Use of proximal hypocotyl segment for high-through put transgenic development of tomato

Achieving high-through put and efficient regeneration are the top priorities of any Agrobacterium mediated plant transformation experiments to develop large numbers of in vitro transformants. The type of explant plays a critical role in shoot regeneration efficiency. In the present investigation, an attempt was made to study the effect of various segments of hypocotyl and abaxial/adaxial orientation of cotyledon explants on regeneration efficiency in tomato. A plant transformation vector, pGRNAi-harboringds RNA expressing construct stargeted to two genes of Helicover paarmigera, serine protease and chymotrypsin independently were used to transform tomato. Of the three segments of hypocotylsobtained from 12 day old seedlings, the proximal (closest to shoot apex) segment had yielded highest regeneration (28.65%)compared to the middle (11.86%)and the distal segments (11.20%). In cotyledon explants, those incubated with their abaxial surface in contact with media exhibited highest regeneration (20.83%) compared to adaxially placed cotyledon explants. However, of the two-explant types, hypocotyls had higher regeneration compared to cotyledons. The molecular characterization of putative transformants through PCR and Southern blot analysis revealed the presence of the transgene. Thus, these results will aid in obtaining high-throughput regeneration in transformation of tomato in particular and other crops in general.

Best explant for tomato transformation is the proximal portion of hypocotyl where highest regeneration efficiency was obtained.

Abaxial incubation of cotyledon yielded higher regeneration than adaxial.