Study of IN VITRO regeneration and transformation parameters for the development of transgenic Tomato (Solanum Lycopersicum L.)

Abstract

Investigation was carried out to develop an in vitro regeneration protocol using three locally grown farmer popular tomato varieties namely BARI Tomato 2, BARI Tomato 9 and Bahar. Later Agrobacterium mediated transformation study was performed to find out the best transformation parameters using these three varieties along with BARI tomato 7 variety. Tomato seeds were sterilized with 24 hours continuous shaking as it took less time for germination and cause less damage to embryo. During in vitro culture BARI tomato 2 and BARI tomato 9 variety showed better response in presence of 1 mg/l BAP (germination percentage 96%, 13 days for germination initiation and 24.8 shoots/explant and germination percentage 93%, 12.6 days for germination initiation and 19 shoots/explants respectively). Bahar variety respond well in 1 mg/l BAP + 0.1 mg/l IAA containing MS media combination (germination percentage 100%, 10 days for germination initiation and 39.6 shoots/explant). All three varieties showed cent percent rooting response in ½ MS + 0.2 mg/l IAA. However, there was no significant difference in rooting response in relation to shooting media. Seeds collected from well ripened fruits of in vitro regenerated plants showed 95-100% viability in germination test. Thus, indicates the reproducibility of the protocol. Transformation efficiency was found to be increased with the increase of optical density of Agrobacterium suspension. All the varieties gave 80-100% GUS positive expression in GUS assay at 0.62 OD600 and minimum 47% was found in BARI tomato 9 variety with 0.49 OD600. In the present study 20 minutes of incubation period with higher OD600 (0.62) gave better transformation in BARI tomato 2, BARI tomato 7 and BARI tomato 9 varieties. Interestingly, Bahar variety produced highest number of GUS positive explants (98%) in 30 minutes incubation period at similar OD. Among the co- cultivation periods 48 hours was found best for all four varieties in transient GUS assay. The highest regeneration percentage in presence of antibiotic (kanamycin, 150 mg/l) following transformation with pBI121 found to be 56% in Bahar variety. Again, when transformation was done by pH7WG2_OsNHX1_1.6 highest regeneration was achieved (23%) in Bahar variety when cultured in hygromycin, more than 4 mg/l.