Development of an efficient method for regeneration and Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) cv. HS6

Cotton (Gossypium hirsutum L.; Family: Malvaceae) is an important industrial crop worldwide. Because of the available non-effective regeneration protocols, transformation efficiency of cotton cultivars has remained poor. In the present study, a highly efficient embryo apex (EA) based Agrobacterium-mediated cotton transformation and regeneration protocol was developed. The EA of cotton cv. HS6, selected as explants, were cultured on Murashige and Skoog (MS) medium containing combinations of various plant growth regulators [6-benzylaminopurine (BAP), kinetin (Kin) & naphthalene acetic acid (NAA)] to induce shoot regeneration. The regeneration percentage was nearly 2-fold higher when MS medium was supplemented with BAP (2.0 mg/L), Kin (1.5 mg/L), proline (250 mg/L) and charcoal (200 mg/L). The age of explants, bacterial inoculum, incubation time of explants in bacterial suspension and co-culture conditions were optimized. A total number of 70 explants were inoculated with Agrobacterium encapsulated with pGreen-GFP construct. After co-cultivation and shoot induction, 14 explants were GFP positive. The presence of transgene (nptII) was confirmed by PCR and the GFP was in situ localized in the transformed cotton by confocal microscopy. The results obtained with this reproducible protocol clearly demonstrated that transformation efficiency and regeneration of G. hirsutum cv. HS6 were much higher compared to the earlier reports.