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.
