Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) via vacuum infiltration

An Agrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirstum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension of Agrobacierium tumefaciens strain GV3101 carrying tDNA with the GUS gene, encoding beta-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochernical GUS assay was done. 46.6% and 20% GUS activity was noted with the vacuum-infiltration and common Agrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis of the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.