In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of D. lotus (Diospyros lotus L.)

In vitro regeneration and Agrobacterium-mediated genetic transformation system of D. lotus (Diospyros lotus L.) has been achieved using leaf explants. Two-step regeneration method was developed. First, the explants cultured in callus induction medium containing 0.1 mg/L naphthalene acetic acid (NAA) and 3.0 mg/L thidiazuron (TDZ) and cultured in darkness for 10 days. Second, leaf discs were introduced on shoot induction medium containing 1 mg/L trans-zeatin (ZT) and 0.1 mg/L indole-3-acetic acid (IAA) after 15 days of inoculation, callus induction rate of Diospyros lotus L. was 100%, average number of adventitious shoots was 4.26, regeneration rate was 94.28% respectively. The highest number root formation was observed in MS(1/2 N) supplemented with 0.5 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L indole-3-butyric acid (IBA). A protocol for genetic transformation of D. lotus via Agrobacterium was established. Agrobacterium strain EHA105 carrying vector pCAMBIA1301-GA2OX2 was used to optimize the transformation conditions, which include pre-culture period, bacterial optical density, infection time, co-culture duration, concentration of acetosyringone (AS) and concentration of hygromycin. Improved transformation rates (14.27 ± 1.32%) were achieved when explants pre-cultured for 4 days were incubated with an Agrobacterium suspension with a culture density of OD600 = 0.5 for 10 min, followed by 1-day co-cultivation. Successful transformation was confirmed by histochemical GUS activity, hpt and gus gene PCR analyses of the regeneration hygromycin resistant plantlets.