In vitro propagation and preliminary results of Agrobacterium-mediated genetic transformation of Cordyline fruticosa

Abstract

A method for in vitro propagation through axillary shoot proliferation and induction of adventitious shoots as well as Agrobacterium-mediated transformation of Cordyline fruticosa is presented. In vitro Cordyline shoots were cultured on MS medium supplemented 0–4mgl−1 thidiazuron (TDZ) for their multiplication. MS medium supplemented with 0.1mgl−1 TDZ proved to be the most effective in which 14 shoots per explants were obtained. Leaf segments were excised from in vitro axillary shoots and cultured on MS medium supplemented with TDZ (0–5mgl−1) for callus induction. Following induction, the non-embryogenic calluses were proliferated on MS medium without plant growth regulators. A rapid and efficient method for producing transgenic plants has been developed for Cordyline via Agrobacterium-mediated transformation. Leaf segments from in vitro shoots were co-cultivated with Agrobacterium tumefaciens EHA105, which harbored the binary vector carrying the bar and β-glucuronidase (GUS) genes in the T-DNA region. The results showed that the number of plants expressing GUS gene first increased with inoculation time and bacterial density (OD600), and then dramatically decreased with the increment of both factors. The highest percentage of transformation efficiency (85%) was obtained when the leaf segment explants were inoculated with Agrobacterium for up to 40min at OD600 of 1.0 followed by 80 and 75% at OD600 of 0.8 and 1.2, respectively. The transgenes were confirmed in transgenic plants using GUS primers and gave the expected band size of 750bp. The results suggested that infection time and Agrobacterium density could have some effects on the transformation efficiency. Southern blot analysis was used for definitive confirmation of tDNA integration in Cordyline and confirmed the results of histochemical GUS assay and PCR amplifications. Regenerated plantlets were acclimatized in greenhouse.