Optimization of Tissue Culturing and Genetic Transformation Protocol for Casuarina equisetifolia

Casuarina equisetifolia is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, C. equisetifolia has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality Casuarina varieties to improve the tolerance and adaptability to cold weather by molecular techniques. The establishment of a rapid and efficient callus induction and regeneration system via tissue culture is pre-requisite for the genetic transformation of C. equisetifolia, which is so far lacking. In this study, we reported an efficient and rapid regeneration system using stem segment explants, in which callus induction was found to be optimal in a basal medium supplemented with 0.1 mg⋅L^-1 TDZ and 0.1 mg⋅L^-1 NAA, and proliferation in a basal medium containing 0.1 mg⋅L^-1 TDZ and 0.5 mg⋅L^-1 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L^-1 6-BA, and a combination of 0.02 mg⋅L^-1 IBA and 0.4 mg⋅L^-1 IAA, respectively. We also optimized genetic a transformation protocol using Agrobacterium tumefaciens harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L^-1 hygromycin, 20 mg L^-1 acetosyringone (As), and 2 days of co-cultivation duration were optimized to improve the transformation efficiency. With these optimized parameters, transgenic plants were obtained in about 4 months. Besides that, Agrobacterium rhizogenes-mediated transformation involving adventitious root induction was also optimized. Our findings will not only increase the transformation efficiency but also shorten the time for developing transgenic C. equisetifolia plants. Taken together, this pioneer study on tissue culturing and genetic transformation of C. equisetifolia will pave the way for further genetic manipulation and functional genomics of C. equisetifolia.