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Research Paper | Biological Engineering | India | Volume 3 Issue 11, November 2014 | Rating: 6.3 / 10
Agrobacterium Mediated Transformation of a Pure Line Variety of Hot Pepper, RCL 59M
P. Raghunathachari | Shashi Kiran Nivas [2] | L. D`Souza [2]
Abstract: Seeds of a pure line variety chilli RCL59M were germinated on sterile media. Intact-hypocotyl explants were prepared from 12-14 days old seedlings and cocultivated with Agrobacterium tumefaciens strain LBA4404 containing the binary vector pRMP9, 770bp of chilli leaf curl virus (ChLCuV) coat protein gene cloned between Hind III and Bgl II sites in the multiple cloning sites (mcs) region. After co-cultivation the explants were transferred to shoot regeneration medium (SRM) supplemented with 250 mg/l of augumentin and kanamycin (25 mg/l). The regenerating shoot buds were rooted. Molecular analysis was carried out. All PCR positive plants were transferred to pots with coco peat. PCR was followed by Southern analysis to confirm the transgenic status of the putative chilli transformants. In the present study, chilli leaf curl virus coat successful transformation was confirmed by Southern hybridization. Seven out of twelve plants tested by Southern blotting, a 0.77 kb band of coat protein gene was released confirming the transgene integration. Rooted plants transferred to soil and the transgenic plants were monitored upto T2 generation. The transgenic plants showed normal phenotype and produced fruits similar to that of non-transgenic plants.
Keywords: Agrobacterium tumefaciens, Southern blotting, transformation, binary vector,
Edition: Volume 3 Issue 11, November 2014,
Pages: 2147 - 2150