High-frequency regeneration via somatic embryogenesis of an elite recalcitrant cotton genotype (Gossypium hirsutum L.) and efficient Agrobacterium-mediated transformation

A highly efficient and reproducible regeneration system based on somatic embryogenesis in Gossypium hirsutum cv. Narasimha (NM), which has superior fiber qualities and is also used as a female parent in several hybrid cottons, has been developed. Embryogenic callus was obtained form both hypocotyls and cotyledonary leaves on Murashige and Skoog (MS) medium containing kinetin and 2,4-dichlorophenoxyacetic acid. Somatic embryogenesis was observed on hormone-free MS medium, but embryos did not grow well beyond globular stage. However, somatic embryos germinated well on MS medium containing B5 vitamins; addition of zeatin was found to be beneficial for their normal development. Most importantly, the media and culture conditions developed for NM were also found to be suitable for high-frequency somatic embryogenesis in Coker 310. In addition, the newly developed regeneration protocol has been successfully tested for genetic transformation through co-cultivation with Agrobacterium using embryogenic calli as explants. Molecular analysis confirmed the stable integration and expression of marker gene, green fluorescent protein (GFP). These results show that it is now possible to introduce foreign gene(s) directly into elite cultivar Narasimha with similar efficiency to in traditionally used Coker lines in a relatively short period of time. Development of efficient regeneration and transformation systems as demonstrated here should augment the introduction of new traits directly into cultivated varieties/hybrids, reducing the time required for back-crossing and the costs for seed production, besides aiding genomic research in cotton.