Genetic transformation is essential for studying gene function and improving agronomic traits in cotton (Gossypium hirsutum L.). In this study, we developed a stable Agrobacterium-mediated transformation protocol using the pKSE401 vector carrying the Cas9 gene. Seven-day-old hypocotyls of cotton cultivar Coker-312 sustained Agrobacterium tumefaciens strain inoculations, with transgenic plants regenerated via somatic embryogenesis. Out of 4,000 hypocotyl explants, seven embryos reached the embryogenesis stage, and five transgenic plants successfully matured. Cas9 integration reached confirmation in T₀ transgenic plants through PCR analysis. Callus induction succeeded using a modified Murashige and Skoog (MS) medium with kinetin (0.1 mg/L), 2,4-D (0.1 mg/L), Kanamycin (50 mg/L) and cefotaxime (400 mg/L). Somatic embryogenesis enhancement using a hormone-free MS medium had supplements with MgCl₂, glutamine, asparagine, CuSO₄, ascorbic acid, and activated charcoal (2 g/L). Shoot and root induction emerged with MS medium containing 1 g/L activated charcoal, BAP (1 mg/L), and kinetin (0.1 mg/L) for shooting, and NAA (1 mg/L) for rooting. Among the Agrobacterium strains tested, EHA101 and LBA4404 exhibited the highest transformation efficiencies (40%). This study establishes a reliable and time-efficient transformation system for cotton with an overall nine months, less regeneration time of 80–90 days, and an optimized range of OD (0.5–0.8) for infection of explant. This protocol offers a valuable tool for molecular breeding and functional genomics research.
Cotton, Cas9 gene, gene transformation, cotton transformation
An optimized Agrobacterium-mediated transformation in cotton was successful by identifying LBA4404 as the most efficient strain and enhancing transformation rates through hormone-free media and an optimized acetosyringone concentration (20 mg/L). Our improved somatic embryogenesis protocol reduced the regeneration timeline to 80–90 days, with activated charcoal (1–2 g/L) playing a key role in embryo development.