Studies On The Genetic Transformation Of Grapevine With Genes Involved In Powdery Mildew Resistance From Chinese Wild Vtitis Pseudoreticulata

Grapevine(Vitis vinifera L.)is one of the world’s most important fruit crops because of their high yielding and value, however, it is sensitive to powdery mildew(PM). To produce high disease-resistance and quality cultivars is one of the objectives of breeding programs. A rapid and highly efficient transformation system for improving disease-resistance in grapevines is urgently needed. In this study, V. vinifera cv. Chardonnay, Merlot, Cabernet Sauvignon, Cabernet Franc and Red Globe were used to establish the somatic embryogenesis. Morever, to obtain higher transformation efficiency, transformation methods and explant types which were critical to genetic transformation via somatic embryogenesis were studied. Based on this Agrobacterium-mediated transformation system, 3 powdery mildew resistant related genes, namely Vp STSg DNA2, Vp UR9, and Vp PR4-1, were transformated into grapevine to expand knowledge of these genes expression and function and obtain disease-resistance grapevines. Vp STSg DNA2 was cloned from Vitis pseudoreticulata accession Baihe-35-1 in preliminary studies. And Vp UR9 and Vp PR4-1 were cloned from c DNA library of high PM resistant Vitis pseudoreticulata accession Baihe-35-1 inoculated by PM. The main results of this study are described as follows:1. Somatic embryogenesis and media optimization: Embryogenic calluses(ECs)were successfully initiated from anthers, ovaries and whole flowers of Chardonnay, Merlot, Cabernet Sauvignon and Cabernet Franc. The average frequency of EC induction of Chardonnay, Merlot was 12.00% and 9.05% and higher than that of Cabernet Sauvignon(0.03%) and Cabernet Franc(0.83%). Whole flowers of Chardonnay produced the most embryogenic calluses(16.4%)on induction media containing Murashige and Skoog’s(MS) basal salts and 3.0 mg L-1 picloram with 0.5 mg L-1 2,4-D and 2.0 mg L-1 6-BA. Proembryonic masses(PEM)of Merlot and Red Globe which was initiated in previous studies could be maintanted and proliferated on X6 medium, and generat into plantlets on MS medium containing 0.2mg L-1 6-BA for 57 and 63 plantlets per 0.1g PEM, respectively, and higher than that of Chardonnay(18 plantlets per 0.1g PEM). However, the best PEM formation of Chardonnay was found with MS medium supplemented with 2.0mg L-1 picloram.The optimal medium for normal plantlets regeneration for Chardonnay was MS medium supplemented with 0.2 mg L-1 kinetin, 0.1 mg L-1 NOA.2. Optimization for transformation methods and transformation with stilbene synthesis gene: GUS reporter gene was transformed into PEM of Chardonnay via Agrobacterium-mediated and particle bombardment transformation system,respectively. Somatic embryos which were transformated via Agrobacterium-mediated method showed higher GUS expression. The ability of 3 explant types, PEM, EC, and somatic embryo(SE), for transformation was then tested by transformed via Agrobacterium-mediated method with a stilbene synthase gene Vp STSg DNA2, cloned from Vitis pseudoreticulata accession Baihe-35-1 in previous studies. EC and SE did not produce transgenic SE lines as they turned brown and died during selection. Then PEM samples produced hygromycin resistance material. PEM was the best source of transgenic material for Agrobacterium-mediated genetic transformation of the three tissue types.3. Molecular analysis of transgenic lines of stilbene synthesis gene: 69 regenerated Chardonnay via Agrobacterium tumefaciens-mediated transformation and 32 of them were confirmed by PCR analysis, 3 regenerated Melot and 2 regenerated Red Globe failed in PCR analysis. Vp STSg DNA2 expression of transgenic Chardonnay comfirmed by southern blotting was significantly induced responsive to PM, while chalcone synthase genes were down regulated then. The positive transgenic grapevine lines exhibited higher levels of trans-resveratrol and ε-viniferin and H2O2 than wild-type vines could slightly reduced the growth of powdery mildew, therefore, Chardonnay transformed with Vp STSg DNA2 had enhanced PM-resistance.4. Transformation of ubiquitin ligase gene and molecular analysis of the transgenic lines: Vp UR9, an ubiquitin ligase gene, which encodes 164 amino acids and possesses a RING conserved motif, was homologous cloned from c DNA library of high PM resistant Vitis pseudoreticulata accession Baihe-35-1 inoculated by PM. The gene is induced responsive to PM and salicylic acid(SA). Vp UR9 controlled by 35 S promoter was transformed into 15 regenerated Red Globe via Agrobacterium tumefaciens-mediated transformation and 12 of them were confirmed by western blotting of FLAG-tag. The PM-resistance of Red Globe transformed with Vp UR9 was weakend.5. Transformation of pathogen related protein gene and molecular analysis of the transgenic lines: Vp PR4-1, a pathogen related protein gene, which encodes 143 amino acids, was homologous cloned from c DNA library of high PM resistant Baihe-35-1 inoculated by PM. The gene is induced responsive to PM, SA and Me JA. Vp PR4-1 possesses DNase activity and localized in nucleus, cytoplasm and cell membrance during subcellular localization analysis. Vp PR4-1 controlled by 35 S promoter was transformed into 30 regenerated Red Globe via Agrobacterium tumefaciens-mediated transformation and 26 of them showed increased level of Vp PR4-1 protein. And the PM-resistance of Red Globe transformed with Vp PR4-1 was enhanced.Above all, it is feasible that transformation of genes involved in powdery mildew resistance from Chinese wild Vitis pseudoreticulata into grapevines, which provides the foundation for improving disease-resistance in grapevines. The study will focus on obtaining higher initiation, maintenance, proliferation, generation of embryogenic tissues and higher transformation efficiency. And the inoculation test and observation of transgenic grapevines will be conducted in the field in future.