SOMATIC EMBRYOGENESIS THROUGH CELL SUSPENSION CULTURE IN INDIAN COMMERCIAL CULTIVARS OF BANANA (MUSA SPP.) THESIS SUBMITTED TO BHARATHIDASAN UNIVERSITY FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY By A. AKBAR P.G. AND RESEARCH DEPARTMENT OF BOTANY JAMAL MOHAMED COLLEGE (AUTONOMOUS) (ACCREDITED AT ‘A’ GRADE BY NAAC- CGPA 3.6 OUT OF 4.0) TIRUCHIRAPPALLI- 620 020 TAMIL NADU, INDIA MARCH - 2011 Date: DECLARATION I do hereby declare that this work entitled “SOMATIC EMBRYOGENESIS THROUGH CELL SUSPENSION CULTURE IN INDIAN COMMERCIAL CULTIVARS OF BANANA (Musa spp.)”, has been originally carried out by me under the guidance of Dr. A. Shajahan, Lecturer (SG), PG and Research Department of Botany, Jamal Mohamed College, Tiruchirappalli- 620 020, and this work has not been submitted elsewhere for any other degree. (A. AKBAR) Dr. A. SHAJAHAN Lecturer (SG) & Principal Investigator (UGC Major Project) P.G. and Research Department of Botany Jamal Mohamed College (Autonomous) (Accredited at ‘A’ Grade by NAAC- CGPA 3.6 Out of 4.0) Tiruchirappalli- 620 020 Tamil Nadu, India Date: CERTIFICATE This is to certify that the thesis entitled “Somatic embryogenesis through cell suspension culture in Indian commercial cultivars of Banana (Musa spp.)”, submitted to Bharathidasan University, Truchirappalli- 620 020, for the award of the degree of Doctor of Philosophy in Botany, is an authentic record of original work carried out by Mr. A. AKBAR under my guidance and supervision during the study period at the PG and Research Department of Botany, Jamal Mohamed College, Thiruchirappalli- 620 020, Tamil Nadu, India. I further certify that no part of this thesis has been submitted any where else for the award of any degree, diploma, associateship fellowship or other similar titles to any candidate. (A. SHAJAHAN) Fax : +91 431 2331235 * Phone : +91 431 2422563 (Resi) * Mobile : + 944387431 * Email : [email protected] GRATITUDE First of all, I would like to thank the Lord Almighty for his benevolent blessing and abundant mercy which enabled me to complete this piece of work in successful manner. An accomplishment requires the effort of many people and this work is not different. I didn’t ask for it to be over, but then again, I never asked for it to begin. For that’s the way it is with life, as some of the most beautiful days come completely by chance. But even the most beautiful days eventually have their sunsets. Now its time to look back and remember those faces with a sense of gratitude. I express my deepest sense of gratitude to my Research Supervisor, Dr. A. Shajahan, Senior Lecturer, PG & Research Dept. of Botany, Jamal Mohamed College, for his encouragement, valuable suggestions and support extended throughout my project work. I thank Dr. S.Uma (Principal Scientist), Head of Crop Improvement Division, National Research Center for Banana (ICAR), Trichy, who has been a backbone to my success in completion of this work. Her untiring help, constant guidance and support extended throughout my work has made this possible. I am profoundly grateful to Janab, M.J. Noorden Sahib, President, Janab. A.K. Khaja Nazeemudeen Sahib, Secretary and Correspondent, Janab Hajee. K.A. Khaleel Ahamed Sahib, Treasurer, Assistant Secretary and the management committee members of Jamal Mohamed College for their encouragement and also providing facilities to complete this research work. I am grateful to Janab. Hajee. Dr. M. Sheik Mohamed, Principal Jamal Mohamed College for this motivation towards research and providing lab facilities to complete this work. I extend my gratitude to Dr. S. Ahmed Johan, Head, PG & Research Dept. of Botany, Jamal Mohamed College for providing lab facilities during my research. I wish to express my heartiest gratitude to Dr. M.N. Abubacker, Head Dept. of Botany, National College for his guidance throughout this research work. My sincere thanks to my professors Prof. A. Syed Ali, Prof. M. Purushothaman, Dr. A. Khaleel Ahamed, Dr. S. Mohamed Salique, Dr. Muhamed Ilyas, Dr. M. Ghouse Basha, Dr. M. Kamaraj, Dr. R. Ravikumar, Dr. K. Mohamed Rafi, Mr. A. Aslam, for their encouragement and constant support during the course of my research. A special thanks to Dr. M.S. Saraswathi (Scientist, SC) and Dr. S. Backiayarani (Senior Scientist), National Research Center for Banana (ICAR), Trichy, for their encouragement and valuable suggestions throughout my project work. I am deeply indebted to several persons for their tireless assistance. Without their aid and support, this research present format of thesis would not have been possible. Here, I would like to express my gratitude to Dr. P. Durai (Technical Officer), Dr. B. Sudhakar (Research Associate) Mr. A. Kaliappan (Research Associate), Mr. R. Mohanraj, Mr. Periasamy, Mr. Peter, Mr. Balu, Mr. S.A. Shafi, Mr. A. Saravana Kumar, Mr. A. Saravanakumar, K.P. Hidayath, Mr. Arun, Mr. K.P. Sajith, Mr. M. Muthusamy, Mr. Punniakotti, Mr. Kannan, Mr. K. Ravi, Mr. C. Sourdar Raju, Mr. Senthil Kumar, Miss. K. Udhayanjali, Miss. Mr. P. Rahul Gandhi, R. Chitra devi, Mrs. Sarasamma, Mrs. Bakiyam and Mrs. Sarasu. Last but not least, I would like to thank to my family members for their constant support to complete my research. ABBREVIATION % - percentage µg - microgram µl - microlitre µM - micromole 2,4-D - 2,4-diclorophenoxy acetic acid ABA - abscisic acid AS-AFLP - allele specific amplified fragment length polymorphism BAP - 6-benzyl adenine BSV - banana streak virus BBMV - banana bract mosaic virus BBTV - banana bunchy top virus bp - base pair (s) C - celsius cm - centimeter CMV - cucumber mosaic virus CTAB - N-Cetyl-N,N,N-trimethyl ammonium bromide cv - cultivar dNTP - deoxy nucleotide triphosphate ECS - embryogenic cell suspension FAO - food and agricultural organization FFF - photosynthetic photon flux G - gram g - gravity (relative centrifugal force) GA3 - gibbrellic acid h - hour INIBAP - international network for the improvement of banana and plantain ISSR - inter simple sequence repeats IEDCs - induced embryogenic determined cells kg - kilo gram Kin - kinetin L - litre m - meter M - molar mg - milligram min - minute ml - millilitre mm - millimeter mM - millimolar MS - murashige & skoog’s medium MW - molecular weight NAA - α – naphthalene acetic acid NAD - nicotinamide adenosine dinucleotide NADP - nicotinamide adenosine dinucleotide phosphate PCR - polymerase chain reaction PCV - packed cell volume PEMs - proembryogenic masses PEDCs - pre-embryogenic determined cells PVP - polyvinyl pyrrolidone RAPD - random amplified polymorphic DNA RFLP - restriction fragment length polymorphism rpm - revolutions per minute RH - relative humidity SE - somatic embryo SH - schenk and hildebrant medium STRs - short tandem repeats SSR - simple sequence repeats s - second TAE - tris acetate ethylene diamine tetra acetic acid TBE - tris borate ethylene diamine tetra acetic acid TE - tris - ethylene diamine tetra acetic acid TDZ - thidiazuron Tris - tris hydroxyl methyl amino methane UV - ultra violet V - volt v - volume w - weight CONTENTS Page No. 1.0. Introduction ...................................................................................................... 1 1.1. Taxonomy........................................................................................................... 2 1.2. Origin, Evolution and Diversity ......................................................................... 2 1.3. Indian commercial cultivars of banana .............................................................. 4 1.4. Production constraints in Banana....................................................................... 6 1.5. Conventional method of propagation ................................................................ 6 1.6. Importance of non conventional methods .......................................................... 7 1.6.1 Importance of in vitro system in improvement of Musa ..................... 8 1.6.2. Somatic embryogenesis ....................................................................... 8 1.6.3. Somaclonal variations in Musa............................................................ 9 1.6.3.1. Identification of genetic variation........................................... 10 1.6.3.2. Use of microsatellite marker................................................... 10 1.7. Objectives.................................................................................................. 11 2.0 Review of literature.................................................................................. 12 2.1. General Description............................................................................................ 12 2.2. Somatic embryogenesis...................................................................................... 13 2.2.1. Phenotypic plasticity in plants ............................................................. 14 2.2.2. Morphogenic competence and morphological expression................... 14 2.2.3. Unicellular and multicellular origin of somatic embryos .................... 15 2.2.4. Application of somatic embryogenesis................................................ 16 2.2.4.1. Important applications ............................................................ 16 2.2.4.2. Other applications ................................................................... 16 2.3. Factors affecting somatic embyrogenesis .......................................................... 17 2.3.1. Effect of genotype ............................................................................... 17 2.3.2. Effect