Optimization of Tissue Culturing and Genetic Transformation Protocol for Casuarina equisetifolia

作者：Huimin Ren, Xu Yan, Xiaohong Zhao, Yan Zhang, Jamshaid Hussain, Fuqiang Cui, Guoning Qi, Shenkui Liu · 发表于：Frontiers in Plant Science · 年份：2022 · DOI：10.3389/fpls.2021.784566 · 被引用次数：14 · 研究领域：Plant tissue culture and regeneration、Transgenic Plants and Applications、Plant Molecular Biology Research

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, t...