Doctor of Philosophy in Botany

Studies on in vitro propagation of Mucuna pruriens L. and evaluation of genetic fidelity through molecular markers)

Thesis

SUBMITTED FOR THE AWARD OF DEGREE OF

Doctor of Philosophy In Botany

Naushad Alam

Under the Supervision of

Professor Mohammad Anis

Department of Botany ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)

ACKNOWLEDGEMENTS

Every result arrived at is a modest beginning for higher goal. My work in the same spirit is just a step in the ladder. It is a drop of ocean. No work can be turned as a one- man show. It needs the close cooperation of friends and colleagues and the guidance of experts in the field to achieve something worthwhile and substantial.

With immense servility, all praise and thanks is for Allah (Subhanahu wa Ta‟ala) and Salutations and Blessings be upon our Beloved Prophet (Sallallaho Alyhi Wasallam), his family and companions, who bestowed me with strength, patience and courage to go through this endeavor

After this it is my pleasant privilege to acknowledge my sense of gratitude for people who have made the completion of this thesis a reality.

With the sense of pride, ecstasy and honesty, I take this privilege to express my heartfelt gratitude to my supervisor, Professor Mohammad Anis (UGC-BSR Faculty Fellow), Department of Botany, Aligarh Muslim University, for his supreme guidance, punctual vigilance, expertise supervisions, constant encouragements, painstaking efforts, constructive criticism and sound execution during the course of investigation and preparation of this manuscript. The „positive attitude‟ inherent to his personality was a learning experience for me and it would definitely help me during „tougher times‟ in my career. I will always remain indebted to him for his benevolence and generosity.

I express my heartfelt gratitude to Professor Nafees Ahmad Khan, Chairman, Department of Botany, AMU, for providing and approving all the necessary facilities for conducting my research work.

I emphatically extend my heartiest thanks to Dr. Naseem Ahmad (Assistant Professor) for his support and help both professionally and personally during the course of this journey.

I would also like to thank Professor Altaf Ahmad, Department of Botany, AMU for his timely advice and valuable suggestion. I am thankful to Dr. Anwar Shahzad (Associate Prof.), Department of Botany, AMU for providing support, motivation and critical appreciation during my research.

I express special gratitude to my seniors Dr. Saad Bin Javed, Dr. Iram Siddiqui (Asstt. Profs.), Dr. Nigar Fatima (Women Scientist, UGC) Department of Botany, AMU for helping me in most needful conditions and supporting me during my whole research work.

The acknowledgment will never be complete without the special mention of my lab seniors who have lived by example. I would like to acknowledge Dr. Ankita Varshney (Former Young Scientist, SERB), Dr. Afshan Naaz (Post Doc Fellow, SERB), Dr. Ruphi Naz (Post Doc Fellow, D S Kothari) for all their support and motivation during my stay in the lab.

I am fortunate to have supportive and wonderful seniors and lab colleagues to help me throughout this endeavour. I am thankful to Dr. Md. Imran Khan (SDM, Manipur Civil Services), Dr. Afsheen Shahid (Post Doc Freiburg University, Germany), Dr. Anees Ahmad, Mr. Sheikh Altaf Hussain, Ms. Mehrun Nisha Khanam and Mr. Waqar Ahmad for creating a healthy work environment in the lab. I am thankful to everyone for their extended support and motivation in the most needful times.

I would like to express my sincere gratitude to my friends Imran K. Khan, Gulwaiz Akhter, Dr. Zeeshan, Faisal, Dr. Taiba Saeed, Dr. Arjumend Shaheen, Mr. Vikas Yadav, Ms. Anamica Upadhyay and Dr. Rakshanda, for their unconditional support and encouragement throughout the period of my doctoral thesis.

I wish to acknowledge Mr. Irfan Mohammad and Mr. Mohd. Athar, lab Attendants for their help during research work.

A special thanks to my family. Words cannot express how grateful I am to my parents, Mr. Mukhtar Ansari and Mrs. Samina Khatoon, sister Shabana Khatoon, brothers Irshad Alam, Tahseen and Ahsaan for all the sacrifices they have made on my behalf. Their prayers for me were what sustained me thus far. Their understanding and their love encouraged me to work hard and to continue pursuing a Ph.D. Their obstinate sacrifices, expectations and blessings have affected me to be steadfast and never bend to difficulty and motivate me to work harder.

Last but not least, I am greatly indebted to my devoted wife “Gul Naaz”. She forms the backbone and origin of my happiness. Her love and support without any complaint or regret has enabled me to complete my work. I owe my every achievement to her.

Needless to say, errors and omissions are all mine.

(NAUSHAD ALAM)

Professor (Dr.) Mohammad Anis Department of Botany PhD (Lko.), FBS, FISG, FISPM Plant Biotechnology Laboratory UGC-BSR Faculty Fellow Aligarh Muslim University Former Chairman, Department of Botany Aligarh 202 002 (INDIA) Former Dean, Faculty of Life Sciences [email protected] Visiting Professor, KSU, Riyadh [email protected]

Dated: ………………….

CERTIFICATE

This is to certify that the thesis entitled “Studies on in vitro propagation of Mucuna pruriens L. and evaluation of genetic fidelity through molecular markers” submitted for the award of the degree of Doctor of Philosophy embodies the original research work carried out in the Plant Biotechnology Laboratory at the Department of Botany, Aligarh Muslim University, Aligarh, India by Mr. NAUSHAD ALAM under my guidance and supervision and that no part of the thesis has been submitted for any degree or diploma of this or any other university.

It is further certified that scholar fulfills all requirements as laid down by the university for the purpose of submission of Ph.D. thesis.

Professor Mohammad Anis (Supervisor)

Tel. Offi. 0571- 2702016; 2700920, 21, 22 (Ext. 3308). Mobile: +91-9837305566 Home address: 07, Rainbow Roofs, Phase-I, Anoopshahar Road, Aligarh- 202 002

ABSTRACT

Because of presence of enormous numbers of plant species, India has earned the title of “the world herbal garden”. With over 45,000 species of herbal medicinal plant spread over hotspots in the Western Ghats, Eastern Himalayas and the Andaman and Nicobar Islands (Anonymous 2003). A significant proportion of these plant species are employed for medicinal purposes both in modern and traditional system of medicine. Recently, plant based products such as dietary supplements and drug manufacturing at industrial level have been improved greatly. A large member of medicinal plant species are at a high risk of extinction because of continuous collection and over exploitation from the wild for commercial uses. We are losing about one potentially vital medicinal plant every two years at present. Depletion occurs due to destructive, unsustainable and continuous harvesting of such potent medicinal plants resulting in the depletion of genetic resource from natural habitat. Foundation of Revitalization of local Health traditions (FRLHT) working under the Ministry of Environment and Forest and the Ministry of Health, Government of India has identified 178 medicinal plant species with high trade potential of M. pruriens. Demand for L-Dopa is largely met by the pharmaceutical industries through extraction of the compound from wild population. The conventional propagation through seed is not an adequate solution to meet the demand. Therefore, there is an urgent need to use alternative techniques for conservation and sustainable utilization. Now-a-days, micropropagation system is mainly used for the bulk production of planting stock material to increase the biomass production and is broadly accepted for mass multiplication, conservation and preservation of elite germplasm. The present investigation was aimed to develop cost effective protocol for conservation and mass production of M. pruriens. The metal tolerance potentiality of the legume plant was also analysed through micropropagation and anti-oxidative enzyme activity (GST) has been measured during in vitro culture practice. Changes in photosynthetic parameters and anti-oxidative enzymes were measured during the ex vitro acclimatization of micropropagated plantlets. To validate the genetic stability among regenerants, the assessment of genetic fidelity was done using ISSR techniques. The main findings are summarized as under.

The best seed germination (95%) was recorded on cotton wetted with double distilled water (DDW) after 7 days of incubation. Cotyledonary node (CN) and nodal explants were excised from 7 days old aseptic seedlings of M. pruriens were employed during the investigation. Direct multiple shoot regeneration was observed from nodal segments inoculated on MS medium augmented with different cytokinins, singly or in combinations with polyamines and auxins. Among the tested cytokinins, mT (6.5 µM) in combination with Putrescine (10.µM) produced the maximum number of shoots. The effect of heavy metals like Cu, Zn, Ni and Cd on direct shoot regeneration from the aseptic nodal explants was also evaluated for standardization of optimal medium for maximum shoot induction and multiplication. NiCl2 (15 µM) when added to MS medium containing 6.5 µM mT and 10.0 µM Put gave best response in term of maximum shoots (34.6 ± 0.56) with mean shoot length (7.22 ± 0.05 cm) in 95% cultures. Glutathione-S-transferase activity of in vitro grown plantlets on metal containing medium showed an increase in GST activity with 100% survival on the optimal concentration of tested metals. An indirect regeneration protocol has been standardized using leaf segments excised from 7 days old axenic seedlings on MS medium supplemented with different concentrations of 2,4-D. Best callogenesis was observed on 0.5 µM 2,4-D enriched MS medium after 28 days on inoculation. The calli when transferred to MS medium containing 6.5 µM mT + 10 µM Put produced maximum shoots (6.60 ± 0.51) with shoot length (5.22 ± 0.11 cm) after 56 days of transfer.

The effect of different basal medium (MS, L2, WPM and B5), different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) and carbon sources were also examined on the optimal MS medium standardized (6.5 μM mT + 10.0 μM Put). The full MS medium, 5.8 pH and 3% sucrose was found to be most suitable for in vitro regeneration and production of maximum shoot after 56 d of culture. Good quality synthetic seeds were produced by encapsulating nodal segments in gelling agent containing sodium alginate and ClCl2.

Fine quality synseeds were produced in 3.0 % sodium alginate and 100 mM CaCl2. MS medium containing 6.5 μM mT and 10.0 μM Put gave maximum shoot conversion frequency of encapsulated nodal segments to plantlets. The synseeds could be stored at low temperature (4 °C) with 37.0 conversion frequency even after 70 days of cold dark storage.

Induction of roots in microshoots (5 cm) was readily achieved with various auxins by both in vitro and ex vitro methods. In vitro rooting was best observed in ½ MS medium supplemented with 0.20 μM IBA where maximum roots were induced after 28 days of culture. However, for ex vitro rooting, proximal end of the shootlets dipped in IBA (90.0 μM) solution for 30 min gave the highest root formation after 28 days of transplantation in Soilrite. The regenerated plantlets were successfully acclimatized in Soilrite inside growth chamber. During the initial days upto (7 days) of acclimatization, the Chl a/b and carotenoid contents got decreased and thereafter increased continuously upto 28 days of acclimatization. The same trend was observed on evaluation of other important enzyme activities such as (SOD, CAT and CA). This increase in photosynthetic and other enzymatic activities predicts better adaptability of regenerants to natural condition. Furthermore, evaluation of genetic fidelity among the regenerants was carried out using genomic DNA and monomorphic banding pattern was observed via RAPD and ISSR analysis with the control (mother) plants. Thus, verifying the clonal stability of the plantlets developed throughout the micropropagation studies in M. pruriens. The protocol provides an alternative viable system for true-to-type production of plantlets as confirmed by the RAPD and ISSR markers. The explored metal tolerant potentiality and bioaccumulation ability of the regenerants of M. pruriens could be fruitful in developing metal tolerant lines of the legume which could be used in the reclamation of polluted and waste lands.

CONTENT

Page No.

List of Figures i-vi List of Tables vii-xii Abbreviations xiii-xiv

CHAPTER 1: INTRODUCTION 1-8

1.1 Plant Description 5 1.1.1 Scientific classification 5 1.1.2 Scientific name 5 1.1.3 English name 5 1.1.4 Common name 5 1.1.5 Propagation 5 1.1.6 Status 5 1.2 Habitat 5 1.3 Botanical description 5 1.4 Phytochemistry 6 1.5 Importance 6 1.5.1 Antivenom and antidiabatic 7 1.5.2 Antioxidant activity 7 1.5.3 Neuroprotective activity 7 1.5.4 As a dietary substance 7 1.5.5 As an aphrodisiac 7 1.5.6 Others uses 7 1.6 Rationale 7

CHAPTER 2: REVIEW OF LITERATURE 9-32

2.1 Micropropagation 12 2.2 In Vitro Morphogenesis and Propagation 13 2.2.1 Explant type 13 2.2.2 Size of the explant 13 2.3 Plant growth regulators (PGRs) 14 2.3.1 Effect of Cytokinins 15 2.3.2 Effect of auxins 16 2.3.3 Effect of meta-topolin (mT) 17 2.3.4 Effect of TDZ 18 2.4 Effect of polyamines (PAs) 19 2.5 Heavy metals 20 2.5.1 Copper 21 2.5.2 Zinc 21 2.5.3 Nickel 22 2.5.4 Cadmium 23 2.6 Effect of Carbon sources 23 2.7 Effect of pH 24 2.8 Rooting 25 2.9 Acclimatization 26 2.10 Physiological studies 27 2.11 Biochemical studies 28 2.12 Synthetic seeds 29 2.13 Genetic fidelity 30 2.14 Tissue culture related work conducted on M. pruriens 31 CHAPTER 3: MATERIALS AND METHODS 33-50

3.1 Source of plant material 33 3.2 Sterilization of seeds 33 3.3 Establishment of aseptic seedlings and explants 33 procurement 3.4 Culture media composition and culture conditions 33 3.4.1 Composition of basal media 33 3.4.2 Stock solution Preparation 34 3.4.3 Plant growth regulators (PGRs), Polyamines and Metals 35 3.4.4 Carbon source, pH and gelling agents of the medium 35 3.5 Media vessel and plugging material 35 3.6 Sterilization 35 3.6.1 Media sterilization 35 3.6.2 Sterilization of glass-wares and instruments 36 3.7 Culture inoculation and incubation 36 3.8 Rooting 36 3.9 Hardening and acclimatization 36 3.10 Synthetic seed production 37 3.10.1 Explant source 37 3.10.2 Encapsulation matrix 37 3.10.3 Encapsulation, planting media and culture conditions 37 3.10.4 Low temperature storage 37 3.11 Physiological and biochemical studies of regenerated 38 plantlets 3.11.1 Pigment contents estimation 38 3.11.1.1 Procedure 38 3.11.1.2 Estimation 38 3.11.2 Estimation of antioxidant enzymes activities 39 3.11.2.1 Estimation of Superoxide dismutase (SOD) 39 3.11.2.1.1 Procedure 39 3.11.2.1.2 Enzymes assay 39 3.11.2.1.3 Reagents preparation 40 3.11.2.1.4 Extraction buffer 40 3.11.2.1.5 Reaction buffer 40 3.11.3 Estimation of Catalase (CAT) activity 41 3.11.3.1 Procedure 41 3.11.3.2 Enzyme assay 41 3.11.3.3 Reagents Preparation 41 3.11.4 Estimation of Glutathione-S-transferase (GST) 42 3.11.4.1 GST enzyme assay 42 3.11.4.1.1 Procedure 42 3.11.4.1.2 Estimation 43 3.11.5 Estimation of carbonic anhydrase (CA) activity 43 3.11.5.1 Procedure 43 3.11.5.1.1 Preparation of Reagents 43 3.12 Molecular marker studies of in vitro raised plantlets 44 3.12.1 Sample source of DNA 44 3.12.2 Isolation of Genomic DNA 44 3.12.3 Preparation of reagents for DNA isolation 44 3.12.4 Extraction and purification protocol 45 3.12.4.1 Purification of genomic DNA 46 3.12.5 DNA assessment (Qualitative and Quantitative) 46 3.12.5.1 Quality analysis 46 3.12.5.2 Quantification analysis 46 3.12.6 Solutions for agarose gel electrophoresis 47 3.12.6.1 TBE (5 X) 500 ml stock solutions 47 3.12.6.2 Running Buffer (1X TBE) 47 3.12.6.3 Gel Loading Dye (6X) 47 3.12.6.4 Gel Staining Dye 47 3.12.7 Agarose gel electrophoresis 47 3.13 RAPD/ISSR primers screening and DNA amplification 48 3.13.1 Reagents for PCR Amplification 48 3.13.2 RAPD and ISSR - PCR based amplification of genomic 48 DNA 3.13.3 Electrophoresis of amplified PCR product 48 3.13.4 Analysis of DNA amplification 48 3.14 Chemicals and Glasswares 49 3.15 Statistical analysis 49

CHAPTER 4: RESULTS 51-152

4.1 Direct shoot regeneration 51 4.1.1 Establishment of aseptic seedlings 51 4.1.2 Regeneration from cotyledonary node explants 52 4.1.2.1 Effect of cytokinins (BA, Kn or 2-iP) 52 4.1.2.1.1 Combined effect of auxin and cytokinin 55 4.1.2.1.2 Combined effect of cytokinin and polyamines 59

4.1.2.1.3 Combined effect of cytokinin, polyamine and CuSO4 63 4.1.2.1.4 Combined effect of cytokinin, polyamine and ZnSO4 64

4.1.2.1.5 Combined effect of cytokinin, polyamine and NiCl2 68 4.1.2.1.6 Combined effect of cytokinin, polyamine and CdCl2 70 4.1.2.2 Effect of meta-topolin (mT) 73 4.1.2.2.1 Combined effect of meta-topoline and auxins 75 4.1.2.2.2 Combined effect of meta-topoline and polyamines 77

4.1.2.2.3 Combined effect of meta-topoline, polyamine and CuSO4 79 4.1.2.2.4 Combined effect of meta-topoline, polyamine and ZnSO4 80 4.1.2.2.5 Combined effect of meta-topoline, polyamine and NiCl2 82 4.1.2.2.6 Combined effect of meta-topoline, polyamine and CdCl2 83 4.1.2.3 Effect of TDZ 85 4.1.3 Nodal explants 88 4.1.3.1 Effect of cytokinins (BA, Kin or 2-iP) 88 4.1.3.1.1 Combined effect of cytokinin and auxin 90 4.1.3.1.2 Combined effect of cytokinin and polyamines 96

4.1.3.1.3 Combined effect of cytokinin, polyamine and CuSO4 100 4.1.3.1.4 Combined effect of cytokinin, polyamine and ZnSO4 103 4.1.3.1.5 Combined effect of cytokinin, polyamine and NiCl2 105 4.1.3.1.6 Combined effect of cytokinins, polyamines and CdCl2 107 4.1.3.2 Effect of meta-topolin 109 4.1.3.2.1 Combined effect of meta-topoline and auxin 111 4.1.3.2.2 Combined effect of meta-topoline and polyamines 113

4.1.3.2.3 Combined effect of meta-topoline, polyamine and CuSO4 113 4.1.3.2.4 Combined effect of meta-topoline, polyamine and ZnSO4 117 4.1.3.2.5 Combined effect of meta-topoline, polyamine and NiCl2 118 4.1.3.2.6 Combined effect of meta-topoline, polyamine and CdCl2 120 4.1.3.3 Effect of TDZ 121 4.1.3.4 GST activity under the regime of heavy metals 125 4.1.3.5 Effect of different culture media 128 4.1.3.6 Effect of carbon source 128 4.1.3.7 Effect of pH 130 4.1.3.8 Subculture passages 131 4.1.3.9 Synthetic seeds 131 4.1.3.9.1 Effect of sodium alginate and calcium chloride 131 4.1.3.9.2 Plant regeneration from alginate encapsulated nodal 132 segments 4.1.3.9.3 Low temperature storage 133 4.2 In direct organogenesis 135 4.2.1 Callus induction and shoot organogenesis 135 4.3 Rooting 137 4.3.1 In vitro rooting 137 4.3.2 Ex vitro rooting 140 4.4 Acclimatization 142 4.4.1 Effect of planting materials for hardening 142 4.4.2 Estimation of physiological parameters during 143 acclimatization 4.4.2.1 Photosynthetic pigments profile 143 4.4.3 Estimation of biochemical parameters during 144 acclimatization 4.4.3.1 Assessment of antioxidant enzymes activities 144 4.5 Genetic stability 146 4.5.1 RAPD analysis 146 4.5.2 ISSR analysis 150

CHAPTER 5: DISCUSSION 153-178

5.1 Seed germination 153 5.2 Direct in vitro regeneration 154 5.2.1 Plant growth regulators 155 5.2.2 Effect of meta-topolin (mT) 156 5.2.3 Combined effect of cytokinin and auxins 157 5.2.4 Effect of TDZ 158 5.2.5 Effect of polyamines 160 5.2.6 Effect of heavy metals on in vitro axillary shoots 161 proliferation

5.2.6.1 Effect of CuSO4 162 5.2.6.2 Effect of ZnSO4 162 5.2.6.3 Effect of NiCl2 163 5.2.6.4 Effect of CdCl2 163 5.2.7 Studies of GST activity in heavy metal treated cultures 164 5.2.8 Indirect organogenesis 165 5.2.9 Effect of subculture on shoot proliferation 166 5.2.10 Effect of different media 166 5.2.11 Effect of media pH on propagation 167 5.2.12 Effect of carbon sources 168 5.2.13 Synthetic seed 168 5.2.14 Rooting 170 5.2.14.1 In vitro rooting 170 5.2.14.2 Ex vitro rooting 171 5.2.15 Acclimatization 172 5.2.15.1 Physiological studies 173 5.2.15.2 Biochemical studies 174 5.2.16 Genetic fidelity 176 CHAPTER 6: SUMMARY AND CONCUSIONS 179-182 CHAPTER 7: REFERENCES 183-250

Publications

LIST OF FIGURES

Title Page No.

Fig. 1. Effect of media on in vitro seed germination, after 28 days of seed 51

germination.

Fig. 2. Effect of BA, Kn and 2-iP on shoot induction from cotyledonary node 52 (CN) explants, after 28 days of inoculation.

Fig. 3. Effect of BA, Kn and 2-iP on shoot induction from cotyledonary node 54 (CN) explants, after 56 days of culture.

Fig. 4. Effect of NAA (0.05 µM) in combination with optimized BA, Kn or 55 2-iP on shoot induction from cotyledonary node (CN) explants, after 28 and 56 days of culture.

Fig. 5. Effect of Polyamines in combination with optimum BA on shoot 59 multiplication from cotyledonary node (CN) explants after 28 days of inoculation.

Fig. 6. Effect of Polyamines in combination with optimum Kn on shoot 61 multiplication from cotyledonary node (CN) explants after 28 days of inoculation.

Fig. 7. Effect of Polyamines in combination with optimum Kn on shoot 62 multiplication from cotyledonary node (CN) explants, after 56 days of culture.

Fig. 8. Effect of CuSO4 in combination with optimum dose of BA + 64 Putrescine on shoot multiplication from cotyledonary node (CN) explants, after 28 and 56 days of culture.

Fig. 9. Effect of ZnSO4 in combination with optimum dose of BA + 66 Putrescine on shoot multiplication from cotyledonary node (CN) explants, after 28 and 56 days of culture.

Fig. 10. Effect of ZnSO4 in combination with optimum dose of Kn + 67 Putrescine on shoot multiplication from cotyledonary node (CN) explants, after 28 and 56 days of culture.

Fig. 11. Effect of NiCl2 in combination with optimum dose of BA + 69 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 12. Effect of NiCl2 in combination with optimum dose of Kn + 69 Putrescine on shoot multiplication from cotyledonary node (CN) explants, after 28 and 56 days of culture.

Fig. 13. Effect of CdCl2 in combination with optimum dose of BA + 71 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 14. Effect of CdCl2 in combination with optimum dose of Kn + 73 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 15. Effect of optimum mT (6.5 µM) on multiple shoot regeneration from 74 cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 16. Effect of NAA (0.05 µM) in combination with optimized mT on 75 shoot induction from cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 17. Effect of Polyamines in combination with optimum mT on shoot 77 multiplication from cotyledonary node (CN) explants, after 56 days of culture.

Fig. 18. Effect of CuSO4 in combination with optimum dose of mT + 80 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days.

Fig. 19. Effect of ZnSO4 in combination with optimum dose of mT + 81 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days.

Fig. 20. Effect of NiCl2 in combination with optimum dose of mT + 83 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days.

Fig. 21. Effect of CdCl2 in combination with optimum dose of mT + 84 Putrescine on shoot multiplication from cotyledonary node (CN) explants after 28 and 56 days.

Fig. 22. Effect of optimized TDZ on multiple shoot regeneration from 86 cotyledonary node (CN) explants after 28 and 56 days of culture.

Fig. 23. Effect of different treatments on multiple shoot regeneration from 88 TDZ (2.5-5.0 μM) exposed (28 d) cotyledonary node (CN) explants, after 56 days of culture.

Fig. 24. Effect of BA, Kn and 2-iP on shoot induction from nodal explants, 90 after 28 days of inoculation.

Fig. 25 Effect of BA, Kn and 2-iP on shoot induction from nodal segment 91 (NS) explants, after 56 days of culture.

Fig. 26. Effect of NAA (0.05 µM) in combination with optimized BA, Kn or 95 2-iP on shoot induction from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 27. Effect of Polyamines in combination with optimum BA on shoot 96 multiplication from nodal segment (NS) explants after 28 days of inoculation.

Fig. 28. Effect of Polyamines in combination with optimum BA on shoot 98 multiplication from nodal segment (NS) explants, after 56 days of culture.

Fig. 29. Effect of Polyamines in combination with optimum Kn on shoot 100 multiplication from nodal segment (NS) explants, after 28 days of inoculation.

Fig. 30. Effect of CuSO4 in combination with optimum dose of BA + 102 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 31. Effect of ZnSO4 in combination with optimum dose of BA + 104 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 32. Effect of ZnSO4 in combination with optimum dose of Kn + 105 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 33. Effect of NiCl2 in combination with optimum dose of BA + 106 Putrescine on shoot multiplication from nodal segment (NS) explants, after 28 and 56 days of culture.

Fig. 34. Effect of CdCl2 in combination with optimum dose of BA + 108 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 35. Effect of optimum mT (6.5 µM) on multiple shoot regeneration from 110 nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 36. Effect of NAA (0.05 µM) in combination with optimized mT on 111 shoot induction from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 37. Effect of Polyamines in combination with optimum mT on shoot 115 multiplication from nodal segment (NS) explants, after 56 days of culture.

Fig. 38. Effect of CuSO4 in combination with optimum mT + Putrescine on 116 shoot multiplication from nodal explants after 28 and 56 days.

iii

Fig. 39. Effect of ZnSO4 in combination with optimum dose of mT + 118 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 40. Effect of NiCl2 in combination with optimum dose of mT + 119 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 41. Effect of CdCl2 in combination with optimum dose of mT + 121 Putrescine on shoot multiplication from nodal segment (NS) explants after 28 and 56 days of culture.

Fig. 42. Effect of optimized TDZ on multiple shoot regeneration from nodal 122 segment (NS) explants after 28 and 56 days of culture.

Fig. 43. Effect of PGRs and adjuvants on multiple shoot regeneration from 123 TDZ (2.5-5.0 μM) exposed (28 d) nodal segment (NS) explants, after 56 days of culture.

Fig. 44. GST Activity of 3 and 6 weeks culture supplemented with optimized 126 concentration of BA (2.5 μM) and Put (10.0 μM) along with varying

concentrations of CuSO4.

Fig. 45. GST Activity of 3 and 6 weeks culture supplemented with optimized 126 concentration of BA (2.5 μM) and Put (10.0 μM) along with varying

concentrations of ZnSO4.

Fig. 46. GST Activity of 3 and 6 weeks culture supplemented with optimized 127 concentration of BA (2.5 μM) and Put (10.0 μM) along with varying

concentrations of NiCl2.

Fig. 47. GST Activity of 3 and 6 weeks culture supplemented with optimized 127 concentration of BA (2.5 μM) and Put (10.0 μM) along with varying

concentrations of CdCl2.

Fig. 48. Effect of different media on multiple shoot regeneration from nodal 128 explants growing on MS medium augmented with mT (6.5 µM) + Put (10.0 µM), after 56 d of culture.

Fig. 49. Effect of different carbon sources on multiple shoots regeneration 129 from nodal segment (NS) explant growing on MS medium augmented with mT (6.5 µM) + Put (1.0 µM), after 56 d of culture.

Fig. 50 Effect of different pH levels of the MS medium on multiple shoot 130 regeneration from nodal segment (NS) explants growing on MS medium augmented with mT (6.5 µM) + Put (1.0 µM).

Fig. 51. Effect of different sub-culture passages on multiple shoot 131 regeneration from nodal explants growing on MS medium augmented with mT (6.5 µM) + Put (1.0 µM).

Fig. 52. (A) Syn-seeds obtained by encapsulation of nodal segments; (B) 134 Syn-seeds inoculated on MS + mT (6.5 µM) + Put (10.0 µM); (C) Germinated seeds on MS + mT (6.5 µM) + Put (10.0 µM) after 14 days; (D) Ibid after 21 days; (E) Individual culture multiplied on MS + mT (6.5 µM) + Put (10.0 µM) after 28 days; (F) Ibid after 56 days of culture.

Fig. 53: (A) Callus induction in leaf explant on MS + 2, 4-D (5.0 μM) after 21 137 days of culture; (B) Ibid after 28 days; (C-D) Shoot and root differentiation in callus on MS + mT (6.5 μM) after 28 days of culture; (D) Ibid after 56 days of culture.

Fig. 54. In vitro rooting in regenerated microshoot. 139

Fig. 55. Ex vitro rooted plantlets in IBA pretreated microshoots after 28 days 141 of culture.

Fig. 56. 28-d-old acclimatized plantlets on Soilrite. 142

Fig. 57. Changes in pigment content (Chlorophyll a/ b, total chlorophyll and 143 carotenoid) (mg/g of FW) and ratio of Chl a/b during acclimatization period (0- 28 days) of in vitro rooted plantlets.

Fig. 58. Changes in SOD (unit mg-1 protein) activity during acclimatization 144 period (0-28 days) of in vitro rooted plantlets.

Fig. 59. Changes in CAT (µmol min-1 mg-1 protein) activity during 145 acclimatization period (0-28 days) of in vitro rooted plantlets.

-1 Fig. 60. Change in carbonic anhydrase (CA) activity [mM CO2 (g ) fresh 145 mass s-1] during acclimatization period (0-28 days) of in vitro rooted plantlets

Fig. 61. RAPD banding profile among regenerants of Mucuna pruriens L. (A) 148-149 Primer OPA 18; (B) Primer OPB 20; (C) Primer OPB 17; (D) Primer OPC 04.

Fig. 62. ISSR banding pattern among regenerants of Mucuna pruriens L. 151-152 (A) Primer UBC 827; (B) Primer UBC 841; (C) Primer UBC 855; (D) Primer UBC 868.

LIST OF TABLES

Title Page No.

Table 1. Effect of various cytokinins on shoot bud induction and 53 multiplication from cotyledonary node explants on MS medium

Table 2. Effect of various concentrations of auxins with the optimum 56 concentrations of BA (2.5 μM) on shoot formation from cotyledonary node explants on MS medium

Table 3. Effect of various concentrations of auxins with the optimum 57 concentrations of Kn (2.5 μM) on shoot formation from cotyledonary node explants on MS medium

Table 4. Effect of various concentrations of auxins with the optimum 58 concentrations of 2-iP (2.5 μM) on shoot formation from cotyledonary node explants on MS medium

Table 5A. Effect of various concentrations of Polyamines with optimum 60 concentration of BA (2.5 μM) on multiple shoot regeneration from cotyledonary node explants on MS medium

Table 5B. Effect of various concentrations of Polyamines with optimum 60 concentration of Kn (2.5 μM) on multiple shoot regeneration from cotyledonary node explants on MS medium

Table 6. Effect of different concentrations of CuSO4 supplied in *OM (MS + 63 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 7. Effect of different concentrations of CuSO4 supplied in *OM (MS + 64 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 8. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 65 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 9. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 67 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 10. Effect of different concentrations of NiCl2 supplied in *OM (MS + 68 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

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Table 11. Effect of different concentrations of NiCl2 supplied in *OM (MS + 70 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 12. Effect of different concentrations of CdCl2 supplied in *OM (MS + 71 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 13. Effect of different concentrations of CdCl2 supplied in *OM (MS + 72 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 14. Effect of various concentration of Meta-topolin (mT) on shoot bud 74 induction and multiplication from cotyledonary node explants on MS medium

Table 15. Effect of various concentrations of auxins with the optimum 76 concentrations of mT (6.5μM) on shoot formation from cotyledonary node explants on MS medium

Table 16. Effect of various concentrations of Polyamines with optimum 78 concentration of mT (6.5 μM) on multiple shoot regeneration from cotyledonary node explants on MS medium

Table 17. Effect of different concentrations of CuSO4 supplied in *OM (MS + 79 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 18. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 81 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 19. Effect of different concentrations of NiCl2 supplied in *OM (MS + 82 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 20. Effect of different concentrations of CdCl2 supplied in *OM (MS + 84 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from cotyledonary node explants

Table 21. Effect of various concentrations of TDZ on multiple shoot 85 regeneration from cotyledonary node explants on MS medium

Table 22. Effect of PGRs and adjuvants on multiple shoot regeneration from 87 TDZ (2.5-5.0 μM) exposed (28 d) cotyledonary node explants

Table 23. Effect of various cytokinins on shoot bud induction and 89 multiplication from nodal explants on MS medium

viii

Table 24. Effect of various concentrations of auxins with the optimum 92 concentrations of BA (2.5 μM) on shoot formation from nodal explants on MS medium

Table 25. Effect of various concentrations of auxins with the optimum 93 concentrations of Kn (2.5 μM) on shoot formation from nodal explants on MS medium

Table 26. Effect of various concentrations of auxins with the optimum 94 concentrations of 2-iP (2.5 μM) on shoot formation from nodal explants on MS medium

Table 27. Effect of various concentrations of Polyamines with optimum 97 concentration of BA (2.5 μM) on multiple shoot regeneration from nodal explants on MS medium

Table 28. Effect of various concentrations of Polyamines with optimum 99 concentration of Kn (2.5 μM) on multiple shoot regeneration from nodal explants on MS medium

Table 29. Effect of different concentrations of CuSO4 supplied in *OM (MS + 101 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 30. Effect of different concentrations of CuSO4 supplied in *OM (MS + 102 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 31. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 103 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 32. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 104 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 33. Effect of different concentrations of NiCl2 supplied in *OM (MS + 106 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 34. Effect of different concentrations of NiCl2 supplied in *OM (MS + 107 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 35. Effect of different concentrations of CdCl2 supplied in *OM (MS + 108 2.5 μM BA + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 36. Effect of different concentrations of CdCl2 supplied in *OM (MS + 109 2.5 μM Kn + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 37. Effect of various concentration of Meta-topolin (mT) on shoot bud 110 induction and multiplication from nodal explants on MS medium

Table 38. Effect of various concentrations of auxins with the optimum 112 concentrations of mT (6.5μM) on shoot formation from nodal explants on MS medium

Table 39. Effect of various concentrations of Polyamines with optimum 114 concentration of mT (6.5 μM) on multiple shoot regeneration from nodal explants on MS medium

Table 40. Effect of different concentrations of CuSO4 supplied in *OM (MS + 116 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 41. Effect of different concentrations of ZnSO4 supplied in *OM (MS + 117 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 42. Effect of different concentrations of NiCl2 supplied in *OM (MS + 119 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 43. Effect of different concentrations of CdCl2 supplied in *OM (MS + 120 6.5 μM mT + 10.0 μM Put) on shoot bud induction and multiplication from nodal explants

Table 44. Effect of various concentrations of TDZ on multiple shoot 122 regeneration from nodal explants on MS medium

Table 45. Effect of PGRs and adjuvants on multiple shoot regeneration from 124 TDZ (2.5-5.0 μM) exposed (28 d) nodal explants

Table 46. Effect of different concentrations of sodium alginate with optimal 132 concentration of calcium chloride on conversion of encapsulated NS on MS medium, after 56 d of culture

Table 47. Effect of different concentrations of calcium chloride with optimal 132 concentration of sodium alginate on conversion of encapsulated NS on MS medium, after 56 d of culture

Table 48. Effect of different treatments on in vitro conversion of synseeds 133 after 56 d of culture

Table 49. Conversion response (CR%) of encapsulated and non-encapsulated 135 nodal segments on MS + mT (6.5 μM) + Put (10.0 μM) at different duration of cold storage (4 °C)

Table 50. Effect of 2,4-D on callus induction in various explants, after 56 days 136 of culture

Table 51. Effect of various concentrations of cytokinins on shoot 136 morphogenesis in leaf callus obtained on 2,4-D (5.0 μM)

Table 52. Effect of various concentration of auxins on MS medium solidified 138 with phytagel on root induction in in vitro raised micro shoots

Table 53. Efficiency of root formation from in vitro raised micro shoots when 140 dipped in IBA at different concentration for 30 min

Table 54. Effect of different planting substrates for hardening 142

Table 55. Randomly amplified polymorphic DNA primers (RAPD) used for 147 screening and evaluation of genetic fidelity

Table 56. List of Inter sequence repeats (ISSR) primers screened and to access 150 the genetic fidelity among regenrants

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ABBREVIATIONS

% Percent ɛ Extinction Coefficient µg Microgram µl Microliter µM Micro molar 2,4-D 2,4-Dichlorophenoxyacetic Acid 3, 5, 6-TPA 4-amino-3, 5, 6-trichloro-2-pyridinyloxyacetic acid AFLP Amplified Fragment Length Polymorphism ANOVA Analysis of variance APX Ascorbate peroxidase B5 Gamborg medium BA 6-Benzyl adenine C Control CA Carbonic anhydrase CaCl2 Calcium chloride Car Carotenoids CAT Catalase Cd Cadmium Chl Chlorophyll Ck Cytokinin cm centimeter CN Cotyledonary node CO2 carbon dioxide CTAB Cetyl Trimethyl Ammonium Bromide Cu Copper d Days DDW Double distilled water Df Dilution factor DMRT Duncan’s multiple range test DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EU Enzyme Units Fig Figure FRLHT Foundation of Revitalization of local Health traditions g Gram GA3 Gibberellic acid GR Glutathione Reductase GST Glutathione- S – Transferase H2O2 Hydrogen peroxide HCl Hydrogen chloride HgCl2 Mercuric chloride HM Heavy metal IAA Indole-3-acetic acid IBA Indole-3-butyric acid ISSR Inter simple sequence repeats Kn Kinetin

xiii

L-DOPA 3, 4-dihydroxyphenyl-L-alanine mg Milligrams min Minute ml Milliliter MS Murashige and Skoog medium mT Meta-topolin NAA α-Naphthalene acetic acid NaCl Sodium chloride NaHCO3 Sodium bicarbonate NaOH Sodium hydroxide NBT Nitroblue tetrazolium Ni Nickel NS Nodal segment - O2 Superoxide radicals ᵒC Degree Celsius OD Optical density OM Optimized medium PAs Polyamines PCR Polymerase chain reaction PGR Plant growth regulator Put Putrescine PVP Polyvinylpyrrolidone RAPD Random amplified polymorphic DNA RFLP Restriction Fragment Length Polymorphism ROS Reactive oxygen species rpm Rotations per minute SCAR Sequenced Characterized Amplified Region SDS Sodium dodecyl sulphate SE Standard error SOD Superoxide dismutase Spd Spermidine Spm Spermine SSR Simple Sequence Repeat TBA Thiobarbituric acid TBARS Thiobarbituric acid reactive substances TCA Trichloroacetic acid TDZ Thidiazuron UV Ultra violet v/v Volume by volume w/v Weight by volume WPM Woody plant medium Zn Zinc

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MATERIALS AND METHODS

3.1 Source of plant material Seeds of Mucuna pruriens were obtained from the Indian Agricultural Research Institute (IARI), New Delhi.

3.2 Sterilization of seeds Healthy, mature seeds were washed thoroughly under runnig tap water for 30 min to remove adherent particles, followed by treatment with labolene (Qualigens, Mumbai, India) detergent 5% (v/v) for 10 min, and then washed with sterile double distilled water for 3-4 times. Surface sterilization was done with 0.1% (w/v) mercuric chloride (HgCl2) solution for 4 min, and finally rinsed 3 times with double distilled water. The sterilized seeds were inoculated in sterilized jam bottles (3 seeds per bottle) lined with cotton moistened with water or on half strength Murashigue and Skoog (MS) (1962), medium solidified with 1% (w/v) agar.

3.3 Establishment of aseptic seedlings and explants procurement Explants were obtained from the seeds germinated aseptically on moist cotton or on ½ MS medium. Cotyledonary nodes (1.50 cm) were excised from 5-days old seedling while the nodal segments (1.0-1.5 cm) were excised from 7 days old aseptic seedlings.

3.4 Culture media composition and culture conditions In plant tissue culture, in vitro morphogenesis and fate of explant are greatly governed by the type of medium and its composition. On the basis of culture practices, nutrient requirements, and explant type, different media have been formulated. We used

Murashigue and Skoog medium as the control media, while, B5 (Gamborg et al. 1968) and WPM (Lloyd and Mc Cown 1980), media were also tested for their effectiveness in induction, multiplication and proliferation of in vitro cultures of M. pruriens.

3.4.1 Composition of basal media

The basal MS, B5 and WPM media differ in their relative salts composition and concentrations. However, these medium are composed of following basic components; i. Essential elements (complex mixture of salts) ii. Organic supplements (vitamins and/ or amino acids) iii. Carbon source (sucrose). Chapter – THREE MATERIALS AND METHODS

For practical purpose, the essential elements are further divided into the following categories; (a) Major or macronutrients (b) Minor or micronutrients

3.4.2 Stock solution Preparation All MS stock solutions were prepared in four different sets, using sterilized double distilled water, described as stock I (20X)- major salts, stock II (200X)- minor salts, stock III (100X)- FeSO4.7H2O and Na2-EDTA and stock IV (100X)- organic nutrients. All the stock solution salts were dissolved separately in required quantity in double distilled water (DDW) to avoid precipitation and final volume is maintained with double distilled water followed by continuous shaking. Working media preparation was done from four deferent stock solutions by taking stock solution I, II, III and IV at 5%, 0.5%, 1% and 1% respectively from each of stock solution. For further use, the stock solutions were stored at 4 ºC in a refrigerator and used carefully to avoid any type of contamination. For the preparation of stock solution of plant growth regulators (PGRs), stock solutions were prepared separately by dissolving the PGRs salt in their relative solvents (NaOH, or alcohol) and maintaining the final volume with sterilised DDW followed by storage at 4 ºC in a refrigerator, and used carefully to avoid any type of contamination. The different concentrations of growth regulators were prepared from stock solutions using the formula.

S1V1 = S2V2

Where,

S1 = Known strength (stock solution)

V1 = Volume (stock solution required)

S2 = Strength (desired solution)

V2 = Volume (desired solution)

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Chapter – THREE MATERIALS AND METHODS

3.4.3 Plant growth regulators (PGRs), Polyamines and Metals On the basis of experimental design, basal media were augmented with different PGRs including cytokinins i.e. 6-benzyladenine (BA), kinetin (Kn), 2-isopentenyladenine (2-iP), meta-topolin (mT) or thidiazuron (TDZ) either singly or in combination with various auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) or 2,4-D (2,4-Dichlorophenoxyacetic acid) and polyamines (Putrescine, Spermine and Spermidine). As per experimental design, the supplementation of micronutrients like Copper sulphate (CuSO4), Zinc sulphate

(ZnSO4) and Nickel Chloride (NiCl2), and heavy metals such as Cadmium Chloride

(CdCl2) were also tried in supplementation with MS basal media. The combination and concentration of PGRs, polyamines, micronutrients and heavy metals was set as specified in the results.

3.4.4 Carbon source, pH and gelling agents of the medium The effect of different percentage (w/v) of Glucose, Fructose and Sucrose on morphogenesis were also considered with optimum combination of PGRs. The MS media augmented with 3% (w/v) sucrose as carbon source was used in all other experiments. NaOH or HCl (1N) was used for adjusting the pH of the media to 5.8 monitored using pH meter (L613, Elico Pvt. Ltd., India). The effects of various, pH levels (4.2, 5.2, 5.8, 6.4 and 7.0) on in vitro propagation were also tested. Agar (Thermo Scientific, India) was used as solidifying agent for media at 0.8 % (w/v), by dissolving it in a microwave oven.

3.5 Media vessel and plugging material About 20 ml molten media was dispensed in glass tubes (25 x 150 mm) and 50 ml in 100 ml wide mouth glass flasks and plugged with cotton plug. All the glass-wares used were heat resistant (Borosil, India). Cotton plugs were prepared with single layer cheese cloth replete with non-absorbent cotton.

3.6 Sterilization 3.6.1 Media sterilization All culture vials containing media were plugged tightly with cotton plugs and wrapped with butter paper and autoclaved at 121 ºC and 1.06 kg cm-3 temperature and pressure respectively for 18 min, carefully to avoid moisture at cotton plugs.

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Chapter – THREE MATERIALS AND METHODS

3.6.2 Sterilization of glass-wares and instruments All the glassware were wrapped in butter paper and sterilized by autoclaving. The instruments viz., forceps, scalpel etc. were wrapped in aluminium foil, sterilized by autoclaving for 20 min. The laminar air flow hood (NSW, Delhi) was sterilized for 20 min by ultraviolet light (UV tube, 30 W, Wipro, India) followed by wiping the working surface in the laminar with 70% ethanol before any operations inside the hood. Flame sterilization was carried out by dipping the stainless steel instruments into rectified sprit followed by their flaming and cooling.

3.7 Culture inoculation and incubation Inoculation was performed under aseptic environment in laminar cabinet. During inoculation or subculturing practices all the instruments were flame sterilized at regular intervals. The surface sterilized seeds or explants were placed/ excised on sterilized petridishes using sterilized forceps, inoculated in culture vessels containing culture media and plugged with cotton plugs. All the culture vessels were incubated in a growth room at 25 ± 2 ºC with a photoperiod of 16 h with a light intensity of 50 μmol m-2 s-1 provided by cool white fluorescent lamp (2x40 W, Philips, India) and 55-60% of relative humidity.

3.8 Rooting For root induction, microshoots (5 cm) with 4-5 leaves were harvested from in vitro grown culture and transferred to medium consisting of different strengths of MS medium (Full, 1/2, 1/3 and 1/4) with or without auxins (IAA, IBA and NAA) at different concentrations (0.05, 0.10, 0.20, 0.50 or 1.00 μM) with 3% w/v sucrose. Data were recorded on rooting percentage, root number and root length after 28 days of inoculation. Ex vitro rooting was accomplished by treating the basal end of the microshoots by dipping in different concentrations (30, 60, 90, 120, 150 or 180 μM) of various auxins for 30 min and cultured in thermocol cups containing Soilrite® (Keltech Energies Pvt. Ltd.) followed by acclimatization.

3.9 Hardening and acclimatization Healthy and well-developed plantlets were removed from the tubes, washed carefully with water and shifted to pots containing different potting materials viz., garden soil: vermicompost (3:1), Soilrite (Keltech Energies Ltd, Bangalore, India) and vermicompost. These pots were exposed to diffuse light with a 16 h photoperiod

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Chapter – THREE MATERIALS AND METHODS

(16/8 h). Potted plantlets were enclosed with transparent plastic bags to maintain high humidity and sprayed every 4th day for 12 days with ½ MS salts excluding vitamins and sucrose. Plastic Bags were removed after 14 days in order to adapt plantlets to natural environment. Well established potted plants were shifted to garden soil and ultimately transferred to greenhouse for their normal growth and development after 42 d of acclimatization.

3.10 Synthetic seeds production

3.10.1 Explant source Aseptic nodal segments (NS), (0.5-0.7 cm) from 56 days old cultures were used as explants source for encapsulation.

3.10.2 Encapsulation matrix Sodium alginate (Qualigens, India) with different strength (2, 3, 4 and 5% w/v) was added to liquid MS medium. For complexation 25, 50, 75, 100 or 200 mM CaCl2.2H2O solution was prepared using liquid MS medium. The pH of the solutions were adjusted to 5.8 before autoclaving at 121 ºC for 20 min.

3.10.3 Encapsulation, planting media and culture conditions Aseptic NS explants were mixed in freshly prepared sterilized sodium alginate solution. Encapsulation was accomplished by dropping sodium alginate droplets in calcium chloride solution. The droplets containing explants were kept for at least 20 min to achieve polymerization to form beads. The sodium alginate beads containing nodal segment were retrieved from the solution and rinsed twice with sterilized water to remove the traces of CaCl2.2H2O and transferred to sterile filter paper in petridishes for 5 min, and incubated on medium containing petridishes or tubes. The encapsulated nodal explants and shoot were transferred to wide mouth flask (Borosil, India) having MS basal medium or MS medium supplemented with different plant growth regulators as explained in result. The alginate beads were incubated under the culture condition as specified in culture inoculation and incubation.

3.10.4 Low temperature storage Synseeds (encapsulated nodal segments) were transferred to petridishes containing agar medium and stored in a laboratory refrigerator at 4 ᵒC. Five different low temperature exposure periods (14, 28, 42, 56 and 70 days) were evaluated for regeneration. After each storage period, encapsulated nodal segments were placed on MS medium with or

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Chapter – THREE MATERIALS AND METHODS without growth regulators for conversion into plantlets. The percentage of encapsulated nodal segments forming shoot were recorded after 56 days of culture on regeneration medium.

3.11 Physiological and biochemical studies of regenerated plantlets A set of tissue culture raised healthy plantlets were transplanted in sterile Soilrite and placed in culture room at 24 ± 2 ᵒC and 16/8 h photoperiod with 55-60% relative humidity under controlled conditions. Leaf samples were taken at transplantation day (day 0, control) and after 7, 14, 21, 28 days and stored in liquid nitrogen for biochemical analysis.

3.11.1 Pigment contents estimation The chlorophyll (Chl a, b and total Chl) and carotenoid (Car) contents of leaf sample were evaluated by the Mackinney (1941) and Maclachan and Zalick (1963) method respectively. 3.11.1.1 Procedure One gram of freshly harvested leaf sample was ground with the help of mortar and pestle in 5 ml freshly prepared 80% acetone solution. The homogenized samples were filtered with Whatman No.1 filter paper. The filtrate was collected in test tubes and volume was maintained upto 10 ml with 80% acetone. 3.11.1.2 Estimation Chlorophyll (Chl) content was determined by observing the change in OD (Optical density) at wavelengths 645 and 663 nm and for carotenoid 480 and 510 nm with the help of spectrophotometer (UV- Pharma spec 1700, Shimadzu, Japan). The Chl contents were expressed as milligram per gram of fresh weight (mg g-1 FW). The calculation for each Chl a/b, total Chl and carotenoid was done using the following formula;

12.7 (O.D. 663 nm) – 2.69 (O.D. 645 nm) Chlorophyll a = x V d × 1000 × W

22.9 (O.D. 645 nm) – 4.68 (O.D. 663 nm) Chlorophyll b = × V d × 1000 × W

7.6 (O.D. 480 nm) – 1.49 (O.D 510nm) Carotenoids = × V d × 1000 × W

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Chapter – THREE MATERIALS AND METHODS

Where,

O.D. = Optical density V = Final volume of extract W = weight of fresh leaf sample d = Length of light path

3.11.2 Estimation of antioxidant enzymes activities 3.11.2.1 Estimation of Superoxide dismutase (SOD) The method proposed by Dhindsa et al. (1981) with slight modifications was used to evaluate Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) activity. 3.11.2.1.1 Procedure Two grams of fresh leaf samples were harvested and homogenized in 2.0 ml of extraction mixture with the help of pre-cooled mortar and pestle. Homogenization was done under cold environmental conditions (4 ᵒC). The homogenate solution was collected and centrifuged at 12,000 rpm for 15 min. at 4 ᵒC. 3.11.2.1.2 Enzymes assay Two gram leaves were taken from 0, 7, 14, 21 and 28 days old micro-propagated plants, homogenized in 2.0 ml of freshly prepared extraction buffer with 1.0% PVP (polyvinylpyrrolidone), 1% Triton X-100 and 0.11 g of EDTA (Ethylene-diamine tetra-acetic acid) using pre chilled mortar and pestle. Homogenized samples were centrifuge for 15 min at 12000 rpm and the supernatant was used for enzyme assay. For SOD activity, freshly prepared 1.5 ml reaction buffer, 0.2 ml methionine, 0.1 ml enzyme extract with equal amount of 1 M Na2CO3, 2.25 mM NBT solution, 3 mM EDTA, 60 µM riboflavin and 1.0 ml of DDW was mixed and incubated in test tubes and kept under the light of 15 W fluorescent lamp for 10 min at room temperature. Blank A containing all the above substances of the reaction mixture, along with the enzyme extract, was placed in light for 10 min and then under dark conditions. Blank B containing all the above substances of reaction mixture except enzyme was placed in light along with the sample. The reaction was dismissed by covering the test tube with a dark colour or black cloth fallowed by dismisses the light source. With the help of spectrophotometer light absorbance of sample along with blank B was read at 560 nm against blank A. The non-radiated solution having enzyme extract did not indicate any sign of blue colour. Data were evaluated on the basis of difference of

Department of Botany, Aligarh Muslim University 39

Chapter – THREE MATERIALS AND METHODS percent reduction in the colour between blank B and sample. Reduction up to 50 % in colour was estimated as one unit of enzyme activity and the unit of activity was expressed as Enzyme Units (EU) mg-1 protein. 3.11.2.1.3 Reagents preparation  Sodium bicarbonate Solution (1 M) 15.9 g of sodium bicarbonate was dissolved in 100 ml DDW.  Methionine solution (200 mM) 2.98 g of methionine was dissolved in 100 ml DDW.  NBT (Nitroblue tetrazolium) solution (2.25 mM) 0.184 g of Nitroblue tetrazolium was dissolved in 100 ml DDW.  EDTA (3 mM) 1.116 mg EDTA was dissolved in 100 ml DDW.  Riboflavin (60 µM) 2.3 mg of riboflavin was dissolved in 100 ml of DDW. 3.11.2.1.4 Extraction buffer  Potassium phosphate buffer (0.5 M at pH 7.3)

It was prepared from 0.5 M phosphate buffer (pH 7.3). The solution of KH2PO4 and

K2HPO4 were first prepared as given bellow:

Solution A (KH2PO4)

3.40 g of KH2PO4 was dissolved in 50 ml DDW.

Solution B (K2HPO4)

8.70 g of K2HPO4 was dissolved in 100 ml DDW. However, the extraction buffer was prepared by mixing solution A and B in an appropriate ratio at pH 7.3 and 1 g of PVP (Polyvinyl pyrrolidone), 0.11 g of EDTA and 1 ml of Triton X-100 were added in 100 ml of the buffer. 3.11.2.1.5 Reaction buffer  Potassium phosphate buffer (0.1 M at pH 7.8) 0.1 M phosphate buffer at pH 7.8 was used as extraction buffer. The solutions of

KH2PO4 and K2HPO4 were prepared separately as given below: Solution A

1.3 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

1.70 g of K2HPO4 was dissolved in 100 ml DDW.

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Chapter – THREE MATERIALS AND METHODS

Both the Solution A and B were mixed in an appropriate ratio at pH 7.8 and 1 g of PVP (Polyvinyl pyrrolidone) was added to 100 ml of the buffer.

3.11.3 Estimation of Catalase (CAT) activity Catalase (CAT: EC 1.11.1.6) activity in leaves of the regenerated plantlets were determined by the method of Aebi (1984) with slight modification.

3.11.3.1 Procedure 5 g of fresh leaf samples of 0, 7, 14, 21 or 28 d old in vitro regenerated plantlets were homogenized using pre chilled mortar and pestle in 5 ml of extraction buffer. The supernatant was immediately used for enzyme assay.

3.11.3.2 Enzyme assay

Catalase activity was observed by determining the disappearance of H2O2, at 240 nm decrease in absorbance using spectrophometer. Reaction was carried in a final volume of 2 ml of reaction mixture containing reaction buffer with 0.1 ml of 3 mM EDTA, 0.1 ml of enzyme extract and 0.1 ml of 3 mM H2O2. The reaction was allowed to run for 5 min. Extinction Coefficient (ɛ) 0.036 mM-1 were used for calculation of activity with units (EU) mg-1 protein. One unit of enzyme regulates the amount necessary to decompose 1 µmol of H2O2 per min at room temperature.

3.11.3.3 Reagents Preparation  Potassium buffer (0.5 M at pH 7.3) Solution A

3.40 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

8.70 g of K2HPO4 was dissolved in 100 ml DDW. Both the Solution A and B were mixed in an appropriate ratio at pH 7.3 and 1 g of PVP (Polyvinyl pyrrolidone), 1.0 ml Triton X- 100 and 0.11 g of EDTA was added to 100 ml of the buffer.  Potassium phosphate buffer (0.5 M at pH 7.2) Solution A

3.40 g of KH2PO4 was dissolved in 50 ml DDW.

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Chapter – THREE MATERIALS AND METHODS

Solution B

8.70/4.35 g of KH2PO4 was dissolved in 100 ml DDW. Both the Solution A and B were mixed in an appropriate ratio at pH 7.2.  Potassium phosphate buffer (0.25 M at pH 7.0) Solution A

1.70 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

8.70 g of KH2PO4 was dissolved in 100 ml DDW. Both the Solution A and B were mixed in an appropriate ratio at pH 7.0.

 H2O2 (3 mM)

0.1 ml of H2O2 was mixed with 9.9 ml of DDW.  EDTA (3 mM) 1.116 mg EDTA was dissolved in 100 ml DDW.

3.11.4 Estimation of Glutathione-S-transferase (GST) This experiment was conducted to check the level of GST specific activity during in vitro culture practice of explants with increasing incubation period under the influence of ZnSO4, CuSO4, NiCl2 and CdCl2 treatments.

3.11.4.1 GST enzyme assay: GST (CDNB) Specific Activity was determined by method described by Habig et al. (1974), with slight modifications, based on following reaction principle; GST G-SH + CDNB G-SDNB Conjugate + HCl

3.11.4.1.1 Procedure One gram leaves were harvested from 3 and 6 weeks old cultures and homogenized in 100 mM phosphate buffer (pH 6.5) with 1.0 mM EDTA at 25 ºC followed by continuous shaking to avoid precipitation. The final volume of this extraction buffer was maintained upto 4 ml and centrifuged at 1000 rpm for 10 min. The supernatant was collected in a fresh tube. The enzymatic reaction containing 75 mM GSH (Glutathione) was prepared in cold phosphate buffer and 30 mM CDNB (1-chloro-2, 4-

Department of Botany, Aligarh Muslim University 42

Chapter – THREE MATERIALS AND METHODS dinitrobenzene, dissolved in 95% ethanol). The GST activity was initiated by the addition of 0.2 ml enzyme leaf extract, 0.1ml GSH, 0.1ml CDNB and 2ml buffer. The reaction was started by GSH conjugation and GST canalization in the mixture

3.11.4.1.2 Estimation The data was evaluated by monitoring increase in absorbance at 340 nm with the help of spectrophotometer (UV-1700 PharmaSpec) for 5 min at the interval of 30 sec. (extinction coefficient, 9.6 mM-1cm-1).

3.11.5 Estimation of carbonic anhydrase (CA) activity Carbonic anhydrase (CA) activity was evaluated following Dwivedi and Randhwa (1974) method with slight modifications. The reversible hydration of carbon dioxide

(CO2) was done to give the bicarbonate ion in the presence of CA catalyse enzyme;

Carbonic anhydrase + -3 H2O + CO2 H + HCO

3.11.5.1 Procedure Plant leaf samples were harvested from in vitro grown plantlet and cut into small pieces. 200 mg leaf pieces were taken and further cut into small pieces (2-3 mm length) in a sterilized petri-dish containing 10 ml 0.2 M cystein at 0 to 4 ºC. After being cut, the solution adhering at their surface was removed with the help of a blotting paper followed by transfer immediately to a test tube, having 4 ml phosphate buffer of pH 6.8.

To this, 3.4 ml 0.2 M sodium bicarbonate (NaHCO3) in 0.02 M sodium hydroxide (NaOH) solution and 0.2 ml 0.002% bromothymol blue indicator was added. After shaking, the tube was kept at 0 - 4 ºC for 20 min.

3.11.5.1.2 Preparation of Reagents  Bromothymol blue (0.002%) indicator in ethanol 0.002 g bromothymol blue was dissolved in 100 ml of ethanol.  Cystein solution (0.2 M) 48 g cystein was dissolved in 1000 ml DDW.  Hydrochloric acid (0.05 N) 4.3 ml pure hydrochloric acid was mixed with 995.7 ml DDW.  Phosphate buffer (0.2 M at pH 6.8)

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This was prepared by dissolving 27.8 g sodium dihydrogen ortho-phosphate and 53.65 g disodium hydrogen orthophosphate in sufficient DDW separately and final volume (1000 ml) was made up with DDW. To obtain pH 6.8, 5 ml of monobasic sodium phosphate solution was mixed with 49 ml of dibasic sodium phosphate solution and diluted to 200 ml with DDW.  Sodium bicarbonate (0.2 M) solution in 0.02 M sodium hydroxide solution 16.8g sodium bicarbonate was dissolved in aqueous sodium hydroxide solution (0.8 g NaOH/100) and final volume was made upto 1 litre with sodium hydroxide solution.

3.12 Molecular marker studies of in vitro raised plantlets

3.12.1 Sample source of DNA Micro-propagated plantlets were used for the isolation of genomic DNA. Young, fresh and green leaves were used for extraction of DNA.

3.12.2 Isolation of Genomic DNA Cetyltrimethylammonium Bromide (CTAB) method (Doyle and Doyle 1990) with minor modifications was used for isolation of genomic DNA from the collected leaves.

3.12.3 Preparation of reagents for DNA isolation  CTAB (10%) 10 g of CTAB (Sigma, USA) was dissolved in 100 ml of sterile DDW for preparation of 100 ml of stock solution. CTAB 2.5% was taken as working concentration.  0.5 M EDTA (pH 8.0) 14.6 g EDTA (Sigma, USA) was dissolved in 50 ml of DDW and the pH of solution was maintained 8.0 with 1N HCl. Final volume was made to 100 ml in DDW. 25 mM EDTA was used as working concentration.  1M Tris- HCl (pH 8.0) 15.76 g Tris-HCl (Sigma, USA) was dissolved in 80 ml of DDW. The pH was adjusted to 8.0 using 1N HCl (Qualigens, India). The final volume was made to 100 ml with DDW. 100 mM Tris was used as working concentration.

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 5M NaCl solution 29.22 g NaCl (Sigma, USA) was dissolved in minimum amount of distilled water and the final volume was made 100 ml using DDW. 1.5 M NaCl solution was taken as working concentration.  Others β-mercaptoethanol 0.2% (v/v) and PVP 1.0% (w/v) were taken before homogenisation.  Preparation of DNA Extraction Buffer For the preparation of 50 ml extraction buffer- Tris- HCl (100 mM) - 5.0 ml EDTA (25 mM) - 2.5 ml NaCl (1.5 M) - 15 ml CTAB (2.5%) – 12.5 ml Be pipette out and dissolved in 14.9 ml of milli Q grade water, add 1% PVP, mix well by inverting the tubes and then incubate the mixture at 65 ºC. Before using add 0.2% (100 µl) β-mercaptoethanol.  TE buffer For the preparation of 10 ml TE, 50 µl of 1M Tris-HCl and 0.5 M, 200 µl EDTA can be dissolving in 9.3 ml of water.

3.12.4 Extraction and purification protocol One gram of young, fresh leaves were taken from the 10 randomly selected micropropagated plants and were washed with tap water. Than these leaves were rinsed in DDW and dried with the help of bloating sheets. Properly dried leaves were grind with the help of mortar and pastle in liquid nitrogen to make fine powder. The powder transferred to 15 ml centrifuge tubes followed by addition of 3 ml DNA extraction buffer, β-mercaptoethanol 0.2% (v/v) and PVP 1.0% (w/v) were homogenized into a fine slurry and the tubes were incubated for 1h at 65 ºC in a water bath. After incubation, equal volume of Chloroform: Isoamylalcohol (24:1, v/v) was added in each of the tube and the samples were mixed properly by inversions till lower part of the tube become dark green. The above suspension was centrifuged at 12000 rpm for 10 min at 4 ºC.

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The aqueous phase was gently collected and transferred to eppendroff tube without disturbing the interphase. To the aqueous phase 170 µl, 5M Sodium chloride solution and double volume of pre-chilled isopropanol was added and the contents were mixed gently and incubated at -20 ºC for 45 min. The precipitated DNA was recovered by centrifugation at 10,000 rpm for 10 min at 4 ºC. The white pellet DNA at the bottom of eppendroff was washed with 500 μl of 70% ethanol and centrifuged at 5000 rpm for five min at 4 ºC. The supernatant was discarded and pellet was air-dried well to avoid excess alcohol. The DNA pellet was dissolved in 300 µl TE buffer and incubated at 4 ºC for 30 min.

3.12.4.1 Purification of genomic DNA 4 µl of RNAse was mixed to the dissolved pellet followed by gently tapping and incubated at 37 ᵒC for 45 min to 1 h. After incubation, 25 µl NaAc (Sodium acetate) mixed gently and 750 µl chilled ethanol was added and mixed properly by inverting the tube, at this stage DNA was precipitated again and incubated at -20 ᵒC for 30 min. The incubated mixture was centrifuged at 10,000 rpm for 10 min at 4 ᵒC. Pellet was retained and supernatant was discarded and 1 ml 70% alcohol was added followed by centrifugation at 10,000 rpm for 5 min. supernatant was decanted off and pellet was air dried and finally dissolved in 200 µl DDW.

3.12.5 DNA assessment (Qualitative and Quantitative) 3.12.5.1 Quality analysis The quality of the DNA was estimated by running the DNA in the agarose (1 %) gel- electrophoresis. 3.12.5.2 Quantification analysis Quantification of the DNA was done using nanophotometer (Implen).The ratio of absorbance at 260 nm (A260) and 280 nm (A280) was measured. The concentration of DNA was calculated on the basis of absorption at 260 nm and DNA purity was check from A260/ A280 ratio as given by Sambrook et al. (1989);

Absorbance ratio (A260/A280) Result Above 1.8 = Contamination of protein Between 1.7 to 1.8 = Best concentration of DNA Less than 1.7 = Contamination of RNA

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3.12.6 Solutions for agarose gel electrophoresis 3.15.6.1 TBE (5 X) 500 ml stock solutions Tris base (27 g), boric acid (13.7 g) and (10 ml) of 0.5 M EDTA were dissolved in 250 ml of DDW. The volume of solution was maintained to 500 ml with DDW. 3.12.6.2 Running Buffer (1X TBE) At the time of gel electrophoresis, 1X TBE buffer was obtained through dilution of 5 X TBE stock buffers with the help of DDW. 3.12.6.3 Gel Loading Dye (6X) To prepare 25 ml of gel loading dye (6X), 30 mg Bromophenol blue dye (Sigma, USA) was mixed with 9.36 ml of 80% Glycerol (Qualigens, India), 30 mg xylene cyanol (Merck) and 300 μl EDTA (0.5 M). The final volume was made with 15.34 ml sterile DDW and the solution was vortexes briefly and stored at room temperature. During loading of DNA gel loading buffer was diluted to 1X with addition of 1X TBE. 3.12.6.4 Gel Staining Dye Ethidium bromide (0.5 µg) was dissolved in 1 ml autoclaved DDW. Due to carcinogenic properties, the solutions were mixed carefully, stored at room temperature and used for staining the DNA gel at a working concentration of 0.5 µg/ml (Sambrook et al. 2001).

3.12.7 Agarose gel electrophoresis Isolated genomic DNA was electrophoresed on 1.0% agarose gel in 1X TBE buffer at 60V to 100V for two hours. To prepare gel, 1gm agarose was dissolve properly in 100 ml 1X TBE buffer to make clear solution followed by addition of 4 μl ethidium bromide at bearable temperature. The gel was allowed to solidify after pouring in gel casting tray. On getting solidification of gel, the stopper and comb was gently removed followed by addition of sufficient volume of 1X TBE buffer in gel running through gel and put gel casting tray in cathode to anode direction. For electrophoresis of genomic DNA sample, 20 μl of DNA sample was properly mixed in 4 μl DNA loading dye and total 24 μl mixture loaded onto each well carefully. The gel was run at a voltage of 50 V for 1 h and evaluates distance travel by DNA with the help of DNA loading dye and then observed on UV transilluminator. Photographs of gel were taken with the help of Gel Doc (Bio Rad, Hercules, USA).

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3.13 RAPD/ISSR primers screening and DNA amplification For screening of genetic variability/integrity, PCR based primer response of sample DNA was analysed, a set of 60 RAPD (OPA, OPB and OPC kit) and 13 ISSR (UBC, Vancouver, BC, Canada) primers were examined.

3.13.1 Reagents for PCR Amplification The sample DNA amplification for initial screening of markers response on single PCR reaction was carried out using various amplification reagents such as master mixture for PCR reaction containing 2.0 μl Taq Buffer (Thermo Scientific, India), 0.4 μl dNTPs

(Thermo Scientific, India), 1.2 μl MgCl2, 0.2 μl Taq DNA polymerase (Thermo Scientific, India), genomic DNA (1.0 μl), 1.0 μl RAPD/ISSR primer, 14.2 μl mQ water.

3.13.2 RAPD and ISSR - PCR based amplification of genomic DNA Amplifications were carried out by mixing the PCR reaction component in a 0.5 ml microfuge tube, mixture content were taken according to the number of PCR reaction required. Total 20 μl volume of reaction mixture was taken in consideration for each of the reaction. DNA amplification program were set according to PCR marker used in the reaction.

3.13.3 Electrophoresis of amplified PCR product The amplified DNA was mixed properly with 6X DNA loading dye and electrophoresed on agarose (1.0%) gel in 1X TAE buffer. 0.5μg/ml ethidium bromide solution was added in the gel. For 2.5 h, gel was run at the rate of 50 V/cm. Gel was visualized under UV light and photographed using gel documenting system (BioRad, Hercules, USA).

3.13.4 Analysis of DNA amplification The bands were counted either as present (1) or absent (0) for each of the RAPD and ISSR markers for 10 plants in comparison with banding pattern. Clear and well-resolved fragments ranging from 100-10,000 bp were taken in consideration for analysis. DNA ladder marker (10 kb in size) were loaded side by side in well of the gel at the time of sample loading and the size of the amplified products were measured in comparison with distance travelled.

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3.14 Chemicals and Glasswares

Most of the chemicals likes (EDTA, PVP, Triton X-100, NBT, H2O2, methionine, TCA, NADH), vitamins (Thiamine HCl, Pyridoxine HCl, Nicotinic acid, Myo-inositol and Glycine) and Plant growth regulators (BA, Kin, 2iP, TDZ, IAA, IBA, NAA) etc. were obtained from Sigma Aldrich Pvt. Ltd., New Delhi, India and/or from Sigma-Aldrich (St. Louis Mo, USA). Other major and minor salts, buffer components were procured from Qualigens, MERCK and/ or SRL. All chemicals used were of analytical grade. Glassware’s, such as, test tube (25 x 150 mm) petri-dishes (17 x 100 mm), wide mouth flasks (100 ml and 250 ml) used during the experiment were procured from Borosil, India.

3.15 Statistical analysis The data on evaluated parameters were exposed to Analysis of Variance (ANOVA) by SPSS version 16 (SPSS Inc., Chicago, USA), and Microsoft Office-Excel 2007 (Microsoft Corp. USA). The significant difference among means was marked out using Duncan’s multiple range test (DMRT) at P = 0.05. The outcomes were shown as means ± standard errors (SE) of 3 repeated experiments, each consisting of 10 replicates per treatment.

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INTRODUCTION

The pyramid of life stands on the strong shoulders of the producers, mainly classified as plants. Since the very beginning of time, humans have exploited plants for getting food, fuel, fibre and medicines. These plants grow in extremely diverse habitat from the cold icy Himalayas to hot burning Sahara, from ocean beds to top of the tallest mountains. This is only possible because of the diversity that exists in life, known as „Biodiversity‟. Biodiversity means the variability among living organisms from all sources. This variability is the genetic wealth of nature and India being of the richest places has earned a place in the 13 mega-biodiversity centers of the world biodiversity. India harbours about 8% of global bio-diversity within only 2.4% of world‟s area. Presence of enormous numbers of plant species, India has earned the title of “the world herbal garden”. With over 45,000 species of herbal medicinal plant spread over hotspots in the Western Ghats, Eastern Himalayas and the Andaman and Nicobar Islands (Anonymous 2003). A significant proportion of these plant species are employed for medicinal purposes both in modern and traditional system of medicine. From the beginning of civilization medicinal plants have been used with great interest as they offer a valuable renewable resource for the numerous drugs and chemicals. In Ayurveda and Unani system of medicine, we find an exhaustive account of medicinal plants, their properties and their practices for supporting and improving different body functions. Nowadays, as compared to synthetic drugs, health care system is dominated by herbal Medicare as it is considered safer. Recently, plant based products such as dietary supplements and drug manufacturing at industrial level have been improved greatly. Diversity of medicinal plants represents major natural wealth of our country. They provide health care facilities to common people and are helpful in economic growth of the country through export of medicinal plants (Bhat and Lone 2017, Van and Prinsloo 2018). A large member of medicinal plant species are at a high risk of extinction because of continuous collection and over exploitation from the wild for commercial uses. We are losing about one potentially vital medicinal plant every two years at present. The loss of possible sources of vitamins, protein rich foods, principal crop and bioactive substrates also represents extinction of plant species from natural habitat. Continuous loss of medicinal plants produce huge fears about income, health care and living safety of the societies in Chapter – one INTRODUCTION developing and under developed countries. Depletion occurs due to destructive, unsustainable and continuously harvesting of such potent medicinal plants resulting in the depletion of genetic resource from natural habitat which may also leads to the serious global shortage of these products (Hamilton 2004, Negi et al. 2018). In India, medicinal plant sector have a significant influence on the socio-cultural and medicinal and economy situation of tribal and rural population. Thus, Indian Government has established a 'Department of Indian system of Medicine and Homeopathy', in addition to 'Medicinal Plant Board' which promotes, regulate and develop the sector for conservation and sustainable consumption. Government and other private agencies have recognized protection of pharmaceutical plants as one of their main goal and have started their conservation programs in the threatened zones. The main aim of conservation programs is the appropriate use of natural resources, encouraging sustainable progress without disturbing the natural species. Sustainable use of pharmaceutical plants includes collection, propagation, evaluation, characterization, disease indexing, exclusion, storage and distribution of medicinal plants by certified organizations (Sharma, 2003, Sarkar et al. 2015). Conservation at ex situ and in situ is two imperative approaches used for management of plant genomic resources. In situ conservation delivers constant protection at natural place. While ex situ conservation deals with conservation away from the native environment and is used to defend the threat of replacement, reduction and damage of exiting habitat (Sharma 2003). Ex situ conservation refers to conservation in botanical gardens, gene bank, pollen and seed banks and in vitro conservation. In vitro propagation and conservation emphasizes on deliberate development and cryopreservation, mainly deals with vegetative propagated and recalcitrant seed plants. Germplasms are reserved as sterilized plantlet on a sterile nutrient medium for short term storage, in slow growth procedures, while in cryopreservation, plant materials are stored in liquid nitrogen (Kasagana and Karumuri 2011). Conservation of germplasm in field condition or as live field gene banks presents various difficulties mainly requirement for vast space, involvements of labour, viability testing and data documentation while much less space is required for in vitro storage. Only minor space is required for conserving the genotype in culture rooms which are sterile and disease free, which removes requirement for long and lengthy quarantine processes at the time of germplasm exchange (Kasagana and Karumuri 2011, Sen and Chakraborty 2017). Moreover, Plant-biotechnology also provides tools and techniques

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Chapter – one INTRODUCTION which are effective in producing novel genetic variability and assortment practice, more precise and reproducible than conventional methods. Plant tissue culture is a key tool of plant biotechnology which exploits the totipotent nature of plant cells, a concept proposed by Haberlandt (1902). It is the science of rising plant cells, tissues or organs isolated from the mother plant on artificial media under aseptic conditions. Plant cell, tissue culture plays a key role in germplasm conservation, rapid clonal multiplication and regeneration of genetically manipulated elite clones, secondary metabolites production and ex vitro conservation of elite and rare valuable plants (Kumar et al. 2018). This technique is not only being used to study different problems in the field of plant physiology, cell biology and genetics but also in agriculture, forestry and horticulture (Padikasan et al. 2018). Micropropagation is an important application of plant tissue culture used for large scale production of elite clones. The conventional method of propagation is time taking, costly and labour intensive. To overcome these difficulties, the micropropagation techniques are being used to produces comparatively more rapidly with reduced cost and labour. Micropropagation is the first important and broadly accepted technique easily applicable and currently has gained the status of a multibillion dollar trade at industry level being practiced in hundreds of biotechnology laboratories and nurseries throughout the world. In vitro mass propagation of medicinal plants has facilitated the breeders in variety of ways; 1. To enhance the proliferation of plants, permits rapid production of threatened plants and species that show poor seed viability. Produces uniform plants of a desired genotype because micropropagated plants are genetically homogeneous. 2. In vitro culture produces disease/virus free plants through shoot tip culture and can also produce plants resistant to insects, diseases and herbicides. 3. Since all the culture conditions required for a plant development including light, temperature and humidity are optimized at control level during the in vitro production, plants are independent of season and other environmental conditions required. 4. A large number of elite plants can be maintained in a small space and for a longer time under controlled culture conditions and proper management. However, micropropagation has certain limitation as well, foremost being higher cost of plant production. Therefore, to overcome this limitation, a number of cost effective approaches have been employed such as, shake culture utilizing liquid culture

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Chapter – one INTRODUCTION medium alone or in combination of semi-solid medium and use of Bioreactors. Along with the reduction of cost during plant propagation by tissue culture and reduce the demanding labour, there is an urgent need to developing scale-up systems and automation (Aitken-Christie 1991, Datta et al. 2017). Commercial exploitation of medicinal plants has resulted in reduction of population of numerous species in their natural habitat. It warrants efforts to conserve these plants in wild and their cultivation for sustainably supply to the industry and reintroduction in threatened areas. Conservation of medicinally and pharmaceutically important threatened plants species through in vitro propagation has been successful for several species (Engelmann 2004). Production of secondary metabolite through plant tissue culture holds several advantages over conventional methods, including simplicity, reliability, predictability and independence from seasonal variations. As compared to extraction from whole plants, isolation of the phytochemicals in cell culture may be more efficient and rapid, and can be controlled for over production of desire compounds. In vitro culture also offers an opportunity for the analysis of metabolic and biochemical pathways involved in production of such compound (Silpa et al. 2018, Atanasov et al. 2015). An important aspect of micropropagation is the genetic uniformity of regenerated plants. For ascertaining the genetic integrity of the regenerants, various molecular markers capable of detecting DNA variation among closely related individuals are employed. Different genetic markers used for detecting genetic stability/variability in plant tissue culture raised plants include Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR), Inter Simple Sequence Repeat (ISSR) and Sequenced Characterized Amplified Region (SCAR) have been used in micropropagated plants. In comparison to AFLP and RFLP, RAPD and ISSR have been used most frequently, because of their simplicity and cost- effectiveness. Genetic stability of micropropagated plants of Gentiana kurroo maintained in vitro for more than 10 years was studied using RAPD markers. RAPD and ISSR markers have been recently used successfully to assess genetic stability among in vitro regenerated plants of many plant species like Ruta graveolens (Faisal et al. 2018a), Lawsonia inermis (Moharana et al. 2018), Ansellia africana (Bhattacharyya et al. 2018), Clitoria ternatea ( Rency et al. 2018), Rauvolfia tetraphylla (Hussain et al. 2018a), Croomia japonica (Jiang et al. 2018), Sesamum indicum (Anandan et al. 2018),

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Chapter – one INTRODUCTION

Brahmi (Faisal et al. 2018b), Cucumis melo (Raji et al. 2018), Digitalis purpurea (Perez-Alonso et al. 2018). In the present investigation, an important medicinal plant species namely, Mucuna pruriens L. has been selected to develop an in vitro regeneration system and to evaluate the effect of heavy metals on the regeneration efficiency of the plant.

1.1 Plant Description

1.1.1 Scientific classification Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Fabales Family: Fabaceae Genus: Mucuna Species: pruriens

1.1.2 Scientific name: Mucuna pruriens L.

1.1.3 English name: Velvet bean, Cowitch, Cowhage

1.1.4 Common name: Kiwanch or Konch

1.1.5 Propagation: Seed

1.1.6 Status: Wild or Cultivated

1.2 Habitat Mucuna pruriens (velvet beans) belonging to Fabaceae family, is a highly valuable climbing legume which consist of about 150 annual or perennial species, some being endemic to India or other tropical regions, found in bushes and hedges at damp places, ravines and scrap jungles throughout the plains of India, Africa and the West Indies (Singh et al. 1996, Anonymous 2003).

1.3 Botanical description M. pruriens is an annual or perennial climbing legume with long vines, plants attain about 10-15 m height having a tap root with numerous lateral root systems. The plant stem body is cylindrical with branches having alternate, trifoliate with rhomboid ovate, slightly pubescent 5-15 cm long with 3-12 cm broad leaflets. Young leaves of plant

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Chapter – one INTRODUCTION have hairs on both the surfaces. Inflorescence is raceme with dark purple color flowers. The axis of raceme is upto 30 cm long with two to many flowers. The axis of flower strand attain a length of upto 5 mm, the bell is long (8-9 mm) and silky. The sepals are generally long. The crown color of flowers is white to purple with 1.5 mm long flag. Fruits are ribbed pod leguminous upto 4-13 cm long and 1-2 cm wide, un-winged, densely covered with persistent pale brown or grey trichomes that cause irritating and blisters, known as husk pods, with 6-7 seeds. Seeds are flattened uniform ellipsoid, white or black in color, and 1-2 cm long, 0.9-1.4 cm wide with 4-6 cm in thickness (Verma et al. 1993, Kavitha and Thangamani 2014).

1.4 Phytochemistry Almost all the plant‟s parts are used for production of medicinal compounds, in which L-dopa (a precursor of dopamine) is one of its main active constituent. Its highest percentage is present in seed (7-10%), while lowest in leaves (1%). It is used in Parkinson‟s disease treatment (Bell and Janzen 1971, Morris and Wang 2018). L-DOPA (3, 4- dihydroxyphenyl-L-alanine) is a non-protein amino acid obtained from its seeds, seeds also contains glutathione, gallic acid and beta-sitosterol. Seeds also possess un- identified tryptamine bases such as mucunadine, mucunine, prurienine and prurieninine (Majumdar and Santra 1953, Kapoor 2017). Chemical compounds like indole-3- alkylamines-N, N-dimethyltryptamine, are obtained from the plant parts, while compound Serotonin is only present in seed pods (Khare 2004, Liu et al. 2016, Upadhyay 2017). The seeds also contain oils including palmitic acid, oleic acid, linoleic acids, nicotinic acid, lecithine, n-hexadecanoic acid, ascorbic acid, squalene and octadecanoic acid (Singh et al. 1996, Upadhyay 2017 ). Apart from these, seed also contains 5-hydroxytryptophan, bufotenine, 5-hydroxytryptamine, 6-methoxyharman, tryptamine, palmitic acid, stearic acid, 3-methoxy-1,1-dimethyl-6,7-dihydroxy-1,2,3,4- tetrahydroquinoline, Indole-3-alkylamines-N, N-dimethyltryptamine, 3-methoxy-1, 1- dimethyl-7,8-dihydroxy-1,2,3.4-tetrahydroquinoline (Liu et al. 2016, Upadhyay 2017).

1.5 Importance The need for L-Dopa is largely met by the pharmaceutical industry through extraction of the compound from wild populations. All parts of plant possess valuable medicinal properties as listed below.

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1.5.1 Antivenom and antidiabatic Protein extracts from seeds of M. pruriens have been shown to be active against snake bites (Tan et al. 2009) and seeds are also used as antidiabetic (Shanmugavel and Krishnamoorthy 2018).

1.5.2 Antioxidant activity

All plant parts are rich in phenolic compound which exhibits significant antioxidant and free radical scavenging capacity (Rajeshwar et al. 2005, Neta et al. 2018).

1.5.3 Neuroprotective activity Mucuna, has a huge global demand for one of its active constituent, L-dopa (a precursor of dopamine), used in the treatment of Parkinson‟s disease (Manyam et al. 2004, Cilia et al. 2017, Cilia et al. 2018).

1.5.4 As a dietary substance Velvet beans are a rich source of carbohydrates and protein and are used as instant energy food to increase muscle mass. At present its demand as a protein supplement for muscle gain as well as a supplementary nutritional has increased many fold (Musthafa et al. 2018, Briguglio et al. 2018).

1.5.5 As an aphrodisiac M. pruriens is commonly used, as a powerful aphrodisiac to increase the sperm count and to increase testosterone levels in the body (Jadhao 2013, Singh et al. 2017c).

1.5.6 Others uses Leaves are harvested as fodder and used as green cover crop. The species is well known for its nematicidal effects and antimicrobial activity when used in rotation with a number of commercial crops (Anonymous 2003).

1.6 Rationale Foundation of Revitalization of local Health traditions (FRLHT) working under the Ministry of Environment and Forest and the ministry of health, Government of India, identified 178 medicinal plant species with high trade potential. M. pruriens found its place among these plants with an estimated trade of annual 1000 metric tons per year. However, the wild population of the species have been decreasing at an alarming rate, due to unsustainable harvesting from the wild and is likely to become endangered. Demand for L-Dopa is largely met by the pharmaceutical industries through extraction

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Chapter – one INTRODUCTION of the compound from wild population. The second reason of its low population is its annual habit; short life span and propagation only through seeds having highly allergic properties causing uncontrolled itching. Apart from these, Mucuna, has a huge global demand for its active constituent. Thus, conventional propagation through seed is not an adequate solution to meet the demand. Therefore, there is an urgent need to use alternative techniques for conservation and sustainable utilization. Now-a-days, micropropagation system is mainly used for the bulk production of planting stock material for further increase in biomass manufacture. Thus, considering the immense possibilities offered by the application of tissue culture techniques and current status of M. pruriens, investigations have been conducted with the following objectives: 1. To establish aseptic cultures and formulate culture condition for in vitro regeneration and proliferation from cotyledonary node and nodal segment explants. 2. To evaluate the optimal medium for rooting in regenerated microshoots and to standardize the hardening and acclimatization procedure. 3. To optimize the technique for synthetic seed production and their conversion into plantlets. 4. To study the physiological and biochemical parameter in regenerants during acclimatization. 5. To establish the genetic fidelity of regenerated plantlets using molecular markers.

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REVIEW OF LITERATURE

Invention of microscope with its application to study the microparticles led to the liberated and practically synchronized progress of the cell theory proposed by Mathlias Jacob Schleiden (1838) and Theodar Schwann (1839), who visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plant termed as totipotency. This was revived and popularized by Virchow in 1858 with his famous aphorism “Omnis Cellula e cellula” on callus induction and on the limitation of plant segments including plant cell to organ and ultimately to form complete plant. On the basis of totipotential principal, the first theoretical and experimental concept of plant tissue culture of single cells was proposed by Gottlieb Haberlandt in his address to the German Academy of Science in 1902 (Haberlandt 1902). He tried to culture isolated mesophyll cells from mature leaf of Lamium purpureum, pith cells from petiole of Eicchornia crassipes, epidermal cells of Ornithogalum, glandular hairy cell of Pulmonari and stamen cells of Tradescantia using Knop's (1865) nutrient solution containing 1-5% sucrose, aspargine and peptone. While, he failed to obtain desire results but clearly established the concept of totipotency by forecasting that one could successfully develop artificial embryos from vegetative cells. The technique of culturing from isolated plant cells in nutrient solution permits the investigation of important breakthrough from a new experimental approach. However, he failed several times because of the poor choice of experimental materials, inadequate nutrient medium, and contamination (Vasil and Vasil 1972). The lack of availability of a nutrient solution that could support the growth of plant cells and tissues hindered further progress. However, he confidently predicted that it should be possible to generate artificial embryos or somatic embryos from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells and were further proved by many authors (Atree and Fowke 1991, Shoyama et al. 1997, Traore et al. 2003, Fki et al. 2011, Mazri et al. 2017). Embryo culture also had its beginning early in the nineteenth century, when Hannig (1904) successfully cultured embryos on mineral salts and sugar solution from nearly mature embryonic tissue of several species of crucifers. Whereas, Simon (1908), regenerated callus, buds and roots from Poplar stem segments and established the concept of callus culture. For about next 30 years Chapter – TWO REVIEW OF LITERATURE

(upto 1934), further there was very little progress in cell culture research. Within this period, discovery of an important vitamin B and natural auxins gave new approach to tissue culture. Initial progress in the culture of plant tissues came from the work of Molliard (1921) in France, Kotte (1922a, 1922b) in Germany, and Robbins (1922a, 1922b) in the United States, who successfully cultured fragments of embryos and excised roots for brief periods of time. In 1926, Fritz Went discovered first plant growth regulator (PGR) namely indol acetic acid (IAA) (Went 1928). It is a naturally occurring member of a class of PGRs termed „auxins‟. Furthermore, White (1934) successfully grew the root tips of tomato (Lycopersicum esculentum) continuously for a prolonged period in liquid medium containing inorganic salts, yeast extract and sucrose. White (1937) replaced yeast extract by vitamin B namely pyridoxine, thiamine and proved their growth promoting effect. Roger J Gautheret (1934) reported the successful culture of cambium cells of several tree species to produce callus and addition of auxin enhanced the proliferation of his cambial cultures. Further research by Nobecourt (1937), who could successfully grow continuous callus cultures of carrot slices, and White (1939) obtained similar results from tumour tissues of hybrid Nicotiana glauca x N langsdorffii. Thus, the possibility of cultivating plant tissues for an unlimited period was independently endorsed (Gautheret 1934). Stem tip cultures yielded success when Ernest Ball (1946) devised a method to identify the exact part of shoot meristem that give rise to whole plant. After 1950, there was an immense advancement in knowledge of effect of PGRs on plant development. Skoog and Tsui (1948) demonstrated induction of cell division and bud formation in tobacco by kinetin. This led to further investigations by Skoog and Miller (1957), isolated „kinetin‟- a derivative of adenine (6- furyl aminopurine). Kinetin and many such other compounds which show bud promoting activities are collectively called cytokinins-a cell division promoter in cells of highly mature and differentiated tissues. Skoog and Miller (1957) worked further to propose the concept of hormonal control on organ formation. Their experiment on tobacco pith cultures showed that high concentration of auxin promoted rooting were higher levels of kinetin induced shoot formation. However, now the concept is altered to multiple factors like source of explant, environmental factors, composition of media, polarity, growth regulators being responsible for determination of organogenesis (Shasmita et al. 2018, Zafar et al. 2019). Besides of PGRs, scientists tried to improve culture media by differing essentially in

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Chapter – TWO REVIEW OF LITERATURE mineral content. In this direction, Murashige and Skoog (1962) formulated a medium by increasing the concentration of salts twenty-five times higher than Knops. Media, which enhanced the growth of tobacco tissues by five times. Even today MS medium has immense commercial applications in tissue culture. Having achieved success and expertise in growth of callus cultures from explants under in vitro conditions, focus was then shifted to preparation of single cell (Anandan et al. 2018). Bergmann (1960) developed a technique for cloning of single cells by filtering suspension cultures. This technique called Plating technique is widely used for cloning isolated single protoplasts. Next step for realization of Haberlandt‟s objectives was the development of whole plant from the proliferated tissue of these cells. Thereafter, Vasil and Hilderbrandt (1965) were first to regenerate plantlets from colonies of isolated cells of hybrid Nicotiana glutinosa x N tabacum. In 1958, the classical work of Steward and his co-workers on induction of somatic embryos from free cells in carrot suspension cultures brought an important breakthrough by finally demonstrating totipotency of somatic cells, thereby validating the ideas of Haberlandt (Steward et al. 1958). This ability of regenerating plants from somatic tissue through normal developmental process had great applications in both plant propagation and also genetic engineering. For instance, micropropagation technique can be utilized for raising thousands of plants from single explant. Morel utilized this application for rapid propagation of orchids. He was also the first scientist to free the orchid and Dahlia plants from virus by cultivating shoot meristem of infected plants (Morel 1952). The role of tissue culture techniques in plant genetic engineering was first exemplified by Guha and Maheshwari (1966). They developed a technique of test tube fertlization which involved growing of excised ovules and pollen grains in the same medium thus overcoming the incompatibility barriers at sexual level. In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos which developed into haploid plants initiating androgenesis. This discovery received a significant attention since plants recovered from doubled haploid cells are homozygous and express all recessive genes thus making them ideal for pure breeding lines. Furthermore, breakthrough in the application of tissue culture came with isolation and regeneration of protoplasts, first demonstrated by Prof. Edward C. Cocking in 1960. Plant protoplasts are naked cells from which cell wall has been removed. Cocking produced large quantities of protoplasts by using cell wall degrading enzymes. After success in regeneration of protoplasts, Carlson et al. (1972) isolated protoplasts from Nicotiana

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Chapter – TWO REVIEW OF LITERATURE glauca x N. langsdorfii and fused them to produce first somatic hybrid. Since then many divergent somatic hybrids have been produced. The most significant breakthrough in the field of plant tissue culture was the development of defined culture medium which was originally devised for the rapid growth and bioassay with tobacco callus (Murashige and Skoog 1962). This was recognized as the most effective and commonly used medium in the area of plant tissue culture. Toshio Murashige, University of California, developed the concept of developmental stages of micopropagation leading to plantlets establishment (Murashige 1974).

2.1 Micropropagation Aseptically grown culture of cells, tissues, organs or whole plants under controlled nutritional and environmental conditions were considered as micropropagation (Thorpe. 2007, Chawla 2018). One of the most incredible discoveries on organogenesis was the role of auxin and cytokinin in determining the fate of plant cells (Skoog and Miller 1957). This pioneering research laid the foundation of the science of organogenesis and culminated into numerous tissue culture systems. Micropropagation is a method for rapid vegetative propagation of plants using very small starting material (explants) under in vitro condition. It is one of the most widely used applications of plant tissue culture. It can serve as a source for important plant species, independent of seasonal variations, with enhanced storage ability and convenient transportability using less energy and space as compared to the conventional ways. It can be accomplished either through the multiplication of shoots from axillary buds or by the formation of adventitious shoots/ somatic embryos via direct or indirect organogenesis. Murashige proposed three different stages of micropropagation techniques; Stage 1: Establishment of axenic cultures Stage 2: Multiplication and proliferation of propagules Stage 3: Re-establishment in natural environment (rooting and transplantation to soil) Deberg and Maene (1981) introduced zero Stage: the care and preparation of stock plant. This stage is considered as important or even indispensable stage in the development of a reliable and repeated tissue culture scheme. Murashige‟s Stage 3 is further divided into 2 sub stages namely, Stage 3a: rooting in regenerated shootlets and stage 3b: acclimatization of plantlets- a crucial stage as the plants gradually acclimatize to lower relative humidity.

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2.2 In Vitro Morphogenesis and Propagation

2.2.1 Explant type Plant tissue culture has played a key role to achieve the goal of micropropagation at large-scale production of genetically uniform plants. The role of explants in achieving direct or indirect morphogenic response was important in determining the success of any micropropagation protocol (Shasmita et al. 2018). Explant is an important independent living entity having all the genetic information that of explant donor plants. Murashige (1974) acknowledged numerous aspects that should be considered in explant collection including source of explant, (origin and nature of the explant), types of explant (shoot apices, hypocotyls, epicotyls, cotyledons, cotyledonary nodes, leaf and stem segments etc.), physiological status , age of organ, the season in which the explant is obtained, the size of explant, quality of the donor plant, the position of the explant on the donor or stock plant, orientation of the explant on the medium and the inoculation density (Bajaj and Gosal 1981). Adventitious buds could be regenerated from different source organs in medicinal herbs such as terminal or lateral buds, bulbils, immature leaf blades, petioles, stem segments and root tips (Gau et al. 1993, Mukhtar et al. 2012). The effect of the nature, type of the explant and culture practices type on establishing and consequent plant regeneration systems through tissue culture has been very well documented recently (Fatima et al. 2015, Shasmita et al. 2018), The physiological nature of an explant is affected by the age of the mother plant which has a direct bearing on the regeneration capability of the explant (Bonga 2017).

2.2.2 Size of the explant The explants with different sizes have been used for micropropagation. The size of the explants determines the future of explant proliferation with great significance on survival frequency of culture (Shen and Hsu 2018). Several scientific reports suggested that smaller explants are most suitable for in vitro propagation because it has minimum chance of infection/contamination (Strosse et al. 2008). In case of meristem tip culture, the size of the explant (0.2-0.5 mm) plays a vital role. However, larger explants are also sometimes used with an advantage: they may consist of a larger part of the shoot apex or stem segments bearing one or more lateral buds; sometimes shoots from other in vitro cultures are employed. The impact of type and size of the explant on in vitro propagation has been significantly enhanced the regeneration (George 2008b). Nodal segments containing axillary buds have quiescent or active meristems depending upon

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Chapter – TWO REVIEW OF LITERATURE the physiological stage of the explant. These buds possess the potential to develop into complete plantlets. In vitro regeneration of various medicinal plants through nodal explants has been discussed by different researchers (Shekhawat et al. 2015a, Rameshkumar et al. 2017). Cotyledonary node explants have also been successfully used for in vitro regeneration of medicinal plants (Venkatachalam et al. 2017, Moharana et al. 2017, Anis and Ahmad 2016).

2.3 Plant growth regulators (PGRs) Growth regulators are those substances that are capable to modifying growth and morphogenesis of plants with exerting large effects in plants in terms of gene expression, growth, and development even though they occur in relatively low concentrations (Davies 2010, Rademacher 2015, Ahmad and Anis 2019). These compounds are naturally synthesized within plant tissues (i.e. endogenously), have a regulatory, rather than a nutritional role in growth and development (Coggins et al. 2014). Augmentation of PGRs to the medium is known to greatly influence the growth and development of plants to be cultured (Faisal et al. 2018a and 2018b, Ahmad et al. 2018a). In tissue culture system, PGRs stimulate the growth of various cultures such as plant cells, tissue, non-zygotic embryo or somatic embryo, callus formation, axillary shoot bud induction and proliferation, along with formation of adventitious roots (Elias et al. 2015, Singh et al. 2016). PGRs are frequently needed to be altered according to the type and stage of culture practice but the type, concentration, and duration of exposure to PGRs typically have the most profound effect (Zafar et al. 2019). PGRs are generally active at very low concentrations in plant tissue culture medium and are used singly or augmented with one or more PGRs as per experimental design (Javed et al. 2017b, Naz et al. 2018). However, the requirement for PGRs varies significantly and it is supposed that it depends on endogenous level in the plants which vary with the type, tissue and the growth phase of the plants and also with the mode of organogenesis, embryogenesis or rhizogenesis (Cosic et al. 2015, Yancheva et al. 2018). The choice of PGR to be used is predicated on what outcome is desired, the sensitivity of the target tissue, the rates of synthesis and degradation within the explant, interactions with endogenous growth substances, and influence of the culture environment. The plant growth regulators commonly used in micropropagation system are, the cytokinins (meta-topolin, BA, Kn or 2-iP), auxins (IAA, IBA, NAA or 2,4-D) and polyamines (putrescine, spermidine or spermin).

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2.3.1 Effect of Cytokinins Cytokinins are plant growth regulators, derivatives of adenine and phenylurea, discovered for their ability to promote cell growth and cell division followed by cell differentiation, and also affect apical dominance, axillary bud growth and leaf senescence (Muller et al. 2015, Martín-Fontecha et al. 2018, Ahmad and Anis 2018b). It showed a regulatory effect on different plant growth parameters and developmental aspects including physiological and biochemical process like nutrient metabolism and mobilization, morphological differentiation and also secondary metabolites production (Murtaza et al. 2016, Brutting et al. 2017). In plant tissue culture system, they are used to alleviate apical dormancy allowing shoot proliferation, and to stimulate cell division often in concert with auxins (Anis and Ahmad 2016, Oliveira et al. 2018, Fatima et al. 2015). Type and concentration of cytokinin, to be used greatly influence the uptake by the explant towards morphogenic response along with transport, metabolism and interaction with the endogenous concentration of PGRs (Javed and Anis 2015, Grzyb et al. 2017). Added to shoot culture media, these compounds overcome apical dominance and release lateral buds from dormancy (Rameau et al. 2015, Noriega and Perez 2017). The influence of PGRs and their interaction in micropropagation of different plant species has been very well described by several plants such as Cassia sp (Anis et al. 2012), Abrus precatorius (Perveen et al. 2013b), Balanites aegyptiaca (Varshney and Anis 2014), Eucomis autumnalis (Masondo et al. 2015), Anoectochilus roxburghii (Zhang et al. 2015b), Lycopodiella inundata (Bienaime et al. 2015), Withania somnifera (Fatima et al. 2015), Cassia alata (Ahmed et al. 2017), Althaea officinalis (Naz et al. 2018). In in vitro tissue culture system most commonly used cytokinins are BAP (6- benzylaminopurine), mT (meta-topolin), Kinetin (6-Furfuryl-aminopurine), 2-iP (isopentenyladenine), Zeatin and Phenylurea based TDZ (thidiazuron) in which BAP (a synthetic cytokinin) is most frequently used (Goyal et al. 2015, Kumar et al. 2017). A large number of scientific reports subjected that cytokinins were found to be the most influencing growth regulators for multiple shoot induction in various medicinal plant species such as Exacum bicolor (Ashwini et al. 2015), Dendrobium thyrsiflorum (Bhattacharyya et al. 2015), Anoectochilus roxburghii (Zhang et al. 2015a), Canscora decussata (Kousalya and Bhai et al. 2016), Rumex nepalensis (Bhattacharyya et al. 2017b), Baptisia australis (Padmanabhan et al 2017), Cunila menthoides (Oliveira et al. 2018), Ajuga bracteosa (Ali et al. 2018), Ocimum basilicum (Monfort et al 2018), Portulaca oleracea (Sedaghati et al. 2019), Hedychium coronarium (Behera et al.

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2018b), Rauvolfia serpentina (Zafar et al. 2019), Prunus cerasifera (Nasri et al. 2019). Faisal et al. 2006a and 2006b established an efficient method for direct plant regeneration in M. pruriens by culturing CN and Nodal explants in MS medium containing BA.

2.3.2 Effect of auxins Auxins play a vital role in many developmental processes including cell elongation, apical dominance, adventitious root formation, and somatic embryogenesis, (Singh et al. 2015). In general a low concentration of auxin favoured root initiation and the higher concentration favours callus formation (Guan et al. 2015, Chawla 2018). Auxins are characterized as having an aromatic ring separated from a carboxyl group and the most common synthetic auxins used are 1-naphthaleneacetic acid (NAA), 2, 4- dichlorophenoxyacetic acid (2, 4-D), and 4-amino-3, 5, 6-trichloro-2-pyridinyloxyacetic acid (3, 5, 6-TPA) (George et al. 2008c, Piotrowska-Niczyporuk and Bajguz 2014, Beyl 2018). Naturally occurring indole-3-acetic acid (IAA) and indol-3-ebutyric acid (IBA) are also used in tissue culture for cell division and root differentiation (Hossain et al. 2016, Dao et al. 2018). Auxins mainly in combination with cytokinins promotes the growth of calli, cell suspensions and morphogenesis and at cellular level controls the basic processes such as cell division and elongation (Singh et al. 2016, Danova et al 2018). During in vitro growth and differentiation, interaction between nutrient and plant growth regulators (PGRs) plays an important role. The PGRs, especially cytokinins in combination with auxins resulted influential effect on developmental processes of explants on culture media. In organized tissues, auxins are involved in the establishment and maintenance of polarity and in whole plants their most marked effect is the maintenance of apical dominance and mediation of tropisms (Guma et al. 2015, Shittu et al. 2017, Ismaini and Surya 2017, Sisodia and Bhatla 2018). Of the various auxins, IAA is least stable in the medium because it is more photoreactive, therefore, synthetic auxins such as NAA and IBA have been preferred for use in tissue culture media so they are widely used for rooting and in combination with a cytokinin for shoot proliferation (Zhang et al. 2015b).

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The successful combinations of auxins and cytokinins have been reported by numerous workers (Faisal et al. 2018a, Oliveira et al. 2018, Jena et al. 2018). Likewise, combined effects of Kn in combination of different auxins showed positive role on shoot proliferation (Moharana et al. 2018). Similar observations were also made in Prunus empyrean (Sadeghi et al. 2015), Momordica dioica (Choudhary et al. 2017), Paederia foetida (Behera et al. 2018a) and Acacia leucophloea (Sharma et al. 2018b). The synergistic effects of BA and auxins has been reported by different workers (Faisal et al. 2006a and 2006b, Faisal et al. 2012a, Ngomuo et al. 2013, Bhojwani and dantu 2013, Abbasi et al. 2013, Phulwaria et al. 2014, Agarwal et al. 2015, Kannan and Agastian 2015, Shekhawat et al. 2015b, Chavan et al. 2015, Saha et al. 2016).

2.3.3 Effect of meta-topolin (mT) Recently, an aromatic cytokinin, meta-topolin (mT)-which is a benzyladenine analog [N 6-(3-hydroxybenzylamino) purine] has been found as a new source of cytokinin that could be suited to promote high morphogenic development (Gentile et al. 2014, da- Silva et al. 2015a). The mT is more active cytokinin than BA because it has a hydroxyl group in the side chain that facilitates the formation of a O-glycoside which helps in rapid conversion to active forms such as nucleosides, nucleotides or free bases thus used in micropropagation to promote more shoot formation (Kubalakova and Strnad 1992, Werbrouck et al. 1996, Strnad et al. 1997, Aremu et al. 2012, Mala et al. 2013, Gentile et al. 2014). The structural difference of mT with other cytokinins could have a profound impact on plant regeneration during micropropagation and can be considered as an alternative to other commonly used cytokinins (Werbrouck et al. 1996, Liu et al. 2017). Use of mT in plant tissue culture has gained increasing interest due to reports on various important parameters such as improved percentage of seed germination (Huyluoglu et al. 2008, Bairu et al. 2009b), enhanced multiple shoot induction (Nas et al. 2010), increased shoot length and quality (Meyer el al. 2009, Niedz and Evens 2010, Vasudevan and van Staden 2011), successful rooting (Magyar-Tabori et al. 2001, Bairu et al. 2007, Gentile et al. 2014), better quality of regenerated plantlets (Bairu et al. 2008, 2009b), easy acclimatization (Bairu et al. 2007, Aracama et al. 2010), alleviation of hyperhydricity (Dobranszki et al. 2005, Bairu et al. 2007), delayed senescence (Catsky et al. 1996, Wojtania 2010), improved histogenic stability (Bogaert et al. 2004, Bairu et al. 2009a), alleviate shoot tip necrosis (Bairu et al. 2009b), improved physiological and biochemical parameters (Mala et al. 2013, Gentile et al 2014), increased yield (Zhang

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Chapter – TWO REVIEW OF LITERATURE and Whiting 2011) and improved biomass content (Aracama et al. 2010, Gentile et al. 2014). The positive account of exogenous application of mT on in vitro regeneration potential has earlier been reported (Bhojwani and Dantu et al. 2013, Esteves et al. 2014, Gantait et al. 2014, Koszeghi et al. 2014, Mirshekar et al. 2014, Joshi et al. 2016, Pramanik et al. 2016, da Silva 2017).

2.3.4 Effect of TDZ The synthetic plant growth regulator TDZ (N-phenyl N-1,2,3-thiazol-5-yl urea), is a phenylurea derivative having potent cytokinin like activity and widely used in propagation (Fatima et al. 2015, Debnath 2018, Ahmad et al. 2018b). It was initially applied on cotton plant as a leaf defoliant, and later on has found to be an effective to break bud dormancy (Arndt 1976, Wang et al. 1986). TDZ is an active cytokinin similar to other cytokinin and auxins in all metabolism aspects and has the ability to work alone or with other PGRs to stimulate in vitro regeneration process (Mok et al. 1982, Murthy et al. 1998). The exploitation of TDZ in many aspects of plant cell, tissue, and organ culture studies, such as callus induction, somatic embryogenesis, shoot organogenesis and proliferation has proved that TDZ is a potent regulator of these morphogenic responses (Shahzad et al. 2017, Deepa et al. 2018). The optimum concentration of TDZ may vary based on the plant species, explants, and duration of exposure (Lata et al. 2013, Ozarowski and Thiem 2013). The TDZ-treated explants with shoot buds subcultured on plant growth regulator (PGR)-free medium resulted in highest rate of shoot proliferation (Siddique and Anis 2007, Aishwariya et al. 2015, Khanam and Anis 2018, Hussain et al. 2018b). Besides, this higher TDZ levels has some limitations in plant tissue culture because they suppressed shoot elongation, produce fascinated shoots, hyperhydricity and other physiological traits (Murch and Saxena 2001, Guo et al. 2017). TDZ can significantly influence the metabolism of other PGRs. It can increase auxin accumulation and translocation (Dinani et al. 2018), cytokinin content (Kodja et al. 2015). TDZ has been effectively used to induce various morphogenic responses in numerous plant systems including medicinal plants (Aishwariya et al. 2015, Tsai et al. 2016, Gondval 2016, Guo et al. 2017, Patial et al. 2017, Dewir et al. 2018, Giridhar et al. 2018, Govindaraj et al. 2018, Dinani et al. 2018, Ahmad et al. 2018a and 2018b). Recently, several scientific reports on TDZ and its applications on various aspects of plant tissue culture including morphogenesis, somatic embryogenesis, and micropropagation of herbaceous and

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Chapter – TWO REVIEW OF LITERATURE woody plant species, highlighting the use of TDZ for the tissue culture of important medicinal plants (Ahmad et al. 2018a and 2018b, Deepa et al. 2018) and its potential use as an elicitor for the production of secondary metabolites (Unal 2018).

2.4 Effect of Polyamines (PAs) Polyamines are ubiquitously present in almost all cells, and due to their stimulatory and regulatory effect on range of metabolic, developmental and physiological activities in plants (Handa et al. 2018). Most of the plants contain aliphatic amine compounds which are necessary for growth and PAs are organic polycation in nature which contained amine group with lower molecular weight (Rivera et al. 2018). In the higher plants, the most common PAs are spermidine (Spd), spermine (Spm) and their diamine precursor putrescine (Put) (Liu et al. 2015b, Nahar et al. 2016). They are a new type of plant growth stimulators that stimulate numerous cellular mechanisms such as synthesis of protein and lipids, replication of DNA, cell cycle control, gene expression and senescence, including biotic / abiotic stresses and secondary metabolism (Tun et al. 2006, Alcazar et al. 2010, Wimalasekera et al. 2011, Sharma et al. 2012, Fan et al. 2013, Gupta et al. 2013, Kostecka-Gugała and Latowski 2018, Paul et al. 2018, Tiburcio and Alcazar 2018). It also has the capability to break down bud dormancy, improve seed germination, organogenesis and somatic embryogenesis, development of flowers and fruits and also regulate leaf senescence (Shi and Chan 2014, Mustafavi et al. 2018). Exogenous application of PAs in plant culture system has been applied both for plant growth enhancement and stress tolerance (Hussain et al. 2011, Tiburcio et al. 2014, Ghassemi et al. 2018). Application of PAs activates proliferation and development of plant cells lead to formation of multiple shoots (Zhu and Chen 2005, Aragao et al 2017, Sathish et al. 2018). Among the three polyamine, Spd enhanced the shoot multiplication in cucumber shoot tip culture but Put and Spm are neutral in differentiation of explants (Vasudevan et al. 2008, Thiruvengadam et al. 2012, Vasudevan et al. 2017). Put is concerned with root initiation, enhanced the production of total peroxidases in the basal part of explant, stimulating the quick development of roots (Rugini et al. 1997, Matam and Parvatam 2017). The exogenously applied Put in the culture medium positively influences the morphogenesis (Bais et al. 2000, Parimalan et al. 2011, Amin et al. 2011, Sarropoulou et al. 2017). A prospective account of the positive role of polyamines on plants development is recognized as the activator of cell division and expansion has

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Chapter – TWO REVIEW OF LITERATURE been reported by many researchers (Cohen 1998, Wallace et al. 2003, Childs et al. 2003, Handa et al. 2018). Reduction in shoot length has been related to internode shortening and this problem was overcome with application of polyamines (Martin-Tanguy 2001, Kumar et al. 2018). Since polyamines can play a significant part in division of cell but not cell expansion in the CN explants, putrescine at moderate concentration and spermidine at higher level improved more the explants capacity to produce multiple shoots than spermine (Purohit et al. 2007, Sarropoulou et al. 2017). Stimulatory effects of PA on both direct and indirect morhogenesis along with rooting experiment were documented in numerous medicinal plants, Nicotiana tobacum L. (Scaramagli et al. 1995), Cynara scolymus L. (Le Guen-Le Saos and Hourmant 2001), Panax ginseng (Monteiro et al. 2002), Picea rubens (Minocha et al. 2004), Berberis buxifolia Lam. (Arena et al. 2005), Nothofagus nervosa (Pastur et al. 2007), Ocotea catharinensis (Santa-Catarina et al. 2007), Araucaria angustifolia (Steiner et al. 2007), Curcuma longa L. (Viu et al. 2009), Withania somnifera L. (Sivanandhan et al. 2011), Saccharum officinalis. L (Shankar et al. 2011), Dendrobium orchid (Kumari and George 2011), Wrightia tomentosa (Joshi et al. 2014), Picea abies (Vondrakova, et al. 2015), Echinacea angustifolia DC. (Chae 2016), cherry rootstocks (Sarropoulou et al. 2017). 2.5 Heavy metals Heavy metals are a group of metals with a density more than 5.0 g cm3 (Antonious 2016). Due to anthropogenic activity, heavy metal (HM) concentration has been increasing in the environment, the levels can reach a concentration that plant cannot tolerate owing to the phytotoxicity of their ions (Cuypers et al. 2013). Heavy metal is one of the biggest problems among the various stresses, affecting directly medicinal as well as dietary plants and their products (Waoo et al. 014). It is difficult to evaluate the stresses and their responses in field condition for a particular plant species and type of metal while in plant tissue culture it is easier to monitor a particular response against the complex stresses, due to controlled environmental conditions and specified media composition used (Sakthivelu et al. 2008, Goncalves et al. 2009, Okem et al. 2016). The primary response of plant towards the exposure of increased concentration of heavy metal is that it produced ROS, leading to oxidative damage and modification of cellular compartment which resulted in abnormal plant growth and development (Ogawa and Iwabuchi 2001, Bhaduri and Fulekar, 2012, Sytar et al. 2013, Evlard et al. 2014). In plant tissue culture some HM at the optimum concentration are used as essential

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Chapter – TWO REVIEW OF LITERATURE mineral nutrients which greatly influence the growth of culture while beyond the threshold limit, it become toxic (Niedz and Evens 2007, Bernabe-Antonio et al. 2015).

2.5.1 Copper

Copper is supplied in MS medium in the form of CuSO4 in trace amount (0.1 µM) as it is an essential micronutrient for normal growth and development of plants in natural habitat (Murashige and Skoog, 1962). Copper (Cu) is an essential redox-active transition metal of plant photosystem, also plays a vital role in iron mobilization, oxidative phosphorylation and signalling of transcription and protein transferring system (Yruela 2005). It regulates biochemical processes in photosynthesis and mitochondrial electron transport (Maksymiec 1998, Yruela 2005). It performs as a cofactor in various enzymes like cytochrome c- oxidase, polyphenol oxidase and amino oxidase (Clemens 2001, Yruela 2005, Clemens 2006). There are many reports towards the multiplication of shoot by manipulating the concentration of copper to optimized concentration beyond the normal level in MS medium e.g, Capsicum annuum (Joshi and Kothari, 2007), Withania somnifera (Fatima et al. 2011), Rauvolfia tetraphylla (Shahid et al. 2016), Sorghum bicolor (Liu et al. 2015a), Stevia rebaudiana (Javed et al. 2017a),

Erythrina variegate (Javed et al. 2017c). By increasing the concentration of CuSO4, beyond the optimal concentration, a sharp decline in various parameters was noticed

Toxic effect of elevated CuSO4 levels on shoot proliferation has been presented in C. annuum (Joshi and Kothari 2007), W. somnifera (Fatima et al. 2011), R. serpentina (Ahmad et al. 2015), Dendrobium kingianum (Prazak and Molas 2015), Dendrocalamus strictus (Singh et al. 2017a).

2.5.2 Zinc Zinc (Zn) regulates the gene expression and plays a significant role in tolerance of environmental stresses (Cakmak, 2000, Broadley et al. 2007, Boonchuay et al. 2013). The Zn ions have a significant role in production of cytochrome, maintenance of ribosomal fractions and plant metabolism by manipulating the actions of enzymes such as hydrogenase and carbonic anhydrase (Tisdale and Nelson1984, Hansch and Mendel 2009, Hafeez et al. 2013). It also plays a significant role in protein synthesis, carbohydrate breakdown, and repairs of the integrity of cellular membranes, pollen development and regulation of auxin production (Tsonev and Cebola Lidon 2012, Mousavi et al. 2013, Mumtaz et al. 2017). Zn is an essential inorganic component of MS medium (Murashige and Skoog 1962). Positive response towards in vitro shoot

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induction and multiplication at modified concentration of ZnSO4 has been reported by several workers (Gatti 2008, Perveen et al. 2012, Bardar et al. 2014, Ahmad et al. 2015, Verma et al. 2016, Shahid et al. 2016, Singh et al. 2017b, Jamal et al. 2018, Das et al. 2018).

2.5.3 Nickel Nickel (Ni) is an essential transition metal for plants and found in natural soils at trace concentrations (Yusuf et al. 2011). The concentration of Ni in plant tissue varies from 0.01-10.0 μg/g of dry weight (Chen et al. 2009). The nickel ion is a component of several plant metalloenzymes like urease, hydrogenases, glyoxalases and dismutases (Dixon et al. 1975, Boer et al. 2014, Fabiano et al. 2015). In leguminous species, it has been found to be an essential micronutrient to activate urease in potato microshoot cultures (Witte et al. 2002). Accumulation of Ni increased many fold in the environment due to anthropogenic cultures and thus plants are defected by its elevated concentration (Yusuf et al. 2011). At higher concentration, Ni is toxic to majority of crop plants and trees where it showed deleterious effect on plant morphology such as necrosis, chlorosis (Gupta et al. 2017). High uptake of Ni in soil induced a decline in water uptake, causes various physiological alterations and diverse toxicity symptoms such as chlorosis and necrosis in different plant species (Pandey and Sharma, 2002, Rahman et al. 2005, Gajewska et al. 2006). In tissue cultures, the presence of 0.1 mM Ni strongly stimulates the growth of soybean cells in a medium containing only urea as a nitrogen source. Slow growth occurs on urea without the deliberate addition of nickel, possibly supported by trace amounts of the element remaining in the cells (Polacco 1976). Cells and tissues are not normally grown with urea as a nitrogen source, and as urease is the only enzyme, which has been shown to have a nickel component, it could be argued that nickel are not essential (Polacco 1976, George et al. 2008d). However, without it soybean plants grown hydroponically accumulate toxic concentrations of urea (2.5%) in necrotic lesions on their leaf tips, whether supplied with inorganic nitrogen, or with nitrogen compounds obtained from bacterial symbiotic nitrogen fixation (Polacco 1976, George et al. 2008d). These symptoms can be alleviated in plants growing in hydroponic culture by adding Nickel to the nutrient solution (Polacco 1976, George et al. 2008d). The positive account on successful application of evaluated concentration of Ni in culture media to enhance biomass production as well as phytoremediation as accumulation capability of micropropagated plants were reported (Rout et al. 1998,

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Bidwell et al. 2001, Vinterhalter and Vinterhalter 2005, Vinterhalter et al. 2008, Yan et al. 2008, Buendia-Gonzalez et al. 2010, Buendia-González et al. 2012, Hand and Reed 2014, Waoo et al. 2014, Katanic et al. 2015, Ghanavatifard et al. 2018,Wiszniewska et al. 2018, Fourati et al. 2019).

2.5.4 Cadmium The regulatory concentration of cadmium in the soil is 100 mg/kg soil (Salt et al. 1995), but due to anthropogenic activity its concentration has increased many fold (Benavides et al. 2005). At higher concentration in soil its probability to defuse in the ground water is increased and gets accumulated in animal and plants species (de-Vries et al. 2007, Bolan et al. 2014). Higher accumulation in plants leads to various morphological changes through disturbing most of the physiological as well as biochemical processes, morphological changes includes chlorosis, growth inhibition, root tip browning accompanied with death of the plants (Wojcik and Tukiendorf 2004, Manciulea and Ramsey 2006, Mohanpuria et al. 2007, Rizwan et al.2017, Gallego and Benavides 2019). Cadmium also reduces the intake capability of water and many other essential elements, interferes in gas exchange, physiological and biochemical cycles and also affects in antioxidant synthesis and plant metabolism (Hossain et al. 2010, Dias et al. 2013, Nazar et al. 2012, Li et al. 2015, Shanmugaraj et al. 2019). In recent year in vitro cultivation of plant at optimal strength of cadmium gave beneficial account on various morphogenic responses, which helps both the production of plant biomass and phytoremediation at contaminated site, apart from toxic effect of Cadmium in natural soil (Xu et al. 2009, Islam et al. 2009, Li et al. 2010, Okem et al. 2016, Wiszniewska et al. 2017, Muszynska et al. 2017, Sofo et al. 2017, Manquian-Cerda et al. 2016, Muszynska et al. 2018).

2.6 Effect of Carbon sources Sucrose is the main carbohydrate, frequently used as a carbon source in the plant tissue culture medium at various concentrations as per the need of experimental set up (Hildebrandt and Riker 1949, Yaseen et al. 2009). For plant tissue culture, in vitro conditions totally depend on exogenously addition of an artificial carbon source in the form of sucrose, lactose, maltose, galactose and of these, sucrose was found to be most effective than others (Jain and Babbar 2003, Hossain et al. 2013) . Sugar has been found to be effective in both to provide carbon as well as to maintain osmotic potential,

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Chapter – TWO REVIEW OF LITERATURE enzyme activity, metabolisms, and proliferation along with gene regulation with expression (Koch 1996, Lemoine et al. 2013, Sami et al. 2016, Hennion et al. 2018). Moreover, sugar plays a crucial role in cell division and expension in growing seedling (Koch 2004, Li et al. 2017). Sugar in the growing media increases the organ multiplication as well as root formation (Gibson 2005, Moriyama et al. 2016). The regulatory role of sugar on in vitro organogenesis of plant species has been proved to be better for shoot proliferation and root formation in several scientific reports (Fotopoulos and Sotiropoulos 2004, Alina et al. 2006, Schmildt et al. 2015, Zahara et al. 2016, Costa et al. 2017, Saenz et al. 2018, Murthy et al. 2018, Regalado et al. 2018, Li et al. 2018b, Adil et al. 2018, Suarez et al. 2019, Kumar et al. 2019).

2.7 Effect of pH The pH of the medium is a very important regulatory factor for the plants in nutrient up take from the environments (Thorpe et al. 2008). Cultures growing in controlled nutrient medium solution is greatly governed by pH to uptake the nutrients and phytoregulation (Raven 1985, George et al. 2008a, Thorpe et al. 2008). Agar is used in the medium as a solidifier which provide a support to growing culture which can be solidified at a basic condition while lower pH of the medium does not solidified the medium properly (Skirvin et al. 1986, Gulsen and Dumanoglu 1991, Gurel and Gulsen 1998, Pasqua et al. 2002, de Klerk et al. 2008, Kovacevic et al. 2013). On high pH, condition of medium becomes so hard to interrupt in uptake of nutrient (Martins et al. 2011). The explants required appropriate pH range for the better growth and development in in vitro condition. Therefore the pH of medium is optimized at the range of 5.5-6.0 in in vitro culture media (Andersone and Ievinsh 2008, Martins et al. 2011, Vuksanovic et al. 2016). The pH 5.8 is generally considered as optimum for growth of cells and tissues because at this range, cells obtain all the salts ion from the solution easily (Da Silva et al. 2015a, Nataraj et al. 2016, Kher et al. 2016, da Silva and Jha 2016, Bayraktar et al. 2016, Gupta et al. 2017, Li et al. 2018b, Ortuno et al. 2018, Vudala et al. 2019, Suarez et al. 2019, Sun et al. 2019).

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2.8 Rooting For the successful establishment of in vitro regenerated shoots in natural conditions, root formation is the most important and essential part. The success and cost effectiveness of micropropagation relies on the rooting percentage and survival of the plantlets in field conditions (Xu et al. 2008, Shekhawat et al. 2015b). Some of the species easily give the rooting response in the simple root inducing medium, while some require additional treatment for rhizogenesis (Larraburu et al. 2016). Adventitious rooting is a complex process and a key step in the vegetative propagation of economically important plant species play a mandatory role in the successful production of in vitro plants. During root induction step, optimization of basal medium, medium strength and plant growth regulators effect is critical (Goncalves et al. 2005, Revathi et al. 2018). One of the remarkable and important observations noticed has been that MS basal medium without PGR accomplished root initiation along with shoot elongation (Wong and Bhalla 2010, Tripathi and Kumari 2010). The capability of full MS basal media to stimulate in vitro root formation has also been reported whereas, sometimes reduced concentration of MS salts (half to more low level) showed more improved root formation in various plant species (Tyagi et al. 2010b, Rai et al. 2010, Phulwaria et al. 2012). Rhizogenesis in MS medium without PGRs may be due to the availability of endogenous auxin concentration in in vitro regenerated microshoots (Minocha 1987, Zhang et al. 2015b, Liu et al. 2017, Frick and Strader 2017). Root formation has been considered and manipulated as a single stage procedure in which auxin is documented to play a key role (Du and Scheres 2017). Generally in tissue culture, auxins are augmented for rooting experiments (Ahmad and Anis 2011, Dewir et al. 2016, Quambusch et al. 2017), whereas, Rout et al. 2000 reported that in some plant species the rooting resulted without auxin exposure, depend on the plant genotype. In most of the rooting experiments, auxin IAA and NAA supplemented media exhibited a significant reduction on rooting in comparison to IBA, because IBA is found to be more stable, with activator of root initiating gene than others (Lodha et al. 2015, Liu et al. 2017). In root initiation, dominance of IBA over other auxins has been well documented in Ruta graveolens (Ahmad et al. 2010a), Althaea officinalis (Naz et al. 2015a), Cocos nucifera L. (Husin et al. 2018). However, exogenously supplied auxins have also been used with or without other growth regulators to induce in vitro rooting in microshoots of Erythrina variegata (Javed and Anis 2015), Prunus empyrean (Sadeghi et al. 2015),

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Basella alba L. (Shekhawat and Manokari 2016b), Prunus avium (Quambusch et al. 2017), Prunus scoparia (Abbasi et al. 2018). Now-a-days many workers directly placed in vitro growing microshoots onto ex vitro root forming medium to reduced labour and material cost (Sharma et al. 2017, Vibha et al. 2014). Ex vitro root formation was taken in consideration on priority basis to minimize both the cost and time spent with good quality of root having capability of easy to acclimatize with high survival rate because ex vitro rooted plantlets do not need any additional acclimatization prior to transplanting in field condition and have more vigour to tolerate environmental stress during hardening (Ahmed and Anis 2014a, Rathore et al. 2015, Lodha et al. 2015. The successful ex vitro rooting has been reported by many authors (Shekhawat and Manokari 2016b, Choudhary et al. 2017, Sharma et al. 2017, Shiji and Siril 2018, Suarez et al. 2018, Silvestri et al. 2018).

2.9 Acclimatization Acclimatization of in vitro regenerated plantlets to green house or field conditions is a crucial step to achieve success towards micropropagation protocol of a desirable genotype (Pospisilova et al. 2007). Acclimatization is an important step of micropropagation to overcome stress which is developed during in vitro to ex vitro condition and regulate homeostatic environment of in vitro raised plants towards natural condition (Pospisilova et al. 2007, Kaur 2015, Chugh et al. 2018). The cultures exposed to outside the vessel leads to biotic / abiotic stresses and these conditions showed abnormal morphology, anatomy and physiology of plantlets (Kozai et al. 1997, Kumar and Rao 2012). So, regenerated plantlets need proper assessment of physiological parameters during hardening process to simplify the development of effective transplantation protocols and help to adjusting environmental conditions (Hazarika 2006, Chandra et al. 2010). Successful hardening protocol delivers suitable environment for the maximum survival percentage of plants and minimizes the percentage of dead and damaged of micropropagated plants to enhance the plant growth and establishment towards natural habitat (Posposilova et al. 1999, Sha Valli Khan 2003, Pospisilova et al. 2007, Aitken-Christie et al. 2013, Cardoso et al. 2013). Efficient acclimatization procedure saves the time, labor, and money and reduces the cost of production of qualified and deliverable products. Dynamics of the process as well as the final percentage of fully acclimatized plants are related to plant species and both in vitro and

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Chapter – TWO REVIEW OF LITERATURE ex vitro culture conditions (Posposilova et al. 1999, Sha Valli Khan 2003, Anis and Ahmad 2016, da Silva et al. 2017, Singh et al. 2017b).

2.10 Physiological studies During acclimatization, improvement of photosynthetic parameters on gradual increasing the hardening time period is a common phenomenon of in vitro-regenerated plants to adapt to the new environmental conditions which allow them to adopt the autotrophic mode of nutrition capability (Posposilova et al. 1999, Shin et al. 2014, Martins et al. 2016, da-Silva et al. 2017). But when regenerated plantlets were exposed to ex vitro condition they must acclimate to higher light intensity, low relative humidity, fluctuating temperatures and lower availability of some mineral nutrients, to ensure overcome the critical acclimatization and mixoheterotrophic nature in adapting to an autotrophic condition (Martins et al. 2016). These conditions result in the formation of plantlets of abnormal morphology, anatomy and physiology which resulted in high percentage of plantlets death because of sudden changes after their ex vitro transfer (Chandra et al. 2010, Kumar and Rao 2012, da Silva et al. 2017). The micropropagated plantlets grow generally under low light intensity, high level of sugar and nutrient to favour heterotrophic growth under high relative humidity (Hazarika 2002, Ko et al. 2018). Because of these factors, tissue culture raised plantlets have low photosynthetic rate with an undeveloped photosynthetic system (Hazarika 2002). After ex vitro transplantation, plantlets usually need some weeks of acclimatization under shade with gradually lowering relative humidity to develop functional photosystem in order to repair the abnormalities (Posposilova et al. 1999, Dewir et al. 2015). After displacement of tissue culture raised plants to ex vitro environments, the improvement in photosynthetic pigment contents have been observed (Schoner and Krause 1990, Kadlecek et al. 2001, Resende et al. 2016). This effect was also evident in originally photo-autotrophically grown Nicotiana plants, but in originally photo-mixotrophically grown plants, an abrupt decrease in chlorophyll „a‟ and chlorophyll „b‟ contents during the first weeks after transplantation was followed by slow increase (Kadlecek et al. 1998, Siddique and Anis 2008, Vadiveloo et al. 2017). The substantial increment in photosynthetic pigment content with high-light intensity suggested that pigment synthesis enzyme is vital for chlorophyll biosynthesis (Coopman et al.2018).

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There are several studies on initial abrupt decrease in photosynthetic pigment contents during the initial days followed by continuous and subsequent increase was reported during acclimatization processes of several plant species (Van Huylenbroeck et al. 2000, Guan et al. 2008, Preece 2009, Faisal and Anis 2009, Varshney and Anis 2012, Uzaribara et al. 2015, Resende et al. 2016, Ferreira et al. 2017). Similar effects were also observed in Rauvolfia tetraphylla L (Faisal et al. 2012a), Albizia lebbeck (L.) (Perveen et al. 2013b), Syzygium cumini L. (Naaz et al. 2014), Rauvolfia serpentina (Ahmad et al. 2015a), Withania somnifera (Dunal) L. (Fatima et al. 2016), Cassia alata L (Ahmed et al. 2017), Vitex species (Ahmad et al. 2018b), Althaea officinalis L. (Naz et al. 2018) and Rauvolfia tetraphylla (L.), (Hussain et al. 2018a). Net photosynthetic rate has been found to decrease in acclimatized plants in the first week after transplantation and increased thereafter (Siddique and Anis 2008, Chandra et al. 2010, Naz et al. 2017). After formation of new roots in microshoots, the increased uptake of water and nutrients improved all the physiological activities, suggesting that the in vitro roots are functional after proper acclimatization (Preece and Sutter 1991, Hazarika et al. 2002, Slesak et al. 2017). The improvement in photosynthetic parameters in regenerated plantlets during acclimatization has been observed in several species (Ahmad and Anis 2010, Jahan et al. 2011, Ahmed and Anis 2012, Khan et al. 2018, Ahmad and Anis 2019). The successful acclimatization of regenerated plantlets is determined by their root growth parameters. Carbonic anhydrase activity was also assessed by various authors during acclimatization process (Ahmad et al. 2012, Ahmad et al. 2018a&b).

2.11 Biochemical studies Plantlets that have grown in vitro have been continuously exposed to a unique environment that has been selected to provide minimal stress and optimum conditions for plant multiplication, while in vitro raised plants face higher mortality rate during acclimatization to greenhouse often limit the commercial approach (Bhattacharyya et al. 2016, Yucesan et al. 2016, Kunakhonnuruk et al. 2018). The greenhouse and field have substantially lower relative humidity, higher light level and septic environment that are stressful to micropropagated plants, and leads to production of reactive oxygen species - (ROS), hydrogen peroxide (H2O2) superoxide radicals (O2 ) and other free radicals (Hazarika 2006). These free radical species are highly reactive /unstable which cause tissue injuries including cell as well as DNA damages thus, affecting the physiological

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Chapter – TWO REVIEW OF LITERATURE and biochemical process by disturbing the function and regulation of various biomolecules (Hazarika 2006, Gill and Tuteja 2010, Kumar and Rao 2012, Sharma et al. 2012, Demidchik 2017). Considerable efforts have been applied to minimize the death rate and to optimize the condition for successful survival of plantltlets during their transfer from in vitro to ex vitro environment via different acclimatization and hardening protocols Ahmed and Anis (2014a,) These efforts have been applied by estimation of different enzymatic or non-enzymatic reaction including GST (Glutathione- S - Transferase) during subculture passages. Plants have shown a natural defense mechanism to counter the free radicals to cope toxic effect through various enzymatic defense mechanism like SOD (superoxide dismutase), CAT (Catalase), APX (Ascorbate peroxidase), GR (Glutathione Reductase) during the acclimatization period (Anderson et al. 1995, Aragon et al. 2010, Dias et al. 2013, Wu et al. 2018, Veerashree and Kumar 2018, Hassanpour et al. 2017) reported previously in many plant species (Ahmad et al. 2010b, Sharma et al. 2012, Wu et al. 2017, Paul et al. 2018).

2.12 Synthetic seeds Synthetic seeds are defined as encapsulated artificially somatic embryos, or non- embryogenic vegetative buds, like axillary buds, nodal segments, apical shoot bud or any other meristemetic tissues that have an ability to germinate into plant in vitro and thus help in storage/preservation of germplasm (Gantait et al. 2015, Ray and Bhattacharya 2008). Synthetic seed production is one of the versatile and advanced technology to explore micropropagation methods and can be applied as an effective means of propagation, conservation, exchange of elite and axenic plant materials as well as distribution of plantlets including germplasm, rare hybrids, genetically modified plants, sterile or choice genotype with non-availability of seeds (Sharma et al. 2013, Perveen and Anis 2014, Gantait et al. 2015). The concept of artificial seeds was specified by Murashige (1977).While successful synseeds were obtained on encapsulation of somatic embroys with alginate hydrogel for the plant alfa-alfa (Redenbaugh et al. 1984). Ever since, a number of researchers have applied these techniques as such or with modification to produce the synthetic seeds (Ahmed et al. 2015, Alatar et al. 2016, Javed et al. 2017b, Naz et al. 2018, Khan et al. 2018). In general, encapsulation is usually done in an appropriate gelling substance (sodium alginate) to produce a synthetic seed coat and the resulting encapsulated

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Chapter – TWO REVIEW OF LITERATURE propagules can be considered like natural seeds (Benelli 2016, Javed et al. 2017b). Recently nutrients, plant hormones, biofertilizers like fungicide or pestisides, antibiotics, nitrogen source as fixing bacteria or other essential additives are also added in to the matrix for easy handling as per needs such as propagation, conservation or exchange programs (Maqsood et al. 2012). The story of successful synthetic seed production and their conversion into complete plantlets have recently been reported in various medicinal as well as commercial plants like Rotula aquatica (Chithra et al. 2005), date palm (Bekheet et al. 2005), Pinus patula (Malabadi and Staden 2005), Tylophora indica (Faisal and Anis 2007), Acca sellowiana (Cangahuala-Inocente et al. 2007), Quercus suber (Pintos et al. 2008), Psidium guajava (Rai et al. 2008), Curculigo orchioides (Nagesh et al. 2009), Rhododendron species (Singh and Gurung 2009), Vitex negundo (Ahmad and Anis 2010), Picrorhiza kurroa (Mishra et al. 2011), Carrizo citrange (Germana et al. 2011), Ruta graveolens (Ahmad et al. 2012b), Withania somnifera (Fatima et al. 2013), Ficus carica (Sharma et al. 2015), Gossypium hirsutum (Hu et al. 2015), Curcuma longa (Babu et al. 2016), Ledebouria revolute (Haque, and Ghosh 2016), Vitex trifolia (Alatar et al. 2016), Limonium hybrid (Bose et al. 2017), Brassica oleracea (Rihan et al. 2017) Date Palm (Bekheet 2017), Erythrina variegata (Javed et al. 2017c), Ansellia africana (Bhattacharyya et al. 2018), Ceropegia barnesii (Ananthan et al. 2018) Gymnema sylvestre (Saeed et al. 2018).

2.13 Genetic fidelity In plant tissue culture the developed protocols are severely hindered due to incidences of somaclonal variations in regenerated plantlets. To obtain elite genotypic plants is one of the most important pre-requisites through micropropagation technique. Several strategies have been developed to monitor the genetic homogeneity or somaclonal variation in micropropagated plantlets such as morphological descriptions, physiological supervisions, cytological studies, isoenzymes etc. (Saker et al. 2000, Jain 2001, Sahijram et al. 2003, Wan and Wang 2012, Slazak et al. 2015, Akdemir et al. 2016, Martínez-Estrada et al. 2017, Delgado-Paredes et al. 2017, Qin et al. 2018, Manchanda et al. 2018). Somaclonal variation mostly occurs in response to the stress imposed on the plant in culture conditions and is manifested in the form of DNA methylations, chromosome rearrangements, and point mutations (Jin et al. 2008, Bairu et al. 2011, Wan and Wang 2012, Neelakandan and Wang 2012, Mujib et al. 2013). Occurrence of somaclonal variation is a potential drawback when the propagation of an

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Chapter – TWO REVIEW OF LITERATURE elite species is intended due to uncontrolled and unpredictable nature of variation (Samantaray and Maiti 2010). The variation may be due to several factors such as genotype, utilization of plant growth regulators at higher concentration and repeated sub-culturing for long periods, hampers maintenance of genetic fidelity (Nwauzoma and Jaja 2013, Matsuda et al. 2014, Sianipar et al. 2015, Regalado et al. 2015, Lestari 2016, Khan et al. 2018). Therefore, there is need to evaluate the genetic stability among regenerants (Bhattacharyya et al. 2017a). Several DNA markers such as random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) are mostly used to detect genetic integrity of micropropagated plants (Phulwaria et al. 2014, Goyal et al. 2015, Chavan et al. 2015, Agarwal et al. 2015, Saha et al. 2016, Ahmed et al. 2017, Thorat et al. 2017, Faisal et al. 2018a,b, Nasri et al. 2019, Seth and Panigrahi 2019). These polymerase chain reaction based techniques are simple, fast, cost-effective, highly discriminative and reliable and require only a small quantity of DNA sample and do not need any prior sequence information to design the primer (Ahmad et al. 2013). Thus, they are suitable for the assessment of genetic fidelity of in vitro raised plants (Dangi et al. 2014). RAPD and ISSR markers have been used successfully to assess genetic stability among several plant species Momordica dioica (Rai et al. 2012a), Arnebia hispidissima (Phulwaria et al. 2013a), Vitex trifolia (Ahmad et al. 2013), Gloriosa superba L (Yadav et al. 2013), Dendrocalamus asper (Singh et al. 2013) Cleome gynandra (Rathore et al. 2014b), Terminalia arjuna (Gupta et al. 2014), Dendrocalamus strictus (Goyal et al. 2015), Ophiorrhiza mungos L. (Kaushik et al. 2015), Cannabis sativa L. (Lata et al. 2016), Artemisia nilagirica (Shinde et al.2016), Cassia alata L.(Ahmed et al. 2017), Curcuma angustifolia (Jena et al. 2018), Lawsonia inermis L. (Moharana et al. 2018), Platanus orientalis L. (Kuzminsky et al. 2018), Polianthes tuberosa L. (Nalousi et al. 2019).

2.14 Tissue culture related work conducted on Mucuna pruriens Lahiri et al. (2006) developed an efficient protocol for callus induction and subsequent proliferation in Mucuna pruriens. Best callusing was obtained on Murashige and Skoog‟s basal medium supplemented with NAA (2.0 mg/L) + BA (2.0 mg/L) at pH 5.8. Faisal et al. (2006a) established an efficient micropropagation system for Mucuna pruriens using CN explants. Of the cytokinins (BA, Kin and 2-iP) tested MS medium augmented with BA (5.0 μM) was found to be optimum for inducing maximum shoots.

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The highest efficiency of shoot proliferation was obtained in 5.0 μM BA and 0.5μM NAA in half-strength MS medium at pH 5.8. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The best condition for rooting was 1/2 MS medium solidified with agar and supplemented with 2.0 μM IBA. After rooting, the plantlets were transferred to plastic pots filled with sterile Soilrite where 90% plant grew well and all exhibited normal development. Faisal et al. (2006b) evaluated the effect of different cytokinins and auxins in different concentrations and combination in MS medium for in vitro regeneration of Mucuna pruriens using aseptic nodal explants. Highest number of shoots (23.3) with average length (5.6 cm) was standardized on ½ strength MS medium supplemented with a combination of 5.0 μM BA and 0.5 μM NAA at pH 5.8. Rooting was best induced in excised shoots on MS medium containing 1.0 μM IBA. The in vitro raised plants showed 90% survival rate. This study provides the first report on in vitro plant regeneration of M. pruriens. Patel et al. (2007) evaluated callus proliferation on cotyledon, leaf and stem explants of Mucuna pruriens cultured on Murashige and Skoog medium (MS) supplemented with 2,4-D, NAA and BAP alone or in combination. Light brown callus formation was followed by formation of milky white callus on the surface of young excised shoot and leaf tissues. Sometimes green callus was also observed. Sathyanarayana et al. (2008) evaluated the in vitro response for auxiliary bud induction on Murashige and Skoog‟s (MS) medium supplemented with different concentrations of cytokinins. During the first culture on BA 3.5 μM, maximum of 6.70 ± 1.15 shoots with an average shoot-length of 1.07 ± 0.21 cm were recorded. The number of shoots increased up to 16.33 ± 0.58 with an average length of 1.16 ± 0.29 cm, when subcultured onto the same hormonal medium. The shoots exhibited adequate elongation (4.00 cm) when treated with on 2.89 μM gibberellic acid (GA). The elongated shoots produced a maximum of 16.67 ± 2.89 roots on 1/2 MS liquid medium supplemented with 16.20 μM NAA. The plantlets were acclimatized by transferring them first to peat moss: compost (1:1) mixture followed by sand: soil (1:1) mixture, recording 95% survival. The genetic fidelity of the regenerated shoots was confirmed using randomly amplified polymorphic DNA (RAPD) analysis employing 15 operon primers. This system provides high fidelity micro-propagation system for an efficient and rapid micro-propagation of this important medicinal herb.

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MATERIALS AND METHODS

3.1 Source of plant material Seeds of Mucuna pruriens were obtained from the Indian Agricultural Research Institute (IARI), New Delhi.

3.4.1 Composition of basal media

For practical purpose, the essential elements are further divided into the following categories; (a) Major or macronutrients (b) Minor or micronutrients

S1V1 = S2V2

Where,

S1 = Known strength (stock solution)

V1 = Volume (stock solution required)

S2 = Strength (desired solution)

V2 = Volume (desired solution)

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(ZnSO4) and Nickel Chloride (NiCl2), and heavy metals such as Cadmium Chloride

(CdCl2) were also tried in supplementation with MS basal media. The combination and concentration of PGRs, polyamines, micronutrients and heavy metals was set as specified in the results.

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3.10 Synthetic seeds production

3.10.1 Explant source Aseptic nodal segments (NS), (0.5-0.7 cm) from 56 days old cultures were used as explants source for encapsulation.

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12.7 (O.D. 663 nm) – 2.69 (O.D. 645 nm) Chlorophyll a = x V d × 1000 × W

22.9 (O.D. 645 nm) – 4.68 (O.D. 663 nm) Chlorophyll b = × V d × 1000 × W

7.6 (O.D. 480 nm) – 1.49 (O.D 510nm) Carotenoids = × V d × 1000 × W

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Where,

O.D. = Optical density V = Final volume of extract W = weight of fresh leaf sample d = Length of light path

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It was prepared from 0.5 M phosphate buffer (pH 7.3). The solution of KH2PO4 and

K2HPO4 were first prepared as given bellow:

Solution A (KH2PO4)

3.40 g of KH2PO4 was dissolved in 50 ml DDW.

Solution B (K2HPO4)

KH2PO4 and K2HPO4 were prepared separately as given below: Solution A

1.3 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

1.70 g of K2HPO4 was dissolved in 100 ml DDW.

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Both the Solution A and B were mixed in an appropriate ratio at pH 7.8 and 1 g of PVP (Polyvinyl pyrrolidone) was added to 100 ml of the buffer.

3.11.3 Estimation of Catalase (CAT) activity Catalase (CAT: EC 1.11.1.6) activity in leaves of the regenerated plantlets were determined by the method of Aebi (1984) with slight modification.

3.11.3.2 Enzyme assay

3.11.3.3 Reagents Preparation  Potassium buffer (0.5 M at pH 7.3) Solution A

3.40 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

3.40 g of KH2PO4 was dissolved in 50 ml DDW.

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Solution B

8.70/4.35 g of KH2PO4 was dissolved in 100 ml DDW. Both the Solution A and B were mixed in an appropriate ratio at pH 7.2.  Potassium phosphate buffer (0.25 M at pH 7.0) Solution A

1.70 g of KH2PO4 was dissolved in 50 ml DDW. Solution B

8.70 g of KH2PO4 was dissolved in 100 ml DDW. Both the Solution A and B were mixed in an appropriate ratio at pH 7.0.

 H2O2 (3 mM)

0.1 ml of H2O2 was mixed with 9.9 ml of DDW.  EDTA (3 mM) 1.116 mg EDTA was dissolved in 100 ml DDW.

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(CO2) was done to give the bicarbonate ion in the presence of CA catalyse enzyme;

Carbonic anhydrase + -3 H2O + CO2 H + HCO

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3.12 Molecular marker studies of in vitro raised plantlets

3.12.1 Sample source of DNA Micro-propagated plantlets were used for the isolation of genomic DNA. Young, fresh and green leaves were used for extraction of DNA.

3.12.2 Isolation of Genomic DNA Cetyltrimethylammonium Bromide (CTAB) method (Doyle and Doyle 1990) with minor modifications was used for isolation of genomic DNA from the collected leaves.

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Absorbance ratio (A260/A280) Result Above 1.8 = Contamination of protein Between 1.7 to 1.8 = Best concentration of DNA Less than 1.7 = Contamination of RNA

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(Thermo Scientific, India), 1.2 μl MgCl2, 0.2 μl Taq DNA polymerase (Thermo Scientific, India), genomic DNA (1.0 μl), 1.0 μl RAPD/ISSR primer, 14.2 μl mQ water.

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3.14 Chemicals and Glasswares

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DISCUSSION

Recent increase in consumption of herbal medicines due to its lower toxicity and side effects compared to allopathic medicines has led to the unexpected rise in the number of herbal drug manufacturers in both the industrial and non-industrial level (Panchal and Tiwari 2017, Yadav et al. 2017, Tiwari et al. 2018). Majority of the pharmaceutically important vegetation are harvested from natural populations as source of raw materials to industrial demands (Lubbe and Verpoorte 2011, Singh 2015, Jamshidi et al. 2018). Over exploitation and unskilful collection of medicinal plants is reducing the natural vegetation with increasing the risk of making a species extinct and in future they may become endangered, so it is necessary to conserve all the valuable plant species by complacency concerning their conservation, cultivation for sustainable demands of supply (Chen et al. 2016, Phondani et al. 2016, Wolff et al. 2017, Negi et al. 2018). During the last two decades, dramatic progress has been made in developing and refining various tissue culture techniques to make them competent enough to meet the growing demand in the global market (Pathak and Abido 2014, Reddy 2015, Ali et al. 2016, Swamy et al. 2018, Iannicelli 2018). Now-a-days micropropagation techniques are of great interest for the conservation of elite germplasm through clonal propagation, along with multiplication, development of new variants, and production of genetically modified plants through genetic transformation (Opabode 2017, Ragavendran and Natarajan 2017, Mahajan et al. 2018, Faisal et al. 2018a, Salma et al. 2018). The economical, ecological and medicinal importance of leguminous forest plants necessitates the application of plant tissue culture techniques for their mass propagation. During the past few years, a number of leguminous plant species have been successfully propagated in vitro (Gatti et al. 2016, Cheeran et al. 2017, Sharma et al. 2017, Aziz et al. 2018, Garay and Dominguez 2018, Pratap et al. 2018, Ahmad et al. 2018a). Therefore, the present study was proposed and carried out to develop in vitro regeneration system for a potential medicinal legume, M. pruriens and assessment of genetic fidelity of in vitro raised plantlets using RAPD and ISSR markers. The results of the investigations have been discussed in this chapter.

5.1 Seed germination In vitro seed germination is the initial steps towards successful in vitro propagation on the basis of selection of suitable explants, which is to be used as the starting material for Chapter – FIVE DISCUSSION the experiments. Using juvenile tissues provide a better understanding on genotypic requirements and its response on the micropropagated plant towards phenotypic expression in in vitro propagation system. In the present findings, seed germination responses were recorded in all the treatments. The highest percent germination was recorded on 1/2 MS medium with 86.2% germination rate after 10 days of inoculation. Reducing the strength of MS medium reduced the germination percent. The results are in accordance with the findings in various medicinal plant species (Gupta and Jatothu 2013, Phulwaria et al. 2013b, Us-Camas et al. 2014, Nayak et al. 2013, Yildirim and Turker 2014, Shekhawat and Manokari 2016a and 2016b, Kirimer et al. 2017, Conger et al. 2018, Haque and Ghosh 2018).

5.2 Direct in vitro regeneration Explant types and its selection are one of the most important and crucial factor affecting in vitro regeneration for the establishment of an efficient micropropagation protocol. Regeneration also significantly influenced by the age of donor plants, concentration and combination of plant growth regulators and their interaction with explant. Different tissues may have different levels of exogenous hormones and therefore, the type of explants source would have a critical impact on the regeneration rates (Fatima et al. 2015, Behera et al. 2018a and 2018b). Propagation through seedling derived explants being juvenile is frequently used for in vitro organogenesis, as they are easy to establish in culture and have higher organogenic ability (Bramhanapalli et al. 2017). Juvenile tissue culture involves culturing of seedling derived explants carrying axillary buds; possess quiescent or active meristem which has the potential of developing into complete plantlets under the influence of plant growth regulators in vitro (Kher et al. 2016). In nature, these buds remain dormant for specific period depending on the growth pattern of plant. The mechanism of dominance has also been demonstrated to be under control of various growth regulators and the proportion of these growth regulators in the culture media can be manipulated to induce the regeneration of each meristem into liable shoots. In vitro protocols using seedling explants have been employed for rapid propagation and manipulation of several legumes, such as Prosopis cineraria (Venkatachalam et al. 2017), Trifolium polymorphum (Castillo et al. 2017), Vigna unguiculata (Jain et al. 2017), Acacia leucophloea (Sharma et al. 2018c) Certain food

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Chapter – FIVE DISCUSSION legumes (Ugandhar 2016), Pterocarpus marsupium (Ahmad et al. 2018a), grain legumes (Pratap et al. 2018). Cotyledonary node and nodal segment explants obtained from axenic seedlings are considered to be the most excellent tissues for attaining maximum frequency of shoot regeneration and multiplication. Between the two explants tested, nodal culture was found to be the best, secure and realistic method of shoot induction and multiplication and assure consistent production of a large number of plants within a short span of time. The mature nodal segments have been proved to be a reliable explant source for large scale plant production under in vitro and are used to maintain clonal fidelity. The observations are in agreement with earlier publications in some medicinal plants (Ahmad et al. 2013, Shekhawat et al. 2015a, Panda et al. 2016, Hussain et al. 2018a and 2018b, Ahmad et al. 2018a and 2018b).

5.2.1 Plant growth regulators Shoot bud induction was not observed in CN explants when inoculated on MS basal medium devoid of PGRs, even after 21 days of incubation. However, when inoculated on MS medium supplemented with different cytokinins with various concentrations of BA, Kn, 2-iP, a differential response with regard to shoot bud initiation, multiplication and elongation was observed. Cytokinins effectively remove the meristemetic shoot apical dominance, so the addition of cytokinin at optimum level in culture media has beneficial effect on shoot induction and proliferation. Similar results have been reported in various earlier scientific reports (Cheruvathur et al. 2015, Ahmed et al. 2017, Adil et al. 2018). Commonly, growth regulators used singly or in combination with auxins for micropropagation, gave a positive correlation on direct organogenesis. There are many reports towards beneficial explanation of combined effect of cytokinins and auxins in plant tissue culture (Hill and Schaller 2013, Faisal et al. 2018a, Monfort et al. 2018, Chawla 2018, Bridgen et al. 2018). Type of cytokinins also affected the morphogenic response among the tested cytokinins. The percentage of the explants forming shoots and shoot numbers per explant was found to be influenced by the type of plant growth regulators used. Among the different cytokinins tested (BA, Kn or 2-iP), BA (2.5 µM) was found to be the best with respect to the induction and subsequent proliferation of shoots. Stimulatory effect of BA has been well documented in M. pruriens (Faisal et al. 2006a and 2006b, Sathyanarayana et al. 2008, Lahir et al. 2011, Lahiri et al. 2012,

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Raaman et al. 2013). Also, the effectiveness of BA on multiple shoot bud differentiation has been demonstrated in many other plants like in Artemisia vulgaris (Sujatha and Kumari 2007), Balanites aegyptiaca (Siddique and Anis 2009a), Euphorbia cotinifolia (Perveen et al. 2013c), Erythrina variegata (Javed and Anis 2015), Curculigo orchioides (Nagesh and Shanthamma 2016), Scadoxus puniceus (Naidoo et al 2017) Cunila menthoides (Oliveira et al. 2018). Cell division requires temporal relation between the S phase and cell division, signifying that auxin and cytokinin concentration in culture media is to be carefully matched. Cells are supposed not to pass in mitosis phase unless cytokinin is present. There are several earlier reports towards beneficial role of cytokinins to promote the axillary bud proliferation and shoot elongation (Ahmad et al. 2013, Naz et al. 2015a, Plihalova et al. 2016, Aremu et al. 2017, Baskaran et al. 2018). Application of cytokinins in tissue culture media varies according to plant species, types of explant used and types of culture practices. MS media in combination with various cytokinins at optimal concentration along with optimized auxins has been most promising and show a mutual regulatory effect on in vitro shoot multiplication and elongation. It means, that exogenous application of phytohormones in MS media at optimal level showed positive effect in many studies (Rathore et al. 2013, Cosic et al. 2015, Patil and Bhalsing 2015, Renuka et al. 2017, Faisal et al. 2018b). The effectiveness of BA over other cytokinins (Kn or 2-iP) at optimum level has been well documented in studies like Withania somnifera (Fatima and Anis 2012), Albizia lebbeck (Perveen and Anis 2015). One of the probable descriptions for better response achieved on BA is that the ribosides and nucleotides are naturally stable in BA compared to other cytokinins (McGaw et al. 1985, Zalabak et al. 2013). However, an increase in concentration beyond the optimal level had a negative effect and the shoot exhibited a stunted nature with the reduction in number of shoot regenerated from each explant. These findings are in consonance with the results obtained in earlier reports (Geng et al. 2016, Aremu et al. 2017, Erland et al. 2017, Oliveira et al. 2018, Foo et al. 2018, Martins et al. 2018, He et al. 2019).

5.2.2 Effect of meta-topolin (mT) The effect of meta-topolin, an aromatic cytokinins on in vitro morphogenesis in CN or NS explant was also evaluated in a separate experiment. These explants failed to induce any shoot bud in meta-topolin free MS medium. Whereas, shoot bud differentiation was promoted only in explants growing on mT supplemented MS media. Cytokinins are

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Chapter – FIVE DISCUSSION frequently used in in vitro propagation as potent PGRs. Among these, mT has been used as a possible substitute of benzyladenine in several plants, such as Spathiphyllum floribundum (Werbrouck et al. 1996), Barleria greenii (Amoo et al. 2011) and Prunus stock (Gentile et al. 2014), Carica papaya (Solorzano-Cascante et al. 2018). The MS medium augmented with mT gave highest regeneration response, shoot multiplication with increase in the shoot length. In the present investigation, higher rate of shoot formation may be because of alterable sequestration of the O-glucosides, which permits for continual availability of cytokinins at a physiologically vital level over a prolonged period (Strnad et al. 1997). Establishment of O-glucosides metabolites are possible through the hydroxyl group in the side chain of mT (Werbrouck et al. 1996). The O-glucosides are considered to be storage form of cytokinins that are stable under certain situations and rapidly convertible to potential cytokinin bases whenever needed (Werbrouck et al. 1996). Therefore, mT is a new alternative and reliable cytokinin in present day to manipulate and utilized in in vitro techniques to better establishment of commercial micropropagation where cost effective plant regeneration protocol are obtained (Bairu et al. 2007). The effectiveness of mT in micropropagation system has been well documented in numerous plants such as Prunus spp. (Monticelli et al. 2015), Cannabis sativa (Lata et al. 2016), Lachenalia montana (Aremu et al. 2017), Corylus colurna (Gentile et al. 2017), Pterocarpus marsupium (Ahmad and Anis 2019).

5.2.3 Combined effect of cytokinin and auxins Plant tissues growing in vitro require exogenous hormones in the nutrient medium for differentiation. The reaction of an isolated tissue to auxin depends upon its endogenous level at the excision time and its genetic capacity during synthesis. In the present work, MS medium was supplemented with various concentrations of different auxins. It was observed that level and type of auxins required for dedifferentiation and optimal callusing varied between the explants. Morphogenesis in micropropagation has been found to be controlled by contact among cytokinins and auxin applications. It is apparent that not only auxins and cytokinin concentration but the magnitudes of one to other are contributing factor in cell cycle, cell division and differentiation control. The process of cell division is supposed to be controlled by the mutual interaction of auxins and cytokinins and both influences different stages of cell cycle. Auxin applies an influence on DNA replication, whereas cytokinin regulates various events of mitosis (Tognetti et al. 2017, Hurny and Benkova 2017). Thus, auxins used are referred to as

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“inducers” and cytokinins as “promoter” of the cell cycle (Richard et al. 2002, Hartig and Beck 2006, Schaller 2015). In the present findings an antagonistic response was recorded on shoot multiplication and elongation when exogenous auxins are supplied in cytokinins optimized medium. The lowest concentration of IAA (0.05 μM) in combination with optimized concentration of cytokinins was most effective for shoot bud induction in M. pruriens. The benefits of adding auxin at lower concentration in the culture media is to nullify the influence of cytokinins on axillary shoot elongation (Hu and Wang 1983, Masondo et al. 2015). While, addition of auxins appears to be non-significant towards regeneration potential of explants. Data revealed that the shoot number did not increase on increasing the auxin concentration. On the basis of our findings auxins had shown adverse effect on shoot regeneration as it stimulated basal callus formation. Thus, the outcome suggests that the MS medium composed to cytokinins singly is sufficient for in vitro propagation of M. pruriens. Similar results have been documented by many workers (Gulati and Jaiwal 1992, Khalafalla and Hattori 2000, Sujatha and Kumari 2007, Ahmad et al. 2012a).

5.2.4 Effect of TDZ TDZ (N-phenyl N-1,2,3-thiazol-5-yl urea) is a synthetic plant growth regulator, and its derivatives has a non-purine structure having potent cytokinin activity, potentially used in micropropagation system (Fatima et al. 2015, Debnath 2018, Ahmad et al. 2018a and 2018b). TDZ may be involved in increasing the biosynthesis or accumulation of endogenous cytokinin (Murthy et al. 1998, Podwyszynska et al. 2014). However, the mechanism of its auxin like activity or its involvement in auxin metabolism remains to be resolved (Dinani et al.2018). The morphogenetic responses in which TDZ has been found to mimic cytokinin- like activity include release of lateral buds from dormancy (Wang et al. 1986, Ahmed and Anis 2012, Singh and Dwivedi 2014, Singh et al. 2017b) and in vitro shoot formation in a wide variety of plant species (Faisal and Anis 2006a, Jahan et al. 2011, Grąbkowska et al. 2014, Faisal et al. 2014). In the present study, both the CN and nodal explants were cultured on different concentration of TDZ to determine the role of TDZ action as cytokinin towards the formation of morphologically well-developed shoots. Of the various concentrations of TDZ tested, 0.8 µM was found to be the most effective in inducing highest percentage (95%) regeneration, number of shoots (11.60 ± 87), and shoot length (3.82 ± 0.09) from

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Chapter – FIVE DISCUSSION nodal explants. Singh et al. (2003) recommended very low concentration of TDZ for high rate of shoot multiplication as obtained from both nodal and cotyledonary node explants in Cajanus cajan. This behaviour is believed to be due to the potency of TDZ to increase the biosynthesis and accumulation of endogenous adenine-type cytokinins, thus, making TDZ as an effective cytokinin for the stimulation of shoot buds. TDZ has been effectively used to induce various morphogenic responses in numerous plant systems including medicinal plants (Aishwariya et al. 2015, Tsai et al. 2016, Gondval et al. 2016, Guo et al. 2017, Patial et al. 2017, Dewir et al. 2018, Giridhar et al. 2018, Govindaraj 2018, Dinani et al. 2018). The mechanism of TDZ action is partly related to the inhibition of cytokinin degradation by cytokinin oxidase, resulting in an increased level of endogenous cytokinin (Hare and Van Staden 1994). Shoot regeneration potential with number of shoots per explant gradually declined on evaluated concentration of TDZ beyond the optimal strength in both the explant. There was a linear correlation between an increase in TDZ concentrations upto an optimal level (0.8 µM) with regard to shoots induced per explant. A reduction in the number of shoots generated from each explant at TDZ concentration higher than the optimal level was earlier reported in Ocimum basilicum (Siddique and Anis 2007), Coleonema pulchellum (Baskaran et al. 2014), Allamanda cathartica (Khanam and Anis 2018), Scutellaria bornmuelleri (Gharari et al. 2019). Continuous exposure with respect to time duration as well as concentration of TDZ more than optimal level gave antagonistic response on regeneration potential of explant with bunching and fasciation of shoots. The inhibitory effect of TDZ on shoot elongation may be due to the presence of phenyl group which on over exposure beyond the optimal level can lead to many drawbacks such as bunching, fasciation, hyperhydricity, poor microshoot quality and loss of rooting capacity (Lu 1993, Singh and Syamal 2001, Ahmad et al. 2018a and 2018b). The deleterious effect on shoot growth and multiplication during prolonged exposure of TDZ has been reported in several plants including Vitex negundo (Ahmad and Anis 2007), Vitex trifolia (Ahmed and Anis 2012), Rauvolfia tetraphylla (Hussain et al. 2018a). Therefore, optimizing the exposure duration and concentration is quite important to overcome deleterious effects of continued presence of TDZ on growth and multiplication of shoots. These problems of shoot elongation were overcome by transferring the cultures on a secondary medium lacking TDZ, or to a medium containing low concentrations of a cytokinin. Similar results on using different medium

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Chapter – FIVE DISCUSSION for TDZ exposed explants have been documented in many reports, Clitoria ternatea (Mukhtar et al. 2012), Salix tetrasperma (Khan and Anis 2012), Cymbidium giganteum (Roy et al. 2012), Bauhinia tomentosa (Naz et al. 2012), Cassia angustifolia (Siddique et al. 2013), Vitex trifolia (Ahmed and Anis 2014b), Harpagophytum procumbens (Grąbkowska et al. 2014), Rhododendron sichotense and Rhododendron catawbiense (Zaytseva et al. 2016), Picrorhiza kurroa (Patial et al. 2017), Rauvolfia tetraphylla (Hussain et al. 2018a) etc.

5.2.5 Effect of polyamines Alteration of the nutrient composition in growth media is one of the effective strategies used to improve the regeneration potential and production of natural product from in vitro regenerants (Isah et al. 2018). In the present investigation, an attempt has been made to evaluate various concentrations of polyamines to find the morphological effect on axillary shoot proliferation and multiplication from CN and NS explants. Among the various concentration of polyamines (Put, Spd or Spm) tested, 10 μM gave the maximum regeneration response. A possible explanation towards positive impact in in vitro shoot multiplication is that polyamines interact with various PGRs and nutrients in the medium. Polyamines are the low molecular weight polycationic molecules, made up of multiple amine groups which are necessary for growth. PAs have the stimulatory effect in plant metabolic processes and regarded as plant biostimulators or PGRs (Sarropoulou et al. 2017). Application of phyto stimulators in micropropagation system are considered as naturally originated non-toxic substance that aim to improve efficiency of nutrient uptake along with abiotic and biotic stress tolerance mechanism due to the change in endogenous phytohormones by which antioxidant content increased (Jardin 2015, Li et al. 2016). The application of PAs have been considered as key signalling molecules in the regulation of various developmental processes in in vitro regeneration and its response to different type of stresses developed during culture practice. It play direct role in scavenging of free radicals and reactive oxygen species to enhance the defence mechanisms towards stresses and considered as most efficient antioxidant (Li et al. 2014, Sheteiwy et al. 2017). The regulatory effect of PAs could be an impact on some of the metabolic pathways of regenerants such as photosynthesis, respiration and also in nucleic acid synthesis and resulted in improved regeneration without changing their natural pathway (Rafiee et al. 2016).

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The most common polyamines used in propagation system are Putrescine (Put), Spermine (Spm) and Spermidine (Spd) which are synthesized in plant or exogenously applied in nutrient medium (Smith et al. 1977, Minocha and Minocha 1995, Piotrowska- Niczyporuk et al. 2012, Gurung et al. 2012, Shi et al. 2013, Xie et al. 2014, Kamiab et al. 2014, Liu et al. 2015b, Stetsenko et al. 2015, Nahar et al. 2016). Exogenous application of PAs is able to form electrostatic linkage with negatively charged molecules, contributed conformational stability of protein, chromatin fibers and even DNA or RNA (Alcazar et al. 2010, Wimalasekera et al. 2011, Chae 2016, Mustafavi et al. 2018). Shi and Chan (2014) reported that PAs play a vital role in a range of developmental and physiological processes such as gene expression, protein and DNA synthesis, along with cell differentiation and division resulted in expression of growth and development process including direct or indirect organogenesis, breaking seed dormancy, seed germination, flower and fruit development and senescence. These aspects are documented in various scientific reports (Tiburcio et al. 2014, Dey et al. 2014). The present investigation on shoot morphogenesis under the influence of polyamines during in vitro culture practice well resembled to many earlier reports in Saccharum officinalis (Shankar et al. 2011), Wrightia tomentosa (Joshi et al. 2014), Picea abies (Vondrakova, et al. 2015), Malus domestica (Tabart et al. 2015), Aechmea distichantha (Tavares et al. 2015) Echinacea angustifolia (Chae 2016), Stevia rebaudiana (Khalil et al. 2016) Cherry rootstocks (Sarropoulou et al. 2017), Cedrela fissilis (Aragao et al. 2017), Phoenix dactylifera (El-Dawayati et al. 2018).

5.2.6 Effect of heavy metals on in vitro axillary shoots proliferation The advantage of adding various metals to culture media was mainly to evaluate the capability of individual elements to improve the growth and differentiation of axillary bud or undifferentiated callus during in vitro morphogenesis. The regeneration potential of explants is known to be greatly influenced by supply of metals in the media. Metals are part of micro or macro nutrients and are indispensable components of several enzymes (Maksymiec 1998, Cuypers et al. 2013). They act as secondary messengers and help in regulating and controlling plant tissue growth (Niedz and Evens 2007). Heavy metals are a group of metals with a density more than 5.0g cm3. In the present investigation, CN and nodal explants obtained from axenic cultures were inoculated on medium as described in materials and methods with additional supply of ZnSO4,

CuSO4, NiCl2 and CdCl2 to assess their effect on in vitro shoot morphogenesis. Axillary

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Chapter – FIVE DISCUSSION shoot proliferation was observed in both the explants used. Improved regeneration as a result of optimized levels of heavy metals has been reported in several plants including Brassica napus and Nicotiana tabacum (Misra and Gedamu 1989), Withania somnifera (Fatima et al. 2011), Ailanthus altissima (Gatti 2008), Myriophyllum alterniflorum (Delmail et al. 2013), Hypoxis hemerocallidea (Okem et al. 2016), Indocalamus latifolius (Emamverdian et al. 2018) etc.

5.2.6.1 Effect of CuSO4 Copper (Cu) is an essential redox-active transition metal of plant photosystem, plays a vital role in iron mobilization, oxidative phosphorylation and signalling of transcription and protein transferring system. A part from this, it is an essential micronutrient and is required for normal growth and development of plants. It is an essential part of various proteins and enzymes involved in photosynthesis and respiration. In the present investigation, 10.0 μM of CuSO4 gave the maximum regeneration with highest number of shoots in 90% of cultures while at elevated concentrations, it showed toxic effects with hampering shoot growth and number. The stimulatory effect of Cu at optimal level for induction and proliferation of shoots has been well documented in plants as depicted by Dahleen 1995, Sahrawat and Chand (1999), Wojnarowiez et al. (2002), Nirwan and Kothari (2003), Tahiliani and Kothari (2004), Joshi and Kothari (2007), Pourvi et al. (2009), Khurana-Kaul et al. (2010), Arunima et al. (2010), Fatima et al. (2011), Al- Mayahi (2014), Ahmad et al. (2015a), Javed et al. (2017c), Trettel et al. (2018), Zhao et al. (2018). The toxic effect may be because increased concentrations of copper catalyse the production of free radicals leading to the damage of proteins and other biomolecules. Toxic effect of elevated CuSO4 levels on shoot proliferation has been recorded in C. annuum (Joshi and Kothari, 2007), W. somnifera (Fatima et al, 2011), R. serpentina (Ahmad et al. 2015a), Dendrobium kingianum (Prazak and Molas 2015), Dendrocalamus strictus (Singh et al. 2017a).

5.2.6.2 Effect of ZnSO4

Of the various concentrations of ZnSO4 tested, 80μM gave highest regeneration response with 95% shoot induction and multiplication. The positive role of ZnSO4 on morphogenesis may be because Zn is an important part of cytochrome enzyme apparatus with properties as maintenance of ribosomal fractions and plant metabolism by manipulating the actions of hydrogenase and carbonic anhydrase (Tisdale et al. 1984, Hansch and Mendel 2009, Hafeez et al. 2013, Kolade 2017). In vitro grown cultures

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Chapter – FIVE DISCUSSION exposed to various types of stresses and Zn has the ability to reduce such stress by modulating gene expression (Cakmak, 2000, Broadley et al. 2007, Boonchuay et al. 2013). Apart from these, Zn also takes a significant role in various biochemical activities like protein synthesis, carbohydrate breakdown, and repairs of cellular membranes and regulation of auxin production (Marschner, 1995, Tsonev and Cebola Lidon 2012, Mousavi et al. 2013, Mumtaz et al. 2017). The positive role of Zn inducing maximum shoot regeneration has also been reported in various plant species such as Aechmea blanchetiana (Giampaoli et al. 2012), Ruta graveolens (Ahmad et al. 2012b), transgenic plum (Faize et al. 2013), banana (Helaly et al. 2014), Rauvolfia serpentina (Ahmad et al. 2015a, Rauvolfia tetraphylla (Shahid et al. 2016), Ocimum basilicum (Verma et al. 2016), Dendrocalamus strictus (Singh et al. 2017a), Coscinium fenestratum (Das et al. 2018), Vigna radiata (Jamal et al. 2018).

5.2.6.3 Effect of NiCl2

Among all the metals tested Ni 15.0 µM showed maximum shoot induction and multiplication response. This promotion in the multiplication and development of shoots by the additional supply of Ni as NiCl2 may be because nickel ions are a component of several plant metalloenzymes like urease, hydrogenases, glyoxalases and dismutases (Dixon et al., 1975, Boer et al. 2014, Fabiano et al. 2015). Recently, it has been found by careful experimentation that nickel is essential for plant growth and development (Gerendas et al. 1999, Rahman et al. 2005). The ion is a component of urease enzymes, which convert urea to ammonia (Dixon et al. 1975, Witte et al. 2002). The positive effects of Ni in culture media to enhance biomass production in micropropagated plants was reported by several workers (Bidwell et al. 2001, Vinterhalter and Vinterhalter 2005, Vinterhalter et al. 2008, Yan et al. 2008, Buendia-Gonzalez et al. 2010, Sarkar et al. 2010, Buendia-Gonzalez et al. 2012, Hand and Reed 2014, Waoo et al. 2014, Katanic et al. 2015, Ghanavatifard et al. 2018, Wiszniewska et al. 2018, Fourati et al. 2019).

5.2.6.4 Effect of CdCl2 Cadmium has been classified as one of the most toxic elements that negatively influence the plant growth and development due to their high toxicity and ubiquitous presence in the environment. Elevated concentration of Cd lead to decline in photosynthesis and alteration of chlorophyll ratio resulting in reduced net CO2 assimilation (Gill et al. 2012,

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Perveen et al. 2012, Mohamed et al. 2012, Wiszniewska et al. 2015). Although sensitivity of plants to Cd varies with species and concentration (Gallego et al. 2012). Some plants have the ability to tolerate elevated level of Cd. These plants are called metallophytes, having greater potential to tolerate and survive in the metal contaminated area (Dresler et al. 2014, Wojcik et al. 2015, Muszynska and Hanus-Fajerska 2017, Sakhanokho and Kelley 2009, Wiszniewska et al. 2017a, Muszynska et al. 2018). Of the various concentrations (0.5-60.0 µM) of CdCl2 tried, 2.5 μM gave positive result on in vitro shoot multiplication and the effectiveness of cadmium on growth in cultures is in accordance to the report in Gypsophila fastigiata (Muszynska et al. 2018). Shoots obtained at higher concentrations (above 2.5 μM) of CdC12 exhibited abnormalities such as stunting of shoots, yellowing of leaves and browning of the explants. Influence of CdCl2 in micropropagation system has been recently observed in many plants such as Vitis vinifera (Cetin et al. 2014), Hypoxis hemerocallidea (Okem et al. 2016), Arabidopsis thaliana (Sofo et al. 2017), Vaccinium corymbosum (Manquian-Cerda et al. 2016).

5.2.7 Studies of GST activity in heavy metal treated cultures Cultures exposed to elevated concentrations of heavy metal greatly influenced the GST enzymatic activity. A significant increase in GST content was recorded upto the optimal concentration. Enhancement in the level of GST showed adaptability of the cultures for these metals. Commonly induced adaptive responses of plants to heavy metal stress is the accumulation of GST to nullify their negative effect (Nepovim et al. 2004, Bittsanszky et al. 2005, Zhang and Liu 2011, Kumar et al. 2013, Xu et al. 2015,

Nianiou-Obeidat et al. 2017). Among the various concentrations of NiCl2, ZnSO4,

CuSO4 and CdCl2 tested, the optimal level of each metal exhibited up to 100 percent survival response with highest GST activity, while on further increasing the concentration, both the survival potential and GST accumulation declined. The primary response of plant towards the exposure of increased concentration of heavy metal is that it produced ROS, leading to oxidative damage and modification of cellular compartment which resulted in abnormal plant growth and development (Ogawa and Iwabuchi 2001). A possible explanation towards the upregulation of GST activity in response to ROS may be that, at the optimal concentration, detoxification capacity of in vitro grown cultures were highest, the plants upregulated GST activity to protect the cells by scavenging ROS (Zhang and Ying 2008, Haluskova et al. 2009,

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Zhang et al. 2013, Li et al. 2018a, Srivastava et al. 2019, Marrs 1996). Our results are in line with various recent studies showing that stress results in several morphogenic changes in vitro, leading to formation of somatic embryo of Lycium barbarum (Kairong Cui et al. 1999), shoot in flax (Mundhara and Rashid 2001), embryo in Gossypium hirsutum (Kumria et al. 2003) and regeneration in triticale (Immonen and Robinson 2000), tolerance to abiotic stress in transgenic Arabidopsis Plants (Xu et al. 2018).

5.2.8 Indirect organogenesis Induction and expression of organogenic calli might be triggered by different factors like choice of explants and use of phytohormones, a critical factor for indirect organogenesis. In the present study, intermodal (IN), leaf (L), cotyledons (CL) and roots (R) segments were used for callus induction, a varied response was observed with respect to the initiating explants. Usually the juvenile explants taken from embryogenic or meristemetic tissue (young inflorescence, leaves and seedlings) are best for callus induction. Calli induced by leaf explants were green and friable, while those produced by internodal explants were brownish cream and compact. The leaf derived calli were found to be organogenic as they produced many shoots in a greater percentage of cultures when transferred to shoot inducing medium. Dhar and Joshi (2005) also found leaf to be better explant for callus induction among various explants in Saussurea obvallata. Owing to its efficiency, leaf explants have been widely used for shoot regeneration and transformation of many important species (Soorni et al. 2012, Dalila et al. 2013, Jayaraman et al. 2014, Fki et al. 2017). Different concentrations of auxin (2,4-D) added to MS basal medium either individually or in combinations with cytokinins (BA or Kin) provoked callus induction response. The effectiveness of 2,4-D for the formation of callus has been well documented in many plants species (Jayaraman et al. 2014, Sen et al. 2014, Upadhyaya et al. 2015, Cueva Agila et al. 2015, Bhati et al. 2017).The most critical stage of indirect organogenesis is the transition from callus to bud induction stage. MS medium containing 2,4-D alone was not sufficient for further differentiation into buds. The association of 2,4-D and cytokinins was important for transition of callus from one stage to another stage. Development of plantlets through indirect organogenesis using 2,4-D along with cytokinins has been reported in plant Phellodendron amurense (Azad et a. 2009). The ratio between the two PGRs (BA or Kn) constitutes the critical factor that triggers developmental events in vitro (Zhao et al. 2018).

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5.2.9 Effect of subculture on shoot proliferation Rapid shoot multiplication depends on the potential of an explant to divide continually to provide secondary new explants for culture initiation on fresh medium or mass propagation through rapid multiplication on same composition of medium. The period from the initiation of a culture or a subculture to the time of its transfer on fresh medium is called a sub-culturing passage. During micropropagation, subculturing often becomes imperative when the density of cells, tissue or organs becomes excessive; to increase the number of shoots. A reason for subculture is that the growth of plant material/explants in a closed vessel eventually leads to the accumulation of toxic metabolites and the exhaustion of the nutrients in the medium. Thus, even to maintain the culture, all or part of it must be transferred onto fresh medium (Apostolo et al. 2005, Pathak and Dhawan 2012, Saha et al 2016). In the present study, effect of subculture passage was evaluated on shoot multiplication for nodal explant on MS medium supplemented with optimum concentration of PGR and polyamines. In the study, rate of shoot multiplication was found highest at 56 days. Rapid rates of plant propagation depend on the ability to sub-culture shoots from proliferating shoot, from cultures giving direct shoot regeneration, capable of reliable shoot regeneration. The potential of shoot multiplication of in vitro cultures is significantly influenced by sub-culture passages. In general, the effect of sub-culturing passages on shoot multiplication of cultures varies from one species to another (Anis et al. 2012, Siddique et al. 2015, Chhajer and Kalia 2016, Shahzad et al. 2017). A similar effect of enhanced rate of shoot multiplication by subsequent subculturing corroborates with the earlier report on Rauvolfia serpentina (Alatar 2015), Boerhaavia diffusa (Patil and Bhalsing 2015), some medicinal and horticultural climbers (Thomas and Hoshino 2016), Nyctanthus arbor-tristis (Shekhawat et al. 2016), Semecarpus anacardium (Panda et al. 2016) Momordica dioica (Choudhary et al. 2017) Dianthus caryophyllus (Thakur and Kanwar 2018), Tylophora indica (Najar et al. 2018), Abutilon indicum (Seth and Panigrahi 2019).

5.2.10 Effect of different media The successful establishment of micropropagation protocol and to achieve maximum regeneration rate of an explant depends on the selection of a medium particularly suited to the species being propagated but adequate results can usually be produced from well-

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Chapter – FIVE DISCUSSION known formulations (Gentile et al. 2014, Arab et al. 2014, Oberschelp and Goncalves

2016, Nikolova 2017). In the present investigation, different basal media i.e. MS, B5, L2 and WPM with optimal concentration of cytokinin in combination with auxin, polyamines and heavy metals were examined to achieve highest and successful regeneration of explant. Of these, full strength MS (1962) medium in combination with optimum dose of mT (6.5 µM) and Put (10.0 µM) was found to be most suitable for highest multiple shoot induction, while lesser shoots induction was observed in other basal media formulation. The highly significant features of MS (1962) medium are its high nitrate, ammonium and potassium contents. Effectiveness of MS medium over other formulations of medium played a significant role in plant regeneration in number of previous reports (Singh et al. 2016, Shekhawat and Manokari 2016a and 2016b, Moharana et al. 2017, Baskaran et al 2017, Rezali et al. 2017, Hussain et al. 2018a).

5.2.11 Effect of media pH on propagation For in vitro propagation, optimal level of pH of a culture medium concerned with numerous processes including salts concentration, uptake of medium ingredients like mineral nutrients and plant growth regulators, chemical reactions especially those catalyzed by enzymes, and gelling efficiency of agar etc. are responsible for desired results (George et al. 2008a, Martins et al. 2013b, Nikolova 2017). The optimal pH regulates the cytoplasmic activity that affects cell division and growth of shoots and it does not interrupt in the functioning of cell membranes and the buffered pH of the cytoplasm (Karim et al. 2007). Therefore, in the present study, evaluation of different pH levels with the optimal growth regulators supplemented to MS medium for maximum shoot formation has been evaluated. Of these, 5.8 pH was found better than other tested pH levels. However, increase and decrease beyond the optimum pH showed lesser performance of shoot multiplication. It was apparent that pH requirements are species specific and developmental stage dependent. These results are in agreement with other findings in many medicinal plants such as Tylophora indica (Faisal et al. 2007), Ruta graveolens (Ahmad et al. 2010a), Withania somnifera (Fatima et al. 2015), Allamanda cathartica (Khanam and Anis 2018). On increasing or decreasing the pH level beyond the optimal level resulted low shoot proliferation which could be due to the unbalance ratio of alkaline and acidic nature of medium because pH of medium depends on relative proportion of nitrate and ammonium ions (Vuksanovic et al. 2017, Sharma et al. 2018a). Generally, the plant tissues will tolerate a range of 5.0-6.0 pH,

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Chapter – FIVE DISCUSSION whereas lower or higher pH will lead to death of tissues (Araujo et al. 2016, Vuksanovic et al. 2017, Shi et al. 2017, Rawat 2017, Trapp et al. 2018).

5.2.12 Effect of carbon sources Carbohydrate (i.e. glucose, sucrose and fructose) is commonly added into the plant tissue culture medium as an energy source as well as osmotic agent because in vitro shoot and root formation are a highly energy requiring process (Yaseen et al. 2013, Waman et al. 2014, Mazri 2014, Bohra et al. 2016). Among the three carbon sources (sucrose, glucose and fructose) tried, 3% (w/v) sucrose was found optimum for better response. The results clearly indicated that the concentration of sucrose in culture medium had a direct influence on morphogenesis, and this was in agreement with the results on several earlier reports (Jayaraman et al. 2014, Warhade et al. 2015, Nataraj et al. 2016, Bohra et al. 2016, Lukatkin et al. 2017, Ahmed et al. 2017, Martinez et al. 2017, Khanam and Anis 2018). Sucrose is the carbohydrate of choice as a carbon for in vitro plant culture probably, because it is the most common carbohydrate in the phloem sap of many plants (Yaseen et al. 2013, Hartmann and Trumbore 2016). Plants growing under in vitro are semi-autotrophic and leaves formed during this condition may not attain photosynthetic ability (Hazarika et al. 2002, Chandra et al. 2010). Besides, sucrose is easily translocatable, maintains osmotic potential and resistant to enzymatic degradation due to its non-reducing nature (de Paiva Neto Otoni 2003, Lipavska and Konradova 2004, Basu et al. 2007, Resende et al. 2019). Therefore, plants growing under control condition have limited accessibility to CO2 inside the vessel (Bishop et al. 2018). Thus, during in vitro regeneration practice, sugar is augmented as a carbon source to maintain an adequate carbon supply for growth of plant tissue, or plantlets (Arencibia et al. 2017).

5.2.13 Synthetic seed Encapsulation of non-embrogenic propagules has been proposed as a low-cost propagation system for obtaining clonal plants. For this reason, it has become a potential tool for mass propagation of elite genotype with high economic value and rare or endangered taxons species (Ara et al. 2000, Reddy et al. 2012). Successful propagation through encapsulation depends on the selection of a suitable plant material, the critical evaluation of the factors affecting the gel matrix formation and optimization of the procedure of conversion for plant recovery. Nodal segments bearing an axillary

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Chapter – FIVE DISCUSSION bud were found suitable for encapsulation of the species studied in the present work as it has great potential for plant development from pre-existing meristemetic tissues. The use of vegetative propagules for development of synthetic seeds has been reported in numerous medicinal plant species, including Tylophora indica (Faisal and Anis 2007), Rauvolfia serpentine (Ray and Bhattacharya 2008, Faisal et al. 2012b), Ocimum basilicum (Siddique and Anis 2009b) and Spilanthes acmella (Singh et al. 2009). Vegitative propagules are very useful in such plant species, where somatic embrogenesis is not well established or else good quality somatic embryos are not produced (Grzegorczyk and Wysokinska 2011). Effective propagation of plants through encapsulation depends on critical evaluation of concentration of sodium alginate and calcium chloride as they influence the gel matrix and capsule quality. Capsule hardiness depends upon optimal ion exchange of Na+ and Ca2+ and it might change with various propagules as well as with different plant species (Rai et al. 2009). In our study, a 3% solution of sodium alginate upon complexation with 100 mM CaCl2 was found to be the best composition which produced firm, clear and isodiametric beads within an ion exchange duration of 20 min. Lower concentrations of sodium alginate (1-2% w/v) and calcium chloride delayed the polymerization duration, as well as resulting in fragile beads, which were too delicate to handle, while at higher concentrations of both, beads were too hard which causes considerable delay in shoot and root emergence. Lower levels of sodium alginate were not suitable for encapsulation, maybe because of reduction in its gelling ability, after exposure to high temperature during autoclaving (Larking et al. 1988). This differential response occurred perhaps, due to the synergistic impact of alginate and calcium concentration on bead formation. Besides the clear, firm and isodiametric structure of beads, their conversion is the most important aspect for synthetic technology (Kozai et al. 1991). Among the various treatments tested, MS medium supplemented with mT (6.5 μΜ) along with Put (10.0 μM) gave the highest frequency of conversion of encapsulated beads into young shootlets. The results obtained are in agreement with finding of Ahmed et al. 2015, Javed et al. 2017b. Encapsulated propagules can be used for preservation of germplasm and exchange between laboratories only if they retain viability in terms of conversion potential after storage for a reasonable period. The encapsulated beads maintained 71.48% viability even after six weeks of cold storage at 4 ºC. However, only 28.00% conversion frequency was observed after six weeks in non-encapsulated nodal segments. This is in accordance to the findings of Naz et al. (2018) and Ahmad et al. (2012b). However

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Chapter – FIVE DISCUSSION beyond four weeks of storage at 4 ºC, a significant decline in percentage sprouting was noticed. It is assumed that any decline in the conversion frequency detected among encapsulated beads stored at low temperatures may be due to oxygen deficiency, inhibition of plant tissue respiration, lack of nutrients, or accumulation of metabolic waste within the alginate capsule (Redenbaugh et al. 1986, Faisal and Anis 2007, Palanyandy et al. 2015). The conversion of encapsulated nodal segments into plantlets after considerable period of storage could be attributed to matrix of synthetic seed which mimics endosperm of natural seed and provide sufficient nutrients to the encapsulated explants for re-growth (Nieves et al. 1998, Germana et al. 2011, Ganapathi et al. 2001).

5.2.14 Rooting Rooting is the next crucial step after successful regeneration and multiplication of shoots for any micropropagation protocol with an aim to establish the regenerated plantlets in the natural soil. Rooting of microshoots was accomplished both under in vitro and ex vitro conditions.

5.2.14.1 In vitro rooting For the successful establishment of regenerated shoots in natural conditions, shoots produced in vitro, were excised and transferred to root inducing medium. Root inducing medium having various strength of MS medium alone or ½ MS media augmented with different concentration of various auxins. Half strength MS was found better in comparison to other strength of MS. During present study, the root initiation took place in the microshoots, when incubated on all the MS treatments except phytagel. The highest percentage (90%) of root formation was recorded on half MS medium containing IBA. However, IAA or NAA containing MS medium exhibited significantly less rooting in comparison to IBA. In root initiation, dominance of IBA over other auxins has been well documented in Vitex trifolia (Ahmad et al. 2013) Althaea officinalis (Naz et al. 2015a), Erythrina variegata (Javed et al. 2015). Optimum rooting response using IBA has also been reported in several leguminous plant species including Dalbergia sissoo (Vibha et al. 2014), Pithecellobium dulce (Goyal et al. 2012), Vigna mungo (Adlinge et al. 2014), Bauhinia racemosa (Sharma et al. 2017), Cicer arietinum (Singh et al. 2019).

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Root formation has been considered as a single stage procedure in which auxin is documented to play a key role. In the present experiment, the frequency of root formation markedly varied on all the treatments. The MS treated microshoots gave less number of roots but the roots were long, healthy and vigorous. Thus it was observed that MS basal medium (PGR free) accomplished root initiation along with shoot elongation. The capability of MS basal media to stimulate in vitro root formation has also been described by some workers (Wong and Bhalla 2010, Tyagi et al. 2010b, Phulwaria et al. 2012).

5.2.14.2 Ex vitro rooting Rooting is an important aspect in micropropagation system to achieve desire healthy plants. Individual healthy microshoots were excise from in vitro grown cultures and given a short pulse treatment of IBA at various concentrations for 30 min followed by their transfer to plastic pots having sterile soilrite. Optimum results with IBA, concerning ex vitro growth, were probably obtained because this phytoregulator is metabolized into IBA aspartate and posteriorly combined with peptides. This combined form serves as storage of this auxin, which is later gradually released, keeping the concentrations at the ideal levels, especially in the final stages of root formation (Rodrigues and Leite 2004, Martins et al. 2013a). It has been reported that ex vitro rooting is the most preferable approach as it involves one step procedure combining both rooting and hardening, hence ex vitro rooted plants are better suited to tolerate environmental stresses (Patel et al. 2014, Shekhawat and Manokari 2016b). Hence, ex vitro rooting is a capable method that can be applied as an alternate to in vitro rooting, reduces the cost, time and also resources for mass propagation along with establishment of plants from laboratory to field (Rathore et al. 2014a, Shekhawat et al. 2015b). Similar strategies for ex vitro root formation has also been utilized in several species including Cadaba fruticosa (Lodha et al. 2015), Jatropha curcas (Rathore et al. 2015), Momordica dioica (Choudhary et al. 2017), Cassia alata (Ahmed et al. 2017), Lawsonia inermis (Shiji and Siril 2018), Tinospora cordifolia (Panwar et al. 2018), Hybanthus enneaspermus (Shekhawat and Manokari 2018), Raspberry (Lebedev et al. 2019).

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5.2.15 Acclimatization Acclimatization of in vitro regenerated plantlets to natural conditions is a crucial step to develop successful micropropagation protocol. The most important and significant step in the acclimatization protocol is their transition during hardening from in vitro to ex vitro conditions along with consequent field performance (Pospisilova et al. 2007, Kaur 2015, Chugh et al. 2018). Successful acclimatization generally depends on several weeks exposer towards transitional environment, for improvement in survival percentage of in vitro raised plants (Yong et al. 2015, Coopman and Kane 2018). The survival percentage of plants was directly influenced by the planting substrates used. The porosity of soilrite enhances the water holding capacity of the substrate as opposed to vermiculite and garden soil, and thus supports development of roots during hardening. Therefore, roots were easily penetrated in Soilrite than in other planting substrates. Among various types of potting materials used for the acclimatization of plantlets, highest (68%) survival was found in Soilrite and lowest (59.5%) in garden soil after 28 days of transplantation. The potted plants grew well without any detectable phenotypic variation. These results showed consistency with the findings of Eucalyptus tereticornis (Aggarwal et al. 2012), Pistacia vera (Benmahioul et al. 2012), Stevia rebaudiana (Chotikadachanarong and Dheeranupattana 2013), Cassia alata (Ahmed and Anis 2014a). Another condition for successful acclimatization with highest regeneration response included fluctuation in temperature, relative humidity, light intensity during in vitro and ex vitro environments. During this period plants are very susceptible to various stresses, because they have not yet developed adequate patterns of resource allocation, morphological and physiological features required for the new environment (Aremu et al. 2012, Osorio et al. 2012). The micropropagated plantlets grow generally under low light intensity, high level of sugar and nutrient to favour heterotrophic growth with high relative humidity (Hazarika 2003, White et al. 2016). A switch to autotrophy and changes in stomata functioning and cuticle compositions are observed during acclimatization (Pospisilova et al. 1999, Van Huylenbroeck et al. 1998, Kundu et al 2017). Because of these factors, tissue culture- raised plantlets have low photosynthetic rate with an undeveloped photosynthetic apparatus. Regenerated plantlets develop functional photosynthetic apparatus when transferred to ex vitro condition, while high light intensity is not linearly translated to an increase in photosynthetic rate (Amancio et al. 1999). The improvement in photosynthetic pigment contents in regenerated plantlets

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Chapter – FIVE DISCUSSION during acclimatization have been observed by several researchers in various plants (Kadlecek et al. 1998, Pospisilova et al. 1999, Faisal and Anis 2010). The substantial increment in photosynthetic pigment content with high-light intensity suggested that pigment synthesizing enzyme is vital for chlorophyll biosynthesis. There are several studies on initial abrupt decrease in photosynthetic pigment contents during the initial days followed by continuous and subsequent increase during acclimatization processes of several plant species (Faisal and Anis 2010, Perveen et al. 2015, Yang et al. 2018). The abnormalities caused by the extreme ex vitro environmental condition in the culture vessel should be overcome. Therefore, considering the importance of acclimatization step various physiological parameters viz., chlorophyll and carotenoid contents, net photosynthetic rate and carbonic anhydrase activity were also studied in relation to progression of acclimatization process. These criteria would be able to more objective information than agronomic parameters or visual assessment when evaluating for component traits of complex characters. Micropropagation protocol depends on successful acclimatization has been well documented in earlier reports such as Baskaran and Van Staden 2013, Perez et al. 2016, da Silva et al. 2017 and Hussain et al. 2018b.

5.2.15.1 Physiological studies In vitro grown cultures were exposed to various types of stresses including change in humidity, temperature, mode of nutrition, and light intensity. Therefore, tissue culture raised plantlets show abnormal functioning of different physiological processes like adjustment to relative humidity, transpiration rate attributed to CO2 conductance in leaves, abnormal stomatal functioning, poor development of photosynthetic system, controlling the water status of the plantlets when they are transferred from in vitro to ex vitro condition for acclimatization (Yang and Yeh 2008, Flexas and Diaz‐Espejo 2015, Han et al. 2018). These are responsible for poor adaptability of most of the micropropagated plantlets to the garden soil. The acclimatization is the climatic adaptation of the plants that have been transferred to a new environment. Low survival of the in vitro raised plants at the time of their transfer in natural conditions, is generally due to the sudden switch off from heterotrophic mode of life in in vitro culture to autotrophic mode of life as when transfer to soil under greenhouse condition. The above consideration on the basis of adaptability leads to the gradual change in physiological activity including pigment contents (Yang et al. 2018). Chlorophyll

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Chapter – FIVE DISCUSSION contents are essential factors in photosynthetic process and are highly sensitive to stresses. Hence in the present investigation, the photosynthetic pigments like chl a, chl b, total chl and carotenoid contents were evaluated during the acclimatization period. During acclimatization the micropropagated plants showed a marked reduction in the chl a, chl b and total chl contents during the initial days, thereafter, chlorophyll contents were increased during the successive days of acclimatization. Similar results have also been documented in earlier reports (Faisal and Anis 2010, Perveen et al. 2015, Yang et al. 2018). Explanation behind fluctuation in chlorophyll content is that in vitro condition of regenerated plants are poor in chlorophyll content and the enzymes responsible for photosynthesis are inactive or absent altogether. Many reports suggested that stomata of in vitro regenerated plantlets are non- functional because they are unable to close under in vitro conditions (Hazarika 2006, Monja-Mio et al. 2015, Maleki 2017). Reduction of photosynthetic traits might be due to stomatal limitations as suggested by the correlation of net photosynthetic rate, stomatal conductance, and inter cellular CO2 concentration (Galmes et al. 2007, Gao et al. 2016, Galmes et al. 2017, Xiong et al. 2018). However, photosynthesis can be correlated with stomatal and non-stomatal factors of regenerated plantlets. The low photosynthetic rate of in vitro plantlets have been largely attributed to non-stomatal factors such as low chlorophyll concentration and decrease in RUBISCO activity or malformation of chloroplast (Oh et al. 2012, Hoang et al. 2017, Yang et al. 2018). Net photosynthetic rate decreased in acclimatized plants in the first week after transplantation and increased thereafter (Siddique and Anis 2008, Chandra et al 2010, Naz et al. 2017). Therefore, successful acclimatization gave better opportunity for regenerated plants to cope with these harsh conditions and developed a better photosynthetic apparatus by various structural and functional modifications in plants for better survival in natural habitat (Siddique and Anis 2009a and 2009b, Shahid et al. 2016, Khanam and Anis 2018, Ahmad et al. 2018a and 2018b).

5.2.15.2 Biochemical studies During acclimatization, micropropagated plantlets showed various physiological and biochemical abnormalities due to the stress they experience from sudden changes in the environment from in vitro to ex vitro. The stresses including change in humidity, temperature, mode of nutrition, and light intensity are probably the main factors promoting production of reactive oxygen species (ROS). ROS are inevitable byproducts

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Chapter – FIVE DISCUSSION of aerobic metabolism which caused lipid peroxidation and consequently membrane injuries, protein degradation, enzyme inactivation, damage of DNA, therefore, ROS production and removal must be controlled (Batkova et al. 2008, Shafi et al. 2015). Plants scavenge ROS through the antioxidant system employing the enzymes and non- enzymatic components of sub cellular compartments. To counter the hazardous effects of ROS, plant cells develop a complex antioxidant defence and enzymatic scavenging system composed of antioxidant enzymes and metabolites such as superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase activity (CA) etc. Such defence system may be inadequate during over production of ROS (Foyer and Mullineaux 1994, Ahmed and Anis 2014a and 2014b, Perveen and Anis 2015). In the present investigation, plantlets of M. pruriens acclimatized in culture room conditions exhibited a significant increase in SOD, CAT and CA activity with the passage of acclimatization period up to 28 days. These results are in accordance with the findings of Fatima et al. (2013) who reported that when SOD, CAT, and CA were consistent and in harmony with each other, stress developed during micropropagation in plantlets could be reduced and kept at a lower concentration which exerted the plant to raise and metabolize naturally hence plants with higher content of antioxidants with increased natural defence mechanisms are usually better adapted to stress. During the detoxification of stress, SOD is thought to be the first line of defence against ROS species. Superoxide is reported to reduce ferric ion to ferrous ion, which then reacts with H2O2 to form DNA reactive hydroxyl group and causes dismutation of superoxide into less harmful H2O2 (Gill and Tuteja 2010, Gill et al. 2015).

At the same time CAT scavenges H2O2 by converting it into O2 and H2O in peroxisomes during photo-respiration of plants (Scandalios 1990, Shankhdhar and Shankhdhar 2017). The increase in SOD activity during acclimatization period resembles with the study of Gerbera jamesonii (Chakrabarty and Datta 2008), Ocimum basilicum (Siddique and Anis 2009b), Rauvolfia tetraphylla and Tylophora indica (Faisal and Anis 2009, 2010), Tecomella undulata (Varshney and Anis 2012), Vitex trifolia and Cassia alata (Ahmed and Anis 2014a), Albizia lebbeck (Perveen and Anis 2015) and Plantago algarbiensis and Plantago almogravensis (Goncalves et al. 2017) and changes in CAT activity were observed by Van Huylenboeck et al. (1998) in Spathiphyllum and Calathea, Racchi et al. (2001) in Quercus robur, Siddique and Anis (2008) in Ocimum basilicum, Faisal and Anis (2009, 2010) in Rauvolfia tetraphylla and Tylophora indica and Varshney and Anis (2012) in Tecomella undulata, Ahmed and

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Anis (2014a and 2014b) in Vitex trifolia and Cassia alata, Perveen and Anis (2015) in Albizia lebbeck, Naz et al. (2015) in Cassia occidentalis, Goncalves et al. (2017) in Plantago algarbiensis and Plantago almogravensis. Similarly CA activity was assessed during acclimatization process and was found to increase during hardening process. The enzymes play a determinant role in transport and exportation of sugar with the plant (Aragon et al. 2005, Ahmad et al. 2012a, Fatima et al. 2013). Therefore, increase in antioxidative enzymes (SOD, CAT and CA) during acclimatization of plantlets reflects the determining ability of the plants to survive against the oxidative stress and its detoxification by the antioxidant defence mechanism and an upregulation of these enzymes in this species would help to reduce the stresses and play an important role for better environmental adaptation of transplanted plantlets from in vitro conditions. It seems that the species studied showed physiological and biochemical behaviour in ex vitro conditions. The results of the study demonstrate that functional photosynthetic machinery developed in micropropagated plants effectively reduced oxidative stress during acclimatization period.

5.2.16 Genetic fidelity Another significant feature of in vitro production procedures of plantlets for marketable purposes is the genetic stability of the plants. Micropropagated plantlets are frequently subjected to genetic variation due to culture stress leading to formation of somaclones (Larkin and Scowcroft 1981, Bairu et al. 2011, Qin et al. 2018). Prolonged culture, explant types with its source of origin and propagation strategies, and repetitive subculturing might resulted in epigenetic or somaclonal variations in the in vitro raised cultures (Mujib et al. 2013). Actual origin of somaclonal variation in micropropagated plants may be caused because of many reasons which include variations in auxin- cytokinin ratio or concentration, time duration, culture condition and stress developed during culture practice (Lestari 2016, Khan et al. 2018). During culture, explants are exposed to various oxidative stresses leading to formation of highly unstable reactive oxygen species (ROS), a DNA damaging agents (Jackson et al.1998, Wilhelm et al. 2005, Krutovsky et al. 2014, Bhattacharyya et al. 2017a and 2017b). Among the regenerated plants recognition of appropriate somclonal variations at the phenotypical, cytological, biochemical and molecular levels have been well documented by many scientists (Akdemir et al. 2016, Martínez-Estrada et al. 2017, Delgado-Paredes et al. 2017, Qin et al. 2018). The occurrence of cryptic genetic effects

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Chapter – FIVE DISCUSSION arising via somaclonal variation in the regenerants seriously limits the broader utility of micropropagation system (Rani and Raina 2000, Bairu et al. 2011, Ahmed et al. 2017, Thorat et al. 2017). Genetic fidelity of micropropagated plants has immense practical utility and commercial implications to develop a suitable protocol for production of genetically identical and stable plants (Fatima et al. 2012, Ahmad et al. 2013, Faisal et al. 2018a and 2018b). Therefore, it is compulsory to asses genetic consistency of in vitro raised plants and establishes the stability of micropropagated plant. To evaluate genetic variations or stability in in vitro regenerants, RAPD and ISSR molecular markers have been used as powerful tool and have become more popular as they don’t require any prior DNA sequence information (Phulwaria et al. 2013a,Yadav et al. 2013, Ahmed et al. 2017, Hussain et al. 2018a). In this study PCR amplification by using a set of RAPD and ISSR primers was carried in 10 randomly selected in vitro raised plants for detecting genetic fidelity among regenerated plantlets of M. pruriens. All the responsive primers tested produced similar bands showing the genetic uniformity in regenerated plantlets. The monomorphic banding pattern of 10 randomly selected regenerants lead to the conclusion that M. pruriens plants are genetically true to the plant of origin. Efficacy of ISSR as a molecular study has been rightly acknowledged in various plant species like Dendrocalamus strictus (Goyal et al. 2015), Cassia occidentalis (Naz et al. 2016), Nothapodytes nimmoniana (Prakash et al. 2016), Cassia alata (Ahmed et al. 2017), Platanus acerifolia (Kuzminsky et al. 2018), Abutilon indicum (Seth and Panigrahi 2019), Prunus cerasifera (Nasri et al. 2019). This established the appropriate protocol development for raising the homogenous population of a desired herb through micropropagation of M. pruriens. These results were further supported by earlier reports where RAPD markers were shown to be a valuable parameter for conservation of genetic integrity amongst plants in Cuphea procumbens (Fatima et al. 2012), Eclipta alba (Singh et al. 2012), Vitex trifolia (Ahmad et al. 2013), Terminalia bellerica (Dangi et al. 2014), Morus alba (Saha et al. 2016), Date Palm (Modi et al. 2017), Lawsonia inermis (Moharana et al. 2018), Olea europaea (Bradai et al. 2019). High multiplication frequency, molecular and phenotypic stability ensures the efficiency of the protocol developed for production and conservation of this commercially important medicinal plant.

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SUMMARY AND CONCLUSIONS

Mucuna pruriens L. (Fabaceae) is an important tropical legume. It is recognized as an aphrodisiac in Ayurvedic medicine. All parts of the plant possess valuable medicinal properties and have found wide application for symptomatic relief of Parkinson’s disease and mental disorder. In nature the species propagates only through seeds. However, propagation via seeds poses problem due to high allergic properties of dense hairs present on pod surface that cause uncontrolled itching while handling the seeds. The conventional propagation through seed is not an adequate solution to meet the demand of this species. Recently there has been an increased interest in in vitro culture techniques which offer a viable tool for mass propagation, germplasm preservation for prized medicinal plants. The current investigation was aimed to develop a reproducible, cost effective protocol for mass production of M. pruriens using nodal and cotyledonary node explants. The metal tolerance potentiality of the legume plant was also analysed through micropropagation and an antioxidative enzyme activity (Glutathione-S- transferase) was measured during in vitro culture practice. Changes in photosynthetic parameters and antioxidative enzymes were also assessed during ex vitro acclimatization of micropropagated plantlets. The genetic stability among regenerants was assessmed using DNA based molecular markers like RAPD and ISSR.

The main findings of the investigation are summarized below;

The best seed germination (95%) was recorded on the moist cotton while media containing half-strength MS resulted in 80% seed germination. Cotyledonary node and nodal segment explants obtained from 7 days old aseptic seedlings were used for in vitro axillary bud differentiation and de novo regeneration via callus phase. Between the cotyledonary and nodal segment, nodal explants produced best response yielding highest number of shoots with maximum shoot length. Direct shoot regeneration was achieved on MS medium augmented with BA, Kn, 2-iP or mT singly or in combinations with polyamines (Putrescine, spermine or spermidine) or auxins (IAA, IBA or NAA). Among the tested cytokinins, mT gave the highest regeneration in both the explant. While combined treatment of mT (6.5 µM) and Putrescine (10.0 µM) produced maximum number of shoots. Chapter – SIX SUMMARY AND CONCLUSIONS

An indirect regeneration system has been standardized using leaf segments excised from 7 days old axenic seedlings on MS medium supplemented with different concentration of 2,4-D (0.5-20 µM). The callus induced on different treatments showed variation in colour and texture and best callogenesis was observed on 5.0 µM 2,4-D after 28 days. The callus when transferred to MS medium supplemented with 6.5µM mT+10 µM Put produced maximum shoot (6.60 ± 0.51) with shoot length (5.22 ± 0.11 cm) per clump after 56 days of transfer. The effect of heavy metals like Cu, Zn, Ni and Cd on direct shoot regeneration from aseptic nodal explant was evaluated on the optimal medium standardized for shoot induction and multiplication. NiCl2 (15µM) was found best as it showed maximum shoots (34.6 ± 0.56) with shoot length (7.22 ± 0.05 cm) in 95% cultures when incubated on MS medium augmented with 6.5 µM mT and 10.0 µM Put. The biochemical observations of the metal raised shootlets showed an increase in GST activity with 100% survival on the optimal concentration of tested metals.

The effect of different basal medium (MS, L2, WPM and B5), different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) and carbon sources was also examined on the optimal MS medium standardized (MS + 6.5 μM mT + 10.0 μM Put). The full MS medium, 5.8 pH and 3% sucrose was found to be most suitable for in vitro regeneration and production of maximum shoots in nodal explants. In order to obtain synthetic seeds, different concentrations of encapsulation matrix (Sodium alginate and complexing agent calcium chloride) were tested for conversion of encapsulated nodal explants into plantlets. Fine quality synseeds were produced in 3.0% sodium alginate and 100 mM CaCl2. MS medium containing mT (6.5 μM) and Put (10.0 μM) gave maximum shoot conversion frequency (91.60 ± 0.81%) of encapsulated nodal segments into plantlets. The encapsulated nodal segments could be stored at low temperature (4 °C) with 37.00 ± 1.58% conversion frequency even after 70 d of cold-dark storage. Induction of roots in regenerated shoots was readily achieved with various auxins (IAA, IBA or NAA) by both in vitro and ex vitro methods. In vitro rooting was best achieved in half-strength MS medium supplemented with IBA (0.20 μM) where maximum roots were induced after 28 d of culture. However, for ex vitro rooting, proximal end of the shootlets dipped in IBA (90.0 μM) solution for 30 min gave the highest root formation prior to their transfer to potting mixture (Soilrite).

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The regenerants were successfully acclimatized and established in pots containing Soilrite with 90% of survival in growth chamber. During initial days of acclimatization, a steady decrease in photosynthetic pigments (Chl a/b and carotenoids) was recorded upto 7 days. However, the contents got increased throughout hardening time under greenhouse conditions. Similarly, the anti-oxidative enzyme activities (SOD, CAT and CA) were also found to be significantly increased. Evaluation of photosynthetic pigments and antioxidants has been shown to be important in determining the ability of the plants to survive oxidative stress and play an essential role for better adaptation of regenerated plantlets to natural environment. RAPD and ISSR markers were used to assess the genetic stability of micropropagated plantlets. The tested primers produced monomorphic banding pattern in the regenerated plants, thus confirming the genetic uniformity of the micropropagated plantlets.

The finding of my study leads to the following conclusions;

1. Best seeds germination (95%) was recorded on cotton wetted with DDW after 7 days of inoculation. 2. Aseptic nodal segment explants responded better in comparison to cotyledonary node explants for achieving maximum shoot regeneration response. 3. MS medium amended with a combination of mT (6.5 µM) + Put (10.0 µM) at pH 5.8 was found to the best media for maximum shoot multiplication from nodal explants. 4. Combined effect of optimized cytokinins (BA, Kn, 2-iP or mT) in combination with auxins (IAA, IBA or NAA) gave antagonistic response towards multiplication of shoots. 5. Among three polyamines tested, 10.0 µM Put in combination with 6.5 µM mT was found to be best for shoot induction and multiplication.

6. Among the various heavy metal tested, NiCl2 (15.0 µM) gave positive effect towards shoot regeneration and growth on MS medium containing mT (6.5 µM) +

Put (10.0 µM) + NiCl2 (15.0 µM) after 56 d of culture. 7. Among the concentrations of TDZ tried, 0.8 µM TDZ supplemented to MS medium was effective for maximum induction of shoots from nodal explant after 56 d of culture.

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8. TDZ exposed explants when transferred to MS medium containing 10.0 µM Put gave better multiplication rate in both explants. 9. The encapsulated nodal segments showed a maximum conversion frequency on MS medium supplemented with mT (6.5 μM) and Put (10.0 μM) and the synseeds retained their viability even after 70 d of storage at 4 ºC. 10. Highest in vitro rooting efficiency in microshoots was observed on ½ MS medium supplemented with 0.20 μM IBA after 28 days. 11. Ex-vitro rooting was successfully achieved when distal end of microshoots were pretreated with 90.0 μM IBA for 30 min followed by their transfer in cups containing Soilrite for 28 days. 12. Regenerated plantlets were successfully acclimatized on Soilrite followed by their transfer to greenhouse where 90% survival rate was observed. 13. The successful acclimatization of micropropagated plantlets suggested the development of functional photosynthetic machinery alongwith auxiliary enzymatic scavenging system. 14. Genetic uniformity of micropropagated plants was confirmed using PCR-based DNA fingerprinting techniques.

The present study describes development of a practicable regeneration system for mass multiplication of genetically stable progenies of Mucuna pruriens which can be reintroduced into the original or favourable habitats for cultivation, conservation and sustainable development. The regenerated protocol is simple, reproducible and efficient which can be explored for mass propagation of valuable medicinal plants and conservation of rare and elite germplasm also. Further, the plantlets developed through this study will help in scavenging the heavy metal contamination in waste and agricultural lands. The results of the investigation can be utilized for commercialization/production of secondary metabolites round the year particularly, the Dopamine for pharmaceutical preparations. This amenable study will provide new innovative ideas with alternate biotechnological strategies for gaining the desired genetic improvements and pharmaceutical discoveries of more important compounds in Mucuna pruriens and other leguminous plant species.

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Cosic T, Motyka V, Raspor M, Savic J, Cingel A, Vinterhalter B and Ninkovic S (2015) In vitro shoot organogenesis and comparative analysis of endogenous phytohormones in kohlrabi (Brassica oleracea var. gongylodes): effects of genotype, explant type and applied cytokinins. Plant Cell Tissue and Organ Culture 121:741-760. Costa MBT, Arruda AS, Vieira MC, Paula MSP and Luz JMQ (2017) In vitro establihment of Ochroma pyramidale in different concentrations of MS medium and sucrose. Revista Agrotecnologia-Agrotec 8:1-9. Cueva-Agila AY, Guachizaca I and Cella R (2015) Combination of 2, 4-D and stress improves indirect somatic embryogenesis in Cattleya maxima Lindl. Plant Biosystems- An International Journal Dealing with all Aspects of Plant Biology 149(2):235-241. Cuypers A, Remans T, Weyens N, Colpaert J, Vassilev A and Vangronsveld J (2013) Soil-plant relationships of heavy metals and metalloids. In Heavy metals in soils. Springer, Dordrecht 161-193. Da Silva JAT and Jha S (2016) Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review. Plant Cell Reports 35: 2207-2225. Da Silva JAT, Cardoso JC, Dobranszki J and Zeng S (2015a) Dendrobium micropropagation: a review. Plant Cell Reports 34:671-704. Da Silva JAT, Hossain MM, Sharma M, Dobranszki J, Cardoso JC and Songjun ZENG (2017) Acclimatization of in vitro-derived Dendrobium. Horticultural Plant Journal 3:110-124. Dahleen LS (1995) Improved plant regeneration from barley callus cultures by increased copper levels. Plant Cell, Tissue and Organ Culture 43:267-269. Dalila ZD, Jaafar H and Manaf AA (2013) Effects of 2,4-D and kinetin on callus induction of Barringtonia racemosa leaf and endosperm explants in different types of basal media. Asian Journal of Plant Sciences 12(1):21-27. Dangi B, Khurana-Kaul V, Kothari SL and Kachhwaha S (2014) Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers. Physiology and Molecular Biology of Plants 20:509-516. Danova K, Motyka V, Todorova M, Trendafilova A, Krumova S, Dobrev P and Evstatieva L (2018) Effect of Cytokinin and Auxin Treatments on Morphogenesis, Terpenoid Biosynthesis, Photosystem Structural Organization, and Endogenous Isoprenoid Cytokinin Profile in Artemisia alba Turra In vitro. Journal of Plant Growth Regulation 37:403-418. Dao GH, Wu GX, Wang XX, Zhuang LL, Zhang TY and Hu HY (2018) Enhanced growth and fatty acid accumulation of microalgae Scenedesmus sp. LX1 by two types of auxin. Bioresource Technology 247:561-567.

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Dewir YH, Murthy HN, Ammar MH, Alghamdi SS, Al-Suhaibani NA, Alsadon AA and Paek KY (2016) In vitro rooting of leguminous plants: difficulties, alternatives, and strategies for improvement. Horticulture, Environment, and Biotechnology 57:311- 322. Dewir YH, Naidoo Y and da Silva JAT (2018) Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Reports 37:1451-1470. Dey A, Gupta K and Gupta B (2014) Role of Polyamines in Plant– Pathogen Interactions. Plant Adaptation to Environmental Change: Significance of Amino Acids and their Derivatives 12:222. Dhar U and Joshi M (2005) Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew (Asteraceae): effect of explant type, age and plant growth regulators. Plant Cell Reports 24(4):195-200. Dhindsa RS, Plumb-Dhindsa P and Thorp TA (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany 32:93- 101 Dias MC, Monteiro C, Moutinho-Pereira J, Correia C, Gonçalves B and Santos C (2013) Cadmium toxicity affects photosynthesis and plant growth at different levels. Acta Physiologiae Plantarum 35:1281-1289. Dinani ET, Shukla MR, Turi CE, Sullivan JA and Saxena PK (2018) Thidiazuron: Modulator of Morphogenesis In vitro. In Thidiazuron: From Urea Derivative to Plant Growth Regulator Springer, Singapore 1-36. Dixon NE, Gazzola C, Blakeley RL and Zerner B (1975) Jack bean urease (EC 3.5. 1.5). Metalloenzyme. Simple biological role for nickel. Journal of the American Chemical Society 97:4131-4133. Dobranszky J, Jambor-Benczur E, Remenyi ML, Magyar-Tabori K, Hudak I, Kiss E and Galli ZS (2005) Effects of aromatic cytokinins on structural characteristics of leaves and their post-effects on subsequent shoot regeneration from in vitro apple leaves of ‗Royal Gala‘. International Journal of Horticultural Science 11:41-46. Dresler S, Bednarek W and Wojcik M (2014) Effect of cadmium on selected physiological and morphological parameters in metallicolous and non-metallicolous populations of Echium vulgare L. Ecotoxicology and Environmental Safety 104:332-338. Du Jardin P (2015) Plant biostimulants: definition, concept, main categories and regulation. Scientia Horticulturae 196:3-14. Du Y, and Scheres B (2017) Lateral root formation and the multiple roles of auxin. Journal of Experimental Botany 69:155-167. Dwivedi RS and Randhawa NS (1974) Evaluation of rapid test for the hidden hunger of Zinc in plants. Plant Soil 40: 445-451.

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El-Dawayati MM, Ghazzawy HS and Munir M (2018) Somatic embryogenesis enhancement of date palm cultivar Sewi using different types of polyamines and glutamine amino acid concentration under in-vitro solid and liquid media conditions. International Journal of Bioscience 12:149-159. Elias H, Taha RM, Hasbullah A, Mohamed N, Manan AA, Mahmad N and Mohajer S (2015) The effects of plant growth regulators on shoot formation, regeneration and coloured callus production in Echinocereus cinerascens in vitro. Plant Cell, Tissue and Organ Culture 120:729-739. Emamverdian A, Ding Y, Mokhberdoran F and Xie Y (2018) Growth Responses and Photosynthetic Indices of Bamboo Plant (Indocalamus latifolius) under Heavy Metal Stress. The Scientific World Journal 2018:1-6. Engelmann F (2004) Plant cryopreservation: progress and prospects. In vitro Cellular & Developmental Biology, Plant 40:427-433. Erland LA, Shukla MR, Glover WB and Saxena PK (2017) A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture. Plant Cell Tissue and Organ Culture 131:459- 470. Esteves P, Clermont I, Marchand S and Belzile F (2014) Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism. Plant Cell Reports 33:871-879. Evlard A, Sergeant K, Ferrandis S, Printz B, Renaut J, Guignard C and Campanella B (2014) Physiological and proteomic responses of different willow clones (Salix fragilis X alba) exposed to dredged sediment contaminated by heavy metals. International Journal of Phytoremediation 16:1148-1169. Fabiano C, Tezotto T, Favarin JL, Polacco J and Mazzafera P (2015) Essentiality of nickel in plants: a role in plant stresses. Frontiers in Plant Science 6:754. Faisal M and Anis M (2007) Regeneration of plants from alginate encapsulated shoots of Tylophora indica (Burm. F.) Merrill., an endangered medicinal plant. Journal of Horticultural Science and Biotechnology 82:351–354 Faisal M and Anis M (2009) Changes in photosynthetic activity, pigment composition, electrolyte leakage, lipid peroxidation, and antioxidant enzymes during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets. Plant Cell, Tissue and Organ Culture 99:125-132. Faisal M and Anis M (2010) Effect of light irradiations on photosynthetic machinery and antioxidative enzymes during ex vitro acclimatization of Tylophora indica plantlets. Journal of Plant Interactions 5:21-27.

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Faisal M, Ahmad N and Anis M (2007) An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant. Plant Biotechnology Reports 1:155-161. Faisal M, Ahmad N, Anis M, Alatar AA and Qahtan AA (2018a) Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems. Saudi Journal of Biological Sciences 25:273-277. Faisal M, Alatar AA, Ahmad N, Anis M and Hegazy AK (2012a) An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers. Applied Biochemistry and Biotechnology 168:1739-1752. Faisal M, Alatar AA, Ahmad N, Anis M and Hegazy AK (2012b) Assessment of genetic fidelity in Rauvolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4 C. Molecules 17:5050-5061. Faisal M, Alatar AA, El-Sheikh MA, Abdel-Salam EM and Qahtan AA (2018b) Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi. Industrial Crops and Products 118:173-179. Faisal M, Alatar AA, Hegazy AK, Alharbi SA, El-Sheikh M and Okla MK (2014) Thidiazuron induced in vitro multiplication of Mentha arvensis and evaluation of genetic stability by flow cytometry and molecular markers. Industrial Crops and Products 62:100-106. Faisal M, Siddique I and Anis M (2006a) An efficient plant regeneration system for Mucuna pruriens L.(DC.) using cotyledonary node explants. In vitro Cellular & Developmental Biology, Plant 42:59-64. Faisal M, Siddique I and Anis M (2006b) In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens–a valuable medicinal plant. Annals of Applied Biology 148:1-6. Faize M, Faize L, Petri C, Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ and Hernandez JA (2013) Cu/Zn superoxide dismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum. Journal of Plant Physiology 170:625-632. Fan H F, Du CX and Guo SR (2013) Nitric oxide enhances salt tolerance in cucumber seedlings by regulating free polyamine content. Environmental and Experimental Botany 86:52-59. Fatima N and Anis M (2012) Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal. Physiology and Molecular Biology of Plants18:59-67. Fatima N, Ahmad N and Anis M (2011) Enhanced in vitro regeneration and change in photosynthetic pigments, biomass and proline content in Withania somnifera L.

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(Dunal) induced by copper and zinc ions. Plant Physiology and Biochemistry 49:1465-1471. Fatima N, Ahmad N and Anis M (2012) In vitro propagation of Cuphea procumbens Orteg. and evaluation of genetic fidelity in plantlets using RAPD marker. Journal of Plant Biochemistry and Biotechnology 21:51-59. Fatima N, Ahmad N and Anis M (2016) In vitro Propagation and Conservation of Withania somnifera (Dunal) L. In Protocols for In vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, Second Edition, Humana Press New York, NY 303-315. Fatima N, Ahmad N, Ahmad I and Anis M (2015) Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L. Applied Biochemistry and Biotechnology 177:118-136. Fatima N, Ahmad N, Anis M and Ahmad I (2013) An improved in vitro encapsulation protocol, biochemical analysis and genetic integrity using DNA based molecular markers in regenerated plants of Withania somnifera L. Industrial Crops and Products 50:468-477. Ferreira LT, de Araujo Silva MM, Ulisses C, Camara TR and Willadino L (2017) Using LED lighting in somatic embryogenesis and micropropagation of an elite sugarcane variety and its effect on redox metabolism during acclimatization. Plant Cell, Tissue and Organ Culture 128:211-221. Fki L, Kriaa W, Nasri A, Baklouti E, Chkir O, Masmoudi RB and Drira N (2017) Indirect somatic embryogenesis of date palm using juvenile leaf explants and low 2, 4-D concentration. In Date Palm Biotechnology Protocols. Humana Press, New York, NY 1:99-106. Fki L, Masmoudi R, Kriaa W, Mahjoub A, Sghaier B, Mzid R and Drira N (2011) Date palm micropropagation via somatic embryogenesis. In Date palm biotechnology, Springer, Dordrecht 47-68. Flexas J and Diaz-Espejo ANTONIO (2015) Interspecific differences in temperature response of mesophyll conductance: food for thought on its origin and regulation. Plant, Cell & Environment 38:625-628. Foo PC, Lee ZH, Chin CK, Subramaniam S and Chew BL (2018) Shoot Induction in White Eggplant (Solanum melongena L. Cv. Bulat Putih) using 6- Benzylaminopurine and Kinetin. Tropical life sciences research 29:119-129. Fotopoulos S and Sotiropoulos T E (2004) In vitro propagation of the peach rootstock: the effect of different carbon sources and types of sealing material on rooting. Biologia Plantarum 48:629-631.

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def

cde

def

cde

def

cdef

0.04

0.02

0.13

0.05

0.07

0.02

0.06

0.24

0.23

0.06

0.09

0.21

0.07

0.13

0.25

0.00

1.04

1.34

1.50

1.44

1.16

1.42

1.66

1.58

1.38

1.46

1.88

2.52

2.12

1.70

0.00

Mean ± SE Mean

(cm)

Shoot length Shoot

bcd

cde

bcd

56 days 56 days

0.37

0.24

0.00

0.20

0.00

0.24

0.20

0.24

0.00

4.20

5.40

6.40

6.00

5.00

4.80

6.00

6.60

6.40

5.80

6.20

6.80

7.80

6.80

6.60

0.00

Mean ± SE Mean

per explant

No. of shoots No.

(2.5 (2.5 μM) on multiple shoot regeneration from

of of

cdefg

defg

efg

cde

cdef

efg

defg

length

0.88 ± 0.08 0.88 ±

1.00 ± 0.00 1.00 ±

1.30 ± 0.13 1.30 ±

1.14 ± 0.06 1.14 ±

0.96 ± 0.04 0.96 ±

0.92 ± 0.04 0.92 ±

1.08 ± 0.04 1.08 ±

1.44 ± 0.26 1.44 ±

1.36 ± 0.19 1.36 ±

1.04 ± 0.04 1.04 ±

1.22 ± 0.06 1.22 ±

1.68 ± 0.20 1.68 ±

2.32 ± 0.08 2.32 ±

1.92 ± 0.14 1.92 ±

1.52 ± 0.24 1.52 ±

0.00 ± 0.00 0.00 ±

Mean ± SE Mean

(cm)

Shoot

significantly different DMRT (p using 0.05) = significantly

28 days

0.24

0.20

0.00

0.24

0.00

0.20

0.24

0.20

0.00

0.24

0.20

0.24

0.20

0.00

3.40

4.20

5.00

4.60

4.00

3.80

4.60

5.20

5.00

4.60

4.80

5.20

6.20

5.40

5.20

0.00

Mean ± SE Mean

per explant

No. of shoots No.

Response

20.0

15.0

10.0

7.5

5.0

0.0

Spd

Effect Effect of various concentrations of Polyamines with optimum concentration

cotyledonary node explants on MS medium explants node cotyledonary

20.0

15.0

10.0

7.5

5.0

0.0

Spm

Polyamines (μM) Polyamines

Values represents means ± SE. Means followed by thenot within letters are same columns by ± SE. followed Values Means represents means

20.0

15.0

10.0

7.5

5.0

0.0

Put Table