Tissue culture-independent in planta transformation strategy: an Agrobacterium tumefaciens-mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.)

Abstract

The major constraint in cotton improvement has been the recalcitrance of cotton varieties to tissue culture. Alternate methods that avoid/ minimize tissue culture would be beneficial for the improvement of cotton. In this report, transgenic cotton plants have been produced by a tissue-culture independent Agrobacterium tumefaciens - mediated transformation procedure. Agrobacterium strain LBA4404 harboring the binary vector pKIWI105 that carries the genes for β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) was used for transformation. Apical meristem of the differentiated embryo of the germinating seedling is infected with Agrobacterium. Since the transgene is integrated into the cells of already differentiated tissues, the T₀ plants will be chimeric and stable integration can be seen only in the T₁ generation. The first proof of transformability in the T₀ generation was indicated by the GUS histochemical analysis of the seedlings, five days after co-cultivation and subsequently in the pollen and lint. T₁ transformants were identified by PCR analysis and subsequently confirmed by Southern. Three plants (T₁) with single copy insertions were selected for continuing into the next generations. Molecular characterization and GUS expression analysis (histochemical and fluorimetric) of the T₁, T₂ and T₃ generation suggested the feasibility of the method to generate transgenic plants in cotton.