Induction of Multiple Shoot Differentiation and Increasing Transformation Frequency in Shoot Tips of Egyptian Cotton
Oct 8, 2018
Multiple shoot differentiation and regeneration from cotton (Gossypium barbadense L.) shoot tips of aseptically-grown seedlings was developed on Murashige and Skoog media supplemented with 100 mg/L myo-inositol, 1.0 mg/L thiamin-HCL, 2% glucose, 1X Gamborg vitamin, 100 mg/L citric acid, 100 mg/L ascorbic acid, 0.1 mg/L kinetin, various levels of BA and TDZ, and 0.2% phytagel. Multiple shoot differentiation and regeneration was genotype independent when tested on four Egyptian cottons, Giza 45 and Giza 87 as extra long staple varieties, and Giza 85 and Giza 89 as long staple varieties. Plant regeneration required a specific concentration of BA and TDZ depending on the origin of the meristems. The frequency of explants forming multiple shoots ranged from 40 % to 65 % among the genotypes and the number of shoots ranged from 2 to 15 per responding explant. More than 80% of the shoots could be rooted. The frequency of genotype-independent transformation of Egyptian cotton varieties by Agrobacterium was increased by microprojectile wounding of the apical meristem. Bombardment was with tungsten (0.5 m) particles and four rupture disk pressures, 650 psi, 900 psi, 1300 psi and 2000 psi. Increasing rupture disk pressure produced a higher transformation frequency by A. tumefaciens but decreased the percentage of recovered plantlets. Regenerated plants were phenotypically normal and all of the mature plants initiated flowers and set variable R1 seeds.