Abstract：In order to develop a high effective gene transformation protocol of cotton hypocotyl pieces via Agrobacterium tumefaciens, an alflafa antifungul peptide gene (alfAFP) was used as a foreign gene, and effect factors of transformation, including Agrobacterium strains, bacterium density, infection time, acetosyringone (AS) concentration in co-culture medium, co-culture time and co-culture temperature, were devalued by the expression detection of GUS and hygromycin resistant gene in transgenic callus. Optimum conditions for transformation was that cotton hypocotyl pieces from 5 to 7 days-old sterile seedlings were dipped into Agrobacterium LBA4404 suspension (OD600=0.5-0.7) for 10-15 min, then were transferred on co-culture medium with 200µmol/L As and were co-cultured at 21℃ for 60h in the dark. The most amount of transgenic calli was gained by culturing hypocotyl pieces on callus introduction medium with 50mg/L hygromycin. 15 regenerative plants were developed from somatic embryos differentiated from transgenic calli on embryogenesis medium. PCR and PCR-southern detection proved that the antifungal gene was introduced in 12 regenerative plants. They might be used as germplasm resources for breeding resistant-fungal disease cotton in the near future.
