Transformation of Egyptian Cotton Tissue (Gossypium barbadense) Using Agrobacterium tumefaciens
Oct 8, 2018
Callus induction from hypocotyl and cotyledonary explants of Egyptian cotton (Gossypium barbadense), extra-long staple (Giza 45, Giza 77) and long staple (Giza 85), were evaluated for in vitro callus induction and maintenance utilising two media formulations (G2) and (CI). Giza 85 produced significantly better callus than Giza 45 and Giza 77. However, induction of callus was genotype dependent and highly variable, not only among varieties, but also among explants of the same variety. Attempts to control tissue contamination and decay of calli were successful. Explants from three varieties were tested for regeneration using G2 and CI media containing different levels of kanamycin to test for tolerance level in callus tissue. Callus induction and growth were inhibited at 25 mg/L kanamycin. Cotyledon and hypocotyl tissues of the three varieties were transformed successfully, and transformation efficiency varied by variety and type of explant. Cotyledon and hypocotyl explants from 12-day-old aseptically germinated seedlings were inoculated with a non-oncogenic Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid vector pBI121 with a chimeric nopaline synthase (NOS) promoter driving the neomycin phosphotransferase (NPT II) gene and a CaMV35S promoter driving the -glucuronidase (GUS) gene. After three days co-cultivation, explants were placed on callus induction medium containing 25 mg/L kanamycin for selection of transformed tissue. The presence of (NPT II) in crude cellular extracts from embryogenic callus tissue was detected by ELISA test. Expression of the GUS gene was confirmed histochemically followed by dot blot analyses to screen for DNA integration using an Xba I- EcoR1 fragment containing GUS gene coding sequences as probe.