High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciens

Abstract

A protocol for consistent production of transgenic cotton plants in three Indian varieties was established utilizing Agrobacterium-mediated transformation. Shoot tip explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA 4404. The strain harbors a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Regeneration potential of explants or different hormones was studied in detail. Among the different combinations of BAP and NAA tested, 0.1 mg l −1 of BAP and NAA in the medium influenced efficient regeneration of shoots by organogenesis. Shoot bud proliferation and elongation was achieved in 3–4 weeks time on medium supplemented with GA 3. The putatively transformed shoots were harvested and placed for rooting on medium containing IBA and 75mg l −1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Molecular analysis of the field established plantlets was carried to confirm the transgenic nature. The presence of GUS and NPTII genes in the transgenic plants was verified by histochemical GUS assay and polymerase chain reaction (PCR) analysis, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. A total of 70–75 transgenic plants were raised in pots. Progeny analysis of these plants showed a classical Mendelian pattern of inheritance.