Studies on the induction of variation through in vitro culture in Jatropha curcas L. Thesis submitted to the Cochin University of Science and Technology under the Faculty of Science in partial fulfilment of the requirement for the degree of Doctor of Philosophy In Biotechnology by JIKKU JOSE Reg. No. 3475 Plant Biotechnology Laboratory Department of Biotechnology Cochin University of Science and Technology Cochin-682022 Kerala, India January 2013 geaw//v”% MM//MW """" N DEPARTMENT OF BIOTECHNOLOGY COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI-682 022. INDIA Phone : 91-484-2576267, Fax: 91 -484-2577595. e-mail: padmanambisanOcusat.ac.in Dr. PADMA NAMBISAN Associate Proflessor (Plant Biotechnology) CERTIFICATE This is to certify that the research work presented in this thesis entitled “Studies on the induction of variation through in vitro culture in Jatropha curcas L.”, is based on the original research work carried out by Ms. Jikku Jose under my guidance and supervision in the Department of Biotechnology, Cochin University of Science and Technology. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and that no part thereof has been presented for the award of any other degree. diploma, associateship or other similar titles or recognition. Drl Padma Nambisan C USAT. 30-0] -20l3. DECLARATION l hereby declare that the work presented in this thesis entitled “Studies on the induction of variation through in vitro culture in Jatropha curcas L.”, is based on original research work carried out by me at the Department of Biotechnology, Cochin University of Science and Technology, Cochin under the guidance of Dr. Padma Nambisan, Associate Professor, Department of Biotechnology, Cochin University of Science and Technology, Cochin, Kerala, India and that no part thereof has been presented for the award of any degree or diploma, associateship or other similar title or recognition. / %<>?Jikku Jose CUSAT, 30-0|-2013. flC7(9VowL£r1>g~£9na&:9v'T ‘I7iis piece qfworli fias 6een accomplis/iedwitli tfie flfmigfity god, His fifiessings and .'His power tflat worliwitfiin me andalfso witli tlie peopfie fie/iind my life for inspiring, guiding and accompanying me tfirougli tfiicliand tfiin. It is a péasant moment to eJQ7ress my fieartfeft gratitude to a[[ I dedicate tfiis page to eacii and everyone wfio fiave Iiefped me to eaqalbre tfie expanses of kpowkdge. I tafie immense pkasure to express my sincere and deep sense of gratitude to my supervising guide and mentor, Dr. Tadma _‘]V'am6isan, flssociate professor, Department of fBiotecfino[ogy, Cocliin ‘University of Science and ‘Tecfinofogy for /ier sustained entfiusiasm, creative suggestions, motivation and exempfizry guidance tfirougfiout tlie course of my doctoraf researcfi. I am deepfi grateful to fierfor providing me necessary facilities and exceiient supervision to complete tfiis worlé Her passion for worfl and zeaf for scientific excellence fias exceptionaffy inspired and enric/ied my growtli as a student and a researc/ier as well" I qfier my profound gratitude to Dr. Sarita Q‘. <Bfuzt, j4ssociate (Professor and Head, Dept. of (Biotec/inofog, Cocfiin ‘University of Science and ‘fecfinofog for Iier vafualik suggestions tfirougfiout my (Pfi.D. I would [i/{e to aclinowledge Dr. C. .5‘. (Paulbse, Trof M. Cflandrasekaran, Dr. ‘Hyas K, K, and Dr. ‘E. ‘Vzjayan,fzcu[ty memfiers of t/ie Department for tlieir advice and support in tfie tenure of my worfi I woufl like to extend my sincere tfianfis to Cocfiin University of Science and Tecfinofogy and ’U_(,‘C 9/louflzna flzadfellbwsfiip, govt. of India for supporting tfiis work wit/i financiaf asistance. I express my gratitude to all" tfie autfiorities and stafi‘ in tfie Department of Diotecfinolbgy andCocliin ‘University of Science and ‘fecfinolbgy for tfieir fiefp and co—operation. I acknowfizifge my tfianlis to stajfs of fltfiufya Ladies 7{oste[ of tfie ’Universityfor tfieir co—operation rendered during my stay. I wisli to extend my gratitude to Dr. Srilianta (Panigrafii, Director general: Car5on Minus India, IISD, for fiis motivation and support tlirougliout my tfiesis work, I woufif like to express my sincere gratitude to Dr. 9r1ariamma ‘Vincent, Department of ‘I-‘orein flmguages, for fier support and encouragement tfirougfiout my (MD study. 1 am wordless in expressing my sincere gratefitfness for constant encouragement of Wlr. Sajit/i (Panyaran, MD of Wtizlag Intemationafgroup, gcc,, wfio fias Iiefped me to see and practice life aftogetlier in a difierent perspective, to stay focused and to go after my dreams. I extend my sincere tfianfis to Dr. Jayapragasam ‘EDT, Coimfiatore, Dr. gopafgumar and Dr. ‘I/idyasagar of 7O4l’U, Tfinssur. Dr. Murafid/iaran and Dr. Wicky Xavier of 7(_<1-"GU, (Peecfii. Dr. Jayac/iandran of M7(’U, Dr. Jefiasingfi, Dr. Manofiaran, Dr. Sujatlia, Dr. Mafialaksfimi qfM7(’U, Madurai, Dr. geetlia of Lafiflznd for tlieir /ielp and encouragement during tfie period of my researcli I would like to express my gratitude to Dr. Latfia, Director qfjawafiarlaf Wefiru ‘fropicaf (Botanic garden Researcfi Institute, (Pabde, ‘Tri'vandrum and Dr. Krisfinan, Head, Department of (Biotecfinobgy, _7W"17B_(,“R_I for pnwizling me facilities to do a part of my work, in your reputed researcfi institute. I am greatfy imflzfited to Dr. Satfieesfikymar, for fiis encouragement, guidance and timely fielp tfirougfiout my study. I also appreciate tfie lielp and pkasant working atmospfiere from alTmem5ers qfDioproduction Eifioratory, ‘TD_(,'fR[. I sincerefy acknowfedge my senior colleagues Dr. ’Unni£nLsfinapi'lEzi, for fiis va[ua6le suggestions and encouragement -w/iicli fiave definitefy fiefped to improve my researcfi work, I also extend my tfianks to Dr. Swapna. I wouldfikg to e;q7ress my speciaftfianks Qf gratitude to my coflegues Mrs. Jasmine (gs/iy, Mrs. /'Inita fPinlieiro, Mrs. .7{a5ee6a K, Ms. ‘Elizalietfi Matfiew, Ms. ‘Ifiresia Regimof, Mrs. /'lrinnia flnto, Mrs. Sudfia, Mrs. Soumya (P5, Ms. 7(_iran Laks/imi and Ms. flnafli for tfieir fiefp, support, interest andvafuafile fiints. I express my tfianks to Dr. Sreeja Cfiefiappan, Dr. Madfiu, Dr. Soorej M Dasfieer, Dr. Jasmin C, Dr. *RajeeuE,umar, Dr. fPra5fiu, Dr. Jissa Q ’I(_risfina, Dr. Deena (PS, Ms. Rpsefin, Mr. G’ Kanfiifieyan, Mr. Cities/i Q’C, Mr. .S'ajan, Mr. Doks Ms. Manjufiz and Ms. Dindftya of Microfiiaf ‘Tecfinoflagy Lafioratory, Mr. Siju M ‘I/argfiese, Mrs flipfionsa ‘Vijaya Ms. Jina jlugustine, Mr. Rag/iu[.S‘u6in .5‘, Ms. ifefvin ‘Vincent, Ms. Smitfia 5, Ms. Mridufiz, Ms. Linda, Ms. .’l{an'sree, Ms. Laacmi, Mr. Wolifir and Mr. Satfieesfi Kumar M1(_qfMicro6ia[ genetics Lafioratory, Mr. Manzur flli <P<P, Mrs. Sapna 7(, Mr. flfiraliam Matfiew, Mr. Ramesfz Kumar and Mrs. ‘Reléfia of I mmuno Teclinolbgy Lafioratory, Dr. Dinoy, Dr. flmee, Dr. flnu, Dr. Sfierine, Dr. flnju, Dr. (Pretty, Dr. _7o6in, Dr. fPeeyusfi, Dr. Jes, Dr. Wandu, Dr. Korali, Dr. Smijin, Mrs. flnita, Mrs. Cliindu, Mr. Jayanarayanan, Mr. Wijif, Ms. ‘Ros/iini, Ms. Sliifpa and Mr. fljayan of Neuroscience Lafioratories and M..S'c. students of tfiis Department for tlieir friendsfiip, lielp and co-operation. ‘ffiankyou a[T for making my days at C"U.S‘fl‘T - a wonferfizf experience witfi your affection amffriemivfiip tfiat eacfi one fias sfiowerezf on me. I woulif like to tfiank_ 94:. Rpsfiny diafiy ‘Ifiomas, for nice compary in tlie liostef and’ making my stay very cfiee1_fii[ It is witfi great pleasure to express my sincere gratituzfe and appreciation to my dear frienzfr Ms. Meera, Mrs. ’Vanz{ana, Ms. flfima, Mrs Lakslimi, Mrs. <Dfianya, 9/ls Siiia, Ms. (Deeptfii, Mr. (Binoy, Mr. ®intu KG’, _‘Mr. .5'i6i, Mr. Krisfinakymar, Mr. flnu flnanzfanzfmr. Sifi[of_79V’17Bg9{I, Mr. Cliristo Rajan of 4{CC,, Mr. Wlieffiu ancf9Wr_7a66er anifms. flnusfia of (Department of Statistics, Mr. Jafieer, Mrs. ‘Teena (Department qffconomics, Mr. _(,'opi, Ms. Renjusfia, Ms. Cassie, Mr. Kiran, Mr. 7(_n'sfinaz{as anz{9Vlrs. Knsfinaveni, wfio motivateifanif stoozf Ey me in a[T my efforts Juring tfie course of my work, I wiff never forget tfie times you fiave spent to make me livefy and clieerfuf even in tfie cfouzfiest of my Jays. I tfiank_ a[T tfie teacliers of my scfioof Jays, graiuation anif post-gratfuation for inspinng ancf encouraging me. 1 was exjtremefy fortunate to Have sucli a fovefy family. Myjoy knew no 6ounz£s in expressing tlie fieartfeft tlianks to my Eefovezf parents, 91/lr. ]ose flosepfl and Mrs. Mariamma Jose. ‘lfiey fizft no stones untumear in making my career successfiif, resuft orientezfamffiumane, witfi enJuring and patient fi1lT manner tliey stoo¢{6y me Juring tfie ups amf downs of my life. ‘Ifiey fiave fieen se_l'fEss in giving me t/ie fiest of everytfiing anzf I express my deep gratitude for tfieir love witliout w/iicli t/its work_wou&{ not fiave Eeen compétear. I am deeply imfefitecf to my cfiar sister, Mrs. Jincy (Biju ‘Ifiomas amf firotlier in law Mr. (Biju (P ‘fliomas for motivating me in ejfectivety working anfaccomplisfiing my goa[ I tIiank_my niece little Maria, wlio was always Eefiinzf me witfi tfiousanir of curious questions on my tfiesis work; am{ my nepfiew C/ins 6a6e. I wisli anJ pray for tfie Blessings of my eternafgranzfparents wfio, /ias afways Been my guiding star tlirougfiout tfle journey of my life. jllro, I acknowfizdge all my refatives anzf fizmify fiiemfs for tfieir goof-will" ancf wisfies. Tinally, I woufif like to tfiank everyfioafy wfio was important to tfie successfuf realization of t/iis tfiesis. Inconvenience Jeeply regrettezffbr not mentioning anyfiozfy in my flcknowédgment. ABBREVIATIONS % Percentage ~< ApproximatelyLess than > Greater than 2 Greater than or equal to ‘C degree Celsius pg/mL Micrograml milliliter uL Microlitre pM Micromolar pm micrometre 2, 4-D 2,4-Dichlorophenoxyacetic acid AFLP Amplified fragment length polymorphism ANOVA Analysis of variance AS Adenine sulphate ASSR Anchored simple sequence repeats ATP Adenine triphosphate BA benzyladenine BAP 6-Benzylaminopurine Cbp ControlBase pair CA Chlorogenic acid cm Centimeter Conc. Concentration CRD Completely randomized design CTAB Cetyl trimethylammonium bromide C uSO.