Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

Abstract

SUMMARYTo facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating rootstocks: Couderc 3309, Vitis riparia 'Gloire de Montpellier', Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N6-benzyladenine (BA) per L, and 0.8% Noble agar]. The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocuhivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of , and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3-4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts 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 rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for 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 Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.

Grapevine Biotechnology: Molecular Approaches Underlying Abiotic and Biotic Stress Responses

“…Biotechnology arises as an alternative to allow the generation of virus-resistant grapevine plants by transgenesis, mainly involving the expression of viral components and exploiting the naturally occurring gene silencing [107][108][109][110][111][112][113][114][115][116][117]. This strategy requires plant transformation with a short sequence of the pathogen genome in a way that a double-strand RNA structure is formed during transcription, initiating gene silencing in the host.…”

Grapevine Crown Gall Suppression Using Biological Control and Genetic Engineering: A Review of Recent Research

“…A total of 928 putative GM grapevine combinations were generated (Xue et al 1999) and tested for resistance to grape CG, grapevine fanleaf virus (GFLV), and grapevine leafrollassociated closterovirus-3 (GLRaV-3). Transformed vines contained a sense-oriented translatable gene of GFLV, an antisense coat protein gene of truncated HSP90-related (heatshock protein 90) gene of GLRaV-3, and a virE2 of B gene (a truncated virE2 that lacks 215 carboxyl-terminal amino acids; Citovsky et al 1994) from A. tumefaciens strain C58.…”

“…Transgenes were present in each case as determined by PCR or Southern Blot analysis. Disease and virus resistance evaluations of the GM vines are in progress (Xue et al 1999).…”

Agrobacterium: A disease-causing bacterium

“…Expression of a mutated virE2 gene lacking the coding sequence for the ssDNA binding domain in transgenic tobacco significantly reduced tumor formation upon inoculation with Agrobacterium (Citovsky et al, 1994). This construct and additional truncated virE2 genes derived from various strains were used to transform grapevines and several of the selected transgenic lines showed reduced susceptibility to crown gall (Xue et al, 1999;Holden et al, 2003). The mutated VirE2 protein probably competes with functional VirE2 molecules in the plant cytoplasm.…”