Development of an Efficient Regeneration and Transformation System for Commercially Grown South African Cotton Cultivars
Oct 8, 2018

Biomol - Biotechnology WCRC WCRC2

While establishing a system for genetic transformation of South African cotton cultivars, a method of regeneration and transformation of the shoot apical meristem of seedlings was developed. Shoot apices from young cotton seedlings (Gossypium hirsutum cvs Sabie, Jassid, Palala and 107/1) were placed onto Murashige and Skoog (MS) basal medium (pH 5.8) containing MS vitamins, 2% sucrose, 0.7% agar and 0.1 mg/L kinetin. Within two weeks of culture, the shoot meristems differentiated into multiple green shoots and spontaneous root regeneration occurred two months later. Plantlets were placed in soil and hardened off in the growth chamber. Four months later the plants had grown to maturity and cotton bolls had formed. This regeneration protocol demonstrated a successful technique for the recovery of cotton plants from tissue culture using the shoot apical meristem. Shoot apical meristems of seedlings were used for transformation and were co-cultivated with Agrobacterium tumefaciens containing the phosphinothricin acetyltransferase (pat) and the neomycin phosphotransferase (npt II) genes. After co-cultivation, selection was carried out on regeneration medium containing 100 mg/L kanamycin and 1 mg/L glufosinate ammonium (PPT). Control explants were completely inhibited from growing on the selection medium, whereas transformed explants gave rise to multiple shoots resistant to PPT and kanamycin after four weeks. The integration of the pat gene in the transformed plant DNA was confirmed by polymerase chain reaction

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