An Efficient Genetic Transformation System For Callus Of Vitis Amurensis

Genetic transformation system is the main platform for improving crop traits and studying gene functions.Grape is one of the most important horticultural crops,but its existing genetic transformation system has many constrains such as time-consuming,low-efficiency and so on,which greatly limite the functional study of quality and stress related gene.Vitis amurensis is a wild Vitis spp.species with remarkable cold and drought tolerance.The research on stress related genes and interpretation of signaling pathways will provide theoretical basis for grape breeding.In this study,Vitis amurensis was used as experimental materials,and Agrobacterium-mediated genetic transformaiton system was initially established and gradually improved by optimizing the parameters including explant types,the concentration of kanamycin,Agrobacterium strains,bacterial densities,infection treatments and co-cultivation time.A cold-stress related transcription factor gene(VaERF057)was transferred into petiole segments of Vitis amurensis and its overexpressed calli were successfully obtained.This efficient transformation system will facilitate the functional analysis of strsss-related genes and agronomic characteristics related genes in V.amurensis.The main results obtained were as follows:1.Selection of suitable explants used for genetic transformation.The differentiation capacity and growth rate of leaf-discs,petiole and stem segments were studied.When compared to calli generated from leaf-discs and stem segments,calli generated from petiole segments were creamish yellow in color and had faster regeneration.Thus,petiole was the optimal explant suitable for genetic transformation and would be used in the subsequent study.2.Petiole segments were used as explants to study the effects of different concentrations of hygromycin and kanamycin on callus formation.The results showed that merely 4 mg/L hygromycin could completely inhibit the callus differentiation and lead to the browning of petiole segments.When kanamycin was added,the callus differentiation decreased with the increasing concentration of kanamycin.The 20 mg/L kanamycin concentration could completely inhibit the differentiation of non-transformed calli and the petiole did not show brown necrosis.Therefore,20 mg/L kanamycin was selected to screen the positive calli.3.The pSAK277-eGFP vector was used as the transformation vector,which contained the NPTII gene(neomycin phosphotransferase gene)and eGFP gene(enhanced green fluorescent protein gene)as double screening markers.In this paper,four parameters for transformation processes were optimized including Agrobacterium stains,bacterial densities,infection treatments and co-cultivation time.The optimum conditions for transformation were:EHA105(0D600=0.5),8 min infection time,2 days co-cultivation,and screening for 6-8 weeks on the culture medium containing 20 mg/L kanamycin.At the same time,more than 20 transformed calli could be obtained by the transformation of the 100 petiole segments.4.Genetic transformation of VaERF057 gene related to cold resistance of Vitis amurensis was carried out using the established transformation system,and the callus was tested by RT-PCR.The results showed that VaERF057 gene was up-regulated in the resistant calli.The acquisition of VaERF057 transgenic calli provides important materials for further study on the function of the genes.