Transformation of five grape rootstocks with plant virus genes and a virE2gene from Agrobacterium tumefaciens

To facilitate the development of transgenic grapevines that are resistant t o grapevine fanleaf virus (CFLV), gapevine leafroll-associated closteroviru s (GLRaV-3) and crown gall diseases, we developed a rapid system for regene rating rootstocks Couderc 3309, Vitis ripanria 'Gloire de Montpellier', Tel eki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryog enesis. Embryo culture and grape regeneration were accomplished with four m edia. Embryogenic calluses from anthers were induced in the initiation medi um [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4- dichlorophen oxyacetic acid (2,4-D) per L, 0.2 mg N-6-benzyladenine (BA) per L, and 0.8% Noble agar]. The percentage of anthers that developed into embryogenic cal li ranged from 2 to 16.3% depending on the rootstock. Calluses with early g lobular stage embryos were cocultivated with Agrobacterium tumefaciens stra in C58Z707 containing the gene constructs of interest. The genes were sense -oriented translatable and antisense coat protein genes from GFLV anti GLRa V-3, a truncated HSP90-related gene of GLRaV-3 (43k), and a virE2 del B gen e from A. tumefaciens strain C58. Twenty independent transformation experim ents were performed on five rootstocks. After 3-4 mo. under kanamycin selec tion, secondary embryos were recovered on differentiation medium (1/2 MS sa lts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Em bryos that were transformed were regenerated on a medium containing MS salt s with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooti ng medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, anti 0.65% Bacto agar A total of 928 independent putative tra nsgenic plants were propagated in the greenhouse. All plants were tested fo r neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Coudere 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to thr virus and crown gall diseases.