Agrobacterium-mediated Genetic Transformation of Cotton and Regeneration via Somatic Embryogenesis.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide. This protocol was validated in: Nat Biotechnol (2016), DOI: 10.1038/nbt.3665.

Efficient production of gossypol from hairy root cultures of cotton (Gossypium hirsutum L.).

A protocol for induction and establishment of Agrobacterium rhizogenes mediated hairy root culture of Gossypium hirsutum was developed through infection with the A4 strain and co-cultivation on hormone-free semi-solid MS medium with B5 vitamins. It resulted in the emergence of hairy roots from the leaf explants, 21 days after infection. The transformation of hairy roots was established by PCR amplification of rol B and rol C genes of the Ri plasmid. All root lines expressed gossypol, although distinct inter-clonal quantitative variations were noticed. Five independent hairy root lines were studied for their growth kinetics as well as gossypol production. The yield potentials of one of them superseded others, as well as the non-transformed, in-vitro grown control roots. The content of gossypol in hairy roots reached a level of 2.43 mg/g DW. It was 4.5 times higher than in vitro and 1.47 times higher than in vivo grown roots of G. hirsutum. Selection of high gossypol producing hairy root lines of G. hirsutum can provide an alternative source for large-scale production of gossypol.