Somatic Embryogenesis And Transformation Of Grapevine Thompson Seedless

In this study,Vitis vinifera cv. Thompson Seedles and Flame Seedless were used asexperimental materials, and somatic embryogenesis was initially established and graduallyimproved by optimizing the parameters including explant types on embryogenic callusfomation, medium effects on embryo formation, sucrose on embryo germination and BAPconcentration on plant conversion. Moreover, embryogenic cultures were maintained andproliferated by secondary embryogenesis in which some abnormal embryos were alsoreused. Based on the generated grapevine somatic embryogenesis, explant type, kanamycinconcentration, selection regimes and methods were further tested and optimized. A stilbenesythase gene (STS) derived from Vitis pseudoreticulata accession Baihe-35-1was transferredinto V. vinifera cv.Thompson Seedless with the aim to obtain new grapevine germplasm withhigh-content resveratrol.Moreover, functional characterization of STS and stilbene contentwere determined in VpSTS-overexpressing lines. The main results obtained were as follows：1. Somatic embryogenesis was established and optimized in V. vinifera cvs. ThompsonSeedless and Flame Seedless. Result demonstrated that the flower buds were more suitablefor primary embryogenic callus induction and is also easy for sterilization. In addition, theinduction rate of flower buds was0.8%-1.9%. The best somatic embryo formation mediumwas X6medium which contain60g/L sucrose. When cultured on MS basic salts mediumwith15g/L sucrose, the germination of somatic embryos was best. The best medium forconversion contained MS basic salts,0.2mg/L BAP and7g/L Agar.2. To increase grapevineconversion efficiency and reduce waste of the abnormal somaticembryos, the influence factors of both the embryo morphology in germination and theembryogenic callus reinduction rate in secondary embryogenesis were further investigated inV. vinifer cv. Thompson Seedless. When embryos germinated on MS basic medium with15g/L sucrose, lower percentage of abnormal morphology was observed on medium withoutplant growth regulator. The fused cotyledons embryo was capable for recycle and they werereinduced into embryogenic callus. Cotyledons showed a higher reinduction rate (72.22%) onECRM1medium.3. A transformation system of V. vinifere cv. Thompson Seedless was established and further optimized. Based on the comparative analysis of grapevine receptor types, antibioticconcentration, selection ways and seedling method, an optimized system for genetictransformation was established. When PEM was used as receptor materials and75mg/Lkanamycin was added for delaying selection of three to four weeks after co-cultivation, it wassupposed to be the most suitable for embryo recovery. It was efficiency for transgenic embryogermination after culturing on medium containing25mg/L kanamycin for15days. Inaddition, plant conversion was improved by an alternative culture on two different media: onecontains0.2mg/L BA and another contains0.25mg/L KT.4.171transgenic lines transformed with ubiquitin lingage genes and81transgenic linestransformed with stilbene synthesis gene were obtaied. Putative grapevine transgenic lineswere further determined by PCR detection, southen blot and RT-PCR. Results showed that allthe tested lines were transgenic grapevines including that70lines transferred with VpPUB23.All the selected lines (70lines) were proved to be transgenic plants as revealed by PCRamplification, Southern blot and RT-PCR.25transgenic lines with VpUU2were proved byPCR,76lines were transformed with VpUR9and all the selected lines (30lines) weretransgenic ones.81positive transgenic lines with stilbene synthesis gene were obtained basedon PCR and RT-PCR assays.5. Functional analysis was performed in STS transgenic lines with RT-PCR and stilbenecontent was tested by HPLC detection. There were two kinds of stilbene content obtained,cis-piceid and trans-piceid. Cis-piceid and trans-piceid increased significantly in transgeniclines compared to wild type. The concentration of trans-piceid in transgenic lines was from22.9-50.4μg/g fresh weight (9.2-21times higher than non-transgenic control) and thecis-piceid was12.1-36.3μg/g fresh weight (2.4-7.4times higher than non-transgenic lines).Comparative analysis of the content of stilbene and composition in the berry skins, flesh,seeds and leaves from21Chinese wild Vitis species indicated that the content of piceid inyoung leaves of transgenic lines was higher than that in mature leaves from all the wildgrapevines and even higher than that in berry skins. Expression analysis of stilbene sythesisgene family showed that some STS genes expressed increased in transgenic lines while CHSgenes were not affected significantly. In addition, increased powdery mildew-resistance intransgenic lines were observed.