Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

Abstract

Toona ciliata has considerable economic value, providing high-quality materials and medicines. However, the growth and sustainable development of T. ciliata is limited due to environmental changes, poor natural regeneration ability, and excessive logging. Therefore, this study used sterile seedling leaves from a good provenance of T. ciliata as explants to establish a highly efficient regeneration and genetic transformation system. The effects of different hormones and concentrations on leaf callus induction were investigated. A genetic transformation system using kanamycin (Kan) as the selective agent was established based on the regeneration system. The results showed that Murashige and Skoog (MS) medium supplemented with 3 mg/L N-(Phenylmethyl)-9H-purin-6-amine (6-BA), 1 mg/L kinetin (KT) and 0.05 mg/L 1-naphthlcetic acid (NAA) (T1 medium) were suitable for callus formation and adventitious bud production from whole leaves at 45 days. In addition, pCAMBIA2301-EYFP was introduced into competent cells of Agrobacterium tumefaciens strain GV3101 and used for genetic transformation. Healthy leaves pre-cultured on T1 medium for three days were directly infected with A. tumefaciens infection solution (OD600 = 0.6) for 20 min through the addition of 50 mmol/L of Acetosyringone (AS). Thereafter, the infected leaves were co-cultivated for one day on T1 medium supplemented with 150 mmol/L AS, and the infected leaves were transferred to fresh T1 medium containing 10 mg/L Kan and 100 mg/L Cefotaxime (Cef). Twenty-four positive transformed plants were selected through histological staining and PCR, laser confocal microscopy of EYFP from 259 putative transformants was conducted, and the transformed plants were obtained after approximately three months.