Molecular characterization of grapevine plants transformed with GFLV resistance genes: I

Abstract

The Grapevine FanLeaf Virus-Coat Protein (GFLV CP) gene was inserted through Agrobacterium-mediated transformation in Vitis vinifera "Nebbiolo", "Lumassina" and "Blaufränkisch". Two plasmids were used: pGA-CP+ (full-length GFLV CP gene with an introduced start codon) and pGA-AS (same gene in antisense orientation). Forty-three transgenic lines were regenerated. As several lines in Southern blots share same hybridization patterns, eight independent line groups resulted for "Nebbiolo", one for "Lumassina", and two for "Blaufränkisch". Inserted T-DNA copies ranged from one to three; one line probably contains an incomplete copy of T-DNA. Except for one "Nebbiolo" line, no evidence for methylation of the transgene at cytosine residues was found by Southern analyses. Specific mRNA was present at variable expression levels; some lines accumulated the coat protein while in others the protein was not detectable by ELISA.

The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange under Well-Watered Conditions But Not under Water Stress

“…The hybridization patterns observed for the different plants in Southern analyses followed a few common motifs, which allowed the clustering of the transgenic lines into groups whose members presumably issued from the same transformation event. A similar situation has previously been reported in other grape transformation experiments (Iocco et al, 2001;Gambino et al, 2005;Maghuly et al, 2006). The transformation protocol we used requires the maintenance of embryogenic cultures, after Agrobacterium tumefaciens coculture, for several months under kanamycin selection pressure before embryo germination.…”

“…Although transgenic plants of cv Nebbiolo have previously been obtained (Gambino et al, 2005), we chose the cv Brachetto for its high capacity to induce somatic embryogenesis and plant regeneration after genetic transformation (G. Gambino and I. Gribaudo, unpublished data). Furthermore, both genotypes show anisohydric behavior when subjected to water stress (Supplemental Fig.…”

“…Embryogenic calli of cv Brachetto were transformed following the protocol by Gambino et al (2005). Single embryo-derived plantlets were micropropagated by repeated subcultural apical cuttings on Murashige and Skoog medium without plant growth regulators and with kanamycin.…”

Non Coding RNAs and Gene Silencing in Grape

“…The soilborne nepovirus Grapevine fanleaf virus (GFLV) causes fanleaf degeneration, characterized by substantial yield losses, low fruit quality, and a progressive decline of infected vines, which can eventually lead to plant mortality. Different research teams inserted the coat protein (CP) gene of GFLV (in sense, antisense orientation, or truncated forms) in several cultivars and rootstocks (Mauro et al 1995;Gambino et al 2005;Maghuly et al 2006). Evaluation of GFLV resistance in these transgenic grapevines has been attempted in natural conditions of infection and by graft inoculation.…”

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.…”