Optimization of Agrobacterium-mediated genetic transformation of shoot tip explants of green gram (Vigna radiata (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0-20 mu M) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 mu M BAP with 100 % shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on A 1/2 MSB5 + 2.46 A mu M IBA and about 90 % of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301 + AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 A mu M acetosyringone in dark at 23 A degrees C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T-0 plants. Stable GUS activity was detected in flowers of T-0 plants and leaves of T-1 plants. PCR analysis of T-1 progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.