Study On Agrobacterium-mediated Transgenic Technology Of Orchid

Phalaenopsis and Hybrid Cymbidium are Orchidaceae plants with high ornamental and economic value.Phalaenopsis enjoys the reputation of"Queen of Orchid"due to its peculiar flower shape,rich flower color and long flowering period.Hybrid Cymbidium is a new type of orchid cultivated by the hybridization of Chinese Cymbidiums and C.hybridum.They possess the delicate and elegant fragrance of Chinese Cymbidiums and the large and bright-colored flowers of C.hybridum.Plant genetic engineering technology is a fast and effective breeding method.So far,no efficient and stable genetic transformation technology system have been established for Phalaenopsis and Hybrid Cymbidium.Therefore,it is meaningful to study and establish the transgenic protocol for promoting the molecular breeding and industrial development of Phalaenopsis and Hybrid Cymbidium.In this study,in order to establish the Agrobacterium-mediated genetic transformation system and obtain transgenic plants of Phalaenopsis and Hybrid Cymbidium,we transfered the At DREB1A gene into the ptocorm-like bodies（PLBs）of‘2017-006’,a new strain of Phalaenopsis,and transferred the overexpression and interference vector of YUCCA6 gene into‘Gongfenjiaren’,a new variety of Hybrid Cymbidium.The main findings were as follows:（1）Establish an efficient and stable genetic transformation receptor system for‘2017-006’.Hormone combination had significant effects on the PLBs induction of‘2017-006’,and the optimum medium was MS+6-BA 10 mg·L^-1+NAA 0.5 mg·L^-1+CW 10%+sucrose 30 g·L^-1+carrageenan 7 g·L^-1.Basic medium had a significant effect on differentiation frequency and rooting and strenghening of plantlets of‘2017-006’,low salt basic medium has better effect,and the optimum proliferation medium was 1/3 MS+6-BA1.0 mg·L^-1+NAA 0.2 mg·L^-1+potato juice 30 g·L^-1+AC 0.5 g·L^-1+sucrose 20 g·L^-1+carrageenan 7 g·L^-1,the optimum differentiation medium was 1/3 MS+6-BA 1.0 mg·L^-1+NAA 0.2 mg·L^-1+potato juice 30 g·L^-1+AC 0.05 g·L^-1+sucrose 20 g·L^-1+carrageenan 7g·L^-1,and the optimum rooting and strenghening medium was 1/3 MS+6-BA 0.1 mg·L^-1+NAA 0.5 mg·L^-1+potato juice 30 g·L^-1+AC 0.5 g·L^-1+sucrose 20 g·L^-1+carrageenan 7g·L^-1.（2）Establish a genetic transformation system in PLBs of‘2017-006’.The concentration of hygromycin had a significant effect on the survival rate of‘2017-006’PLBs,and the appropriate concentration of hygromycin for the transformant screening was 10 mg·L^-1.When the cephalosporin concentration was 200 mg·L^-1can completely inhibit the proliferation of Agrobacterium.The size of the receptor material,the concentration of the bacterial solution,the infection time and the co-cultivation time had significant effect on the transformation efficiency of the At DREB1A gene in PLBs of’2017-006’.The suitable transformation conditions for PLBs of‘2017-006’were as follows:the OD₆₀₀value of the engineering bacterial solution was 0.4,the infection time was 40 min,the co-cultivation time was 3 days,and the diameter of the recipient material was 0.3 cm.Using the transformation system,we gained 18 transgenic positive plants from 182regenerated plants with a conversion rate of 9.89%.（3）Genetic transformation of YUCCA6 gene in PLBs of C.‘Gongfenjiaren’A genetic transformation system with OD₆₀₀=0.4,40 min of infection time,3 days of co-cultivation time,and 0.3 cm of PLBs diamete was used to tranfer the overexpression and interference vector of YUCCA6 into the PLBs of‘Gongfenjiaren’.The results showed that there were 28 plants successfully transformed with HptⅡmarker gene among 165regenerated plants,but no transgene-positive plants were detected using the GUS reporter gene by PCR amplification.In this study,we established the genetic transformation technological protocol of At DREB1A gene for PLBs of‘2017-006 in Phalaenopsis,and obtained 18 transgenic positive plantlets,which would provide a technological reference for transgenic breeding.