Construction Of PLBs Transgenic System Mediated By Agrobacterium Tumefaciens In Phalaenopsis Orchid

Phalaenopsis is one of the most popular orchids for its rich colour, beautiful butterfly pattern and the long flowering period of 3 to 4 months. It is widely popular in the world market as it has high ornamental and economic value.Firstly, leaf blade of Phalaenopsis plantlets was used as the material to construct a stable PLBs induction system. Secondly, the PLBs and plantlets leaf blade from the established induction system were used for transformation system research. The infect liquid concentrations, infection time, additives were compared to find the optimal condition for successful transgenic system in Phalaenopsis.The results are shown below:(1) Construction of PLB induction systemBlade selection: the blade with the growth period of 4 to 5 weeks, size of 8 ~ 12 mm × 10 ~ 13mm(width × length) was good for induction PLBs. It was found that the performance of leaf base is better that the leaf opex and leaf middle, which has more PLBs, with high PLB induction rate and bud induction rate. Therefore, leaf base is good for induction of PLB. Besides, we also found the 10mm×3mm blade has the most number of PLB and its PLB induction rate reached up to 52.78%; followed by 10mm×5mm blade with PLB induction rate of 22.22%.As a result, the best medium for induction PLB in this study is: 1/2MS+TDZ 0.2mg/L+BA 4mg/L+CW 20%+PVP 3g/L+ sucrose 15g/L,Phytagel 3g/L with the pH 5.5. The PLB induction rate was 47.21% and the average number of PLB induced from each blade was 8.15 in this condition. The result manifests that both 0~1mg/L TDZ and 0~4mg/L BA have positive effect on induction PLB.(2) Proliferation and differentiation of PLBOptimal proliferation medium:1/2MS+ZT 3mg/L+ adenine sulfate 10mg/L+CW 15%+ peptone 2g/L+ sucrose 15g/L+AC 1g/L, Phytagel 3g/L and with the pH 5.5.Optimal differentiation medium:Hyponex+BA 3mg/L+CW 15%+ sucrose 15g/L+PVP 2g/L, Phytagel 3g/L, and with the pH 5.5.(3) Construction of Agrobacterium-mediated transgenic systemPhalaenopsis PLBs transgenic system mediated by the Agrobacterium tumefaciens was successfully constructed. A plant expression vector pCAMBIA1301, which was under the control of the cauliflower mosaic virus(CaMV) 35 S promoter, contained a GUS reporter gene and hygromycin resistance gene, and was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. The best transformation efficiency was achieved when pre-cultured time was 2 to 3 days, OD=0.3, infection time was 60 min, co-cultivation time is 44~52h.The selected cultivation time was 55 days in the selection medium containing 10mg/L hygromycin and 300mg/L carbenicillin. Under this condition, the resistance PLBs survival rate was 40.82%, percentage of GUS-positive reached 9.91% and percentage of PCRpositive was up to 19.24%.