Agricultural Universities, CSIR institute, DBT, DST, DRDO etc. • “Genebank App” provides a dashboard view of indigenous collections (state-wise), exotic collections for germplasm trait-specific evaluation and utilization. (country-wise), addition of accessions to genebank, etc. The app also helps generate routine genebank reports. The app uses databases live on the backend and hence always gives updated information. • Memorandum of Understanding (MoU) was signed between the Indian Council of Agricultural Research (ICAR) and the Royal Botanic Gardens (RBG), Kew, UK to enhance capacities of both the institutions in • “PGR Map App” offers three benefits: “What's around me” helps user to obtain quickly the accessions research on conservation biology. that have been collected and conserved in the genebank from a particular location in India where the user is located at the moment; “Search the map” helps user to list the accessions that have been • ICAR-National Bureau of Plant Genetic Resources and Forest Research Institute, Dehradun had signed a collected and conserved in the genebank from any selected location in India; “Search for species” helps MoU for the conservation of seed bearing trees species of forestry importance. user to map the collection sites of a crop species. • MoU of ICAR-NBPGR with CPCRI, Kasaragod and NRC Orchids has been operationalized for cryo- • Establishment of geo-informatics portal in PGR: A study to link germplasm to changing climatic regimes conservation of coconut and orchids germplasm, respectively, at the Cryogenebank, ICAR-NBPGR. was earlier carried out with the funding of the CGIAR Research Program on Climate Change, Agriculture ICAR-NBPGR for Safeguarding Nation's Future Food and Nutritional Security: Safety Duplicates and Food Security (CCAFS). A web interface named PGR CLiM was also developed to access information (www.nbpgr.ernet.in:8080/climate). ICAR has taken a step forward in securing its crop genetic diversity by depositing 25 accessions of pigeonpea in the Svalbard Global Seed Vault (SGSV) Norway. This was the first such deposit by India as safety duplicates Germplasm Registration in the global genebank which is jointly maintained and managed by Norway's Department of Agriculture Recognizing the importance of PGR with novel, unique, distinct and high heritability traits of value that could and the Global Crop Diversity Trust under the ITPGRFA. A second deposit of 100 samples each of rice and be used in crop improvement, and to facilitate flow of germplasm to users, ICAR-NBPGR plays a vital role in sorghum was sent to SGSV for safety duplication in 2016. germplasm registration. More than 900 potentially valuable germplasm of over 120 species of various crops With the successful initiatives in the recent past, like collecting wild and trait-specific germplasm, multi- registered so far. To facilitate smooth registration process, a fully online system of filing registration location evaluation, on-farm conservation, genomic resource development and trait identification, the applications, their scrutiny, review and communications at every stage has been developed ICAR-NBPGR has positioned itself strategically as a national custodian of PGR. The NGB is a 'genetic (http://www.nbpgr.ernet.in:8080/registration/). Details of the registered germplasm could accessed at insurance' for food and nutritional security of the country. The effective utilization of its germplasm would http://www.nbpgr.ernet.in:8080/ircg/index.htm. ensure meeting of Sustainable Development Goals and preparedness for climate change, at national and global levels. All India Coordinated Network Project Potential Crops During the past couple of years the Bureau has made great progress in this direction through a paradigm This network program is located at ICAR-NBPGR, New Delhi and has 13 main centers in different parts of the shift in its evaluation strategy of germplasm being followed by integration of large scale phenotyping and country. The major functions are introduction, evaluation, conservation, and popularization of new high throughput genomics of all germplasm in NGB in various crop groups like, minor legumes, minor potential and useful plant species for acclimatization to local condition. Grain amaranth, buckwheat, oilseeds, chickpea, rice and wheat being undertaken through externally funded projects. The Bureau has ricebean, jatropha and simarouba have been developed and popularized under this project. mobilized about Rs 280 crores involving several ICAR institutes, SAUs for undertaking this mammoth task. Grain amaranth (Amaranthus spp.), normally grown in the hilly region, was tested for cultivation in the NBPGR is also partner in National/ Global Earth Bio-genome project. plains after the inception of the network in mid 1980s in Gujarat. The crop of grain amaranth acclimatized Conservation of Genetic Resources of Horticultural Crops well and was found suitable for cultivation during rabi season. Three varieties, namely, GA-1, GA-2 and GA-3 were released for cultivation in north Gujarat. Conservation of genetic resources of wild species and domesticated diversity of horticultural crops is Likewise, germplasm for some other potential crops like quinoa (Chenopodium quinoa), Faba beans (Vicia imminent. Biological resources once lost may not be replaced. Therefore, their conservation is of prime faba), etc, have been introduced and distributed to farmers. importance and of great significance for present and future use. Conservation of genetic resources can be achieved either in situ or ex situ. Both the approaches are complementary which can be judiciously

24 25 NBPGR is involved in the release of about 100 varieties in the past in different agri-horticultural crops either Human Resources Development through direct introduction or by selection from the introduced germplasm and popularized several such introductions for commercial cultivation. Also many temperate fruits including kiwi, hops and several • NBPGR faculty conducts M.Sc and Ph.D. courses in PGR under the Post-Graduate School of Indian medicinal and aromatic plants like rose geranium are getting increasingly popular in Himalayan states, Agriculture Research Institute (IARI), New Delhi. Uttarakhand and HP. • National and international training program conducted routinely on various aspects of PGR management. PGR Documentation • NBPGR is designated as the Centre of Excellence since 2006 by ICAR and Bioversity International to A PGR Portal has been hosted on NBPGR website, which is a gateway to information on plant genetic impart training on in vitro conservation and cryopreservation of PGR and more than eight trainings resources conserved. The Portal contains information on about 0.4 million accessions belonging to about conducted. So far, more than 120 researchers from 40 countries have benefitted from this training 1800 species. PGR documentation is done in various forms including printing of books, crop catalogues, course. inventories, research papers, popular articles, pamphlets etc. In addition, NBPGR has developed mobile apps Genebank and PGR map in PGR Informatics which can accessed through NBPGR web pages, National and International Linkages genebank.nbpgr.ernet.in and http://pgrinformatics.nbpgr.ernet.in/pgrmap/ • Close collaborations with Bioversity International, ICARDA, IRRI, CIMMYT and other countries on • Two mobile apps “Genebank” and “PGR Map” have been developed to enhance access to PGR genetic resources management and utilization. information with an easy user interface. The apps have been hosted on Google Play and App Store. • Collaborations with all ICAR institute, State Agricultural Universities, CSIR institute, DBT, DST, DRDO etc. • “Genebank App” provides a dashboard view of indigenous collections (state-wise), exotic collections for germplasm trait-specific evaluation and utilization. (country-wise), addition of accessions to genebank, etc. The app also helps generate routine genebank reports. The app uses databases live on the backend and hence always gives updated information. • Memorandum of Understanding (MoU) was signed between the Indian Council of Agricultural Research (ICAR) and the Royal Botanic Gardens (RBG), Kew, UK to enhance capacities of both the institutions in • “PGR Map App” offers three benefits: “What's around me” helps user to obtain quickly the accessions research on conservation biology. that have been collected and conserved in the genebank from a particular location in India where the user is located at the moment; “Search the map” helps user to list the accessions that have been • ICAR-National Bureau of Plant Genetic Resources and Forest Research Institute, Dehradun had signed a collected and conserved in the genebank from any selected location in India; “Search for species” helps MoU for the conservation of seed bearing trees species of forestry importance. user to map the collection sites of a crop species. • MoU of ICAR-NBPGR with CPCRI, Kasaragod and NRC Orchids has been operationalized for cryo- • Establishment of geo-informatics portal in PGR: A study to link germplasm to changing climatic regimes conservation of coconut and orchids germplasm, respectively, at the Cryogenebank, ICAR-NBPGR. was earlier carried out with the funding of the CGIAR Research Program on Climate Change, Agriculture ICAR-NBPGR for Safeguarding Nation's Future Food and Nutritional Security: Safety Duplicates and Food Security (CCAFS). A web interface named PGR CLiM was also developed to access information (www.nbpgr.ernet.in:8080/climate). ICAR has taken a step forward in securing its crop genetic diversity by depositing 25 accessions of pigeonpea in the Svalbard Global Seed Vault (SGSV) Norway. This was the first such deposit by India as safety duplicates Germplasm Registration in the global genebank which is jointly maintained and managed by Norway's Department of Agriculture Recognizing the importance of PGR with novel, unique, distinct and high heritability traits of value that could and the Global Crop Diversity Trust under the ITPGRFA. A second deposit of 100 samples each of rice and be used in crop improvement, and to facilitate flow of germplasm to users, ICAR-NBPGR plays a vital role in sorghum was sent to SGSV for safety duplication in 2016. germplasm registration. More than 900 potentially valuable germplasm of over 120 species of various crops With the successful initiatives in the recent past, like collecting wild and trait-specific germplasm, multi- registered so far. To facilitate smooth registration process, a fully online system of filing registration location evaluation, on-farm conservation, genomic resource development and trait identification, the applications, their scrutiny, review and communications at every stage has been developed ICAR-NBPGR has positioned itself strategically as a national custodian of PGR. The NGB is a 'genetic (http://www.nbpgr.ernet.in:8080/registration/). Details of the registered germplasm could accessed at insurance' for food and nutritional security of the country. The effective utilization of its germplasm would http://www.nbpgr.ernet.in:8080/ircg/index.htm. ensure meeting of Sustainable Development Goals and preparedness for climate change, at national and global levels. All India Coordinated Network Project Potential Crops During the past couple of years the Bureau has made great progress in this direction through a paradigm This network program is located at ICAR-NBPGR, New Delhi and has 13 main centers in different parts of the shift in its evaluation strategy of germplasm being followed by integration of large scale phenotyping and country. The major functions are introduction, evaluation, conservation, and popularization of new high throughput genomics of all germplasm in NGB in various crop groups like, minor legumes, minor potential and useful plant species for acclimatization to local condition. Grain amaranth, buckwheat, oilseeds, chickpea, rice and wheat being undertaken through externally funded projects. The Bureau has ricebean, jatropha and simarouba have been developed and popularized under this project. mobilized about Rs 280 crores involving several ICAR institutes, SAUs for undertaking this mammoth task. Grain amaranth (Amaranthus spp.), normally grown in the hilly region, was tested for cultivation in the NBPGR is also partner in National/ Global Earth Bio-genome project. plains after the inception of the network in mid 1980s in Gujarat. The crop of grain amaranth acclimatized Conservation of Genetic Resources of Horticultural Crops well and was found suitable for cultivation during rabi season. Three varieties, namely, GA-1, GA-2 and GA-3 were released for cultivation in north Gujarat. Conservation of genetic resources of wild species and domesticated diversity of horticultural crops is Likewise, germplasm for some other potential crops like quinoa (Chenopodium quinoa), Faba beans (Vicia imminent. Biological resources once lost may not be replaced. Therefore, their conservation is of prime faba), etc, have been introduced and distributed to farmers. importance and of great significance for present and future use. Conservation of genetic resources can be achieved either in situ or ex situ. Both the approaches are complementary which can be judiciously

24 25 integrated so that the conservation efforts are effectively realized for sustainable use of variability. Complementary conservation strategies employing in situ and ex situ approaches have been applied to Exploration and Collecting Fruit Genetic conserve horticultural crops diversity. Field gene bank status at Regional stations of ICAR- NBPGR (collections includes Fruit crops, Medicinal & Resources for Food and Agriculture Aromatic Plants, Tuber crops, Spices and Perennial wild relatives) S. P. Ahlawat The main goal to be achieved by conservation of genetic resources of Division of Plant Exploration and Germplasm Collection Regional Stations No. of ICAR-National Bureau of Plant Genetic Resources, New Delhi horticultural crops is their present and future utilization for the benefit accessions of mankind. Some significant results have been obtained by using wild Email: [email protected] Bhowali 1252 relatives of domesticated plants in breeding programmes, exploiting Thrissur 1614 the traits acquired by co-evolving of wild relatives with pest and Introduction diseases. For example in tomato, several wild species have been used Cuttack 519 Tropical Asian countries are the centre of origin and diversity of many globally important tropical fruit tree as gene donors for fungus resistance, virus resistance and fruit quality. Jodhpur 423 This underlines the importance of wild horticultural genetic resources species and their wild relatives. These species contribute to the well being of human communities by in crop improvement programmes. Under the scenario of changing Ranchi 617 providing a source of supplementary food, sustaining healthy diets and enhancing both household income climate of regions, there is the need to identify, develop or introduce Shimla 1023 and national revenues (Arora and Ramnanatha Rao, 1998). India is an important centre of diversity and has varieties or cultivars against biotic and abiotic stresses such as drought rich variability of several fruits. There is a large diversity in several native fruit crop species like mango, citrus, resistant germplasm etc. It is also necessary to observe migration of cropping areas and wild populations banana, and underutilized fruits namely aonla, ber, bael, chironjee, jackfruit, jamun, karonda, lasoda, due to changing climatic conditions, especially in the temperate regions. phalsa, tamarind and woodapple etc. A number of superior clones and varieties in these fruits have been selected through exploitation of available variability. Simultaneously, exotic introduction of new fruit Further research on the assessment of horticultural biodiversity, with focus on its economic evaluation, use species and varieties have been made in several fruits adding to the existing variability. As a result, a rich and conservation needs is to be carried out to lay future priorities. There is need to strengthen existing reservoir of species and cultivars of several fruit crops has been built up at various research centres listed programmes and to take new initiatives that secure the future of the crop diversity for mankind. The below: conservation of horticultural genetic resources can be made more secure through increased protection of biodiversity rich areas, active farmer and local community participation in on-farm conservation, and by Apple & Temperate fruits CITH, Srinagar; YSPUHF (Solan), NBPGR RS, Shimla strengthening of infrastructure, scientific capabilities and improved coordination between various national institutes involved in conservation of plant genetic resources in horticultural crops. Conservation Banana NRC Banana, Trichy horticulture is a positive step to meet the forthcoming challenges before us and must be popularized and Ber CIAH, Bikaner; HAU, Hissar, CAZRI, Jodhpur emphasized among the farmers, researchers, general public and policy makers. Citrus CCRI, Nagpur, IARI, New Delhi, APAU, Tirupati, CRS, Tinsukia Future Thrusts for ICAR-NBPGR Grape NRC Grape, Pune, IARI, New Delhi; IIHR, Bengaluru • Geo-referencing of all the indigenous germplasm and superimpose it with soil and climate maps. Guava CISH, Lucknow and IIHR, Bengaluru, JNKVV, Rewa • Targeted germplasm collection based on gap analysis. Mango CISH, Lucknow; IIHR, Bengaluru; IARI, New Delhi; APAU Sangareddy, CHES, Ranchi, TNAU, Coimbatore • Evaluation of germplasm for target traits including biotic, abiotic stress, nutritional and processing traits. Pomegranate NRC on Pomegranate, Sholapur, MPKV, Rahuri, • Strengthening of post-entry quarantine. All crops cultivated now were originally wild plants. Wild plants of food and economic use have been • Geo-referencing of perennial crop (fruit and forest tree) germplasm. transformed into the crops that we grow and use now through the process of domestication and cultivation. Maxted et al. (2006) defined crop wild relative (CWR) as 'a wild plant taxon that has an indirect use derived • Establishment of Field Genebank for semi-arid fruits at Issapur farm of ICAR-NBPGR. from its close genetic relationship to a crop'. They may include wild forms/populations of crops, wild • Establishment of clonal repository of temperate fruits. progenitors and other closely related taxa. Role of CWR in crop improvement (pre-breeding, possessing biotic resistance, adapted traits in changing climate) are well known. Need for novel genes, genes for • Generation of robust cores based on high throughput genomics resources and phenotypic traits in all climate resilience, the breakdown of barriers to introgression through biotechnological tools, increasing the major crops. pressure on wild species population, meager collection in genebanks, signifies the importance of collecting Acknowledgements germplasm of wild relatives. Important cultivated and wild species of fruit plants are given in Table 1 and their diversity rich areas are presented in Table 2. This compilation is a contribution of hundreds of scientists who worked for ICAR-NBPGR and other institutes of ICAR and State Agricultural Universities. The authors are indebted to all of them and acknowledge their contribution.

26 27 integrated so that the conservation efforts are effectively realized for sustainable use of variability. Complementary conservation strategies employing in situ and ex situ approaches have been applied to Exploration and Collecting Fruit Genetic conserve horticultural crops diversity. Field gene bank status at Regional stations of ICAR- NBPGR (collections includes Fruit crops, Medicinal & Resources for Food and Agriculture Aromatic Plants, Tuber crops, Spices and Perennial wild relatives) S. P. Ahlawat The main goal to be achieved by conservation of genetic resources of Division of Plant Exploration and Germplasm Collection Regional Stations No. of ICAR-National Bureau of Plant Genetic Resources, New Delhi horticultural crops is their present and future utilization for the benefit accessions of mankind. Some significant results have been obtained by using wild Email: [email protected] Bhowali 1252 relatives of domesticated plants in breeding programmes, exploiting Thrissur 1614 the traits acquired by co-evolving of wild relatives with pest and Introduction diseases. For example in tomato, several wild species have been used Cuttack 519 Tropical Asian countries are the centre of origin and diversity of many globally important tropical fruit tree as gene donors for fungus resistance, virus resistance and fruit quality. Jodhpur 423 This underlines the importance of wild horticultural genetic resources species and their wild relatives. These species contribute to the well being of human communities by in crop improvement programmes. Under the scenario of changing Ranchi 617 providing a source of supplementary food, sustaining healthy diets and enhancing both household income climate of regions, there is the need to identify, develop or introduce Shimla 1023 and national revenues (Arora and Ramnanatha Rao, 1998). India is an important centre of diversity and has varieties or cultivars against biotic and abiotic stresses such as drought rich variability of several fruits. There is a large diversity in several native fruit crop species like mango, citrus, resistant germplasm etc. It is also necessary to observe migration of cropping areas and wild populations banana, and underutilized fruits namely aonla, ber, bael, chironjee, jackfruit, jamun, karonda, lasoda, due to changing climatic conditions, especially in the temperate regions. phalsa, tamarind and woodapple etc. A number of superior clones and varieties in these fruits have been selected through exploitation of available variability. Simultaneously, exotic introduction of new fruit Further research on the assessment of horticultural biodiversity, with focus on its economic evaluation, use species and varieties have been made in several fruits adding to the existing variability. As a result, a rich and conservation needs is to be carried out to lay future priorities. There is need to strengthen existing reservoir of species and cultivars of several fruit crops has been built up at various research centres listed programmes and to take new initiatives that secure the future of the crop diversity for mankind. The below: conservation of horticultural genetic resources can be made more secure through increased protection of biodiversity rich areas, active farmer and local community participation in on-farm conservation, and by Apple & Temperate fruits CITH, Srinagar; YSPUHF (Solan), NBPGR RS, Shimla strengthening of infrastructure, scientific capabilities and improved coordination between various national institutes involved in conservation of plant genetic resources in horticultural crops. Conservation Banana NRC Banana, Trichy horticulture is a positive step to meet the forthcoming challenges before us and must be popularized and Ber CIAH, Bikaner; HAU, Hissar, CAZRI, Jodhpur emphasized among the farmers, researchers, general public and policy makers. Citrus CCRI, Nagpur, IARI, New Delhi, APAU, Tirupati, CRS, Tinsukia Future Thrusts for ICAR-NBPGR Grape NRC Grape, Pune, IARI, New Delhi; IIHR, Bengaluru • Geo-referencing of all the indigenous germplasm and superimpose it with soil and climate maps. Guava CISH, Lucknow and IIHR, Bengaluru, JNKVV, Rewa • Targeted germplasm collection based on gap analysis. Mango CISH, Lucknow; IIHR, Bengaluru; IARI, New Delhi; APAU Sangareddy, CHES, Ranchi, TNAU, Coimbatore • Evaluation of germplasm for target traits including biotic, abiotic stress, nutritional and processing traits. Pomegranate NRC on Pomegranate, Sholapur, MPKV, Rahuri, • Strengthening of post-entry quarantine. All crops cultivated now were originally wild plants. Wild plants of food and economic use have been • Geo-referencing of perennial crop (fruit and forest tree) germplasm. transformed into the crops that we grow and use now through the process of domestication and cultivation. Maxted et al. (2006) defined crop wild relative (CWR) as 'a wild plant taxon that has an indirect use derived • Establishment of Field Genebank for semi-arid fruits at Issapur farm of ICAR-NBPGR. from its close genetic relationship to a crop'. They may include wild forms/populations of crops, wild • Establishment of clonal repository of temperate fruits. progenitors and other closely related taxa. Role of CWR in crop improvement (pre-breeding, possessing biotic resistance, adapted traits in changing climate) are well known. Need for novel genes, genes for • Generation of robust cores based on high throughput genomics resources and phenotypic traits in all climate resilience, the breakdown of barriers to introgression through biotechnological tools, increasing the major crops. pressure on wild species population, meager collection in genebanks, signifies the importance of collecting Acknowledgements germplasm of wild relatives. Important cultivated and wild species of fruit plants are given in Table 1 and their diversity rich areas are presented in Table 2. This compilation is a contribution of hundreds of scientists who worked for ICAR-NBPGR and other institutes of ICAR and State Agricultural Universities. The authors are indebted to all of them and acknowledge their contribution.

26 27 Table 1: Number of cultivated and wild species of genera belonging to fruit crops in India Table 2: Wild relatives of fruit crops occurring in different regions of India

Genus Cultivated Wild Important cultivated and wild species Region Diversity in major fruit crops Mangifera 1 7 Mangifera indica L., M. sylvatica Roxb., M. gedebe (syn. M. North-eastern region Banana, garcinia, mango, ber, jamun, and Citrus spp. camptosperma Pierre), M. griffithii, M. andamanica King, M. Western Himalayas Pome, stone, soft and nut fruits, Punica and Carisa spp. nicobarica Kosterm, M. sikkimensis Eastern Himalayas Pome and stone, citrus fruits Musa 2 25 Musa balbisiana var. Balbisiana, var. andamanica, M. Eastern peninsular region Banana, bael, aonla, coconut, jamun, jackfruit, mango, lemon/lime, acuminata Colla subsp. Acuminato, M. sikkimensis , and var. tamarind, Carambola and Spondias simmondsii (endemic to Manipur), M. kamengensis, M. nagensium and var. hongii, M. itinerans, M. ochracea, M. Gangetic plains Jackfruit, mango, lemon/lime, orange, jujube, Indian gooseberry, jamun and cheesmanii, M. thomsonii, M. flaviflora, M. sanguine, M. mulberry kattuvazhana, M. aurantiaca, M. velutina, M. laterita, M. Indus plains Khirni and phalsa rubra, M. ornata, M. mannii, M. shankarii, M. chunii, M. Western Peninsular region Jackfruit, banana, lemon/lime, orange, jamun and coconut puspanjaliae, M. sabuana, M. markkui, M. indandamanensis, M. nagalandiana, M. argentii, M. Western Arid Zizyphus, Phoenis, Carissa, Cordia, Capparis, Prosopis, Grewia spp. cylindrica Island regions Coconut, breadfruit Citrus 8 23 Citrus medica, C. cavaleriei (syn. C. ichangensis), C. hystrix, C. indica, C. macroptera, C. latipes Tanaka, C. assamensis, C. Collecting germplasm megaloxycarpa. Germplasm collection is the first and foremost activity of organizations dealing with management of plant Artocarpus Artocarpus heterophyllus, A. hirsutus, A. lakoocha genetic resources (PGR). This is a prerequisite to conserve, utilize these valuable resources in crop improvement and analyze temporal changes. The requirement for PGR in general/ or in specific is Ficus 1 11 Ficus glumerata, F. carica, F. bengalensis, unpredictable and dynamic. Besides germplasm, a voucher herbarium specimen is collected, pressed, plant Grewia 1 39 Grewia asiatica, G. subinaequalis, G. elastica sample deposited for future reference particularly for the variant type and wild relatives. It supports research work and may be examined to verify the identity of the specific plant used in a study. In addition to Phyllanthus 1 2 Phyllanthus emblica L. syn. Emblica officinalis Gaertn. their taxonomic importance, herbaria are commonly used in the fields of ecology, plant anatomy and P. acidus, P. amarus morphology, conservation biology, biogeography, ethnobotany, and paleobotany. Ziziphus 3 15 Ziziphus mauritiana Lam. var. mauritiana, var. pedunculata, Collection mission for germplasm sample and herbarium specimen requires almost similar and meticulous var. fruticosa, Z. jujube, Z. nummularia, Z. rotundifolia preparations from finding the target species and populations to capturing maximum number of species, Juglans 1 1 Juglans regia L., J. sigillata Dode diversity for the amount of material collected and resources invested (Guarino et al., 1995a). In a vast and diverse country having temperate to tropical climate, great specific and intra-specific variation is expected Phoenix 1 13 Phoenix sylvestris, P. dactylifera, P. roebelenii, P. acaulis, P. in various agro-ecological systems and forests. It is essential to get a fair idea of the extant floristic and humalis genetic diversity, their distribution across geographical and ecological niches before embarking on actual Morus 1 3 Morus alba, M. indica, M. nigra, M. serrata collection expeditions. Conducting an ecogeographic survey has been a canonical method for such a preparation (Maxted et al., 1995). Such surveys help in increasing the emphasis on localized floristic and Syzygium 3 72 Syzygium cuminii, S. jumbos, S. aromaticum, S. germplasm collecting with a focus on specific traits. Step-wise activities of exploration mission is depicted andamanicum by flow chart in Fig.1. Vitis 1 3 Vitis flexuosa Thunb., V. heyneana Roem. & Schult. var. Broadly two types of explorations are formulated based on priority of crops and the area/region to be heyneana, var. glabra, V. lanata, V. oblecta, V. perviflora, surveyed. They are (a) crop specific and (b) region specific/multicrop collection. However, in case of fruit Garcinia 2 15 Garcinia cambogia, G. indica, G. andamanica, G. cowa, crops, crop specific explorations are conducted unless some specific area to be surveyed. Malus 1 2 Malus baccata (L.) Borkh. var. himalaica (Maxim.). M. Planning Exploration Mission: Principles and Practices sikkimensis Exploration and germplasm collection of fruit trees is more difficult than cereals, pulses and field crops, as it Pyrus 1 13 requires a different strategy, items and planning due to the nature of plants and material being dealt with. Prunus 5 17 Since this activity is being undertaken by organizations with different mandates, the guidelines have been framed to help the explorers in maintaining standard methods and procedures in collecting PGR (Arora, Rubus 1 33 1981; Gautam et al., 1998, Guriano et al. 1995b, Hawkes, 1976, 1980; Jain and Rao, 1977). An excerpt of guidelines made by Division of Plant Exploration and Germplasm Collection at ICAR-NBPGR are given here for training purpose (ICAR-NBPGR, 2016). Before proceeding on exploration, the scientist should have proper understanding and knowledge on:

28 29 Table 1: Number of cultivated and wild species of genera belonging to fruit crops in India Table 2: Wild relatives of fruit crops occurring in different regions of India

28 29 • Knowledge of fruit crops, diversity distribution, landraces/genotypes • Types of survey: Coarse grid survey should be conducted in unexplored areas to capture the overall variability, while fine grid survey is carried out to build-up more collections for specific trait(s) known to • Knowledge of its wild relatives exist in identified pockets in previously explored areas. • Understanding for observing the variation in plants and environments • Multi-crop/ crop-specific explorations: Fruit crop-specific exploration are undertaken to collect the • Knowledge on mode of reproduction, morphological traits of fruit varieties and their landraces variability in a particular crop and its genepool. The samples collected must be representative of the diversity that exists within each crop/ crop groups in a given area. Multi-crop exploration is carried out • Knowledge of genepool sampling concept to collect the diversity in general of a given region (also referred as region-specific exploration) or in • Knowledge of random and non-random sampling; Coarse-grid and fine-grid sampling strategy unexplored areas. Conducting Ecogeographic Survey • Permission of collection from protected/restricted areas: Prior permission should be obtained from the concerned authorities for exploring and collecting in protected (biosphere reserves, sanctuaries, Prior to explorations, information about distribution of species and on diversity in morphology of tree and national parks) and restricted areas (international border) and particularly for foreign missions. quality of fruit is essential to study from secondary sources, including local markets and fruit mandi. Ecogeographic survey is prerequisite before collecting genetic resources of cultivated and wild plants and to • Tour itinerary: A tentative tour itinerary should be drawn up at an early stage of the planning, showing plan in situ conservation (IBPGR, 1985). An ecogeographic survey is defined as information gathering and the main target areas (or even precise localities) to be visited within the overall target region, the synthesis process on geographical, ecological, taxonomic and genetic diversity data (Maxted et al., 1995; roads/tracks to be followed and the proposed timings of each visit. The mode of transport should also be Castañeda Álvarez et al., 2011). The survey generally consists of collating information from herbarium specified. Letters of introduction to local government officials are often useful, and their preparation specimens, genebank accessions, PGR databases, in addition to published as well as informal literature etc. again will require some rough idea of the itinerary to be followed. Maps will clearly be needed in Outcome of such a survey is predictive and can be used in formulation of collection priorities. Generally, an planning the itinerary, but local contacts are essential for advice on the feasibility of following particular ecogeographic survey and analysis results in i. delineation of priority crop species, ii. identification of areas routes at different times of the year (Hawkes, 1980). for germplasm, herbarium collecting and in situ conservation, iii. identification of populations of cultivated • Period of collection: For germplasm collection of vegetatively propagated crops/species, the targeted and CWR species which are not conserved in genebank, iv. planning and execution of collection missions. areas should be surveyed first for identification and marking of elite types at the time of Ecogeographic surveys provide information on PGR to infer about history of their evolution and adaptation, flowering/fruiting and subsequently the collections are made at appropriate time. For seed producing to assess status of conservation and to prioritize areas for conservation. crops and species, exploration should be undertaken when these are physiologically mature and ready Prioritization of Species and Areas after Gap Analysis for harvest. In case of species with shattering nature, missions are executed rather earlier (7-10 days depending on crop/ species) before their maturity. Further, longer duration (2-3 weeks) mission and • Depending upon the priorities, gathering knowledge of the crops/species before launching a mission is repeat visits are suggested for collection of wild species. For taxonomic and herbarium collection, the important. flowering season of species is best. Period of explorations being organized within the country should be • The areas to be explored and crops/ species to be collected should be prioritized after thorough gap of at least 10-15 working days (excluding journey period) and more than a month when organised in analysis based on information from different sources including database of National Genebank/field foreign countries. genebank. However, for foreign explorations guidelines of the agricultural research department • Team composition: The exploration and collecting team should be familiar with basics of /ministry in the country to be followed to develop and finalize the mission. agriculture/plant genetic resources to meet the objective of the mission. Team consisting of 2-3 • The explorer should be well-versed with the nature and extent of diversity and breeding behaviour of members including a crop breeder, a collaborator and need-based local-aid may be formed preferably a crop/species to be collected and plan well in advance to facilitate preparations of proposed missions botanist/ breeder as team leader. Team of more than three persons is not desirable due to practical except those to be carried out under special situations like rescue collecting. Though majority of fruit problems that create hesitation in farmers, villagers. trees are cross pollinated resulting variability in seeds, except those are grafted or clones. • Area and route of exploration: This should be fine-tuned in consultation with the subject experts of • Visit to herbaria should be made to know the range of distribution, localities, diversity pattern and local bodies, staff of forest, agriculture departments, as soon as the team reaches to the starting point period of collection particularly for wild species. Flora of targeted area, R&D institutions and experts in keeping in view the targeted species and areas of the proposed mission. area should be consulted. • Items and equipments required: As per the nature of the germplasm to be collected (fruit/ seed/ • Collaborator(s) should be identified and communicated to join the mission well in advance with details vegetative propagule/ in vitro/ live plants) and the area(s) to be explored, several items and equipments of preparations, if required, particularly in case of vegetatively propagated and recalcitrant material. are required. Herbarium press, secateur and large size poly bags (1.5x2 m) are essential items required for herbarium specimen collection. • Phytosanitary regulations should be followed in case the material is transported from foreign country. • Domestic quarantine: All precautions including need-based domestic quarantine should be followed for Finalization of Mission pest-free collection and its transportation. • Gathering eco-geographic information: Information on topography, climatic conditions, vegetation, crops in cultivation and their maturity, etc. needs to be gathered to finalize the itinerary of collecting mission. Besides, explorers should establish local contacts especially at grass root level to seek the social, cultural, ethnic and other information of interest.

30 31 • Knowledge of fruit crops, diversity distribution, landraces/genotypes • Types of survey: Coarse grid survey should be conducted in unexplored areas to capture the overall variability, while fine grid survey is carried out to build-up more collections for specific trait(s) known to • Knowledge of its wild relatives exist in identified pockets in previously explored areas. • Understanding for observing the variation in plants and environments • Multi-crop/ crop-specific explorations: Fruit crop-specific exploration are undertaken to collect the • Knowledge on mode of reproduction, morphological traits of fruit varieties and their landraces variability in a particular crop and its genepool. The samples collected must be representative of the diversity that exists within each crop/ crop groups in a given area. Multi-crop exploration is carried out • Knowledge of genepool sampling concept to collect the diversity in general of a given region (also referred as region-specific exploration) or in • Knowledge of random and non-random sampling; Coarse-grid and fine-grid sampling strategy unexplored areas. Conducting Ecogeographic Survey • Permission of collection from protected/restricted areas: Prior permission should be obtained from the concerned authorities for exploring and collecting in protected (biosphere reserves, sanctuaries, Prior to explorations, information about distribution of species and on diversity in morphology of tree and national parks) and restricted areas (international border) and particularly for foreign missions. quality of fruit is essential to study from secondary sources, including local markets and fruit mandi. Ecogeographic survey is prerequisite before collecting genetic resources of cultivated and wild plants and to • Tour itinerary: A tentative tour itinerary should be drawn up at an early stage of the planning, showing plan in situ conservation (IBPGR, 1985). An ecogeographic survey is defined as information gathering and the main target areas (or even precise localities) to be visited within the overall target region, the synthesis process on geographical, ecological, taxonomic and genetic diversity data (Maxted et al., 1995; roads/tracks to be followed and the proposed timings of each visit. The mode of transport should also be Castañeda Álvarez et al., 2011). The survey generally consists of collating information from herbarium specified. Letters of introduction to local government officials are often useful, and their preparation specimens, genebank accessions, PGR databases, in addition to published as well as informal literature etc. again will require some rough idea of the itinerary to be followed. Maps will clearly be needed in Outcome of such a survey is predictive and can be used in formulation of collection priorities. Generally, an planning the itinerary, but local contacts are essential for advice on the feasibility of following particular ecogeographic survey and analysis results in i. delineation of priority crop species, ii. identification of areas routes at different times of the year (Hawkes, 1980). for germplasm, herbarium collecting and in situ conservation, iii. identification of populations of cultivated • Period of collection: For germplasm collection of vegetatively propagated crops/species, the targeted and CWR species which are not conserved in genebank, iv. planning and execution of collection missions. areas should be surveyed first for identification and marking of elite types at the time of Ecogeographic surveys provide information on PGR to infer about history of their evolution and adaptation, flowering/fruiting and subsequently the collections are made at appropriate time. For seed producing to assess status of conservation and to prioritize areas for conservation. crops and species, exploration should be undertaken when these are physiologically mature and ready Prioritization of Species and Areas after Gap Analysis for harvest. In case of species with shattering nature, missions are executed rather earlier (7-10 days depending on crop/ species) before their maturity. Further, longer duration (2-3 weeks) mission and • Depending upon the priorities, gathering knowledge of the crops/species before launching a mission is repeat visits are suggested for collection of wild species. For taxonomic and herbarium collection, the important. flowering season of species is best. Period of explorations being organized within the country should be • The areas to be explored and crops/ species to be collected should be prioritized after thorough gap of at least 10-15 working days (excluding journey period) and more than a month when organised in analysis based on information from different sources including database of National Genebank/field foreign countries. genebank. However, for foreign explorations guidelines of the agricultural research department • Team composition: The exploration and collecting team should be familiar with basics of /ministry in the country to be followed to develop and finalize the mission. agriculture/plant genetic resources to meet the objective of the mission. Team consisting of 2-3 • The explorer should be well-versed with the nature and extent of diversity and breeding behaviour of members including a crop breeder, a collaborator and need-based local-aid may be formed preferably a crop/species to be collected and plan well in advance to facilitate preparations of proposed missions botanist/ breeder as team leader. Team of more than three persons is not desirable due to practical except those to be carried out under special situations like rescue collecting. Though majority of fruit problems that create hesitation in farmers, villagers. trees are cross pollinated resulting variability in seeds, except those are grafted or clones. • Area and route of exploration: This should be fine-tuned in consultation with the subject experts of • Visit to herbaria should be made to know the range of distribution, localities, diversity pattern and local bodies, staff of forest, agriculture departments, as soon as the team reaches to the starting point period of collection particularly for wild species. Flora of targeted area, R&D institutions and experts in keeping in view the targeted species and areas of the proposed mission. area should be consulted. • Items and equipments required: As per the nature of the germplasm to be collected (fruit/ seed/ • Collaborator(s) should be identified and communicated to join the mission well in advance with details vegetative propagule/ in vitro/ live plants) and the area(s) to be explored, several items and equipments of preparations, if required, particularly in case of vegetatively propagated and recalcitrant material. are required. Herbarium press, secateur and large size poly bags (1.5x2 m) are essential items required for herbarium specimen collection. • Phytosanitary regulations should be followed in case the material is transported from foreign country. • Domestic quarantine: All precautions including need-based domestic quarantine should be followed for Finalization of Mission pest-free collection and its transportation. • Gathering eco-geographic information: Information on topography, climatic conditions, vegetation, crops in cultivation and their maturity, etc. needs to be gathered to finalize the itinerary of collecting mission. Besides, explorers should establish local contacts especially at grass root level to seek the social, cultural, ethnic and other information of interest.

30 31 List of items and equipments for collecting • The optimum sample size per site would be the number of plants required to obtain, with 95 percent certainty, all the alleles at a random locus occurring in the target population with frequency greater than Survey / collecting items Global Positioning System (GPS), digital camera with additional 0.05 (Hawkes, 1976; Marshall and Brown, 1975). memory card, binocular, magnifying glasses, handheld microscope, digital Vernier calliper and portable balance, haversack/ kitbag, seed • In case of extremely variable populations like in cross-pollinated fruits, one can either take the envelopes, cloth bags, polythene bags, aluminium & tag labels, drying vegetative propagule of mother trees or make larger samples (bulking of fruits for genepool collection), sheets, old newspapers, plant press, moss, rubber bands, packing or take as sub-samples if observed interesting variants, and be given separate collecting numbers. tape, sutli (thick and thin), secateurs, scissors, knife, digger, torch • In general, targeted sampling of elite type trees are best due to possibility of variation in matured fruits light, measuring tape, passport data book, field note book, pencil, and limitation of land for conservation in field genebank. However, random sampling should be made by ballpoint pen and permanent marker. collecting single fruit/berry/pod from at least 50 plants along a number of transects throughout the field Reference material Regional/ national flora, digital herbarium, lap-top and accessories, (Hawkes, 1976 & 1980) to obtain a representative and adequate sample. Plant population at border of list of local names of plants, road-map, vegetation/climate map, list field should be avoided. of rest-houses/ lodges, hotels, resting/ stay places and list of local • In a situation when wild population with few individuals occur, one should better collect from all the contacts (phone, fax, e-mail). plants so as to make the representative sample from that site. In case of certain wild and semi- First Aid-Box Anti-malaria pills, anti-allergen tablets, pain killers, anti- amoebic and domesticated species occurring in small pocket with scattered populations (treated as sampling site) anti-diarrhoeal tablets, mosquito repellent, antifungal/ antibacterial/ having specific use/traits, the seed should be bulked. However one should not deplete the populations antiseptic creams or lotions, cotton-packs, band-aid, anti-septic, of farmers' planting stocks or wild species, or remove significant genetic variation. dressing gauze, water-purifying tablets, etc. • In case of species with extremely small-sized seeds, low seed-set, asynchronous maturity and low seed viability, care should be taken to collect adequate sample size. Collecting strategy and Sampling Methodology • While collecting the seed, the explorer should keep in mind the required quantity of material to be At the actual collecting sites, there will be need to apply sampling method to ensure that the genetic sampled for long term conservation (2000 and 4000 seeds for self and cross pollinated crops, diversity of crop/species is adequately represented in the sample collected. The following points are given respectively) besides meeting the requirement of characterization, evaluation and related studies. to take care depending upon the situations. Establishing Taxonomic Identity Sampling Sites • Material with dubious identity, unidentified material and only vernacular name should be collected • For cultivated fruit species, sampling sites in order of preference should be orchard, farmers' field, along with herbarium specimen and photographs for authentication. In case when herbarium backyard/ kitchen garden, wastelands in villages and forests. Inaccessible areas of valleys, isolated hills, specimens are not available, efforts should be made by the explorer to raise plants to establish its villages at the edge of deserts, forests, mountains and isolated coastal belts may hold rich genetic correct identity. diversity, potential/ trait-specific germplasm and wild relatives. • Normally 4-5 individual plants/parts having representation of all parts especially flowers, or fruits or • Collecting mission should be started first from warm, drier tracts (vs. humid), un-irrigated areas (vs. both should be collected for preparing herbarium specimen. Locality, date of collection and field notes irrigated), valleys (vs. hills) to capture maximum available diversity in a planned manner. should be clearly recorded. Characters which are lost on drying, or which may not be represented in the herbarium specimen (plant height), flower colour, leaflets (which may be shed on drying) should be • The fruit crops often vary with ethnic diversity and different array of materials may be collected even mentioned in field notes. The detailed guidelines for preparation and processing herbarium specimen from contiguous belts occupied by different tribes. should be followed as per Jain and Rao (1977). • Sites having stress situations viz. saline habitat, un-irrigated/ drought conditions, desert (cold and hot), Type of Material flood prone areas should be identified as target areas for collection of respective trait-specific material. In such cases, selective sampling of promising genotypes should be done. • Majority of tropical fruits have recalcitrant seeds (sensitive to desiccation and freezing), hence difficult to conserve ex-situ in seed genebank and in-vitro. Cryo-preservation protocols are not available for • For biotic stress tolerant material, hot-spot areas should be visited to collect healthy plants in fields mango, jamun, jackfruit, some species of garcinia etc. and are hence maintained in field genebanks. where severe pest damage is evident. Further, most of these are cross pollinated and therefore, vegetative parts are required to collect true to • The frequency of sampling (number of samples per site) should be decided based on on-the-spot type germplasm. For genepool collecting, their seeds and sampling method of cross pollinated crops observations on the variability available. In general, more sites per target area are preferred to sample would be applied, but number of trees and total samples would be high. the targeted species rather than sampling from a few sites. • Depending on the objective of the collection mission, seed, vegetative propagule, in vitro material and Sampling Method (Self, cross pollinated and vegetatively propagated material) pollen are collected. For herbarium specimens, all possible parts of plants including root, stem, leaves, buds, flowers and fruits are collected. Herbarium specimens, in general and especially of the wild types • In case of scion collection for budding and grafting the sample size will depend upon the number of and wild relatives should always be collected to help in identification/ authentication. Wherever rootstocks available but not less than ten per sample so that at least eight grafts may survive. In case of possible efforts should be made to collect economic products of local and wider use as supportive cuttings and rooted suckers (e.g. grapes, ornamentals, passion fruits, black pepper, beetle vine, banana, material. cardamom, etc.) 15-20 cuttings may be sufficient.

32 33 List of items and equipments for collecting • The optimum sample size per site would be the number of plants required to obtain, with 95 percent certainty, all the alleles at a random locus occurring in the target population with frequency greater than Survey / collecting items Global Positioning System (GPS), digital camera with additional 0.05 (Hawkes, 1976; Marshall and Brown, 1975). memory card, binocular, magnifying glasses, handheld microscope, digital Vernier calliper and portable balance, haversack/ kitbag, seed • In case of extremely variable populations like in cross-pollinated fruits, one can either take the envelopes, cloth bags, polythene bags, aluminium & tag labels, drying vegetative propagule of mother trees or make larger samples (bulking of fruits for genepool collection), sheets, old newspapers, plant press, moss, rubber bands, packing or take as sub-samples if observed interesting variants, and be given separate collecting numbers. tape, sutli (thick and thin), secateurs, scissors, knife, digger, torch • In general, targeted sampling of elite type trees are best due to possibility of variation in matured fruits light, measuring tape, passport data book, field note book, pencil, and limitation of land for conservation in field genebank. However, random sampling should be made by ballpoint pen and permanent marker. collecting single fruit/berry/pod from at least 50 plants along a number of transects throughout the field Reference material Regional/ national flora, digital herbarium, lap-top and accessories, (Hawkes, 1976 & 1980) to obtain a representative and adequate sample. Plant population at border of list of local names of plants, road-map, vegetation/climate map, list field should be avoided. of rest-houses/ lodges, hotels, resting/ stay places and list of local • In a situation when wild population with few individuals occur, one should better collect from all the contacts (phone, fax, e-mail). plants so as to make the representative sample from that site. In case of certain wild and semi- First Aid-Box Anti-malaria pills, anti-allergen tablets, pain killers, anti- amoebic and domesticated species occurring in small pocket with scattered populations (treated as sampling site) anti-diarrhoeal tablets, mosquito repellent, antifungal/ antibacterial/ having specific use/traits, the seed should be bulked. However one should not deplete the populations antiseptic creams or lotions, cotton-packs, band-aid, anti-septic, of farmers' planting stocks or wild species, or remove significant genetic variation. dressing gauze, water-purifying tablets, etc. • In case of species with extremely small-sized seeds, low seed-set, asynchronous maturity and low seed viability, care should be taken to collect adequate sample size. Collecting strategy and Sampling Methodology • While collecting the seed, the explorer should keep in mind the required quantity of material to be At the actual collecting sites, there will be need to apply sampling method to ensure that the genetic sampled for long term conservation (2000 and 4000 seeds for self and cross pollinated crops, diversity of crop/species is adequately represented in the sample collected. The following points are given respectively) besides meeting the requirement of characterization, evaluation and related studies. to take care depending upon the situations. Establishing Taxonomic Identity Sampling Sites • Material with dubious identity, unidentified material and only vernacular name should be collected • For cultivated fruit species, sampling sites in order of preference should be orchard, farmers' field, along with herbarium specimen and photographs for authentication. In case when herbarium backyard/ kitchen garden, wastelands in villages and forests. Inaccessible areas of valleys, isolated hills, specimens are not available, efforts should be made by the explorer to raise plants to establish its villages at the edge of deserts, forests, mountains and isolated coastal belts may hold rich genetic correct identity. diversity, potential/ trait-specific germplasm and wild relatives. • Normally 4-5 individual plants/parts having representation of all parts especially flowers, or fruits or • Collecting mission should be started first from warm, drier tracts (vs. humid), un-irrigated areas (vs. both should be collected for preparing herbarium specimen. Locality, date of collection and field notes irrigated), valleys (vs. hills) to capture maximum available diversity in a planned manner. should be clearly recorded. Characters which are lost on drying, or which may not be represented in the herbarium specimen (plant height), flower colour, leaflets (which may be shed on drying) should be • The fruit crops often vary with ethnic diversity and different array of materials may be collected even mentioned in field notes. The detailed guidelines for preparation and processing herbarium specimen from contiguous belts occupied by different tribes. should be followed as per Jain and Rao (1977). • Sites having stress situations viz. saline habitat, un-irrigated/ drought conditions, desert (cold and hot), Type of Material flood prone areas should be identified as target areas for collection of respective trait-specific material. In such cases, selective sampling of promising genotypes should be done. • Majority of tropical fruits have recalcitrant seeds (sensitive to desiccation and freezing), hence difficult to conserve ex-situ in seed genebank and in-vitro. Cryo-preservation protocols are not available for • For biotic stress tolerant material, hot-spot areas should be visited to collect healthy plants in fields mango, jamun, jackfruit, some species of garcinia etc. and are hence maintained in field genebanks. where severe pest damage is evident. Further, most of these are cross pollinated and therefore, vegetative parts are required to collect true to • The frequency of sampling (number of samples per site) should be decided based on on-the-spot type germplasm. For genepool collecting, their seeds and sampling method of cross pollinated crops observations on the variability available. In general, more sites per target area are preferred to sample would be applied, but number of trees and total samples would be high. the targeted species rather than sampling from a few sites. • Depending on the objective of the collection mission, seed, vegetative propagule, in vitro material and Sampling Method (Self, cross pollinated and vegetatively propagated material) pollen are collected. For herbarium specimens, all possible parts of plants including root, stem, leaves, buds, flowers and fruits are collected. Herbarium specimens, in general and especially of the wild types • In case of scion collection for budding and grafting the sample size will depend upon the number of and wild relatives should always be collected to help in identification/ authentication. Wherever rootstocks available but not less than ten per sample so that at least eight grafts may survive. In case of possible efforts should be made to collect economic products of local and wider use as supportive cuttings and rooted suckers (e.g. grapes, ornamentals, passion fruits, black pepper, beetle vine, banana, material. cardamom, etc.) 15-20 cuttings may be sufficient.

32 33 Transportation Packaging and Labelling (Sharing, Accessioning, Multiplication and Conservation) • In case of fruits and vegetative propagules and in long duration missions, if required, the explorer should The clean and dried material should be kept in envelopes with proper labels specifying its botanical name make prior arrangements for en-route transportation of collected material to the place of its and collector number. One set of the material along with passport data should be sent for accessioning, establishment/ maintenance to avoid deterioration. Daily checking of collected material, change of conservation (LTS/MTS) and another set be sent to the collaborating institute for initial seed increase (if blotting papers of herbarium sheets, room drying of collected material after reaching at place of halt is required), maintenance, characterization and evaluation. essential. Establishment / Maintenance for Vegetatively Propagated Material Recording Information Vegetatively propagated material should be sent for establishment/ maintenance in field genebank or at Passport Data suitable site. The material for in vitro and cryo-genebank should be handed over to the concerned curators. The elite material, if any, should be studied in detail to generate supporting information as well as for Passport data is important for database management, documentation, enhanced utilization of PGR and validation of the known trait(s) for its registration with concerned agency. studying the variation in distributional pattern with respect to ecological and socio-economic factors. It is advisable to record information on both the essential and optional fields in the passport data sheet or field Report Writing and Publication book at the site of the collection itself by the explorer. Sample content of passport data book of NBPGR, New After completing the mission of herbarium and germplasm collection from a target area and processing the Delhi is given in Annexure-I. However, in any circumstances, the explorer should not leave the information collected material, it is important to write the comprehensive report to fulfil the mission's objectives. This blank on essential fields namely sample labelling (name of organization(s) and collectors, collectors' helps in follow-up collecting(s) and allows users to know the availability of the germplasm as well as in number, date and type of material); sample identification (botanical identity, vernacular name, its biological publications. The information on the samples collected can be entered into database for its access to users. status); sampling information (sampling type, method and source) and collecting site localization (state, The report on the exploration and collection should broadly include: district, village, latitude, longitude and altitude). This information is important for herbarium specimens and record in field books. • Name of the organisation(s) Since, characterization and evaluation of perennial tree germplasm takes quite long andis difficult due to • Name of the scientist(s)/person(s) involved limitation of resources, land and time, hence, recording the descriptors of fruit tree on the site itself would • Collaborating organisation(s) be the best. Though it cannot provide data expected from evaluation trials at research farm with uniform treatments to different genotypes. Qualitative and highly inherited traits are least affected by the • Objectives of the collecting mission environment and hence correct information is expected from this data. PPV&FRA has also adopted this • A description of the environment, flora, people of the target area method in case of perennial trees. So, there is need to use additional passport data consisting descriptors of species to record individual tree data. • An account of the logistics and scientific planning Related Information • Details of the execution of the mission (timing, itinerary, sampling strategy and collecting techniques) Information on genetic erosion should be gathered from aged-farmers particularly on the depletion of • A summary of the results (areas surveyed along with route maps, germplasm and herbarium specimens landraces cultivated over the time and the reasons for their loss in general and native crops in particular. collected, indigenous knowledge documented and extent and magnitude of diversity collected with Indigenous traditional knowledge on plants, their use, agricultural technologies etc. are also asked and elite germplasm, if any) recorded. Meaning of the name of landraces and their properties should be asked from farmers and • Role of women in conservation of diversity recorded. Observations on the distribution pattern and frequency status of crop wild relatives, rare, endangered and threatened species of PGR importance should be recorded for their sustainable • Details of sharing germplasm and information management. Ethnobotanical observations and new uses of plants, especially those collected from tribal • Photographs dominated tracts, are currently recorded as a database which would be available for reference in future collections. • An account on loss of germplasm and ITK, if any Post Collection Handling • Difficulties encountered during collecting mission Seed Extraction, Cleaning/Drying • Recommendations for follow-up action(s) The extraction and cleaning of seed should be done preferably on the same day or immediately after • Acknowledgement completing the expedition and processed for their drying under shade/ sun/ controlled conditions. Seeds DO'S AND DON'TS with short longevity should be processed at the earliest and care should be taken during threshing/ cleaning to avoid damage. In a situation, when delay in processing is anticipated, all precautions should be taken to In addition to above guidelines for exploration, germplasm and herbarium collection, the collector(s) should maintain its viability. The observations on variability parameters on fruit/ pod/ seed should be recorded observe a well-defined code of conduct as well as take necessary precautionary measures in its smooth along with photographs for report writing, documentation and publication. execution as mentioned below:

34 35 Transportation Packaging and Labelling (Sharing, Accessioning, Multiplication and Conservation) • In case of fruits and vegetative propagules and in long duration missions, if required, the explorer should The clean and dried material should be kept in envelopes with proper labels specifying its botanical name make prior arrangements for en-route transportation of collected material to the place of its and collector number. One set of the material along with passport data should be sent for accessioning, establishment/ maintenance to avoid deterioration. Daily checking of collected material, change of conservation (LTS/MTS) and another set be sent to the collaborating institute for initial seed increase (if blotting papers of herbarium sheets, room drying of collected material after reaching at place of halt is required), maintenance, characterization and evaluation. essential. Establishment / Maintenance for Vegetatively Propagated Material Recording Information Vegetatively propagated material should be sent for establishment/ maintenance in field genebank or at Passport Data suitable site. The material for in vitro and cryo-genebank should be handed over to the concerned curators. The elite material, if any, should be studied in detail to generate supporting information as well as for Passport data is important for database management, documentation, enhanced utilization of PGR and validation of the known trait(s) for its registration with concerned agency. studying the variation in distributional pattern with respect to ecological and socio-economic factors. It is advisable to record information on both the essential and optional fields in the passport data sheet or field Report Writing and Publication book at the site of the collection itself by the explorer. Sample content of passport data book of NBPGR, New After completing the mission of herbarium and germplasm collection from a target area and processing the Delhi is given in Annexure-I. However, in any circumstances, the explorer should not leave the information collected material, it is important to write the comprehensive report to fulfil the mission's objectives. This blank on essential fields namely sample labelling (name of organization(s) and collectors, collectors' helps in follow-up collecting(s) and allows users to know the availability of the germplasm as well as in number, date and type of material); sample identification (botanical identity, vernacular name, its biological publications. The information on the samples collected can be entered into database for its access to users. status); sampling information (sampling type, method and source) and collecting site localization (state, The report on the exploration and collection should broadly include: district, village, latitude, longitude and altitude). This information is important for herbarium specimens and record in field books. • Name of the organisation(s) Since, characterization and evaluation of perennial tree germplasm takes quite long andis difficult due to • Name of the scientist(s)/person(s) involved limitation of resources, land and time, hence, recording the descriptors of fruit tree on the site itself would • Collaborating organisation(s) be the best. Though it cannot provide data expected from evaluation trials at research farm with uniform treatments to different genotypes. Qualitative and highly inherited traits are least affected by the • Objectives of the collecting mission environment and hence correct information is expected from this data. PPV&FRA has also adopted this • A description of the environment, flora, people of the target area method in case of perennial trees. So, there is need to use additional passport data consisting descriptors of species to record individual tree data. • An account of the logistics and scientific planning Related Information • Details of the execution of the mission (timing, itinerary, sampling strategy and collecting techniques) Information on genetic erosion should be gathered from aged-farmers particularly on the depletion of • A summary of the results (areas surveyed along with route maps, germplasm and herbarium specimens landraces cultivated over the time and the reasons for their loss in general and native crops in particular. collected, indigenous knowledge documented and extent and magnitude of diversity collected with Indigenous traditional knowledge on plants, their use, agricultural technologies etc. are also asked and elite germplasm, if any) recorded. Meaning of the name of landraces and their properties should be asked from farmers and • Role of women in conservation of diversity recorded. Observations on the distribution pattern and frequency status of crop wild relatives, rare, endangered and threatened species of PGR importance should be recorded for their sustainable • Details of sharing germplasm and information management. Ethnobotanical observations and new uses of plants, especially those collected from tribal • Photographs dominated tracts, are currently recorded as a database which would be available for reference in future collections. • An account on loss of germplasm and ITK, if any Post Collection Handling • Difficulties encountered during collecting mission Seed Extraction, Cleaning/Drying • Recommendations for follow-up action(s) The extraction and cleaning of seed should be done preferably on the same day or immediately after • Acknowledgement completing the expedition and processed for their drying under shade/ sun/ controlled conditions. Seeds DO'S AND DON'TS with short longevity should be processed at the earliest and care should be taken during threshing/ cleaning to avoid damage. In a situation, when delay in processing is anticipated, all precautions should be taken to In addition to above guidelines for exploration, germplasm and herbarium collection, the collector(s) should maintain its viability. The observations on variability parameters on fruit/ pod/ seed should be recorded observe a well-defined code of conduct as well as take necessary precautionary measures in its smooth along with photographs for report writing, documentation and publication. execution as mentioned below:

34 35 Do's • Get acquainted with the International Code of Conduct for Plant Germplasm Collecting and Transfer developed by FAO (1993). • Always keep a route map of the target area with list of important places and the distance covered during travel to facilitate report writing. • Before entering into a forest take the help of forest guards to have forehand knowledge of possible dangers in the target area. If needed, help of a gunman is taken during survey in dense forest. • Explain the purpose and get consent from the farmers for collecting germplasm. • Keep important telephone numbers of concerned officers including district authorities, hospitals, dispensaries and police station. • Keep your identity card and a certificate from Head of Organization for proposed mission. • Honour social customs of local inhabitants of the target area. • While talking and discussing with ladies, be polite and respectful to them. • After day's collection and before retiring to bed, have a glance at your equipments, passport data and collected material for need-based updating. Don'ts • Do not provide lift to strangers in your vehicle under any pretext. • Do not indulge in unnecessary discussion related to politics, religion and local beliefs with the local people. • Do not make false promises with donors. • Do not plan the expedition during important festivals and peak election campaign in the target area. • Do not enter any house for seed collection in absence of male members of the family. • Do not eat unknown wild fruits since some of them may be toxic or internally infected. • Do not collect the seed in large quantities from any household if the farmers wish so. • Over-collecting of the genetic diversity with similar attributes should be avoided to save time and energy in collection and evaluation and to save space in the genebank.

Fig. 1. Flow chart of Exploration for germplasm and herbarium collection activities 36 37 Do's • Get acquainted with the International Code of Conduct for Plant Germplasm Collecting and Transfer developed by FAO (1993). • Always keep a route map of the target area with list of important places and the distance covered during travel to facilitate report writing. • Before entering into a forest take the help of forest guards to have forehand knowledge of possible dangers in the target area. If needed, help of a gunman is taken during survey in dense forest. • Explain the purpose and get consent from the farmers for collecting germplasm. • Keep important telephone numbers of concerned officers including district authorities, hospitals, dispensaries and police station. • Keep your identity card and a certificate from Head of Organization for proposed mission. • Honour social customs of local inhabitants of the target area. • While talking and discussing with ladies, be polite and respectful to them. • After day's collection and before retiring to bed, have a glance at your equipments, passport data and collected material for need-based updating. Don'ts • Do not provide lift to strangers in your vehicle under any pretext. • Do not indulge in unnecessary discussion related to politics, religion and local beliefs with the local people. • Do not make false promises with donors. • Do not plan the expedition during important festivals and peak election campaign in the target area. • Do not enter any house for seed collection in absence of male members of the family. • Do not eat unknown wild fruits since some of them may be toxic or internally infected. • Do not collect the seed in large quantities from any household if the farmers wish so. • Over-collecting of the genetic diversity with similar attributes should be avoided to save time and energy in collection and evaluation and to save space in the genebank.

Fig. 1. Flow chart of Exploration for germplasm and herbarium collection activities 36 37 References Annexure-I 1. Arora RK (1981) Plant genetic resources exploration and collection: planning and logistics. In: Mehra NATIONAL BUREAU OF PLANT GENETIC RESOURCES, NEW DELHI -12 KL, RK Arora and SR Wadhi (eds) Plant exploration and collection, NBPGR Sci. Monograph 3, National Bureau of Plant Genetic Resources, New Delhi, pp 46-54. PASSPORT DATA SHEET 2. Arora RK and V Ramanatha Rao (eds) (1998) Tropical fruits in Asia. Diversity, maintenance, conservation and use, Proceedings of the IPGRI-ICAR_UTFANET, regional training course on the Date...... Collector's No...... Accession No...... conservation and use of germplasm of Tropical fruits in Asia, held at IIHR, 18-31 May 1997, Bangalore, Botanical Name...... Common Name (English)...... India; IPGRI Rome Italy. 3. Castañeda Álvarez NP, HA Vincent, SP Kell, RJ Eastwood and N Maxted (2011) Ecogeographic surveys. Crop/Vern. Name...... Cultivar name...... In: Guarino L, V Ramanatha Rao and E Goldberg (eds). Collecting Plant Genetic Diversity: Technical Region Explored...... Guidelines. 2011 update. Bioversity International, Rome. 4. FAO (1993) International Code of Conduct for Germplasm Collecting and Transfer. FAO, Rome. Village/Block...... District...... State...... www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en Latitude…..……………0N Longitude…..………………0E Altitude….………..m 5. FAO (1997). The state of the world's plant genetic resources for food and agriculture. FAO, Rome. PP 444. http://www.fao.org/3/a-w7324e.pdf Temp...... Rainfall ...... 6. Frankel OH and AHD Brown (1984) Plant genetic resources today: a critical appraisal. In: Holden JHW COLLECTION SITE 1. Natural wild 2.Disturbed wild 3.Farmer's field and JT Williams (eds.) Crop genetic resources: Conservation and evaluation. Allen and Unwin, 4.Threshing yard 5.Fallow 6.Farm store 7.Market Winchester p 249–257. 8.Garden 9.Institute 10...... 7. Gautam PL, BS Dabas, U Srivastava and SS Duhoon (1998) Plant Germplasm Collecting: Principles and BIOLOGICAL STATUS 1.Wild 2.Weed 3.Landrace 4.Primitive cultivar 5. Procedures. NBPGR, New Delhi, 218 p. Breeder's line 8. Greene SL and TC Hart (1999) Implementing a geographic analysis in germplasm conservation. In: Greene SL, L Guarino (eds.) Linking genetic resources and geography: emerging strategies for FREQUENCY 1.Abundant 2.Frequent 3.Occassional 4.Rare conserving and using crop biodiversity. American Society of Agronomy; Crop Science Society of MATERIAL 1. Seeds 2.Fruits 3.Inflorescence 4.Roots 5.Tubers 6. America, Madison, pp 25–38. Rhizomes 7. Suckers 8.Live plants 9.Herbarium 9. Guarino L, V Ramanatha Rao, R Reid (1995a) A brief history of plant germplasm collecting. In: Guarino 10………….. L, Ramanatha Rao V and Reid R (eds.) Collecting plant genetic diversity. Technical guidelines. CAB International, Wallingford, UK, pp 1-11. BREEDING SYSTEM 1.Self-pollinated 2.Cross-pollinated 3.Vegetatively 10. Guarino L, V Ramanatha Rao and R Reid (1995b). Collecting plant genetic diversity: Technical propagated guidelines. International Plant Genetic Resources Institute (IPGRI), Rome, Italy; Plant Production and SAMPLE TYPE 1.Population 2.Pure line 3.Individual plant Protection Division, FAO, Rome, Italy; World Conservation Union (IUCN), Gland, Switzerland; CABI, Wallingford, UK, 748 p. SAMPLE METHOD 1.Bulk 2.Random 3.Selective (non-random) 11. Hawkes JG (1976). Manual for field collectors (Seed crops), International Board for Plant Genetic HABITAT 1. Cultivated 2.Disturbed 3.Partly disturbed Resources, FAO, Rome, Italy. 4.Rangeland 5……………….. 12. Hawkes JG (1980). Crop genetic resources - A field collection manual, IBPGR/EUCARPIA, Univ. of DISEASE SYMPTOMS 1.Susceptible 2.Mildly susceptible 3.Tolerant Birmingham, UK. 4.Resistant 5.Immune 13. Howard A and GLC Howard (1910) Wheat in India, its Production, Varieties and Improvement. Thacker Spinn & Co., Calcutta. INSECT/ PEST/ NEMATODE INFECTION 1. Mild 2.Moderate 3. High 14. IBPGR (1985) Ecogeogrpahic surveying and in situ conservation of crop relatives. Report of an IBPGR CULTURAL PRACTICE SEASON 1. Irrigated 2.Rainfed 3.Arid 4.Wet 5...... task force meeting held at Washington DC. IBPGR, Rome, 33 p. 1.Kharif 2.Rabi 3.Spring-summer 4.Perennial type 15. ICAR-NBPGR (2016) Guidelines for Management of Plant Genetic Resources in India. ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. ASSOCIATED FLORA 1. Sole 2.Mixed with……………………… 16. Jain S.K and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow SOIL COLOUR 1. Black 2.Yellow 3.Red 4.Brown 5…….. Printers and Publishers, New Delhi, p. 157. SOIL TEXTURE 1.Sandy 2.Sandy loam 3.Loam 4.Silt loam 5.Clay 6.Silt 17. Marshall, DR and AHD Brown (1975). Optimum sampling strategies in genetic conservation. In: Frankel OH and JG Hawkes (eds). Genetic resources for today and tomorrow. Cambridge Univ. Press, TOPOGRAPHY 1.Swamp 2.Flood plain 3.Level 4.Undulating 5.Hilly Cambridge, pp. 53-80. dissected 6.Steeply dissected 7.Mountainous 8.Valley 18. Maxted N, MW van Slageren and JR Rihan (1995) Ecogeographic surveys. In: Guarino L, V Ramanatha AGRONOMIC SCORE 1.Very poor 2. Poor 3. Average 4. Good 5. Very good Rao and R Reid (eds.) Collecting Plant Genetic Diversity. CABI International, Wallingford, UK, pp. 255–285. ETHNOBOTANICAL USES PART(S) 1. Stem 2. Leaf 3.Root 4. Fruit 5.Flower 6.Whole plant 19. Maxted N, BV Ford-Lloyd, SL Jury, SP Kell and MA Scholten (2006) Towards a definition of a crop wild 7.Seed 8.Others relative. Biodiversity and Conservation 14: 1-13. 38 39 References Annexure-I 1. Arora RK (1981) Plant genetic resources exploration and collection: planning and logistics. In: Mehra NATIONAL BUREAU OF PLANT GENETIC RESOURCES, NEW DELHI -12 KL, RK Arora and SR Wadhi (eds) Plant exploration and collection, NBPGR Sci. Monograph 3, National Bureau of Plant Genetic Resources, New Delhi, pp 46-54. PASSPORT DATA SHEET 2. Arora RK and V Ramanatha Rao (eds) (1998) Tropical fruits in Asia. Diversity, maintenance, conservation and use, Proceedings of the IPGRI-ICAR_UTFANET, regional training course on the Date...... Collector's No...... Accession No...... conservation and use of germplasm of Tropical fruits in Asia, held at IIHR, 18-31 May 1997, Bangalore, Botanical Name...... Common Name (English)...... India; IPGRI Rome Italy. 3. Castañeda Álvarez NP, HA Vincent, SP Kell, RJ Eastwood and N Maxted (2011) Ecogeographic surveys. Crop/Vern. Name...... Cultivar name...... In: Guarino L, V Ramanatha Rao and E Goldberg (eds). Collecting Plant Genetic Diversity: Technical Region Explored...... Guidelines. 2011 update. Bioversity International, Rome. 4. FAO (1993) International Code of Conduct for Germplasm Collecting and Transfer. FAO, Rome. Village/Block...... District...... State...... www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en Latitude…..……………0N Longitude…..………………0E Altitude….………..m 5. FAO (1997). The state of the world's plant genetic resources for food and agriculture. FAO, Rome. PP 444. http://www.fao.org/3/a-w7324e.pdf Temp...... Rainfall ...... 6. Frankel OH and AHD Brown (1984) Plant genetic resources today: a critical appraisal. In: Holden JHW COLLECTION SITE 1. Natural wild 2.Disturbed wild 3.Farmer's field and JT Williams (eds.) Crop genetic resources: Conservation and evaluation. Allen and Unwin, 4.Threshing yard 5.Fallow 6.Farm store 7.Market Winchester p 249–257. 8.Garden 9.Institute 10...... 7. Gautam PL, BS Dabas, U Srivastava and SS Duhoon (1998) Plant Germplasm Collecting: Principles and BIOLOGICAL STATUS 1.Wild 2.Weed 3.Landrace 4.Primitive cultivar 5. Procedures. NBPGR, New Delhi, 218 p. Breeder's line 8. Greene SL and TC Hart (1999) Implementing a geographic analysis in germplasm conservation. In: Greene SL, L Guarino (eds.) Linking genetic resources and geography: emerging strategies for FREQUENCY 1.Abundant 2.Frequent 3.Occassional 4.Rare conserving and using crop biodiversity. American Society of Agronomy; Crop Science Society of MATERIAL 1. Seeds 2.Fruits 3.Inflorescence 4.Roots 5.Tubers 6. America, Madison, pp 25–38. Rhizomes 7. Suckers 8.Live plants 9.Herbarium 9. Guarino L, V Ramanatha Rao, R Reid (1995a) A brief history of plant germplasm collecting. In: Guarino 10………….. L, Ramanatha Rao V and Reid R (eds.) Collecting plant genetic diversity. Technical guidelines. CAB International, Wallingford, UK, pp 1-11. BREEDING SYSTEM 1.Self-pollinated 2.Cross-pollinated 3.Vegetatively 10. Guarino L, V Ramanatha Rao and R Reid (1995b). Collecting plant genetic diversity: Technical propagated guidelines. International Plant Genetic Resources Institute (IPGRI), Rome, Italy; Plant Production and SAMPLE TYPE 1.Population 2.Pure line 3.Individual plant Protection Division, FAO, Rome, Italy; World Conservation Union (IUCN), Gland, Switzerland; CABI, Wallingford, UK, 748 p. SAMPLE METHOD 1.Bulk 2.Random 3.Selective (non-random) 11. Hawkes JG (1976). Manual for field collectors (Seed crops), International Board for Plant Genetic HABITAT 1. Cultivated 2.Disturbed 3.Partly disturbed Resources, FAO, Rome, Italy. 4.Rangeland 5……………….. 12. Hawkes JG (1980). Crop genetic resources - A field collection manual, IBPGR/EUCARPIA, Univ. of DISEASE SYMPTOMS 1.Susceptible 2.Mildly susceptible 3.Tolerant Birmingham, UK. 4.Resistant 5.Immune 13. Howard A and GLC Howard (1910) Wheat in India, its Production, Varieties and Improvement. Thacker Spinn & Co., Calcutta. INSECT/ PEST/ NEMATODE INFECTION 1. Mild 2.Moderate 3. High 14. IBPGR (1985) Ecogeogrpahic surveying and in situ conservation of crop relatives. Report of an IBPGR CULTURAL PRACTICE SEASON 1. Irrigated 2.Rainfed 3.Arid 4.Wet 5...... task force meeting held at Washington DC. IBPGR, Rome, 33 p. 1.Kharif 2.Rabi 3.Spring-summer 4.Perennial type 15. ICAR-NBPGR (2016) Guidelines for Management of Plant Genetic Resources in India. ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. ASSOCIATED FLORA 1. Sole 2.Mixed with……………………… 16. Jain S.K and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow SOIL COLOUR 1. Black 2.Yellow 3.Red 4.Brown 5…….. Printers and Publishers, New Delhi, p. 157. SOIL TEXTURE 1.Sandy 2.Sandy loam 3.Loam 4.Silt loam 5.Clay 6.Silt 17. Marshall, DR and AHD Brown (1975). Optimum sampling strategies in genetic conservation. In: Frankel OH and JG Hawkes (eds). Genetic resources for today and tomorrow. Cambridge Univ. Press, TOPOGRAPHY 1.Swamp 2.Flood plain 3.Level 4.Undulating 5.Hilly Cambridge, pp. 53-80. dissected 6.Steeply dissected 7.Mountainous 8.Valley 18. Maxted N, MW van Slageren and JR Rihan (1995) Ecogeographic surveys. In: Guarino L, V Ramanatha AGRONOMIC SCORE 1.Very poor 2. Poor 3. Average 4. Good 5. Very good Rao and R Reid (eds.) Collecting Plant Genetic Diversity. CABI International, Wallingford, UK, pp. 255–285. ETHNOBOTANICAL USES PART(S) 1. Stem 2. Leaf 3.Root 4. Fruit 5.Flower 6.Whole plant 19. Maxted N, BV Ford-Lloyd, SL Jury, SP Kell and MA Scholten (2006) Towards a definition of a crop wild 7.Seed 8.Others relative. Biodiversity and Conservation 14: 1-13. 38 39 KIND 1.Food 2.Medicine 3.Fibre 4.Timber 5.Fodder 6.Fuel 7.Insecticide/ Pesticide 8.Others Plant Exploration and Germplasm HOW USED ...... INFORMANT(S) 1.Local Vaidya 2.Housewife 3.Old folk 4.Graziers Collecting in Fruit Crops – /Shepherds 5.Others PHOTOGRAPH 1.Colour/Video Practical Considerations Joseph John K.* and Suma A. FARMER'S/ DONOR'S NAME ...... ICAR-National Bureau of Plant Genetic Resources ETHNIC GROUP ...... Regional Station, Thrissur, Kerala, India *Email: [email protected] MOBILE NO ………………………

ADDRESS Introduction PLANT CHARACTERISTICS/ India has a rich and diverse agro-biodiversity heritage comprising an array of habitat diversity ranging from USES ADDL. NOTES tropical to subtropical and temperate, coastal low lands and even below sea level farm lands. Over 167 cultivated crops and nearly 500 crop wild relatives (CWRs) and Indigenous technical knowledge of agrarian tribes and ethnic diversity hold the key to agro-biodiversity of the country (Pradheep et al., 2014). India shares or holds a sizable part of four of the world mega biodiversity hotspots, (1) the Western Ghats and Sri Lanka (2) Himalayas (3) Indo-Malayan (Andaman Islands) and (4) Sundaland (Nicobar group of islands) which are home to many endemic CWRs, some of them yet to be discovered. The role of Indian gene center in agricultural development and sustainability was well recognized by eminent and world renowned crop geneticists like Vavilov and Harlan. Plant genetic resources (PGRs) refer to genetic material of actual or potential value and are the building blocks for any sustainable crop improvement programs. Germplasm are live tissues such as seeds, suckers, budwood, scion etc. maintained for the purpose of crop improvement. It includes landraces and traditional cultivars, obsolete varieties, released varieties, parental lines, hybrids and genetic stocks with specific traits, wild relatives and wild and feral forms of the crop. It is estimated that nearly 5000 species of plants are being used by humans of which only 500 contribute to the food basket and 20-30 crops are the major contributors. Fruit crops comprise an assortment of various species, mostly woody perennials, few biennials or annuals. According to Arora (1998), there are about 500 species of tropical fruit trees under 30 families and 59 genera in Asia Pacific Oceana region. The Vavilovian centre of South East Asia alone is estimated to have 120 major fruit species and 275 minor fruit species (Verheij and Coronel, 1992), most of which are still growing in wild or semi-wild state. Citrus, banana, mango, jackfruit, litchi and durian occupy 80 per cent of total fruit production in the region (Malik et al., 2010). Coming to fruit crops, India is endowed with suitable agro-ecological conditions for cultivation of various tropical, subtropical and temperate fruits. Mango, Banana, Jackfruit, Kokum, Aonla etc. had their centre of origin and diversity in India. While Kokum and Jackfruit have origin and diversity in Western Ghats, mango and banana have a diffused origin extending to parts of South and South East Asia. Further many of the wild gathered or underutilized fruits eg. Salacia fruticosa, Artocarpus hirsutus, A. lakoocha, Cordia dichotoma are endemic to some regions of the country. History says that over 1,000 varieties of Mango were maintained by Mugals in their gardens. Exotic fruit species have varied introduction history in the country, while pineapple, papaya, guava have a direct link to Portuguese rule, the other exotic fruits like Rambutan, Durian, Mangosteen, Avocado, Kiwi etc were introduced to the country by travelers, missionaries, Indian Diaspora, Government agencies and enthusiastic growers. Human and natural selection dating back to the history of agriculture has led to the evolution and development of numerous landraces in many of our fruit crops. In vegetatively propagated crops like banana, it has been easy to fix such accumulated micro and macro mutations leading to clonal selection and landrace diversity. While nucellar polyembryony (mango) and parthenocarpy induced

40 41 KIND 1.Food 2.Medicine 3.Fibre 4.Timber 5.Fodder 6.Fuel 7.Insecticide/ Pesticide 8.Others Plant Exploration and Germplasm HOW USED ...... INFORMANT(S) 1.Local Vaidya 2.Housewife 3.Old folk 4.Graziers Collecting in Fruit Crops – /Shepherds 5.Others PHOTOGRAPH 1.Colour/Video Practical Considerations Joseph John K.* and Suma A. FARMER'S/ DONOR'S NAME ...... ICAR-National Bureau of Plant Genetic Resources ETHNIC GROUP ...... Regional Station, Thrissur, Kerala, India *Email: [email protected] MOBILE NO ………………………

40 41 vegetative reproduction (banana) enabled perpetuation of chance superior combinations as a named sampling is to be practiced in tree crops due to space constraint and perennial nature of trees with long landrace, in crops like jackfruit, because of absence of natural vegetative propagation (assisted vegetative gestation period. So maximum diversity is to be captured within minimum samples as handling issues exist propagation has a short history) landrace diversity is very less even though extremely high heterozygosity from collection site to field genebank (FGB) establishment. Biased sampling of unique plus trees mostly initially identified by user and verified by the collector, from predetermined sites (AIR/DD/Social Table 1: Some of the landraces in fruit crops in Western Ghats region media/press appeal-response) save energy/time/decision making.

Crop Landraces Collection sites Plantain Nendran-Attunendran, Nedunendran, Changalikkodan, Wayanadan, Site of collection may vary from crop to crop or sample to sample. It can be farmers field viz., Manjery, Changanacherry homestead/backyard garden, orchard, field or wild habitat (forest, road side) and in some cases markets/nurseries. In any case the collector should try to fill as many columns in the passport data with Banana Monthan (vegetable), Kali, Kunnan, Kannan, Kudappanilla kunnan, compulsory GPS recording and ITK on the specimen including landrace/vernacular name. Information on Kadali, Matti, Poovan/Rasabala, Palayamkodan/Mysore, vernacular name of the desired material as well as a ready to show/forward picture of the plant/plant Njalipoovan/Elakki, Karpooravalli, Padatti product will help to detect the species easily especially in the case of rare CWR of tropical fruits. Mango (polyembryonic) Komavu/Vattan, Kolambu, Moovandan, Kilichundan, Chandrakaran, Sample size and strategy Tholikaypan, Olor, Kurukkan, Nambiarmanga, Mylappoo, Mailpeeli, Priour (Parekka manga) The aim of the explorer in a broader perspective determine the sample size, whether to conserve the entire variability or to target the specific traits i.e., to capture at least single or multiple copies of the rare alleles or Exploration and collection is the first step in PGR management of any crop, leading to conservation and to capture at least single or multiple copies of the all the alleles. It is known that genetic diversity depends on utilization. PGR exploration still depends heavily on information buildup of predecessors like published the number and frequency of all the alleles across all loci in a population as well as the population structure literature, reports and memoirs. The basic idea of PGR exploration is to assemble maximum possible (homogenous vs. heterogeneous) which is in turn determined by the distribution and mating behaviour of diversity within minimum samples and for this purpose random sampling is very important. Thus maximum the species. Some optimization is needed for representing the di-allelic in mono locus to multi- allelic and recovery of genetic variation requires random sampling, however in perennial tree crops practical issues multi loci systems in addition to the resources needed for their augmentation. As proposed by Marshall and need to be addressed. Germplasm collection strategies vary for temperate and tropical fruits and between Brown (1975), most important measure of genetic diversity is the frequency of alleles i.e., average number species due to their unique biology. While dormant buds (scions) are ideal for temperate (stone/pome of alleles per locus. In the context of genetic diversity conservation, property of allelic richness is more fruits) fruits, tropical plants give results in active growth phase (Mango, Jack etc. in humid growth season). important than property of allelic evenness. As fruit crops are numerous (Jack, Mango, Aonla, Rambutan, Pulasan, Durian, Mangosteen, Citrus, Kokkum, Sampling strategy depends upon factors like crop specific/trait specific or multi-crop/general exploration, Mulberry, Avocado, Bael, Ber, Phalsa, Sweetsop, Soursop, Dragon fruit, Pineapple, Litchi, Persimon, Longan, breeding behavior of the species; extend of gene exchange between the populations and pattern and Sweet tamarind, Pomegranate, Fig, Dates, Loquat, Apple, Pear, Plum, Cherry, Strawberry, Banana, Jamun, distribution of genetic variation. Ecological and climatic parameters lead to clonal variations and ecotypes Caronda, Roseapple, Sapota, Eggfruit, Guava, Papaya, Passion fruit, Grapes, Hogplum, Khoonphal, Burmese (low elevation fruiting Avocado, high altitude- cool climate adapted Banana). grape and like, the list can be very long), individual collector has to modify the general guidelines according to the situation, keeping the broad genetic principles and recovery of maximum alleles in mind. Number of loci, alleles and allelic frequencies, ploidy level, degree of certainty Collector should have a basic understanding of the crop species, its phenology and should be able to The sample size increases with the number of loci. It is the rare alleles that affect the sample size rather than distinguish between heritable and environment induced variations in the target species. A critical the common alleles. It was proposed by Singh and Srivastava (2004) that the sample size increases at an understanding of the descriptor and descriptor states of the target species is required. It is also important to exponential rate, as the allele is progressively getting rarer, eg. for rare allele frequencies of 0.05, 0.03, 0.01 explore variability/diversity rich area which can be decided based on perusal of eco-geographic distribution and 0.001, the respective sample sizes will be 59, 99, 299 and 2999 in the population. Further, the number of data/previous germplasm collection reports/flora (in the case of CWR) etc. Visit should coincide with fruit individuals to be sampled in a tetraploid population is almost half as compared to sampling from diploid ripening. In the case of fruit trees, often two visits are required, the former a coarse grid survey to locate population. The higher the degree of certainty, more the sample size i.e., when the certainty level to include superior mother plants and if possible in situ characterization and the latter at fruit ripening/bud a rare allele in diallelic condition increases from 95% to 99.99%, the number of individuals to be sampled dormancy/best time for propagule collection, to ensure trait specific germplasm with minimum increases from 59 to 180. However, Lawrence et al. (1995) suggested a sample size of about 172 plants for evaluation/characterization data. A collector has to take into account future potential value as unforeseen conserving all or very nearly all the polymorphic genes with high probability provided their frequency is not climate changes and new disease and pest strains may affect our crop production. CWR and primitive forms less than 0.05, irrespective of whether the individuals in the species are self or cross breeding or both. Sapra of the crop (truly wild forms/populations of Jack, Mango, Hogplum in the Western Ghats) assume et al. (1998) proposed a 2k-polyploid model for multi-allelic and, multi-loci situation. significance as gene donor as well as adaptive root stock. Strategy has to be species specific as breeding Sampling from several populations behavior, biology, mode of propagation etc. vary from species to species. Barring a couple of well-known species, most of the tropical fruits of Indian origin are less understood genetically and agronomically. Status Common alleles are those alleles with frequency above 0.05 and rare alleles below 0.05, which may be of genetic erosion, between and within population variation etc. are poorly studied and understood in most either widespread (occurring in many populations) or locally distributed (occurring in one or a few adjacent of our fruit crops. populations in the targeted area). Hence, according to Marshall and Brown (1975), there are four kinds of alleles. Objectives 1. Common, widely distributed 2. Common, locally distributed The main objectives are to maximize genetic diversity between populations (vegetative propagation/clones) or within populations (annuals-watermelon, muskmelon). Hence naturally, selective 3. Rare, widely distributed 4. Rare, locally distributed

42 43 vegetative reproduction (banana) enabled perpetuation of chance superior combinations as a named sampling is to be practiced in tree crops due to space constraint and perennial nature of trees with long landrace, in crops like jackfruit, because of absence of natural vegetative propagation (assisted vegetative gestation period. So maximum diversity is to be captured within minimum samples as handling issues exist propagation has a short history) landrace diversity is very less even though extremely high heterozygosity from collection site to field genebank (FGB) establishment. Biased sampling of unique plus trees mostly initially identified by user and verified by the collector, from predetermined sites (AIR/DD/Social Table 1: Some of the landraces in fruit crops in Western Ghats region media/press appeal-response) save energy/time/decision making.

42 43 The explorer normally aims for common and locally distributed alleles. Collection of common wide spread • Polyembryony - an understanding of the diversity available in existing collection to avoid duplicate and rare wide spread alleles are independent of the sampling strategy whereas rare local alleles seem collection. practically impossible. Further, when little or no information is available on the distribution of variation in • Gap analysis- area wise diversity/variability, trait-wise gaps in the target species (eg. Rhizome weevil nature, the optimal strategy is (a) to collect 50-100 plants per site (b) to sample as many sites as possible resistance in banana, yellow rind in kokum). within the time available (c) to ensure that sampling sites represent a broad range of environments. Thumb rule suggested by Marshall and Brown (1975) is that the objective of random sampling is to capture at least • Geographical distribution, areas of maximum occurrence/cultivation and ecological amplitudes (low one copy of 95% of the locally common alleles occurring at frequencies higher than 0.05. altitude avocado). General considerations • A perusal of memoirs of past explorers. The number of samples per site and size of the sample depends on many factors and on the species. The • Herbarium consultation/critical study of herbarium labels/GPS information, altitude, description of collector has to take a judicious decision based on site situation and practicality. In a crop like banana, variability, vernacular name, ITK, specific traits etc. Mangosteen – apospory/specific ecological niche – number of samples as well as sample size can be small. Maturity index, be it for seed or sucker as in the case riverbeds - well drained deep soil - fruit size may be collected - environmental variation- less scope for of banana, should be taken into account while sampling. Extra-large size/weight of the sample also needs to genetic variability. be taken care. Collecting explants like male buds for micropropagation will be feasible for banana or Logistics seeds/ripe fruits for wild/seed setting Musa. Assisted ripening of fruits for seed extraction can be hastened in the case of wild Musa by placing the harvested bunch/hands in a cupboard/box and keeping a burning After finalizing a tentative itinerary of collection sites (broad) necessary logistic planning may be made. If insense stick inside the closed chamber. For wild Musa, the general sample size may be a little over 500 collection (entry) in any protected area is involved, permission for collection needs to be taken (applied well seeds for conservation and 3-6 male cobs/suckers per spotting (subject to availability and ease of harvest). in advance, in the prescribed format) from PCCF/Wildlife Warden. Further entry into any tribal reserves (A&N) needs permission from District Magistrate and for many North Eastern region states, inner line Other general guidelines in ideal situations permit is required. To the extent possible team composition, transport (vehicle hiring quotation), Annual fruit crops (muskmelon/water melon etc.) accommodation (remote areas) should be planned in advance. Ground access hook/knife attached poles (preferably foldable), refractometer (TSS recording), cutting knife (secateurs) are a must for fruit crops PGR • Random collection from 50 individuals, pool and reduce size to 3,000-6,000 seeds. collection. Provision for engaging an experienced tree climber, well versed with maturity index will be ideal • Collect physiologically mature fruits, special care for viviparous/sprouting seeds, eg. Artocarpus for fruit harvest/scion collection. cammensi. Practical considerations • Also make biased samples of unique morphological variants (utilization purpose). Primary source of information on PGR wealth may be diverse. Visiting seasonal exhibitions, local fruit • Voucher herbarium specimens (CWR) for identification, good digital images. markets, hiring the service of a local informant, advanced planned use of press, AIR, DD, social media for eliciting response of custodian farmers etc. should be part of a multipronged approach. Often village level • In situ characterization : TSS etc., field notes recording/ITK/ donor mobile number. procurers and wholesale vendors are good source of information for locating plus trees and superior Vegetatively propagated crops/CWR genotypes. In CWR, especially mast fruiting wild Mangifera, sometimes the collector has to entrust some reliable local accompanying person, the responsibility to collect and courier late maturing samples at • Sample each distinct morphotype/plus tree. appropriate time. Nocturnal feeding of fallen nuts/fruits of rare Mangifera has been observed. In such • Collect 10-15 individuals as bulk samples from a site (strawberry, pineapple suckers). cases, fallen ripe fruits should be collected before dusk (Mangifera andamanica). Ensure rootstock availability, good misting facility, skilled nursery technician etc. before venturing scion collection. In • Seed setting fruit trees like Rambutan, Durian, Jackfruit, Guava, Avocado etc. may be collected as scions/ multipurpose fruit crops like Mango, Jack, banana etc. end use is very important. The collector should be approach graft/ layer according to species behavior. clear about the material he is looking for i.e., pickling/table fruit, cooking/vegetable type/ chips and the like • Polyembryonic mango may be taken as scions supplemented with nuts. purpose as there are specific varieties/ genotypes for specific use. • In the case of CWR of perennial fruit trees, collection is a compromise of what is available in the field Random sampling issues in a perennial tree crop (remote areas like A&N forests, seedlings may be pulled out and properly packed, nuts may be collected) The primary aim of any exploration mission is rescue and conservation of threatened germplasm and its • Variation between populations/landraces as well within populations (eg. Plantain, Nendran, utilization. Requirements from breeders for the effective utilization i.e., conservation and utilization are the Changalikkodan, Nedunendran, Aattunendran, Waynadan, Manjery etc, Mango-Kilichundan, motivating forces. Nevertheless in fruit trees considering long gestation period and land requirement for Kochukilichundan, Karuthakilichundan, Valiyakilichundan etc. based on specific heritable traits) FGB, the need for biased sampling and handpicking only trait specific germplasm cannot be ruled out. An ideal option may be to accommodate random samples especially in crops of Indian origin in forest • GI (Geographical Indication) tagged variations/ landraces e.g. Changalikkodan plantain, Vazakkulam land/afforestation plots and germplasm of immediate use in the FGB of research institutes. Thus, jackfruit pineapple may be collected as seeds and seedling plots established in afforestation/social forestry/roadside • Polymorphic forms (allelic series) adaptive variations, heterozygocity (jackfruit) plantations and chance superior trees collected as scions/approach graft and established in the FGB. Appropriate plant quarantine procedure/sample hygiene to be observed while collecting vegetative • Basic understanding of the morphology of the genus- for new CWR detection. eg. Musa, Artocarpus, propagules (possibility of spread of Banana pseudostem weevil, Fusarium wilt, Bunchy top virus, Root knot Garcinia, Mangifera and such indigenous genera. nematodes, Cuscuta infestation etc.). Convenient packets of seed dressing fungicides, wound healing paste, • Mode of propagation/reproduction/levels of gene exchange/cross pollination are also to be understood perlite and vermiculite (inert media for rooting pulled out seedlings), silica gel (seed drying), full size (eg. Jack - highly heterozygous). polybags for keeping scions in turgid and desiccation free conditions (vasculam) etc. may be kept handy. 44 45 The explorer normally aims for common and locally distributed alleles. Collection of common wide spread • Polyembryony - an understanding of the diversity available in existing collection to avoid duplicate and rare wide spread alleles are independent of the sampling strategy whereas rare local alleles seem collection. practically impossible. Further, when little or no information is available on the distribution of variation in • Gap analysis- area wise diversity/variability, trait-wise gaps in the target species (eg. Rhizome weevil nature, the optimal strategy is (a) to collect 50-100 plants per site (b) to sample as many sites as possible resistance in banana, yellow rind in kokum). within the time available (c) to ensure that sampling sites represent a broad range of environments. Thumb rule suggested by Marshall and Brown (1975) is that the objective of random sampling is to capture at least • Geographical distribution, areas of maximum occurrence/cultivation and ecological amplitudes (low one copy of 95% of the locally common alleles occurring at frequencies higher than 0.05. altitude avocado). General considerations • A perusal of memoirs of past explorers. The number of samples per site and size of the sample depends on many factors and on the species. The • Herbarium consultation/critical study of herbarium labels/GPS information, altitude, description of collector has to take a judicious decision based on site situation and practicality. In a crop like banana, variability, vernacular name, ITK, specific traits etc. Mangosteen – apospory/specific ecological niche – number of samples as well as sample size can be small. Maturity index, be it for seed or sucker as in the case riverbeds - well drained deep soil - fruit size may be collected - environmental variation- less scope for of banana, should be taken into account while sampling. Extra-large size/weight of the sample also needs to genetic variability. be taken care. Collecting explants like male buds for micropropagation will be feasible for banana or Logistics seeds/ripe fruits for wild/seed setting Musa. Assisted ripening of fruits for seed extraction can be hastened in the case of wild Musa by placing the harvested bunch/hands in a cupboard/box and keeping a burning After finalizing a tentative itinerary of collection sites (broad) necessary logistic planning may be made. If insense stick inside the closed chamber. For wild Musa, the general sample size may be a little over 500 collection (entry) in any protected area is involved, permission for collection needs to be taken (applied well seeds for conservation and 3-6 male cobs/suckers per spotting (subject to availability and ease of harvest). in advance, in the prescribed format) from PCCF/Wildlife Warden. Further entry into any tribal reserves (A&N) needs permission from District Magistrate and for many North Eastern region states, inner line Other general guidelines in ideal situations permit is required. To the extent possible team composition, transport (vehicle hiring quotation), Annual fruit crops (muskmelon/water melon etc.) accommodation (remote areas) should be planned in advance. Ground access hook/knife attached poles (preferably foldable), refractometer (TSS recording), cutting knife (secateurs) are a must for fruit crops PGR • Random collection from 50 individuals, pool and reduce size to 3,000-6,000 seeds. collection. Provision for engaging an experienced tree climber, well versed with maturity index will be ideal • Collect physiologically mature fruits, special care for viviparous/sprouting seeds, eg. Artocarpus for fruit harvest/scion collection. cammensi. Practical considerations • Also make biased samples of unique morphological variants (utilization purpose). Primary source of information on PGR wealth may be diverse. Visiting seasonal exhibitions, local fruit • Voucher herbarium specimens (CWR) for identification, good digital images. markets, hiring the service of a local informant, advanced planned use of press, AIR, DD, social media for eliciting response of custodian farmers etc. should be part of a multipronged approach. Often village level • In situ characterization : TSS etc., field notes recording/ITK/ donor mobile number. procurers and wholesale vendors are good source of information for locating plus trees and superior Vegetatively propagated crops/CWR genotypes. In CWR, especially mast fruiting wild Mangifera, sometimes the collector has to entrust some reliable local accompanying person, the responsibility to collect and courier late maturing samples at • Sample each distinct morphotype/plus tree. appropriate time. Nocturnal feeding of fallen nuts/fruits of rare Mangifera has been observed. In such • Collect 10-15 individuals as bulk samples from a site (strawberry, pineapple suckers). cases, fallen ripe fruits should be collected before dusk (Mangifera andamanica). Ensure rootstock availability, good misting facility, skilled nursery technician etc. before venturing scion collection. In • Seed setting fruit trees like Rambutan, Durian, Jackfruit, Guava, Avocado etc. may be collected as scions/ multipurpose fruit crops like Mango, Jack, banana etc. end use is very important. The collector should be approach graft/ layer according to species behavior. clear about the material he is looking for i.e., pickling/table fruit, cooking/vegetable type/ chips and the like • Polyembryonic mango may be taken as scions supplemented with nuts. purpose as there are specific varieties/ genotypes for specific use. • In the case of CWR of perennial fruit trees, collection is a compromise of what is available in the field Random sampling issues in a perennial tree crop (remote areas like A&N forests, seedlings may be pulled out and properly packed, nuts may be collected) The primary aim of any exploration mission is rescue and conservation of threatened germplasm and its • Variation between populations/landraces as well within populations (eg. Plantain, Nendran, utilization. Requirements from breeders for the effective utilization i.e., conservation and utilization are the Changalikkodan, Nedunendran, Aattunendran, Waynadan, Manjery etc, Mango-Kilichundan, motivating forces. Nevertheless in fruit trees considering long gestation period and land requirement for Kochukilichundan, Karuthakilichundan, Valiyakilichundan etc. based on specific heritable traits) FGB, the need for biased sampling and handpicking only trait specific germplasm cannot be ruled out. An ideal option may be to accommodate random samples especially in crops of Indian origin in forest • GI (Geographical Indication) tagged variations/ landraces e.g. Changalikkodan plantain, Vazakkulam land/afforestation plots and germplasm of immediate use in the FGB of research institutes. Thus, jackfruit pineapple may be collected as seeds and seedling plots established in afforestation/social forestry/roadside • Polymorphic forms (allelic series) adaptive variations, heterozygocity (jackfruit) plantations and chance superior trees collected as scions/approach graft and established in the FGB. Appropriate plant quarantine procedure/sample hygiene to be observed while collecting vegetative • Basic understanding of the morphology of the genus- for new CWR detection. eg. Musa, Artocarpus, propagules (possibility of spread of Banana pseudostem weevil, Fusarium wilt, Bunchy top virus, Root knot Garcinia, Mangifera and such indigenous genera. nematodes, Cuscuta infestation etc.). Convenient packets of seed dressing fungicides, wound healing paste, • Mode of propagation/reproduction/levels of gene exchange/cross pollination are also to be understood perlite and vermiculite (inert media for rooting pulled out seedlings), silica gel (seed drying), full size (eg. Jack - highly heterozygous). polybags for keeping scions in turgid and desiccation free conditions (vasculam) etc. may be kept handy. 44 45 Some constraints awaiting explorer and practical tips • Collectors may face resistance from ill-informed activists regarding benefit sharing and in such cases, the collector should be aware of the rights of genuine research as per the provisions of Biological Diversity Act (2002). • Collector should have a general idea of the geographic pattern of distribution of variability in the crop of his choice (eg. Red fleshed Jackfruit to be searched in Thumkur part of Karnataka, not in Panruthi, Tamil Nadu or Kerala or Tripura.) • In order to extend viability of recalcitrant seeds, seed extraction from fresh fruits may be delayed to the last leg of the trip. Seeds should be washed, cleaned of pulp, wiped with cotton cloth and packed with perlite/vermiculite mix or sawdust and transported for nursery raising. • In protected areas, the collector should prepare and submit list of collections at the respective range office and get transfer permit issued to avoid difficulties at checking points. • In some cases, custodians may hesitate to share germplasm citing superstitions regarding non- auspicious day for seed sharing etc., and in all such cases, the team should deal with the situation very tactically. The team should not engage into any heated arguments or controversy and the local traditions should be respected and followed. • Team should be careful not to over collect or endanger rare populations, especially CWR. References 1. Arora R (1998) Genetic resources of native tropical fruits in Asia: Diversity, distribution and IPGRI's emphasis on their conservation and use. In Tropical Fruits in Asia: Diversity, Maintenance, Conservation and Use (R.K. Arora and V. Ramanatha Rao, Eds). IPGRI Office for South Asia, New Delhi, India. Available online: http://cropgenebank. sgrp. cgiar. org/ index. Php. Pp. 42-53 2. Pradheep K, DC Bhandari and KC Bansal (2014) Wild Relatives of Cultivated Plants in India. Indian Council of Agricultural Research, New Delhi. 3. Lawrence MJ, DF Marshall and P Davies (1995) Genetics of genetic conservation II; Sample size when collecting seed of cross pollinating species and the information that can be obtained from the evaluation of material held in gene banks. Euphytica 84:101–107. 4. Malik SK, R Chaudhury, OP Dhariwal and DC Bhandari (2010) Genetic Resources of Tropical Underutilized Fruits in India. NBPGR, New Delhi, p.168. 5. Marshall DR and AHD Brown (1975) Optimum sampling strategies in genetic conservation. In: OH Frankel and JG Hawkes, (eds) Genetic Resources for Today and Tomorrow. Cambridge University Press, Cambridge, pp 53–80. 6. Sapra RL, Prem Narain and SVS Chauhan (1998) A general model for sample size determination for collecting germplasm. J. Biosci. 23: 647–652 7. Singh BP and U Srivastava (2004) Plant Genetic Resources in Indian Perspective: Theory and Practice. Directorate of Information and Publications of Agriculture, Indian Council of Agricultural Research, New Delhi, 715 p. 8. Verheij, EWM and RE Coronel (1992) Plant Resources of South East Asia. Edible Fruits and Nuts. PROSEA Foundation, Bogor, Indonesia.

46 Access to Germplasm: Policies, Guidelines and Procedures Pratibha Brahmi* and Pragya Principal Scientist, Germplasm Exchange and Policy Unit ICAR-National Bureau of Plant Genetic Resources, New Delhi *Email: [email protected]

Plant genetics resources (PGR) include all plant species of actual or potential value, provide the raw material for crop improvement and help adapt to ever changing climate. PGR are acquired by either collecting the crop diversity from the farmers' fields or from the centers of diversity of crop plants distributed throughout the world. No country or organization is self-sufficient in its PGR requirements. Access to PGR and their utilization is crucial for the world's food and nutritional requirements. Certain principles govern the exchange and continued availability of PGR. The international conventions and agreements that affect the current scenario of regulations for sharing and exchange of PGR at national and international land are described in Table 1 and 2. Table 1. National/International legal instruments related to access and use of genetic resources

International Legal Scope/ Applicability Status / Jurisdiction Instruments (Relevant Indian legislation )

International Treaty on • Covers all PGRFA (does not • Adopted by FAO Conference Plant Genetic Resources regulate non-food and non- in 2001. for Food and Agriculture agricultural uses) and addresses • Entry into force after the 40th (ITPGRFA) diverse topics, including country ratified in November conservation use, international 2004. cooperation, technical assistance • Legally binding for all and farmers' rights. countries that ratify and not • Establishes multilateral system applicable to those that do for selected crops (over 35 food not. crop/29 forages species). • Countries that ratify will be • Sets rules for access and benefit- required to bring national sharing for these materials, both laws and regulations into ex situ and in situ, while conformity with the Treaty respecting property rights (except • CGIAR Centers are likely to for CGIAR holdings of sign agreements with the agreements) Treaty's Governing Body in Order to adhere to the Treaty formally.

• Legally binding for countries Convention on Biological Covers all biodiversity, provides that have ratified (183 as on Diversity (CBD) (Biological general principles for access and March 2002). Diversity Act 2002) benefit-sharing concerning materials collected after the coming of CBD • Ratified countries must and not covered by the International adopt appropriate Treaty on PGRFA. legislation/regulations and/or bring existing ones into harmony with the Convention.

47 relation to a legally binding International Plant • An instrument and forum for • Established in 1952 (last agreement. Protection Convention harmonizing Phytosanitary revised in 1997). Plant Quarantine measures used in regulating • Adopted in January 2000. FAO Code of Conduct for • Deals with the etiquette of • Adopted by FAO Conference (Regulation of Import into international trade. Plant Germplasm Collecting collecting and transfer of PGRFA. in 1993. India) Order 2003 issued • Provides international regulatory and Transfer • Has a provision on collectors, • Not legally binding. under the Destructive framework for trade and sponsors, curators and users, as Insects and Pests (DIP) protection of environment from well as on reporting, monitoring Act 1914) adverse effects of products of and evaluating observance of the modern biotechnology. Code. CBD/International Members State must comply with FAO-CGIAR Agreements Agreements apply to the 11 Centers (those having ex situ Protocol on Biosafety minimum standards of protection of Placing collections under management, availability and collections) have signed binding (Cartagena Protocol) IP. the auspices of FAO transfer of specifically designated agreements with FAO in 2011. (Guidelines for safety of germplasm. Biotechnology by DBT, 2002 under the Associated documents/ Joint statement address concern Agreements coming into fold of environment Protection instruments include: regarding the practical the ITPGRFA. Treaty's Governing Act 2005 ) a) 2 joint FAO-CGIAR implementation of the agreement; Body and Centers to develop Statement on the the agreed MTA (which has the force new agreements in line with WTO-TRIPS Agreement Must ensure protection of • Entered into force in January Agreement of a binding contract on recipients) is Article 15 of the Treaty. (Protection of Plant microorganisms, non- biological and 1995 after the Marrakesh b) standard Material used in the transfer of designated Varieties and Farmer microbiological processes and plant Ministerial Meeting in April Transfer Agreement germplasm. Right Act 2001 and Patent varieties that meet protection 1994. (MTA) (Amendments) Act 2005 criteria. • Legally binding on all WTO Members. Nagoya Protocol on Access Internationally accepted protocol on Entered into Force: 12 October • Has possibility for trade and Benefit Sharing ABS. 2014. sanctions for those found not in compliance. Table 2. National legislations with implication on access to plant genetic resources WTO/Agreement on Covers measures adopted by Adopted by 167 WTO members Application of Sanitary countries to protect human or animal presently Acts/Guidelines Objectives Scope and Phytosanitary life from foodborne risks: animal or Measures (Plant plant-carried diseases; plant pests Protection of Plant To provide for an effective system of New varieties to be protected Quarantine (Regulation of and diseases to ensure food safety Varieties and Farmer protection of plant varieties: and (registered) in the Act, should be Import into India) Order and prevent the spread of pests Rights right of farmers and plant breeding; novel, distinct, stable and 2003 issued under the DIP among animals and plant. (PPVFR Act, 2001) ensure availability of high quality uniform (NDUS), and shall be Act 1914) seeds improved varieties to farmers. subjected to tests by the PPVFR Authority. UPOV (Plant Breeders' UPOV aims to maximize plant • Four versions: only 1991 is Effective from January a) Application for new varieties Rights) breeding efforts by proving a model still open for joining. 2005 should be accomplished India is not member of for securing protection under UPOV • Legally binding on 50 Parties with complete passport data UPOV for plant breeders' rights for plant to the Treaty. of all the genetic resources varieties. used in the development of new material FAO Global plan of Action • Covers all PGRFA. • Adopted in 1996 by the 4th Intl b) Proof of lawful acquisition • Contains specific “activates” on in Technical Conference on of all materials used in situ conservation and PGRFA (150 counties). development of new development, ex situ • Legally non-binding. varieties to be furnished. conservation, utilization and • Served as a framework, guide (Brahmi et al., 2004) institutions and capacity building. and catalyst for PGRFA, and thus a bearing on and a

48 49 relation to a legally binding International Plant • An instrument and forum for • Established in 1952 (last agreement. Protection Convention harmonizing Phytosanitary revised in 1997). Plant Quarantine measures used in regulating • Adopted in January 2000. FAO Code of Conduct for • Deals with the etiquette of • Adopted by FAO Conference (Regulation of Import into international trade. Plant Germplasm Collecting collecting and transfer of PGRFA. in 1993. India) Order 2003 issued • Provides international regulatory and Transfer • Has a provision on collectors, • Not legally binding. under the Destructive framework for trade and sponsors, curators and users, as Insects and Pests (DIP) protection of environment from well as on reporting, monitoring Act 1914) adverse effects of products of and evaluating observance of the modern biotechnology. Code. CBD/International Members State must comply with FAO-CGIAR Agreements Agreements apply to the 11 Centers (those having ex situ Protocol on Biosafety minimum standards of protection of Placing collections under management, availability and collections) have signed binding (Cartagena Protocol) IP. the auspices of FAO transfer of specifically designated agreements with FAO in 2011. (Guidelines for safety of germplasm. Biotechnology by DBT, 2002 under the Associated documents/ Joint statement address concern Agreements coming into fold of environment Protection instruments include: regarding the practical the ITPGRFA. Treaty's Governing Act 2005 ) a) 2 joint FAO-CGIAR implementation of the agreement; Body and Centers to develop Statement on the the agreed MTA (which has the force new agreements in line with WTO-TRIPS Agreement Must ensure protection of • Entered into force in January Agreement of a binding contract on recipients) is Article 15 of the Treaty. (Protection of Plant microorganisms, non- biological and 1995 after the Marrakesh b) standard Material used in the transfer of designated Varieties and Farmer microbiological processes and plant Ministerial Meeting in April Transfer Agreement germplasm. Right Act 2001 and Patent varieties that meet protection 1994. (MTA) (Amendments) Act 2005 criteria. • Legally binding on all WTO Members. Nagoya Protocol on Access Internationally accepted protocol on Entered into Force: 12 October • Has possibility for trade and Benefit Sharing ABS. 2014. sanctions for those found not in compliance. Table 2. National legislations with implication on access to plant genetic resources WTO/Agreement on Covers measures adopted by Adopted by 167 WTO members Application of Sanitary countries to protect human or animal presently Acts/Guidelines Objectives Scope and Phytosanitary life from foodborne risks: animal or Measures (Plant plant-carried diseases; plant pests Protection of Plant To provide for an effective system of New varieties to be protected Quarantine (Regulation of and diseases to ensure food safety Varieties and Farmer protection of plant varieties: and (registered) in the Act, should be Import into India) Order and prevent the spread of pests Rights right of farmers and plant breeding; novel, distinct, stable and 2003 issued under the DIP among animals and plant. (PPVFR Act, 2001) ensure availability of high quality uniform (NDUS), and shall be Act 1914) seeds improved varieties to farmers. subjected to tests by the PPVFR Authority. UPOV (Plant Breeders' UPOV aims to maximize plant • Four versions: only 1991 is Effective from January a) Application for new varieties Rights) breeding efforts by proving a model still open for joining. 2005 should be accomplished India is not member of for securing protection under UPOV • Legally binding on 50 Parties with complete passport data UPOV for plant breeders' rights for plant to the Treaty. of all the genetic resources varieties. used in the development of new material FAO Global plan of Action • Covers all PGRFA. • Adopted in 1996 by the 4th Intl b) Proof of lawful acquisition • Contains specific “activates” on in Technical Conference on of all materials used in situ conservation and PGRFA (150 counties). development of new development, ex situ • Legally non-binding. varieties to be furnished. conservation, utilization and • Served as a framework, guide (Brahmi et al., 2004) institutions and capacity building. and catalyst for PGRFA, and thus a bearing on and a

48 49 Biological Diversity Act Conservation and sustainable use of The access to Indian biological These two documents must accompany every consignment of seed/planting material imported from biological diversity; regulation of resources by non-Indians shall foreign countries. access to biological resources; secure be subject to the approval of Issuance of import permit (IP) Effective from April 2004 sharing of benefits out of use if National Biodiversity Authority. Indian biological resources/ use of These conditions do not apply to • Researcher/user desirous of importing seed/planting material into India has to apply to the Director, Patent (Amendments) traditional knowledge transfer of genetic resources NBPGR, New Delhi, on a prescribed application form (PQ 08) available at NBPGR website Act 2005 New amendment include patenting under any project approved by www.nbpgr.ernet.in (Annexure 1). on newly developed (GM) micro- the Govt. of India, and also • Along with the application form PQ 08, the applicable processing fee for issuance of an Import Permit Effective from January organisms and micro biological transfer of results under (Annexure 2) should be sent in the form of a demand draft in favour of Director, NBPGR, New Delhi. The 2005 process that conform to the criteria approved projects. fee is non-refundable and subject to revision. of novelty, inventive step and utility. • Private organizations/seed companies requesting for IP must be certified R&D organizations. They must Geographical Indications Applicable to goods such as a) Sources of biological provide a copy of valid R&D recognition certificate issued from Department of Scientific and Industrial of Goods agricultural goods, natural goods or resources used in the Research (DSIR), Ministry of Science and Technology, Government of India. (Registration and manufactured goods as originating development of patents is to • After all information provided in the application is validated, the IP is issued in form PQ 09 in triplicate, Protection) Act 1999 and manufactured in a territory of a be acknowledged. with a yellow/green tag for germplasm and a red/white tag for transgenics/ GMOs. country where the given quality/ b) All biological resources used reputation is essentially attributable in development of patents to • The prescribed quantity of the seed and the planting material permitted for import should be for its geographic region (Land races of be deposited in the experimental use only. Details of quantities allowed for research purpose are available at NBPGR website crop plants specific to certain agro- designated repositories (Annexure 3). climatic regions of the country). before sealing of patents. • For specific crops, the requests for import permit are to be routed first through Directors of respective ICAR crop-based institutes for non-transgenic as well as for transgenic crops (as per schedule V, clause 3 Plant Quarantine Rules for Regulating the Import of a) The Order includes (3)(6) and 10 and 11 (3) of PQ Order 2003). This applies to all indents from NARS/SAUs/Private Sector/any (Regulation of Import Plants etc. into India, including prohibition on import of other. into India) Order 2003 import of germplasm/genetically commodities with under the DIP Act 1914 modified organisms weed/alien species • IP is valid for six months from the date of issue and valid for successive shipment provided the exporter (GMOs/transgenic planting material; contamination and and importer, bill of entry, country of origin and phytosanitary certificate are the same for the entire Effective from April, 2004 live insects/ fungi including bio- restriction on import of consignment. control agents, soil, peat, and packaging material of plant • Validity of IP may be extended up to one year on request before the expiry of permit and payment of sphagnum moss, timber and wooden origin unless treated. prescribed fee, if adequate reasons are provided in writing and are justified. logs b) Additional Declarations to be given on phytosanitary • The import permit issued is non-transferable and no amendments to the permit shall be issued except certificates have been for change of point of entry subject to justified reasons to be recorded in writing. specified and notified points Mandatory requirement of phytosanitary certificate of entry have been increased. • Phytosanitary certificate (PC), a document regarding the health status of consignment (original copy) Guidelines for Safety of National framework for development Gives guidelines for import and issued by Govt. official from country of origin in the prescribed format of Food and Agriculture Biotechnology by DBT of genetically modified plants and shipment of genetically Organization (FAO). under the EPA 1986 their commercial exploitation. modified plants only for toxicity • Every consignment should be accompanied by PC issued by authorized officer at country of origin/ and allergenicity evaluation of supplier country with additional declarations for freedom from specific pests and diseases as specified or Effective from 1990 (last transgenic seeds, plants and that the pests specified do not occur in the country or state of origin as supported by documentary revised in 1998) plant parts. evidence thereof. • Specific pests for the crops are mentioned in Schedule V and VI of PQ Order 2003. II Procedures for germplasm exchange with reference to fruit crops (www.plantquarantineindia.org). It obligatory for all plant breeders and researchers intending to import seed/planting materials, • NBPGR retains up to 5% of the seeds or minimum of 50 seeds in its facility as voucher samples and keep from other countries to fulfill the following two mandatory requirements under the Plant them under safe custody. Quarantine (Regulation of Import into India) Order 2003 (www.plantquarantineindia.org), which came into force on the first day of January 2004. Import of transgenic seed/planting materials (i) Import permit before import of any material • Under the provisions of clause 6(1) (2) of Plant Quarantine (Regulation of Import into India) Order 2003, import of transgenic seeds, genetically modified organism is also permitted for research purposes (ii) Phytosanitary certificate from the country of origin. subject to the clearance from Department of Biotechnology (DBT).

50 51 Biological Diversity Act Conservation and sustainable use of The access to Indian biological These two documents must accompany every consignment of seed/planting material imported from biological diversity; regulation of resources by non-Indians shall foreign countries. access to biological resources; secure be subject to the approval of Issuance of import permit (IP) Effective from April 2004 sharing of benefits out of use if National Biodiversity Authority. Indian biological resources/ use of These conditions do not apply to • Researcher/user desirous of importing seed/planting material into India has to apply to the Director, Patent (Amendments) traditional knowledge transfer of genetic resources NBPGR, New Delhi, on a prescribed application form (PQ 08) available at NBPGR website Act 2005 New amendment include patenting under any project approved by www.nbpgr.ernet.in (Annexure 1). on newly developed (GM) micro- the Govt. of India, and also • Along with the application form PQ 08, the applicable processing fee for issuance of an Import Permit Effective from January organisms and micro biological transfer of results under (Annexure 2) should be sent in the form of a demand draft in favour of Director, NBPGR, New Delhi. The 2005 process that conform to the criteria approved projects. fee is non-refundable and subject to revision. of novelty, inventive step and utility. • Private organizations/seed companies requesting for IP must be certified R&D organizations. They must Geographical Indications Applicable to goods such as a) Sources of biological provide a copy of valid R&D recognition certificate issued from Department of Scientific and Industrial of Goods agricultural goods, natural goods or resources used in the Research (DSIR), Ministry of Science and Technology, Government of India. (Registration and manufactured goods as originating development of patents is to • After all information provided in the application is validated, the IP is issued in form PQ 09 in triplicate, Protection) Act 1999 and manufactured in a territory of a be acknowledged. with a yellow/green tag for germplasm and a red/white tag for transgenics/ GMOs. country where the given quality/ b) All biological resources used reputation is essentially attributable in development of patents to • The prescribed quantity of the seed and the planting material permitted for import should be for its geographic region (Land races of be deposited in the experimental use only. Details of quantities allowed for research purpose are available at NBPGR website crop plants specific to certain agro- designated repositories (Annexure 3). climatic regions of the country). before sealing of patents. • For specific crops, the requests for import permit are to be routed first through Directors of respective ICAR crop-based institutes for non-transgenic as well as for transgenic crops (as per schedule V, clause 3 Plant Quarantine Rules for Regulating the Import of a) The Order includes (3)(6) and 10 and 11 (3) of PQ Order 2003). This applies to all indents from NARS/SAUs/Private Sector/any (Regulation of Import Plants etc. into India, including prohibition on import of other. into India) Order 2003 import of germplasm/genetically commodities with under the DIP Act 1914 modified organisms weed/alien species • IP is valid for six months from the date of issue and valid for successive shipment provided the exporter (GMOs/transgenic planting material; contamination and and importer, bill of entry, country of origin and phytosanitary certificate are the same for the entire Effective from April, 2004 live insects/ fungi including bio- restriction on import of consignment. control agents, soil, peat, and packaging material of plant • Validity of IP may be extended up to one year on request before the expiry of permit and payment of sphagnum moss, timber and wooden origin unless treated. prescribed fee, if adequate reasons are provided in writing and are justified. logs b) Additional Declarations to be given on phytosanitary • The import permit issued is non-transferable and no amendments to the permit shall be issued except certificates have been for change of point of entry subject to justified reasons to be recorded in writing. specified and notified points Mandatory requirement of phytosanitary certificate of entry have been increased. • Phytosanitary certificate (PC), a document regarding the health status of consignment (original copy) Guidelines for Safety of National framework for development Gives guidelines for import and issued by Govt. official from country of origin in the prescribed format of Food and Agriculture Biotechnology by DBT of genetically modified plants and shipment of genetically Organization (FAO). under the EPA 1986 their commercial exploitation. modified plants only for toxicity • Every consignment should be accompanied by PC issued by authorized officer at country of origin/ and allergenicity evaluation of supplier country with additional declarations for freedom from specific pests and diseases as specified or Effective from 1990 (last transgenic seeds, plants and that the pests specified do not occur in the country or state of origin as supported by documentary revised in 1998) plant parts. evidence thereof. • Specific pests for the crops are mentioned in Schedule V and VI of PQ Order 2003. II Procedures for germplasm exchange with reference to fruit crops (www.plantquarantineindia.org). It obligatory for all plant breeders and researchers intending to import seed/planting materials, • NBPGR retains up to 5% of the seeds or minimum of 50 seeds in its facility as voucher samples and keep from other countries to fulfill the following two mandatory requirements under the Plant them under safe custody. Quarantine (Regulation of Import into India) Order 2003 (www.plantquarantineindia.org), which came into force on the first day of January 2004. Import of transgenic seed/planting materials (i) Import permit before import of any material • Under the provisions of clause 6(1) (2) of Plant Quarantine (Regulation of Import into India) Order 2003, import of transgenic seeds, genetically modified organism is also permitted for research purposes (ii) Phytosanitary certificate from the country of origin. subject to the clearance from Department of Biotechnology (DBT).

50 51 • DBT and the Ministry of Environment and Forest (MoEF) have separate set of prescribed procedures for Germplasm Export providing permission for import of transgenic material into India for research/experimental purposes The export of germplasm is dealt under four categories: (for further details log on to http://dbtindia.nic.in/uniquepage.asp). • Review Committee on Genetic Manipulation (RCGM), constituted by DBT examines the desirability of Category 1 Export of germplasm not covered under any collaborative research project with import of transgenic line, from the biosafety point of view under the Environment (Protection) Act, 1986. research institutes/counter parts, public-private transfer, private entities In accordance with this Act, all transgenic plants are regulated items. (Indian Citizen/Non-Indian), as per Section 3 (2) of the BDA, 2002 • For transgenic material/Genetically Modified Organisms (GMOs), IP is issued subject to the approval of Category 2 Export of germplasm under collaborative research projects/work plans, under RCGM. After getting approval, the applicant shall apply to the Director, NBPGR, New Delhi in the Section 5 of the BDA, 2002 prescribed form (PQ 08) for the issuance of import permit. The applicant is required to enclose import Category 3 Export of Annex 1 crops under ITPGRFA and FAO designated accessions of CG clearance letter from DBT and demand draft of prescribed fee (Annexure 2) for issuance of IP. Centres located in India • Along with the application form, the indenter is also required to furnish the information and follow the Category 4 Indian researcher/ Government institution to carry/send germplasm for non- instructions as per para 6 of permit letter for authorization to import (subject to revision). commercial research/ or research for emergency purposes other than • NBPGR retains up to 5% of the transgenic seeds or minimum of 50 seeds as voucher sample in its facility collaborative research under safe custody, as per the directives of RCGM. NBPGR issues a receipt of deposition of voucher samples to the applicant. Export of Germplasm under Category 1 (Not covered under Collaborative Research Project) • The voucher sample retained by the NBPGR may be used by the Government for future reference. • For export of germplasm not covered under any collaborative research project, applicants are required • The transgenic material shall be handled, packaged and transported as specified in “R-DNA safety to seek written approval from the NBA, in the prescribed format (Form 1 under Rule 14 of the Biological guidelines-1990” of the DBT, Government of India (http://dbtindia.nic.in/uniquepage.asp). Diversity Rules, 2004) along with the prescribed fee (http://nbaindia.org/). Shipment of the Consignment • After written approval from NBA, applicant should seek an IP from the appropriate authority in the country of germplasm export. • The applicant is required to send two copies the import permit to the concerned official/supplier (abroad) that has agreed to supply the required germplasm for use in research. • After obtaining the IP, Plant Quarantine Officials should inspect the germplasm meant for export and issue a PC to the applicant. • The import permit in duplicate must be enclosed with the seed/planting material for custom clearance. • The consignment for export should be sent through suitable postal services along with IP, PC and other • The supplier should be instructed to send back both the copies of IP (one pasted outside the seed parcel relevant documents, either by the supplier of germplasm or through NBPGR. and other inside the seed parcel) and other relevant documents with the consignment. Always ensure that the consignment must be addressed to the Director, NBPGR, New Delhi. Export of Germplasm under Category 2 (Collaborative Research Project) • The material so introduced is assigned an exotic collection (EC) number after quarantine clearance which • Prior to export of germplasm under collaborative research projects, following documents need to be remains unchanged during the course of its utilization in crop improvement programmes. assembled by the indenter: • The instructions should be clear to the sender/supplier that consignment should be addressed only to - A request letter from foreign institution submitted to ICAR/research institutions/ researcher in the Director, NBPGR, New Delhi. The port of entry of germplasm is New Delhi Airport only. India for access to germplasm. • Seed/planting material meant for export to India should not be treated with any chemical. - A copy of the collaborative research agreement, bilateral/multilateral agreement, Memorandum of Understanding, work plans, etc. approved by the concerned Ministry/ Department of Accessioning and dispatch of consignment Central/State Governments. • The accessioning of samples after quarantine clearance should be done online and each accession - Details of the seed/planting material for export. should be assigned national identity i.e. exotic collection (EC) number. - IP of the recipient country. • Never release a variety directly from Introduced accessions. Always maintain original national identity IC/EC) for obvious national and international obligations of access and benefit sharing. - Signed copy of ICAR/DARE approved Material Transfer Agreement (MTA) or SMTA, as applicable. • Voucher sample is to be kept in the National Genebank. • Export of germplasm under Category 2 shall be as per the extant relevant national legislations-the provisions contained in the Biological Diversity Act, 2002, the Biological Diversity Rules, 2014, the • After due payment of handling charges and quarantine examination fee the consignment is dispatched Ministry of Environment & Forests & CC (MoEF&CC) Notification S.O.1911(E)dated 8th November, 2006 to the inventor. and GSR 8267 dated 21st Nov, 2014 and any further guidelines notified by the Central Government in this regard. • After approval of DARE, the consignment for export should be submitted to Director NBPGR, where it should be registered and all details (crop name, no. of packages and the receipt country) should be documented.

52 53 • DBT and the Ministry of Environment and Forest (MoEF) have separate set of prescribed procedures for Germplasm Export providing permission for import of transgenic material into India for research/experimental purposes The export of germplasm is dealt under four categories: (for further details log on to http://dbtindia.nic.in/uniquepage.asp). • Review Committee on Genetic Manipulation (RCGM), constituted by DBT examines the desirability of Category 1 Export of germplasm not covered under any collaborative research project with import of transgenic line, from the biosafety point of view under the Environment (Protection) Act, 1986. research institutes/counter parts, public-private transfer, private entities In accordance with this Act, all transgenic plants are regulated items. (Indian Citizen/Non-Indian), as per Section 3 (2) of the BDA, 2002 • For transgenic material/Genetically Modified Organisms (GMOs), IP is issued subject to the approval of Category 2 Export of germplasm under collaborative research projects/work plans, under RCGM. After getting approval, the applicant shall apply to the Director, NBPGR, New Delhi in the Section 5 of the BDA, 2002 prescribed form (PQ 08) for the issuance of import permit. The applicant is required to enclose import Category 3 Export of Annex 1 crops under ITPGRFA and FAO designated accessions of CG clearance letter from DBT and demand draft of prescribed fee (Annexure 2) for issuance of IP. Centres located in India • Along with the application form, the indenter is also required to furnish the information and follow the Category 4 Indian researcher/ Government institution to carry/send germplasm for non- instructions as per para 6 of permit letter for authorization to import (subject to revision). commercial research/ or research for emergency purposes other than • NBPGR retains up to 5% of the transgenic seeds or minimum of 50 seeds as voucher sample in its facility collaborative research under safe custody, as per the directives of RCGM. NBPGR issues a receipt of deposition of voucher samples to the applicant. Export of Germplasm under Category 1 (Not covered under Collaborative Research Project) • The voucher sample retained by the NBPGR may be used by the Government for future reference. • For export of germplasm not covered under any collaborative research project, applicants are required • The transgenic material shall be handled, packaged and transported as specified in “R-DNA safety to seek written approval from the NBA, in the prescribed format (Form 1 under Rule 14 of the Biological guidelines-1990” of the DBT, Government of India (http://dbtindia.nic.in/uniquepage.asp). Diversity Rules, 2004) along with the prescribed fee (http://nbaindia.org/). Shipment of the Consignment • After written approval from NBA, applicant should seek an IP from the appropriate authority in the country of germplasm export. • The applicant is required to send two copies the import permit to the concerned official/supplier (abroad) that has agreed to supply the required germplasm for use in research. • After obtaining the IP, Plant Quarantine Officials should inspect the germplasm meant for export and issue a PC to the applicant. • The import permit in duplicate must be enclosed with the seed/planting material for custom clearance. • The consignment for export should be sent through suitable postal services along with IP, PC and other • The supplier should be instructed to send back both the copies of IP (one pasted outside the seed parcel relevant documents, either by the supplier of germplasm or through NBPGR. and other inside the seed parcel) and other relevant documents with the consignment. Always ensure that the consignment must be addressed to the Director, NBPGR, New Delhi. Export of Germplasm under Category 2 (Collaborative Research Project) • The material so introduced is assigned an exotic collection (EC) number after quarantine clearance which • Prior to export of germplasm under collaborative research projects, following documents need to be remains unchanged during the course of its utilization in crop improvement programmes. assembled by the indenter: • The instructions should be clear to the sender/supplier that consignment should be addressed only to - A request letter from foreign institution submitted to ICAR/research institutions/ researcher in the Director, NBPGR, New Delhi. The port of entry of germplasm is New Delhi Airport only. India for access to germplasm. • Seed/planting material meant for export to India should not be treated with any chemical. - A copy of the collaborative research agreement, bilateral/multilateral agreement, Memorandum of Understanding, work plans, etc. approved by the concerned Ministry/ Department of Accessioning and dispatch of consignment Central/State Governments. • The accessioning of samples after quarantine clearance should be done online and each accession - Details of the seed/planting material for export. should be assigned national identity i.e. exotic collection (EC) number. - IP of the recipient country. • Never release a variety directly from Introduced accessions. Always maintain original national identity IC/EC) for obvious national and international obligations of access and benefit sharing. - Signed copy of ICAR/DARE approved Material Transfer Agreement (MTA) or SMTA, as applicable. • Voucher sample is to be kept in the National Genebank. • Export of germplasm under Category 2 shall be as per the extant relevant national legislations-the provisions contained in the Biological Diversity Act, 2002, the Biological Diversity Rules, 2014, the • After due payment of handling charges and quarantine examination fee the consignment is dispatched Ministry of Environment & Forests & CC (MoEF&CC) Notification S.O.1911(E)dated 8th November, 2006 to the inventor. and GSR 8267 dated 21st Nov, 2014 and any further guidelines notified by the Central Government in this regard. • After approval of DARE, the consignment for export should be submitted to Director NBPGR, where it should be registered and all details (crop name, no. of packages and the receipt country) should be documented.

52 53 • The seed/planting material meant for export should be then submitted to Division of Plant Quarantine, Export of Germplasm for Non-Commercial Research/ or Research for Emergency Purposes Other than NBPGR for quarantine inspection as per the IP instructions of the importing country, for issuance of PC. Collaborative Research • The seed/planting material, along with IP and PC should be dispatched to the receiving country either by • Any Indian researcher/ Government institution who intends to carry/ send the biological resources the indentor or by NBPGR. outside India to undertake basic research other than collaborative research as referred to in section 5 of the Biological Diversity Act , 2002 shall apply to the NBA in Form 'B'. Export of Germplasm under Category 3 (Annex-I crops under ITGRFA and FAO designated accessions of CG centres located in India) • Any Government Institution which intends to send biological resources to carry out certain urgent studies to avert emergencies like epidemics, etc., shall apply in Form 'B'. • Export of germplasm under category 3 shall be as per the MoEF & CC Notification S.O. 3232 (E) 17.12.2014 and Department of Agriculture & Cooperation O.M. No. 13-5/2013-SD-V dated 16.2.2015. • The NBA shall, on being satisfied with the application accord its approval within a period of 45 days from the date of receipt of the application. • The Department of Agricutral Cooperation (DAC), MoA&FW, GOI is the Nodal Department and Joint Secretary (Seeds) is the National Focal Point (NFP) for access to PGRFA of designated accessions in • On receipt of approval of the NBA, the applicant shall deposit voucher specimens in the designated accordance with the provisions of the ITPGRFA to which India is a party, and relevant extant national national repositories before carrying / sending the biological resources outside India and a copy of proof legislations. of such deposits shall be endorsed to NBA. • All requests for access to PGRFA listed in Annex 1 of the ITPGRFA (as per Article 12.3 c of ITPGRFA) should National (Domestic) Supply of Germplasm be addressed to NFP, either directly or through NBPGR. Access to PGRFA requested shall be granted only • Requests for samples of plant germplasm available with/maintained by NBPGR/NAGS should be sent to with the approval of NFP. the Director, NBPGR, Pusa Campus, New Delhi 110012, in the prescribed GEX 01 form (Annexure 4) along • On receipt of request for export, the Germplasm Export Facilitation Committee (GEFC) constituted for with a signed MTA (Annexure 5) by the indenter of germplasm. The GEX 01 and MTA can also be the purpose shall examine on case-to-case basis and submit its recommendations to NFP. downloaded from NBPGR website (www.nbpgr.ernet.in). • After the approval of NFP, NBPGR shall coordinate the supply of PGRFA as per established norms. • Acknowledgement of receipt of germplasm from NBPGR should be provided by the indenter immediately after receipt of material. • The access shall be provided to the natural and legal persons from the Contracting Parties (countries signatory to ITPGRFA), only. • Indenter should retain the unique identity no. assigned by NBPGR (IC/EC/ET) while using the germplasm. • Facilitated access shall be provided only for the PGRFA designated by GOI under the multilateral System (MLS) of the ITPGRFA. • Feedback information (in the prescribed format) on the performance or utilization of material should be sent to the Director, NBPGR, New Delhi. • In addition to the above-mentioned PGRFA, the germplasm of mandate crops available with International Agricultural Research Centres (IARCs) collected before 1993 (also known as 'FAO • Whenever requested, sufficient quantity of multiplied seed must be sent back to NBPGR at the earliest. designated' accessions of 'in trust' material), shall also be facilitated for access (as per Article 15 of In Vitro Cultures for National (Domestic) Supply ITPGRFA). • Distribution of germplasm for national supply shall be done from in vitro active collections and not from • Facilitated access to PGRFA as notified by GOI under MLS of the Treaty should be provided only for the base collections, keeping the national and international guidelines in view. purpose of utilization and conservation for research, breeding and training for food and agriculture, provided that such purpose does not include chemical, pharmaceutical and/or other non-food/feed • Consignment need to prepared in advance, if cultures are under slow growth or if there is less number of industrial uses (as per Article 12.3a of ITPGRFA). cultures of a species. • All requests for other purposes including non food/feed use of PGRFA shall be in accordance with the • If cultures are stored as alternate explants (in vitro storage organs/encapsulated explants), it is desirable provisions of extant relevant national legislations. to send the request in advance for providing shoots/plantlets. • Access by other countries, which are not Contracting Parties, will be in accordance with the extant • Recipient should enquire in advance the details of the culture medium and the physical conditions relevant national legislations, on a bilateral basis. required for maintaining the cultures in vitro. • Voucher samples along with passport data of all PGRFA accessed/ supplied, shall be conserved and • Procedure for in vitro multiplication/regeneration should accompany the in vitro cultures for recipients documented by NBPGR. to initiate fresh cultures. • All germplasm shall be exported to the requesting country only under the provisions of Standard • In case of usage of glass vessels for transporting in vitro cultures, proper stuffing of the vessel be ensured Material Transfer Agreement (SMTA) adopted by Governing Body (GB) of the ITPGRFA. Specific along with declaration of fragile nature of the vessel. conditions may be added in SMTA in case of any material 'under development' as per provisions of the • The glass or plastic containers being used for sending in vitro cultures need to have correct orientation of ITPGRFA. SMTA shall be signed prior to transfer of PGRFA only as hard copies and the Director, NBPGR containers, indicated. shall be the authorized signatory. • A regular feedback regarding the growth and establishment of received cultures be provided to the • For export of PGRFA, the IP from the importing country shall be required. The transfer of material donor. remains subject to normal phytosanitary inspection and clearance.

54 55 • The seed/planting material meant for export should be then submitted to Division of Plant Quarantine, Export of Germplasm for Non-Commercial Research/ or Research for Emergency Purposes Other than NBPGR for quarantine inspection as per the IP instructions of the importing country, for issuance of PC. Collaborative Research • The seed/planting material, along with IP and PC should be dispatched to the receiving country either by • Any Indian researcher/ Government institution who intends to carry/ send the biological resources the indentor or by NBPGR. outside India to undertake basic research other than collaborative research as referred to in section 5 of the Biological Diversity Act , 2002 shall apply to the NBA in Form 'B'. Export of Germplasm under Category 3 (Annex-I crops under ITGRFA and FAO designated accessions of CG centres located in India) • Any Government Institution which intends to send biological resources to carry out certain urgent studies to avert emergencies like epidemics, etc., shall apply in Form 'B'. • Export of germplasm under category 3 shall be as per the MoEF & CC Notification S.O. 3232 (E) 17.12.2014 and Department of Agriculture & Cooperation O.M. No. 13-5/2013-SD-V dated 16.2.2015. • The NBA shall, on being satisfied with the application accord its approval within a period of 45 days from the date of receipt of the application. • The Department of Agricutral Cooperation (DAC), MoA&FW, GOI is the Nodal Department and Joint Secretary (Seeds) is the National Focal Point (NFP) for access to PGRFA of designated accessions in • On receipt of approval of the NBA, the applicant shall deposit voucher specimens in the designated accordance with the provisions of the ITPGRFA to which India is a party, and relevant extant national national repositories before carrying / sending the biological resources outside India and a copy of proof legislations. of such deposits shall be endorsed to NBA. • All requests for access to PGRFA listed in Annex 1 of the ITPGRFA (as per Article 12.3 c of ITPGRFA) should National (Domestic) Supply of Germplasm be addressed to NFP, either directly or through NBPGR. Access to PGRFA requested shall be granted only • Requests for samples of plant germplasm available with/maintained by NBPGR/NAGS should be sent to with the approval of NFP. the Director, NBPGR, Pusa Campus, New Delhi 110012, in the prescribed GEX 01 form (Annexure 4) along • On receipt of request for export, the Germplasm Export Facilitation Committee (GEFC) constituted for with a signed MTA (Annexure 5) by the indenter of germplasm. The GEX 01 and MTA can also be the purpose shall examine on case-to-case basis and submit its recommendations to NFP. downloaded from NBPGR website (www.nbpgr.ernet.in). • After the approval of NFP, NBPGR shall coordinate the supply of PGRFA as per established norms. • Acknowledgement of receipt of germplasm from NBPGR should be provided by the indenter immediately after receipt of material. • The access shall be provided to the natural and legal persons from the Contracting Parties (countries signatory to ITPGRFA), only. • Indenter should retain the unique identity no. assigned by NBPGR (IC/EC/ET) while using the germplasm. • Facilitated access shall be provided only for the PGRFA designated by GOI under the multilateral System (MLS) of the ITPGRFA. • Feedback information (in the prescribed format) on the performance or utilization of material should be sent to the Director, NBPGR, New Delhi. • In addition to the above-mentioned PGRFA, the germplasm of mandate crops available with International Agricultural Research Centres (IARCs) collected before 1993 (also known as 'FAO • Whenever requested, sufficient quantity of multiplied seed must be sent back to NBPGR at the earliest. designated' accessions of 'in trust' material), shall also be facilitated for access (as per Article 15 of In Vitro Cultures for National (Domestic) Supply ITPGRFA). • Distribution of germplasm for national supply shall be done from in vitro active collections and not from • Facilitated access to PGRFA as notified by GOI under MLS of the Treaty should be provided only for the base collections, keeping the national and international guidelines in view. purpose of utilization and conservation for research, breeding and training for food and agriculture, provided that such purpose does not include chemical, pharmaceutical and/or other non-food/feed • Consignment need to prepared in advance, if cultures are under slow growth or if there is less number of industrial uses (as per Article 12.3a of ITPGRFA). cultures of a species. • All requests for other purposes including non food/feed use of PGRFA shall be in accordance with the • If cultures are stored as alternate explants (in vitro storage organs/encapsulated explants), it is desirable provisions of extant relevant national legislations. to send the request in advance for providing shoots/plantlets. • Access by other countries, which are not Contracting Parties, will be in accordance with the extant • Recipient should enquire in advance the details of the culture medium and the physical conditions relevant national legislations, on a bilateral basis. required for maintaining the cultures in vitro. • Voucher samples along with passport data of all PGRFA accessed/ supplied, shall be conserved and • Procedure for in vitro multiplication/regeneration should accompany the in vitro cultures for recipients documented by NBPGR. to initiate fresh cultures. • All germplasm shall be exported to the requesting country only under the provisions of Standard • In case of usage of glass vessels for transporting in vitro cultures, proper stuffing of the vessel be ensured Material Transfer Agreement (SMTA) adopted by Governing Body (GB) of the ITPGRFA. Specific along with declaration of fragile nature of the vessel. conditions may be added in SMTA in case of any material 'under development' as per provisions of the • The glass or plastic containers being used for sending in vitro cultures need to have correct orientation of ITPGRFA. SMTA shall be signed prior to transfer of PGRFA only as hard copies and the Director, NBPGR containers, indicated. shall be the authorized signatory. • A regular feedback regarding the growth and establishment of received cultures be provided to the • For export of PGRFA, the IP from the importing country shall be required. The transfer of material donor. remains subject to normal phytosanitary inspection and clearance.

54 55 Access to Fruit genetic resources PQ Form 08 Annexure 1

In general, fruit crops are conserved as field collections (>80%) or through vegetative propagules i.e. in vitro NATIONAL BUREAU OF PLANT GENETIC RESOURCES derived shoot-tips and dormant buds conserved/ cryopreserved in liquid nitrogen (LN). Major genebanks INDIAN COUNCIL OF AGRICULTURAL RESEARCH involved in conservation of fruit germplasm include 11 USDA National Plant Germplasm System (NPGS) NEW DELHI, INDIA repositories devoted to clonally propagated, horticultural fruit and nut crops. Some of the important genebanks from where request for tropical and sub-tropical fruit crops can be made for fruit genetic resources are: Application for Permit to Import Germplasm/ Transgenics/ Genetically Modified Organisms (GMO's) • USDA-ARS-NCGR, Hilo, Hawai: https://www.ars.usda.gov/pacific-west-area/hilo-hi/ (For Research Purpose) • USDA-ARS-NCGR, Miami, Florida: https://www.ars.usda.gov/southeast-area/miami-fl The Director, • USDA-ARS-TARS, Mayaguez, Puerto Rico: https://www.ars.usda.gov/southeast-area/mayaguez-pr/ National Bureau of Plant Genetic Resources • USDA-ARS-NCGR, Riverside, California: https://www.ars.usda.gov/pacific-west-area/riverside-ca Pusa Campus, New Delhi-110012 References I hereby apply for a permit in accordance with provisions of clause 6 (2) of the Plant 1. Brahmi P and V Tyagi (2019) IPR issues related to access and use of genetic resources. Indian J. Plant Quarantine (Regulation of Import into India) Order, 2003 issued under the Sub-section (1) of Genet. Resou. 79: 315-319. Section (3) of the Destructive Insects & Pests Act, 1914 (2 of 1914), authorizing the import of plants/planting materials for research purposes as per details given below: 2. Brahmi P, RK Khetarpal and BS Dhillon (2005) Access to Plant Genetic Resources: The Changing Scenario. In: Tandon P, M Sharma and R Swaroop (eds.) Biodiversity: Status and Prospects. Narosa Publishing 1. Name and address of the applicant : House, New Delhi, India, pp 124-133. along with e-mail & mobile No. 3. Brahmi P, RP Dua and BS Dhillon (2004) The Biological Diversity Act of India and agro-biodiversity management. Curr. Sci. 86:659 -664. 2. Research and Development (R&D) status : 4. Postman JD, KE Hummer, EW Stover, R Krueger, PL Forsline, LJ Grauke, FT Zee, T Ayala Silva, BM Irish /affiliations of the organisation (2006) Fruit and nut genebanks in the US National Plant Germplasm System. HortScience 41(5):1188- [Please attach relevant documents] 1194. 5. Tyagi RK, A Agrawal, R Pandey, V Celia Chalam and B Mal (eds) (2016) Guidelines for Management of 3. Exact description of Seeds/Planting Materials : Plant Genetic Resources in India. ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New to be imported: Delhi, 142 +xxiv p. a) Common and botanical name : b) Germplasm/variety/hybrid/composite : 6. http://nbaindia.org/ (National Biodiversity Authority) /synthetic/provenance/clone/others 7. http://nbaindia.org/content/25/19/1/act.html (Biological Diversity Act) c) Form of material required (seed/rooted : 8. https://www.wto.org/english/tratop_e/trips_e/intel2_e.htm (WTO- TRIPS) plants/ scions/ tubers/cuttings/bulbs in vitro cultures 9. http://plantquarantineindia.nic.in/pqispub/html/pqo_amendments.htm (Plant Quarantine Order, (d) Parentage, if known : 2003) 4. Place of collection/origin of material to be : 10. http://www.fao.org/cgrfa/en/ (CGRFA) imported (country/state) 11. http://www.fao.org/plant-treaty/en/(ITPGRFA) http://www.ipindia.nic.in/acts-patents.htm 5. Whether transgenic/GMO or not? : 12. (Patent Act) [If yes, attach the approval letter issued by RCGM 13. https://www.upov.int/upovlex/en/upov_convention.html (UPOV) /(DBT) in original] 6. Name and address of the organization/institution : 14. http://www.ipindia.nic.in/registered-gls.htm (Geographical Indications Registry) producing the material. 15. http://www.plantauthority.gov.in/ (PPVFRA) 7. Number of samples to be imported : 16. https://www.ars-grin.gov/ 8. Quantity to be imported (separately for each : 17. https://www.cbd.int/ (CBD home page) accession/ variety/ hybrid/ transgenic/ GMO) 18. https://www.cbd.int/abs/(Nagoya Protocol on ABS)

56 57 Access to Fruit genetic resources PQ Form 08 Annexure 1

56 57 Annexure 2 9. Suggested source of availability of material including : published reference, if known (a) Whether the aforesaid germplasm/ variety/ : Processing fee applicable for issuance of Import permit w.e.f. July 2017 hybrid was imported by you earlier? S.No. Organization/ Import Fee amount GST Total Fee per If so, details thereof (year, quantity, source, etc.) Institution* Permit in Rs. @18% permit in Rs. (b) Was the material shared with other scientists/ : National Gene Bank at NBPGR? 1. Public Non Transgenic 150.00 27.00 177.00 2. Public Transgenic 250.00 45.00 295.00 10. Expected date and arrival in India : 3. Private Non Transgenic 300.00 54.00 354.00 11. Mode of shipment (Airmail/Air freight/ : accompanied baggage) 4. Private Transgenic 550.00 99.00 649.00 12. Place where imported seeds/ planting material : will be grown and scientists under whose supervision Fee to be paid as Demand Draft in favour of Director, ICAR-NBPGR payable at Delhi the seeds/ planting material will be grown Revalidation fee for Extension of validity or revision of Import Permit

Declaration S.No. Category Public Sector Private Sector 1. I hereby declare that the germplasm under import has no commercial value/exclusive 1. Revalidation of existing Import Permit for a further ownership and may be shared freely for research purposes. period of six months for import of non-transgenic 2. The germplasm does not contain any terminator genes. germplasm Rs. 25/- Rs. 50/- 3. I undertake that the material is exclusively for research purposes. 2. Revalidation of existing Import Permit for a further period of six months for import of transgenic germplasm Rs. 50/- Rs. 100/- Place: *Organizations/ Institution qualifying for import of germplasm/ research material Date: The government notifications recognizing the R&D status of an organization as issued by the Department of Scientific and Industrial Research/ Department of Science and Technology/ State Government Department Signature of the Applicant & Address (s), will be considered as the criterion for issuing the import permit to a given organization. ______* GST @ 18% applicable (subject to revision) For further information contact Tel. No. 91-11-25843697 or Fax. 91-11 25844295 or E-Mail - [email protected] and Web Address- http://nbpgr.ernet.in

58 59 Annexure 2 9. Suggested source of availability of material including : published reference, if known (a) Whether the aforesaid germplasm/ variety/ : Processing fee applicable for issuance of Import permit w.e.f. July 2017 hybrid was imported by you earlier? S.No. Organization/ Import Fee amount GST Total Fee per If so, details thereof (year, quantity, source, etc.) Institution* Permit in Rs. @18% permit in Rs. (b) Was the material shared with other scientists/ : National Gene Bank at NBPGR? 1. Public Non Transgenic 150.00 27.00 177.00 2. Public Transgenic 250.00 45.00 295.00 10. Expected date and arrival in India : 3. Private Non Transgenic 300.00 54.00 354.00 11. Mode of shipment (Airmail/Air freight/ : accompanied baggage) 4. Private Transgenic 550.00 99.00 649.00 12. Place where imported seeds/ planting material : will be grown and scientists under whose supervision Fee to be paid as Demand Draft in favour of Director, ICAR-NBPGR payable at Delhi the seeds/ planting material will be grown Revalidation fee for Extension of validity or revision of Import Permit

58 59 Annexure 3 Annexure 4

Maximum quantity permitted for Import and Export, per sample/accession/ To variety for research purposes The Director National Bureau of Plant Genetic Resources (NBPGR) S.No. Plants parts Quantity Permitted per Pusa Campus, New Delhi-110 012 sample/ accession/variety Ph: 011-25843697, 25841129 ; Fax: 011-25842495 E mail: : [email protected], [email protected] 1. Seed Large seeded crop species viz; Zea mays, Helianthus spp, Requisition for supply of seed/ planting material for research from/ through Carthamus spp. Phaseolus spp. Arachis spp., Dolichos spp., NBPGR, New Delhi Mucuna spp., Pisum sativum, Cicer sp., Vicia spp.,

Cajanus spp., Canavalia spp., Cyamopsis spp., Palms 1. Details of seed/planting material required for research and others Up to 1kg

Small seeded crop species viz; Allium spp; Brassica spp; S.No. Botanical Crop No. of Seed Purpose Capsicum spp; Solanum melongena, Carica papaya and name name accessions Quantity (screening/breeding/ others Up to 100g (IC/EC ) (per accession/ evaluation augmentation/ Very small/ light weight seeded crop species viz; Tobacco, per sample) multiplication) Tomato, Grasses, Eucalyptus etc. Up to 25g All other species of plants viz; Rice, Wheat, Barley etc Up to 200g 2. Vegetative Propagules Number of rooted cuttings/ plants Up to 25 in numbers Number of other vegetative propagules Up to 50 in numbers 2. Thesis/ Project title for which request is made (name the funding agency)------3. Objective / Activity for utilization of indented material: (Please attach sheet if required): ------4. Material Transfer Agreement (enclosed): Yes/ No 5. Feedback report submitted on germplasm received earlier (if applicable): Yes/No 6. Have you or your Institute developed any variety based on germplasm supplied by NBPGR? Yes/No (If yes, please let us know the details) 7. If required by NBPGR, will you be able to send viable multiplied seed material of the above seed in sufficient quantity for conservation: Yes/No 8. Signature of the indentor:

Name: ------Designation: ------Address of the Institute ------Phone (with STD code) ------M ------, Fax ------, E-mail ------

60 61 Annexure 3 Annexure 4

Cajanus spp., Canavalia spp., Cyamopsis spp., Palms 1. Details of seed/planting material required for research and others Up to 1kg

Name: ------Designation: ------Address of the Institute ------Phone (with STD code) ------M ------, Fax ------, E-mail ------

60 61 Annexure 5 I/We agree to abide by the following terms of the MTA and certify that: i) The germplasm material and associated knowledge transferred herein as listed in annexure shall be Material Transfer Agreement for Research Use within Country for used only for the purpose of research under my/ our close supervision and will not be used for Public and Private Entities commercial purposes or for profit making whatsoever. The germplasm material accessed shall not be used for chemical, pharmaceutical and/ or other non-food/feed, industrial uses. PREAMBLE ii) All the information and the material supplied by ICAR shall be made available to the recipient in confidence. The recipient agrees to maintain the confidential status of the material and the Being signatory to the Convention on Biological Diversity, 19931 (CBD), the Government of India enacted the information. Biological Diversity Act, 2002 (BDA) hereinafter referred to as BDA, 2002 and notified the Biological Diversity Rules, 2004. The access to biological resources of India is now regulated by BDA, 2002. iii) The Recipient shall not claim any intellectual property or other rights on the material provided and associated information under this agreement 'in the form received.' Whereas, the National Bureaux of Genetic Resources under the aegis of Indian Council of Agricultural Research2 hereinafter called ICAR have the mandate for collecting, conservation, characterization, iv) Access to GR protected by intellectual and other property rights shall be consistent with the extant 5 evaluation and exchange of genetic resources (GR) in a network mode, the Bureaux encourage the national laws . researchers in the country to make use of germplasm for their effective utilization. Reiterating the fact that v) The intellectual property protection or benefit sharing in respect of derivatives of the material(s) GR are the essential raw materials for all improvement programmes and, hence, extremely important for received/ accessed, wherever applicable, shall be as per the Biological Diversity Act 2002 and food and nutritional security, their exchange and utilization need to be promoted in accordance with Guidelines on ABS Regulations, 20148. national laws and regulations and in compliance with international agreements. vi) Permission from National Biodiversity Authority (NBA) shall be sought through ICAR/ Department of Emphasizing the fact that the purpose of supply of GR under this agreement would be solely for research Agricultural Research and Education (DARE), Ministry of Agriculture and Farmers Welfare (MoAFW), and no deviation from the proposed objectives is permitted. Access shall be provided for the germplasm Government of India, if the accessed germplasm is intended to be transferred to any third party for available with the National Agricultural Research System (NARS), which is duly designated by concerned commercial utilization institute/ National Active Germplasm Sites (NAGS). Such exchange shall be done under the conditions of the following Material Transfer Agreement (MTA). The private entities falling under Section 3 (2) of BDA, 20023 vii) The recipient shall not claim any intellectual property right over the products derived from the can access germplasm after signing the MTA, subject to approval of National Biodiversity Authority (NBA)4. material accessed including its related information and knowledge without prior written approval of the NBA, India. MTA agreed between viii) Commercialization of any product(s) based on this material shall be undertaken with the prior ICAR, Krishi Bhawan, New Delhi-110001, a Society registered under the Registration of Societies Act (Act XXI approval of NBA/ concerned State Biodiversity Boards (SBBs). Such permission shall be sought only of 1860) which shall include its successors or assignees) being the First Party (Provider of the Material) through ICAR/ DARE. A separate Memorandum of Agreement (MoA) shall have to be entered into …………………………………………………………………………………………………...... with conditions of mutually agreed terms for benefit sharing with the owner/ developer of the material as per ICAR6 and NBA Guidelines7, 8. And ix) The recipient agrees to acknowledge explicitly the name, original identity and source in all …………………………………………………………………………………………………...... publications brought out from the work carried out from the accessed material. Being the Second Party (Recipient of the Material) x) The recipient agrees to supply the feedback information on the performance/ utilization/ research outcome of the material to the provider institute depending on crop on a seasonal/yearly basis. xi) The recipient agrees to pay the handling and processing charges for material received/accessed as Description of the material (Annexure): decided on case-to-case basis. xii) On completion/ suspension/ termination of the research involving, the material accessed, wherever 1 th th Convention on Biological Diversity signed at Rio deJanerio, vide NA92-7807, dated 5 June, 1992 and came into force on 29 available shall be conserved adopting suitable measures including deposition with the supplier December, 1993 (https://www.cbd.int/) 2 http://www.icar.org.in/en/node/325 xiii) The MTA is non-assignable and non-transferable. 3 Entities listed at Section 3 (2) of Biological Diversity Act : a person who is not a citizen of India; (b) a citizen of India, who is a non-resident as defined in clause (30) of section 2 of the Income tax Act, 196 1; (c) a body corporate, association or xiv) Every dispute, difference, or question which may at any time arise between the parties hereto or any organization- (i) not incorporated or registered in India; or (ii) incorporated or registered in India under any law for the time person claiming under them, touching or arising out of or in respect of this agreement or the subject being in force which has any non-Indian participation in its share capital or management. matter thereof, shall be amicably settled between the parties. In case the same is not amicably 4 National Biodiversity Authority established under sub-section 1 of section 8, BDA, 2002 settled, the dispute shall be referred to the Sole Arbitrator to be appointed by the Secretary DARE, Government of India. The decision of the Sole Arbitrator shall be final and binding on the parties. The seat of the Arbitration shall be at New Delhi, India and the proceedings shall be governed by the Arbitration and Conciliation Act, 1996 as amended from time to time and the substantive Indian Law will apply.

62 63 Annexure 5 I/We agree to abide by the following terms of the MTA and certify that: i) The germplasm material and associated knowledge transferred herein as listed in annexure shall be Material Transfer Agreement for Research Use within Country for used only for the purpose of research under my/ our close supervision and will not be used for Public and Private Entities commercial purposes or for profit making whatsoever. The germplasm material accessed shall not be used for chemical, pharmaceutical and/ or other non-food/feed, industrial uses. PREAMBLE ii) All the information and the material supplied by ICAR shall be made available to the recipient in confidence. The recipient agrees to maintain the confidential status of the material and the Being signatory to the Convention on Biological Diversity, 19931 (CBD), the Government of India enacted the information. Biological Diversity Act, 2002 (BDA) hereinafter referred to as BDA, 2002 and notified the Biological Diversity Rules, 2004. The access to biological resources of India is now regulated by BDA, 2002. iii) The Recipient shall not claim any intellectual property or other rights on the material provided and associated information under this agreement 'in the form received.' Whereas, the National Bureaux of Genetic Resources under the aegis of Indian Council of Agricultural Research2 hereinafter called ICAR have the mandate for collecting, conservation, characterization, iv) Access to GR protected by intellectual and other property rights shall be consistent with the extant 5 evaluation and exchange of genetic resources (GR) in a network mode, the Bureaux encourage the national laws . researchers in the country to make use of germplasm for their effective utilization. Reiterating the fact that v) The intellectual property protection or benefit sharing in respect of derivatives of the material(s) GR are the essential raw materials for all improvement programmes and, hence, extremely important for received/ accessed, wherever applicable, shall be as per the Biological Diversity Act 2002 and food and nutritional security, their exchange and utilization need to be promoted in accordance with Guidelines on ABS Regulations, 20148. national laws and regulations and in compliance with international agreements. vi) Permission from National Biodiversity Authority (NBA) shall be sought through ICAR/ Department of Emphasizing the fact that the purpose of supply of GR under this agreement would be solely for research Agricultural Research and Education (DARE), Ministry of Agriculture and Farmers Welfare (MoAFW), and no deviation from the proposed objectives is permitted. Access shall be provided for the germplasm Government of India, if the accessed germplasm is intended to be transferred to any third party for available with the National Agricultural Research System (NARS), which is duly designated by concerned commercial utilization institute/ National Active Germplasm Sites (NAGS). Such exchange shall be done under the conditions of the following Material Transfer Agreement (MTA). The private entities falling under Section 3 (2) of BDA, 20023 vii) The recipient shall not claim any intellectual property right over the products derived from the can access germplasm after signing the MTA, subject to approval of National Biodiversity Authority (NBA)4. material accessed including its related information and knowledge without prior written approval of the NBA, India. MTA agreed between viii) Commercialization of any product(s) based on this material shall be undertaken with the prior ICAR, Krishi Bhawan, New Delhi-110001, a Society registered under the Registration of Societies Act (Act XXI approval of NBA/ concerned State Biodiversity Boards (SBBs). Such permission shall be sought only of 1860) which shall include its successors or assignees) being the First Party (Provider of the Material) through ICAR/ DARE. A separate Memorandum of Agreement (MoA) shall have to be entered into …………………………………………………………………………………………………...... with conditions of mutually agreed terms for benefit sharing with the owner/ developer of the material as per ICAR6 and NBA Guidelines7, 8. And ix) The recipient agrees to acknowledge explicitly the name, original identity and source in all …………………………………………………………………………………………………...... publications brought out from the work carried out from the accessed material. Being the Second Party (Recipient of the Material) x) The recipient agrees to supply the feedback information on the performance/ utilization/ research outcome of the material to the provider institute depending on crop on a seasonal/yearly basis. xi) The recipient agrees to pay the handling and processing charges for material received/accessed as Description of the material (Annexure): decided on case-to-case basis. xii) On completion/ suspension/ termination of the research involving, the material accessed, wherever 1 th th Convention on Biological Diversity signed at Rio deJanerio, vide NA92-7807, dated 5 June, 1992 and came into force on 29 available shall be conserved adopting suitable measures including deposition with the supplier December, 1993 (https://www.cbd.int/) 2 http://www.icar.org.in/en/node/325 xiii) The MTA is non-assignable and non-transferable. 3 Entities listed at Section 3 (2) of Biological Diversity Act : a person who is not a citizen of India; (b) a citizen of India, who is a non-resident as defined in clause (30) of section 2 of the Income tax Act, 196 1; (c) a body corporate, association or xiv) Every dispute, difference, or question which may at any time arise between the parties hereto or any organization- (i) not incorporated or registered in India; or (ii) incorporated or registered in India under any law for the time person claiming under them, touching or arising out of or in respect of this agreement or the subject being in force which has any non-Indian participation in its share capital or management. matter thereof, shall be amicably settled between the parties. In case the same is not amicably 4 National Biodiversity Authority established under sub-section 1 of section 8, BDA, 2002 settled, the dispute shall be referred to the Sole Arbitrator to be appointed by the Secretary DARE, Government of India. The decision of the Sole Arbitrator shall be final and binding on the parties. The seat of the Arbitration shall be at New Delhi, India and the proceedings shall be governed by the Arbitration and Conciliation Act, 1996 as amended from time to time and the substantive Indian Law will apply.

62 63 xv) In case of misuse/transfer of material by the recipient and use other than intended purpose, as stated DEFINITIONS under Clause (i) of MTA, the recipient shall be liable for penalties as defined under Section 55 of BDA, In this Agreement, the expressions set out below shall have the following meaning: 2002. “Benefit sharing,” means sharing of benefits arising from use of genetic resources.

5 Patent Act 2005, The Protection of Plant Varieties and Farmers' Rights Act, 2001 “Germplasm” means whole plant, animal, insect, fish, fungi, microbes or in parts, and their propagules 6 ICAR (2006). ICAR Guidelines for Intellectual Property Management and Technology Transfer/ Commercialization, Indian including seeds, vegetative parts, tissue cultures, embryo, ova, semen, spawn, colonies, cultures, cell lines, Council of Agricultural Research, New Delhi. 7 genes and DNA based sequences etc, that are held in a repository or collected from wild as the case may be Guidelines for International Collaboration Research Projects involving Transfer or Exchange of Biological Resources or and are utilized in research, genetic studies or breeding programmes for improvement of relevant information relating thereto between institutions including Government sponsored Institutions and such Institutions in other countries S.O.1911 (E) dated 08.11.2006 agricultural – biodiversity component. 8 Guidelines on Access to Biological Resources and associated Knowledge and Benefit Sharing, 2014. “Intellectual Property Rights” refer to inventions, new products, processes derived from use of genetic resources. Agreed and Accepted “Genetic Resources” means any genetic material of actual or potential value. “Product” means genetic resource that incorporates the material provided under the MTA or any of its (To be signed in duplicate) genetic parts or components that are ready for commercialization. “To commercialize,” means to sell a Product or Products for monetary benefits in the open market, and RECIPIENT PROVIDER “commercialization” has a corresponding meaning.

Name : Name : Designation: Designation: Institution/ Organization: Institution/ Organization:

Full Address with PIN Code: Full Address with PIN Code:

Phone/ Fax/ E –mail: Phone/ Fax/ E –mail: Signature: Signature: Date: Date:

Authorized Institutional Official Authorized Institutional Official

Name : Name : Designation: Designation: Institution/ Organization: Institution/ Organization: Full Address with PIN Code: Full Address with PIN Code: Phone/ Fax/ E –mail: Phone/ Fax/ E –mail: Signature: Signature: Date: Date:

Official Seal Official Seal

64 65 xv) In case of misuse/transfer of material by the recipient and use other than intended purpose, as stated DEFINITIONS under Clause (i) of MTA, the recipient shall be liable for penalties as defined under Section 55 of BDA, In this Agreement, the expressions set out below shall have the following meaning: 2002. “Benefit sharing,” means sharing of benefits arising from use of genetic resources.

Name : Name : Designation: Designation: Institution/ Organization: Institution/ Organization:

Full Address with PIN Code: Full Address with PIN Code:

Phone/ Fax/ E –mail: Phone/ Fax/ E –mail: Signature: Signature: Date: Date:

Authorized Institutional Official Authorized Institutional Official

Official Seal Official Seal

64 65 Annexure 6 National Plant Quarantine System List and Description of the GR provided under this S. C. Dubey and Kavita Gupta* Material Transfer Agreement Division of Plant Quarantine National Bureau of Plant Genetic Resources, New Delhi *Email: [email protected] 1. Type of the Genetic Resource (plant/ animal/fish/microbe/insect etc.,): Introduction 2. Common name and Scientific name: Exchange of plant genetic resources (PGR) has contributed significantly towards crop improvement and 3. Type of propagule (depending on the type of resource) : increased crop production in the country. However, many pests have also moved across the countries along with planting material. Plant quarantine is a government endeavor enforced through legislative measures to 4. Number of accessions: regulate the introduction of planting material, plant products, soil and living organisms, etc. in order to 5. Details of the accessions : prevent inadvertent introduction of pests (including fungi, bacteria, viruses, nematodes, insects and weeds) harmful to the agriculture of a country/ state/ region, and if introduced, prevent their establishment and 6. Quantity required (per accession/per sample): further spread (Kahn et al., 1989). The historical Irish famine of 1845, caused by late blight of potato (Phytophthora infestans) introduced from 7. Purpose (screening/ breeding/ evaluation/ augmentation/ multiplication/ others (please specify). Central America; powdery mildew (Uncinula nacator), root eating aphid (Phylloxera vitifolia) and downy th 8. Enclose a copy of approval of NBA (if applicable): mildew (Plasmopara viticola) of grapes into France in quick succession in mid 19 Century from America; coffee rust into Sri Lanka in 1875 and its subsequent introduction into India in 1876 are prominent examples that clearly demonstrate that introduction and establishment of quarantine pests into new areas can Recipient's Signature...... Date...... severely damage the crop production and economy of a region/ country (Khetarpal et al.,2006). Like-wise, in India also, a number of exotic pests got introduced along with imported planting material causing serious crop losses from time to time. These include the recently introduced Spodoptera littoralis fall armyworm on maize in 2018; Puccinia horiana causing white rust in chrysanthemum in 2016; tomato pin worm Tuta absoluta in 2014, Jackbeardsley mealybug (Pseudococcus jackbeardsleyi) in 2012, papaya mealy bug (Paracoccus marginatus) in 2007, fluted scale on citrus introduced from Sri Lanka in 1928; San Jose scale in apple in 1930s; bunchy top of banana introduced from Sri Lanka in 1943; the golden nematode infesting potatoes introduced in 1960s from UK and the noxious weed, Lantana camara introduced in 1809 from Central America are glaring examples that clearly demonstrate that introduction and establishment of quarantine pests including weeds into new areas can severely damage the crop production and economy of a region/ country (Dubey and Gupta, 2016). These introductions highlighted the fact that increased international travel and trade had exposed the country to the danger of infiltration of exotic pests harmful to our agriculture. With the liberalization of trade under World Trade Organization (WTO), the quarantine set-up including legislation and infrastructure of the country has been reviewed. As far as legislation is concerned, the Destructive Insects and Pests (DIP) Act was legislated by the British government ruling India in 1914 which was retained revising it as per requirements over the years through various amendments. However, after the WTO came into force, India legislated the Plant Quarantine (Regulation of Import into India) Order in 2003, henceforth referred to as the PQ Order. The Directorate of Plant Protection Quarantine and Storage (DPPQS) of the Ministry of Agriculture and Farmers Welfare is the nodal agency for implementation of PQ Order. ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR is the nodal agency for PGR management in the country, has been empowered under the PQ Order for issuance of Import Permit and to undertake quarantine processing of all imported PGR including transgenics and trial material meant for research. Besides, NBPGR also tests samples of bulk imports sent by DPPQS and it's Regional Plant Quarantine Stations for presence of exotic pests. ICAR-NBPGR is well equipped with most modern quarantine facilities including a Containment Facility of Level 4 (CL- 4) for quarantine processing of transgenic germplasm in a risk-free manner. ICAR-NBPGR also has a well-equipped quarantine station at Hyderabad, which mainly deals with the quarantine processing of PGR meant for Southern India including State Agricultural Universities, ICAR institutes, private industry and international institutes viz., International Crop Research Institute for Semi-arid Tropics (ICRISAT), CIMMYT and AVRDC. 66 67 Annexure 6 National Plant Quarantine System List and Description of the GR provided under this S. C. Dubey and Kavita Gupta* Material Transfer Agreement Division of Plant Quarantine National Bureau of Plant Genetic Resources, New Delhi *Email: [email protected] 1. Type of the Genetic Resource (plant/ animal/fish/microbe/insect etc.,): Introduction 2. Common name and Scientific name: Exchange of plant genetic resources (PGR) has contributed significantly towards crop improvement and 3. Type of propagule (depending on the type of resource) : increased crop production in the country. However, many pests have also moved across the countries along with planting material. Plant quarantine is a government endeavor enforced through legislative measures to 4. Number of accessions: regulate the introduction of planting material, plant products, soil and living organisms, etc. in order to 5. Details of the accessions : prevent inadvertent introduction of pests (including fungi, bacteria, viruses, nematodes, insects and weeds) harmful to the agriculture of a country/ state/ region, and if introduced, prevent their establishment and 6. Quantity required (per accession/per sample): further spread (Kahn et al., 1989). The historical Irish famine of 1845, caused by late blight of potato (Phytophthora infestans) introduced from 7. Purpose (screening/ breeding/ evaluation/ augmentation/ multiplication/ others (please specify). Central America; powdery mildew (Uncinula nacator), root eating aphid (Phylloxera vitifolia) and downy th 8. Enclose a copy of approval of NBA (if applicable): mildew (Plasmopara viticola) of grapes into France in quick succession in mid 19 Century from America; coffee rust into Sri Lanka in 1875 and its subsequent introduction into India in 1876 are prominent examples that clearly demonstrate that introduction and establishment of quarantine pests into new areas can Recipient's Signature...... Date...... severely damage the crop production and economy of a region/ country (Khetarpal et al.,2006). Like-wise, in India also, a number of exotic pests got introduced along with imported planting material causing serious crop losses from time to time. These include the recently introduced Spodoptera littoralis fall armyworm on maize in 2018; Puccinia horiana causing white rust in chrysanthemum in 2016; tomato pin worm Tuta absoluta in 2014, Jackbeardsley mealybug (Pseudococcus jackbeardsleyi) in 2012, papaya mealy bug (Paracoccus marginatus) in 2007, fluted scale on citrus introduced from Sri Lanka in 1928; San Jose scale in apple in 1930s; bunchy top of banana introduced from Sri Lanka in 1943; the golden nematode infesting potatoes introduced in 1960s from UK and the noxious weed, Lantana camara introduced in 1809 from Central America are glaring examples that clearly demonstrate that introduction and establishment of quarantine pests including weeds into new areas can severely damage the crop production and economy of a region/ country (Dubey and Gupta, 2016). These introductions highlighted the fact that increased international travel and trade had exposed the country to the danger of infiltration of exotic pests harmful to our agriculture. With the liberalization of trade under World Trade Organization (WTO), the quarantine set-up including legislation and infrastructure of the country has been reviewed. As far as legislation is concerned, the Destructive Insects and Pests (DIP) Act was legislated by the British government ruling India in 1914 which was retained revising it as per requirements over the years through various amendments. However, after the WTO came into force, India legislated the Plant Quarantine (Regulation of Import into India) Order in 2003, henceforth referred to as the PQ Order. The Directorate of Plant Protection Quarantine and Storage (DPPQS) of the Ministry of Agriculture and Farmers Welfare is the nodal agency for implementation of PQ Order. ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR is the nodal agency for PGR management in the country, has been empowered under the PQ Order for issuance of Import Permit and to undertake quarantine processing of all imported PGR including transgenics and trial material meant for research. Besides, NBPGR also tests samples of bulk imports sent by DPPQS and it's Regional Plant Quarantine Stations for presence of exotic pests. ICAR-NBPGR is well equipped with most modern quarantine facilities including a Containment Facility of Level 4 (CL- 4) for quarantine processing of transgenic germplasm in a risk-free manner. ICAR-NBPGR also has a well-equipped quarantine station at Hyderabad, which mainly deals with the quarantine processing of PGR meant for Southern India including State Agricultural Universities, ICAR institutes, private industry and international institutes viz., International Crop Research Institute for Semi-arid Tropics (ICRISAT), CIMMYT and AVRDC. 66 67 Plant Quarantine: Legislation purposes. The PQ Stations under the DPPQS undertake quarantine processing and clearance of consignments of the first two categories. However, ICAR-National Bureau of Plant Genetic Resources (ICAR- The quarantine measures are of utmost relevance to a country like India whose economy is largely th NBPGR) undertakes the quarantine processing of all plant germplasm and transgenic planting material agriculture based. The awareness to quarantine measures in India started in early 20 century when the under exchange. It has developed well- equipped laboratories and green house complex (Bhalla et al., Indian Government in 1906 ordered compulsory fumigation of imported cotton bales to prevent 2018). A containment facility of CL-4 level has been recently established for processing transgenics (Gupta et introduction of Mexican cotton boll weevil (Anthonomus grandis). With a view to restrict the entry of exotic al., 2007). pests, pathogens and weeds through regulation of imports, the Government of India legislated the Destructive Insects and Pests (DIP) Act in 1914 (http://plantquarantineindia.nic.in/ Bulk Consignments for Consumption/ Propagation pqispub/docfiles/dip_act.htm). This Act has been amended through various notifications issued from time The DPPQS needs to be approached for import permit (IP) and quarantine clearance. The Plant Quarantine to time also restricted the movement of certain planting material from one state to another state within the Order 2003 stipulates two kinds of conditions for the import of horticultural planting material. These are (a) country through domestic quarantine. In 1984, a notification was issued under this Act namely Plants, Fruits General Conditions and (b) Special conditions and post-entry quarantine. and Seeds (Regulation of Import into India) Order popularly known as the PFS Order which was revised in 1989 after the announcement of the New Policy on Seed Development by the Government of India in 1988, (a) General Conditions for Imports proposing major modifications for smooth quarantine functioning. This Order has now been superseded by Import of seeds and plants for sowing or planting of fruit crops is permitted by the Export and Import (EXIM) the Plant Quarantine (Regulation for Import into India) Order 2003 which came into force as there was an Committee of the Department of Agriculture and Cooperation. Import permit (IP) needs to be procured urgent need to fill-up the gaps in existing PFS order regarding import of germplasm/ GMO's/ transgenic before importing any consignment. It is valid for a period of six months and extendable further for a period plant material/ bio-control agents etc., to fulfill India's legal obligations under the international of another six months. The Phytosanitary Certificate (PC) issued by the quarantine authority of the country Agreements, to protect the interest of the farmers of the country by preventing the entry, establishment of export should accompany the consignment. All consignments should be imported only through entry and spread of destructive pests, and to safeguard the national bio-diversity from threats of invasions by points notified by Central Government from time to time. The Plant Protection Advisor to the Govt. of India alien species. Under this Order, the need for incorporation of Additional/ Special declarations for freedom of or the official authorized by him may, after inspection, fumigation, disinfection/ disinfestation accord import commodities from quarantine and invasive alien species (IAS), on the basis of standardized pest risk quarantine clearance or order for destruction of consignment or return the same to the source country. In analysis (PRA), particularly for seed/ planting materials is also dealt with. Further, the scope of plant addition, PRA which has been made mandatory for all materials except for those listed under Schedule VII of quarantine activities has been widened with incorporation of additional definitions. The other salient the Plant Quarantine Order 2003 has to be conducted as per norms. features of the Order are: (b) Special Conditions of Imports • Prohibition on import of commodities with weed/ alien species contamination as per Schedule VIII; & restriction on import of packaging material of plant origin unless treated. Horticultural planting material is prohibited from certain parts of the world because of prevalence of pests • Provisions included for regulating the import of soil, peat & sphagnum moss; germplasm/ GMOs/ are listed at Schedule IV of the PQ Order (Annex I). Materials listed under Schedule V and VI needs to meet transgenic material for research; live insects/ microbial cultures & biocontrol agents and import of certain additional declarations and special conditions. timber & wooden logs. Germplasm • Agricultural imports have been classified as (a) prohibited plant species (Schedule IV); (b) restricted Research institutions of public and private sector interested in importing plants or planting material should species where import permitted only by authorized institutions (Schedule V); (c) restricted species request NBPGR for IP which is not transferable. The point of entry of all germplasm consignments is New permitted only with additional declarations of freedoms from quarantine/ regulated pests and subject Delhi airport only. On arrival, the quarantine scientists carefully process the material. In case material is to specified treatment certifications (Schedule VI) and; (d) plant material imported for consumption/ found to be infected/ infested with pests, all efforts are made to salvage the material. Only in rare cases, industrial processing permitted with normal Phytosanitary Certificate (Schedule VII). when the material cannot be salvaged it is incinerated. In case post-entry quarantine examination of the • Additional declarations being specified in the Order for import of 699 agricultural commodities with imported material is required, it is done at quarantine greenhouse facility, at NBPGR, New Delhi and its specific lists of more than > 1000 quarantine pests and 57 weed species. Regional Station, Bhowali for temperate fruits and other horticultural crops. • Notified points of entry increased to 182. Quarantine Procedures for Import of Horticultural Crops • Certification fee and inspection charges have been rationalized. Pre-entry Plant Quarantine Requirements So far, i.e., till July 2020, 84 amendments of the Plant Quarantine (PQ) Order 2003 have been notified to the WTO revising definitions, clarifications regarding specific queries raised by quarantine authorities of various (a) Import Permit is a statutory requirement from the country of import and the conditions/ additional countries, with revised lists of crops under the Schedules IV, V VI and VII. The revised list under Schedule VI declarations laid on it need to be fulfilled by the country of export. and VII now include 699 and 519 crops/ commodities, respectively. (b) Phytosanitary Certificate is also a statutory requirement and is a proof that the consignment has been National Plant Quarantine Set-up for import of Horticultural Material examined according to the requirements of the importing country and found to be free from the quarantine pests mentioned in the IP. The Directorate of Plant Protection, Quarantine and Storage (DPPQS) of Ministry of Agriculture and Farmers Welfare is the apex body for implementation of plant quarantine regulation and the PQ order forms the (c) PRA carried out by the country of import for which desired information is supplied by the country of basis of the functioning of the Directorate. It has a national network of 72 plant quarantine stations at export. different airports (24), seaports (24) and land frontiers (24). In all, three categories of materials are being (d) Approval of Post-entry Quarantine (PEQ) Growing Facility is essential as all the horticultural planting imported: (a) bulk consignments of grains/ pulses for consumption, (b) bulk consignments of seeds/ material is subjected to PEQ inspection. The material cannot be imported unless planting materials for sowing/ planting, and (c) samples of germplasm in small quantities for research

68 69 Plant Quarantine: Legislation purposes. The PQ Stations under the DPPQS undertake quarantine processing and clearance of consignments of the first two categories. However, ICAR-National Bureau of Plant Genetic Resources (ICAR- The quarantine measures are of utmost relevance to a country like India whose economy is largely th NBPGR) undertakes the quarantine processing of all plant germplasm and transgenic planting material agriculture based. The awareness to quarantine measures in India started in early 20 century when the under exchange. It has developed well- equipped laboratories and green house complex (Bhalla et al., Indian Government in 1906 ordered compulsory fumigation of imported cotton bales to prevent 2018). A containment facility of CL-4 level has been recently established for processing transgenics (Gupta et introduction of Mexican cotton boll weevil (Anthonomus grandis). With a view to restrict the entry of exotic al., 2007). pests, pathogens and weeds through regulation of imports, the Government of India legislated the Destructive Insects and Pests (DIP) Act in 1914 (http://plantquarantineindia.nic.in/ Bulk Consignments for Consumption/ Propagation pqispub/docfiles/dip_act.htm). This Act has been amended through various notifications issued from time The DPPQS needs to be approached for import permit (IP) and quarantine clearance. The Plant Quarantine to time also restricted the movement of certain planting material from one state to another state within the Order 2003 stipulates two kinds of conditions for the import of horticultural planting material. These are (a) country through domestic quarantine. In 1984, a notification was issued under this Act namely Plants, Fruits General Conditions and (b) Special conditions and post-entry quarantine. and Seeds (Regulation of Import into India) Order popularly known as the PFS Order which was revised in 1989 after the announcement of the New Policy on Seed Development by the Government of India in 1988, (a) General Conditions for Imports proposing major modifications for smooth quarantine functioning. This Order has now been superseded by Import of seeds and plants for sowing or planting of fruit crops is permitted by the Export and Import (EXIM) the Plant Quarantine (Regulation for Import into India) Order 2003 which came into force as there was an Committee of the Department of Agriculture and Cooperation. Import permit (IP) needs to be procured urgent need to fill-up the gaps in existing PFS order regarding import of germplasm/ GMO's/ transgenic before importing any consignment. It is valid for a period of six months and extendable further for a period plant material/ bio-control agents etc., to fulfill India's legal obligations under the international of another six months. The Phytosanitary Certificate (PC) issued by the quarantine authority of the country Agreements, to protect the interest of the farmers of the country by preventing the entry, establishment of export should accompany the consignment. All consignments should be imported only through entry and spread of destructive pests, and to safeguard the national bio-diversity from threats of invasions by points notified by Central Government from time to time. The Plant Protection Advisor to the Govt. of India alien species. Under this Order, the need for incorporation of Additional/ Special declarations for freedom of or the official authorized by him may, after inspection, fumigation, disinfection/ disinfestation accord import commodities from quarantine and invasive alien species (IAS), on the basis of standardized pest risk quarantine clearance or order for destruction of consignment or return the same to the source country. In analysis (PRA), particularly for seed/ planting materials is also dealt with. Further, the scope of plant addition, PRA which has been made mandatory for all materials except for those listed under Schedule VII of quarantine activities has been widened with incorporation of additional definitions. The other salient the Plant Quarantine Order 2003 has to be conducted as per norms. features of the Order are: (b) Special Conditions of Imports • Prohibition on import of commodities with weed/ alien species contamination as per Schedule VIII; & restriction on import of packaging material of plant origin unless treated. Horticultural planting material is prohibited from certain parts of the world because of prevalence of pests • Provisions included for regulating the import of soil, peat & sphagnum moss; germplasm/ GMOs/ are listed at Schedule IV of the PQ Order (Annex I). Materials listed under Schedule V and VI needs to meet transgenic material for research; live insects/ microbial cultures & biocontrol agents and import of certain additional declarations and special conditions. timber & wooden logs. Germplasm • Agricultural imports have been classified as (a) prohibited plant species (Schedule IV); (b) restricted Research institutions of public and private sector interested in importing plants or planting material should species where import permitted only by authorized institutions (Schedule V); (c) restricted species request NBPGR for IP which is not transferable. The point of entry of all germplasm consignments is New permitted only with additional declarations of freedoms from quarantine/ regulated pests and subject Delhi airport only. On arrival, the quarantine scientists carefully process the material. In case material is to specified treatment certifications (Schedule VI) and; (d) plant material imported for consumption/ found to be infected/ infested with pests, all efforts are made to salvage the material. Only in rare cases, industrial processing permitted with normal Phytosanitary Certificate (Schedule VII). when the material cannot be salvaged it is incinerated. In case post-entry quarantine examination of the • Additional declarations being specified in the Order for import of 699 agricultural commodities with imported material is required, it is done at quarantine greenhouse facility, at NBPGR, New Delhi and its specific lists of more than > 1000 quarantine pests and 57 weed species. Regional Station, Bhowali for temperate fruits and other horticultural crops. • Notified points of entry increased to 182. Quarantine Procedures for Import of Horticultural Crops • Certification fee and inspection charges have been rationalized. Pre-entry Plant Quarantine Requirements So far, i.e., till July 2020, 84 amendments of the Plant Quarantine (PQ) Order 2003 have been notified to the WTO revising definitions, clarifications regarding specific queries raised by quarantine authorities of various (a) Import Permit is a statutory requirement from the country of import and the conditions/ additional countries, with revised lists of crops under the Schedules IV, V VI and VII. The revised list under Schedule VI declarations laid on it need to be fulfilled by the country of export. and VII now include 699 and 519 crops/ commodities, respectively. (b) Phytosanitary Certificate is also a statutory requirement and is a proof that the consignment has been National Plant Quarantine Set-up for import of Horticultural Material examined according to the requirements of the importing country and found to be free from the quarantine pests mentioned in the IP. The Directorate of Plant Protection, Quarantine and Storage (DPPQS) of Ministry of Agriculture and Farmers Welfare is the apex body for implementation of plant quarantine regulation and the PQ order forms the (c) PRA carried out by the country of import for which desired information is supplied by the country of basis of the functioning of the Directorate. It has a national network of 72 plant quarantine stations at export. different airports (24), seaports (24) and land frontiers (24). In all, three categories of materials are being (d) Approval of Post-entry Quarantine (PEQ) Growing Facility is essential as all the horticultural planting imported: (a) bulk consignments of grains/ pulses for consumption, (b) bulk consignments of seeds/ material is subjected to PEQ inspection. The material cannot be imported unless planting materials for sowing/ planting, and (c) samples of germplasm in small quantities for research

68 69 Table 1. Important interceptions in horticultural crops P o s t - e n t r y Q u a r a n t i n e Requirements Pests Host Origin/Source (a) Screening at the port of Insects/ Mites entry- the examination is Anthonomus grandis* Hibiscus sp. Italy, USA, Zambia carried out to make sure t h a t p h y t o s a n i t a r y A. pullus* Hibiscus sp. Nigeria, Zambia conditions laid down in the Aonidiella orientalis Vitis vinifera (cuttings) Brazil import permit have been taken care of and material Bruchus dentipes Vicia faba Syria, Iraq is free from exotic pests. Ephestia elutella Macadamia (nuts) USA (b) Examination of the material Parasessatia nigra Persea americana Vietnam after entry, PEQ growing and treatment if required. Polyphagotarsonemus latus Processing of Material for Pachymerus lacerdae Orbynya phalerata nuts Brazil Detection of Pests Rhizopertha dominica Allium sativum Bhutan, Egypt The Quarantine processing Beta vulgaris involves examination of material Fungi under exchange by naked eye or with the help of magnifying glass Alternaria amaranthi Amaranthus sp. Taiwan (visual inspection) for detecting A. dauci Daucus carota France, Poland, UK, USA the presence of insect damaged seeds, dead or actively moving A. helianthi Lycopersicon esculentum Taiwan larval and adult stages, flour, A. porri Allium sp. France webbing, presence of excreta, soil clods, plant debris, Botrytis allii Allium sp. France, Germany d i s c o l o u r e d , d e f o r m e d , Lycopersicon esculentum Taiwan malformed seeds, bunt balls/ Colletotrichum lagenarium Cucumis melo USA spores, ergot sclerotia, rust Drechslera maydis Capsicum spp. USA pustules/ spores, crust of downy mildew/ spores, weed seeds, Nematodes p r e s e n c e o f y e l l o w Aphelenchoides fragariae* Fragaria sp. (rooted plants) Australia, Holland discolouration around the hilum, nematode galls, etc. Ditylenchus destructor* Humulus lupulus (rooted plants) Australia, Germany, Samples are then processed Philippines, UK, USA through various specialized Solanum tuberosum (tubers) Peru techniques for each discipline Pratylenchus brachyurus* Fragaria sp. (plants) USA n a m e l y , E n t o m o l o g y , N e m a t o l o g y a n d P l a n t Helicotylenchus pseudorobustus Prunus spp. France, Netherlands pathology. On detection of a pest the course of action is decided depending upon the type/ category of pest Pratylenchus penetrans Rooted cuttings detected. The material is salvaged to the extent possible using physical, chemical, physico-chemical Xiphinema diversicaudatum* treatments including Hot Water Treatment (HWT). Germplasm of vegetable legumes is grown in Green *Pest not yet reported from India House/ Screen House for virus indexing. Material found chemically treated with pesticides is grown in Post- Entry Quarantine Nursery (PEQN) for expression of disease symptoms. Only harvest from disease free The pests intercepted can be categorized as material is released to the indenter. A flow chart of different steps involved in quarantine processing of exotic (i) those which are not known to occur in India; germplasm at NBPGR is given in Fig. 1. (ii) have different races/ biotypes/ strains not known to occur in India; Interception of Important Pests in Imported Horticultural PGR (iii) are present on a new host or are from a country from where they were never reported before; Several pests of quarantine significance have been intercepted from imported seeds and planting material (iv) an entirely new pest species hitherto unreported in science or of horticultural crops. Some of the important pests intercepted in horticultural crops are presented in Table 1. (v) are reported to be present in India but with a wide host range 70 71 Table 1. Important interceptions in horticultural crops P o s t - e n t r y Q u a r a n t i n e Requirements Pests Host Origin/Source (a) Screening at the port of Insects/ Mites entry- the examination is Anthonomus grandis* Hibiscus sp. Italy, USA, Zambia carried out to make sure t h a t p h y t o s a n i t a r y A. pullus* Hibiscus sp. Nigeria, Zambia conditions laid down in the Aonidiella orientalis Vitis vinifera (cuttings) Brazil import permit have been taken care of and material Bruchus dentipes Vicia faba Syria, Iraq is free from exotic pests. Ephestia elutella Macadamia (nuts) USA (b) Examination of the material Parasessatia nigra Persea americana Vietnam after entry, PEQ growing and treatment if required. Polyphagotarsonemus latus Processing of Material for Pachymerus lacerdae Orbynya phalerata nuts Brazil Detection of Pests Rhizopertha dominica Allium sativum Bhutan, Egypt The Quarantine processing Beta vulgaris involves examination of material Fungi under exchange by naked eye or with the help of magnifying glass Alternaria amaranthi Amaranthus sp. Taiwan (visual inspection) for detecting A. dauci Daucus carota France, Poland, UK, USA the presence of insect damaged seeds, dead or actively moving A. helianthi Lycopersicon esculentum Taiwan larval and adult stages, flour, A. porri Allium sp. France webbing, presence of excreta, soil clods, plant debris, Botrytis allii Allium sp. France, Germany d i s c o l o u r e d , d e f o r m e d , Lycopersicon esculentum Taiwan malformed seeds, bunt balls/ Colletotrichum lagenarium Cucumis melo USA spores, ergot sclerotia, rust Drechslera maydis Capsicum spp. USA pustules/ spores, crust of downy mildew/ spores, weed seeds, Nematodes p r e s e n c e o f y e l l o w Aphelenchoides fragariae* Fragaria sp. (rooted plants) Australia, Holland discolouration around the hilum, nematode galls, etc. Ditylenchus destructor* Humulus lupulus (rooted plants) Australia, Germany, Samples are then processed Philippines, UK, USA through various specialized Solanum tuberosum (tubers) Peru techniques for each discipline Pratylenchus brachyurus* Fragaria sp. (plants) USA n a m e l y , E n t o m o l o g y , N e m a t o l o g y a n d P l a n t Helicotylenchus pseudorobustus Prunus spp. France, Netherlands pathology. On detection of a pest the course of action is decided depending upon the type/ category of pest Pratylenchus penetrans Rooted cuttings detected. The material is salvaged to the extent possible using physical, chemical, physico-chemical Xiphinema diversicaudatum* treatments including Hot Water Treatment (HWT). Germplasm of vegetable legumes is grown in Green *Pest not yet reported from India House/ Screen House for virus indexing. Material found chemically treated with pesticides is grown in Post- Entry Quarantine Nursery (PEQN) for expression of disease symptoms. Only harvest from disease free The pests intercepted can be categorized as material is released to the indenter. A flow chart of different steps involved in quarantine processing of exotic (i) those which are not known to occur in India; germplasm at NBPGR is given in Fig. 1. (ii) have different races/ biotypes/ strains not known to occur in India; Interception of Important Pests in Imported Horticultural PGR (iii) are present on a new host or are from a country from where they were never reported before; Several pests of quarantine significance have been intercepted from imported seeds and planting material (iv) an entirely new pest species hitherto unreported in science or of horticultural crops. Some of the important pests intercepted in horticultural crops are presented in Table 1. (v) are reported to be present in India but with a wide host range 70 71 Interceptions, especially of pests and their variability not yet reported from India signify the importance of Under the PQ order, PRA has been made mandatory for all material being imported into the country other quarantine in preventing the introduction of destructive exotic pests. The third and fourth category of pests than those present in Schedules V, VI and VII. The various schedules V, VI and VII of the PQ Order give lists of are not expected in the sample as per the risk analysis which is literature-based and since no records are crops for which a generic PRA is given and detailed PRA is not required. In case of PGR, a large number of available on the pest/ host their presence is unexpected and important from quarantine view point. The last species of cultivated crops (and their wild relatives/ land races) with useful traits are imported. Such wild category - pests with a wide host range are critical and could become invasive in case they find suitable biotic relatives, land races of germplasm whose pest profile is not adequately recorded hampers PRA preparation and abiotic environment (Khetarpal and Gupta, 2008). Such interceptions signify the success of quarantine; and consequently their import. However, in 2007 the legislation has been amended whereby ICAR-NBPGR is otherwise, these pests would have entered the country and played havoc with the plant biodiversity and empowered to undertake PRA for germplasm material for pest free import. This is more relevant in the agriculture. present context when access to germplasm is becoming more and more difficult under the Convention on Biological Diversity, 1992. Salvaging of Infested/ Infected/ Contaminated Material Another issue, which is faced during import of certain material, is the additional declarations being sought The introduced germplasm is disinfested/ disinfected/ decontaminated using various methods. under the Schedule VI. Many countries from where a pest is not reported are unable to certify in the Mechanical cleaning: The seed lots are cleaned mechanically (hand picking) by removing the soil clods, phytosanitary certificate the freedom from those pests. The recent amendments of the PQ Order has plant debris, weeds, discoloured, deformed and shrivelled seeds. Infected portion of vegetative propagules resolved this problem to an extent by giving country specific requirements under additional declarations. is excised. This would greatly help the indentors in procuring germplasm of their interest from varied sources. Besides, NBPGR has also been empowered to relax (under delegation of powers Clause 14) certain conditions for X-ray radiography: The seeds of 340 plant genera known to carry hidden infestation are subjected to X-ray import in specific cases where the material being imported is of utmost importance for the country. radiography (Bhalla et al., 2018) or transparency test with lactophenol to detect hidden infestation in seeds due to seed beetles (bruchids)/ seed wasps (phytophagous chalcidoids) or weevils including detection of The technical issues include issues pertaining to quarantine processing are as follows: mango stone weevil, Sternochaetus mangiferae in the large seeds of mango. Infested seeds in the • Sample size based non-destructive procedures are required for detection developed X-ray films can be easily distinguished and are then handpicked from the seed geometry of sample kept undisturbed. • A repository of antisera needs to be established as diagnostic reagents such as antisera for viruses/ bacteria are often not available for exotic pests. Hot water treatment (HWT): It is used for eliminating fungi, bacteria and nematodes; material is treated in specialized hot water tanks with temperature control by using various combinations of temperature and • Expertise is also required in the field of taxonomy and biosystematics to identify unknown/ new pests or time. strains. Pesticidal treatment: Seed dressing with chemicals is generally given for eradicating common seed-borne • New reports of pests need to be authenticated by another organization prior to publication. fungi and bacteria; while vegetatively propagated material is dipped in pesticide solution for insects and • Highly sensitive and practically feasible molecular techniques are required for the detection of new nematodes for 10-15 minutes before release. pests/ races/ biotypes/ strains/ pathotypes etc. Fumigation: The most effective method used in quarantine for eliminating insects, mites and nematodes is • Need to consolidate the taxonomic information and prepare digitized keys for quick and reliable fumigation. Atmospheric fumigation is done at normal air pressure in an airtight container to eliminate identification of insect pests is reference collections for exotic insect pests and identification keys are external/ surface feeding insects and mites. Ethylene dichloride - carbon tetrachloride (EDCT) and not readily available. phosphine (aluminium phosphide) have been found to be effective for most of the common stored grain insect pests. The operation takes minimum 24 h. Other fumigants used are ethylene oxide and carbon • Non-destructive and eco-friendly salvaging techniques are required. dioxide mixture, hydrogen cyanide gas and methyl bromide. Some plant material viz., rooted plants, • Post-entry quarantine testing at NBPGR and release from indexed virus-free plants may take one crop cuttings, tubers and other vegetatively propagated material which are not tolerant to fumigation doses season. required for eliminating the infestation are given pesticidal dip or spray treatment. It is effective against surface feeding insects and nematodes. • Strengthening of post-entry quarantine facilities at public and private stakeholders. Salvaging through In vitro culture: It is used mainly for eliminating virus infection from vegetatively Websites like http://www.plantquarantineindia.nic.in, consisting of national database on legislation, propagated material. Viruses are eliminated through culturing abscised meristems in vitro. quarantine procedures, methodologies, plant quarantine alerts, etc. designed by Directorate of Plant Protection, Quarantine and Storage (DPPQS) are available. However, an internet-based portal mechanism Issues in International Exchange of PGR for exchange of official information to facilitate communication among countries needs to be developed. There are a number of issues related to quarantine in exchange of plant germplasm both legislative and Also, availability of databases on quarantine pests and endemic pests would simplify the work of the quarantine methodology. The national quarantine legislation has classified all imports as: quarantine personnel. Such list of potential quarantine and endemic pests of different crop groups is being compiled at ICAR-NBPGR which would act as a ready reckoner. (a) Prohibited plant species (Schedule IV); It is clear that under the present international scenario, the quarantine specialists have a major role to play (b) Restricted plant species where import is permitted only by authorized institutions (Schedule V); not only in promoting and facilitating the export and import in the interest of their respective nations but (c) Restricted plant species permitted only with additional declarations of freedom from quarantine/ also in protecting the environment from the onslaughts of invasive alien species. The importance of regulated pests and subject to specified treatment certifications (Schedule VI) and; quarantine has increased manifold in the WTO regime and adopting not only the appropriate technique but also the right strategy for pest detection and diagnosis would go a long way in ensuring pest-free exchange (d) Plant material imported for consumption/ industrial processing permitted with normal Phytosanitary of germplasm and transparency in international exchange, and is considered the best strategy for managing Certificate (Schedule VII). transboundary movement of pests.

72 73 Interceptions, especially of pests and their variability not yet reported from India signify the importance of Under the PQ order, PRA has been made mandatory for all material being imported into the country other quarantine in preventing the introduction of destructive exotic pests. The third and fourth category of pests than those present in Schedules V, VI and VII. The various schedules V, VI and VII of the PQ Order give lists of are not expected in the sample as per the risk analysis which is literature-based and since no records are crops for which a generic PRA is given and detailed PRA is not required. In case of PGR, a large number of available on the pest/ host their presence is unexpected and important from quarantine view point. The last species of cultivated crops (and their wild relatives/ land races) with useful traits are imported. Such wild category - pests with a wide host range are critical and could become invasive in case they find suitable biotic relatives, land races of germplasm whose pest profile is not adequately recorded hampers PRA preparation and abiotic environment (Khetarpal and Gupta, 2008). Such interceptions signify the success of quarantine; and consequently their import. However, in 2007 the legislation has been amended whereby ICAR-NBPGR is otherwise, these pests would have entered the country and played havoc with the plant biodiversity and empowered to undertake PRA for germplasm material for pest free import. This is more relevant in the agriculture. present context when access to germplasm is becoming more and more difficult under the Convention on Biological Diversity, 1992. Salvaging of Infested/ Infected/ Contaminated Material Another issue, which is faced during import of certain material, is the additional declarations being sought The introduced germplasm is disinfested/ disinfected/ decontaminated using various methods. under the Schedule VI. Many countries from where a pest is not reported are unable to certify in the Mechanical cleaning: The seed lots are cleaned mechanically (hand picking) by removing the soil clods, phytosanitary certificate the freedom from those pests. The recent amendments of the PQ Order has plant debris, weeds, discoloured, deformed and shrivelled seeds. Infected portion of vegetative propagules resolved this problem to an extent by giving country specific requirements under additional declarations. is excised. This would greatly help the indentors in procuring germplasm of their interest from varied sources. Besides, NBPGR has also been empowered to relax (under delegation of powers Clause 14) certain conditions for X-ray radiography: The seeds of 340 plant genera known to carry hidden infestation are subjected to X-ray import in specific cases where the material being imported is of utmost importance for the country. radiography (Bhalla et al., 2018) or transparency test with lactophenol to detect hidden infestation in seeds due to seed beetles (bruchids)/ seed wasps (phytophagous chalcidoids) or weevils including detection of The technical issues include issues pertaining to quarantine processing are as follows: mango stone weevil, Sternochaetus mangiferae in the large seeds of mango. Infested seeds in the • Sample size based non-destructive procedures are required for detection developed X-ray films can be easily distinguished and are then handpicked from the seed geometry of sample kept undisturbed. • A repository of antisera needs to be established as diagnostic reagents such as antisera for viruses/ bacteria are often not available for exotic pests. Hot water treatment (HWT): It is used for eliminating fungi, bacteria and nematodes; material is treated in specialized hot water tanks with temperature control by using various combinations of temperature and • Expertise is also required in the field of taxonomy and biosystematics to identify unknown/ new pests or time. strains. Pesticidal treatment: Seed dressing with chemicals is generally given for eradicating common seed-borne • New reports of pests need to be authenticated by another organization prior to publication. fungi and bacteria; while vegetatively propagated material is dipped in pesticide solution for insects and • Highly sensitive and practically feasible molecular techniques are required for the detection of new nematodes for 10-15 minutes before release. pests/ races/ biotypes/ strains/ pathotypes etc. Fumigation: The most effective method used in quarantine for eliminating insects, mites and nematodes is • Need to consolidate the taxonomic information and prepare digitized keys for quick and reliable fumigation. Atmospheric fumigation is done at normal air pressure in an airtight container to eliminate identification of insect pests is reference collections for exotic insect pests and identification keys are external/ surface feeding insects and mites. Ethylene dichloride - carbon tetrachloride (EDCT) and not readily available. phosphine (aluminium phosphide) have been found to be effective for most of the common stored grain insect pests. The operation takes minimum 24 h. Other fumigants used are ethylene oxide and carbon • Non-destructive and eco-friendly salvaging techniques are required. dioxide mixture, hydrogen cyanide gas and methyl bromide. Some plant material viz., rooted plants, • Post-entry quarantine testing at NBPGR and release from indexed virus-free plants may take one crop cuttings, tubers and other vegetatively propagated material which are not tolerant to fumigation doses season. required for eliminating the infestation are given pesticidal dip or spray treatment. It is effective against surface feeding insects and nematodes. • Strengthening of post-entry quarantine facilities at public and private stakeholders. Salvaging through In vitro culture: It is used mainly for eliminating virus infection from vegetatively Websites like http://www.plantquarantineindia.nic.in, consisting of national database on legislation, propagated material. Viruses are eliminated through culturing abscised meristems in vitro. quarantine procedures, methodologies, plant quarantine alerts, etc. designed by Directorate of Plant Protection, Quarantine and Storage (DPPQS) are available. However, an internet-based portal mechanism Issues in International Exchange of PGR for exchange of official information to facilitate communication among countries needs to be developed. There are a number of issues related to quarantine in exchange of plant germplasm both legislative and Also, availability of databases on quarantine pests and endemic pests would simplify the work of the quarantine methodology. The national quarantine legislation has classified all imports as: quarantine personnel. Such list of potential quarantine and endemic pests of different crop groups is being compiled at ICAR-NBPGR which would act as a ready reckoner. (a) Prohibited plant species (Schedule IV); It is clear that under the present international scenario, the quarantine specialists have a major role to play (b) Restricted plant species where import is permitted only by authorized institutions (Schedule V); not only in promoting and facilitating the export and import in the interest of their respective nations but (c) Restricted plant species permitted only with additional declarations of freedom from quarantine/ also in protecting the environment from the onslaughts of invasive alien species. The importance of regulated pests and subject to specified treatment certifications (Schedule VI) and; quarantine has increased manifold in the WTO regime and adopting not only the appropriate technique but also the right strategy for pest detection and diagnosis would go a long way in ensuring pest-free exchange (d) Plant material imported for consumption/ industrial processing permitted with normal Phytosanitary of germplasm and transparency in international exchange, and is considered the best strategy for managing Certificate (Schedule VII). transboundary movement of pests.

72 73 Acknowledgement: Annex I The authors duly acknowledge the contribution of all past and present staff of Division of Plant Quarantine List of horticultural plants/ planting materials and countries from where import for their dedicated support. is prohibited along with justifications (Schedule-IV; P.Q. Order 2003) References: S. Plant species/ Categories of Prohibited from Justification for Prohibition 1. Bhalla S, VC Chalam, B Singh, K Gupta and SC Dubey (2018) Biosecuring Plant Genetic Resources in India: No. variety plant material the countries Role of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 216 p +vi (ISBN 978-81-937111-1-8) 1. Banana, Plantain and Rhizomes/ Central & South Due to incidence of destructive pests Abaca (Musa spp.) Suckers America, Hawaii, such as Moko wilt (Burkholderia 2. Dubey SC and K Gupta (2016) Plant Quarantine system for PGR in India. Indian J. Plant Genet. Resour. 29: Philippines and solanacearum) race 2 and Cameroon 410-413. Cameroon marbling (phytoplasmas) 3. Gupta K, M Lata Kapur and RK Khetarpal (2007) Safe Transboundary Movement of Transgenic Planting 2. Cassava or tapioca Seeds/ Stem Africa & South Due to incidence of destructive pests Material. In: Randhawa GJ, S Bhalla, V Celia Chalam and SK Sharma (eds.) Cartagena Protocol on (Manihot esculenta) cuttings America such as: Super elongation (Sphaceloma Biosafety Decisions to Diagnostics. National Bureau of Plant Genetic Resources, New Delhi, India pp 57- manihoticola), Cassava bacterial blight (Xanthomonas campestris pv. 73. manihotis) - American strains, Cassava 4. Kahn RP (1989) Plant protection and quarantine: selected pest and pathogens of quarantine witches' broom (phytoplasma) and significance Vol II. CRC Press Inc. Florida, 265 p. several cassava viruses 5. Khetarpal RK and K Gupta (2008) Plant quarantine in India in the wake of international agreements: A 3. Cocoa (Theobroma Fresh beans/ West Africa, Due to incidence of destructive pests cacao) and plants Pods/Bud wood/ Tropical America such as: Swollen shoot virus and review. In: Reddy SM and HN Gour (eds) Review of Plant Pathology Vol 4 Scientific Publishers (India), species belong to Grafts/ Root and Sri Lanka. related virus strains of cocoa, Witches' Jodhpur, pp: 367-391. Sterculiaceae, stocks/ Saplings broom (Crinipellis ( Marasmius) 6. Khetarpal RK, A Lal, KS Varaprasad, PC Agarwal, S Bhalla, VC Chalam and K Gupta (2006) Quarantine for Bombacaceae and perniciosa Watery pod rot ( Monilia Safe Exchange of Plant Genetic Resources. In: AK Singh, Sanjeev Saxena, Kalyani Srinivasan and BS Tiliaceae. (Moniliopthora) roreri) Mealy pod Dhillon (eds) 100 years of PGR Management in India National Bureau of Plant Genetic Resources, New (Trachysphaera fructigena) Mirids (Sahlbergia singularis & Delhi, India, pp. 83-108. Distantiella theobroma), Cocoa moth 7. Plant Quarantine (Regulation of Import into India) Order. (2003). The Gazette of India Extraordinary, (Acorocercops cramerella), Cocoa Part II Section 3 Sub-section (ii) No. 1037 Published by Authority. p 105. capsid (Sahlbergiella theobroma), Cocoa beetle (Steirastoma brevi), 8. http://plantquarantineindia.nic.in/pqispub/docfiles/dip_act.htm Seedling damping-off (Phytophthora cactorum), Chestnut downy mildew 9. http://www.plantquarantineindia.nic.in (Phytophthora katsurae) and Black pod 10. https://www.ippc.int/en/ of cocoa (Phytophthora megakarya) 4. Cocoyam or Dasheen Plants/Corms/ Cook Islands, Due to incidence of destructive pests or Taro (Arvi) Cormlets/ Papua New such as Alomae land Bobone (Rhabdo (Colocasia esculenta) Suckers Guinea, Solomon viruses), Dasheen mosaic virus (South and other edible aroids Islands and South Pacific strains) and Bacterial blight ` Pacific countries (Xanthomonas campestric pv. dieffenbachiae) 5. Potato (Solanum Tubers and South America Due to incidence of destructive pests tuberosum) and other other planting such as Potato smut [Thecaphora tuber bearing species material (Angiosorus) solani], Potato purple-top of Solanaceae wilt & stolbur (phytoplasmas), Potato viruses viz. Andean potato latent, Andean potato mottle, Arracacha B virus, Potato deforming mosaic, Potato T (capillo virus), Potato yellow dwarf, Potato yellow vein, Potato calico strain of Tobacco ring spot virus, Potato strain of Tobacco streak virus and Andean potato weevil (Premnotrypes spp.)

74 75 Acknowledgement: Annex I The authors duly acknowledge the contribution of all past and present staff of Division of Plant Quarantine List of horticultural plants/ planting materials and countries from where import for their dedicated support. is prohibited along with justifications (Schedule-IV; P.Q. Order 2003) References: S. Plant species/ Categories of Prohibited from Justification for Prohibition 1. Bhalla S, VC Chalam, B Singh, K Gupta and SC Dubey (2018) Biosecuring Plant Genetic Resources in India: No. variety plant material the countries Role of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 216 p +vi (ISBN 978-81-937111-1-8) 1. Banana, Plantain and Rhizomes/ Central & South Due to incidence of destructive pests Abaca (Musa spp.) Suckers America, Hawaii, such as Moko wilt (Burkholderia 2. Dubey SC and K Gupta (2016) Plant Quarantine system for PGR in India. Indian J. Plant Genet. Resour. 29: Philippines and solanacearum) race 2 and Cameroon 410-413. Cameroon marbling (phytoplasmas) 3. Gupta K, M Lata Kapur and RK Khetarpal (2007) Safe Transboundary Movement of Transgenic Planting 2. Cassava or tapioca Seeds/ Stem Africa & South Due to incidence of destructive pests Material. In: Randhawa GJ, S Bhalla, V Celia Chalam and SK Sharma (eds.) Cartagena Protocol on (Manihot esculenta) cuttings America such as: Super elongation (Sphaceloma Biosafety Decisions to Diagnostics. National Bureau of Plant Genetic Resources, New Delhi, India pp 57- manihoticola), Cassava bacterial blight (Xanthomonas campestris pv. 73. manihotis) - American strains, Cassava 4. Kahn RP (1989) Plant protection and quarantine: selected pest and pathogens of quarantine witches' broom (phytoplasma) and significance Vol II. CRC Press Inc. Florida, 265 p. several cassava viruses 5. Khetarpal RK and K Gupta (2008) Plant quarantine in India in the wake of international agreements: A 3. Cocoa (Theobroma Fresh beans/ West Africa, Due to incidence of destructive pests cacao) and plants Pods/Bud wood/ Tropical America such as: Swollen shoot virus and review. In: Reddy SM and HN Gour (eds) Review of Plant Pathology Vol 4 Scientific Publishers (India), species belong to Grafts/ Root and Sri Lanka. related virus strains of cocoa, Witches' Jodhpur, pp: 367-391. Sterculiaceae, stocks/ Saplings broom (Crinipellis ( Marasmius) 6. Khetarpal RK, A Lal, KS Varaprasad, PC Agarwal, S Bhalla, VC Chalam and K Gupta (2006) Quarantine for Bombacaceae and perniciosa Watery pod rot ( Monilia Safe Exchange of Plant Genetic Resources. In: AK Singh, Sanjeev Saxena, Kalyani Srinivasan and BS Tiliaceae. (Moniliopthora) roreri) Mealy pod Dhillon (eds) 100 years of PGR Management in India National Bureau of Plant Genetic Resources, New (Trachysphaera fructigena) Mirids (Sahlbergia singularis & Delhi, India, pp. 83-108. Distantiella theobroma), Cocoa moth 7. Plant Quarantine (Regulation of Import into India) Order. (2003). The Gazette of India Extraordinary, (Acorocercops cramerella), Cocoa Part II Section 3 Sub-section (ii) No. 1037 Published by Authority. p 105. capsid (Sahlbergiella theobroma), Cocoa beetle (Steirastoma brevi), 8. http://plantquarantineindia.nic.in/pqispub/docfiles/dip_act.htm Seedling damping-off (Phytophthora cactorum), Chestnut downy mildew 9. http://www.plantquarantineindia.nic.in (Phytophthora katsurae) and Black pod 10. https://www.ippc.int/en/ of cocoa (Phytophthora megakarya) 4. Cocoyam or Dasheen Plants/Corms/ Cook Islands, Due to incidence of destructive pests or Taro (Arvi) Cormlets/ Papua New such as Alomae land Bobone (Rhabdo (Colocasia esculenta) Suckers Guinea, Solomon viruses), Dasheen mosaic virus (South and other edible aroids Islands and South Pacific strains) and Bacterial blight ` Pacific countries (Xanthomonas campestric pv. dieffenbachiae) 5. Potato (Solanum Tubers and South America Due to incidence of destructive pests tuberosum) and other other planting such as Potato smut [Thecaphora tuber bearing species material (Angiosorus) solani], Potato purple-top of Solanaceae wilt & stolbur (phytoplasmas), Potato viruses viz. Andean potato latent, Andean potato mottle, Arracacha B virus, Potato deforming mosaic, Potato T (capillo virus), Potato yellow dwarf, Potato yellow vein, Potato calico strain of Tobacco ring spot virus, Potato strain of Tobacco streak virus and Andean potato weevil (Premnotrypes spp.)

74 75 6. Sweet potato Stem (Vine) South Africa, Due to incidence of destructive (Ipomoea spp.) cuttings rooted East Africa, New pests such as: Scab (Elsinoe PGR Informatics and its utility in or un-rooted/ Zealand, Nigeria, batatas), Scurf (Moniliochaetes tubers USA, Argentina infuscans), Foot rot (Plenodomus and Israel. destruens), Soil rot (Streptomyces conservation and utilization of genetic ipomoeae), Bacteria wilt (Pseudomonas batatae), Sweet potato viruses viz. Russet crack; resources of Fruits and Vegetables feathery mottle; internal cork; Sunil Archak* and Rajeev Gambhir chlorotic leaf spot; vein mosaic; Agriculture Knowledge Management Unit mild mottle and yellow dwarf, vein ICAR- National Bureau of Plant Genetic Resources, New Delhi clearing; chlorotic stunt; Sheffied's *Email - [email protected] virus A and B etc., Sweet potato witches' broom (phytoplasmas) and seed bruchid (Mimosestes mimosae) United Nations' International Year of Fruits and Vegetables 2021 is an apt year, if not too late, to look at the status of PGR documentation in these crops and initiate action in this regard. In this section of the training, 7. Yam (Dioscorea Tubers for West Africa and Due to incidence of destructive we aim to learn about PGR Informatics and its utility in conservation and utilization of genetic resources of spp.) planting or Caribbean region Yam mosaic virus/ green banding Fruits and Vegetables. propagation virus What is PGR Informatics? PGR Informatics is the management (creation, storage, retrieval and presentation) and analyses (discovery, exploration and extraction) of diverse information (facts, figures, statistics, knowledge and news). PGR Informatics has assumed significance because of the following factors: (i) Increased awareness about PGRFA (ii) Various international agreements (CBD, GPA, ITPGRFA) coming into force (iii) Availability of information in text, images, maps, videos, etc. (iv) Technologies to record, link and archive such diverse types of information (v) Growing power (and falling costs) of computers and internet to facilitate access and retrieval Fundamental merit of an organized digital information system is that it provides fair and just opportunity for all to access. On-line portals, as a consequence of PGR Informatics, enable non-exclusive access to PGR information to a large number of users involved in overlapping research areas on PGR management. Typically information is collected on details of multitude of Passport data including taxonomy, biogeography, and ethnobotany of the germplasm acquisitions (domestic collections and exotic introductions), their Seed Health, multiplication for Supply and Conservation, Regeneration, experimental data on Characterization and Evaluation leading to Utilization. In addition to field data, it also includes biochemical and genomic data as well as publications. Once the information is digitized and stored, computer technologies allow management and analysis irrespective of the scale and types of data leading better visualization and predictions. Relevance of PGR informatics Global assessment indicates that many of the world's PGR are insufficiently and poorly documented. The passport information and characterization and evaluation data on genebank accessions conserved in genebanks are either lacking or poorly recorded or scattered at different places, such as passport data sheets, reports of collection and exploration missions, crop catalogues, published articles, etc. In addition, there exist informal or non-coded knowledge held by traditional farmers and indigenous people. To use this information efficiently and effectively, the valuable information need to be collected, collated, maintained and exchanged with the help of PGR Informatics. Global initiatives on PGR informatics include database systems and online portals associated with genebanks such as Germplasm Resources Information Network (GRIN); European Search Catalogue for Plant Genetic Resources (EURISCO); Portal od the Japanese Genebank of National Agriculture and Food 76 77 6. Sweet potato Stem (Vine) South Africa, Due to incidence of destructive (Ipomoea spp.) cuttings rooted East Africa, New pests such as: Scab (Elsinoe PGR Informatics and its utility in or un-rooted/ Zealand, Nigeria, batatas), Scurf (Moniliochaetes tubers USA, Argentina infuscans), Foot rot (Plenodomus and Israel. destruens), Soil rot (Streptomyces conservation and utilization of genetic ipomoeae), Bacteria wilt (Pseudomonas batatae), Sweet potato viruses viz. Russet crack; resources of Fruits and Vegetables feathery mottle; internal cork; Sunil Archak* and Rajeev Gambhir chlorotic leaf spot; vein mosaic; Agriculture Knowledge Management Unit mild mottle and yellow dwarf, vein ICAR- National Bureau of Plant Genetic Resources, New Delhi clearing; chlorotic stunt; Sheffied's *Email - [email protected] virus A and B etc., Sweet potato witches' broom (phytoplasmas) and seed bruchid (Mimosestes mimosae) United Nations' International Year of Fruits and Vegetables 2021 is an apt year, if not too late, to look at the status of PGR documentation in these crops and initiate action in this regard. In this section of the training, 7. Yam (Dioscorea Tubers for West Africa and Due to incidence of destructive we aim to learn about PGR Informatics and its utility in conservation and utilization of genetic resources of spp.) planting or Caribbean region Yam mosaic virus/ green banding Fruits and Vegetables. propagation virus What is PGR Informatics? PGR Informatics is the management (creation, storage, retrieval and presentation) and analyses (discovery, exploration and extraction) of diverse information (facts, figures, statistics, knowledge and news). PGR Informatics has assumed significance because of the following factors: (i) Increased awareness about PGRFA (ii) Various international agreements (CBD, GPA, ITPGRFA) coming into force (iii) Availability of information in text, images, maps, videos, etc. (iv) Technologies to record, link and archive such diverse types of information (v) Growing power (and falling costs) of computers and internet to facilitate access and retrieval Fundamental merit of an organized digital information system is that it provides fair and just opportunity for all to access. On-line portals, as a consequence of PGR Informatics, enable non-exclusive access to PGR information to a large number of users involved in overlapping research areas on PGR management. Typically information is collected on details of multitude of Passport data including taxonomy, biogeography, and ethnobotany of the germplasm acquisitions (domestic collections and exotic introductions), their Seed Health, multiplication for Supply and Conservation, Regeneration, experimental data on Characterization and Evaluation leading to Utilization. In addition to field data, it also includes biochemical and genomic data as well as publications. Once the information is digitized and stored, computer technologies allow management and analysis irrespective of the scale and types of data leading better visualization and predictions. Relevance of PGR informatics Global assessment indicates that many of the world's PGR are insufficiently and poorly documented. The passport information and characterization and evaluation data on genebank accessions conserved in genebanks are either lacking or poorly recorded or scattered at different places, such as passport data sheets, reports of collection and exploration missions, crop catalogues, published articles, etc. In addition, there exist informal or non-coded knowledge held by traditional farmers and indigenous people. To use this information efficiently and effectively, the valuable information need to be collected, collated, maintained and exchanged with the help of PGR Informatics. Global initiatives on PGR informatics include database systems and online portals associated with genebanks such as Germplasm Resources Information Network (GRIN); European Search Catalogue for Plant Genetic Resources (EURISCO); Portal od the Japanese Genebank of National Agriculture and Food 76 77 Research Organization (NARO); GENESYS, a global portal to information about PGR, from which information PGR Map (http://pgrinformatics.nbpgr.ernet.in/pgrmap/) on germplasm accessions from genebanks around the world can be found; and PGR Portal, the gateway to information on PGR conserved in the Indian National Genebank housed at ICAR-NBPGR, New Delhi, with Map-based data retrieval provides easy and intuitive access to PGR information. The process is vital for information on developing mobile apps. Computational algorithms for map based applications in PGR were developed and were implemented as an interactive application called “PGR Map”. Table 1: PGR Informatics databases, portals and websites

Information resource Web address

Japanese genebank portal www.gene.affrc.go.jp/databases_en.php European genebanks portal eurisco.ipk-gatersleben.de Genesys portal www.genesys-pgr.org Indian genebank portal pgrportal.nbpgr.ernet.in Barcode of Life www.barcodeoflife.org Convention on Biological Diversity www.cbd.int Encyclopedia of Life www.eol.org Global Biodiversity Information Forum www.gbif.org Indian Bio-resources Information Network www.ibin.gov.in International Legume Database www.ildis.org International variety protection database www.upov.int National Plant Germplasm System of USDA www.ars-grin.gov/npgs PGR Map offers three benefits Species 2000 www.sp2000.org World Information and Early Warning System www.fao.org/wiews/en o “What's around me” helps user to obtain quickly the accessions that have been collected and conserved in the genebank from a particular location in India where the user is located at the moment Details of the PGR Informatics applications including list of web-applications and mobile apps, are available at: http://pgrinformatics.nbpgr.ernet.in o “Search the map” helps user to list the accessions that have been collected and conserved in the genebank from any selected location in India All the PGR Informatics products developed in ICAR- o “Search for species” helps user to map the collection sites of a crop species National Fellow (NF) project are Geo-informatics portal in PGR (http://pgrinformatics.nbpgr.ernet.in/pgrclim) listed here. These were d e v e l o p e d b a s e d o n A GIS-server based interactive application was implemented comprising of layers of germplasm accessions novel/improved algorithms. of ten major crops; soil type; AEZ; temperature and rainfall (current, 2020 and 2030). The data for the PGR Many of them are first of their Clim was generated in a CCAFS funded project to link germplasm to changing climatic regimes. kind in the world and are accessed world over. Use of these web-apps and mobile- apps by researchers in many countries demonstrate the s i g n i f i c a n c e o f t h e s e applications and the immense impact of the ICAR-NF project. Usage statistics of PGR Informatics Portal (left) and locations from where a ccessed (map above).

Source: Google Analytics (1 Oct 2014 to 07 Aug 2019).

Note the impetus provided by the ICAR National Fellow Project adding value to PGR Portal with multiple web -apps and mobile - apps.

78 79 Research Organization (NARO); GENESYS, a global portal to information about PGR, from which information PGR Map (http://pgrinformatics.nbpgr.ernet.in/pgrmap/) on germplasm accessions from genebanks around the world can be found; and PGR Portal, the gateway to information on PGR conserved in the Indian National Genebank housed at ICAR-NBPGR, New Delhi, with Map-based data retrieval provides easy and intuitive access to PGR information. The process is vital for information on developing mobile apps. Computational algorithms for map based applications in PGR were developed and were implemented as an interactive application called “PGR Map”. Table 1: PGR Informatics databases, portals and websites

Information resource Web address

Source: Google Analytics (1 Oct 2014 to 07 Aug 2019).

Note the impetus provided by the ICAR National Fellow Project adding value to PGR Portal with multiple web -apps and mobile - apps.

78 79 Genebank applications In vitro genebank (pgrinformatics.nbpgr.ernet.in/invitro) A quick access to the information on number of germplasm accessions conserved in the National Genebank has been a long pending demand. User friendly dashboards were designed, developed and implemented to provide a personalized quick access to genebank information to PGR workers, breeders, students, research managers and administrators. The dashboard figures are dynamically updated as and when genebank databases are updated. Seed Genebank (genebank.nbpgr.ernet.in)

Germplasm Import Version 2.0 (exchange.nbpgr.ernet.in)

Cryo genebank (pgrinformatics.nbpgr.ernet.in/cryobank)

Germplasm Registration Information System (www.nbpgr.ernet.in/registration) The Germplasm Registration Information System has been developed to make the entire process of germplasm registration—submission of application, evaluation by experts and decision by Plant Germplasm Registration Committee—online, easy and fast. The system is expected to provide genebank managers, breeders and plant researchers with a hands-on tool for management of germplasm registration process, and to policy makers with a reliable source of information. With the advent of this system, it is expected that the entire process of germplasm registration is made simple and transparent.

80 81 Genebank applications In vitro genebank (pgrinformatics.nbpgr.ernet.in/invitro) A quick access to the information on number of germplasm accessions conserved in the National Genebank has been a long pending demand. User friendly dashboards were designed, developed and implemented to provide a personalized quick access to genebank information to PGR workers, breeders, students, research managers and administrators. The dashboard figures are dynamically updated as and when genebank databases are updated. Seed Genebank (genebank.nbpgr.ernet.in)

Germplasm Import Version 2.0 (exchange.nbpgr.ernet.in)

Cryo genebank (pgrinformatics.nbpgr.ernet.in/cryobank)

80 81 Gap Analysis Version 2.0 (Meant for internal use of NBPGR scientists)

Genebank to Genbank G2G (http://pgrinformatics.nbpgr.ernet.in/g2g) G2G links information on genomic resources of crop plants available in the GenBank with associated plant genetic resources belonging to 263 countries conserved in 194 genebanks located in 68 countries around the world. G2G connects GenBank depositions—of 1,407,145 nucleotide, 6,72,796 protein and 1,606,302 EST sequences of 2889 cultivars/ genotypes/ landraces belonging to 50 crop species—to genebanks from Virtual Herbarium of the National Herbarium of Crop Plants where the seeds/propagules can be accessed legitimately as per extant international regulatory framework. The motto of G2G is to present access seekers as well as access providers a common platform to enhance (http://pgrinformatics.nbpgr.ernet.in/nhcp) utilization of germplasm and associated knowledge. National Herbarium of Crop Plants (NHCP), established in 1985 at NBPGR, has 23,665 specimens representing 267 families, 1,521 genera and 4,271 species. In order to make students and researchers across the world to access the invaluable information by the click of a button, ICAR-NF collaborated with NHCP to develop an online application creating a virtual herbarium with over 3,500 species of crop genepools complete with taxonomic information and about 7,000 images.

82 83 Gap Analysis Version 2.0 (Meant for internal use of NBPGR scientists)

82 83 Mobile Apps in PGR Informatics Genebank and PGR Map are developed as mobile apps for both Android and iOS users. Both the apps are first of their kind among all the genebanks.

Acknowledgments The authors acknowledge the contributions made by colleagues at NBPGR particularly Mr. Ratnesh Kumar Tiwari and Mr. Firoz Ahmad. Financial support by ICAR-National Fellowship is also deeply acknowledged.

84 Principles of Germplasm Characterization and Evaluation Ashok Kumar and Kuldeep Tripathi Division of Germplasm Evaluation ICAR-National Bureau of Plant Genetic Resources, New Delhi *Email - [email protected]

Introduction Characterization, evaluation and regeneration of germplasm are an integral component of Plant Genetic Resources management. Characterization and evaluation of germplasm is the key to accelerate utilization in crop improvement programme by exposing the actual value of germplasm. The characterization of germplasm deals with the understanding and recording of highly heritable characters which may be used in establishing taxonomic identity, while, the germplasm evaluation deals with assessing the agronomic potential of an accession including quality parameters and response to various abiotic and biotic stresses. Maintenance of germplasm without losing genetic integrity is also a prime objective in PGR management. For germplasm to be of use to breeders it must be characterised and evaluated. There is often a delay between collection of germplasm and its evaluation, particularly of fruit trees because of the time required for them to reach maturity. Evaluation is most useful if it considers the traits wanted by plant breeders. Preliminary evaluation can indicate those accessions that warrant more detailed evaluation, but those that appear not to be of immediate value should not be simply discarded. Germplasm Characterization The characterization of germplasm deals with the recording of easily observable traits which are generally expressed in all the environments, therefore, it can be performed in a single environment. The characterization should be carried out during the initial stage of acquisition of germplasm and may be completed in a suitable environment preferably at a site nearest to the site of collection of the germplasm or under similar agro-climatic conditions. The internationally/nationally accepted descriptors and descriptor states' should be used to record observation such as those developed by UPOV, USDA, Bioversity International (formerly known as IPGRI/IBPGR) or NBPGR Minimal Descriptors for Characterization and Evaluation of Field Crops (Mahajan et al., 2000). Wherever, descriptors and descriptors' states are not available these should be developed by germplasm curators in consultation with crop advisory committee and crop experts for a given crop species (Bioversity International, 2007). Biological status of the germplasm should also be known in advance to determine the characterization strategy. With the advancement of techniques, the molecular and biochemical markers are being used. The highly reproducible molecular markers like Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs) should be used for characterization. The field experiment should be conducted with statistically sound experimental design depending upon the quantity and number of germplasm accessions under trial. Wherever, the number of accessions is more, the characterization data is generally utilized to develop core set comprising 10 per cent of the entire collection representing the total variability in the germplasm to bring them to a manageable level. Further, if the number of accessions is large in core set which becomes difficult to handle, a mini core set comprising 10 per cent of the core set representing the total variability in the core set is developed for facilitating germplasm utilization in crop improvement programs. Germplasm Evaluation Evaluation of genetic resources is necessary to identify the appropriate germplasm with a target trait for their further utilization. Genetic resources are invaluable sources of variation for improving agricultural productivity. However, the conservation of a resource only becomes important if it acquires recognized value which can be assigned only through detailed evaluation of the germplasm for the critical genetic material. Evaluation should be undertaken in germplasm accessions which are already characterized and 85 where enough quantity of planting material is available. They should confirm to standardized and calibrated ii) Management descriptors: These provide the basis for management of accessions in a genebank and measuring formats. Evaluation of germplasm is a multi-disciplinary approach and it should be done in assist for multiplication and regeneration of the accessions. collaborative mode involving germplasm curator, plant breeder, physiologist, pathologist, entomologist, iii) Environment and site descriptors: These describe the environmental and site-specific parameters that biochemist and other experts. A network/coordinated approach at multiple locations under different agro- are important when characterization and evaluation trials are held. They can be important for the climatic zones in collaboration with crop-based institutes, Project Directors/coordinators and AICRP centres interpretation of the results of those trials. Site descriptors for germplasm collecting are also included is recommended. The accessions should be evaluated in the area of their adaptation or under similar here. environmental condition considering the breeding behavior and biological status of germplasm. Standard agronomic practices prescribed for raising a good crop alongwith plant protection measures need to be iv) Characterization descriptors: Phenotypic differentiation becomes easier and quicker with these followed. Evaluation experiments should be conducted with proper experimental design, depending upon descriptors. the number of accessions to be evaluated. The blocks should be laid out across the soil fertility gradient of v) Evaluation descriptors: These are environment sensitive so that assessment of these descriptors needs the experimental plot. In general, ABD is being practised in large number of accessions. Three or more special experimental designs and techniques. Their assessment may also require complex biochemical checks in which one national as well as one locally adapted check used for comparative assessment of or molecular characterization methods. germplasm. For few or promising accessions and less soil heterogeneity, evaluation should be done in RBD wherein the checks should be randomized along with the accessions in each replication. The experimental Descriptor's states plot should have at least three rows of 3-5 m length for each accession with recommended gap in between A descriptor state represents relative state of occurrence of a descriptor. Descriptor states are usually pre- the accessions The number and row length should be more for cross pollinated species than those for self- defined as a set of alternative discrete classes of expression one of which can describe a germplasm pollinated ones. The observations should be recorded on the plants from the middle row to avoid the border accession more closely. In order to avoid ambiguity among various descriptor states, the list of minimal effects. After evaluation, the promising accessions should be further validated through multi-locational, descriptors are accompanied by the standards of qualitative state, measurement technique, time and unit multi-season and multi-year evaluations. Majority of agronomic descriptors are polygenic in nature and of measurement for quantitative descriptors, like descriptors, the descriptors states are also developed by highly influenced by the environment. Therefore, evaluation trails should be carried out in at least three the scientists, curators and experts who are working on the particular species. They have to decide on diverse environments to minimize Genotype x Environment (G x E) interaction. which descriptor and descriptor's states to include for a given plant species. Now, a number of measures Germplasm Maintenance have been taken to improve the characterization and evaluation of germplasm collection involving breeders and also adopting new techniques including molecular markers. Germplasm maintenance poses the greatest risks to the genetic integrity of germplasm, due to selection, out-crossing or mechanical mixing. The risk of losing genetic integrity is especially high when raising Guidelines for recording descriptors genetically heterogeneous germplasm accessions of out-crossing crops. The purpose of maintenance is to Following are the guidelines for scoring and recording of descriptors for characterization and preliminary keep the original genetic diversity and structure of the accession or collection. Maintenance of genetic evaluation. These may be strictly followed. integrity involves maintaining the joint frequency distribution of all alleles at all loci. In fact, one should try to preserve the genotypic structure of the populations as well as the allelic frequencies. The appropriate a) Observations are to be recorded in the centimetre–gram–second system (CGS). The units are population size of an accession is an important factor for their maintenance. It will depend upon the mentioned in parenthesis after descriptor name. breeding behavior of the crop as well as the biological status of the material. The population size should be b) Presence/absence of characters are scored as 1 (present) and 0 (absent); large enough in cross and often cross pollinated crops to avoid genetic drift. Cross-pollinating species usually require more plants to maintain the genetic variation that exists within the population than self- c) Many quantitative descriptors which are continuously variable are recorded on a 0-9 scale, where: 0- pollinating species. In order to minimize the loss of heterozygosity, inbreeding depression and change in Immune are the expression of a character. For some characters, only a selected scores i.e. 1,3,5,7,9 are allelic frequency, a minimum number of 50-100 plants are required in a population. In case of wild species, described. Where this has occurred, it shows the gradient (from very low to very high) for that particular where few seeds are available and difficult to germinate and grow, special treatments wherever required character. should be given for their proper germination and less number of plants may be considered. i. Very low Descriptors and Descriptor States ii. Very low to low Depending upon the individual circumstances, the list of these descriptors may be developed by the iii. Low organization working on PGR, crop curators/plant breeders with his experience and with the help of the iv. Low to intermediate crop advisory committee and several experts of different related fields like chemistry, pathology, entomology etc. Bioversity International, Rome, in collaboration with international partners, has developed v. Intermediate various crop descriptors which are being used globally and ICAR-NBPGR has also developed minimal vi. Intermediate or high descriptors at national level. The use of uniform descriptors and descriptor states facilitate the utilization of vii. High germplasm by different researchers. Various kinds of descriptors have been identified as essential parameters to facilitate the management and use of germplasm available in different forms. viii. High to very high Types of Descriptors ix. Very high I) Passport descriptors: These provide the basic information recorded at the time of original collection. It d) For recording quantitative characters, the average of 5-10 plants is considered. Further, in case of cross- is used for the identification of germplasm and required for registration at the genebank. pollinated /often cross-pollinated species, the number of plants selected may be more (10-25 plants) depending upon the species and available resources.

86 87 where enough quantity of planting material is available. They should confirm to standardized and calibrated ii) Management descriptors: These provide the basis for management of accessions in a genebank and measuring formats. Evaluation of germplasm is a multi-disciplinary approach and it should be done in assist for multiplication and regeneration of the accessions. collaborative mode involving germplasm curator, plant breeder, physiologist, pathologist, entomologist, iii) Environment and site descriptors: These describe the environmental and site-specific parameters that biochemist and other experts. A network/coordinated approach at multiple locations under different agro- are important when characterization and evaluation trials are held. They can be important for the climatic zones in collaboration with crop-based institutes, Project Directors/coordinators and AICRP centres interpretation of the results of those trials. Site descriptors for germplasm collecting are also included is recommended. The accessions should be evaluated in the area of their adaptation or under similar here. environmental condition considering the breeding behavior and biological status of germplasm. Standard agronomic practices prescribed for raising a good crop alongwith plant protection measures need to be iv) Characterization descriptors: Phenotypic differentiation becomes easier and quicker with these followed. Evaluation experiments should be conducted with proper experimental design, depending upon descriptors. the number of accessions to be evaluated. The blocks should be laid out across the soil fertility gradient of v) Evaluation descriptors: These are environment sensitive so that assessment of these descriptors needs the experimental plot. In general, ABD is being practised in large number of accessions. Three or more special experimental designs and techniques. Their assessment may also require complex biochemical checks in which one national as well as one locally adapted check used for comparative assessment of or molecular characterization methods. germplasm. For few or promising accessions and less soil heterogeneity, evaluation should be done in RBD wherein the checks should be randomized along with the accessions in each replication. The experimental Descriptor's states plot should have at least three rows of 3-5 m length for each accession with recommended gap in between A descriptor state represents relative state of occurrence of a descriptor. Descriptor states are usually pre- the accessions The number and row length should be more for cross pollinated species than those for self- defined as a set of alternative discrete classes of expression one of which can describe a germplasm pollinated ones. The observations should be recorded on the plants from the middle row to avoid the border accession more closely. In order to avoid ambiguity among various descriptor states, the list of minimal effects. After evaluation, the promising accessions should be further validated through multi-locational, descriptors are accompanied by the standards of qualitative state, measurement technique, time and unit multi-season and multi-year evaluations. Majority of agronomic descriptors are polygenic in nature and of measurement for quantitative descriptors, like descriptors, the descriptors states are also developed by highly influenced by the environment. Therefore, evaluation trails should be carried out in at least three the scientists, curators and experts who are working on the particular species. They have to decide on diverse environments to minimize Genotype x Environment (G x E) interaction. which descriptor and descriptor's states to include for a given plant species. Now, a number of measures Germplasm Maintenance have been taken to improve the characterization and evaluation of germplasm collection involving breeders and also adopting new techniques including molecular markers. Germplasm maintenance poses the greatest risks to the genetic integrity of germplasm, due to selection, out-crossing or mechanical mixing. The risk of losing genetic integrity is especially high when raising Guidelines for recording descriptors genetically heterogeneous germplasm accessions of out-crossing crops. The purpose of maintenance is to Following are the guidelines for scoring and recording of descriptors for characterization and preliminary keep the original genetic diversity and structure of the accession or collection. Maintenance of genetic evaluation. These may be strictly followed. integrity involves maintaining the joint frequency distribution of all alleles at all loci. In fact, one should try to preserve the genotypic structure of the populations as well as the allelic frequencies. The appropriate a) Observations are to be recorded in the centimetre–gram–second system (CGS). The units are population size of an accession is an important factor for their maintenance. It will depend upon the mentioned in parenthesis after descriptor name. breeding behavior of the crop as well as the biological status of the material. The population size should be b) Presence/absence of characters are scored as 1 (present) and 0 (absent); large enough in cross and often cross pollinated crops to avoid genetic drift. Cross-pollinating species usually require more plants to maintain the genetic variation that exists within the population than self- c) Many quantitative descriptors which are continuously variable are recorded on a 0-9 scale, where: 0- pollinating species. In order to minimize the loss of heterozygosity, inbreeding depression and change in Immune are the expression of a character. For some characters, only a selected scores i.e. 1,3,5,7,9 are allelic frequency, a minimum number of 50-100 plants are required in a population. In case of wild species, described. Where this has occurred, it shows the gradient (from very low to very high) for that particular where few seeds are available and difficult to germinate and grow, special treatments wherever required character. should be given for their proper germination and less number of plants may be considered. i. Very low Descriptors and Descriptor States ii. Very low to low Depending upon the individual circumstances, the list of these descriptors may be developed by the iii. Low organization working on PGR, crop curators/plant breeders with his experience and with the help of the iv. Low to intermediate crop advisory committee and several experts of different related fields like chemistry, pathology, entomology etc. Bioversity International, Rome, in collaboration with international partners, has developed v. Intermediate various crop descriptors which are being used globally and ICAR-NBPGR has also developed minimal vi. Intermediate or high descriptors at national level. The use of uniform descriptors and descriptor states facilitate the utilization of vii. High germplasm by different researchers. Various kinds of descriptors have been identified as essential parameters to facilitate the management and use of germplasm available in different forms. viii. High to very high Types of Descriptors ix. Very high I) Passport descriptors: These provide the basic information recorded at the time of original collection. It d) For recording quantitative characters, the average of 5-10 plants is considered. Further, in case of cross- is used for the identification of germplasm and required for registration at the genebank. pollinated /often cross-pollinated species, the number of plants selected may be more (10-25 plants) depending upon the species and available resources.

86 87 e) For most of the qualitative characters the last state is 'Others', if a particular state is not present in the a specific set of characteristics. Seeds from one or a few plants are unlikely to contain a representative descriptor states, write the code '99' for 'Others'. Additional information for that descriptor may be sample of the genes of the population and, therefore, cannot reproduce its structure. Thus, seeds appended in the 'Remarks' descriptor as follows: from many plants of a cross-pollinated species should be collected in order to sample adequately its genes and retain its population structure. Likewise, further multiplication of such accessions must i. If the red colour state is not listed, then the red colour is to be given in the Remarks column along involve many plants from seed of the original collections (Burton and Davies, 1984). Further, with the name of the descriptor. collections of cross-pollinated crops, when planted for the first time after collection, fail to reveal most ii. If a particular descriptor is inapplicable, e.g., when the plant does not bear pods, the pods related of their recessive alleles because they are suppressed by dominant alleles. Such accessions cannot be descriptor(s) become inapplicable. Such state(s) are to be mentioned as 'Inapplicable' along with effectively screened for their recessive alleles until they are selfed. In general, genes for short stature the notes under the Remarks column. and many of those for pest and disease resistance are recessive. The seed derived from the first selfed generation (S1) from an accession can be screened immediately for both dominant and recessive iii. For descriptors which are not generally uniform throughout the accession (e.g. mixed collection, genes. For many crops, the production of S1 seed is less laborious than sibbing. Further, increasing an genetic segregation), all the states should be mentioned in descending order of occurrence under accession by selfing is also less likely to result in loss of alleles, and reduces exposure to genetic the Remarks column (e.g. 50% red, 30% white, 10% yellow and 10% brown). contamination compared to increasing it by sibbing. f) The severity or extent of infection or infestation may be recorded on 1-9 scale under the descriptor 3. Population size and genetic drift: After germplasm is collected from nature or from farmer's field and 'Biotic Stress Susceptibility”. However the detailed information in respect of name(s) of disease(s) placed in a gene bank or regenerated, loss of genetic variation or change in the genetic structure of the pest(s) and causal organism (s) may be appended in the Biotic notes; descriptor. collection may occur. One of the most important duties of curators is, therefore, to minimize such g) Dates should be expressed numerically in the format DD/MM/YYYY where genetic changes. In order to do so, a sufficiently large effective population size must be preserved and, whenever the population is regenerated, a sufficiently large number of plants must be grown and DD - 2 digits to represent the Days enough pollinations should be made or facilitated to maintain large effective populations. MM-2 digits to represent the Month In small populations, allelic frequencies are subject to random fluctuations arising from the sampling YYYY-4 digits to represent the Year of gametes. Therefore, small populations drift toward fixation of particular alleles - 'genetic drift'. This will lead in increasing the frequency of homozygotes at the expense of heterozygotes. Many of the h) Standard colour chart e.g. Royal Horticultural Society Colour Charts or computer simulated colour deleterious alleles, which are otherwise masked in the heterozygotes, will become fixed and will charts should be used to determine colour of the flower/other plant parts. ultimately be eliminated from the population. Although, the change of gene frequency resulting from Table 1: Types of measurement data (Dillon and Goldstein, 1984) sampling is random in the sense that its direction is unpredictable, its magnitude can be predicted in terms of variance of the change (Falconer, 1960). Since the fixation of alleles is a function of initial gene Type Scale Measurement Typical descriptor frequency, the size of the population and the number of generations of sexual reproduction are Quantitative Interval Direct measure of a trait Height, yield components, days important factors in reducing genetic drift. Therefore, larger the population size, the slower will be the to flowering, maturity and fixation of genes (Wright, 1952). The genetic drift towards fixation occurs at the same rate in small harvest, protein contents populations whether the mating system is outbreeding or predominantly selfing (Allard and Hansche, Ratio Combining two traits measures Harvest index, content 1964). Therefore, it is suggested that, in order to minimize loss of heterozygosity, inbreeding into a single descriptor percentage, length/width ratio depression and allele loss, a minimum effective population number of 50-70 plants is required to Qualitative Ordinal Relative value assigned within Host plant resistance (from 1: constitute a population. Nevertheless, population genetic theory and practical experience have a standard scale highly tolerant to 9: highly shown that the changes do occur in allelic frequencies. Hence, practical compromises are necessary - susceptible) the objective will be to choose options that cause minimum change. Nominal Qualitative state assigned into Colour, seed pattern, 4. Optimum environment for multiplication and evaluation: The strategy and tactics of growing genetic arbitrarily number-scale growth habit resources for multiplication, maintenance and evaluation have been widely discussed (Allard, 1970; Binary Trait presence or absence Pubescence, colour spot Sevchuk, 1973; Erskine and Williams, 1980; Frankel and Soulé, 1981). According to them, the environmental conditions of the multiplication site(s) should be as near as possible to those under which the accession evolved or was cultivated for a long period. Since the distribution ranges of There is no rigid rule about characterization/preliminary evaluation and further evaluation. The principles accessions of all major crops vary greatly, it is likely that two or more multiplication sites will be and procedures to be applied should be flexible, since the diverse groups of crops have different problems. necessary to determine adaptability and site x genotype interaction. The advantage of choosing such a However, the following considerations should be taken into account during multiplication, characterization range lies in reducing the evaluation period, because the complete range of climatic factors may be and evaluation. encountered over a shorter period of time. 1. Factors affecting seed multiplication: A pure line can be multiplied by growing only a few plants and When the germplasm is grown across locations and over years, a knowledge of the nature and relative the actual number will depend on the multiplication rate and the seed quantity required, whereas, a magnitude of various types of genotype-environment interaction is important in making decisions heterozygous population would need to be multiplied from a much larger population sample and regarding ranking of accessions. Several investigators have highlighted the importance of these much care should be taken to ensure the maintenance of genetic integrity. interactions as well as shown their use in setting up selection and testing regimes for applied breeding 2. Germplasm maintenance of cross-pollinated crops: A cross-pollinated population consists of a and genotype testing programmes (Comstock and Moll, 1963; Allard and Barawshaw, 1964; Knight, mixture of genotypes, no two of which are alike. Collectively, these genotypes form a population with 1970; Paroda and Hayes, 1971; Brown et al., 1983).

88 89 e) For most of the qualitative characters the last state is 'Others', if a particular state is not present in the a specific set of characteristics. Seeds from one or a few plants are unlikely to contain a representative descriptor states, write the code '99' for 'Others'. Additional information for that descriptor may be sample of the genes of the population and, therefore, cannot reproduce its structure. Thus, seeds appended in the 'Remarks' descriptor as follows: from many plants of a cross-pollinated species should be collected in order to sample adequately its genes and retain its population structure. Likewise, further multiplication of such accessions must i. If the red colour state is not listed, then the red colour is to be given in the Remarks column along involve many plants from seed of the original collections (Burton and Davies, 1984). Further, with the name of the descriptor. collections of cross-pollinated crops, when planted for the first time after collection, fail to reveal most ii. If a particular descriptor is inapplicable, e.g., when the plant does not bear pods, the pods related of their recessive alleles because they are suppressed by dominant alleles. Such accessions cannot be descriptor(s) become inapplicable. Such state(s) are to be mentioned as 'Inapplicable' along with effectively screened for their recessive alleles until they are selfed. In general, genes for short stature the notes under the Remarks column. and many of those for pest and disease resistance are recessive. The seed derived from the first selfed generation (S1) from an accession can be screened immediately for both dominant and recessive iii. For descriptors which are not generally uniform throughout the accession (e.g. mixed collection, genes. For many crops, the production of S1 seed is less laborious than sibbing. Further, increasing an genetic segregation), all the states should be mentioned in descending order of occurrence under accession by selfing is also less likely to result in loss of alleles, and reduces exposure to genetic the Remarks column (e.g. 50% red, 30% white, 10% yellow and 10% brown). contamination compared to increasing it by sibbing. f) The severity or extent of infection or infestation may be recorded on 1-9 scale under the descriptor 3. Population size and genetic drift: After germplasm is collected from nature or from farmer's field and 'Biotic Stress Susceptibility”. However the detailed information in respect of name(s) of disease(s) placed in a gene bank or regenerated, loss of genetic variation or change in the genetic structure of the pest(s) and causal organism (s) may be appended in the Biotic notes; descriptor. collection may occur. One of the most important duties of curators is, therefore, to minimize such g) Dates should be expressed numerically in the format DD/MM/YYYY where genetic changes. In order to do so, a sufficiently large effective population size must be preserved and, whenever the population is regenerated, a sufficiently large number of plants must be grown and DD - 2 digits to represent the Days enough pollinations should be made or facilitated to maintain large effective populations. MM-2 digits to represent the Month In small populations, allelic frequencies are subject to random fluctuations arising from the sampling YYYY-4 digits to represent the Year of gametes. Therefore, small populations drift toward fixation of particular alleles - 'genetic drift'. This will lead in increasing the frequency of homozygotes at the expense of heterozygotes. Many of the h) Standard colour chart e.g. Royal Horticultural Society Colour Charts or computer simulated colour deleterious alleles, which are otherwise masked in the heterozygotes, will become fixed and will charts should be used to determine colour of the flower/other plant parts. ultimately be eliminated from the population. Although, the change of gene frequency resulting from Table 1: Types of measurement data (Dillon and Goldstein, 1984) sampling is random in the sense that its direction is unpredictable, its magnitude can be predicted in terms of variance of the change (Falconer, 1960). Since the fixation of alleles is a function of initial gene Type Scale Measurement Typical descriptor frequency, the size of the population and the number of generations of sexual reproduction are Quantitative Interval Direct measure of a trait Height, yield components, days important factors in reducing genetic drift. Therefore, larger the population size, the slower will be the to flowering, maturity and fixation of genes (Wright, 1952). The genetic drift towards fixation occurs at the same rate in small harvest, protein contents populations whether the mating system is outbreeding or predominantly selfing (Allard and Hansche, Ratio Combining two traits measures Harvest index, content 1964). Therefore, it is suggested that, in order to minimize loss of heterozygosity, inbreeding into a single descriptor percentage, length/width ratio depression and allele loss, a minimum effective population number of 50-70 plants is required to Qualitative Ordinal Relative value assigned within Host plant resistance (from 1: constitute a population. Nevertheless, population genetic theory and practical experience have a standard scale highly tolerant to 9: highly shown that the changes do occur in allelic frequencies. Hence, practical compromises are necessary - susceptible) the objective will be to choose options that cause minimum change. Nominal Qualitative state assigned into Colour, seed pattern, 4. Optimum environment for multiplication and evaluation: The strategy and tactics of growing genetic arbitrarily number-scale growth habit resources for multiplication, maintenance and evaluation have been widely discussed (Allard, 1970; Binary Trait presence or absence Pubescence, colour spot Sevchuk, 1973; Erskine and Williams, 1980; Frankel and Soulé, 1981). According to them, the environmental conditions of the multiplication site(s) should be as near as possible to those under which the accession evolved or was cultivated for a long period. Since the distribution ranges of There is no rigid rule about characterization/preliminary evaluation and further evaluation. The principles accessions of all major crops vary greatly, it is likely that two or more multiplication sites will be and procedures to be applied should be flexible, since the diverse groups of crops have different problems. necessary to determine adaptability and site x genotype interaction. The advantage of choosing such a However, the following considerations should be taken into account during multiplication, characterization range lies in reducing the evaluation period, because the complete range of climatic factors may be and evaluation. encountered over a shorter period of time. 1. Factors affecting seed multiplication: A pure line can be multiplied by growing only a few plants and When the germplasm is grown across locations and over years, a knowledge of the nature and relative the actual number will depend on the multiplication rate and the seed quantity required, whereas, a magnitude of various types of genotype-environment interaction is important in making decisions heterozygous population would need to be multiplied from a much larger population sample and regarding ranking of accessions. Several investigators have highlighted the importance of these much care should be taken to ensure the maintenance of genetic integrity. interactions as well as shown their use in setting up selection and testing regimes for applied breeding 2. Germplasm maintenance of cross-pollinated crops: A cross-pollinated population consists of a and genotype testing programmes (Comstock and Moll, 1963; Allard and Barawshaw, 1964; Knight, mixture of genotypes, no two of which are alike. Collectively, these genotypes form a population with 1970; Paroda and Hayes, 1971; Brown et al., 1983).

88 89 However, the identification of testing sites which give results with wide applicability has not been 6. Burton GW and W Ellis Davies (1984) Handling germplasm of cross-pollinated forage crops, pp. 180- given the attention it deserves. Knowledge of the correlation between the value of a genotype relative 190. In: Holden JHW and JT Williams (eds) Crop genetic resources: Conservation and evaluation. to a test environment and its value relative to the whole population of environments where it is to George Alien and Unwin, London. perform is necessary for such optimum sites to be identified. 7. Comstock RE and RH Moll (1963) Genotype-environment interactions. In: Statistical genetics and 5. Experimental design: Single row (3-5 m) plot or small plots of more than one row, depending upon the plant breeding, Nat. Acad. Sci. Pub., Washington, D.C. pp. 164-196. quantity of seeds available and the nature of plant species, are generally grown for germplasm 8. Dillon WR and M Goldstein (1984) Multivariate analysis. Methods and applications. John Wiley Inc. evaluation. Enough space should be kept between rows and between plants to permit them to express New York. their differences and avoid competition. Accessions belonging to the same maturity group should be planted together on one date of sowing. Accessions suspected to be duplicates should be grown side 9. Erskine W and JT Williams (1980) The principles, problems and responsibilities of the preliminary by side to facilitate comparison while evaluating in the field. During the process of growing, attention evaluation of genetic resources samples of seed-propagated crops. Plant Genet. Resour. Newslett. 41: should be given to minimise natural cross pollination, contamination and erroneous labelling. Some of 19-33. the basic requirements for field evaluation are field of rectangular shape, uniform fertility, 10. Falconer DC (ed.) (1960) Introduction to quantitative genetics. New York, Ronald Press. dependable water supply, drainage facility, equipments for seed processing, and supply of agricultural chemicals and other materials such as bags, labels, stationery, etc. 11. Federer TW (1956) Augmented (or Hoonuiaku) designs. The Hawaiian Planters' Record, vol. IV, second issue. 191-208. When the germplasm collections are large, it is always preferable to grow and evaluate the germplasm in an augmented block design (Federer, 1956). The augmented block design provides a welcome 12. Frankel OH and M Soulé (eds.) (1981) Conservation and evolution. Cambridge Univ. Press, Cambridge. flexibility to block design where not all treatments appear to warrant full replication. By reducing 13. Knight RC (1970) The measurement and interpretation of genotype x environment interaction. replication, representative size may be reduced. A major advantage of this design is that such Euphytica. 19: 225-235. treatments, no matter how discripant their values may be, do not contribute to the experimental error of the experiment. 14. Mahajan RK, Sapra RL, Umesh S, Singh M, Sharma GD (2000) Minimal Descriptors (For Characterization And Evaluation) Of Agri-Horticultural Crops. National Bureau Of Plant Genetic 6. Multiplication/rejuvenation of germplasm: The need for multiplication/rejuvenation of germplasm Resources; New Delhi is a function of size of the initial sample, user demand and seed longevity under the condition of storage. As mentioned earlier, the aim during rejuvenation should be to retain the essential genetic 15. Paroda RS and JD Hayes (1971) Investigation of genotype - environment interactions for rate of ear characteristics of the accession and obtain sufficient quantities of high quality seed to satisfy emergence in spring barley. Heredity. 26: 157-176. requirements for storage and user demand. It is also necessary during the regeneration process to 16. Sevchuk T (1973) Evaluation of plant collections. Plant Genet. Resour. Newslett. 29: 2-6. consider how best to reduce changes due to contamination through mutation, foreign pollen or seed, and to minimize genetic drift or shift by ensuring sufficient population size and reducing opportunities 17. Wright S (1952) The theoretical variance within and among sub-division of a population that is in a for natural selection. Mutation rates are too low to pose a serious problem. The main dangers are due steady state. Genetics. 37: 312-321. to inappropriate seed handling during harvesting, threshing, cleaning, sub-sampling and packing. For the outbreeding populations, another important factor is to maintain proper isolation distance. Maintaining proper isolation distance becomes difficult when a large number of accessions have to be multiplied simultaneously and, under such conditions, controlled hand pollination is the only correct approach. However, in any case, regeneration cannot avoid natural selection which, in turn, leads to genetic shift. It is therefore, advisable that regeneration should be carried out as infrequently as possible. In the recent years, better storage conditions of the germplasm have greatly extended the storage life of seeds, thus reducing the need for frequent rejuvenation. References 1. Allard RW (1970) Population structure and sampling methods, pp. 97-107. In: Frankel OH and E Bennett (eds) Genetic resources in plants - Their exploration and conservation. Oxford, Blackwell. 2. Allard RW and AD Barawshaw (1964) Implications of genotype-environmental interactions in applied plant breeding. Crop Sci. 4: 503-508. 3. Allard RW and PE Hansche (1964) Some parameters of population variability and their implications in plant breeding. Adv. Agron. 16: 281-325. 4. Bioversity International (2007) Guidelines for the development of crop descriptor lists. Bioversity Technical Bulletin Series. Bioversity International, Rome, Italy. xii+72p. 5. Brown KD, ME Sorrells and WR Coffman (1983) A method for classification and evaluation of testing environments. Crop. Sci. 23: 889-893.

90 91 However, the identification of testing sites which give results with wide applicability has not been 6. Burton GW and W Ellis Davies (1984) Handling germplasm of cross-pollinated forage crops, pp. 180- given the attention it deserves. Knowledge of the correlation between the value of a genotype relative 190. In: Holden JHW and JT Williams (eds) Crop genetic resources: Conservation and evaluation. to a test environment and its value relative to the whole population of environments where it is to George Alien and Unwin, London. perform is necessary for such optimum sites to be identified. 7. Comstock RE and RH Moll (1963) Genotype-environment interactions. In: Statistical genetics and 5. Experimental design: Single row (3-5 m) plot or small plots of more than one row, depending upon the plant breeding, Nat. Acad. Sci. Pub., Washington, D.C. pp. 164-196. quantity of seeds available and the nature of plant species, are generally grown for germplasm 8. Dillon WR and M Goldstein (1984) Multivariate analysis. Methods and applications. John Wiley Inc. evaluation. Enough space should be kept between rows and between plants to permit them to express New York. their differences and avoid competition. Accessions belonging to the same maturity group should be planted together on one date of sowing. Accessions suspected to be duplicates should be grown side 9. Erskine W and JT Williams (1980) The principles, problems and responsibilities of the preliminary by side to facilitate comparison while evaluating in the field. During the process of growing, attention evaluation of genetic resources samples of seed-propagated crops. Plant Genet. Resour. Newslett. 41: should be given to minimise natural cross pollination, contamination and erroneous labelling. Some of 19-33. the basic requirements for field evaluation are field of rectangular shape, uniform fertility, 10. Falconer DC (ed.) (1960) Introduction to quantitative genetics. New York, Ronald Press. dependable water supply, drainage facility, equipments for seed processing, and supply of agricultural chemicals and other materials such as bags, labels, stationery, etc. 11. Federer TW (1956) Augmented (or Hoonuiaku) designs. The Hawaiian Planters' Record, vol. IV, second issue. 191-208. When the germplasm collections are large, it is always preferable to grow and evaluate the germplasm in an augmented block design (Federer, 1956). The augmented block design provides a welcome 12. Frankel OH and M Soulé (eds.) (1981) Conservation and evolution. Cambridge Univ. Press, Cambridge. flexibility to block design where not all treatments appear to warrant full replication. By reducing 13. Knight RC (1970) The measurement and interpretation of genotype x environment interaction. replication, representative size may be reduced. A major advantage of this design is that such Euphytica. 19: 225-235. treatments, no matter how discripant their values may be, do not contribute to the experimental error of the experiment. 14. Mahajan RK, Sapra RL, Umesh S, Singh M, Sharma GD (2000) Minimal Descriptors (For Characterization And Evaluation) Of Agri-Horticultural Crops. National Bureau Of Plant Genetic 6. Multiplication/rejuvenation of germplasm: The need for multiplication/rejuvenation of germplasm Resources; New Delhi is a function of size of the initial sample, user demand and seed longevity under the condition of storage. As mentioned earlier, the aim during rejuvenation should be to retain the essential genetic 15. Paroda RS and JD Hayes (1971) Investigation of genotype - environment interactions for rate of ear characteristics of the accession and obtain sufficient quantities of high quality seed to satisfy emergence in spring barley. Heredity. 26: 157-176. requirements for storage and user demand. It is also necessary during the regeneration process to 16. Sevchuk T (1973) Evaluation of plant collections. Plant Genet. Resour. Newslett. 29: 2-6. consider how best to reduce changes due to contamination through mutation, foreign pollen or seed, and to minimize genetic drift or shift by ensuring sufficient population size and reducing opportunities 17. Wright S (1952) The theoretical variance within and among sub-division of a population that is in a for natural selection. Mutation rates are too low to pose a serious problem. The main dangers are due steady state. Genetics. 37: 312-321. to inappropriate seed handling during harvesting, threshing, cleaning, sub-sampling and packing. For the outbreeding populations, another important factor is to maintain proper isolation distance. Maintaining proper isolation distance becomes difficult when a large number of accessions have to be multiplied simultaneously and, under such conditions, controlled hand pollination is the only correct approach. However, in any case, regeneration cannot avoid natural selection which, in turn, leads to genetic shift. It is therefore, advisable that regeneration should be carried out as infrequently as possible. In the recent years, better storage conditions of the germplasm have greatly extended the storage life of seeds, thus reducing the need for frequent rejuvenation. References 1. Allard RW (1970) Population structure and sampling methods, pp. 97-107. In: Frankel OH and E Bennett (eds) Genetic resources in plants - Their exploration and conservation. Oxford, Blackwell. 2. Allard RW and AD Barawshaw (1964) Implications of genotype-environmental interactions in applied plant breeding. Crop Sci. 4: 503-508. 3. Allard RW and PE Hansche (1964) Some parameters of population variability and their implications in plant breeding. Adv. Agron. 16: 281-325. 4. Bioversity International (2007) Guidelines for the development of crop descriptor lists. Bioversity Technical Bulletin Series. Bioversity International, Rome, Italy. xii+72p. 5. Brown KD, ME Sorrells and WR Coffman (1983) A method for classification and evaluation of testing environments. Crop. Sci. 23: 889-893.

90 91 Characterization and Evaluation Site for evaluation To get reasonable information about the genetic potential of the germplasm for different parameters, namely agronomic and quality parameters, the accessions should be evaluated in the area of their of Fruit Crops – Role of Descriptors adaptation or under similar environmental condition. The target traits must be prioritized and suitable number of locations should be selected keeping in view the similar environment matching with the major agro-ecological zones of the crop/germplasm. The optimum number of locations will depend upon available and Other Practical Considerations resources and the relative magnitude of interaction of environmental factors with genetic material. Narender Negi*, Rahul Chandora, Badal Singh, Dayal Singh, Evaluation for biotic and abiotic stresses is to be carried out in specific environment i.e. hot spots or over a Ram Chander and Mohar Singh range of environments (locations, years etc). But it is very difficult for a curator to conduct multi-location testing as elaborately undertaken by plant breeders for a limited number of entries. ICAR-NBPGR Regional Station Phagli, Shimla Himachal Pradesh *Email - [email protected] The International Plant Genetic Resources Institute (IPGRI) now Bioversity International makes a distinction for practical purposes between characterization and detailed evaluation (Table 1). The descriptors Introduction distinguish those traits, which can be considered highly heritable and environmentally stable from those Characterization and evaluation of germplasm is pre-requisite in any crop improvement programme. It is whose expression generally depends on the conditions in which they are assessed. The practice of the description of germplasm in respect of highly heritable traits and useful for identification of inter- evaluation begins with the adoption of a descriptor list. Based on our requirements and feedback from accession diversity for establishing true values, while, evaluation provides meaningful information on different Crop Germplasm Advisory Committees and discussions in Annual Crop Workshops, the National germplasm generally required by the breeders for their utilization. Until a collection has been properly Bureau of Plant Genetic Resources (NBPGR), has developed Minimal Crop Descriptors for over 140 agri- evaluated and its attributes become known to researchers, it has little practical use. Germplasm evaluation, horticultural crops which includes (27 tropical and subtropical fruits) and 9 (temperate fruits and nut crops). in the broad sense and in the context of genetic resources, is the description of the material in a genepool. It Table 1: Distinction between characterization and evaluation covers the whole range of activities starting from the receipt of the new samples by the curator and growing these for seed increase, characterization and preliminary evaluation, and also for further or comprehensive Characterization Evaluation evaluation and documentation, as well. The description of attributes, which for a The assessment in described environments or Characterisation given genotype, may be considered invariant under specified conditions of traits that are known to be environmentally sensitive It refers to description of the qualitative traits, which are highly heritable and generally expressed in all Qualitative, environmentally stable, mono-> Quantitative, environmentally influenced, environments. Characterization of germplasm should be carried out at the initial stage preferably within 2-4 oligogenic, easily manipulated in breeding oligo->polygenic, not so easily manipulated years of its acquisition (varies for different fruit crops). The germplasm accessions which are to be characterized should contain the national identity along with complete passport information for deciding For example Seed pattern, flower pattern, Plant height, protein content, time to maturity the characterization strategies. For example, the germplasm accessions should be grouped as per their isozyme pattern growth habit and maturity to avoid the competition among accessions. Biological status of the material is to be considered to chalk out strategy for their characterization. Internationally and nationally accepted Descriptor and descriptor states descriptors are used for characterization with standardized and calibrated format. In the era of A descriptor is defined as an attribute, characteristic or measurable trait that is observed in an accession of a advancement at molecular level highly reproducible markers can also be used for characterization, purpose. gene bank. It is used to facilitate data classification, storage, retrieval, exchange and use. The under Site of characterization utilization of germplasm by the breeders for their genetic improvement programme is due to the lack of basic characterization data with proper passport information. A descriptor list is a set of individual The characterization should be completed in a suitable environment preferably at a site nearest to the site of descriptors used for the description of germplasm of a particular crop or species (list of descriptors for collection of germplasm or under identical agro-climatic condition to have proper expression of descriptors mango). So the descriptors (information) are very much essential due to following reasons: and to avoid any genetic shift. 1. The proper management of the resources in the gene banks by gene bank managers/crop curators. Evaluation 2. Accurate documentation of information about the origin, characterization and performance of Evaluation being a multidisciplinary approach, should involve a germplasm curator, plant breeder, genepool is essential for safe conservation and use. physiologist, pathologist, entomologist, biochemist etc., for a meaningful study. A network/ coordinated approach at multiple locations under different agro-climatic zones in collaboration with crop based 3. Developing international standards such as descriptor lists is to make the most of biodiversity by institutes and other institutes working in same direction can be recommended for evaluation. Dissimilarity describing crops and species in a lingua franca. in the incidence of pests, severity of abiotic stresses and the fluctuation in environmental and climatic 4. Increasing access to this diversity by the users of plant genetic resources, who in turn will benefit from factors in the field impact the accuracy of data should be mitigated through reasonably replicated, multi- their economic and social values. location, multi-season and multiyear expression. The referral laboratories should be involved for the evaluation of quality traits like oil, protein, secondary metabolites etc., as estimation of these parameters requires specialized instruments, consumables and trained manpower etc. After the collection of germplasm, there is need for its systematic evaluation in order to know its various morphological, physiological and developmental attributes including some special features, such as stress tolerance, pest and disease resistance etc. 92 93 Characterization and Evaluation Site for evaluation To get reasonable information about the genetic potential of the germplasm for different parameters, namely agronomic and quality parameters, the accessions should be evaluated in the area of their of Fruit Crops – Role of Descriptors adaptation or under similar environmental condition. The target traits must be prioritized and suitable number of locations should be selected keeping in view the similar environment matching with the major agro-ecological zones of the crop/germplasm. The optimum number of locations will depend upon available and Other Practical Considerations resources and the relative magnitude of interaction of environmental factors with genetic material. Narender Negi*, Rahul Chandora, Badal Singh, Dayal Singh, Evaluation for biotic and abiotic stresses is to be carried out in specific environment i.e. hot spots or over a Ram Chander and Mohar Singh range of environments (locations, years etc). But it is very difficult for a curator to conduct multi-location testing as elaborately undertaken by plant breeders for a limited number of entries. ICAR-NBPGR Regional Station Phagli, Shimla Himachal Pradesh *Email - [email protected] The International Plant Genetic Resources Institute (IPGRI) now Bioversity International makes a distinction for practical purposes between characterization and detailed evaluation (Table 1). The descriptors Introduction distinguish those traits, which can be considered highly heritable and environmentally stable from those Characterization and evaluation of germplasm is pre-requisite in any crop improvement programme. It is whose expression generally depends on the conditions in which they are assessed. The practice of the description of germplasm in respect of highly heritable traits and useful for identification of inter- evaluation begins with the adoption of a descriptor list. Based on our requirements and feedback from accession diversity for establishing true values, while, evaluation provides meaningful information on different Crop Germplasm Advisory Committees and discussions in Annual Crop Workshops, the National germplasm generally required by the breeders for their utilization. Until a collection has been properly Bureau of Plant Genetic Resources (NBPGR), has developed Minimal Crop Descriptors for over 140 agri- evaluated and its attributes become known to researchers, it has little practical use. Germplasm evaluation, horticultural crops which includes (27 tropical and subtropical fruits) and 9 (temperate fruits and nut crops). in the broad sense and in the context of genetic resources, is the description of the material in a genepool. It Table 1: Distinction between characterization and evaluation covers the whole range of activities starting from the receipt of the new samples by the curator and growing these for seed increase, characterization and preliminary evaluation, and also for further or comprehensive Characterization Evaluation evaluation and documentation, as well. The description of attributes, which for a The assessment in described environments or Characterisation given genotype, may be considered invariant under specified conditions of traits that are known to be environmentally sensitive It refers to description of the qualitative traits, which are highly heritable and generally expressed in all Qualitative, environmentally stable, mono-> Quantitative, environmentally influenced, environments. Characterization of germplasm should be carried out at the initial stage preferably within 2-4 oligogenic, easily manipulated in breeding oligo->polygenic, not so easily manipulated years of its acquisition (varies for different fruit crops). The germplasm accessions which are to be characterized should contain the national identity along with complete passport information for deciding For example Seed pattern, flower pattern, Plant height, protein content, time to maturity the characterization strategies. For example, the germplasm accessions should be grouped as per their isozyme pattern growth habit and maturity to avoid the competition among accessions. Biological status of the material is to be considered to chalk out strategy for their characterization. Internationally and nationally accepted Descriptor and descriptor states descriptors are used for characterization with standardized and calibrated format. In the era of A descriptor is defined as an attribute, characteristic or measurable trait that is observed in an accession of a advancement at molecular level highly reproducible markers can also be used for characterization, purpose. gene bank. It is used to facilitate data classification, storage, retrieval, exchange and use. The under Site of characterization utilization of germplasm by the breeders for their genetic improvement programme is due to the lack of basic characterization data with proper passport information. A descriptor list is a set of individual The characterization should be completed in a suitable environment preferably at a site nearest to the site of descriptors used for the description of germplasm of a particular crop or species (list of descriptors for collection of germplasm or under identical agro-climatic condition to have proper expression of descriptors mango). So the descriptors (information) are very much essential due to following reasons: and to avoid any genetic shift. 1. The proper management of the resources in the gene banks by gene bank managers/crop curators. Evaluation 2. Accurate documentation of information about the origin, characterization and performance of Evaluation being a multidisciplinary approach, should involve a germplasm curator, plant breeder, genepool is essential for safe conservation and use. physiologist, pathologist, entomologist, biochemist etc., for a meaningful study. A network/ coordinated approach at multiple locations under different agro-climatic zones in collaboration with crop based 3. Developing international standards such as descriptor lists is to make the most of biodiversity by institutes and other institutes working in same direction can be recommended for evaluation. Dissimilarity describing crops and species in a lingua franca. in the incidence of pests, severity of abiotic stresses and the fluctuation in environmental and climatic 4. Increasing access to this diversity by the users of plant genetic resources, who in turn will benefit from factors in the field impact the accuracy of data should be mitigated through reasonably replicated, multi- their economic and social values. location, multi-season and multiyear expression. The referral laboratories should be involved for the evaluation of quality traits like oil, protein, secondary metabolites etc., as estimation of these parameters requires specialized instruments, consumables and trained manpower etc. After the collection of germplasm, there is need for its systematic evaluation in order to know its various morphological, physiological and developmental attributes including some special features, such as stress tolerance, pest and disease resistance etc. 92 93 Characterization and evaluation carried out using standard descriptors adds following useful advantages: Beyond the basic characterization for several agro-botanical and economic characters, some other characters are also of great interest to breeders. Typically these include stress tolerance, disease and pest 1. Even though different programmes or institutions use different documentation systems (i.e. hardware resistance including quality parameters. Evaluation for many of these traits is outside the resources of most and software) or languages, if they use a common descriptor system the exchange of information is curators. They may require specialized laboratory or greenhouse work and the assistance of an expert facilitated. familiar with the specific character and procedure of testing. Depending on circumstances, this work is 2. The crop descriptor lists have an internationally accepted format and have been developed by and are collaborated with concerned scientists of the same institute or collaborated with other organizations or may shared among scientists worldwide. be contracted out to other institutes. The extent of this work depends on perceived needs of breeders. However, a provision has been made in the descriptor list to record preliminary informationon reaction to 3. The utilization of a 'universal language' in well defined and thoroughly-tested descriptor lists for biotic stresses, namely incidence of diseases and insects/pests along with recording of details of these characterizing germplasm simplifies data recording, updating, modification, retrieval, exchange and under the head biotic notes. analysis. The approach to recording parameters for characterization and preliminary evaluation work is a 4. Germplasm conservers and users adopting the same descriptors at different locations are helpful to fundamental matter. The developing of descriptor lists has been done much to standardize practices to exchange and interpret each other's data. record data with consequent improvements by the breeders/curators. There are four type of data Characterization and preliminary evaluation of fruit crops measurement which covers the range of quantitative to qualitative characters (Dillon and Goldstein, 1984) (table 2). Both interval and ratio scales depend upon real unit (e.g., gram, meter) or are derived from them. This is the basic description of work and is considered to be the direct responsibility of the curator. It includes Ordinal data generally require the construction of a standard scale, frequently on a 0-9 basis and definition basic morphological description of accessions (characterization) which may enable any subsequent in words and/or diagrams of what each of the scores mean. Here for most of the descriptors only five contamination or mix-up of accessions to be identified. The extent of this description depends very much on character states have been described and for most practical purposes this is probably sufficient. However, the species in question. wherever justified, the intermediate values were also used. For nominal scale, the score has no meaning as a Germplasm evaluation in broad sense and in the context of genetic resources work is the description of the number in either absolute or relative terms with the descriptor state, but it may be the most convenient way material in a genepool. It involves whole range of activities starting from the receipt of new samples by the to represent the data. curator and growing these for seed/plant increase, characterization and preliminary evaluation, and also for Table 2: Different types of data measurement further detailed evaluation and documentation. During evaluation it is important that attributes scored have some general and uniform basis, so that the traits described are passed from generation to generation Scale Basis of observation Example within an accession or can be expected to appear in the progeny of a breeder's cross. Interval Direct measurement of an attribute Plant height, days to flower, tuber weight, Germplasm collected in the form of vegetative propagules (especially in case of fruit crops) are subject to number of flowers per plant quick drying and deterioration. The fruit crops germplasm should not be collected in the form of seeds Ratio Combination of two direct measurement Harvest index, % protein, oil or sugar except the germplasm/ species having non-availability of vegetative propagules. The budwood of fruit trees or inference from a single measurement content should be utilized for propagation as early as possible, particularly of tropical and subtropical fruits, on Ordinal Assigning, sometimes subjectively, a Susceptibility to pests and diseases, suitable rootstock. Similarly, other propagules viz., cuttings for rooting, layers, suckers, slips, crowns, relative value from a standard scale overall processing quality, leaf or seed shapes runners etc. for establishment and multiplication of germplasm should also be utilized as of fruit crops. The fruit crops germplasm grown through seeds are papaya, phalsa, karonda and to some extent jackfruit Nominal Assignment of qualitative character Flower colour, seed patterns although the resultant seedling may not be true to type. When a population is classified into distinct states into arbitrarily numbered classes phenotypic classes for a given character, it is discontinuous variation and called as qualitative type. Such characters are under the control of one or few genes. Such variations can be analysed by count and ratios like The following useful descriptors are also required to be undertaken: presence and absence of a character etc. When a character shows continuous variation, it is called as I. What variation exists within a trial and what confidence can be placed on the score value quantitative variation. Such characters are controlled by many genes with each gene contributing to the total phenotypic variation. Under qualitative characters a) plant data, namely plant growth habit, stem II. The variation among trials and shape, leaf lamina shape, ii) inflorescence and fruit data, namely, sex type, flower colour, fruit shape, fruit III. Whether a scored value can be considered as 'good' or 'bad'. In some cases, they will also be needed as ridges per fruit, flesh texture etc. and in iii) seed data, such as seediness, seed cavity (small/medium/large) controls to verify that a particular test e.g., disease inoculation, has functioned correctly. In connection are scored in comparison to checks to determine variation within and among the traits. Quantitative with disease observation in the field based on chance outbreaks it is worth saying that non-incidence characters such as plant height, days to flowering, number of fruiting nodes per plant, number of pods per of disease may be due to either resistance or disease escape. cluster, number of fruits, days to maturity are influenced by the environment. The choice of check line will depend very much on circumstances. For preliminary evaluation, locally Evaluation of fruit crops germplasm often needs inclusion or planting of a pollinator in the germplasm lines. adapted cultivars provide understandable comparisons and a dependable way of monitoring trial (often In several cases 1-9 code rating should be followed. In case of petal colour, fruit skin colour or pulp colour etc. year to year) variation. For detailed evaluation which usually addresses one trait at a time, there will often an ascending order should be followed (e.g. white, yellow, pink, brown, red, gray, blue, violet and black). be a well-recognized set of checks that cover the likely range of scores e.g., known resistant and susceptible Similarly for fruit shape an order of flat, round, globose, oval, oblong, cordate, elliptic, cylindrical, and cultivars or accessions for disease reaction. arched should be followed. However, scope must be left to suitably incorporate specific positions like short- elliptical, broad-elliptical, and narrowly-elliptical. The same will apply to other morphological descriptors An additional problem is the question of Genotype x Environment (G x E) interaction.This has the practical like leaf shape, leaf apex, leaf base, fruits base, fruit apex, fruit beak and stone etc. Fruit taste, total soluble consequence of significantly changing the expression of accessions. In practice, the number of accessions solids content, flesh-stone ratio are highly important characters in fruit crops characterization. Overall processed in most genetic resources work is extensive and anyone accession is likely to be evaluated only a productivity status of the germplasm will reflect its use in breeding and commerce. 94 95 Characterization and evaluation carried out using standard descriptors adds following useful advantages: Beyond the basic characterization for several agro-botanical and economic characters, some other characters are also of great interest to breeders. Typically these include stress tolerance, disease and pest 1. Even though different programmes or institutions use different documentation systems (i.e. hardware resistance including quality parameters. Evaluation for many of these traits is outside the resources of most and software) or languages, if they use a common descriptor system the exchange of information is curators. They may require specialized laboratory or greenhouse work and the assistance of an expert facilitated. familiar with the specific character and procedure of testing. Depending on circumstances, this work is 2. The crop descriptor lists have an internationally accepted format and have been developed by and are collaborated with concerned scientists of the same institute or collaborated with other organizations or may shared among scientists worldwide. be contracted out to other institutes. The extent of this work depends on perceived needs of breeders. However, a provision has been made in the descriptor list to record preliminary informationon reaction to 3. The utilization of a 'universal language' in well defined and thoroughly-tested descriptor lists for biotic stresses, namely incidence of diseases and insects/pests along with recording of details of these characterizing germplasm simplifies data recording, updating, modification, retrieval, exchange and under the head biotic notes. analysis. The approach to recording parameters for characterization and preliminary evaluation work is a 4. Germplasm conservers and users adopting the same descriptors at different locations are helpful to fundamental matter. The developing of descriptor lists has been done much to standardize practices to exchange and interpret each other's data. record data with consequent improvements by the breeders/curators. There are four type of data Characterization and preliminary evaluation of fruit crops measurement which covers the range of quantitative to qualitative characters (Dillon and Goldstein, 1984) (table 2). Both interval and ratio scales depend upon real unit (e.g., gram, meter) or are derived from them. This is the basic description of work and is considered to be the direct responsibility of the curator. It includes Ordinal data generally require the construction of a standard scale, frequently on a 0-9 basis and definition basic morphological description of accessions (characterization) which may enable any subsequent in words and/or diagrams of what each of the scores mean. Here for most of the descriptors only five contamination or mix-up of accessions to be identified. The extent of this description depends very much on character states have been described and for most practical purposes this is probably sufficient. However, the species in question. wherever justified, the intermediate values were also used. For nominal scale, the score has no meaning as a Germplasm evaluation in broad sense and in the context of genetic resources work is the description of the number in either absolute or relative terms with the descriptor state, but it may be the most convenient way material in a genepool. It involves whole range of activities starting from the receipt of new samples by the to represent the data. curator and growing these for seed/plant increase, characterization and preliminary evaluation, and also for Table 2: Different types of data measurement further detailed evaluation and documentation. During evaluation it is important that attributes scored have some general and uniform basis, so that the traits described are passed from generation to generation Scale Basis of observation Example within an accession or can be expected to appear in the progeny of a breeder's cross. Interval Direct measurement of an attribute Plant height, days to flower, tuber weight, Germplasm collected in the form of vegetative propagules (especially in case of fruit crops) are subject to number of flowers per plant quick drying and deterioration. The fruit crops germplasm should not be collected in the form of seeds Ratio Combination of two direct measurement Harvest index, % protein, oil or sugar except the germplasm/ species having non-availability of vegetative propagules. The budwood of fruit trees or inference from a single measurement content should be utilized for propagation as early as possible, particularly of tropical and subtropical fruits, on Ordinal Assigning, sometimes subjectively, a Susceptibility to pests and diseases, suitable rootstock. Similarly, other propagules viz., cuttings for rooting, layers, suckers, slips, crowns, relative value from a standard scale overall processing quality, leaf or seed shapes runners etc. for establishment and multiplication of germplasm should also be utilized as of fruit crops. The fruit crops germplasm grown through seeds are papaya, phalsa, karonda and to some extent jackfruit Nominal Assignment of qualitative character Flower colour, seed patterns although the resultant seedling may not be true to type. When a population is classified into distinct states into arbitrarily numbered classes phenotypic classes for a given character, it is discontinuous variation and called as qualitative type. Such characters are under the control of one or few genes. Such variations can be analysed by count and ratios like The following useful descriptors are also required to be undertaken: presence and absence of a character etc. When a character shows continuous variation, it is called as I. What variation exists within a trial and what confidence can be placed on the score value quantitative variation. Such characters are controlled by many genes with each gene contributing to the total phenotypic variation. Under qualitative characters a) plant data, namely plant growth habit, stem II. The variation among trials and shape, leaf lamina shape, ii) inflorescence and fruit data, namely, sex type, flower colour, fruit shape, fruit III. Whether a scored value can be considered as 'good' or 'bad'. In some cases, they will also be needed as ridges per fruit, flesh texture etc. and in iii) seed data, such as seediness, seed cavity (small/medium/large) controls to verify that a particular test e.g., disease inoculation, has functioned correctly. In connection are scored in comparison to checks to determine variation within and among the traits. Quantitative with disease observation in the field based on chance outbreaks it is worth saying that non-incidence characters such as plant height, days to flowering, number of fruiting nodes per plant, number of pods per of disease may be due to either resistance or disease escape. cluster, number of fruits, days to maturity are influenced by the environment. The choice of check line will depend very much on circumstances. For preliminary evaluation, locally Evaluation of fruit crops germplasm often needs inclusion or planting of a pollinator in the germplasm lines. adapted cultivars provide understandable comparisons and a dependable way of monitoring trial (often In several cases 1-9 code rating should be followed. In case of petal colour, fruit skin colour or pulp colour etc. year to year) variation. For detailed evaluation which usually addresses one trait at a time, there will often an ascending order should be followed (e.g. white, yellow, pink, brown, red, gray, blue, violet and black). be a well-recognized set of checks that cover the likely range of scores e.g., known resistant and susceptible Similarly for fruit shape an order of flat, round, globose, oval, oblong, cordate, elliptic, cylindrical, and cultivars or accessions for disease reaction. arched should be followed. However, scope must be left to suitably incorporate specific positions like short- elliptical, broad-elliptical, and narrowly-elliptical. The same will apply to other morphological descriptors An additional problem is the question of Genotype x Environment (G x E) interaction.This has the practical like leaf shape, leaf apex, leaf base, fruits base, fruit apex, fruit beak and stone etc. Fruit taste, total soluble consequence of significantly changing the expression of accessions. In practice, the number of accessions solids content, flesh-stone ratio are highly important characters in fruit crops characterization. Overall processed in most genetic resources work is extensive and anyone accession is likely to be evaluated only a productivity status of the germplasm will reflect its use in breeding and commerce. 94 95 few times, perhaps only once, for anyone trait. It is, however, useful to monitor with the help of the check Pollination varieties for the occurrence of G x E interaction among trials so as to realize the extent it can occur for anyone The pollinator facilitates fruit set in self-incompatible and self-unfruitful types. This will also enable the character. An estimate can then be made as to the reliability of scores. curator to identify cross-incompatible germplasm. However, this involves floral biology studies and usual Methods of propagation of fruit crops pollination techniques. Such studies can be taken up as soon as the germplasm starts flowering. There are different methods used for the multiplication/propagation of fruit crops viz., seed; layering (air For recording observation on vegetative growth parameters, only untrained, non-juvenile germplasm layering (gootee), stool (mound) layering and trench layering); cutting (hardwood, semi hardwood and soft trees/plants are to be studied for characterization and evaluation. The normal practices of training and wood); grafting (tongue, cleft, inarching, veneer and whip grafting); budding (shield, patch, chip, forkert and pruning can be adopted when the tree comes in fruiting stage. The flower and fruiting characters can be ring budding); suckers (slips, crowns, stem discs); runner and micropropagation (tissue culture and shoot tip recorded after about 5 years of tree growth except in banana, papaya, pine apple, strawberry etc. culture). The fruit crops are perennial in nature. Majority of the fruit crops come into flowering and fruiting after 3-5 In vegetative propagation care must be taken for proper establishment of the material so that it may not be years, if grown through grafting and budding. The fruit plants grown through seed (seedling) or on own roots lost due to poor adaptation, biotic and abiotic stresses. Therefore, the plants must be multiplied in sufficient (cutting, layering, sucker, runner, micro-propagation etc.) may take even more time to fruit. Moreover, many number. It is also important that the site for characterization should be as close as possible to that of its of them reach full (commercial) bearing after 10-15 years except in crops like banana, papaya, pineapple, original collection site to avoid climate effect on growth and fruiting behaviour of the germplasm. strawberry etc. which start bearing after 1·2 years. The fruit crops trees also take 8-15 years to attain their full size. As such it is not even possible to describe their growth habit in early stage. It is, therefore, desirable Rootstocks used for fruit crops to arrive at a compromising age of the tree when characterization and evaluation can be taken up without In situations where the germplasm has been propagated/multiplied through grafting and budding, it is waiting for the tree to reach in full bearing capacity. In many cases it has been suggested herein that the apparent that a rootstock has been used. Two types of rootstocks are commonly used. characterization and evaluation of fruit crops be done in the 3rd year of bearing. However, if the fruiting starts in 1-2 years, these can be evaluated accordingly. I. Seedling rootstock: Grown from the seeds of the same fruit crop or the seeds ofany other related species which is graft compatible. Guidelines for recording descriptors II. Standard/clonal rootstock: These are normally available for some of the temperatefruit crops and are Following are the useful guidelines for scoring and recording of characterization and evaluation descriptors. well defined for their characteristics viz. vigour, precocity, biotic and abiotic stress resistance etc. These may be strictly followed. It is important that a particular fruit crop germplasm be grafted/budded on the samerootstock. If seedlings a) Observations are to be recorded in the CGS system. The units are mentioned in parenthesis with the are used as rootstock, the seeds must be collected from a single treeand grown to raise the seedlings to descriptor name; bring uniformity in the scion. On the other hand the clonal rootstock produce uniform scion. However, few important factors are to be kept in mind while using clonal rootstocks. b) Presence/absence of characters are scored as 0 (absent) and 1 (present); I. Do not use the rootstocks which produce very vigorous or very dwarf trees, c) Many quantitative descriptors which are continuously variable are recorded on a 1 to 9 scale, where, 1 is for very low; 2 very low to low; 3 low; 4 low to intermediate; 5 intermediate; 6 intermediate to high; 7 II. Use only those clonal rootstock whose graft compatibility is well known, high; 8 high to very high; and 9 is for very high are the expression of a character. If the character is not III. The rootstocks which (a) produce a uniform graft union with scion or'(b) even growsover the scion be expressed, '0' should be recorded. For some characters, only a selected scores i.e., 1, 3, 5, 7, 9 are preferred. Do not use rootstock where Scion over grows the root stock. described. Where this has occurred, it shows the gradient (from very low to very high) for that particular descriptor; IV. Also avoid those rootstocks whose delayed incompatibility character has been reported. V. Same rootstock (seedling or clonal) is to be used for preparing the material forevaluation at all the sites. d) When the descriptor state is inapplicable, '0' is used as the descriptor value, e.g. if an accession does not have a central leaf, '0' would be scored for the descriptor. Methodology for Germplasm Characterisation/Evaluation Trials: Experimental design e) For most at the qualitative characters the last state is 'Others', if a particular state is not present in the The fruit trees occupy large area vertically as well as horizontally due to their growth habit. For the proper descriptor states, write the code '99' for 'Others'. Additional information for that descriptor may be characterisation and evaluation it is important to provide enough space to express it in full potential appended in the 'Remarks' descriptor. however the ultimate vigour of the tree depends on the tree character. A Randomized Block Design for most of the fruit crops germplasm evaluation may be therefore impracticable due to paucity of land in case of f) The severity of extent of infection or infestation may be recorded on 1·9 scale under the descriptor large number of accessions. In such circumstances "Row Trial System" can be an ideal to be followed. Thus 5- 'Biotic Stress Susceptibility'. However the detailed information in respect of name(s) of 10 plants of an accession of fruit crops germplasm be grown in a single row. The single tree/plant may be disease(s)/pest(s) and causal organism(s) may be appended in the 'Biotic notes' descriptor. considered as one replication. The spacing between row to row and plant to plant depends on the tree g) Dates should be expressed numerically in the format DD/MM/YYYY vigour and its growth habit. h) Standard colour chart e.g. Royal Horticultural Society colour charts should be used to determine the Use of Standard Checks colour of flower/other organs. In order to ascertain the comparative performance of each accession, it is advisable to include at least one standard variety while evaluation. Qualitative and quantitative characters showing range of expression can only be meaningfully assessed in comparison with standard variety. As such locally adopted cultivars along with national recommended checks must be included in the germplasm lines for effective comparative study. 96 97 few times, perhaps only once, for anyone trait. It is, however, useful to monitor with the help of the check Pollination varieties for the occurrence of G x E interaction among trials so as to realize the extent it can occur for anyone The pollinator facilitates fruit set in self-incompatible and self-unfruitful types. This will also enable the character. An estimate can then be made as to the reliability of scores. curator to identify cross-incompatible germplasm. However, this involves floral biology studies and usual Methods of propagation of fruit crops pollination techniques. Such studies can be taken up as soon as the germplasm starts flowering. There are different methods used for the multiplication/propagation of fruit crops viz., seed; layering (air For recording observation on vegetative growth parameters, only untrained, non-juvenile germplasm layering (gootee), stool (mound) layering and trench layering); cutting (hardwood, semi hardwood and soft trees/plants are to be studied for characterization and evaluation. The normal practices of training and wood); grafting (tongue, cleft, inarching, veneer and whip grafting); budding (shield, patch, chip, forkert and pruning can be adopted when the tree comes in fruiting stage. The flower and fruiting characters can be ring budding); suckers (slips, crowns, stem discs); runner and micropropagation (tissue culture and shoot tip recorded after about 5 years of tree growth except in banana, papaya, pine apple, strawberry etc. culture). The fruit crops are perennial in nature. Majority of the fruit crops come into flowering and fruiting after 3-5 In vegetative propagation care must be taken for proper establishment of the material so that it may not be years, if grown through grafting and budding. The fruit plants grown through seed (seedling) or on own roots lost due to poor adaptation, biotic and abiotic stresses. Therefore, the plants must be multiplied in sufficient (cutting, layering, sucker, runner, micro-propagation etc.) may take even more time to fruit. Moreover, many number. It is also important that the site for characterization should be as close as possible to that of its of them reach full (commercial) bearing after 10-15 years except in crops like banana, papaya, pineapple, original collection site to avoid climate effect on growth and fruiting behaviour of the germplasm. strawberry etc. which start bearing after 1·2 years. The fruit crops trees also take 8-15 years to attain their full size. As such it is not even possible to describe their growth habit in early stage. It is, therefore, desirable Rootstocks used for fruit crops to arrive at a compromising age of the tree when characterization and evaluation can be taken up without In situations where the germplasm has been propagated/multiplied through grafting and budding, it is waiting for the tree to reach in full bearing capacity. In many cases it has been suggested herein that the apparent that a rootstock has been used. Two types of rootstocks are commonly used. characterization and evaluation of fruit crops be done in the 3rd year of bearing. However, if the fruiting starts in 1-2 years, these can be evaluated accordingly. I. Seedling rootstock: Grown from the seeds of the same fruit crop or the seeds ofany other related species which is graft compatible. Guidelines for recording descriptors II. Standard/clonal rootstock: These are normally available for some of the temperatefruit crops and are Following are the useful guidelines for scoring and recording of characterization and evaluation descriptors. well defined for their characteristics viz. vigour, precocity, biotic and abiotic stress resistance etc. These may be strictly followed. It is important that a particular fruit crop germplasm be grafted/budded on the samerootstock. If seedlings a) Observations are to be recorded in the CGS system. The units are mentioned in parenthesis with the are used as rootstock, the seeds must be collected from a single treeand grown to raise the seedlings to descriptor name; bring uniformity in the scion. On the other hand the clonal rootstock produce uniform scion. However, few important factors are to be kept in mind while using clonal rootstocks. b) Presence/absence of characters are scored as 0 (absent) and 1 (present); I. Do not use the rootstocks which produce very vigorous or very dwarf trees, c) Many quantitative descriptors which are continuously variable are recorded on a 1 to 9 scale, where, 1 is for very low; 2 very low to low; 3 low; 4 low to intermediate; 5 intermediate; 6 intermediate to high; 7 II. Use only those clonal rootstock whose graft compatibility is well known, high; 8 high to very high; and 9 is for very high are the expression of a character. If the character is not III. The rootstocks which (a) produce a uniform graft union with scion or'(b) even growsover the scion be expressed, '0' should be recorded. For some characters, only a selected scores i.e., 1, 3, 5, 7, 9 are preferred. Do not use rootstock where Scion over grows the root stock. described. Where this has occurred, it shows the gradient (from very low to very high) for that particular descriptor; IV. Also avoid those rootstocks whose delayed incompatibility character has been reported. V. Same rootstock (seedling or clonal) is to be used for preparing the material forevaluation at all the sites. d) When the descriptor state is inapplicable, '0' is used as the descriptor value, e.g. if an accession does not have a central leaf, '0' would be scored for the descriptor. Methodology for Germplasm Characterisation/Evaluation Trials: Experimental design e) For most at the qualitative characters the last state is 'Others', if a particular state is not present in the The fruit trees occupy large area vertically as well as horizontally due to their growth habit. For the proper descriptor states, write the code '99' for 'Others'. Additional information for that descriptor may be characterisation and evaluation it is important to provide enough space to express it in full potential appended in the 'Remarks' descriptor. however the ultimate vigour of the tree depends on the tree character. A Randomized Block Design for most of the fruit crops germplasm evaluation may be therefore impracticable due to paucity of land in case of f) The severity of extent of infection or infestation may be recorded on 1·9 scale under the descriptor large number of accessions. In such circumstances "Row Trial System" can be an ideal to be followed. Thus 5- 'Biotic Stress Susceptibility'. However the detailed information in respect of name(s) of 10 plants of an accession of fruit crops germplasm be grown in a single row. The single tree/plant may be disease(s)/pest(s) and causal organism(s) may be appended in the 'Biotic notes' descriptor. considered as one replication. The spacing between row to row and plant to plant depends on the tree g) Dates should be expressed numerically in the format DD/MM/YYYY vigour and its growth habit. h) Standard colour chart e.g. Royal Horticultural Society colour charts should be used to determine the Use of Standard Checks colour of flower/other organs. In order to ascertain the comparative performance of each accession, it is advisable to include at least one standard variety while evaluation. Qualitative and quantitative characters showing range of expression can only be meaningfully assessed in comparison with standard variety. As such locally adopted cultivars along with national recommended checks must be included in the germplasm lines for effective comparative study. 96 97 Summary Germplasm Core collections- Utility of germplasm depends on the information generated through characterization and evaluation. It starts from the germplasm augmentation, characterization, preliminary and detailed evaluation for particular traits, documentation and utilization for crop improvement programmes and maintenance. In Principles and Methodologies this chapter the characterization and evaluation with respect to the fruit crops has been covered by Rakesh Singh* and D. R. Choudhury following standard method and guidelines framed by NBPGR and Bioversity International. Division of Genomic Resources References ICAR-National Bureau of Plant Genetic Resources, New Delhi. *Email: [email protected] 1. Dillon WR and M Goldstein (1984) Multivariate analysis. Methods and applications. John Wiley Inc. New York. Germplasm 2. ICAR-NBPGR (2016) Guidelines for management of plant genetic resources in India. ICAR-National Germplasm is a living tissue from which it is possible to produce new plants. It can be a seed or a root, a piece Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. of stem, pollen, or even only a few cells may be converted into an entire plant. Germplasm provides data on 3. Mahajan RK, KK Gangopadhyay, G Kumar, VK Dobhal, U Srivastava, PN Gupta and SK Pareek (2002) the genetic composition of a population, a valuable natural resource for plant diversity study. Agriculture Minimal Descriptors of Agri-Horticultural Crops. Part III: Fruit Crops. National Bureau of Plant Genetic benefits from uniformity among crop plants within a variety, which ensures consistent yields and make Resources, Pusa Campus, New Delhi, x + 242 p. management easier. However, genetic uniformity leaves crop vulnerable to new pests and stresses. Genetic diversity of germplasm gives plant breeders the sustained ability to develop new high yielding varieties that 4. Rana JC (2017) Characterisation and evaluation of plant genetic resources – guidelines. Training can resist constantly evolving pests, diseases and environmental stresses. Sexually compatible wild species manaual, National Training Course on Management of Plant Genetic Resources of fruit crops. and landraces – ancestral varieties of crop species- are the key to genetic diversity, but due to continuous Organised by ICAR-NBPGR New Delhi in Collaboration with ICAR-AICRP on fruits from 22nd – 25th March, land shrinking due to human activities many plant species and varieties are disappearing. Therefore, to 2017. gather, preserve, evaluate, catalogue and distribute germplasm for people all over the world to use, the 5. Singh BP and U Srivastava (2004) Plant Genetic Resources in Indian Perspective, Theory and Practices. plant science community has developed conservation programs. Ex-situ germplasm collections have Indian Council of Agricultural Research, New Delhi, 715 p. increased enormously in number and size over the last three to four decades as a result of global efforts to conserve plant genetic resources for food and agriculture. The large sizes of many of these collections, either individually or collectively for a given species complicate the characterization, evaluation, utilization and maintenance of the conserved germplasm. The approach of forming core collections was introduced to increase the efficiency of characterization and utilization of collections stored in the gene banks, while preserving as much as possible the genetic diversity of the entire collection (Frankel, 1984; Brown, 1989a). Frankel (1984) defined a core collection as a limited set of accessions representing, with minimum repetitiveness, the genetic diversity of a crop species and its wild relatives. From the original definition, several operational definitions have been coined (Brown, 1995 and van Hintum et al., 2000). Plant Genetic Resources The full spectrum of PGR consists of diverse type of collections such as those derived from centers of diversity, centers of domestication and from breeding programs. A functional classification of the PGR given by Gautam et al., 2006, broadly include landraces, modern varieties, wild and weedy relatives of cultivated plants, and potential domesticates such as wild species used or potential value. Landraces: these are the conventional and invaluable legacy of crop germplasm. The diversity in thousands of years of deliberate selection, exposure to a range of natural conditions, natural hybridization and other modifications which farmers have attempted. These conventional landraces are regularly profoundly variable in appearance, but each is identifiable, having specific properties or characteristics, such as early or late developing, versatility to specific soil. Utilization of Landraces often have survived long among various biotic and abiotic stresses in cultivation and thus offer a good source of genes and potential resistance, making them important for modern plant breeding. Modern varieties: These are also referred to as high yielding varieties, particularly, developed during 1950s and 60s. Their spread was more rapid and dramatic than anything that ever happened in agriculture before. Within a decade, the new varieties of wheat and rice were grown on nearly 55 million ha in the third world. It has been contended that these modern varieties, with advanced recombination have a narrow genetic base. There is however, a divergent view that many of the modern cultivars possessing multiple disease and insect-pest resistance have been evolved from varied sources in the agronomically adapted landraces or varieties and have the plasma of a large number of genetic stocks.

98 99 Summary Germplasm Core collections- Utility of germplasm depends on the information generated through characterization and evaluation. It starts from the germplasm augmentation, characterization, preliminary and detailed evaluation for particular traits, documentation and utilization for crop improvement programmes and maintenance. In Principles and Methodologies this chapter the characterization and evaluation with respect to the fruit crops has been covered by Rakesh Singh* and D. R. Choudhury following standard method and guidelines framed by NBPGR and Bioversity International. Division of Genomic Resources References ICAR-National Bureau of Plant Genetic Resources, New Delhi. *Email: [email protected] 1. Dillon WR and M Goldstein (1984) Multivariate analysis. Methods and applications. John Wiley Inc. New York. Germplasm 2. ICAR-NBPGR (2016) Guidelines for management of plant genetic resources in India. ICAR-National Germplasm is a living tissue from which it is possible to produce new plants. It can be a seed or a root, a piece Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. of stem, pollen, or even only a few cells may be converted into an entire plant. Germplasm provides data on 3. Mahajan RK, KK Gangopadhyay, G Kumar, VK Dobhal, U Srivastava, PN Gupta and SK Pareek (2002) the genetic composition of a population, a valuable natural resource for plant diversity study. Agriculture Minimal Descriptors of Agri-Horticultural Crops. Part III: Fruit Crops. National Bureau of Plant Genetic benefits from uniformity among crop plants within a variety, which ensures consistent yields and make Resources, Pusa Campus, New Delhi, x + 242 p. management easier. However, genetic uniformity leaves crop vulnerable to new pests and stresses. Genetic diversity of germplasm gives plant breeders the sustained ability to develop new high yielding varieties that 4. Rana JC (2017) Characterisation and evaluation of plant genetic resources – guidelines. Training can resist constantly evolving pests, diseases and environmental stresses. Sexually compatible wild species manaual, National Training Course on Management of Plant Genetic Resources of fruit crops. and landraces – ancestral varieties of crop species- are the key to genetic diversity, but due to continuous Organised by ICAR-NBPGR New Delhi in Collaboration with ICAR-AICRP on fruits from 22nd – 25th March, land shrinking due to human activities many plant species and varieties are disappearing. Therefore, to 2017. gather, preserve, evaluate, catalogue and distribute germplasm for people all over the world to use, the 5. Singh BP and U Srivastava (2004) Plant Genetic Resources in Indian Perspective, Theory and Practices. plant science community has developed conservation programs. Ex-situ germplasm collections have Indian Council of Agricultural Research, New Delhi, 715 p. increased enormously in number and size over the last three to four decades as a result of global efforts to conserve plant genetic resources for food and agriculture. The large sizes of many of these collections, either individually or collectively for a given species complicate the characterization, evaluation, utilization and maintenance of the conserved germplasm. The approach of forming core collections was introduced to increase the efficiency of characterization and utilization of collections stored in the gene banks, while preserving as much as possible the genetic diversity of the entire collection (Frankel, 1984; Brown, 1989a). Frankel (1984) defined a core collection as a limited set of accessions representing, with minimum repetitiveness, the genetic diversity of a crop species and its wild relatives. From the original definition, several operational definitions have been coined (Brown, 1995 and van Hintum et al., 2000). Plant Genetic Resources The full spectrum of PGR consists of diverse type of collections such as those derived from centers of diversity, centers of domestication and from breeding programs. A functional classification of the PGR given by Gautam et al., 2006, broadly include landraces, modern varieties, wild and weedy relatives of cultivated plants, and potential domesticates such as wild species used or potential value. Landraces: these are the conventional and invaluable legacy of crop germplasm. The diversity in thousands of years of deliberate selection, exposure to a range of natural conditions, natural hybridization and other modifications which farmers have attempted. These conventional landraces are regularly profoundly variable in appearance, but each is identifiable, having specific properties or characteristics, such as early or late developing, versatility to specific soil. Utilization of Landraces often have survived long among various biotic and abiotic stresses in cultivation and thus offer a good source of genes and potential resistance, making them important for modern plant breeding. Modern varieties: These are also referred to as high yielding varieties, particularly, developed during 1950s and 60s. Their spread was more rapid and dramatic than anything that ever happened in agriculture before. Within a decade, the new varieties of wheat and rice were grown on nearly 55 million ha in the third world. It has been contended that these modern varieties, with advanced recombination have a narrow genetic base. There is however, a divergent view that many of the modern cultivars possessing multiple disease and insect-pest resistance have been evolved from varied sources in the agronomically adapted landraces or varieties and have the plasma of a large number of genetic stocks.

98 99 Elite germplasm are the materials generated in a breeding programme and identified to be promising. It includes elite finished products that could not come under cultivation. Additionally, it also includes genetic redundancy and is crucial for exploiting germplasm via genome stocks and novel types developed through pre-breeding. Plant breeders preferably and almost exclusively research. These might consist of sets that constitute the broad use this elite germplasm to produce new commercials cultivars. genetic version available for a crop genome. The accessions or entries excluded from the pattern would be retained as a reserve Wild and weedy relatives the PGR also encompass wild progenitors of crop plants and weedy races. The collection. The main purpose of core fraction is to provide efficient wild and weedy races interact genetically with cultivated as well as truly wild species. The diversity of the access to the whole collection that should be representative of the wild and weedy relatives has enabled them to survive longer than the oldest cultivated type, and to survive diversity. The accessions in core collection should not be selected without human assistance. Thus, as sources of resistance/tolerance, these are a treasure and are used in on the basis of percentage of the entire collection but should be plant breeding programs. Working with them is very difficult, as along with every desirable characteristic selected to have a manageable collection scaled down to the needs that is transferred to cultivated types, a number of linked but undesirable characters are also transferred. In of the breeder and to include the widest possible range of an increasing number of crops the genes for resistance / tolerance stored in the wild relatives play an variability. Researchers have outlined the procedure to develop important role and, in some instances, the only resources available. Almost exclusively use this elite core collection by using information on origin and characteristics of germplasm to produce new commercials cultivars. the accessions. According the guiding principles to constitute a Figure 1: Schematic representation Obsolete Cultivar- Improved varieties of the recent past are known as Obsolete cultivars. These are varieties core collection are (i) the entire collection is a large taxonomic of Core Collection Concept that were popular earlier and now has been replaced by new varieties. For eg. K 68, K65 and Pb 591 were entity, (ii) the core collection has a greatly reduced size, (iii) the core popular tall varieties of Wheat which has been replaced by high yielding dwarf Mexican Wheat variety. is a true representative of entire collection and (iv) the core too is nearly as diverse as the entire collection. However obsolete cultivars still come under Plant Genetic resources. Core collection is available mostly in all the crop plants. Core collections are established to improve the conservation and use of genetic resources. Core can help in gene bank management, in the decisions that Biodiversity in fruit crops need to be taken on what should be conserved and in the improved use of material held in gene banks. The overall objective is to get a collection conserved better and used more effectively. It would be There is greater tolerance of fruit species to variable agro-ecological conditions. Nature has produced counterproductive to have a core so large that it suffers the same problems as the whole collection. On the countless species of plants, of which only about 5,000 plant species are used globally by human beings. other hand, a core that failed to contain a significant fraction of the whole collection's diversity would not Today, only about 150 plant species are essential to mankind in terms of food. There is greater dependence serve its purpose There are currently three different strategies for generating a core collection from a large of few species of plants, i.e. about 20-30 worldwide. This eventually led to the loss of native genetic population using molecular marker data (Odong et al., 2013). Firstly, it is possible to build up a core resources, which are otherwise important as genetic diversity building blocks. It is estimated that there are collection that represents the individual accessions (CC-I), e.g., a uniform representation of the original about 500 species of tropical fruit trees under 30 families and 59 genera in Asia Pacific Oceana region (Arora population. Second, it is possible to select a core collection based on accessions that represents the & Rao, 1998). In Southeast Asia alone, there are 120 major fruit species and 275 minor fruit species (Verheij distribution of all relevant traits (CC-D), e.g., if the majority of the original population contains allele A at a and Coronel ,1991). In Asia 50-60 species belong to the most important indigenous fruits (Arora and Rao, given locus, then the core collection should imitate this behavior. Thirdly, accessions can be selected that 1996). Citrus, Banana, Mango, Jackfruit, Litchi and Durian occupy 80 per cent of total fruit production in the represent the extremes of all relevant traits (CC-X), e.g., different entries into the core collection should be region. India is an important centre of origin and diversity of many horticultural crops including fruit crops as diverse as possible with regard to the selected traits. Depending on which strategy is used, there are like Mango, Citrus and Banana. The sub-continent has tropical, subtropical and temperate climate. disadvantages in terms of working with the whole population. For example, trait customized core Therefore, a variety of fruits originating in tropics, subtropics and temperate regions of the world have been collections (CC-X), which aim to maximize diversity for that particular trait, would be better suited to finding introduced in India and many of them are commercially grown in the country. rare alleles than a core collection that is designed to represent the original population (CC-I). The loss of rare It becomes mandate to have a strategy to conserve these plant genetic resources for future use. A large alleles, especially in plant and resistance breeding, is one of the main concerns when working with core number of accessions of a wide range of crop species are stored by Gene banks. By the middle of the collections (Odong et al., 2013). twentieth century, Gene banks were developed to conserve cultivated biodiversity as landraces started to be replaced by modern varieties. In general, this change was agreed as a crucial measure to safeguard the Methods of creating core collection future. Gene banks conserves and makes available genetic material to consumers such as breeders, The method of creating a core collection is essentially very simple. Even a random selection from a set might researchers and even farmers. Germplasm collection includes collecting samples of a species from field be called a core collection, albeit less effective than other techniques. A random selection would reflect populations or natural environments for protection and eventual use. Depending on the breeding scheme genetic diversity better than a sequential collection by simple equidistant sampling of accessions., of the species, the collection unit can be botanic seeds or vegetative propagules. However, it becomes (Spagnoletti Zeuli and Qualset , 1993; Vaughan, 1991). For instance, all accessions with accession numbers problematic when seeds are unavailable or nonviable due to: damage of plants by grazing or diseases; large ending with zero could be included in a 10 percent core. But it is much more difficult to pick a core selection and fleshy seeds that are difficult to transport; or where samples are not likely to remain viable during to ensure representative diversity. A general procedure for the selection of a core collection can be divided transportation due to remoteness of the collecting site from the gene bank. into four steps, which will be briefly presented here, and discussed in detail in the following sections. Core collection: an approach of efficient germplasm conservation 1. The domain description: In constructing a core collection, the first step is to identify the material to be represented for eg. Passport data Since the inception of the idea of core collections, a body of literature on the theory and practice of core collections has accumulated. Very many approaches for selecting core collections have been proposed and 2. A division into different classes: This requires the division of the domain into classes that should be as used (e.g. M-Strat (Gouesnard et al., 2001), Genetic distance sampling (Jansen and van Hintum, 2007), genetically separate as possible, for eg in rice one can divide the domain into different classes by their PowerCore (Kim et al., 2007) and CoreHunter (Thachuk et al., 2009). grain length, grain width, grain color etc. “Core collection” has been proposed earlier as a collection that encompasses a representative sample of the 3. Assignment of entries across classes: First, it is important to decide the total size of the core set, and entire collection with minimal repetitiveness and maximum genetic range. A core collection reduces then to choose the number of entries per category. 100 101 Elite germplasm are the materials generated in a breeding programme and identified to be promising. It includes elite finished products that could not come under cultivation. Additionally, it also includes genetic redundancy and is crucial for exploiting germplasm via genome stocks and novel types developed through pre-breeding. Plant breeders preferably and almost exclusively research. These might consist of sets that constitute the broad use this elite germplasm to produce new commercials cultivars. genetic version available for a crop genome. The accessions or entries excluded from the pattern would be retained as a reserve Wild and weedy relatives the PGR also encompass wild progenitors of crop plants and weedy races. The collection. The main purpose of core fraction is to provide efficient wild and weedy races interact genetically with cultivated as well as truly wild species. The diversity of the access to the whole collection that should be representative of the wild and weedy relatives has enabled them to survive longer than the oldest cultivated type, and to survive diversity. The accessions in core collection should not be selected without human assistance. Thus, as sources of resistance/tolerance, these are a treasure and are used in on the basis of percentage of the entire collection but should be plant breeding programs. Working with them is very difficult, as along with every desirable characteristic selected to have a manageable collection scaled down to the needs that is transferred to cultivated types, a number of linked but undesirable characters are also transferred. In of the breeder and to include the widest possible range of an increasing number of crops the genes for resistance / tolerance stored in the wild relatives play an variability. Researchers have outlined the procedure to develop important role and, in some instances, the only resources available. Almost exclusively use this elite core collection by using information on origin and characteristics of germplasm to produce new commercials cultivars. the accessions. According the guiding principles to constitute a Figure 1: Schematic representation Obsolete Cultivar- Improved varieties of the recent past are known as Obsolete cultivars. These are varieties core collection are (i) the entire collection is a large taxonomic of Core Collection Concept that were popular earlier and now has been replaced by new varieties. For eg. K 68, K65 and Pb 591 were entity, (ii) the core collection has a greatly reduced size, (iii) the core popular tall varieties of Wheat which has been replaced by high yielding dwarf Mexican Wheat variety. is a true representative of entire collection and (iv) the core too is nearly as diverse as the entire collection. However obsolete cultivars still come under Plant Genetic resources. Core collection is available mostly in all the crop plants. Core collections are established to improve the conservation and use of genetic resources. Core can help in gene bank management, in the decisions that Biodiversity in fruit crops need to be taken on what should be conserved and in the improved use of material held in gene banks. The overall objective is to get a collection conserved better and used more effectively. It would be There is greater tolerance of fruit species to variable agro-ecological conditions. Nature has produced counterproductive to have a core so large that it suffers the same problems as the whole collection. On the countless species of plants, of which only about 5,000 plant species are used globally by human beings. other hand, a core that failed to contain a significant fraction of the whole collection's diversity would not Today, only about 150 plant species are essential to mankind in terms of food. There is greater dependence serve its purpose There are currently three different strategies for generating a core collection from a large of few species of plants, i.e. about 20-30 worldwide. This eventually led to the loss of native genetic population using molecular marker data (Odong et al., 2013). Firstly, it is possible to build up a core resources, which are otherwise important as genetic diversity building blocks. It is estimated that there are collection that represents the individual accessions (CC-I), e.g., a uniform representation of the original about 500 species of tropical fruit trees under 30 families and 59 genera in Asia Pacific Oceana region (Arora population. Second, it is possible to select a core collection based on accessions that represents the & Rao, 1998). In Southeast Asia alone, there are 120 major fruit species and 275 minor fruit species (Verheij distribution of all relevant traits (CC-D), e.g., if the majority of the original population contains allele A at a and Coronel ,1991). In Asia 50-60 species belong to the most important indigenous fruits (Arora and Rao, given locus, then the core collection should imitate this behavior. Thirdly, accessions can be selected that 1996). Citrus, Banana, Mango, Jackfruit, Litchi and Durian occupy 80 per cent of total fruit production in the represent the extremes of all relevant traits (CC-X), e.g., different entries into the core collection should be region. India is an important centre of origin and diversity of many horticultural crops including fruit crops as diverse as possible with regard to the selected traits. Depending on which strategy is used, there are like Mango, Citrus and Banana. The sub-continent has tropical, subtropical and temperate climate. disadvantages in terms of working with the whole population. For example, trait customized core Therefore, a variety of fruits originating in tropics, subtropics and temperate regions of the world have been collections (CC-X), which aim to maximize diversity for that particular trait, would be better suited to finding introduced in India and many of them are commercially grown in the country. rare alleles than a core collection that is designed to represent the original population (CC-I). The loss of rare It becomes mandate to have a strategy to conserve these plant genetic resources for future use. A large alleles, especially in plant and resistance breeding, is one of the main concerns when working with core number of accessions of a wide range of crop species are stored by Gene banks. By the middle of the collections (Odong et al., 2013). twentieth century, Gene banks were developed to conserve cultivated biodiversity as landraces started to be replaced by modern varieties. In general, this change was agreed as a crucial measure to safeguard the Methods of creating core collection future. Gene banks conserves and makes available genetic material to consumers such as breeders, The method of creating a core collection is essentially very simple. Even a random selection from a set might researchers and even farmers. Germplasm collection includes collecting samples of a species from field be called a core collection, albeit less effective than other techniques. A random selection would reflect populations or natural environments for protection and eventual use. Depending on the breeding scheme genetic diversity better than a sequential collection by simple equidistant sampling of accessions., of the species, the collection unit can be botanic seeds or vegetative propagules. However, it becomes (Spagnoletti Zeuli and Qualset , 1993; Vaughan, 1991). For instance, all accessions with accession numbers problematic when seeds are unavailable or nonviable due to: damage of plants by grazing or diseases; large ending with zero could be included in a 10 percent core. But it is much more difficult to pick a core selection and fleshy seeds that are difficult to transport; or where samples are not likely to remain viable during to ensure representative diversity. A general procedure for the selection of a core collection can be divided transportation due to remoteness of the collecting site from the gene bank. into four steps, which will be briefly presented here, and discussed in detail in the following sections. Core collection: an approach of efficient germplasm conservation 1. The domain description: In constructing a core collection, the first step is to identify the material to be represented for eg. Passport data Since the inception of the idea of core collections, a body of literature on the theory and practice of core collections has accumulated. Very many approaches for selecting core collections have been proposed and 2. A division into different classes: This requires the division of the domain into classes that should be as used (e.g. M-Strat (Gouesnard et al., 2001), Genetic distance sampling (Jansen and van Hintum, 2007), genetically separate as possible, for eg in rice one can divide the domain into different classes by their PowerCore (Kim et al., 2007) and CoreHunter (Thachuk et al., 2009). grain length, grain width, grain color etc. “Core collection” has been proposed earlier as a collection that encompasses a representative sample of the 3. Assignment of entries across classes: First, it is important to decide the total size of the core set, and entire collection with minimal repetitiveness and maximum genetic range. A core collection reduces then to choose the number of entries per category. 100 101 4. Selection of entries: The last step is the option of entries that are to be included in the core from each is important to have readily accessible, authentic and well-documented content at the core, practical category. considerations may also play a role in this decision. This includes such factors as the availability of the seeds and the reliability and quantity of the accession data. In the case of the 'random core' and 'equidistant core' the domain is not divided into genetically different classes, and the choice of entries is either random or equidistant. If the domain could be divided into classes, If extensive and complete molecular marker data or other characterization data are available, it is possible an increase in the consistency of the core would be expected (Erskine and Muehlbauer, 1991) to base the choice of entries on the result of a multivariate analysis. This would allow an optimal representation of diversity within the group. Examples of variations of such an approach are Spagnoletti Definition of the domain Zeuli and Qualset (1993), who first clustered durum wheat accessions on the basis of morphological data The content that the core should represent - that is, the core selection domain - can vary from core to core. It and then stratified on the basis of country of origin, and Charmet and Balfourier (1995) who clustered their will rely on the available material and the collection's intent. The domain could include, for example, the French perennial ryegrass populations on the basis of agronomic traits with the constraint that populations CSIRO collection of perennial glycine (Brown et al., 1987), the complete US peanut germplasm collection had to be collected in the same area to be clustered together. If the pedigrees of material in a certain group (Holbrook et al., 1993), the Peruvian quinoa collection (Ortiz et al., 1998), the small annual Medicago are known, it is also possible to use these to maximize the allelic richness in the entries selected from this collection (Diwan et al., 1994), or the entire genepool of Hordeum (Knüpffer and van Hintum, 1995). group (van Hintum, 1994b; van Hintum and Haalman, 1994). However, material with unique characteristics, such as local maize populations with a strong combining capacity (Radovic and Jelovac, 1994) or Pisum sativum germplasm with disease resistance (Matthews and Why to go for core development? The examples given epitomize its importance Ambrose, 1994) can also form a part of the domain. Core development is suggested for now to enhance the use of germplasm collections available in gene bank Division in groups (Frankel, 1984). Core Collection are an expeditious means to intensively characterize, explore, and use genetic resources conserved in gene bank as well as monitor genetic drift during preservation and identify The division of the domain into separate groups can be done stepwise; that is, a group of accessions is gaps in genetic diversity for further development of the gene bank (Frankel, 1984; Frankel and Brown, 1984, divided into as genetically separate subgroups as possible at every step. This process of branching, which Brown, 1989a). The idea of core collection was proposed by Frankel and Brown, 1984 is a small set of can be done on the basis of taxonomy, domestication, distribution, breeding history and utilization, or on accessions (usually 10 % of the population) chosen to represent the genetic spectrum of the whole the basis of molecular marker or other characterization details, was done by Van Hintum (1994a). A collection (Ebana et al., 2008). The sampling percentage of the core collection has long been under debate 'diversity tree' can be developed which reflects the domain's genetic structure. The branching exercise ends (Wang et al., 2014); 20%-30% of the sampling percentage has been suggested by Yonezawa et al., 1995. Mini when no further subdivision can be made into genetically distinct classes or adequate branching has been core collection of about 1% has also been suggested for very large collection (Upadhaya et al., 2010; Jiang et achieved. Such studies have been done in 'lentil landraces from Greece and Turkey' (Erskine and al., 2011; Sharma et al., 2012). A mini core subset from USDA rice gene bank has been developed using 26 Muehlbauer, 1991), 'Medicago arabica' (Diwan et al., 1994) or 'pea landraces from Mongolia' (Matthews phenotypic trait and 70 molecular markers (Agrama et al., 2009). Still a perfect ratio and fixed size for all and Ambrose, 1994). core collection does not exist as different crops or different constructing goal needs different sampling Allocation of entries percentage (Wang et al., 2014). Several strategies have been successfully deployed for developing a core collection. Stratified random sampling method, in contrast to simple random sampling has been The choice of the number of entries to be included in the core depends on the purpose of the core set and is successfully applied to the development of numerous core collections (Balfourier et al., 1998; Hu et al., subjective. In general, the number of entries chosen is slightly lower than the number of domain accessions, 2000; Chandra et al., 2002; Franco et al., 2006). One common approach for constructing core collection is typically in the range of 5 to 20 percent. grouping the whole collections into major ecotypes and then selecting representatives from each ecotype A stepwise process can again be followed for the distribution of the entries through the classes. When each (Reeves et al., 2012; Wang et al., 2014). A core collection formed for the purpose of capturing accessions group is divided into subgroups, it is determined how the group entries should be divided into subgroups. In with rare or extreme values of the desired trait(s) (e.g., high resistance to pest or high yield) should be this phase, there are many techniques to use. If no information on the significance or diversity of the evaluated differently from one formed with the intention of representing the genetic diversity in the subgroups is available, the number of accessions in the subgroup can be used for this decision. In this case collection. the constant (C), proportional (P) and logarithmic (L) strategies are available (Brown, 1989b). If quantitative Genetic stocks and reference material, including standard or reference material and well-defined marker information about the diversity in the subgroups is available the gene diversity (H) or maximization (M) materials, are to be represented but the number of entries in this group still has to be decided. The final strategy can be used (Schoen and Brown, 1995). And finally, if there is an idea of the relative importance of choice of entries for each group was made by scientists and curators with specific knowledge and the subgroups this can also be used for the allocation. experience with the groups. Their approaches differed as per group for example the representatives of the The strategy for subgroup allocation of entries will depend on the available information for the group and its wild species can be chosen directly by an expert in the field. Similarly, in the case of 'Cultivated material from subgroups and which vary by group. For example, if no additional information on accessions is available in a Europe', a preliminary selection of landraces and cultivars was made by an expert and this selection was group of wild materials divided into subgroups by country of origin, the number of entries for the core characterized using molecular markers and from the result of this investigation, it was concluded that the selection from each subgroup can be chosen based on the number of accessions in the subgroups. If there is selection had to be adjusted and material from southern Europe was added, and northwestern material was detailed molecular marker information available on the majority of accessions in a group of cultivars, one removed. The selection for the group 'Cultivated material from the Americas' was made after close might use a diversity-based strategy, and if a decision has to be made concerning the division in wild and consultation with American barley breeders and researchers. cultivated subgroups, generally a strategy based on the weight or importance of groups for the core Types of core collection collection will be followed. Global core collection: A core collection developed from these global germplasm collections could be Choice of accessions termed as global core collection. The final choice of individual community entries can be made randomly or, where available, on the basis of Regional core collection: Ecological environments influence adaptation of germplasm accessions. For additional data. The goal should be for the diversity of the category to be better reflected by the entries. As it example, groundnut is cultivated in over 113 countries, but it is an important crop in 25 countries in the

102 103 4. Selection of entries: The last step is the option of entries that are to be included in the core from each is important to have readily accessible, authentic and well-documented content at the core, practical category. considerations may also play a role in this decision. This includes such factors as the availability of the seeds and the reliability and quantity of the accession data. In the case of the 'random core' and 'equidistant core' the domain is not divided into genetically different classes, and the choice of entries is either random or equidistant. If the domain could be divided into classes, If extensive and complete molecular marker data or other characterization data are available, it is possible an increase in the consistency of the core would be expected (Erskine and Muehlbauer, 1991) to base the choice of entries on the result of a multivariate analysis. This would allow an optimal representation of diversity within the group. Examples of variations of such an approach are Spagnoletti Definition of the domain Zeuli and Qualset (1993), who first clustered durum wheat accessions on the basis of morphological data The content that the core should represent - that is, the core selection domain - can vary from core to core. It and then stratified on the basis of country of origin, and Charmet and Balfourier (1995) who clustered their will rely on the available material and the collection's intent. The domain could include, for example, the French perennial ryegrass populations on the basis of agronomic traits with the constraint that populations CSIRO collection of perennial glycine (Brown et al., 1987), the complete US peanut germplasm collection had to be collected in the same area to be clustered together. If the pedigrees of material in a certain group (Holbrook et al., 1993), the Peruvian quinoa collection (Ortiz et al., 1998), the small annual Medicago are known, it is also possible to use these to maximize the allelic richness in the entries selected from this collection (Diwan et al., 1994), or the entire genepool of Hordeum (Knüpffer and van Hintum, 1995). group (van Hintum, 1994b; van Hintum and Haalman, 1994). However, material with unique characteristics, such as local maize populations with a strong combining capacity (Radovic and Jelovac, 1994) or Pisum sativum germplasm with disease resistance (Matthews and Why to go for core development? The examples given epitomize its importance Ambrose, 1994) can also form a part of the domain. Core development is suggested for now to enhance the use of germplasm collections available in gene bank Division in groups (Frankel, 1984). Core Collection are an expeditious means to intensively characterize, explore, and use genetic resources conserved in gene bank as well as monitor genetic drift during preservation and identify The division of the domain into separate groups can be done stepwise; that is, a group of accessions is gaps in genetic diversity for further development of the gene bank (Frankel, 1984; Frankel and Brown, 1984, divided into as genetically separate subgroups as possible at every step. This process of branching, which Brown, 1989a). The idea of core collection was proposed by Frankel and Brown, 1984 is a small set of can be done on the basis of taxonomy, domestication, distribution, breeding history and utilization, or on accessions (usually 10 % of the population) chosen to represent the genetic spectrum of the whole the basis of molecular marker or other characterization details, was done by Van Hintum (1994a). A collection (Ebana et al., 2008). The sampling percentage of the core collection has long been under debate 'diversity tree' can be developed which reflects the domain's genetic structure. The branching exercise ends (Wang et al., 2014); 20%-30% of the sampling percentage has been suggested by Yonezawa et al., 1995. Mini when no further subdivision can be made into genetically distinct classes or adequate branching has been core collection of about 1% has also been suggested for very large collection (Upadhaya et al., 2010; Jiang et achieved. Such studies have been done in 'lentil landraces from Greece and Turkey' (Erskine and al., 2011; Sharma et al., 2012). A mini core subset from USDA rice gene bank has been developed using 26 Muehlbauer, 1991), 'Medicago arabica' (Diwan et al., 1994) or 'pea landraces from Mongolia' (Matthews phenotypic trait and 70 molecular markers (Agrama et al., 2009). Still a perfect ratio and fixed size for all and Ambrose, 1994). core collection does not exist as different crops or different constructing goal needs different sampling Allocation of entries percentage (Wang et al., 2014). Several strategies have been successfully deployed for developing a core collection. Stratified random sampling method, in contrast to simple random sampling has been The choice of the number of entries to be included in the core depends on the purpose of the core set and is successfully applied to the development of numerous core collections (Balfourier et al., 1998; Hu et al., subjective. In general, the number of entries chosen is slightly lower than the number of domain accessions, 2000; Chandra et al., 2002; Franco et al., 2006). One common approach for constructing core collection is typically in the range of 5 to 20 percent. grouping the whole collections into major ecotypes and then selecting representatives from each ecotype A stepwise process can again be followed for the distribution of the entries through the classes. When each (Reeves et al., 2012; Wang et al., 2014). A core collection formed for the purpose of capturing accessions group is divided into subgroups, it is determined how the group entries should be divided into subgroups. In with rare or extreme values of the desired trait(s) (e.g., high resistance to pest or high yield) should be this phase, there are many techniques to use. If no information on the significance or diversity of the evaluated differently from one formed with the intention of representing the genetic diversity in the subgroups is available, the number of accessions in the subgroup can be used for this decision. In this case collection. the constant (C), proportional (P) and logarithmic (L) strategies are available (Brown, 1989b). If quantitative Genetic stocks and reference material, including standard or reference material and well-defined marker information about the diversity in the subgroups is available the gene diversity (H) or maximization (M) materials, are to be represented but the number of entries in this group still has to be decided. The final strategy can be used (Schoen and Brown, 1995). And finally, if there is an idea of the relative importance of choice of entries for each group was made by scientists and curators with specific knowledge and the subgroups this can also be used for the allocation. experience with the groups. Their approaches differed as per group for example the representatives of the The strategy for subgroup allocation of entries will depend on the available information for the group and its wild species can be chosen directly by an expert in the field. Similarly, in the case of 'Cultivated material from subgroups and which vary by group. For example, if no additional information on accessions is available in a Europe', a preliminary selection of landraces and cultivars was made by an expert and this selection was group of wild materials divided into subgroups by country of origin, the number of entries for the core characterized using molecular markers and from the result of this investigation, it was concluded that the selection from each subgroup can be chosen based on the number of accessions in the subgroups. If there is selection had to be adjusted and material from southern Europe was added, and northwestern material was detailed molecular marker information available on the majority of accessions in a group of cultivars, one removed. The selection for the group 'Cultivated material from the Americas' was made after close might use a diversity-based strategy, and if a decision has to be made concerning the division in wild and consultation with American barley breeders and researchers. cultivated subgroups, generally a strategy based on the weight or importance of groups for the core Types of core collection collection will be followed. Global core collection: A core collection developed from these global germplasm collections could be Choice of accessions termed as global core collection. The final choice of individual community entries can be made randomly or, where available, on the basis of Regional core collection: Ecological environments influence adaptation of germplasm accessions. For additional data. The goal should be for the diversity of the category to be better reflected by the entries. As it example, groundnut is cultivated in over 113 countries, but it is an important crop in 25 countries in the

102 103 Asian continent. The core developed from the accessions involving accessions from these countries might opportunity not only to determine an optimal Core Collection size, but also to choose the representative be more beneficial to users in Asia than a global core collection. individuals for a given sized Core Collection (Gouesnard et al., 2001). Trait-specific core collection: Some research programs might have special focus on developing a trait- Validation of a core collection specific core collection, for example, early maturity of the crop or long grains of the crop, giving rise to Trait Once a core collection has been established, an important question for genebank managers is the extent to based core. which it meets its original objectives in terms of the representation of diversity and lack of repetition. Morphological core collection: Characterization and evaluation of core collection using morphological (Noirot et al., 1996, Ortiz,et al.,1999). characters is what we call a morphological core. Initially morphological core used to be the basis of core Once the strategy and the software to be used for generation of core collection have been decided the core collection. After the advent of molecular markers molecular core has been given the choice. Nevertheless; set generation becomes easy. There are parameters which are given by the software used to substantiate both types of core are being used today. A morphological core has been recently made by Adbi et al. in 2020 the core collection . For example in PowerCore: Mean difference, Varience difference, Confidence ratio, in subterranean clover (Trifolium subterraneum L.). Morphological core collection has been developed in Variable rate, Coverage can be calculated to authenticate the core collection. Below given are the relevant many crops (Erskine and Muehlbauer 1991; Tohme et al., 1995; Knu¨pffer and van Hintum, 1995; Cordeiro et formulas for the same. al., 1995; Bisht et al., 1998; Huama´n et al., 1999; Upadhyaya et al. 2001, 2002, 2003).

Molecular core: With the advent of molecular markers core collection has been more simplified and Mean Difference %, approachable. An increasing number of germplasm collections are being genotyped for marker loci such as allozymes, RFLPs, RAPD and SSR markers. Schoen and Brown (1993) proposed two strategies that can use Me: Mean of entire collection marker diversity to allocate sampling effort for the construction of the core collection. A core set of rice Mc: Mean of core collection germplasm from North-East region of India has been developed using SNP markers using ICAR-NBPGR gene bank accessions (Choudhury et al., 2014). Composite core: A composite core collection is a representative of the complete range of phenotypic and Variance Difference % genetic variability of the crop. When phenotypic and genotypic data are merged and analyzed by various Ve: Variance of entire collection strategies for core development is called composite core. A composite core has been made in Wheat using ICAR-NBPGR gene bank collection (Phogat et al., 2020). Vc: Variance of core collection Algorithms for Core Collection Core Hunter, Power Core, MSTART, Power Marker, STRUCTURE Confidence Ratio % The H strategy seeks to maximize the total number of alleles in the core collection by sampling accessions Re: Range of entire collection from groups in pro portion to their within-group genetic diversity. The M strategy examines all possible core Rc: Range of core collection collections and singles out those that maximize the number of observed alleles at the marker loci. These can then be chosen as final candidates for the core. The expected superiority of this marker-based method is based on the correlation between observed allelic richness at the marker loci and allelic richness on other Variable Rate % loci. Such a correlation (or linkage disequilibrium between marker and target alleles) is expected on theoretical grounds either because of (1) shared coancestry of populations, (2) the mating system of the CVe: coefficient of variation of entire collection species considered, or (3) episodes of selection where by selected (target) and neutral (marker) alleles CVc: coefficient of variation of core collection, m: number of traits become associated through hitchhiking. Example of selection of core sub set based on marker data Coverage % De: Classes in entire collection Dc: Classes in core collection, m: number of traits

Uses of Core collections: • Several new sources of variation for use in crop improvement programs • Allele mining • Limited size of the core is the key reason for its overall manageability • Reference set of accessions for the whole collection

The M strategy aims at selecting the highest diversity among subsets and is expected to perform well in • Pre-breeding Programs marker-based grouping. The MSTRAT program was able to implement the M strategy, providing the

104 105 Asian continent. The core developed from the accessions involving accessions from these countries might opportunity not only to determine an optimal Core Collection size, but also to choose the representative be more beneficial to users in Asia than a global core collection. individuals for a given sized Core Collection (Gouesnard et al., 2001). Trait-specific core collection: Some research programs might have special focus on developing a trait- Validation of a core collection specific core collection, for example, early maturity of the crop or long grains of the crop, giving rise to Trait Once a core collection has been established, an important question for genebank managers is the extent to based core. which it meets its original objectives in terms of the representation of diversity and lack of repetition. Morphological core collection: Characterization and evaluation of core collection using morphological (Noirot et al., 1996, Ortiz,et al.,1999). characters is what we call a morphological core. Initially morphological core used to be the basis of core Once the strategy and the software to be used for generation of core collection have been decided the core collection. After the advent of molecular markers molecular core has been given the choice. Nevertheless; set generation becomes easy. There are parameters which are given by the software used to substantiate both types of core are being used today. A morphological core has been recently made by Adbi et al. in 2020 the core collection . For example in PowerCore: Mean difference, Varience difference, Confidence ratio, in subterranean clover (Trifolium subterraneum L.). Morphological core collection has been developed in Variable rate, Coverage can be calculated to authenticate the core collection. Below given are the relevant many crops (Erskine and Muehlbauer 1991; Tohme et al., 1995; Knu¨pffer and van Hintum, 1995; Cordeiro et formulas for the same. al., 1995; Bisht et al., 1998; Huama´n et al., 1999; Upadhyaya et al. 2001, 2002, 2003).

104 105 • Greater information value of the collection, reduced costs and increased efficiency in evaluation 7. Brown AHD (1989a) The case for core collections. In: Brown AHD, OH Frankel, DR Marshall and JT Williams (eds.) The Use of Plant Genetic Resources. Cambridge University Press, Cambridge, UK, pp. Maintaining the core collection 136-155. First question to be asked is whether sets of the core collection entries need to be maintained separately 8. Brown AHD (1995) The core collection at the crossroads. In: Hodgkin T, AHD Brown, TJL van Hintum from the rest of the genebank accessions or whether, even if maintained as part of the whole collection, and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John Wiley and Sons, Chichester, they need any special treatment. In genebanks with good facilities and ample storage or maintenance pp. 3–19. capacity, it may seem unnecessary to maintain core entries separately from the rest of the collection. However, even in these situations, additional quantities of seed or propagating material of the core entries 9. Brown AHD (1989b) Core collections: a practical approach to genetic resources management. will probably be required and space and facilities allocated accordingly. (van hintum et. al. 2000) Genome 31:818-824. 10. Brown AHD, JP Grace and SS Speer (1987) Designation of a "core" collection of perennial Glycine. There are a number of reasons why this may be desirable: Soybean Genet. Newsl. 14: 59-70. • Minimizing the possibility of errors by ensuring that a single standard source is used which is 11. Chandra S, Z Huaman, S Hari Krishna and R Ortiz (2002) Optimal sampling strategy and core collection maintained in one place as the core collection size of Andean tetraploid potato based on isozyme data: A simulation study. Theor. Appl. Genet. 104: • Simplifying distribution and use by retaining all the core collection in the same physical location 1325–1334. • Simplifying the additional monitoring that is desirable for a core collection for example, the amounts 12. Charmet G and F Balfourier (1995) The use of geostatistics for sampling a core collection of perennial and quality of the entries ryegrass populations. Genet. Resour. & Crop Evol. 42: 303-309. • Providing added security by maintaining the core collection in a more secure site (e.g. under close 13. Cordeiro CMT, EAV Morales, P Ferreira, DMS Rocha, IRS Costa, ACC Valois and S Silva (1995) Towards a supervision near appropriate facilities in the case of field-maintained material) Brazilian core collection of cassava. In: Hodkin T, AHD Brown, TJL van Hintum, and BAV Morales (eds.) Core Collection of Plant Genetic Resources, International Plant Genetic Resources Institute (IPGRI). • Meeting the expected needs for increased quantities of the core collection entries John Wiley and Sons, New York, USA, pp. 155–168. Information management 14. Diwan N, GR Bauchan and MS McIntosh (1994) A core collection for the United States annual Medicago germplasm collection. Crop Sci. 34: 279-285. As the core collection is developed and used, more and more information will become available on the entries. No special procedures may be required for this and the genebank's normal documentation systems 15. Ebana K, Y Kojima, S Fukuoka, T Nagamine and M Kawase (2008) Development of mini core collection will be appropriate. Normally, the core collection will be fully characterized and, where possible, it should be of Japanese rice landrace. Breed. Sci. 58: 281-291. the first material used when new characters (e.g. biochemical or molecular markers) are assessed. The 16. Erskine W and FJ Muehlbauer (1991) Allozyme and morphological variability, outcrossing rate and information available on the core entries will therefore soon become more extensive than that available for core collection formation in lentil germplasm. Theor. Appl. Genet. 83: 119–125. other accessions. This is likely to be particularly the case for information on the agronomically important characteristics that constitute evaluation data. Consequently, population conservation programmes are an 17. Franco J, J Crossa, ML Warburton and S Taba (2006) Sampling strategies for conserving maize diversity increasingly relevant way of maintaining adaptive capacity to cope with climate change or increasing UV when forming core subsets using genetic markers. Crop Sci. 46: 854–864. radiation. 18. Frankel OH (1984) Genetic perspective of germplasm conservation. In: Arber WK, WL Peacock, P Starlinger (eds.) Genetic manipulation: Impact on man and society.Cambridge University Press, References Cambridge, England, pp. 161-170. 1. Abdi AI, PGH Nichols, P Kaur, BJ Wintle and W Erskine (2020) Morphological diversity within a core 19. Frankel OH and AHD Brown (1984) Plant genetic resources today: a critical appraisal. In: Holden JHW collection of subterranean clover (Trifolium subterraneum L.): Lessons in pasture adaptation from the and JT Williams (eds.) Crop Genetic Resources: Conservation & Evaluation. George Alien & Unwin wild. PLOS ONE 15(1): e0223699. Ltd., London, pp. 249-257. 2. Agrama HA, WG Yan, F Lee, R Fjellstrom, MH Chen et al.. (2009) Genetic assessment of a mini-core 20. Gautam PL, S Saxena and A Pandey (2006) Plant Genetic Resources: Concerns for Management and subset developed from USDA Rice Genebank. Crop Sci. 49: 1336-1346. Conservation. In: Dhillon BS, S Saxena, A Agrawal and RK Tyagi (eds.) Plant Genetic Resources: 3. Arora RK and VR Rao (1998) Tropical Fruits in Asia, Diversity, Maintenance, Conservation and Use, Foodgrain Crops. Narosa Publishing House, New Delhi, India, pp.1-26. Proceeding of the IPGRI/ICAR/UTFANET Regional Training Course on the Conservation and Use of 21. Gouesnard B, TM Bataillon, G Decoux, C Rozale, DJ Schoen and JL David (2001) MSTRAT: An algorithm Germplasm of Tropical Fruit in Asia. IPGRI Office for South Asia, New Delhi, India. for building germplasm core collections by maximizing allelic or phenotypic richness. J. Hered. 92: 4. Arora RK, VR Rao and AN Rao (1996) Status Reports on Genetic Resources of Mango in South, 93–94. Southeast and East Asia. IPGRI Regional Office for Asia, the Pacific and Oceania, Singapore. 22. Holbrook CC, WF Anderson and RN Pittman (1993) Selection of a core collection from the U.S. 5. Balfourier F, G Charmet, JM Prosperi, M Goulard and P Monestiez (1998) Comparison different spatial germplasm collection of Peanut. Crop Sci. 33: 859-861. strategies for sampling a core collection of natural populations of fodder crops. Genet. Sel. Evol. 30: 23. Huama´n Z, C Aguilar and R Ortiz (1999) Selecting a Peruvian sweet potato core collection on the basis 215–235. of morphological, ecogeographical, and disease and pest reaction data. Theor. Appl. Genet. 98: 6. Bisht IS, RK Mahajan, TR Loknathan and RC Agrawal (1998) Diversity in Indian sesame collection and 840–844. stratification of germplasm accessions in different diversity groups. Genet. Resour. & Crop Evol. 45: 325–335.

106 107 • Greater information value of the collection, reduced costs and increased efficiency in evaluation 7. Brown AHD (1989a) The case for core collections. In: Brown AHD, OH Frankel, DR Marshall and JT Williams (eds.) The Use of Plant Genetic Resources. Cambridge University Press, Cambridge, UK, pp. Maintaining the core collection 136-155. First question to be asked is whether sets of the core collection entries need to be maintained separately 8. Brown AHD (1995) The core collection at the crossroads. In: Hodgkin T, AHD Brown, TJL van Hintum from the rest of the genebank accessions or whether, even if maintained as part of the whole collection, and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John Wiley and Sons, Chichester, they need any special treatment. In genebanks with good facilities and ample storage or maintenance pp. 3–19. capacity, it may seem unnecessary to maintain core entries separately from the rest of the collection. However, even in these situations, additional quantities of seed or propagating material of the core entries 9. Brown AHD (1989b) Core collections: a practical approach to genetic resources management. will probably be required and space and facilities allocated accordingly. (van hintum et. al. 2000) Genome 31:818-824. 10. Brown AHD, JP Grace and SS Speer (1987) Designation of a "core" collection of perennial Glycine. There are a number of reasons why this may be desirable: Soybean Genet. Newsl. 14: 59-70. • Minimizing the possibility of errors by ensuring that a single standard source is used which is 11. Chandra S, Z Huaman, S Hari Krishna and R Ortiz (2002) Optimal sampling strategy and core collection maintained in one place as the core collection size of Andean tetraploid potato based on isozyme data: A simulation study. Theor. Appl. Genet. 104: • Simplifying distribution and use by retaining all the core collection in the same physical location 1325–1334. • Simplifying the additional monitoring that is desirable for a core collection for example, the amounts 12. Charmet G and F Balfourier (1995) The use of geostatistics for sampling a core collection of perennial and quality of the entries ryegrass populations. Genet. Resour. & Crop Evol. 42: 303-309. • Providing added security by maintaining the core collection in a more secure site (e.g. under close 13. Cordeiro CMT, EAV Morales, P Ferreira, DMS Rocha, IRS Costa, ACC Valois and S Silva (1995) Towards a supervision near appropriate facilities in the case of field-maintained material) Brazilian core collection of cassava. In: Hodkin T, AHD Brown, TJL van Hintum, and BAV Morales (eds.) Core Collection of Plant Genetic Resources, International Plant Genetic Resources Institute (IPGRI). • Meeting the expected needs for increased quantities of the core collection entries John Wiley and Sons, New York, USA, pp. 155–168. Information management 14. Diwan N, GR Bauchan and MS McIntosh (1994) A core collection for the United States annual Medicago germplasm collection. Crop Sci. 34: 279-285. As the core collection is developed and used, more and more information will become available on the entries. No special procedures may be required for this and the genebank's normal documentation systems 15. Ebana K, Y Kojima, S Fukuoka, T Nagamine and M Kawase (2008) Development of mini core collection will be appropriate. Normally, the core collection will be fully characterized and, where possible, it should be of Japanese rice landrace. Breed. Sci. 58: 281-291. the first material used when new characters (e.g. biochemical or molecular markers) are assessed. The 16. Erskine W and FJ Muehlbauer (1991) Allozyme and morphological variability, outcrossing rate and information available on the core entries will therefore soon become more extensive than that available for core collection formation in lentil germplasm. Theor. Appl. Genet. 83: 119–125. other accessions. This is likely to be particularly the case for information on the agronomically important characteristics that constitute evaluation data. Consequently, population conservation programmes are an 17. Franco J, J Crossa, ML Warburton and S Taba (2006) Sampling strategies for conserving maize diversity increasingly relevant way of maintaining adaptive capacity to cope with climate change or increasing UV when forming core subsets using genetic markers. Crop Sci. 46: 854–864. radiation. 18. Frankel OH (1984) Genetic perspective of germplasm conservation. In: Arber WK, WL Peacock, P Starlinger (eds.) Genetic manipulation: Impact on man and society.Cambridge University Press, References Cambridge, England, pp. 161-170. 1. Abdi AI, PGH Nichols, P Kaur, BJ Wintle and W Erskine (2020) Morphological diversity within a core 19. Frankel OH and AHD Brown (1984) Plant genetic resources today: a critical appraisal. In: Holden JHW collection of subterranean clover (Trifolium subterraneum L.): Lessons in pasture adaptation from the and JT Williams (eds.) Crop Genetic Resources: Conservation & Evaluation. George Alien & Unwin wild. PLOS ONE 15(1): e0223699. Ltd., London, pp. 249-257. 2. Agrama HA, WG Yan, F Lee, R Fjellstrom, MH Chen et al.. (2009) Genetic assessment of a mini-core 20. Gautam PL, S Saxena and A Pandey (2006) Plant Genetic Resources: Concerns for Management and subset developed from USDA Rice Genebank. Crop Sci. 49: 1336-1346. Conservation. In: Dhillon BS, S Saxena, A Agrawal and RK Tyagi (eds.) Plant Genetic Resources: 3. Arora RK and VR Rao (1998) Tropical Fruits in Asia, Diversity, Maintenance, Conservation and Use, Foodgrain Crops. Narosa Publishing House, New Delhi, India, pp.1-26. Proceeding of the IPGRI/ICAR/UTFANET Regional Training Course on the Conservation and Use of 21. Gouesnard B, TM Bataillon, G Decoux, C Rozale, DJ Schoen and JL David (2001) MSTRAT: An algorithm Germplasm of Tropical Fruit in Asia. IPGRI Office for South Asia, New Delhi, India. for building germplasm core collections by maximizing allelic or phenotypic richness. J. Hered. 92: 4. Arora RK, VR Rao and AN Rao (1996) Status Reports on Genetic Resources of Mango in South, 93–94. Southeast and East Asia. IPGRI Regional Office for Asia, the Pacific and Oceania, Singapore. 22. Holbrook CC, WF Anderson and RN Pittman (1993) Selection of a core collection from the U.S. 5. Balfourier F, G Charmet, JM Prosperi, M Goulard and P Monestiez (1998) Comparison different spatial germplasm collection of Peanut. Crop Sci. 33: 859-861. strategies for sampling a core collection of natural populations of fodder crops. Genet. Sel. Evol. 30: 23. Huama´n Z, C Aguilar and R Ortiz (1999) Selecting a Peruvian sweet potato core collection on the basis 215–235. of morphological, ecogeographical, and disease and pest reaction data. Theor. Appl. Genet. 98: 6. Bisht IS, RK Mahajan, TR Loknathan and RC Agrawal (1998) Diversity in Indian sesame collection and 840–844. stratification of germplasm accessions in different diversity groups. Genet. Resour. & Crop Evol. 45: 325–335.

106 107 24. Jansen J and van Hintum TJL (2007) Genetic distance sampling: a novel sampling method for obtaining 41. Tohme J, P Jones, S Beebe and M Ibanga (1995) The combined use of agroecological and core collections using genetic distances with an application to cultivated lettuce. Theor. Appl. Genet. characterization data to establish the CIAT Phaseolus vulgaris core collection. In: Hodgkin T, AHD 114(3): 421–428. Brown, TJL van Hintum and BAV Morales (eds.) Core Collection of Plant Genetic Resources. 25. Jiang Q, J Hou, C Hao, L Wang, H Ge et al.. (2011) The wheat (T. aestivum) sucrose synthase 2 gene International Plant Genetic Resources Institute (IPGRI). John Wiley and Sons, New York, USA, pp. (TaSus2) active in endosperm development is associated with yield traits. Funct. Integr. Genomics 11: 95–108 49-61. 42. Upadhyaya HD, PJ Bramel and S Singh (2001) Development of a chickpea core subset using geographic 26. Kim KW, HK Chung et al. (2007) Power Core: a program applying the advanced M strategy with a distribution and quantitative traits. Crop Sci. 41: 206–210. heuristic search for establishing core sets. Bioinformatics 23(16): 2155–2162. 43. Upadhyaya HD, PJ Bramel, R Ortiz and S Singh (2002) Developing a mini core of peanut for utilization of 27. Knüpffer H and van Hintum TJL (1995) The barley core collection: an international effort. In: Hodgkin T, genetic resources. Crop Sci. 42: 2150–2156. AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John 44. Upadhyaya HD, PJ Bramel, R Ortiz and S Singh (2003) Development of a groundnut core collection Wiley and Sons, Chichester, UK, pp. 171-178. using taxonomical, geographical and morphological descriptors. Genet. Resour. and Crop Evol. 50: 28. Matthews P and MJ Ambrose (1994) Development and use of a 'core' collection for the John Innes 139–148. Pisum collection. In: Balfourier F and MR Perretant (eds.) Evaluation and Exploitation of Genetic 45. Upadhyaya HD, D Yadav, N Dronavalli, CLL Gowda and S Singh (2010) Mini core germplasm collections Resources, Pre-Breeding. Proceedings of the Genetic Resources Section Meeting of Eucarpia, for infusing genetic diversity in plant breeding programs. Electronic J. Plant Breed. 1: 1294-1309. Clermont-Ferrand, pp. 99-107. 46. van Hintum TJL, AHD Brown, C Spillane and T Hodgkin (2000) Core collections of plant genetic 29. Noirot M, S Hamon and F Anthony (1996) The principal component scoring: a new method of resources. Technical Bulletin. 3: International Plant Genetic Resources Institute, Rome Italy. constituting a core collection using quantitative data. Genet. Resour. Crop Evol. 43: 1–6. 47. van Hintum TJL (1994a) Hierarchical approaches to the analysis of genetic diversity in crop plants. In: 30. Odong TL, J Jansen, FA van Eeuwijk and TJL van Hintum (2013) Quality of core collections for effective Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds) Core Collections of Plant Genetic utilization of genetic resources review, discussion and interpretation. Theor. Appl. Genet. 126: 289- Resources. John Wiley and Sons, Chichester, UK. pp. 23-34. 305. 48. van Hintum TJL (1994b) Comparison of marker systems and construction of a core collection in a 31. Ortiz R, EN Ruiz-Tapia and A Mujica-Sanchez (1998) Sampling strategy for a core collection of Peruvian pedigree of European spring barley. Theor. Appl. Genet. 89:991-997. quinoa germplasm. Theor. Appl. Genet. 96: 475-483. 49. van Hintum TJL and D Haalman (1994) Pedigree analysis for composing a core collection of modern 32. Phogat BS, S Kumar, J Kumari, N Kumar, A Pandey (2020) Characterization of wheat germplasm cultivars, with examples from barley (Hordeum vulgare s. lat.). Theor. Appl. Genet. 88: 70-74. conserved in the Indian National Genebank and establishment of a composite core collection. Crop 50. Vaughan DA (1991) Choosing rice germplasm for evaluation. Euphytica 54: 147-154. Sci. doi: 10.1002/csc2.20285. 51. Verheij EWM and RE Coronel (1991) Plant Resources of South-East Asia No. 2. Edible Fruits and Nuts. 33. Radovic G and D Jelovac (1994) The possible approach in maize 'core collection' development. In: Pudoc. Wageningen, the Netherlands. Balfourier F and MR Perretant (eds.) Evaluation and Exploitation of Genetic Resources, Pre-Breeding. Proceedings of the Genetic Resources Section Meeting of Eucarpia, Clermont-Ferrand. pp. 109-115. 52. Wang J, Y Guan, Y Wang, L Zhu and Q Wang (2014) A Strategy for finding the optimal scale of plant core collection based on Monte Carlo simulation. Sci. World J. doi: 10.1155/2014/503473. 34. Reeves PA, LW Panella and CM Richards (2012) Retention of agronomically important variation in germplasm core collections: implications for allele mining. Theor. Appl. Genet. 124: 1155-1171. 53. Yonezawa K, T Nomura and H Morishima (1995) Sampling strategies for use in stratified germplasm collections. In: Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of 35. Choudhury DR, N Singh, AK Singh, S Kumar, K Srinivasan, RK Tyagi, A Ahmad, NK Singh and R Singh Plant Genetic Resources. John Wiley and Sons, UK. pp. 35-54. (2014) Analysis of genetic diversity and population structure of rice germplasm from north-eastern region of india and development of a core germplasm set. PLoS ONE. 9(11):1–12. 36. Schoen DJ and AHD Brown (1995) Maximising genetic diversity in core collections of wild relatives of crop species. In: Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John Wiley and Sons, Chichester, UK. pp. 55-76. 37. Schoen DJ and AHD Brown (1993) Conservation of allelic richness in wild crop relatives is aided by assessment of genetic markers. Proc. Natl. Acad. Sci. USA 90: 10623– 10627. 38. Sharma M, A Rathore, U Naga Mangala, R Ghosh, S Sharma et al.. (2012) New sources of resistance to Fusarium wilt and sterility mosaic disease in a mini-core collection of pigeonpea germplasm. Eur. J. Plant Pathol. 133: 707-714. 39. Spagnoletti Zeuli PL and CO Qualset (1993) Evaluation of five strategies for obtaining a core subset from a large genetic resource collection of durum wheat. Theor. Appl. Genet. 87: 295-304. 40. Thachuk C, J Crossa, J Franco, S Dreisigacker, M Warburton and GF Davenport (2009) Core Hunter: an algorithm for sampling genetic resources based on multiple genetic measures. BMC Bioinformatics 10: 243

108 109 24. Jansen J and van Hintum TJL (2007) Genetic distance sampling: a novel sampling method for obtaining 41. Tohme J, P Jones, S Beebe and M Ibanga (1995) The combined use of agroecological and core collections using genetic distances with an application to cultivated lettuce. Theor. Appl. Genet. characterization data to establish the CIAT Phaseolus vulgaris core collection. In: Hodgkin T, AHD 114(3): 421–428. Brown, TJL van Hintum and BAV Morales (eds.) Core Collection of Plant Genetic Resources. 25. Jiang Q, J Hou, C Hao, L Wang, H Ge et al.. (2011) The wheat (T. aestivum) sucrose synthase 2 gene International Plant Genetic Resources Institute (IPGRI). John Wiley and Sons, New York, USA, pp. (TaSus2) active in endosperm development is associated with yield traits. Funct. Integr. Genomics 11: 95–108 49-61. 42. Upadhyaya HD, PJ Bramel and S Singh (2001) Development of a chickpea core subset using geographic 26. Kim KW, HK Chung et al. (2007) Power Core: a program applying the advanced M strategy with a distribution and quantitative traits. Crop Sci. 41: 206–210. heuristic search for establishing core sets. Bioinformatics 23(16): 2155–2162. 43. Upadhyaya HD, PJ Bramel, R Ortiz and S Singh (2002) Developing a mini core of peanut for utilization of 27. Knüpffer H and van Hintum TJL (1995) The barley core collection: an international effort. In: Hodgkin T, genetic resources. Crop Sci. 42: 2150–2156. AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John 44. Upadhyaya HD, PJ Bramel, R Ortiz and S Singh (2003) Development of a groundnut core collection Wiley and Sons, Chichester, UK, pp. 171-178. using taxonomical, geographical and morphological descriptors. Genet. Resour. and Crop Evol. 50: 28. Matthews P and MJ Ambrose (1994) Development and use of a 'core' collection for the John Innes 139–148. Pisum collection. In: Balfourier F and MR Perretant (eds.) Evaluation and Exploitation of Genetic 45. Upadhyaya HD, D Yadav, N Dronavalli, CLL Gowda and S Singh (2010) Mini core germplasm collections Resources, Pre-Breeding. Proceedings of the Genetic Resources Section Meeting of Eucarpia, for infusing genetic diversity in plant breeding programs. Electronic J. Plant Breed. 1: 1294-1309. Clermont-Ferrand, pp. 99-107. 46. van Hintum TJL, AHD Brown, C Spillane and T Hodgkin (2000) Core collections of plant genetic 29. Noirot M, S Hamon and F Anthony (1996) The principal component scoring: a new method of resources. Technical Bulletin. 3: International Plant Genetic Resources Institute, Rome Italy. constituting a core collection using quantitative data. Genet. Resour. Crop Evol. 43: 1–6. 47. van Hintum TJL (1994a) Hierarchical approaches to the analysis of genetic diversity in crop plants. In: 30. Odong TL, J Jansen, FA van Eeuwijk and TJL van Hintum (2013) Quality of core collections for effective Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds) Core Collections of Plant Genetic utilization of genetic resources review, discussion and interpretation. Theor. Appl. Genet. 126: 289- Resources. John Wiley and Sons, Chichester, UK. pp. 23-34. 305. 48. van Hintum TJL (1994b) Comparison of marker systems and construction of a core collection in a 31. Ortiz R, EN Ruiz-Tapia and A Mujica-Sanchez (1998) Sampling strategy for a core collection of Peruvian pedigree of European spring barley. Theor. Appl. Genet. 89:991-997. quinoa germplasm. Theor. Appl. Genet. 96: 475-483. 49. van Hintum TJL and D Haalman (1994) Pedigree analysis for composing a core collection of modern 32. Phogat BS, S Kumar, J Kumari, N Kumar, A Pandey (2020) Characterization of wheat germplasm cultivars, with examples from barley (Hordeum vulgare s. lat.). Theor. Appl. Genet. 88: 70-74. conserved in the Indian National Genebank and establishment of a composite core collection. Crop 50. Vaughan DA (1991) Choosing rice germplasm for evaluation. Euphytica 54: 147-154. Sci. doi: 10.1002/csc2.20285. 51. Verheij EWM and RE Coronel (1991) Plant Resources of South-East Asia No. 2. Edible Fruits and Nuts. 33. Radovic G and D Jelovac (1994) The possible approach in maize 'core collection' development. In: Pudoc. Wageningen, the Netherlands. Balfourier F and MR Perretant (eds.) Evaluation and Exploitation of Genetic Resources, Pre-Breeding. Proceedings of the Genetic Resources Section Meeting of Eucarpia, Clermont-Ferrand. pp. 109-115. 52. Wang J, Y Guan, Y Wang, L Zhu and Q Wang (2014) A Strategy for finding the optimal scale of plant core collection based on Monte Carlo simulation. Sci. World J. doi: 10.1155/2014/503473. 34. Reeves PA, LW Panella and CM Richards (2012) Retention of agronomically important variation in germplasm core collections: implications for allele mining. Theor. Appl. Genet. 124: 1155-1171. 53. Yonezawa K, T Nomura and H Morishima (1995) Sampling strategies for use in stratified germplasm collections. In: Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of 35. Choudhury DR, N Singh, AK Singh, S Kumar, K Srinivasan, RK Tyagi, A Ahmad, NK Singh and R Singh Plant Genetic Resources. John Wiley and Sons, UK. pp. 35-54. (2014) Analysis of genetic diversity and population structure of rice germplasm from north-eastern region of india and development of a core germplasm set. PLoS ONE. 9(11):1–12. 36. Schoen DJ and AHD Brown (1995) Maximising genetic diversity in core collections of wild relatives of crop species. In: Hodgkin T, AHD Brown, TJL van Hintum and EAV Morales (eds.) Core Collections of Plant Genetic Resources. John Wiley and Sons, Chichester, UK. pp. 55-76. 37. Schoen DJ and AHD Brown (1993) Conservation of allelic richness in wild crop relatives is aided by assessment of genetic markers. Proc. Natl. Acad. Sci. USA 90: 10623– 10627. 38. Sharma M, A Rathore, U Naga Mangala, R Ghosh, S Sharma et al.. (2012) New sources of resistance to Fusarium wilt and sterility mosaic disease in a mini-core collection of pigeonpea germplasm. Eur. J. Plant Pathol. 133: 707-714. 39. Spagnoletti Zeuli PL and CO Qualset (1993) Evaluation of five strategies for obtaining a core subset from a large genetic resource collection of durum wheat. Theor. Appl. Genet. 87: 295-304. 40. Thachuk C, J Crossa, J Franco, S Dreisigacker, M Warburton and GF Davenport (2009) Core Hunter: an algorithm for sampling genetic resources based on multiple genetic measures. BMC Bioinformatics 10: 243

108 109 Registration of Trait Specific Flow Chart of Plant Germplasm Registration Activity Plant Germplasm Registration Germplasm at ICAR-NBPGR Seed material Proposal Vegetatively Propogagules Anjali Kak Koul* and Veena Gupta NAGS Division of Germplasm Conservation NBPGR Establishment & ICAR-National Bureau of Plant Genetic Resources, New Delhi Issue a Certificate Untreated *Email: [email protected] Proposal sent to the expert for comments Seed Material Introduction PC/PD ADG India has a very strong crop improvement programme undertaken by the Indian Council of Agricultural Seed Health Testing (Joint Inspection) PGRC MEETING Entomology Nematology Pathology Research (ICAR) in the form of All India Co-ordinated Crop Improvement Projects (for various crops). This has Elimination of weeds, soil, plant debris, discoloured and deformed seeds, nematode galls, fungal fructifications, etc. paid rich dividends in the form of development and release of improved crop varieties and hybrids that played a pivotal role in enhancing the national food production. During the process of germplasm Seed Viability Testing manipulations, research and experimentation to develop superior genotypes for specific or multiple traits, GERMPLASM REGISTERED (>85%) many useful materials are developed, which may not qualify for notification and release. Such material may Allotment of National Identity have resistance/tolerance to biotic and abiotic stresses, and other useful traits and may be novel, unique and distinct with academic, scientific and applied value or others having commercial importance. ICAR has established a mechanism to register the trait-specific germplasm through National Bureau of Plant Genetic Conservation in the National Geneb ank Resources (NBPGR) to address the above concerns and recognizing researchers who had Certificates issued developed/identified the trait-specific germplasm. One-page note published in IJPGR Since the inception of the mechanism in 1996, a total of 42 meetings of Plant Germplasm Registration Committee have been held till 2020 and about 4000 proposals were considered so far. Out of 4000 In an effort towards promoting the registration of horticultural crops the data requirement for the perennial proposals 1,643 potentially valuable germplasm/genetic stocks, belonging to 233 crop species, have been horticultural crops was reduced from four environments to only two environment data in the revised registered. The guidelines for registration of germplasm are available at NBPGR website guidelines as per the recommendation of the Expert Committee on Horticultural Crops. (http://www.nbpgr.ernet.in:8080/registration/Guidelines.aspx). The information about registered germplasm is published in five bulletins. One-page notes are also published in the Indian Journal of Plant Data requirement for registration of trait specific germplasm of perennial horticultural crops Genetic Resources (IJPGR, an official publication of the Indian Society of Plant Genetic Resources, New Delhi) to disseminate information among scientists/users. All claims concerning the germplasm submitted for registration should accompany scientific evidence for uniqueness, reproducibility and value in the form of: An online Germplasm Registration Information System (GRIS) has been developed to digitize the entire process of germplasm registration, right from submission of application to evaluation by experts and I Performance (yield contributing traits, adaptation traits, quality traits) data for at least two decision by Plant Germplasm Registration Committee. An organized digital information system provides environments/seasons (location and year combination) under All India Coordinated Research Project fair and just opportunity for all to access; along with facilitating efficient PGR management and enhanced (AICRP) trial/nursery tests supported with relevant extracts of the documents (e.g. comparative data utilization. The system is expected to provide genebank managers, breeders and plant researchers with a of all entries tested) or verification by concerned Project Director/Project Co-ordinator (PD/PC) or hands-on tool for management of germplasm registration process, and to policy makers with a reliable under any other relevant system verified by Competent Authority. For qualitative traits (e.g. flower source of information. With the advent of this system the entire process of germplasm registration has colour, pulp colour, fruit/ pod colour and seed colour etc) data of two environments/ seasons duly become simple and fast. supported by documents. The application is to be filled online by registering on to (http://www.nbpgr.ernet.in:8080/registration/). ii. For resistance/tolerance to biotic and abiotic stresses, data should be obtained from at least two Once the application is filled and documentary evidence uploaded, the progress of the application can be environments/seasons under established hot spot locations/under artificial screening (epiphytotic) tracked using the personal dashboard. The review process is also online, making the whole cycle of conditions. All the proposers of the germplasm/genetic stock should sign declaration that standard application-to-registration quick. Once approved by Plant Germplasm Registration Committee the procedures were followed for testing/screening. registration certificate too can be obtained online. iii Supporting biochemical evaluation data should be obtained from at least two environments/seasons. iv. The proposed genetic stock/germplasm should also be evaluated along with already registered genetic stock(s)/germplasm(s), if available. v. Supporting documentary evidence on (i), (ii) and (iii) either in Institute Annual report/ AICRP Report/peer reviewed journals. vi. Recommendation of Institute Germplasm Identification Committee (IGIC) regarding the uniqueness of germplasm for trait(s) claimed. 110 111 Registration of Trait Specific Flow Chart of Plant Germplasm Registration Activity Plant Germplasm Registration Germplasm at ICAR-NBPGR Seed material Proposal Vegetatively Propogagules Anjali Kak Koul* and Veena Gupta NAGS Division of Germplasm Conservation NBPGR Establishment & ICAR-National Bureau of Plant Genetic Resources, New Delhi Issue a Certificate Untreated *Email: [email protected] Proposal sent to the expert for comments Seed Material Introduction PC/PD ADG India has a very strong crop improvement programme undertaken by the Indian Council of Agricultural Seed Health Testing (Joint Inspection) PGRC MEETING Entomology Nematology Pathology Research (ICAR) in the form of All India Co-ordinated Crop Improvement Projects (for various crops). This has Elimination of weeds, soil, plant debris, discoloured and deformed seeds, nematode galls, fungal fructifications, etc. paid rich dividends in the form of development and release of improved crop varieties and hybrids that played a pivotal role in enhancing the national food production. During the process of germplasm Seed Viability Testing manipulations, research and experimentation to develop superior genotypes for specific or multiple traits, GERMPLASM REGISTERED (>85%) many useful materials are developed, which may not qualify for notification and release. Such material may Allotment of National Identity have resistance/tolerance to biotic and abiotic stresses, and other useful traits and may be novel, unique and distinct with academic, scientific and applied value or others having commercial importance. ICAR has established a mechanism to register the trait-specific germplasm through National Bureau of Plant Genetic Conservation in the National Geneb ank Resources (NBPGR) to address the above concerns and recognizing researchers who had Certificates issued developed/identified the trait-specific germplasm. One-page note published in IJPGR Since the inception of the mechanism in 1996, a total of 42 meetings of Plant Germplasm Registration Committee have been held till 2020 and about 4000 proposals were considered so far. Out of 4000 In an effort towards promoting the registration of horticultural crops the data requirement for the perennial proposals 1,643 potentially valuable germplasm/genetic stocks, belonging to 233 crop species, have been horticultural crops was reduced from four environments to only two environment data in the revised registered. The guidelines for registration of germplasm are available at NBPGR website guidelines as per the recommendation of the Expert Committee on Horticultural Crops. (http://www.nbpgr.ernet.in:8080/registration/Guidelines.aspx). The information about registered germplasm is published in five bulletins. One-page notes are also published in the Indian Journal of Plant Data requirement for registration of trait specific germplasm of perennial horticultural crops Genetic Resources (IJPGR, an official publication of the Indian Society of Plant Genetic Resources, New Delhi) to disseminate information among scientists/users. All claims concerning the germplasm submitted for registration should accompany scientific evidence for uniqueness, reproducibility and value in the form of: An online Germplasm Registration Information System (GRIS) has been developed to digitize the entire process of germplasm registration, right from submission of application to evaluation by experts and I Performance (yield contributing traits, adaptation traits, quality traits) data for at least two decision by Plant Germplasm Registration Committee. An organized digital information system provides environments/seasons (location and year combination) under All India Coordinated Research Project fair and just opportunity for all to access; along with facilitating efficient PGR management and enhanced (AICRP) trial/nursery tests supported with relevant extracts of the documents (e.g. comparative data utilization. The system is expected to provide genebank managers, breeders and plant researchers with a of all entries tested) or verification by concerned Project Director/Project Co-ordinator (PD/PC) or hands-on tool for management of germplasm registration process, and to policy makers with a reliable under any other relevant system verified by Competent Authority. For qualitative traits (e.g. flower source of information. With the advent of this system the entire process of germplasm registration has colour, pulp colour, fruit/ pod colour and seed colour etc) data of two environments/ seasons duly become simple and fast. supported by documents. The application is to be filled online by registering on to (http://www.nbpgr.ernet.in:8080/registration/). ii. For resistance/tolerance to biotic and abiotic stresses, data should be obtained from at least two Once the application is filled and documentary evidence uploaded, the progress of the application can be environments/seasons under established hot spot locations/under artificial screening (epiphytotic) tracked using the personal dashboard. The review process is also online, making the whole cycle of conditions. All the proposers of the germplasm/genetic stock should sign declaration that standard application-to-registration quick. Once approved by Plant Germplasm Registration Committee the procedures were followed for testing/screening. registration certificate too can be obtained online. iii Supporting biochemical evaluation data should be obtained from at least two environments/seasons. iv. The proposed genetic stock/germplasm should also be evaluated along with already registered genetic stock(s)/germplasm(s), if available. v. Supporting documentary evidence on (i), (ii) and (iii) either in Institute Annual report/ AICRP Report/peer reviewed journals. vi. Recommendation of Institute Germplasm Identification Committee (IGIC) regarding the uniqueness of germplasm for trait(s) claimed. 110 111 Submission of propagating material 11. Temperate ICAR-CITH, ICAR-Central Institute for 0194-2305044 Since most of the horticultural crops are vegetatively propagated the germplasm material/propagules horticultural Srinagar Temerate Horticulture (CITH), crops Old AirPort Road, Rangreth, (tubers, bulbs, rhizomes, cuttings etc.) have to be submitted at the concerned crop-based designated NAGS Srinagar, Kashmir-190 007 (Table 1) and the establishment certificate obtained. This certificate has to be uploaded along with application form. ICAR-NBPGR ICAR-National Bureau of Plant 0177-2835459 RS, Shimla Genetic Resources, Regional Station, Table 1. List of National Active Germplasm Sites for Fruit crops Phagli, Shimla-171 004, Himachal Pradesh S.No. Crop(s) Institute Address Phone 12. Tropical fruits ICAR-IIHR, ICAR-Indian Institute of 080- 28466471, 1. Crops of North ICAR RC NEH, ICAR Research Complex for 0364- 2570257 Bangalore Horticultural Research (IIHR), 28466353 East Meghalaya NEH Region, Umroi Road, Hessaraghatta lake post, Umiam-793 103, Meghalaya Bangaluru-560 089, Karnataka 2. Arid fruits ICAR-CIAH, ICAR-Central Institute for 0151-2250960, AICRP, ICAR- All India Coordinated Research 080- 28466471, Bikaner Arid Horticulture (CIAH), 2250147 IIHR, Projection Tropical Fruits, ICAR- 28466353 Sri Ganganagar Road, NH-15, Bangalore Indian Institute of Horticultural Beechwal, Bikaner-334 006, Research (IIHR), Hessaraghatta Rajasthan lake post, Bangalore-560 089, 3. Banana ICAR-NRCB, ICAR-National Research 0431-2618104, Karnataka Tiruchirapalli Centre for Banana (NRCB), 2618106 Thogamalai Road, Thayanur Post, Status of registration of trait specific genetic stocks of fruits crops Tiruchirapalli -620 102, Tamil Nadu In fruit crops, 46 genotypes belonging to 15 genera and 21 species have been registered 4. Cashew ICAR-DCR, ICAR-Directorate of Cashew 08251-231530, (http://www.nbpgr.ernet.in:8080/registration/InventoryofGermplasm.aspx). The major genera are Puttur Research (DCR), Post Darbe, 230902, 236490 mango, cashew, ber and guava. India is an important centre of diversity of mango, having large number of Puttur-574 202, Dakshina Kannada, wild and cultivated types. The wild endemic species Mangifera andamanica and M. griffithi from Andaman Karnataka Islands have been registered for disease resistance and wood value. The cultivated species M. indica (8 5. Citrus species ICAR-NRC, ICAR-National Research Centre 0712-2500249, acc.) have been registered mostly for dwarf plant type, less fibrous fruit, deep yellow pulp and good taste Nagpur for Citrus, Amravati Road, 2500615 (Fig. 1). Among the fruit crops registered, Sapindus mukorossi registered by NBPGR, RS Bhowali is unique in Nagpur-440 010, Maharastra having sweet and fleshy fruit. In addition, wild and cultivated species of cashew are also registered. 6. Grapes ICAR-NRC, ICAR-National Research Centre 020-26956000 Wild relatives should get priority for registration as they are sources of many desirable genes for biotic and Pune for Grapes, P.B. No. 3, P.O. Manjri Farm, Solapur Road, Pune-412 307, abiotic stresses. Priority should be the registration of trait specific temperate fruits and rootstocks as no Maharashtra such germplasm has been registered in 7. Litchi, Bael, ICAR-NRC, ICAR-National Research Centre for 0621 - 2289475, these crops. Traits for Aonla & Jackfruit Muzaffarpur Litchi, Mushahari, P.O. Ramna, 2281160 value addition and Muzaffarpur-842 002, Bihar better shelf life need to 8. Mango ICAR-CISH, ICAR-Central Institute for 0522-2841022, be identified and Lucknow Subtropical Horticulture (CISH), 2841023 registered, particularly Rehmankhera, Kakori- i n p l a n t s h a v i n g Lucknow-227 107, Uttar Pradesh perishable fruits. 9. Sub tropical AICRP, ICAR- ICAR-Central Institute for 0522- 2841115 Directorate of Cashew fruits CISH, Lucknow Subtropcal Horticulture (CISH), Research (DCR), Puttur, Rehmankhera, Kakori- IIHR Bengaluru and Lucknow-227 107, Uttar Pradesh CCS HAU, Hissar are 10. Mulberry CSGRC, Hosur Central Sericultural Germplasm 04344-222013, the major contributors Resources Centre, Central Silk 221148, 221149 f o r f r u i t c r o p Board, Ministry of Textile, registration (Kak and Government of India, P.B.-44, Gupta, 2021). Thally Road, Hosur-635 109, Dharmapuri, Tamil Nadu

Fig. 1. Trait Specific Germplasm of Fruit Crops registered with ICAR-NBPGR

112 113 Submission of propagating material 11. Temperate ICAR-CITH, ICAR-Central Institute for 0194-2305044 Since most of the horticultural crops are vegetatively propagated the germplasm material/propagules horticultural Srinagar Temerate Horticulture (CITH), crops Old AirPort Road, Rangreth, (tubers, bulbs, rhizomes, cuttings etc.) have to be submitted at the concerned crop-based designated NAGS Srinagar, Kashmir-190 007 (Table 1) and the establishment certificate obtained. This certificate has to be uploaded along with application form. ICAR-NBPGR ICAR-National Bureau of Plant 0177-2835459 RS, Shimla Genetic Resources, Regional Station, Table 1. List of National Active Germplasm Sites for Fruit crops Phagli, Shimla-171 004, Himachal Pradesh S.No. Crop(s) Institute Address Phone 12. Tropical fruits ICAR-IIHR, ICAR-Indian Institute of 080- 28466471, 1. Crops of North ICAR RC NEH, ICAR Research Complex for 0364- 2570257 Bangalore Horticultural Research (IIHR), 28466353 East Meghalaya NEH Region, Umroi Road, Hessaraghatta lake post, Umiam-793 103, Meghalaya Bangaluru-560 089, Karnataka 2. Arid fruits ICAR-CIAH, ICAR-Central Institute for 0151-2250960, AICRP, ICAR- All India Coordinated Research 080- 28466471, Bikaner Arid Horticulture (CIAH), 2250147 IIHR, Projection Tropical Fruits, ICAR- 28466353 Sri Ganganagar Road, NH-15, Bangalore Indian Institute of Horticultural Beechwal, Bikaner-334 006, Research (IIHR), Hessaraghatta Rajasthan lake post, Bangalore-560 089, 3. Banana ICAR-NRCB, ICAR-National Research 0431-2618104, Karnataka Tiruchirapalli Centre for Banana (NRCB), 2618106 Thogamalai Road, Thayanur Post, Status of registration of trait specific genetic stocks of fruits crops Tiruchirapalli -620 102, Tamil Nadu In fruit crops, 46 genotypes belonging to 15 genera and 21 species have been registered 4. Cashew ICAR-DCR, ICAR-Directorate of Cashew 08251-231530, (http://www.nbpgr.ernet.in:8080/registration/InventoryofGermplasm.aspx). The major genera are Puttur Research (DCR), Post Darbe, 230902, 236490 mango, cashew, ber and guava. India is an important centre of diversity of mango, having large number of Puttur-574 202, Dakshina Kannada, wild and cultivated types. The wild endemic species Mangifera andamanica and M. griffithi from Andaman Karnataka Islands have been registered for disease resistance and wood value. The cultivated species M. indica (8 5. Citrus species ICAR-NRC, ICAR-National Research Centre 0712-2500249, acc.) have been registered mostly for dwarf plant type, less fibrous fruit, deep yellow pulp and good taste Nagpur for Citrus, Amravati Road, 2500615 (Fig. 1). Among the fruit crops registered, Sapindus mukorossi registered by NBPGR, RS Bhowali is unique in Nagpur-440 010, Maharastra having sweet and fleshy fruit. In addition, wild and cultivated species of cashew are also registered. 6. Grapes ICAR-NRC, ICAR-National Research Centre 020-26956000 Wild relatives should get priority for registration as they are sources of many desirable genes for biotic and Pune for Grapes, P.B. No. 3, P.O. Manjri Farm, Solapur Road, Pune-412 307, abiotic stresses. Priority should be the registration of trait specific temperate fruits and rootstocks as no Maharashtra such germplasm has been registered in 7. Litchi, Bael, ICAR-NRC, ICAR-National Research Centre for 0621 - 2289475, these crops. Traits for Aonla & Jackfruit Muzaffarpur Litchi, Mushahari, P.O. Ramna, 2281160 value addition and Muzaffarpur-842 002, Bihar better shelf life need to 8. Mango ICAR-CISH, ICAR-Central Institute for 0522-2841022, be identified and Lucknow Subtropical Horticulture (CISH), 2841023 registered, particularly Rehmankhera, Kakori- i n p l a n t s h a v i n g Lucknow-227 107, Uttar Pradesh perishable fruits. 9. Sub tropical AICRP, ICAR- ICAR-Central Institute for 0522- 2841115 Directorate of Cashew fruits CISH, Lucknow Subtropcal Horticulture (CISH), Research (DCR), Puttur, Rehmankhera, Kakori- IIHR Bengaluru and Lucknow-227 107, Uttar Pradesh CCS HAU, Hissar are 10. Mulberry CSGRC, Hosur Central Sericultural Germplasm 04344-222013, the major contributors Resources Centre, Central Silk 221148, 221149 f o r f r u i t c r o p Board, Ministry of Textile, registration (Kak and Government of India, P.B.-44, Gupta, 2021). Thally Road, Hosur-635 109, Dharmapuri, Tamil Nadu

Fig. 1. Trait Specific Germplasm of Fruit Crops registered with ICAR-NBPGR

112 113 References Holistic Strategy for Germplasm 1. http://www.nbpgr.ernet.in:8080/registration/ 2. http://www.nbpgr.ernet.in:8080/registration/Guidelines.aspx Conservation of Fruit Crops 3. http://www.nbpgr.ernet.in:8080/registration/InventoryofGermplasm.aspx Anuradha Agrawal*, Vartika Srivastava, Era Vaidya Malhotra, Sandhya Gupta, Sangita Bansal 4. Kak A and RK Tyagi (2010) Inventory of Registered Crop Germplasm (2009-2010). ICAR-National Bureau Tissue Culture and Cryopreservation Unit of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 75p. *Email: [email protected] 5. Kak A and RK Tyagi (2015) Inventory of registered Crop Germplasm (2010-2014) ICAR-National Bureau Introduction of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, 103p. Collections of plant genetic resources (PGR), representing the genetic 6. Kak A and V Gupta (2018) Inventory of registered Crop Germplasm (2015-2017). ICAR-National Bureau diversity of any crop or species, are of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, fundamentally required to ensure 74p. food security in a progressively 7. Kak A and V Gupta (2021) Registration of Trait specific Genetic Resources of Horticultural Crops in India. unpredictable world. The Oxford In: KV Peter (ed.) Innovative Techniques for Horticulture Developments, Brillion Publishing, New Delhi, English Dictionary defines the term pp-79-94. 'conservation' as 'keeping from harm, decay, or loss, especially with the view 8. Kak A, K Srinivasan and SK Sharma (2009) Plant Germplasm Registration (2005-2008). ICAR-National to later 'use' and 'to maintain constant Bureau of Plant Genetic Resources, Pusa Campus. Indian Council of Agricultural Research (ICAR).New quantity' (Tyagi and Agrawal, 2015). It Delhi-110012. has been acknowledged since long 9. Kak A, S Archak and RK Tyagi (2017) Online system for plant Germplasm Registration. ICAR-NBPGR. that any conservation strategy alone is not sufficient to conserve the entire 10. NBPGR- Guidelines for registration of Plant Germplasm (Revised, 2014) (2014) ICAR-National Bureau of genetic diversity or genepool of a Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi -110012. particular species (Hoyt, 1988; 11. Plant Germplasm Registration (2006) ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, Maxted et al., 1997). Complementary Indian Council of Agricultural Research, New Delhi-110012. conservation strategies are strongly Fig 1. Complimentary strategies for holistic conservation of PGR recommended to ensure effective and optimum conservation of maximum genepool of a given species (UNCED, 1992). Hence, current trends for germplasm conservation advocate safeguarding PGR diversity both ex situ (outside their natural setting) and also facilitate their continued evolution in situ, in their natural habitats (Fig. 1). In Situ Conservation In situ conservation refers to the maintenance of ecosystems and natural habitats, and recovery of viable populations of species in their natural surroundings. In the case of domesticated or cultivated species, it refers to the surroundings where crops have developed their distinctive properties. For agriculturally important plant species, the greatest diversity in landraces and in crop wild relatives (CWR) is typically found near regions where they were first domesticated. 'On-farm conservation' of PGR is a part of strategy for in situ conservation of plant varieties developed and protected by farmers for centuries in certain areas (Tyagi and Agrawal, 2013). It involves continued cultivation and management by farmers of a diverse set of crop populations in the agro-systems where the crop has evolved or in secondary centres of diversity. The strategy allows for crop populations to continue their evolution in response to natural and human selection. On-farm conservation requires a multidisciplinary approach. It involves (i) socio-economic research to understand and analyze farmers' knowledge; (ii) population and conservation biology; (iii) studies on the dynamics of the local landraces and farmer's varieties to understand population differences, gene flow, degree of inbreeding, selection pressure etc.; (iv) crop improvement research in mass selection and simple breeding methods and (v) extension studies for lesser known crops and varieties for their seed production, marketing and distribution (Tyagi and Agrawal, 2015). In situ approach is the most appropriate conservation strategy for native and endemic species besides CWR. On-farm conservation is more important for landraces and farmers' varieties.

114 115 References Holistic Strategy for Germplasm 1. http://www.nbpgr.ernet.in:8080/registration/ 2. http://www.nbpgr.ernet.in:8080/registration/Guidelines.aspx Conservation of Fruit Crops 3. http://www.nbpgr.ernet.in:8080/registration/InventoryofGermplasm.aspx Anuradha Agrawal*, Vartika Srivastava, Era Vaidya Malhotra, Sandhya Gupta, Sangita Bansal 4. Kak A and RK Tyagi (2010) Inventory of Registered Crop Germplasm (2009-2010). ICAR-National Bureau Tissue Culture and Cryopreservation Unit of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 75p. *Email: [email protected] 5. Kak A and RK Tyagi (2015) Inventory of registered Crop Germplasm (2010-2014) ICAR-National Bureau Introduction of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, 103p. Collections of plant genetic resources (PGR), representing the genetic 6. Kak A and V Gupta (2018) Inventory of registered Crop Germplasm (2015-2017). ICAR-National Bureau diversity of any crop or species, are of Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi-110012, fundamentally required to ensure 74p. food security in a progressively 7. Kak A and V Gupta (2021) Registration of Trait specific Genetic Resources of Horticultural Crops in India. unpredictable world. The Oxford In: KV Peter (ed.) Innovative Techniques for Horticulture Developments, Brillion Publishing, New Delhi, English Dictionary defines the term pp-79-94. 'conservation' as 'keeping from harm, decay, or loss, especially with the view 8. Kak A, K Srinivasan and SK Sharma (2009) Plant Germplasm Registration (2005-2008). ICAR-National to later 'use' and 'to maintain constant Bureau of Plant Genetic Resources, Pusa Campus. Indian Council of Agricultural Research (ICAR).New quantity' (Tyagi and Agrawal, 2015). It Delhi-110012. has been acknowledged since long 9. Kak A, S Archak and RK Tyagi (2017) Online system for plant Germplasm Registration. ICAR-NBPGR. that any conservation strategy alone is not sufficient to conserve the entire 10. NBPGR- Guidelines for registration of Plant Germplasm (Revised, 2014) (2014) ICAR-National Bureau of genetic diversity or genepool of a Plant Genetic Resources, Pusa Campus, Indian Council of Agricultural Research, New Delhi -110012. particular species (Hoyt, 1988; 11. Plant Germplasm Registration (2006) ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, Maxted et al., 1997). Complementary Indian Council of Agricultural Research, New Delhi-110012. conservation strategies are strongly Fig 1. Complimentary strategies for holistic conservation of PGR recommended to ensure effective and optimum conservation of maximum genepool of a given species (UNCED, 1992). Hence, current trends for germplasm conservation advocate safeguarding PGR diversity both ex situ (outside their natural setting) and also facilitate their continued evolution in situ, in their natural habitats (Fig. 1). In Situ Conservation In situ conservation refers to the maintenance of ecosystems and natural habitats, and recovery of viable populations of species in their natural surroundings. In the case of domesticated or cultivated species, it refers to the surroundings where crops have developed their distinctive properties. For agriculturally important plant species, the greatest diversity in landraces and in crop wild relatives (CWR) is typically found near regions where they were first domesticated. 'On-farm conservation' of PGR is a part of strategy for in situ conservation of plant varieties developed and protected by farmers for centuries in certain areas (Tyagi and Agrawal, 2013). It involves continued cultivation and management by farmers of a diverse set of crop populations in the agro-systems where the crop has evolved or in secondary centres of diversity. The strategy allows for crop populations to continue their evolution in response to natural and human selection. On-farm conservation requires a multidisciplinary approach. It involves (i) socio-economic research to understand and analyze farmers' knowledge; (ii) population and conservation biology; (iii) studies on the dynamics of the local landraces and farmer's varieties to understand population differences, gene flow, degree of inbreeding, selection pressure etc.; (iv) crop improvement research in mass selection and simple breeding methods and (v) extension studies for lesser known crops and varieties for their seed production, marketing and distribution (Tyagi and Agrawal, 2015). In situ approach is the most appropriate conservation strategy for native and endemic species besides CWR. On-farm conservation is more important for landraces and farmers' varieties.

114 115 In situ conservation is fraught with danger of a species becoming extinct or its population declining due to genetic equipment, high budget and highly skilled personnel to prepare and store genomic resources. However, drift, inbreeding, demographic and environmental variations, habitat loss, competition from exotic species, pest with increasing awareness, establishment of DNA banks is progressing worldwide (Table 1). At ICAR-NBPGR, incidence or over-exploitation and human disturbances. In such conditions or even otherwise, ex situ approach New Delhi, genomic DNAs of 2,194 accessions of different plant species have been conserved in liquid offers a feasible choice to conserve diversity of species and genotypes (Tyagi and Agrawal, 2015). nitrogen (-196ºC). Ex Situ Conservation Thus, there are a number of options for conserving PGR ex situ, and the choice is more often based on its merits in utility, security, complementarity and the advantages over the other available techniques. The use The ex situ strategy entails conservation of biological diversity outside its natural habitat, under suitable conditions of alternative conservation methods should not only be considered for vegetatively propagated and non- to prolong their lives. Broadly this may be in botanical gardens, arboreta, or gene banks (Fig. 2). Ex situ orthodox seeded species. In some cases, in addition to traditional conservation methods, it is necessary to conservation is particularly important for PGR. The term 'gene bank' is used to describe an organizational unit implement complementary strategies to increase the chances of survival even for species with orthodox which has the objective of managing PGR collections for their conservation and to facilitate their use (Tyagi and seeds that are easily preserved in seed banks (ICAR-NBPGR 2016). Agrawal, 2015). Gene banks are typically establishments where routine operations such as management of plant collections in fields, the storage of seed, plant tissue, pollen and/or DNA sequences are carried out. Thus, there In the following section, guidelines and standard operating procedures (SOPs) developed by ICAR-NBPGR exist seed gene banks (SGB), in vitro gene banks (IVGB), cryo gene banks (CGB), DNA banks or live collections in for in vitro conservation and cryopreservation are reproduced, which are relevant to tropical fruit genetic field gene banks ((FGB) for conserving different types of PGR, depending on the biology of the plant and the end resources (ICAR-NBPGR, 2016). user need (Agrawal et al., 2019). Ex situ conservation also appealing because it allows plant breeders easy access to PGR than is provided by in situ conservation. However, PGR first must be collected, and samples of only a small fraction of the world's PGR have been collected so far. Stored plant materials must be kept under controlled conditions, and periodically regenerated (planted and grown) in order to maintain viability (Tyagi and Agrawal, 2015). Conventional SGB conservation (dry storage at -18 to -20°C) is the simplest and most efficient method for ex situ conservation of plant germplasm, and it is also the best choice because it preserves genetically diverse material. However, not all plant species can be preserved by this technique (Benson, 2002). Species with recalcitrant (desiccation-sensitive) and intermediate (relatively desiccation-tolerant) seeds cannot stand the desiccation conditions and cold storage without losing viability; only orthodox seeds (desiccation-tolerant) are able to do so. Further, there are species that do not produce seeds and are propagated vegetatively or clonally, such as several fruit crops. For these plant species, the traditional ex situ conservation method is in the form of field collections. But this method also has its limitation, as it usually requires large areas of land, is labor intensive, and collections are unprotected from natural disasters. The alternative approaches for the conservation of these species, is the application of biotechnology (Pence et al., 2020). Techniques like in vitro conservation and cryopreservation have made it possible to collect and conserve genetic resources, especially of species that are difficult to conserve in form of seeds (Benson et al., 2011a, b, c). Pollen storage also contribute to ex situ conservation. Conservation of plant genomic resources in form of DNA is an emerging complementary strategy for sustainable utilization of PGR. Genomic resources become even more useful in case of recalcitrant and difficult-to-conserve species or the species that are highly threatened in the wild. Though currently it is neither possible to regenerate the whole plant nor recover original genotypes from genomic resources, but they are a valuable source of important traits of an organism that can be utilized in different molecular breeding programs. With rapid advancement in molecular techniques and biotechnology, genomic Table 1. DNA Banks in the World resources are becoming increasingly important in study and Fig 2: Schematic representation of ex situ conservation of genetic and management of PGR. Genomic resources in form of genomic DNAs, 1. The Royal Botanic Gardens, Kew, UK genomic resources in genebanks DNA fragments, genes, cDNAs, gene libraries, RNAs etc. are 2. The US Missouri Botanical Garden routinely prepared and stored for short-term in different molecular 3. The Australian Plant DNA Bank, Guidelines of In Vitro Conservation and Cryopreservation of Fruit Genetic Resources biology laboratories. Options are available for conserving them for Southern Cross University long-term in DNA banks. DNAs can be stored at -20ºC for mid-term 4. The DNA bank at Leslie Hill Molecular In Vitro Culture Initiation and Multiplication storage and for long-term storage, ultra-low temperature deep Systematics Laboratory, National Germplasm acquisition for conservation freezers (-80ºC) and liquid nitrogen (LN) is used (Ehsan et al., 2006). Botanical Institute (NBI), Kirstenbosch, The advantages of conserving genomic resources are (i) DNAs are South Africa • The germplasm for in IVGB should be obtained from an authentic source (e.g. FGB, greenhouse, relatively stable when stored at low temperatures; (ii) sample size is 5. National Institute of Agrobiological glasshouse, nethouse, or other IVG) along with its correct taxonomic identity. small and storage requires less space; (iii) efficient and long-term Sciences (NIAS), Ibaraki, Japan • It should always be accompanied with passport information. Characterization and/or evaluation data method for conserving genetic information of an organism. The 6. National Herbarium, Netherlands are desirable. limtations with genomic resource conservation are (i) It is not 7. DNA Bank Brazilian Flora Species Plant possible to regenerate whole plant from genomic resources; (ii) DNA Bank, Korea • Samples for IVG may be obtained as whole plants, vegetative propagules, in vitro cultures and in RNA is less stable during storage; (iii) require sophisticated Trinity College DNA Bank, Dublin exceptional cases as seeds/embryos (e.g. threatened species, crop wild relatives). 116 117 In situ conservation is fraught with danger of a species becoming extinct or its population declining due to genetic equipment, high budget and highly skilled personnel to prepare and store genomic resources. However, drift, inbreeding, demographic and environmental variations, habitat loss, competition from exotic species, pest with increasing awareness, establishment of DNA banks is progressing worldwide (Table 1). At ICAR-NBPGR, incidence or over-exploitation and human disturbances. In such conditions or even otherwise, ex situ approach New Delhi, genomic DNAs of 2,194 accessions of different plant species have been conserved in liquid offers a feasible choice to conserve diversity of species and genotypes (Tyagi and Agrawal, 2015). nitrogen (-196ºC). Ex Situ Conservation Thus, there are a number of options for conserving PGR ex situ, and the choice is more often based on its merits in utility, security, complementarity and the advantages over the other available techniques. The use The ex situ strategy entails conservation of biological diversity outside its natural habitat, under suitable conditions of alternative conservation methods should not only be considered for vegetatively propagated and non- to prolong their lives. Broadly this may be in botanical gardens, arboreta, or gene banks (Fig. 2). Ex situ orthodox seeded species. In some cases, in addition to traditional conservation methods, it is necessary to conservation is particularly important for PGR. The term 'gene bank' is used to describe an organizational unit implement complementary strategies to increase the chances of survival even for species with orthodox which has the objective of managing PGR collections for their conservation and to facilitate their use (Tyagi and seeds that are easily preserved in seed banks (ICAR-NBPGR 2016). Agrawal, 2015). Gene banks are typically establishments where routine operations such as management of plant collections in fields, the storage of seed, plant tissue, pollen and/or DNA sequences are carried out. Thus, there In the following section, guidelines and standard operating procedures (SOPs) developed by ICAR-NBPGR exist seed gene banks (SGB), in vitro gene banks (IVGB), cryo gene banks (CGB), DNA banks or live collections in for in vitro conservation and cryopreservation are reproduced, which are relevant to tropical fruit genetic field gene banks ((FGB) for conserving different types of PGR, depending on the biology of the plant and the end resources (ICAR-NBPGR, 2016). user need (Agrawal et al., 2019). Ex situ conservation also appealing because it allows plant breeders easy access to PGR than is provided by in situ conservation. However, PGR first must be collected, and samples of only a small fraction of the world's PGR have been collected so far. Stored plant materials must be kept under controlled conditions, and periodically regenerated (planted and grown) in order to maintain viability (Tyagi and Agrawal, 2015). Conventional SGB conservation (dry storage at -18 to -20°C) is the simplest and most efficient method for ex situ conservation of plant germplasm, and it is also the best choice because it preserves genetically diverse material. However, not all plant species can be preserved by this technique (Benson, 2002). Species with recalcitrant (desiccation-sensitive) and intermediate (relatively desiccation-tolerant) seeds cannot stand the desiccation conditions and cold storage without losing viability; only orthodox seeds (desiccation-tolerant) are able to do so. Further, there are species that do not produce seeds and are propagated vegetatively or clonally, such as several fruit crops. For these plant species, the traditional ex situ conservation method is in the form of field collections. But this method also has its limitation, as it usually requires large areas of land, is labor intensive, and collections are unprotected from natural disasters. The alternative approaches for the conservation of these species, is the application of biotechnology (Pence et al., 2020). Techniques like in vitro conservation and cryopreservation have made it possible to collect and conserve genetic resources, especially of species that are difficult to conserve in form of seeds (Benson et al., 2011a, b, c). Pollen storage also contribute to ex situ conservation. Conservation of plant genomic resources in form of DNA is an emerging complementary strategy for sustainable utilization of PGR. Genomic resources become even more useful in case of recalcitrant and difficult-to-conserve species or the species that are highly threatened in the wild. Though currently it is neither possible to regenerate the whole plant nor recover original genotypes from genomic resources, but they are a valuable source of important traits of an organism that can be utilized in different molecular breeding programs. With rapid advancement in molecular techniques and biotechnology, genomic Table 1. DNA Banks in the World resources are becoming increasingly important in study and Fig 2: Schematic representation of ex situ conservation of genetic and management of PGR. Genomic resources in form of genomic DNAs, 1. The Royal Botanic Gardens, Kew, UK genomic resources in genebanks DNA fragments, genes, cDNAs, gene libraries, RNAs etc. are 2. The US Missouri Botanical Garden routinely prepared and stored for short-term in different molecular 3. The Australian Plant DNA Bank, Guidelines of In Vitro Conservation and Cryopreservation of Fruit Genetic Resources biology laboratories. Options are available for conserving them for Southern Cross University long-term in DNA banks. DNAs can be stored at -20ºC for mid-term 4. The DNA bank at Leslie Hill Molecular In Vitro Culture Initiation and Multiplication storage and for long-term storage, ultra-low temperature deep Systematics Laboratory, National Germplasm acquisition for conservation freezers (-80ºC) and liquid nitrogen (LN) is used (Ehsan et al., 2006). Botanical Institute (NBI), Kirstenbosch, The advantages of conserving genomic resources are (i) DNAs are South Africa • The germplasm for in IVGB should be obtained from an authentic source (e.g. FGB, greenhouse, relatively stable when stored at low temperatures; (ii) sample size is 5. National Institute of Agrobiological glasshouse, nethouse, or other IVG) along with its correct taxonomic identity. small and storage requires less space; (iii) efficient and long-term Sciences (NIAS), Ibaraki, Japan • It should always be accompanied with passport information. Characterization and/or evaluation data method for conserving genetic information of an organism. The 6. National Herbarium, Netherlands are desirable. limtations with genomic resource conservation are (i) It is not 7. DNA Bank Brazilian Flora Species Plant possible to regenerate whole plant from genomic resources; (ii) DNA Bank, Korea • Samples for IVG may be obtained as whole plants, vegetative propagules, in vitro cultures and in RNA is less stable during storage; (iii) require sophisticated Trinity College DNA Bank, Dublin exceptional cases as seeds/embryos (e.g. threatened species, crop wild relatives). 116 117 • The number of explants for culture initiation should be determined based on the reproductive biology • Obvious signs of growth are visible within 1-2 weeks following in vitro introduction. of the species, season and propagation rate. • It is good practice to test new cultures for systemic infection (endogeous bacteria), 4-6 months after Quarantine/ health testing of samples initiation using bacteriological indexing procedures. • Germplasm samples should ideally be sourced from disease-free mother plants/propagules. • Bacteria-free cultures should be indexed for viruses using standard serological (ELISA), molecular (PCR based) and ultrastructural (Electron microscopy) techniques; virus-free material should preferably be • Exotic collections should be accompanied with suitable Phytosanitary Certificate from the source used for subsequent steps. country, and have undergone suitable quarantine checks in the host country. In vitro multiplication • In case of indigenous collections obtained as live plants/cuttings, they should be suitably planted in screen house or in growth chamber for establishment and growth. • Only aseptic cultures, established in vitro, should be selected for multiplication and generating adequate number of explants for subsequent IVAG conservation. • Prior to in vitro introduction, the disease status, especially endogenous bacteria and viruses should be checked in the samples, as per guidelines and/or standard operating procedures available (Srivastava et • Shoots should be removed aseptically under a laminar air flow (LAF) cabinet from the culture vessel and al., 2020). micro-cuttings of apical and/or nodal sections be transferred to a new medium. Individual test-tube should be labelled clearly using a standardized format either manually or using a data logger Establishment of in vitro cultures barcode/QR code. • Depending on the species to be established, suitable explants need to be selected for its introduction in • The medium required for culture initiation may be different from that required for shoot multiplication, tissue culture. These may be meritems, shoot tips, nodal cuttings, young shoots derived from whole plantlet regeneration and storage. This should be determined based on available literature. plants or vegetative propagules like budwood, suckers and rhizomes; in rare cases like triploid/seed- sterile plants, floral buds may also be used. • Cultures must be transferred to a regulated culture room at the appropriate crop-specific, temperature/light regime. • Type, source and physiological stage of explant are the important factors in optimizing the tissue culture protocol. The material for in vitro conservation should be transported from the site of collection to IVG • Perodically cultures should be monitored for contamination, hyperhydricity, growth abnormalities in the shortest possible time and under the best possible conditions. (somaclonal variation, loss of regenerability etc.) after each subculture cycle. • The explants must be disinfected to remove microbial contaminants on the surface. Using one or more In Vitro Conservation Using Slow-Growth Strategies disinfectants, like ethanol (70%), sodium/calcium hypochlorite, commercial bleach/ chlorine water and Well-established aseptic cultures required for medium-term conservation in IVAG are normally subjected to antibiotics, in appropriate concentration. Pre-soaking in an anti-oxidant solution (e.g. ascorbic acid) a suitable slow-growth storage strategy to save time, energy, costs and space. Slow growth strategies are may be useful for woody plants. Explants drawn from field need to be checked for presence of mites, based on extending subculture duration, without risking germplasm loss and compromising genetic stability thrips and other insects. When working with new explant type, it is better to run preliminary cleaning through stressful treatments. Species/genotype response to growth limiting treatments can be highly tests to determine the longest exposure time sustained by explants. variable. For species which are naturally slow growing, there is no need for manipulation of media or of • Using sterilized forceps, explants are transferred into a sterile container and rinsed 2-3 times with sterile culture conditions. Details of various slow growth strategies are given below. In general two or more distilled water. Edges of the explant are cut with the help of a scalpel (with sterile blade). techniques are normally combined to obtain slow growth in cultures under in vitro conservation. Stored cultures need to be scored for viability, chlorosis, defoliation, browning, tip necrosis, hyperhydricity and • Care should be taken to avoid dehydration of delicate explants such as shoot tips. To minimize the contamination. Depending on the method applied, cultures can remain viable from 6-30 months, without chances of cross-contamination in explants derived from underground storage organs, each explant subculture. should be cultured in individual tubes. Growth retardation by changing physical parameters • Prior to culture initiation, base of the explants should be streaked on a medium (mostly nutrient agar) for testing any microbial contaminant. • Decreasing temperature at which cultures are maintained (e.g. from 25˚C to 4-15˚C). It is the most commonly used and single method for restricting the growth of tissues, found suitable for temperate • With sterile forceps, explants are planted on a suitable culture medium (e.g. Murashige and Skoog). crops. Tropical plant species are generally cold-sensitive and may exhibit chilling injury. Each tube should be covered with a plastic cap and sealed with parafilm/cling film; in some cases, cotton plugs give better growth. Labelling in each tube is done for an accession number, the date of • Reducing light intensity at which cultures are maintained. It has been found beneficial for reducing introduction and line number. growth in several species, especially temperate crops. • Culture tubes are transferred to a growth chamber (temperature 25±2oC) under suitable photoperiod • Changing the culture vessel enclosures influences the rate of evaporation of water content of the and light intensity, depending species. medium. Use of polypropylene caps instead of cotton plugs as has been found to significantly increase storage period of the cultures. • The crop/genotype specific media should be pre-determined, as also the additives such as charcoal, plant growth regulators, antioxidants etc. to obtain the desired response from explants. • Size and type of the culture vessels also plays a very important role. e.g. sterile, heat sealable polypropylene bags and large glass bottles have proven to be better than glass test tubes. • Culture initiation media should be gelled with a clear gelling agent (e.g. gellan gum rather than agar) for easier detection of bacteria and other contaminants. • Minimal growth can be achieved by lowering the available oxygen level to tissues. The simplest method is to cover the tissues with mineral oil layer. However, some associated problems with decreased oxygen • Seasonal, physiological and developmental factors of the explants can also affect success of culture concentration may be encountered (e.g. vitrification, partial or complete necrosis), which depends on initiation. species/genotypes. 118 119 • The number of explants for culture initiation should be determined based on the reproductive biology • Obvious signs of growth are visible within 1-2 weeks following in vitro introduction. of the species, season and propagation rate. • It is good practice to test new cultures for systemic infection (endogeous bacteria), 4-6 months after Quarantine/ health testing of samples initiation using bacteriological indexing procedures. • Germplasm samples should ideally be sourced from disease-free mother plants/propagules. • Bacteria-free cultures should be indexed for viruses using standard serological (ELISA), molecular (PCR based) and ultrastructural (Electron microscopy) techniques; virus-free material should preferably be • Exotic collections should be accompanied with suitable Phytosanitary Certificate from the source used for subsequent steps. country, and have undergone suitable quarantine checks in the host country. In vitro multiplication • In case of indigenous collections obtained as live plants/cuttings, they should be suitably planted in screen house or in growth chamber for establishment and growth. • Only aseptic cultures, established in vitro, should be selected for multiplication and generating adequate number of explants for subsequent IVAG conservation. • Prior to in vitro introduction, the disease status, especially endogenous bacteria and viruses should be checked in the samples, as per guidelines and/or standard operating procedures available (Srivastava et • Shoots should be removed aseptically under a laminar air flow (LAF) cabinet from the culture vessel and al., 2020). micro-cuttings of apical and/or nodal sections be transferred to a new medium. Individual test-tube should be labelled clearly using a standardized format either manually or using a data logger Establishment of in vitro cultures barcode/QR code. • Depending on the species to be established, suitable explants need to be selected for its introduction in • The medium required for culture initiation may be different from that required for shoot multiplication, tissue culture. These may be meritems, shoot tips, nodal cuttings, young shoots derived from whole plantlet regeneration and storage. This should be determined based on available literature. plants or vegetative propagules like budwood, suckers and rhizomes; in rare cases like triploid/seed- sterile plants, floral buds may also be used. • Cultures must be transferred to a regulated culture room at the appropriate crop-specific, temperature/light regime. • Type, source and physiological stage of explant are the important factors in optimizing the tissue culture protocol. The material for in vitro conservation should be transported from the site of collection to IVG • Perodically cultures should be monitored for contamination, hyperhydricity, growth abnormalities in the shortest possible time and under the best possible conditions. (somaclonal variation, loss of regenerability etc.) after each subculture cycle. • The explants must be disinfected to remove microbial contaminants on the surface. Using one or more In Vitro Conservation Using Slow-Growth Strategies disinfectants, like ethanol (70%), sodium/calcium hypochlorite, commercial bleach/ chlorine water and Well-established aseptic cultures required for medium-term conservation in IVAG are normally subjected to antibiotics, in appropriate concentration. Pre-soaking in an anti-oxidant solution (e.g. ascorbic acid) a suitable slow-growth storage strategy to save time, energy, costs and space. Slow growth strategies are may be useful for woody plants. Explants drawn from field need to be checked for presence of mites, based on extending subculture duration, without risking germplasm loss and compromising genetic stability thrips and other insects. When working with new explant type, it is better to run preliminary cleaning through stressful treatments. Species/genotype response to growth limiting treatments can be highly tests to determine the longest exposure time sustained by explants. variable. For species which are naturally slow growing, there is no need for manipulation of media or of • Using sterilized forceps, explants are transferred into a sterile container and rinsed 2-3 times with sterile culture conditions. Details of various slow growth strategies are given below. In general two or more distilled water. Edges of the explant are cut with the help of a scalpel (with sterile blade). techniques are normally combined to obtain slow growth in cultures under in vitro conservation. Stored cultures need to be scored for viability, chlorosis, defoliation, browning, tip necrosis, hyperhydricity and • Care should be taken to avoid dehydration of delicate explants such as shoot tips. To minimize the contamination. Depending on the method applied, cultures can remain viable from 6-30 months, without chances of cross-contamination in explants derived from underground storage organs, each explant subculture. should be cultured in individual tubes. Growth retardation by changing physical parameters • Prior to culture initiation, base of the explants should be streaked on a medium (mostly nutrient agar) for testing any microbial contaminant. • Decreasing temperature at which cultures are maintained (e.g. from 25˚C to 4-15˚C). It is the most commonly used and single method for restricting the growth of tissues, found suitable for temperate • With sterile forceps, explants are planted on a suitable culture medium (e.g. Murashige and Skoog). crops. Tropical plant species are generally cold-sensitive and may exhibit chilling injury. Each tube should be covered with a plastic cap and sealed with parafilm/cling film; in some cases, cotton plugs give better growth. Labelling in each tube is done for an accession number, the date of • Reducing light intensity at which cultures are maintained. It has been found beneficial for reducing introduction and line number. growth in several species, especially temperate crops. • Culture tubes are transferred to a growth chamber (temperature 25±2oC) under suitable photoperiod • Changing the culture vessel enclosures influences the rate of evaporation of water content of the and light intensity, depending species. medium. Use of polypropylene caps instead of cotton plugs as has been found to significantly increase storage period of the cultures. • The crop/genotype specific media should be pre-determined, as also the additives such as charcoal, plant growth regulators, antioxidants etc. to obtain the desired response from explants. • Size and type of the culture vessels also plays a very important role. e.g. sterile, heat sealable polypropylene bags and large glass bottles have proven to be better than glass test tubes. • Culture initiation media should be gelled with a clear gelling agent (e.g. gellan gum rather than agar) for easier detection of bacteria and other contaminants. • Minimal growth can be achieved by lowering the available oxygen level to tissues. The simplest method is to cover the tissues with mineral oil layer. However, some associated problems with decreased oxygen • Seasonal, physiological and developmental factors of the explants can also affect success of culture concentration may be encountered (e.g. vitrification, partial or complete necrosis), which depends on initiation. species/genotypes. 118 119 Growth retardation by chemical changes in medium Classical cryopreservation techniques • Decreasing the nutrient contents in the culture medium and/or sucrose restricts the growth of cultures, This method follow protocols which induces a freeze-dehydration process using a slow freezing regime. Ice is thereby increasing the subculture duration. initially formed in the extracellular solution, during slow temperature decrease and this external crystallization • Use of osmotic regulators like mannitol and sorbitol (3-6% w/w) minimizes growth by imposing osmotic promotes the efflux of water from the cytoplasm and vacuoles to the outside of the cells. Therefore, cell stress on the cultures. dehydration will depend on the cooling rate and the prefreezing temperature set up before immersion of samples to LN. Classical cryo-techniques have been successfully applied to cell suspensions and calluses of different plant • Use of inhibitory level of growth retardants (2-50 mg/l) such as maleic hydrazide, abscicic acid, species, apices of cold-tolerant plants as well as shoot-apices from tropical species. Following successive steps n-dimethyl succinamic acid, cycocel and phosphine-D to the culture medium leads to slow growth. need to be defined for each species. However, use of growth retardants is generally not preferred due to its effects on genetic integrity of the cultures. • Selection of material and pretreatment of donor plants: Generally, rapidly growing meristems which are small, relatively resistant to freezing, possessing fewer or smaller vacuoled cells and dense cytoplasm are Monitoring Genetic Stability of In Vitro Conserved Germplasm preferred for cryopreservation. The material can be sampled preferably from in vitro fresh active cultures to In vitro selection pressure can potentially generate variants or mutants. Also some genotypes have a avoid contamination. propensity for producing off-types and variants (due to natural chimeras). Maintaining shoot and plantlet • Pre-treatment of isolated explants: It refers to the period in culture before cryopreservation begins. It offers to regeneration through preformed (original) meristems (apical and axillary buds) and avoiding adventitious induce physiological changes in tissues to increase freeze-tolerance. Following the aseptic excision of the shoots greatly aids the maintenance of trueness-to-type and genetic stability and offset the risks of SCV. explants and culture on high sucrose medium (0.1-0.5 M) the plant tissues can be cold-hardened by using However, some slow growth treatments can aggravate abnormal morphogenetic responses (Agrawal et al., effective pre-growth additives such as mannitol, sorbitol, proline or dimethylsulphoxide (DMSO) in 2011). Thus, in vitro conserved cultures should be periodically monitored for genetic stability by suitable pre-growth culture medium. methods (phenotypic, chromosomal, cytological, epigenetic and genetic molecular tests, RAPD, DNA methylation, RFLP, AFLP, SSR, etc (Agrawal et al., 2014; Malhotra et al., 2020; Sharma et al., 2021). • Application of cryoprotectant: Just before freezing, the effective compounds as cryoprotectants are applied which includes DMSO, glycerol, proline, sucrose, sorbitol and polyethylene glycol (PEG). These compounds IVBG - Long-Term Conservation Using Cryopreservation can be used individually or in combination. Possible cryoprotectant must be screened for toxicity without Cryopreservation is the technique utilized for long-term storage of living cell, tissues, organs and other freezing using concentration and time as variables for the investigation. biological resources at ultra-low temperatures (−196°C) usually in liquid nitrogen (LN). Under such a low • Freezing, storage and thawing: These steps are followed very carefully, otherwise damage during freezing may temperature, the metabolic activities of the tissue virtually comes to a halt, and thus, it can be conserved occur. Slow freezing is carried out by using controlled freezing apparatus and which help in extra-cellular ice without significant change in its viability (Wang et al., 2020). Furthermore, the cryopreserved tissues are formation. The frozen specimen is then transferred to storage in liquid nitrogen. Specimens are thawed stored in a small volume, require very low maintenance (replenishment of LN only), samples are free from rapidly or slowly to avoid the risk of damage to cellular integrity due to recrystallization of any residual ice the risk of contamination and other operational errors. This technique provides safe, cost-effective formed during freezing. conservation of almost all biological material including non-orthodox seeds, vegetatively propagated species and biotechnologically important plant cell lines. • Recovery growth: It is important that viability level achieved on thawing should be high. Usually recovery growth is carried out on a defined medium supplemented with activated charcoal to adsorb toxins released Tissues from the field that are amenable to cryopreservation include seeds, embryos, embryonic axes, by damaged cells and growth substances to stimulate the desired responses. It is sometimes necessary to budwood and pollen. In vitro derived tissues normally cryopreserved are meristems, shoot tips, nodal avoid the osmotic shock caused by an intermediate transfer onto a medium with low potential by successive cuttings, cell suspensions and somatic embryos. Seeds of various orthodox species are usually desiccation transfer of material to progressively less concentrated medium and to avoid photo-oxidation which may be and freezing tolerant and thus can be easily conserved at cryogenic temperature (-196˚C) with good harmful, recovery can be made in dark. regeneration after thawing. However, seeds of recalcitrant nature (eg. mango, litchi, jackfruit, sapota etc.) are very sensitive to desiccation and freezing, hence, the embryo or embryonic axes (EA) of these seeds are New cryopreservation techniques observed to be the best propagules for developing long-term conservation strategy. Embryos or EA may be These are vitrification-based procedures that involve cell dehydration prior to cooling by exposure of samples to cryopreserved either directly or after desiccation to desired moisture levels or treated with cryoprotectants highly concentrated cryoprotectant solution (usually called plant vitrification solutions, PVS) and/or by air after osmotic dehydration followed by cryopreservation. To conserve the vegetatively propagated crops, desiccation. Physical process of vitrification per se is definite as the transition of the liquid phase to an amorphous either dormant buds or explants derived from healthy aseptic in vitro cultures are used as starting material glassy solid at the glass transition (Tg) temperature (Mandal et al., 2003). When the explants undergo desiccation, for cryopreservation. The explants usually employed in in vitro cryopreservation contain high amounts of rapid or ultra-rapid cooling and thawing process, the glass transition may help in preventing tissue collapse, solute cellular water and are extremely sensitive to freezing injury during liquid nitrogen (LN) exposure. Thus, cells concentration and pH alterations. Different techniques of cryopreservation are given in Table 2. need to be dehydrated artificially to protect them from the damages caused by the crystallization of intracellular water. The cellular water can be dehydrated till the required extent using various techniques, for successful cryopreservation. Over the past 40 years a range of cryopreservation techniques have been developed and tested for conserving plant cells and tissues. Cryopreservation simply follows the process of removal of water from the cells, which plays an important role in preventing cellular damage due to freezing injury. Cryopreservation can be attempted using classical (freeze induced in which cooling is performed in the presence of ice) or new cryopreservation (vitrification-based in which cooling normally takes place without ice formation) techniques, as detailed hereunder.

120 121 Growth retardation by chemical changes in medium Classical cryopreservation techniques • Decreasing the nutrient contents in the culture medium and/or sucrose restricts the growth of cultures, This method follow protocols which induces a freeze-dehydration process using a slow freezing regime. Ice is thereby increasing the subculture duration. initially formed in the extracellular solution, during slow temperature decrease and this external crystallization • Use of osmotic regulators like mannitol and sorbitol (3-6% w/w) minimizes growth by imposing osmotic promotes the efflux of water from the cytoplasm and vacuoles to the outside of the cells. Therefore, cell stress on the cultures. dehydration will depend on the cooling rate and the prefreezing temperature set up before immersion of samples to LN. Classical cryo-techniques have been successfully applied to cell suspensions and calluses of different plant • Use of inhibitory level of growth retardants (2-50 mg/l) such as maleic hydrazide, abscicic acid, species, apices of cold-tolerant plants as well as shoot-apices from tropical species. Following successive steps n-dimethyl succinamic acid, cycocel and phosphine-D to the culture medium leads to slow growth. need to be defined for each species. However, use of growth retardants is generally not preferred due to its effects on genetic integrity of the cultures. • Selection of material and pretreatment of donor plants: Generally, rapidly growing meristems which are small, relatively resistant to freezing, possessing fewer or smaller vacuoled cells and dense cytoplasm are Monitoring Genetic Stability of In Vitro Conserved Germplasm preferred for cryopreservation. The material can be sampled preferably from in vitro fresh active cultures to In vitro selection pressure can potentially generate variants or mutants. Also some genotypes have a avoid contamination. propensity for producing off-types and variants (due to natural chimeras). Maintaining shoot and plantlet • Pre-treatment of isolated explants: It refers to the period in culture before cryopreservation begins. It offers to regeneration through preformed (original) meristems (apical and axillary buds) and avoiding adventitious induce physiological changes in tissues to increase freeze-tolerance. Following the aseptic excision of the shoots greatly aids the maintenance of trueness-to-type and genetic stability and offset the risks of SCV. explants and culture on high sucrose medium (0.1-0.5 M) the plant tissues can be cold-hardened by using However, some slow growth treatments can aggravate abnormal morphogenetic responses (Agrawal et al., effective pre-growth additives such as mannitol, sorbitol, proline or dimethylsulphoxide (DMSO) in 2011). Thus, in vitro conserved cultures should be periodically monitored for genetic stability by suitable pre-growth culture medium. methods (phenotypic, chromosomal, cytological, epigenetic and genetic molecular tests, RAPD, DNA methylation, RFLP, AFLP, SSR, etc (Agrawal et al., 2014; Malhotra et al., 2020; Sharma et al., 2021). • Application of cryoprotectant: Just before freezing, the effective compounds as cryoprotectants are applied which includes DMSO, glycerol, proline, sucrose, sorbitol and polyethylene glycol (PEG). These compounds IVBG - Long-Term Conservation Using Cryopreservation can be used individually or in combination. Possible cryoprotectant must be screened for toxicity without Cryopreservation is the technique utilized for long-term storage of living cell, tissues, organs and other freezing using concentration and time as variables for the investigation. biological resources at ultra-low temperatures (−196°C) usually in liquid nitrogen (LN). Under such a low • Freezing, storage and thawing: These steps are followed very carefully, otherwise damage during freezing may temperature, the metabolic activities of the tissue virtually comes to a halt, and thus, it can be conserved occur. Slow freezing is carried out by using controlled freezing apparatus and which help in extra-cellular ice without significant change in its viability (Wang et al., 2020). Furthermore, the cryopreserved tissues are formation. The frozen specimen is then transferred to storage in liquid nitrogen. Specimens are thawed stored in a small volume, require very low maintenance (replenishment of LN only), samples are free from rapidly or slowly to avoid the risk of damage to cellular integrity due to recrystallization of any residual ice the risk of contamination and other operational errors. This technique provides safe, cost-effective formed during freezing. conservation of almost all biological material including non-orthodox seeds, vegetatively propagated species and biotechnologically important plant cell lines. • Recovery growth: It is important that viability level achieved on thawing should be high. Usually recovery growth is carried out on a defined medium supplemented with activated charcoal to adsorb toxins released Tissues from the field that are amenable to cryopreservation include seeds, embryos, embryonic axes, by damaged cells and growth substances to stimulate the desired responses. It is sometimes necessary to budwood and pollen. In vitro derived tissues normally cryopreserved are meristems, shoot tips, nodal avoid the osmotic shock caused by an intermediate transfer onto a medium with low potential by successive cuttings, cell suspensions and somatic embryos. Seeds of various orthodox species are usually desiccation transfer of material to progressively less concentrated medium and to avoid photo-oxidation which may be and freezing tolerant and thus can be easily conserved at cryogenic temperature (-196˚C) with good harmful, recovery can be made in dark. regeneration after thawing. However, seeds of recalcitrant nature (eg. mango, litchi, jackfruit, sapota etc.) are very sensitive to desiccation and freezing, hence, the embryo or embryonic axes (EA) of these seeds are New cryopreservation techniques observed to be the best propagules for developing long-term conservation strategy. Embryos or EA may be These are vitrification-based procedures that involve cell dehydration prior to cooling by exposure of samples to cryopreserved either directly or after desiccation to desired moisture levels or treated with cryoprotectants highly concentrated cryoprotectant solution (usually called plant vitrification solutions, PVS) and/or by air after osmotic dehydration followed by cryopreservation. To conserve the vegetatively propagated crops, desiccation. Physical process of vitrification per se is definite as the transition of the liquid phase to an amorphous either dormant buds or explants derived from healthy aseptic in vitro cultures are used as starting material glassy solid at the glass transition (Tg) temperature (Mandal et al., 2003). When the explants undergo desiccation, for cryopreservation. The explants usually employed in in vitro cryopreservation contain high amounts of rapid or ultra-rapid cooling and thawing process, the glass transition may help in preventing tissue collapse, solute cellular water and are extremely sensitive to freezing injury during liquid nitrogen (LN) exposure. Thus, cells concentration and pH alterations. Different techniques of cryopreservation are given in Table 2. need to be dehydrated artificially to protect them from the damages caused by the crystallization of intracellular water. The cellular water can be dehydrated till the required extent using various techniques, for successful cryopreservation. Over the past 40 years a range of cryopreservation techniques have been developed and tested for conserving plant cells and tissues. Cryopreservation simply follows the process of removal of water from the cells, which plays an important role in preventing cellular damage due to freezing injury. Cryopreservation can be attempted using classical (freeze induced in which cooling is performed in the presence of ice) or new cryopreservation (vitrification-based in which cooling normally takes place without ice formation) techniques, as detailed hereunder.

120 121 Table 2: Techniques of Cryopreservation

Technique Explants Procedure Remarks 1. Pregrowth Meristems Tissue/explants are cultured in Applicable to very limited Shoot tips/buds presence of cryoprotectants species and response is Somatic embryos (DMSO, PEG, Sucrose etc.) for genotype dependant, e.g. few days/weeks followed by Musa rapid freezing in LN 2. Pregrowth- Shoot tips/buds Tissue/explants are cultured in Applicable to very limited desiccation Nodal cuttings presence of cryoprotectants species (asparagus, oil Somatic embryo (DMSO, PEG, Sucrose etc.) for palm, melon, coconut) cultures Zygotic few days followed by air/silica gel embryos desiccation and rapid freezing in LN 3. Encapsulation- Meristems Explants are encapsulated in sodium/ Although several # dehydration Shoot tips/buds calcium alginate The beads parameters need to be Fig. 2. Pollen collection on filter paper disc using cyclohexane method. Nodal cuttings precultured with high sucrose (0.3 to standardized, has been Somatic embryos 1.2 M) for 1-7 days. Beads successfully been in many Zygotic/microspore desiccated to 13-35% moisture clonally propagated crops Application of Cryotechniques in Tropical Fruits Crops embryos Cell content in air or silica gel, followed Cryopreservation has been demonstrated as a safe and effective method for conserving desiccation tolerant suspensions by rapid freezing in LN short-lived seeds, zygotic embryos, dormant buds, spores, pollen, shoot tips, somatic embryos, 4. Vitrification$ Meristems Tissue/explants may be pretreated Protocol has been applied to gametophytes, and algae, with a growing number of studies indicating that viability can be maintained over Shoot tips/buds (cold /simulated acclimation.) a wide range of culture types at least 2–3 decades. The ideal explants for long-term conservation in tropical fruit crops vary depending on Nodal cuttings followed by loading, cryoprotection, and plant species the species (Chaudhury et al., 2004). For instance, embryos isolated from seeds are the best propagules to Somatic embryos rapid freezing in LN, and deloading cryopreserve intermediate seeds of citrus, papaya and recalcitrant seeds of jackfruit, as the seeds of these Zygotic embryos crops are sensitive to desiccation (Table 3). In contrast, only the pollen grains of mango and litchi are Cell suspensions amenable for conservation for long duration at cryogenic temperatures, so far. Vegetative propagules in 5. Droplet Meristems Same as vitrification, expect Protocol has been applied to banana and grapes (in vitro derived shoot tips/meristems) are easier to conserve both under in vitro vitrification* Shoot tips/buds freezing is carried out directly in a wide range of culture types conditions and cryopreservation, and possible to conserve the clonal genotypes (Van den houwe et al., Nodal cuttings LN at ultra rapid rates, by exposing and plant species with higher 2020; Panis et al., 2020). Recently it has been shown that genepool of Musaceae (Musa and Ensete spp.) can Somatic embryos the explants directly to LN, placed success rate than vitrification also be conserved for long-term using seeds or embryonic axes (Kallow et al., 2020; Singh et al., 2021a,b) Zygotic embryos on aluminum foil strips (Table 3). Cell suspensions 6. Encapsulation- Meristems A combination of techniques 3 and Used in a few plant Table 3: Application of different cryopreservation techniques for long-term conservation some vitrification Shoot tips/buds 4. Encapsulated explants are systems tropical fruit crops Nodal cuttings dehydrated with cryoprotectant Species Propagule Strategy Results Reference Somatic embryos solutions. Rapid freezing in LN cryopreserved Zygotic embryos followed by rapid thawing and Cell suspensions de-loading SEEDS AND EMBRYONIC AXES 7. V-Cryo-plate Meristems Similar to techniques 5 and 6, A relatively new technique Artocarpus Embryonic Axes Preculture on Woody Plant 50% survival Thammasiri and Shoot tips/buds except use of aluminum cryoplates successfully applied in large heterophyllus Medium with 0.3 M sucrose (1989) D-Cryoplate Nodal cuttings (which have wells, dia 1.5 mm, number of species and 0.5 M glycerol at 25˚C vitrification Somatic embryos depth 0.75 mm). for 16 h followed by PVS2 exposure at 25˚C for 50 min. (Source: Agrawal et al., 2013) an cooling in LN Artocarpus Embryonic Drying to 14% MC and rapid 30% survival Chandel et al. Pollen cryopreservation (haploid genepool) heterophyllus Axes cooling in LN2 (1995) Conservation of pollen grains is a complementary strategy to conserve the genepool of highly recalcitrant Artocarpus Embryonic Drying to 41-65% MC and No survival Hor et al. species. Its greater use is as an aid to breeding programmes. Protocols for pollen storage in LN have been integer Axes rapid cooling in LN (1990) reported for more than 170 species, including fruit, forest trees, staple crops, vegetables, forage grasses, and ornamentals, economic and medicinal plants. The ICAR-NBPGR Cryogenebank so far has a total of 591 Carica papaya Seeds Desiccation to 9-10% MC and Viabile Chin and accessions in the form of pollen grains belonging to different crops. This includes major collections of rapid cooling in LN Krishnapillay (1989) Mangifera indica (371) and Litchi chinensis (30) pollen collected using an improved method of collection using cyclohexane (Chaudhury et al., 2010, Fig 2).

122 123 Table 2: Techniques of Cryopreservation

122 123 Citrus Embryonic Preculture in MS medium 55-92% Cho et al. Musa spp. 1,600 In vitro conservation Bioversity International, aurantifolia axes containing 0.1-0.7 M sucrose viability (2002) Leuven, Belgium for 24 h; desiccation at 7.3% MC Musa spp. 1,100 Cryopreservation Bioversity International, followed by rapid cooling in LN Leuven, Belgium C.macroptera Embryonic Air desiccation followed by 87% ©. Malik and Musa spp. 443 In vitro conservation ICAR-NBPGR, New Delhi and C. latipes axes rapid cooling in LN macroptera) Chaudhury India 64% ©. latipes) (2006) Blackberry Rubus hybrid 6 Cryopreservation ICAR-NBPGR, New Delhi Poncirus Embryonic Desiccation at 14% MC 68% Radhamani and India trifoliata axes followed by rapid cooling in LN regeneration Chandel (1992) Blackberry, Rubus spp. 62 In vitro conservation ICAR-NBPGR, New Delhi Citrus grandis Embryonic axes Desiccation at 13% MC followed 93% Wen et al. Raspberry India by rapid cooling in LN regeneration (2010) Blueberry Vaccinium spp. 21 In vitro conservation ICAR-NBPGR, New Delhi Citrus Embryonic Desiccation at 13% MC Recovery of Zoubi and India suhuiensis, axes followed by rapid cooling in LN2 `plantlets Normah Citrumelo, \80-83% (Citrus (2012) Vaccinium spp. 7 Cryopreservation ICAR-NBPGR, New Delhi Fortunella suhuiensis) India polyandra 97-100% Apricot & Prunus spp. 15 In vitro conservation ICAR-NBPGR, New Delhi (Citrumelo) others India 50% (Fortunella Citrus Citrus spp. 451 Cryopreservation USDA-ARS, Ford polyandra) Collins and Corvallis, USA SHOOT TIPS/DORMANT BUDS/POLLEN Citrus spp. 642 Cryopreservation ICAR-NBPGR, New Delhi Vitis labrusca Shoot tip India (dormant buds) Two step cooling 90-100% Esensee and Kiwifruit Actinidia spp. 11 In vitro conservation ICAR-NBPGR, New Delhi Stushnoff India (1990) Monkey jack Artocarpus 1 In vitro conservation ICAR-NBPGR, New Delhi Vitis vinifera Shoot tip Encapsulation-dehydration + 15-40% Zhao et al. lakoocha India two step cooling (2001) Mulberry Morus spp. 1,236 Cryopreservation National Institute of Vitis vinifera Shoot tip Droplet vitrification 40-76% Bi (2017) Agrobiological Sciences Musa spp. Shoot tip Droplet vitrification 52.9% Panis et al. (NIAS), Tsukuba, Japan (2005) Morus spp. 329 Cryopreservation ICAR-NBPGR, New Delhi Vitis vinifera Pollen Two step cooling 54.7-77.3% Ganeshan India (1985) Pear Pyrus spp. 250 In vitro conservation USDA-ARS, NCGR, Corvallis, Mangifera Pollen Extraction in cyclohexane and 70-80% Chaudhury USA indica, Litchi cooling in LN et al. (2010) Pyrus communis 73 In vitro conservation ICAR-NBPGR, New Delhi chinensis India Strawberry Fragaria spp. 81 In vitro conservation ICAR-NBPGR, New Delhi Table 4: Status of fruit germplasm in various gene banks India

Crop Crop Botanical Accessions Conservation Genebank/ Fragaria spp. 194 Cryopreservation Julius Kühn-Institut (JKI), name (no.) Strategy Institutes Institut für Züchtungsforschung Apple Malus domestica 33 In vitro conservation ICAR-NBPGR, New Delhi an Obst, Dresden, Germany India Malus spp. 2,155 Cryopreservation USDA-ARS, Ford Collins and Corvallis, USA Bael Aegle marmelos 2 In vitro conservation ICAR-NBPGR, New Delhi India Banana Musa spp. 100 Cryopreservation ICAR-NBPGR, New Delhi India

124 125 Citrus Embryonic Preculture in MS medium 55-92% Cho et al. Musa spp. 1,600 In vitro conservation Bioversity International, aurantifolia axes containing 0.1-0.7 M sucrose viability (2002) Leuven, Belgium for 24 h; desiccation at 7.3% MC Musa spp. 1,100 Cryopreservation Bioversity International, followed by rapid cooling in LN Leuven, Belgium C.macroptera Embryonic Air desiccation followed by 87% ©. Malik and Musa spp. 443 In vitro conservation ICAR-NBPGR, New Delhi and C. latipes axes rapid cooling in LN macroptera) Chaudhury India 64% ©. latipes) (2006) Blackberry Rubus hybrid 6 Cryopreservation ICAR-NBPGR, New Delhi Poncirus Embryonic Desiccation at 14% MC 68% Radhamani and India trifoliata axes followed by rapid cooling in LN regeneration Chandel (1992) Blackberry, Rubus spp. 62 In vitro conservation ICAR-NBPGR, New Delhi Citrus grandis Embryonic axes Desiccation at 13% MC followed 93% Wen et al. Raspberry India by rapid cooling in LN regeneration (2010) Blueberry Vaccinium spp. 21 In vitro conservation ICAR-NBPGR, New Delhi Citrus Embryonic Desiccation at 13% MC Recovery of Zoubi and India suhuiensis, axes followed by rapid cooling in LN2 `plantlets Normah Citrumelo, \80-83% (Citrus (2012) Vaccinium spp. 7 Cryopreservation ICAR-NBPGR, New Delhi Fortunella suhuiensis) India polyandra 97-100% Apricot & Prunus spp. 15 In vitro conservation ICAR-NBPGR, New Delhi (Citrumelo) others India 50% (Fortunella Citrus Citrus spp. 451 Cryopreservation USDA-ARS, Ford polyandra) Collins and Corvallis, USA SHOOT TIPS/DORMANT BUDS/POLLEN Citrus spp. 642 Cryopreservation ICAR-NBPGR, New Delhi Vitis labrusca Shoot tip India (dormant buds) Two step cooling 90-100% Esensee and Kiwifruit Actinidia spp. 11 In vitro conservation ICAR-NBPGR, New Delhi Stushnoff India (1990) Monkey jack Artocarpus 1 In vitro conservation ICAR-NBPGR, New Delhi Vitis vinifera Shoot tip Encapsulation-dehydration + 15-40% Zhao et al. lakoocha India two step cooling (2001) Mulberry Morus spp. 1,236 Cryopreservation National Institute of Vitis vinifera Shoot tip Droplet vitrification 40-76% Bi (2017) Agrobiological Sciences Musa spp. Shoot tip Droplet vitrification 52.9% Panis et al. (NIAS), Tsukuba, Japan (2005) Morus spp. 329 Cryopreservation ICAR-NBPGR, New Delhi Vitis vinifera Pollen Two step cooling 54.7-77.3% Ganeshan India (1985) Pear Pyrus spp. 250 In vitro conservation USDA-ARS, NCGR, Corvallis, Mangifera Pollen Extraction in cyclohexane and 70-80% Chaudhury USA indica, Litchi cooling in LN et al. (2010) Pyrus communis 73 In vitro conservation ICAR-NBPGR, New Delhi chinensis India Strawberry Fragaria spp. 81 In vitro conservation ICAR-NBPGR, New Delhi Table 4: Status of fruit germplasm in various gene banks India

124 125 Banana (Musa spp.) spp., Poncirus trifoliata and Severinia species including rootstocks from exotic and indigenous sources are maintained at Central Citrus Research Institute (CCRI), Nagpur. Clonal repository of citrus at ICAR-NBPGR, Musa species (Musaceae), which include bananas and plantains, are grown in more than 130 countries across the Regional Station Bhowali maintains citrus (86), lime (20), lemon (8), malta (15) accessions including many world. India is the largest producer, contributing 29% to the global production of banana. In India, 32 Musa wild exotic types (Fig. 3). As older FGBs are facing serious problems of virus diseases and die-back syndrome species are reported to be distributed in the Eastern Ghats, Western Ghats, North-Eastern India and in Andaman causing loss of many accessions, conserving citrus genepool for long-term using alternative techniques has and Nicobar Islands, including 20 endemic species. Conservation of Musa germplasm has received great priority at become imperative. At ICAR-NBPGR, New Delhi, large collection of Citrus spp. (642 accessions) including global, regional and national levels, including India. As most cultivated bananas do not produce seeds, accessions wild and endemic types are conserved safely for long-term in cryobank as seeds or embryonic axes. have to be conserved as tissue cultures in vitro and in FGB. As per database available at MusaNet, 2016, Musa germplasm accessions are conserved ex situ in field gene banks (9,051), greenhouses (898), in vitro gene banks (4,507) and Cryo Gene Banks (926). These collections predominantly consist of cultivated genotypes, raised from vegetative explants such as suckers/shoot tips/floral explants. More recently seed and embryos conservation through cryopreservation has been emphasised for conservation of germplasm CWR. In India, largest collection of banana (410) is maintained by the National Research Center for Banana, Tiruchirapalli, Tamil Nadu. A safety duplicate of the collection (443) is conserved in the IVGB at ICAR-NBPGR, New Delhi including many wild Musa species. Mango (Mangifera indica) The mango belonging to the family Anacardiaceae is considered to be one of the most important fruit crop of the world particularly in Asia. The mango has a natural distribution throughout Southeast Asia. Indian peninsula, the most important centre of diversity for M. indica has a large array of cultivated varieties as well as wild types. Other Mangifera species viz., M. andamanica, M. khasiana, M. sylvatica and M. camptosperma have been reported from Fig 3. View of Citrus FGB in ICAR-NBPGR, RS, Bhowali India. Mangifera sylvatica occurs in the Eastern Himalayas in Sikkim, Darjeeling district, Khasi Hills, Upper Assam and Surma valley; and in the Andaman Islands. Mangifera andamanica, an endemic and rare species is widespread in tropical, wet evergreen forests of Andaman Islands. Owing to recalcitrant nature of the seeds, mango Guava (Psidium guajava) germplasm conservation is mostly accomplished in FGB and pollen cryobanks. Though, in vitro conservation Guava is indigenous to tropical America and naturalized in many countries including India. It is found through induction of somatic embryos derived from nucellar region of the immature seeds and its subsequent growing throughout the length and breadth of the country and huge variability is observed in the states of cryopreservation with good rate of success is also reported, its effective utilization of long-term conservation of Uttar Pradesh, Bihar, Madhya Pradesh and Maharashtra. Around 205 accessions of guava are maintained in Indian mango cultivars need to be studied and standardized. Mango pollen and DNA cryobanking is already the FGB of CISH, Lucknow and IIHR, Bangalore. Few accessions are also conserved in the Cryogenebank at followed in ICAR-NBPGR and IIHR independently. ICAR-NBPGR as seed samples for long-term conservation. Citrus (Citrus spp.) Papaya (Carica papaya ) India has a significant diversity in citrus genetic resources, both in cultivated and wild species. In addition to the Papaya is a popular tropical fruit crop susceptible to numerous diseases including detrimental Papaya frequently cultivated species/hybrids, viz., citron, grapefruit, lemon, lime, mandarin, pummel, sour and sweet Ring Spot Virus affecting the crop in many countries. Conservation of the papaya genepool can be done ex oranges; four species, viz. Indian wild orange, Khasi papeda, Ichang papeda, and Melanesian papeda were situ in the field or seed gene banks. Papaya seeds are generally classified as intermediate seed storage recorded to occur in wild or semi-wild state in Northeast India. Citrus genetic wealth of India includes several type; thus storage for periods greater than 5 years is difficult. Loss of viability has been reported at distinct indigenous cultivars/land races/ natural hybrids known under C. assamensis (Ada- Jamir or Ginger lemon moisture content (MC) below 8 to 10% (Ellis et al., 1991) although viability has been reported when of Assam), C. jambhiri (rough lemon), C. karna (Karna Orange or Karna- khatta), C. limetta (Sweet lime), C. seed was desiccated to 5% (Magill et al., 1994). Cryopreservation of whole seeds at 9-10% MC has found limettioides (Sweet lime), C. megaloxycarpa (Sour pummelo), C. pseudolimon (Hill lemon), etc., which occur to be promising for long-term storage of the gnepool. mostly in North-East India and in the North-western Himalayas. These indigenous genetic resources have great utility in citrus improvement programmes. Sincere in situ conservation efforts are urgently required to safeguard Grape (Vitis vinifera) existing genetic diversity for posterity and utilization in Citrus improvement programmes. To avoid the depletion of Vitis genetic resources, the long-term conservation of plant material can be Initiative on citrus genetic resources conservation was taken by establishing the “Citrus Gene Sanctuary” in the accomplished in gene banks, and conserved material can be utilized as breeding material for future research Garo hills of Meghalaya in the year 1981 by ICAR-NBPGR. Citrus species growing in the gene sanctuary are safe, work. In India, the FGB of grape is mainly established at National Research Center for Grapes at Pune however, prone to genetic erosion due to increasing human interventions. conserving around 437 accessions including 110 indigenous and 305 exotic collections. Protocols for long term conservation using the in vitro regenerated shoot tips are already available and needs to be On-farm conservation is an important component of genetic resources conservation especially in marginal and implemented for conserving available genepool of grape in India. diverse agro-ecosystems. Important species of North-eastern India which are being conserved on-farm are C. reticulata (Khasi mandarin and Darjeeling mandarin), C. sinensis (Tasi and Soh-nairange), C. medica, C. jambhiri, C. Jackfruit (Artocarpus heterophyllus) grandis, C. limon (Assam lemon), C. latipes, C. macroptera and C. assamensis. Similarly, diversity of C. grandis, C. Jackfruit is one of the most useful tropical fruit tree native to India. It is widely distributed in the states of jambhiri, C. karna, C. rugulosa and one hybrid of C. grandis locally known as Kimb is being maintained in foot hills of Jharkhand, Bihar, Chhatisgarh, Assam, Manipur, West Bengal, Uttar Pradesh, Maharashtra, Kerala, Tamil North-western Himalayas especially in parts of Uttaranchal, Himachal Pradesh, Uttar Pradesh and Punjab. Nadu, and Karnataka and considered to be the “poor man's food”. Like other fruit crops, most of the germplasm of Citrus species are being maintained and conserved in around 20 FGBs throughout India as 1,500 accessions (indigenous and exotic). Largest collection of 645 accessions of Citrus 126 127 Banana (Musa spp.) spp., Poncirus trifoliata and Severinia species including rootstocks from exotic and indigenous sources are maintained at Central Citrus Research Institute (CCRI), Nagpur. Clonal repository of citrus at ICAR-NBPGR, Musa species (Musaceae), which include bananas and plantains, are grown in more than 130 countries across the Regional Station Bhowali maintains citrus (86), lime (20), lemon (8), malta (15) accessions including many world. India is the largest producer, contributing 29% to the global production of banana. In India, 32 Musa wild exotic types (Fig. 3). As older FGBs are facing serious problems of virus diseases and die-back syndrome species are reported to be distributed in the Eastern Ghats, Western Ghats, North-Eastern India and in Andaman causing loss of many accessions, conserving citrus genepool for long-term using alternative techniques has and Nicobar Islands, including 20 endemic species. Conservation of Musa germplasm has received great priority at become imperative. At ICAR-NBPGR, New Delhi, large collection of Citrus spp. (642 accessions) including global, regional and national levels, including India. As most cultivated bananas do not produce seeds, accessions wild and endemic types are conserved safely for long-term in cryobank as seeds or embryonic axes. have to be conserved as tissue cultures in vitro and in FGB. As per database available at MusaNet, 2016, Musa germplasm accessions are conserved ex situ in field gene banks (9,051), greenhouses (898), in vitro gene banks (4,507) and Cryo Gene Banks (926). These collections predominantly consist of cultivated genotypes, raised from vegetative explants such as suckers/shoot tips/floral explants. More recently seed and embryos conservation through cryopreservation has been emphasised for conservation of germplasm CWR. In India, largest collection of banana (410) is maintained by the National Research Center for Banana, Tiruchirapalli, Tamil Nadu. A safety duplicate of the collection (443) is conserved in the IVGB at ICAR-NBPGR, New Delhi including many wild Musa species. Mango (Mangifera indica) The mango belonging to the family Anacardiaceae is considered to be one of the most important fruit crop of the world particularly in Asia. The mango has a natural distribution throughout Southeast Asia. Indian peninsula, the most important centre of diversity for M. indica has a large array of cultivated varieties as well as wild types. Other Mangifera species viz., M. andamanica, M. khasiana, M. sylvatica and M. camptosperma have been reported from Fig 3. View of Citrus FGB in ICAR-NBPGR, RS, Bhowali India. Mangifera sylvatica occurs in the Eastern Himalayas in Sikkim, Darjeeling district, Khasi Hills, Upper Assam and Surma valley; and in the Andaman Islands. Mangifera andamanica, an endemic and rare species is widespread in tropical, wet evergreen forests of Andaman Islands. Owing to recalcitrant nature of the seeds, mango Guava (Psidium guajava) germplasm conservation is mostly accomplished in FGB and pollen cryobanks. Though, in vitro conservation Guava is indigenous to tropical America and naturalized in many countries including India. It is found through induction of somatic embryos derived from nucellar region of the immature seeds and its subsequent growing throughout the length and breadth of the country and huge variability is observed in the states of cryopreservation with good rate of success is also reported, its effective utilization of long-term conservation of Uttar Pradesh, Bihar, Madhya Pradesh and Maharashtra. Around 205 accessions of guava are maintained in Indian mango cultivars need to be studied and standardized. Mango pollen and DNA cryobanking is already the FGB of CISH, Lucknow and IIHR, Bangalore. Few accessions are also conserved in the Cryogenebank at followed in ICAR-NBPGR and IIHR independently. ICAR-NBPGR as seed samples for long-term conservation. Citrus (Citrus spp.) Papaya (Carica papaya ) India has a significant diversity in citrus genetic resources, both in cultivated and wild species. In addition to the Papaya is a popular tropical fruit crop susceptible to numerous diseases including detrimental Papaya frequently cultivated species/hybrids, viz., citron, grapefruit, lemon, lime, mandarin, pummel, sour and sweet Ring Spot Virus affecting the crop in many countries. Conservation of the papaya genepool can be done ex oranges; four species, viz. Indian wild orange, Khasi papeda, Ichang papeda, and Melanesian papeda were situ in the field or seed gene banks. Papaya seeds are generally classified as intermediate seed storage recorded to occur in wild or semi-wild state in Northeast India. Citrus genetic wealth of India includes several type; thus storage for periods greater than 5 years is difficult. Loss of viability has been reported at distinct indigenous cultivars/land races/ natural hybrids known under C. assamensis (Ada- Jamir or Ginger lemon moisture content (MC) below 8 to 10% (Ellis et al., 1991) although viability has been reported when of Assam), C. jambhiri (rough lemon), C. karna (Karna Orange or Karna- khatta), C. limetta (Sweet lime), C. seed was desiccated to 5% (Magill et al., 1994). Cryopreservation of whole seeds at 9-10% MC has found limettioides (Sweet lime), C. megaloxycarpa (Sour pummelo), C. pseudolimon (Hill lemon), etc., which occur to be promising for long-term storage of the gnepool. mostly in North-East India and in the North-western Himalayas. These indigenous genetic resources have great utility in citrus improvement programmes. Sincere in situ conservation efforts are urgently required to safeguard Grape (Vitis vinifera) existing genetic diversity for posterity and utilization in Citrus improvement programmes. To avoid the depletion of Vitis genetic resources, the long-term conservation of plant material can be Initiative on citrus genetic resources conservation was taken by establishing the “Citrus Gene Sanctuary” in the accomplished in gene banks, and conserved material can be utilized as breeding material for future research Garo hills of Meghalaya in the year 1981 by ICAR-NBPGR. Citrus species growing in the gene sanctuary are safe, work. In India, the FGB of grape is mainly established at National Research Center for Grapes at Pune however, prone to genetic erosion due to increasing human interventions. conserving around 437 accessions including 110 indigenous and 305 exotic collections. Protocols for long term conservation using the in vitro regenerated shoot tips are already available and needs to be On-farm conservation is an important component of genetic resources conservation especially in marginal and implemented for conserving available genepool of grape in India. diverse agro-ecosystems. Important species of North-eastern India which are being conserved on-farm are C. reticulata (Khasi mandarin and Darjeeling mandarin), C. sinensis (Tasi and Soh-nairange), C. medica, C. jambhiri, C. Jackfruit (Artocarpus heterophyllus) grandis, C. limon (Assam lemon), C. latipes, C. macroptera and C. assamensis. Similarly, diversity of C. grandis, C. Jackfruit is one of the most useful tropical fruit tree native to India. It is widely distributed in the states of jambhiri, C. karna, C. rugulosa and one hybrid of C. grandis locally known as Kimb is being maintained in foot hills of Jharkhand, Bihar, Chhatisgarh, Assam, Manipur, West Bengal, Uttar Pradesh, Maharashtra, Kerala, Tamil North-western Himalayas especially in parts of Uttaranchal, Himachal Pradesh, Uttar Pradesh and Punjab. Nadu, and Karnataka and considered to be the “poor man's food”. Like other fruit crops, most of the germplasm of Citrus species are being maintained and conserved in around 20 FGBs throughout India as 1,500 accessions (indigenous and exotic). Largest collection of 645 accessions of Citrus 126 127 India is endowed with rich biodiversity of the species that naturalized across habitats. A total of 246 3. Agrawal A, RK Tyagi, R Goswami, S Uma, MS Saraswathi, P Durai (2011) Cryobanking of banana (Musa accessions of jackfruit collected from six states namely Bihar, Jharkhand, Odisha, West Bengal, Uttar sp.) germplasm in India: Evaluation of agronomic and molecular traits of cryopreserved plants. Acta Pradesh and Assam are being ex situ conserved in FGB of ICAR-NBPGR, Regional Station, Ranchi, Jharkhand. Hortic. 908: 129-138. Around 75 accessions are being maintained at FGB of IIHR-CHES Hirehalli. The seeds of jackfruit are highly 4. Agrawal A, S Verma, DPS Meena and RK Tyagi (2013) In vitro conservation of clonally propagated crops. recalcitrant and thus not amenable for normal seed genebank storage. Studies with isolated embryonic axes resulted in limited success with cryopreservation. In vitro conservation strategies in addition to protocol In: Jacob SR, N Singh, K Srinivasan, V Gupta, J Radhamani, A Kak, C Pandey, S Pandey, J Aravind, RK Tyagi development of valuable jackfruit germplasm need to be devised for its long-term conservation. (eds) Training Manual for Management of Plant Genetic Resources, September 16-25. Conclusion 5. Benson E (ed) (2002) Plant Conservation Biotechnology, Taylor and Francis, London. 6. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den India has a great wealth of fruit genetic resources, and there is pressing need that greater emphasis be given for safe and healthy germplasm conservation. Table 5 summarizes the current status of major tropical fruits houwe, N Roux (2011a) Refinement and standardization of storage procedures for clonal crops - Global conserved in variuos gene banks in India. While FGBs are sufficiently carrying out conservation of large Public Goods Phase 2: Part I. Project landscape and general status of clonal crop in vitro conservation number of germplasm (7,980), a safety duplication using either conventional (e.g. clonal repository) or technologies. System-wide Genetic Resources Programme, Rome, Italy. biotechnological methods (in vitro, cryo, pollen and DNA) is the need of the hour. This is prescribed as per 7. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den the currently practised global gene bank standards (FAO, 2013). Basic research is also required to develop houwe, N Roux (2011b) Refinement and standardization of storage procedures for clonal crops - Global robust long-term conservation protocols in recalcitrant species like jackfruit, litchi, mango, sapota, guava, Public Goods Phase 2: Part II. Status of in vitro conservation technologies for: Andean root and tuber which are highly remunerative and show excellent genepool diversity in the country. Equally important is crops, cassava, Musa, potato, sweetpotato and yam. System-wide Genetic Resources Programme, the conservation of germplasm through utilization. Genetic diversity captured in variuos collections need to Rome, Italy. be fully utilized for developing climate-resilient and nutritous varieites for the ever-increasing human population. 8. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den houwe, N Roux (2011c) Refinement and standardization of storage procedures for clonal crops - Global Table 5: Fruit crop germplasm conserved in different ex situ collections in India (NAGS, SAUs, Public Goods Phase 2: Part III. Multi-crop guidelines for developing in vitro conservation best practices ICAR-NBPGR-RS, National Genebank, ICAR-NBPGR) for clonal crops. System-wide Genetic Resources Programme, Rome, Italy. Crop Field Gene Bank* In Vitro In Vitro Base Seed/ EA/ Pollen/ 9. Bi WL (2017) Cryopreservation of shoot tips of grapevine (Vitis spp.) and cryotherapy for eradication of Gene Bank Gene Bank Cryo Gene Bank grapevine leafroll-associated virus 3. PhD thesis, Northwest A&F University, Yangling, China. Mango 2,733 - - 371 (Pollen) 10. Chandel KPS, R Chaudhury, J Radhamani and SK Malik (1995) Desiccation and freezing sensitivity in Banana 2,450 443 57 43 (Seed/EA) recalcitrant seeds of tea, cocoa and jackfruit. Ann Bot. 76: 443–450. Citrus 1,000 - - 642 (Seed/EA) 11. Chaudhury R, R Pandey, SK Malik and Bhag Mal (eds) (2004) In Vitro Conservation and Cryopreservation 16 (Pollen) of Tropical Fruit Species. IPGRI Office for South Asia, New Delhi, India/ National Bureau of Plant Genetic Guava 350 - - 6 (Seed) Resources, New Delhi. Grapes 600 - - 9 (Seed) 12. Chaudhury R, SK Malik and S Rajan (2010) An improved pollen collection and cryopreservation method Jackfruit 400 - - 9 (EA) for highly recalcitrant tropical fruit species of mango (Mangifera indica L.) and litchi (Litchi chinensis Litchi 165 - - 30 (Pollen) Sonn.). CryoLetters 31: 268–278. Sapota 166 - - - 13. Chin HF and B Krishnapillay (1989) Cryogenic storage of some horticultural species. Acta Hort. 253:107- Papaya 116 - - 87 (Seed/EA) 112. 7,980 443 57 1213 14. Cho EG, MN Normah, HK Haeng, VR Rao and F Engelmann (2002) Cryopreservation of Citrus

Source: www.cish.icar.gov.in; www.iihr.res.in; www. ccri.icar.gov.in; www.nrcb.res.in; nrcgrapes.icar.gov.in; aurantifolia seeds and embryonic axes using a desiccation protocol. CryoLetters 23: 309-316. www.yspuniversity.ac.in; www.nbpgr.ernet.in 15. Ehsan D, Y Nagamura and O Ryder (2006) DNA storage as a complementary conservation strategy. In: *Approximate values M.C. de Vicente and M.S. Andersson (eds) DNA banks-providing novel options for genebanks? References International Plant Genetic Resources Institute, Rome, Italy, pp. 11-24. 1. Agrawal A, R Sanayaima, R Singh, R Tandon, S Verma and RK Tyagi (2014) Phenotypic and molecular 16. Ellis RH, TD Hong and EH Roberts (1991) Effect of storage temperature and moisture on the studies for genetic stability assessment of cryopreserved banana meristems derived from field and in germination of papaya seeds. Seed Sci. Res. 1:69-72. vitro explants sources. In Vitro Cellular Develop Biol Plant. 50: 345-356. 17. Esensee V and C Stushnoff (1990) Cryoconservation of dormant grape (Vitis sp.) buds. Contributed 2. Agrawal A, Shivani, EV Malhotra, DPS Meena and RK Tyagi (2019) In vitro conservation and papers (oral and poster) 87th Annual Meeting of the American Society for Horticultural Science 25: cryopreservation of clonally propagated horticultural species. In: Rajasekharan PE, Ramanatha Rao V Abstract #190. (eds) Conservation and Utilization of Horticultural Genetic Resources. Springer India, pp 529-578. doi 10.1007/978-981-13-3669-0. 128 129 India is endowed with rich biodiversity of the species that naturalized across habitats. A total of 246 3. Agrawal A, RK Tyagi, R Goswami, S Uma, MS Saraswathi, P Durai (2011) Cryobanking of banana (Musa accessions of jackfruit collected from six states namely Bihar, Jharkhand, Odisha, West Bengal, Uttar sp.) germplasm in India: Evaluation of agronomic and molecular traits of cryopreserved plants. Acta Pradesh and Assam are being ex situ conserved in FGB of ICAR-NBPGR, Regional Station, Ranchi, Jharkhand. Hortic. 908: 129-138. Around 75 accessions are being maintained at FGB of IIHR-CHES Hirehalli. The seeds of jackfruit are highly 4. Agrawal A, S Verma, DPS Meena and RK Tyagi (2013) In vitro conservation of clonally propagated crops. recalcitrant and thus not amenable for normal seed genebank storage. Studies with isolated embryonic axes resulted in limited success with cryopreservation. In vitro conservation strategies in addition to protocol In: Jacob SR, N Singh, K Srinivasan, V Gupta, J Radhamani, A Kak, C Pandey, S Pandey, J Aravind, RK Tyagi development of valuable jackfruit germplasm need to be devised for its long-term conservation. (eds) Training Manual for Management of Plant Genetic Resources, September 16-25. Conclusion 5. Benson E (ed) (2002) Plant Conservation Biotechnology, Taylor and Francis, London. 6. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den India has a great wealth of fruit genetic resources, and there is pressing need that greater emphasis be given for safe and healthy germplasm conservation. Table 5 summarizes the current status of major tropical fruits houwe, N Roux (2011a) Refinement and standardization of storage procedures for clonal crops - Global conserved in variuos gene banks in India. While FGBs are sufficiently carrying out conservation of large Public Goods Phase 2: Part I. Project landscape and general status of clonal crop in vitro conservation number of germplasm (7,980), a safety duplication using either conventional (e.g. clonal repository) or technologies. System-wide Genetic Resources Programme, Rome, Italy. biotechnological methods (in vitro, cryo, pollen and DNA) is the need of the hour. This is prescribed as per 7. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den the currently practised global gene bank standards (FAO, 2013). Basic research is also required to develop houwe, N Roux (2011b) Refinement and standardization of storage procedures for clonal crops - Global robust long-term conservation protocols in recalcitrant species like jackfruit, litchi, mango, sapota, guava, Public Goods Phase 2: Part II. Status of in vitro conservation technologies for: Andean root and tuber which are highly remunerative and show excellent genepool diversity in the country. Equally important is crops, cassava, Musa, potato, sweetpotato and yam. System-wide Genetic Resources Programme, the conservation of germplasm through utilization. Genetic diversity captured in variuos collections need to Rome, Italy. be fully utilized for developing climate-resilient and nutritous varieites for the ever-increasing human population. 8. Benson EE, K Harding, D Debouck, D Dumet, R Escobar, G Mafla, B Panis, A Panta, D Tay, I Van den houwe, N Roux (2011c) Refinement and standardization of storage procedures for clonal crops - Global Table 5: Fruit crop germplasm conserved in different ex situ collections in India (NAGS, SAUs, Public Goods Phase 2: Part III. Multi-crop guidelines for developing in vitro conservation best practices ICAR-NBPGR-RS, National Genebank, ICAR-NBPGR) for clonal crops. System-wide Genetic Resources Programme, Rome, Italy. Crop Field Gene Bank* In Vitro In Vitro Base Seed/ EA/ Pollen/ 9. Bi WL (2017) Cryopreservation of shoot tips of grapevine (Vitis spp.) and cryotherapy for eradication of Gene Bank Gene Bank Cryo Gene Bank grapevine leafroll-associated virus 3. PhD thesis, Northwest A&F University, Yangling, China. Mango 2,733 - - 371 (Pollen) 10. Chandel KPS, R Chaudhury, J Radhamani and SK Malik (1995) Desiccation and freezing sensitivity in Banana 2,450 443 57 43 (Seed/EA) recalcitrant seeds of tea, cocoa and jackfruit. Ann Bot. 76: 443–450. Citrus 1,000 - - 642 (Seed/EA) 11. Chaudhury R, R Pandey, SK Malik and Bhag Mal (eds) (2004) In Vitro Conservation and Cryopreservation 16 (Pollen) of Tropical Fruit Species. IPGRI Office for South Asia, New Delhi, India/ National Bureau of Plant Genetic Guava 350 - - 6 (Seed) Resources, New Delhi. Grapes 600 - - 9 (Seed) 12. Chaudhury R, SK Malik and S Rajan (2010) An improved pollen collection and cryopreservation method Jackfruit 400 - - 9 (EA) for highly recalcitrant tropical fruit species of mango (Mangifera indica L.) and litchi (Litchi chinensis Litchi 165 - - 30 (Pollen) Sonn.). CryoLetters 31: 268–278. Sapota 166 - - - 13. Chin HF and B Krishnapillay (1989) Cryogenic storage of some horticultural species. Acta Hort. 253:107- Papaya 116 - - 87 (Seed/EA) 112. 7,980 443 57 1213 14. Cho EG, MN Normah, HK Haeng, VR Rao and F Engelmann (2002) Cryopreservation of Citrus

Source: www.cish.icar.gov.in; www.iihr.res.in; www. ccri.icar.gov.in; www.nrcb.res.in; nrcgrapes.icar.gov.in; aurantifolia seeds and embryonic axes using a desiccation protocol. CryoLetters 23: 309-316. www.yspuniversity.ac.in; www.nbpgr.ernet.in 15. Ehsan D, Y Nagamura and O Ryder (2006) DNA storage as a complementary conservation strategy. In: *Approximate values M.C. de Vicente and M.S. Andersson (eds) DNA banks-providing novel options for genebanks? References International Plant Genetic Resources Institute, Rome, Italy, pp. 11-24. 1. Agrawal A, R Sanayaima, R Singh, R Tandon, S Verma and RK Tyagi (2014) Phenotypic and molecular 16. Ellis RH, TD Hong and EH Roberts (1991) Effect of storage temperature and moisture on the studies for genetic stability assessment of cryopreserved banana meristems derived from field and in germination of papaya seeds. Seed Sci. Res. 1:69-72. vitro explants sources. In Vitro Cellular Develop Biol Plant. 50: 345-356. 17. Esensee V and C Stushnoff (1990) Cryoconservation of dormant grape (Vitis sp.) buds. Contributed 2. Agrawal A, Shivani, EV Malhotra, DPS Meena and RK Tyagi (2019) In vitro conservation and papers (oral and poster) 87th Annual Meeting of the American Society for Horticultural Science 25: cryopreservation of clonally propagated horticultural species. In: Rajasekharan PE, Ramanatha Rao V Abstract #190. (eds) Conservation and Utilization of Horticultural Genetic Resources. Springer India, pp 529-578. doi 10.1007/978-981-13-3669-0. 128 129 18. Eyland D, C Breton, J Sardos, S Kallow, B Panis, R Swennen, J Paofa, F Tardieu, C Welcker, SB Janssens, SC 35. Singh S, R Thangjam, GD Harish, H Singh, R Kumar, DPS Meena and A Agrawal (2021a) Conservation Carpentier (2020) Filling the gaps in gene banks: Collecting, characterizing, and phenotyping wild protocols for Ensete glaucum, a crop wild relative of banana, using plant tissue culture and banana relatives of Papua New Guinea. Crop Science. 2020. cryopreservation techniques on seeds and zygotic embryos. Plant Cell, Tissue and Organ Culture. 144: 19. FAO (2013) Genebank Standards for Plant Genetic Resources for Food and Agriculture. Food and 195–209. https://doi.org/10.1007/s11240-020-01881-8 Agriculture Organization of the United Nations, Rome, 166 p 36. Srivastava V, DK Nerwal, A Kandan, J Akhtar, N Sharma, R Kiran, S Bansal, A Agrawal (2020) Management 20. Ganeshan S (1985) Cryogenic preservation of grape (Vitis vinifera L.), pollen. Vitis. 173:169–173. of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott]. In Vitro Cellular & Developmental Biology-Plant. https://doi.org/10.1007/s11627-020- 21. Gupta S, K Singh and V Gupta (2019) Genetic resource conservation of horticultural crops in India - 10125-5 Achievements and issues. Prog. Hort. 51(1): 16-29. 37. Thammasiri K (1998) Cryopreservation of embryonic axes of jackfruit by vitrification. In: "Proceedings 22. Hor YL, PC Stanwood and HF Chin (1990) Effects of dehydration on freezing characteristics and survival of the twenty-four congress on science and technology of Thailand: Programme and abstracts, Mahidol in liquid nitrogen of three recalcitrant seeds. Pertanika. 13:309–314. Univ., Bangkok (Thailand). Faculty of Science, Chulalongkorn Univ., Bangkok (Thailand). Faculty of 23. ICAR-NBPGR (2016) Guidelines for Management of Plant Genetic Resources in India. ICAR-National Science- Bangkok (Thailand) p. 714-715. Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. 38. Tyagi RK and A Agrawal (2015) Conservation of plant genetic resources: Current trends and future 24. ICAR-NBPGR (2020) Annual Report 2019, ICAR-National Bureau of Plant Genetic Resources, New Delhi, thrusts. In : Jacob SR, Singh N, Srinivasan K, Gupta V, Radhamani J, Kak A, Pandey C, Pandey S, Aravind J, India, 239 p. Bisht IS, Tyagi RK (eds) Management of Plant Genetic Resources. Teaching Manual. NBPGR, New Delhi, pp. 1-8. 25. Kallow S, K Longin, NF Sleziak, SB Janssens, F Vandelook, J Dickie, R Swennen, J Paofa, S Carpentier and B Panis (2020) Challenges for Ex Situ Conservation of Wild Bananas: Seeds Collected in Papua New 39. Van den houwe, I, R Chase, J Sardos, M Ruas, E Kempenaers, V Guignon, S Massart, S Carpentier, B Guinea Have Variable Levels of Desiccation Tolerance. Plants. 9(9):1243. Panis, M Rouard and N Roux (2020) Safeguarding and using global banana diversity: a holistic approach. CABI Agric Biosci 1, 15. https://doi.org/10.1186/s43170-020-00015-6 26. Magill W, N Deighton, HW Pritchard, EE Benson and BA Goodman (1994) Physiological and biochemical studies of seed storage parameters in Carica papaya. Proceeding of the Royal Society 40. Wang MR, M Lambardi, F Engelmann, R Pathirana, B Panis, GM Volk and QC Wang (2020) Advances in of Edinburgh. 102B: 439-442. cryopreservation of in vitro-derived propagules: technologies and explant sources. Plant Cell, Tissue and Organ Cult. 19:1-4. 27. Malhotra EV, M Kamalapriya, S Bansal, DP Meena and A Agrawal (2020) Improved protocol for 41. Wen B, C Cai, R Wang, Y Tan and Q Lan (2010) Critical moisture content windows differ for the micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cryopreservation of pomelo (Citrus grandis) seeds and embryonic axes. CryoLetters. 31(1): 29-39. c a r d a m o m u m M a t o n ) . I n V i t r o C e l l u l a r & D e v e l o p m e n t a l B i o l o g y - P l a n t https://doi.org/10.1007/s11627-020-10131-7 42. Zhao YH, YJ Wu, F Engelmann and M Zhou (2001) Cryopreservation of axillary buds of grape (Vitis vinifera) in vitro plantlets. CryoLetters. 22:321–328. 28. Malik SK and R Chaudhury (2006) The cryopreservation of embryonic axes of two wild and endangered Citrus species. Plant Genet Res Charact Utiliz. 4: 204–209. 43. Zoubi OM and MN Normah (2012) Desiccation sensitivity and cryopreservation of excised embryonic axes of Citrus suhuiensis cv. Limau Madu, Citrumelo [Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] 29. Mandal BB, R Chaudhury, F Engelmann, Bhagmal, KL Tao and BS Dhillon (eds) (2003) Conservation and Fortunella polyandra, CryoLetters. 33 (3): 240-250. Biotechnology of Plant Germplasm. National Bureau of Plant Genetic Resources, New Delhi, India. 30. Panis B, B Piette and R Swennen (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci. 168:45–5. 31. Panis B, M Nagel and I Van den houwe (2020) Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen. Plants. 9(1634): 1-22; doi:10.3390/plants9121634. 32. Pence VC, D Ballesteros, C Walters, BM Reed, M Philpott, KW Dixon, HW Pritchard, TM Culley and AC Vanhove (2020) Cryobiotechnologies: Tools for expanding long-term ex situ conservation to all plant species. Biological Conservation 250:108736. 33. Sharma N, R Gowthami, SV Devi, EV Malhotra, R Pandey and A Agrawal (2021) Cryopreservation of shoot tips of Gentiana kurroo Royle – a critically endangered medicinal plant of India. Plant Cell Tissue Organ Cult. 144, 67–72. https://doi.org/10.1007/s11240-020-01879-2 34. Singh S, A Agrawal, R Kumar, R Thangjam and K Joseph John (2021b) Seed storage behavior of Musa balbisiana Colla, a wild progenitor of bananas and plantains - Implications for ex situ germplasm conservation. Scientia Horticulturae. 280: 109926. https://doi.org/10.1016/j.scienta.2021.109926

130 131 18. Eyland D, C Breton, J Sardos, S Kallow, B Panis, R Swennen, J Paofa, F Tardieu, C Welcker, SB Janssens, SC 35. Singh S, R Thangjam, GD Harish, H Singh, R Kumar, DPS Meena and A Agrawal (2021a) Conservation Carpentier (2020) Filling the gaps in gene banks: Collecting, characterizing, and phenotyping wild protocols for Ensete glaucum, a crop wild relative of banana, using plant tissue culture and banana relatives of Papua New Guinea. Crop Science. 2020. cryopreservation techniques on seeds and zygotic embryos. Plant Cell, Tissue and Organ Culture. 144: 19. FAO (2013) Genebank Standards for Plant Genetic Resources for Food and Agriculture. Food and 195–209. https://doi.org/10.1007/s11240-020-01881-8 Agriculture Organization of the United Nations, Rome, 166 p 36. Srivastava V, DK Nerwal, A Kandan, J Akhtar, N Sharma, R Kiran, S Bansal, A Agrawal (2020) Management 20. Ganeshan S (1985) Cryogenic preservation of grape (Vitis vinifera L.), pollen. Vitis. 173:169–173. of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott]. In Vitro Cellular & Developmental Biology-Plant. https://doi.org/10.1007/s11627-020- 21. Gupta S, K Singh and V Gupta (2019) Genetic resource conservation of horticultural crops in India - 10125-5 Achievements and issues. Prog. Hort. 51(1): 16-29. 37. Thammasiri K (1998) Cryopreservation of embryonic axes of jackfruit by vitrification. In: "Proceedings 22. Hor YL, PC Stanwood and HF Chin (1990) Effects of dehydration on freezing characteristics and survival of the twenty-four congress on science and technology of Thailand: Programme and abstracts, Mahidol in liquid nitrogen of three recalcitrant seeds. Pertanika. 13:309–314. Univ., Bangkok (Thailand). Faculty of Science, Chulalongkorn Univ., Bangkok (Thailand). Faculty of 23. ICAR-NBPGR (2016) Guidelines for Management of Plant Genetic Resources in India. ICAR-National Science- Bangkok (Thailand) p. 714-715. Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India, 142 + xxiv p. 38. Tyagi RK and A Agrawal (2015) Conservation of plant genetic resources: Current trends and future 24. ICAR-NBPGR (2020) Annual Report 2019, ICAR-National Bureau of Plant Genetic Resources, New Delhi, thrusts. In : Jacob SR, Singh N, Srinivasan K, Gupta V, Radhamani J, Kak A, Pandey C, Pandey S, Aravind J, India, 239 p. Bisht IS, Tyagi RK (eds) Management of Plant Genetic Resources. Teaching Manual. NBPGR, New Delhi, pp. 1-8. 25. Kallow S, K Longin, NF Sleziak, SB Janssens, F Vandelook, J Dickie, R Swennen, J Paofa, S Carpentier and B Panis (2020) Challenges for Ex Situ Conservation of Wild Bananas: Seeds Collected in Papua New 39. Van den houwe, I, R Chase, J Sardos, M Ruas, E Kempenaers, V Guignon, S Massart, S Carpentier, B Guinea Have Variable Levels of Desiccation Tolerance. Plants. 9(9):1243. Panis, M Rouard and N Roux (2020) Safeguarding and using global banana diversity: a holistic approach. CABI Agric Biosci 1, 15. https://doi.org/10.1186/s43170-020-00015-6 26. Magill W, N Deighton, HW Pritchard, EE Benson and BA Goodman (1994) Physiological and biochemical studies of seed storage parameters in Carica papaya. Proceeding of the Royal Society 40. Wang MR, M Lambardi, F Engelmann, R Pathirana, B Panis, GM Volk and QC Wang (2020) Advances in of Edinburgh. 102B: 439-442. cryopreservation of in vitro-derived propagules: technologies and explant sources. Plant Cell, Tissue and Organ Cult. 19:1-4. 27. Malhotra EV, M Kamalapriya, S Bansal, DP Meena and A Agrawal (2020) Improved protocol for 41. Wen B, C Cai, R Wang, Y Tan and Q Lan (2010) Critical moisture content windows differ for the micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cryopreservation of pomelo (Citrus grandis) seeds and embryonic axes. CryoLetters. 31(1): 29-39. c a r d a m o m u m M a t o n ) . I n V i t r o C e l l u l a r & D e v e l o p m e n t a l B i o l o g y - P l a n t https://doi.org/10.1007/s11627-020-10131-7 42. Zhao YH, YJ Wu, F Engelmann and M Zhou (2001) Cryopreservation of axillary buds of grape (Vitis vinifera) in vitro plantlets. CryoLetters. 22:321–328. 28. Malik SK and R Chaudhury (2006) The cryopreservation of embryonic axes of two wild and endangered Citrus species. Plant Genet Res Charact Utiliz. 4: 204–209. 43. Zoubi OM and MN Normah (2012) Desiccation sensitivity and cryopreservation of excised embryonic axes of Citrus suhuiensis cv. Limau Madu, Citrumelo [Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] 29. Mandal BB, R Chaudhury, F Engelmann, Bhagmal, KL Tao and BS Dhillon (eds) (2003) Conservation and Fortunella polyandra, CryoLetters. 33 (3): 240-250. Biotechnology of Plant Germplasm. National Bureau of Plant Genetic Resources, New Delhi, India. 30. Panis B, B Piette and R Swennen (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci. 168:45–5. 31. Panis B, M Nagel and I Van den houwe (2020) Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen. Plants. 9(1634): 1-22; doi:10.3390/plants9121634. 32. Pence VC, D Ballesteros, C Walters, BM Reed, M Philpott, KW Dixon, HW Pritchard, TM Culley and AC Vanhove (2020) Cryobiotechnologies: Tools for expanding long-term ex situ conservation to all plant species. Biological Conservation 250:108736. 33. Sharma N, R Gowthami, SV Devi, EV Malhotra, R Pandey and A Agrawal (2021) Cryopreservation of shoot tips of Gentiana kurroo Royle – a critically endangered medicinal plant of India. Plant Cell Tissue Organ Cult. 144, 67–72. https://doi.org/10.1007/s11240-020-01879-2 34. Singh S, A Agrawal, R Kumar, R Thangjam and K Joseph John (2021b) Seed storage behavior of Musa balbisiana Colla, a wild progenitor of bananas and plantains - Implications for ex situ germplasm conservation. Scientia Horticulturae. 280: 109926. https://doi.org/10.1016/j.scienta.2021.109926

130 131 In 1985 the concept of a "DNA fingerprinting" was introduced as a means of evaluating human identity and DNA Fingerprinting and Use of relatedness (Jeffreys et al., 1985). The possible forensic and legal applications of DNA evidence were quickly appreciated and such data are now frequently presented in court cases involving serious crimes such as murder and rape. DNA evidence is also used in establishing paternity, in determining relatedness in Genomics in Plant Genetic immigration and inheritance disputes, and in identifying disaster victims. Such cases, especially those involving famous people, are widely reported in the media and are of interest to the general population. Resources Management DNA fingerprinting technology has now been extended from humans to even plants. In plants, identification of cultivars is one area where DNA fingerprinting is being used routinely and has applications Mukesh Kumar Rana* and Amit Kumar Singh in protection of plant breeders' and farmers rights. Division of Genomic Resources, ICAR- National Bureau of Plant Genetic Resources, New Delhi The question underlying the use of DNA fingerprinting data is what is the probability that two or more *Email: [email protected] cultivars have the same genetic fingerprint/ profile? If we could sequence and compare the DNA of each cultivar, we would find that all cultivars are different. Even natural clones, such as identical twins in humans, Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and will have acquired a few differences that are mutations in their DNA, during their growth from a single cell to editing of genomes. Genomic DNA constitutes the total genetic information of an organism. The genomes of an adult. When it is not possible to sequence the entire individual's genome then instead, we rely on almost all organisms are DNA, the only exceptions being some viruses that have RNA genomes. Genomic differences in length of short stretches of DNA at different loci commonly called markers. These stretches of DNA molecules are generally large, and in most organisms are organized into DNA–protein complexes called DNA can be visualized as shown in the figure. Based on this figure can we say that any two cultivars are chromosomes. The size, number of chromosomes, and nature of genomic DNA varies between different identical? Can we assign a numerical value to our organisms. Viral DNA genomes are relatively small and can be single- or double-stranded, linear, or circular. answer? Our answer to this question depends on the All other organisms have double-stranded DNA genomes. Bacteria have a single, circular chromosome. In probability of two or more cultivars having the same eukaryotes, most genomic DNA is located within the nucleus (nuclear DNA) as multiple linear chromosomes pattern of these DNA fragments, the same sized SSR of different sizes. Eukaryotic cells additionally contain genomic DNA in the mitochondria and, in plants and markers. The SSRs chosen for DNA fingerprinting lower eukaryotes, the chloroplasts. This DNA is usually a circular molecule and is present as multiple copies purposes are inherited independently of each other. within these organelles. They are on different chromosomes. Therefore, in theory, if the frequency of SSR1 in a population is 1 in 50, The assessment of extent and pattern of genetic diversity in crop genetic resources conserved in the and SSR2 has a frequency of 1 in 1000, then only 1 in national and international genebank collections is a critical step for their efficient utilization in breeding 50,000 cultivars would be expected to have both these program. Until recently the genetic diversity in gene bank collections was primarily assessed based on the SSRs. More SSRs (not two as in the present case) are morphological traits which are difficult to record and dependent on the environmental conditions and thus usually monitored in each "DNA fingerprint" and are Figure: DNA profile, with SSR markers, are not suitable to provide a comprehensive picture on the actual genomic/ gene level diversity available in chosen so that the probability of a false positive match is of six cultivars with two primers gene pool of a species. Moreover, the evaluation of huge size of collections in various species for the extremely low. desirable traits is very laborious and resources intensive that has deterred breeders to utilize them in breeding program. In recent years, the developments in the field of genomics particularly next generation Criteria for an appropriate methodology for DNA fingerprinting sequencing and high throughput genotyping technologies have made it possible to characterize germplasm There are different methodologies available for DNA fingerprinting such as RFLP, RAPD, ISSR, AFLP, SSR, SNP reliably at the level of DNA and cost effectively identify genes and genomic regions associated with various etc. The important criteria for choosing a methodology depends on the reproducibility of the data between economic important traits. These technologies have given a boost to DNA fingerprinting and DNA barcoding laboratories, accessibility of detection platforms, repeatability over time, discrimination power and ease in of crop genetic resources for their unequivocally identification with high precision and efficiency. The other creating database. applications of genomics in germplasm collection and utilization include species identification using DNA barcoding, construction and characterization of core sets and trait discovery using association mapping Database generation: As advancements in methodologies and new equipment are routinely happening, which are going to be discussed here. the continued sustainability of database should be an important consideration. This is particularly true for DNA sequencing data. Also, earlier radioactively labeled primers and sequencing gels were used to produce DNA fingerprinting DNA fingerprinting data which now can be done using fluorescent labeling followed by separation on high throughput detection systems. Repeatability and reproducibility are important in the construction, DNA fingerprinting involves the generation of a set of distinct DNA fragments from a single DNA sample. The operation and longevity of databases and is very important in generating a centrally maintained database, generated DNA fragments are then used as a source of genotypic information. A variety of techniques can be populated with verified data from different sources. used to generate DNA fingerprinting patterns. The choice of the technique depends on the organism being studied and on the question being addressed. All DNA fingerprinting techniques study patterns associated Mapped markers: With the availability of robust techniques, the trend has been to use mapped SSRs with genetic markers; however, individual techniques differ in terms of the number and type of genetic (microsatellites) and may be sequencing (Single Nucleotide Polymorphisms, SNPs) in near future. Other markers examined. For example, some approaches allow the examination of a marker at a single locus genic or functional marker techniques which rely on DNA sequence information may also fulfill the above (called single-locus markers), whereas others allow the simultaneous investigation of multiple loci (called criteria but their use in DNA profiling of plant varieties needs to be explored. A marker or set of markers multi-locus markers). Some approaches focus on co-dominant markers, which provide information about selected for DNA profiling should be polymorphic and have repeatability within and reproducibility both alleles present at a given locus. In contrast, other techniques are concerned with dominant markers, between, laboratories in terms of scoring data. To help avoid markers that may be linked, it is desirable to which only report the presence or absence of a given allele and cannot provide information about whether know the map position of the markers. It is also advisable to avoid markers with “null” alleles. an individual is homozygous for that allele.

132 133 In 1985 the concept of a "DNA fingerprinting" was introduced as a means of evaluating human identity and DNA Fingerprinting and Use of relatedness (Jeffreys et al., 1985). The possible forensic and legal applications of DNA evidence were quickly appreciated and such data are now frequently presented in court cases involving serious crimes such as murder and rape. DNA evidence is also used in establishing paternity, in determining relatedness in Genomics in Plant Genetic immigration and inheritance disputes, and in identifying disaster victims. Such cases, especially those involving famous people, are widely reported in the media and are of interest to the general population. Resources Management DNA fingerprinting technology has now been extended from humans to even plants. In plants, identification of cultivars is one area where DNA fingerprinting is being used routinely and has applications Mukesh Kumar Rana* and Amit Kumar Singh in protection of plant breeders' and farmers rights. Division of Genomic Resources, ICAR- National Bureau of Plant Genetic Resources, New Delhi The question underlying the use of DNA fingerprinting data is what is the probability that two or more *Email: [email protected] cultivars have the same genetic fingerprint/ profile? If we could sequence and compare the DNA of each cultivar, we would find that all cultivars are different. Even natural clones, such as identical twins in humans, Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and will have acquired a few differences that are mutations in their DNA, during their growth from a single cell to editing of genomes. Genomic DNA constitutes the total genetic information of an organism. The genomes of an adult. When it is not possible to sequence the entire individual's genome then instead, we rely on almost all organisms are DNA, the only exceptions being some viruses that have RNA genomes. Genomic differences in length of short stretches of DNA at different loci commonly called markers. These stretches of DNA molecules are generally large, and in most organisms are organized into DNA–protein complexes called DNA can be visualized as shown in the figure. Based on this figure can we say that any two cultivars are chromosomes. The size, number of chromosomes, and nature of genomic DNA varies between different identical? Can we assign a numerical value to our organisms. Viral DNA genomes are relatively small and can be single- or double-stranded, linear, or circular. answer? Our answer to this question depends on the All other organisms have double-stranded DNA genomes. Bacteria have a single, circular chromosome. In probability of two or more cultivars having the same eukaryotes, most genomic DNA is located within the nucleus (nuclear DNA) as multiple linear chromosomes pattern of these DNA fragments, the same sized SSR of different sizes. Eukaryotic cells additionally contain genomic DNA in the mitochondria and, in plants and markers. The SSRs chosen for DNA fingerprinting lower eukaryotes, the chloroplasts. This DNA is usually a circular molecule and is present as multiple copies purposes are inherited independently of each other. within these organelles. They are on different chromosomes. Therefore, in theory, if the frequency of SSR1 in a population is 1 in 50, The assessment of extent and pattern of genetic diversity in crop genetic resources conserved in the and SSR2 has a frequency of 1 in 1000, then only 1 in national and international genebank collections is a critical step for their efficient utilization in breeding 50,000 cultivars would be expected to have both these program. Until recently the genetic diversity in gene bank collections was primarily assessed based on the SSRs. More SSRs (not two as in the present case) are morphological traits which are difficult to record and dependent on the environmental conditions and thus usually monitored in each "DNA fingerprint" and are Figure: DNA profile, with SSR markers, are not suitable to provide a comprehensive picture on the actual genomic/ gene level diversity available in chosen so that the probability of a false positive match is of six cultivars with two primers gene pool of a species. Moreover, the evaluation of huge size of collections in various species for the extremely low. desirable traits is very laborious and resources intensive that has deterred breeders to utilize them in breeding program. In recent years, the developments in the field of genomics particularly next generation Criteria for an appropriate methodology for DNA fingerprinting sequencing and high throughput genotyping technologies have made it possible to characterize germplasm There are different methodologies available for DNA fingerprinting such as RFLP, RAPD, ISSR, AFLP, SSR, SNP reliably at the level of DNA and cost effectively identify genes and genomic regions associated with various etc. The important criteria for choosing a methodology depends on the reproducibility of the data between economic important traits. These technologies have given a boost to DNA fingerprinting and DNA barcoding laboratories, accessibility of detection platforms, repeatability over time, discrimination power and ease in of crop genetic resources for their unequivocally identification with high precision and efficiency. The other creating database. applications of genomics in germplasm collection and utilization include species identification using DNA barcoding, construction and characterization of core sets and trait discovery using association mapping Database generation: As advancements in methodologies and new equipment are routinely happening, which are going to be discussed here. the continued sustainability of database should be an important consideration. This is particularly true for DNA sequencing data. Also, earlier radioactively labeled primers and sequencing gels were used to produce DNA fingerprinting DNA fingerprinting data which now can be done using fluorescent labeling followed by separation on high throughput detection systems. Repeatability and reproducibility are important in the construction, DNA fingerprinting involves the generation of a set of distinct DNA fragments from a single DNA sample. The operation and longevity of databases and is very important in generating a centrally maintained database, generated DNA fragments are then used as a source of genotypic information. A variety of techniques can be populated with verified data from different sources. used to generate DNA fingerprinting patterns. The choice of the technique depends on the organism being studied and on the question being addressed. All DNA fingerprinting techniques study patterns associated Mapped markers: With the availability of robust techniques, the trend has been to use mapped SSRs with genetic markers; however, individual techniques differ in terms of the number and type of genetic (microsatellites) and may be sequencing (Single Nucleotide Polymorphisms, SNPs) in near future. Other markers examined. For example, some approaches allow the examination of a marker at a single locus genic or functional marker techniques which rely on DNA sequence information may also fulfill the above (called single-locus markers), whereas others allow the simultaneous investigation of multiple loci (called criteria but their use in DNA profiling of plant varieties needs to be explored. A marker or set of markers multi-locus markers). Some approaches focus on co-dominant markers, which provide information about selected for DNA profiling should be polymorphic and have repeatability within and reproducibility both alleles present at a given locus. In contrast, other techniques are concerned with dominant markers, between, laboratories in terms of scoring data. To help avoid markers that may be linked, it is desirable to which only report the presence or absence of a given allele and cannot provide information about whether know the map position of the markers. It is also advisable to avoid markers with “null” alleles. an individual is homozygous for that allele.

132 133 Microsatellite markers: Microsatellite or Simple Sequence Repeats (SSRs) make use of polymerase chain Constitution of germplasm core set reaction (PCR) and has several advantages. SSR markers are expressed co-dominantly, are generally robust, repeatable, easy to score (record), and easy to automate for high-throughput detection. Moreover, several Frankel (1984) introduced the term core collection as a concept. Germplasm core set refers to small set of SSRs can be multiplexed and detected by non-radioactive DNA sequences or on gel electrophoresis or germplasm that represent total diversity present in the original collection of a species. The core set with capillary electrophoresis. With recent advancements in genomics, mapped SSRs are now available in several small number of accessions representing maximum diversity for various traits can be thoroughly evaluated crops. For effective microsatellite analysis, selecting high quality SSR markers that are unlinked, for various desirable traits, thereby it can facilitate efficient utilization of germplasm in breeding programs. polymorphic, reliable and reproducible and having no stuttering is essential. Primers for any kind of marker Conventionally, breeders have used morphological and agronomical parameters to constitute core sets. The system should be synthesized by an assured supplier. Some SSRs are publicly available. main limitation of this approach is that morphological traits are influenced by environmental condition as a result core set constituted based on the morphological parameters might not always represent total Plant material: The source and type of the material and how many samples need to be analyzed are the diversity present in entire collection. main issues with regard to the material to be analyzed. The plant material to be analyzed should be an authentic, representative sample of the variety and, where possible, should be obtained from the sample of Currently, high throughput cost effective genotyping methods such as low coverage whole genome re- the variety used for examination for the purposes of Plant Breeders' Rights or for official registration. Use of sequencing, genotyping by sequencing (GBS), SNP arrays etc. offer the possibility to obtain genotyping data samples of material submitted for examination for the purposes of Plant Breeders' Rights or for official of entire germplasm of a species enabling constitution of core set solely based on the genotyping data. The registration will require the permission of the relevant authority, breeder and/or maintainer, as appropriate. genotype information of the germplasm lines could then be used for identification of core set using various The plant material from which the samples are taken should be traceable in case some of the samples marker-based statistics such as pairwise dissimilarity, allelic richness, or heterozygosity. Some of the subsequently prove not to be representative of the variety. important software programs that can be used to constitute core sets are PowerCore, Core Hunter 3 etc. The PowerCore program selects core set of accession based on genetic data by an extension of a heuristic Sampling strategies: The type of plant material to be sampled and the procedure for sampling the material algorithm using an advanced maximization strategy. The PowerCore analysis allows for the retention of all for DNA extraction will, to a large extent, depend on the crop or plant species concerned. For example, in alleles in a core collection with the minimum possible number of accessions as possible. seed-propagated varieties, seed or seedlings may be used as the source of DNA, whereas, in vegetative propagated varieties, the DNA may be extracted from leaf material. Whatever the source of material, the Association mapping for trait discovery method for sampling and DNA extraction should be standardized and documented. Furthermore, it should Conventionally, trait mapping has been done using bi-parental mapping/ QTL mapping approach that uses be verified that the sampling and extraction methods produce consistent results by DNA analysis. It is mapping populations to identify genomic regions/ QTLs associated with a trait. Although this approach is essential that the samples taken for analysis are representative of the variety. With regard to being powerful in unravelling genes/g enomic regions controlling a targeted trait, it suffers from two major representative of the variety, consideration should be given to the mode of propagation i.e. whether self- limitation 1) needs generation of mapping populations which are very laborious and time intensive to be pollinated or cross-pollinated. The size of the sample should be determined taking into account suitable generated, and 2) generates poor resolution maps as the mapping population capture only one few rounds statistical procedures. of recombination events. Association mapping is an alternative approach of trait mapping that exploits Reference set: A DNA reference sample set of varieties can be created for use in different detection linkage disequilibrium at adjacent loci in diverse set of genotypes, to establish correlation between equipment in different laboratories and for appropriate size calling as molecular size standards behave genotype and phenotype. Since the diverse set of genotypes capture hundreds of historical recombinant differently in the various detection systems. This DNA reference sample set can be stored and also supplied events it can provide high resolution maps of the targeted traits. The large set of germplasm lines conserved to other laboratories. in the national and international genebanks could be used to conduct association analysis to discover novel genes/ alleles associated with various useful traits. Depending on the region of the genome used for DNA barcoding for species identification association analysis, association mapping is grouped into two types: Candidate gene (CG) based association The utilization and conservation of the germplasm resources requires their proper identification at the mapping and Genome-wide association mapping (GWAS). Both these approaches are briefly described species level. Traditionally a range of parameters such as morphological, cytological and chemotaxonomic below. have been used for species identification but these mostly failed to resolve species complexes. In the 21st a. Candidate gene-based association mapping: As the name suggests, this approach considers already Century, these approaches were supplemented with DNA barcoding that uses one or more loci (rbcL, matK, known candidate genes for a target trait for association mapping. This approach is based on the fact bcL gene, rpoB gene, rpoC1, psbK–psbI spacer, and trnH–psbA spacer) of chloroplast genome for the that sequence variation in regulatory/ coding region can affect their functionality and in turn the purpose of resolving species complexes (Group et al., 2009). However, locus-based approach failed to associated trait. CG approach involves sequencing of candidate genes controlling the targeted trait resolves several complex species and also there is no universally agreed locus for DNA barcoding of plant from the diverse set of lines to discover the identify SNPs/ Indels which are then tested for their species. Recently, with the decreasing sequencing cost emphasis has been laid on using whole chloroplast association with the targeted trait using various association models. The approach can facilitate DNA sequence rather than just one or two loci that could be much useful for the purpose of DNA barcoding. discovery of novel allelic variants for various useful traits. In plants, first candidate gene-based The use of whole chloroplast sequences eliminates the need to have a priori information on a locus of association study was conducted for flowering trait in maize (Remington et al., 2001) and, thereafter, choice, a difficulty that acts as a major hindrance to single or multi-locus DNA barcoding studies (Wambugu CG association analyses have been conducted for a wide range of traits in many species. A study by et al., 2018). The advantage of using entire chloroplast sequence is that long DNA sequence is compared Yumnam et al. (2015) reported novel haplotypes in two low phosphorus tolerance of genes STOL1 and which may have more variations in the form of SNPs and Indels that can help discriminate closely related PupK20-2 from the rice genotypes adopted to acidic conditions. Mishra et al. (2016) reported allele species. Besides chloroplast DNA, with the ever-decreasing cost, it is now feasible to compare entire nuclear mining in eight members of HKT gene family from Indian wild rice and identified salt tolerant allele of genome sequence for establishing species relationships. Therefore, routine sequencing of complete nuclear HKT2;3 and HKT1;5 genes. genomes might make whole-genome sequences of species to be a very important tool in the future for use b. Genome-wide association mapping: GWAS is a powerful approach for trait mapping in plants in which in plant systematics. genome-wide markers are scanned for their association with desired trait in a diverse panel of genotypes. Unlike QTL mapping approach, it does not require development of mapping populations

134 135 Microsatellite markers: Microsatellite or Simple Sequence Repeats (SSRs) make use of polymerase chain Constitution of germplasm core set reaction (PCR) and has several advantages. SSR markers are expressed co-dominantly, are generally robust, repeatable, easy to score (record), and easy to automate for high-throughput detection. Moreover, several Frankel (1984) introduced the term core collection as a concept. Germplasm core set refers to small set of SSRs can be multiplexed and detected by non-radioactive DNA sequences or on gel electrophoresis or germplasm that represent total diversity present in the original collection of a species. The core set with capillary electrophoresis. With recent advancements in genomics, mapped SSRs are now available in several small number of accessions representing maximum diversity for various traits can be thoroughly evaluated crops. For effective microsatellite analysis, selecting high quality SSR markers that are unlinked, for various desirable traits, thereby it can facilitate efficient utilization of germplasm in breeding programs. polymorphic, reliable and reproducible and having no stuttering is essential. Primers for any kind of marker Conventionally, breeders have used morphological and agronomical parameters to constitute core sets. The system should be synthesized by an assured supplier. Some SSRs are publicly available. main limitation of this approach is that morphological traits are influenced by environmental condition as a result core set constituted based on the morphological parameters might not always represent total Plant material: The source and type of the material and how many samples need to be analyzed are the diversity present in entire collection. main issues with regard to the material to be analyzed. The plant material to be analyzed should be an authentic, representative sample of the variety and, where possible, should be obtained from the sample of Currently, high throughput cost effective genotyping methods such as low coverage whole genome re- the variety used for examination for the purposes of Plant Breeders' Rights or for official registration. Use of sequencing, genotyping by sequencing (GBS), SNP arrays etc. offer the possibility to obtain genotyping data samples of material submitted for examination for the purposes of Plant Breeders' Rights or for official of entire germplasm of a species enabling constitution of core set solely based on the genotyping data. The registration will require the permission of the relevant authority, breeder and/or maintainer, as appropriate. genotype information of the germplasm lines could then be used for identification of core set using various The plant material from which the samples are taken should be traceable in case some of the samples marker-based statistics such as pairwise dissimilarity, allelic richness, or heterozygosity. Some of the subsequently prove not to be representative of the variety. important software programs that can be used to constitute core sets are PowerCore, Core Hunter 3 etc. The PowerCore program selects core set of accession based on genetic data by an extension of a heuristic Sampling strategies: The type of plant material to be sampled and the procedure for sampling the material algorithm using an advanced maximization strategy. The PowerCore analysis allows for the retention of all for DNA extraction will, to a large extent, depend on the crop or plant species concerned. For example, in alleles in a core collection with the minimum possible number of accessions as possible. seed-propagated varieties, seed or seedlings may be used as the source of DNA, whereas, in vegetative propagated varieties, the DNA may be extracted from leaf material. Whatever the source of material, the Association mapping for trait discovery method for sampling and DNA extraction should be standardized and documented. Furthermore, it should Conventionally, trait mapping has been done using bi-parental mapping/ QTL mapping approach that uses be verified that the sampling and extraction methods produce consistent results by DNA analysis. It is mapping populations to identify genomic regions/ QTLs associated with a trait. Although this approach is essential that the samples taken for analysis are representative of the variety. With regard to being powerful in unravelling genes/g enomic regions controlling a targeted trait, it suffers from two major representative of the variety, consideration should be given to the mode of propagation i.e. whether self- limitation 1) needs generation of mapping populations which are very laborious and time intensive to be pollinated or cross-pollinated. The size of the sample should be determined taking into account suitable generated, and 2) generates poor resolution maps as the mapping population capture only one few rounds statistical procedures. of recombination events. Association mapping is an alternative approach of trait mapping that exploits Reference set: A DNA reference sample set of varieties can be created for use in different detection linkage disequilibrium at adjacent loci in diverse set of genotypes, to establish correlation between equipment in different laboratories and for appropriate size calling as molecular size standards behave genotype and phenotype. Since the diverse set of genotypes capture hundreds of historical recombinant differently in the various detection systems. This DNA reference sample set can be stored and also supplied events it can provide high resolution maps of the targeted traits. The large set of germplasm lines conserved to other laboratories. in the national and international genebanks could be used to conduct association analysis to discover novel genes/ alleles associated with various useful traits. Depending on the region of the genome used for DNA barcoding for species identification association analysis, association mapping is grouped into two types: Candidate gene (CG) based association The utilization and conservation of the germplasm resources requires their proper identification at the mapping and Genome-wide association mapping (GWAS). Both these approaches are briefly described species level. Traditionally a range of parameters such as morphological, cytological and chemotaxonomic below. have been used for species identification but these mostly failed to resolve species complexes. In the 21st a. Candidate gene-based association mapping: As the name suggests, this approach considers already Century, these approaches were supplemented with DNA barcoding that uses one or more loci (rbcL, matK, known candidate genes for a target trait for association mapping. This approach is based on the fact bcL gene, rpoB gene, rpoC1, psbK–psbI spacer, and trnH–psbA spacer) of chloroplast genome for the that sequence variation in regulatory/ coding region can affect their functionality and in turn the purpose of resolving species complexes (Group et al., 2009). However, locus-based approach failed to associated trait. CG approach involves sequencing of candidate genes controlling the targeted trait resolves several complex species and also there is no universally agreed locus for DNA barcoding of plant from the diverse set of lines to discover the identify SNPs/ Indels which are then tested for their species. Recently, with the decreasing sequencing cost emphasis has been laid on using whole chloroplast association with the targeted trait using various association models. The approach can facilitate DNA sequence rather than just one or two loci that could be much useful for the purpose of DNA barcoding. discovery of novel allelic variants for various useful traits. In plants, first candidate gene-based The use of whole chloroplast sequences eliminates the need to have a priori information on a locus of association study was conducted for flowering trait in maize (Remington et al., 2001) and, thereafter, choice, a difficulty that acts as a major hindrance to single or multi-locus DNA barcoding studies (Wambugu CG association analyses have been conducted for a wide range of traits in many species. A study by et al., 2018). The advantage of using entire chloroplast sequence is that long DNA sequence is compared Yumnam et al. (2015) reported novel haplotypes in two low phosphorus tolerance of genes STOL1 and which may have more variations in the form of SNPs and Indels that can help discriminate closely related PupK20-2 from the rice genotypes adopted to acidic conditions. Mishra et al. (2016) reported allele species. Besides chloroplast DNA, with the ever-decreasing cost, it is now feasible to compare entire nuclear mining in eight members of HKT gene family from Indian wild rice and identified salt tolerant allele of genome sequence for establishing species relationships. Therefore, routine sequencing of complete nuclear HKT2;3 and HKT1;5 genes. genomes might make whole-genome sequences of species to be a very important tool in the future for use b. Genome-wide association mapping: GWAS is a powerful approach for trait mapping in plants in which in plant systematics. genome-wide markers are scanned for their association with desired trait in a diverse panel of genotypes. Unlike QTL mapping approach, it does not require development of mapping populations

134 135 which is a laborious and time intensive exercise. GWAS approach leverages recombination events Number of markers: The number of markers significantly affects GWAS resolution. The minimum across the generations, thereby, enabling fine resolution mapping of the targeted trait. It can provide number of markers for GWAS analysis primarily depends on the extent of LD in association panel. If LD very high resolution up to 100-200 Kb and may even identify causative genes associated with the target decay is faster, a greater number of markers would be needed to identify the causal variants for the trait trait. Some of the important association mapping studies include identification of agronomic traits and and vice versa. ionomic variation in rice (Huang et al., 2010), flowering time in Arabidopsis (Brachi et al., 2010), rust Quality of the phenotyping data: For complex traits that are influenced by environmental factors, resistance in wheat (Pradhan et al., 2020), salt tolerance traits in wheat (Chaurasia et al., 2020) etc. phenotyping data should be obtained from multiple location/ year experiments. Normalized trait c. Association mapping steps values derived from multi-location/ year data should be used in GWAS analysis for detection of reliable association regions. Further precise phenotype is necessary for the identification of reliable causal Selection of association panel: Association panel for GWAS analysis may consists of diverse set of variants associated with a trait. germplasm lines or multi-parent populations such as nested association mapping population (NAM) and multi-parent advanced generation intercross (MAGIC) populations. The core and mini-core The advanced genomics approaches can play crucial role in genetic dissection of complex traits. An germplasm set of crops constituted from germplasm collection in genebanks are genetically very integrated strategy involving association mapping, QTL mapping and other genomics approaches such diverse as they represent total genetic variability of the collection. So, these have been considered as as transcriptomics, proteomics and metabolomics, is expected to greatly accelerate discovery of good starting material for conducting association analyses in crops. genes/QTLs for various economically important traits in plants. Generation of genotyping data: Association mapping panel should be densely genotyped in order to Suggested Readings find markers very closely linked to the causal genes for the trait. The genotyping density of an association panel essentially depends on LD decay distance which varies from species to species. In 1. Brachi B, N Faure, M Horton, E Flahauw, A Vazquez, M Nordborg, J Bergelson, J Cuguen and Roux F cross-pollinated crops such as maize and pearl millet, LD decays faster and hence a greater number of (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLos Genet. 6 markers would be needed for the high-resolution mapping of a trait. A variety of SNP assays have been (5): e1000940. https://doi.org/10.1371/journal.pgen.1000940. developed in different crops. Recently, genotyping by sequencing (GBS) has been widely used for 2. Chaurasia S, AK Singh, LS Songachan, AD Sharma, R Bhardwaj and Singh K (2020) Multi-locus genome- generation of genotyping data. The GBS is becoming a common genotyping approach as the species wide association studies reveal novel genomic regions associated with vegetative stage salt tolerance without a sequenced genome can also genotyped using this approach. in bread wheat (Triticum aestivum L.). Genomics. 112: 4608-21. Association analysis: A number of statistical models have been developed for calculating marker-trait 3. Frankel OH (1984) Genetic perspective of germplasm conservation. In: Arber W, K Llimensee, WJ associations. Initial GWAS studies used generalized linear model (GLM) that considered only two Peacock and P Starlinger (eds) Genetic manipulations: Impact on man and society. Univ. Press, variables; genotype and phenotype. However, this model does not take into account the population Cambridge, England, pp. 161–170. structure and genetic relatedness in association panel that might have confounding effect on marker trait association; generating more number of spurious association. To, address this limitation, Yu et al. 4. Group CP, PM Hollingsworth, L Forrest, JL Spouge, M Hajibabaei, S Ratnasingham, M van der Bank, MW (2006) developed mixed linear model (MLM) that uses population structure (K) and kinship matrix (Q). Chase, RS Cowan, DL Erickson AJ and Fazekas (2009) A DNA barcode for land plants. Proc Natl Acad Sci. The MLM model was further improved using additional criteria and have been widely used for 106: 12794-7. conducting GWAS analysis. However, both GLM and MLM are single locus models; and as one marker is 5. Huang X, X Wei, T Sang, Q Zhao, Q Feng, Y Zhao et al (2010) Genome-wide association studies of 14 tested at a time, so these cannot be efficiently utilised for mapping complex traits that are governed by agronomic traits in rice landraces. Nat Genet. 42: 961–967 multiple genes with small effects. In order to overcome this problem, several advanced multi-locus models have been designed such as mrMLM (Wang et al., 2016), FASTmrMLM (Zhang and Tamba, 6. Jeffreys AJ, V Wilson, et al. (1985) Individual-specific 'fingerprints' of human DNA. Nature 316 (6023): 2018), FASTmrEMMA (Wen et al., 2018). Since in multi-locus models, all markers effects on a trait are 76-79. simultaneously estimated, application of these models can facilitate accurate estimation of marker 7. Juliana P, J Poland, J Huerta-Espino et al. (2019) Improving grain yield, stress resilience and quality of effects in case of complex traits. bread wheat using large-scale genomics. Nat Genet. 51: 1530–1539 d. Factors affecting GWAS 8. Larson S (2002) Plant Genotyping: The DNA Fingerprinting of Plants. Heredity 88: 220-220. Though conducting GWAS analysis is very straight forward; genotyping, phenotyping and marker-trait 9. Lind BM, M Menon, CE Bolte, TM Faske and AJ Eckert (2018) The genomics of local adaptation in trees: association analysis and detection power depends on various factors (Lind et al., 2018) some of which Are we out of the woods yet? Tree Genet Genomes 14:29. are listed below. 10. Mishra S, B Singh, K Panda, BP Singh, N Singh, P Misra, V Rai and NK Singh (2016) Association of SNP Association panel size: Larger the size of association panel, more the chance to detect loci controlling haplotypes of HKT family genes with salt tolerance in Indian wild rice germplasm. Rice (1):1-3. targeted trait. Generally, a diverse association panel of about 200 accessions is considered suitable for detecting important loci controlling most of the trait subjected to condition that significant phenotypic 11. Nybom H, K Weising and B Rotter (2014) DNA fingerprinting in botany: past, present, future. Investig variation for the targeted trait is also available in this panel. Genet. 5:1. doi:10.1186/2041-2223-5-1. Trait heritability: Higher the heritability of the trait analyzed, greater would be the chance to detect 12. Rana MK and KV Bhat (2017) DNA Fingerprinting in Plants: Standard Operating Methods and Protocols, the loci responsible for variation in that trait. ICAR-National Bureau of Plant Genetic Resources, New Delhi, India, 73 pp. (http://www.nbpgr.ernet.in/Divisions_and_Units/ Downloadfile.aspx? EntryId=7432) Inheritance pattern of the trait: Complex traits such as yield and abiotic stress tolerance are governed by multiple genes each having very small effect. The association signal for these traits would be weaker 13. Wambugu PW, MN Ndjiondjop and Henry RJ (2018) Role of genomics in promoting the utilization of as compared to traits that are governed by major genes. plant genetic resources in genebanks. Brief Funct. Genomics 17:198-206.

136 137 which is a laborious and time intensive exercise. GWAS approach leverages recombination events Number of markers: The number of markers significantly affects GWAS resolution. The minimum across the generations, thereby, enabling fine resolution mapping of the targeted trait. It can provide number of markers for GWAS analysis primarily depends on the extent of LD in association panel. If LD very high resolution up to 100-200 Kb and may even identify causative genes associated with the target decay is faster, a greater number of markers would be needed to identify the causal variants for the trait trait. Some of the important association mapping studies include identification of agronomic traits and and vice versa. ionomic variation in rice (Huang et al., 2010), flowering time in Arabidopsis (Brachi et al., 2010), rust Quality of the phenotyping data: For complex traits that are influenced by environmental factors, resistance in wheat (Pradhan et al., 2020), salt tolerance traits in wheat (Chaurasia et al., 2020) etc. phenotyping data should be obtained from multiple location/ year experiments. Normalized trait c. Association mapping steps values derived from multi-location/ year data should be used in GWAS analysis for detection of reliable association regions. Further precise phenotype is necessary for the identification of reliable causal Selection of association panel: Association panel for GWAS analysis may consists of diverse set of variants associated with a trait. germplasm lines or multi-parent populations such as nested association mapping population (NAM) and multi-parent advanced generation intercross (MAGIC) populations. The core and mini-core The advanced genomics approaches can play crucial role in genetic dissection of complex traits. An germplasm set of crops constituted from germplasm collection in genebanks are genetically very integrated strategy involving association mapping, QTL mapping and other genomics approaches such diverse as they represent total genetic variability of the collection. So, these have been considered as as transcriptomics, proteomics and metabolomics, is expected to greatly accelerate discovery of good starting material for conducting association analyses in crops. genes/QTLs for various economically important traits in plants. Generation of genotyping data: Association mapping panel should be densely genotyped in order to Suggested Readings find markers very closely linked to the causal genes for the trait. The genotyping density of an association panel essentially depends on LD decay distance which varies from species to species. In 1. Brachi B, N Faure, M Horton, E Flahauw, A Vazquez, M Nordborg, J Bergelson, J Cuguen and Roux F cross-pollinated crops such as maize and pearl millet, LD decays faster and hence a greater number of (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLos Genet. 6 markers would be needed for the high-resolution mapping of a trait. A variety of SNP assays have been (5): e1000940. https://doi.org/10.1371/journal.pgen.1000940. developed in different crops. Recently, genotyping by sequencing (GBS) has been widely used for 2. Chaurasia S, AK Singh, LS Songachan, AD Sharma, R Bhardwaj and Singh K (2020) Multi-locus genome- generation of genotyping data. The GBS is becoming a common genotyping approach as the species wide association studies reveal novel genomic regions associated with vegetative stage salt tolerance without a sequenced genome can also genotyped using this approach. in bread wheat (Triticum aestivum L.). Genomics. 112: 4608-21. Association analysis: A number of statistical models have been developed for calculating marker-trait 3. Frankel OH (1984) Genetic perspective of germplasm conservation. In: Arber W, K Llimensee, WJ associations. Initial GWAS studies used generalized linear model (GLM) that considered only two Peacock and P Starlinger (eds) Genetic manipulations: Impact on man and society. Univ. Press, variables; genotype and phenotype. However, this model does not take into account the population Cambridge, England, pp. 161–170. structure and genetic relatedness in association panel that might have confounding effect on marker trait association; generating more number of spurious association. To, address this limitation, Yu et al. 4. Group CP, PM Hollingsworth, L Forrest, JL Spouge, M Hajibabaei, S Ratnasingham, M van der Bank, MW (2006) developed mixed linear model (MLM) that uses population structure (K) and kinship matrix (Q). Chase, RS Cowan, DL Erickson AJ and Fazekas (2009) A DNA barcode for land plants. Proc Natl Acad Sci. The MLM model was further improved using additional criteria and have been widely used for 106: 12794-7. conducting GWAS analysis. However, both GLM and MLM are single locus models; and as one marker is 5. Huang X, X Wei, T Sang, Q Zhao, Q Feng, Y Zhao et al (2010) Genome-wide association studies of 14 tested at a time, so these cannot be efficiently utilised for mapping complex traits that are governed by agronomic traits in rice landraces. Nat Genet. 42: 961–967 multiple genes with small effects. In order to overcome this problem, several advanced multi-locus models have been designed such as mrMLM (Wang et al., 2016), FASTmrMLM (Zhang and Tamba, 6. Jeffreys AJ, V Wilson, et al. (1985) Individual-specific 'fingerprints' of human DNA. Nature 316 (6023): 2018), FASTmrEMMA (Wen et al., 2018). Since in multi-locus models, all markers effects on a trait are 76-79. simultaneously estimated, application of these models can facilitate accurate estimation of marker 7. Juliana P, J Poland, J Huerta-Espino et al. (2019) Improving grain yield, stress resilience and quality of effects in case of complex traits. bread wheat using large-scale genomics. Nat Genet. 51: 1530–1539 d. Factors affecting GWAS 8. Larson S (2002) Plant Genotyping: The DNA Fingerprinting of Plants. Heredity 88: 220-220. Though conducting GWAS analysis is very straight forward; genotyping, phenotyping and marker-trait 9. Lind BM, M Menon, CE Bolte, TM Faske and AJ Eckert (2018) The genomics of local adaptation in trees: association analysis and detection power depends on various factors (Lind et al., 2018) some of which Are we out of the woods yet? Tree Genet Genomes 14:29. are listed below. 10. Mishra S, B Singh, K Panda, BP Singh, N Singh, P Misra, V Rai and NK Singh (2016) Association of SNP Association panel size: Larger the size of association panel, more the chance to detect loci controlling haplotypes of HKT family genes with salt tolerance in Indian wild rice germplasm. Rice (1):1-3. targeted trait. Generally, a diverse association panel of about 200 accessions is considered suitable for detecting important loci controlling most of the trait subjected to condition that significant phenotypic 11. Nybom H, K Weising and B Rotter (2014) DNA fingerprinting in botany: past, present, future. Investig variation for the targeted trait is also available in this panel. Genet. 5:1. doi:10.1186/2041-2223-5-1. Trait heritability: Higher the heritability of the trait analyzed, greater would be the chance to detect 12. Rana MK and KV Bhat (2017) DNA Fingerprinting in Plants: Standard Operating Methods and Protocols, the loci responsible for variation in that trait. ICAR-National Bureau of Plant Genetic Resources, New Delhi, India, 73 pp. (http://www.nbpgr.ernet.in/Divisions_and_Units/ Downloadfile.aspx? EntryId=7432) Inheritance pattern of the trait: Complex traits such as yield and abiotic stress tolerance are governed by multiple genes each having very small effect. The association signal for these traits would be weaker 13. Wambugu PW, MN Ndjiondjop and Henry RJ (2018) Role of genomics in promoting the utilization of as compared to traits that are governed by major genes. plant genetic resources in genebanks. Brief Funct. Genomics 17:198-206.

136 137 14. Wang SB, JY Feng, WL Ren, B Huang, L Zhou, YJ Wen, J Zhang, JM Dunwell, S Xu and YM Zhang (2016) Improving power and accuracy of genome-wide association studies via a multi-locus mixed linear Organizing Committee model methodology. Sci. Rep. 6 : 19444. 15. Weising K, H Nybom, K Wolff and G Kahl (2005) DNA fingerprinting in plants: principles, methods, and applications, 2nd edn. CRC Press, 472 p. Training Director 16. Wen YJ, H Zhang, YL Ni, B Huang, J Zhang, JY Feng, SB Wang, JM Dunwell, YM Zhang and R Wu (2018) Dr Kuldeep Singh, Methodological implementation of mixed linear models in multi-locus genome-wide association Director, studies. Brief Bioinform. 19: 700–712. ICAR - National Bureau of Plant Genetic Resources (NBPGR), New Delhi 17. Yu J, G Pressoir, WH Briggs, IV Bi, M Yamasaki, JF Doebley, MD McMullen, BS Gaut, DM Nielsen, JB Training Coordinator Holland and S Kresovich (2006) A unified mixed-model method for association mapping that accounts Dr Prakash Patil, for multiple levels of relatedness. Nature Genet 38: 203-208. Project Coordinator (Fruits), 18. Yumnam JS, M Rai and W Tyagi (2017) Allele mining across two low-P tolerant genes PSTOL1 and ICAR-All India Coordinated Research Projects(AICRP)on Fruits, ICAR - Indian Institute of PupK20-2 reveals novel haplotypes in rice genotypes adapted to acidic soils. Plant Genet Resour. 15: Horticultural Research (IIHR), Bengaluru 221. Training Convenor 19. Zhang YM and CL Tamba (2018) A fast mrMLM algorithm for multi-locus genome-wide association Dr Anuradha Agrawal, studies. bioRxiv: 341784. Principal Scientist and Officer-In-Charge, Tissue Culture and Cryopreservation Unit (TCCU), ICAR - NBPGR, New Delhi

Training Co-Convenors 1. Dr Vartika Srivastava, Scientist, TCCU, ICAR-NBPGR, New Delhi 2. Dr Era Vaidya Malhotra, Scientist, TCCU, ICAR-NBPGR, New Delhi 3. Dr K.S. Hooda, HRD Nodal Officer, ICAR-NBPGR, New Delhi 4. Dr Sridhar Gutam, Senior Scientist, AICRP on Fruits, ICAR-IIHR, Bengaluru

Computer Assistance Mr Vijay Kumar Mandal, Technical Assistant., Agriculture Knowledge Management Unit, ICAR-NBPGR, New Delhi

138 139 14. Wang SB, JY Feng, WL Ren, B Huang, L Zhou, YJ Wen, J Zhang, JM Dunwell, S Xu and YM Zhang (2016) Improving power and accuracy of genome-wide association studies via a multi-locus mixed linear Organizing Committee model methodology. Sci. Rep. 6 : 19444. 15. Weising K, H Nybom, K Wolff and G Kahl (2005) DNA fingerprinting in plants: principles, methods, and applications, 2nd edn. CRC Press, 472 p. Training Director 16. Wen YJ, H Zhang, YL Ni, B Huang, J Zhang, JY Feng, SB Wang, JM Dunwell, YM Zhang and R Wu (2018) Dr Kuldeep Singh Methodological implementation of mixed linear models in multi-locus genome-wide association Director studies. Brief Bioinform. 19: 700–712. ICAR - National Bureau of Plant Genetic Resources (NBPGR), New Delhi 17. Yu J, G Pressoir, WH Briggs, IV Bi, M Yamasaki, JF Doebley, MD McMullen, BS Gaut, DM Nielsen, JB Training Coordinator Holland and S Kresovich (2006) A unified mixed-model method for association mapping that accounts Dr Prakash Patil for multiple levels of relatedness. Nature Genet 38: 203-208. Project Coordinator 18. Yumnam JS, M Rai and W Tyagi (2017) Allele mining across two low-P tolerant genes PSTOL1 and ICAR – All India Coordinated Research Project (AICRP) on Fruits PupK20-2 reveals novel haplotypes in rice genotypes adapted to acidic soils. Plant Genet Resour. 15: ICAR – Indian Institute of Horticultural Research (IIHR), Bengaluru 221. Training Convenor 19. Zhang YM and CL Tamba (2018) A fast mrMLM algorithm for multi-locus genome-wide association Dr Anuradha Agrawal studies. bioRxiv: 341784. Principal Scientist and Officer-In-Charge Tissue Culture and Cryopreservation Unit (TCCU), ICAR - NBPGR, New Delhi

Training Co-Convenors 1. Dr Vartika Srivastava Scientist TCCU, ICAR-NBPGR, New Delhi 2. Dr Era Vaidya Malhotra Scientist TCCU, ICAR-NBPGR, New Delhi 3. Dr K. S. Hooda HRD Nodal Officer ICAR-NBPGR, New Delhi 4. Dr Sridhar Gutam Senior Scientist AICRP on Fruits, ICAR-IIHR, Bengaluru

Computer Assistance Mr Vijay Kumar Mandal Senior Technical Assistant Agriculture Knowledge Management Unit, ICAR-NBPGR, New Delhi

138 139 NOTES

140