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initially maintained in ~onviron"" growth cllaniber 2nd li~ter in tlie P2 facility especially
designed for growlng rra~isgi.nics. A total of I I plants w~lli UiC,?;lAb ,ind 9 pla~its w1t11
SB77 genes were obta~iicd. Molec~~lar analysis of tlic>e pi~ti~t~ve tr,ins!'o~nia~its was done
irlitinlly by GUS hisrocliemicnl assay followed by conlir~untion by PCR and Souther11
blot analy~is. Soutlicrn blotting \\as done by 11o1i-rad~o~ict~vc ~iicthod by us~ng tile
coniniercially available no~l-rnd~o;ict~ve AlklJlios clirect 1nbtl111g iir fro111 A~ncrsl~a~n
(USA). PCll a~iiplificat~o~l of tlpill, uiiiA, B/Ci),lilD and SB1'1 gc~ics \r;;is carried out for
preliminary screening of the putatlvc transformar~ts, About (10% of [lie Iputall\e
transformants sho\ved positivc reaction for PCR for ~rpiil, 70% for ~iidi\ genes, 30% for
BtCrylAb and 10% for SET1 gciic. Factors affecting restriction of geno~nic DNA such as
enzyme conci'ntration, watcr and BSA were opt~~ilizcd. Southern blot analysis of
B~CrylAb piarits sliowsd 70% ofpla~~ts ivill~ /lp/Il and BIC/yMb gene integrat~ons.
In co~iclus~on tlic rcgencratio~i protocol developed during tlic course of this study,
was tbi111d to be very eflic~cnt slncc culli~re coiid~r~ons for all tile stages of rcge~~cration
and recovery of 111 vim grown plants tllat incl~ded seed geriiiirlatio~~, rnultiple shoot
induction, elongatio~~, rooting hardening ;ind tra~isplantatio~i were optinlized, l'h~s
protocol w:is effect~\,cly i~scd for succeasl'i~l geilctic trn~~ifor~~iatiu~~ of cllickpca w1t11
insecticidal genes like BiCrjIAb and .SUTI genes. Follow~~ig tliis protocol, 40% of the
sclccted putat~\c transgenic plants can be obtn111cd 111 per~ud of 90 to 100 days. In the
present study, over 30tr;1nsgen1c plants carrqing U/C',),IAb wcrc obtained. Eight of these
pl:iiits arc being 11in111taincd Sor cspcri~~ic~ir;~l iuorl< 011 illsect b~oassayi and field trials.