AUTHOR=Ren Huimin , Xu Yan , Zhao Xiaohong , Zhang Yan , Hussain Jamshaid , Cui Fuqiang , Qi Guoning , Liu Shenkui 

TITLE=Optimization of Tissue Culturing and Genetic Transformation Protocol for Casuarina equisetifolia

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.784566

DOI=10.3389/fpls.2021.784566

ISSN=1664-462X

ABSTRACT=<p><italic>Casuarina equisetifolia</italic> is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, <italic>C. equisetifolia</italic> has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality <italic>Casuarina</italic> 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 <italic>via</italic> tissue culture is pre-requisite for the genetic transformation of <italic>C. equisetifolia</italic>, 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<sup>–1</sup> TDZ and 0.1 mg⋅L<sup>–1</sup> NAA, and proliferation in a basal medium containing 0.1 mg⋅L<sup>–1</sup> TDZ and 0.5 mg⋅L<sup>–1</sup> 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L<sup>–1</sup> 6-BA, and a combination of 0.02 mg⋅L<sup>–1</sup> IBA and 0.4 mg⋅L<sup>–1</sup> IAA, respectively. We also optimized genetic a transformation protocol using <italic>Agrobacterium tumefaciens</italic> harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L<sup>–1</sup> hygromycin, 20 mg L<sup>–1</sup> 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, <italic>Agrobacterium rhizogenes</italic>-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 <italic>C. equisetifolia</italic> plants. Taken together, this pioneer study on tissue culturing and genetic transformation of <italic>C. equisetifolia</italic> will pave the way for further genetic manipulation and functional genomics of <italic>C. equisetifolia</italic>.</p>