Articles
TRANSFORMATION OF VITIS VINIFERA L. CV NEBBIOLO WITH THE COAT PROTEIN GENE OF GRAPEVINE FANLEAF VIRUS (GFLV)
Article number
603_40
Pages
309 – 314
Language
English
Abstract
Grapevine FanLeaf Virus (GFLV) is the main causal agent of the grapevine fanleaf disease, one of the most damaging and widespread viral diseases affecting grapevine.
As new truly resistant grapes has not been developed yet by traditional breeding methods, genetic engineered virus-resistant grape plants have been proposed as an alternative approach to control this important disease.
The effectiveness of the adopted strategy in conferring virus resistance has to be carefully investigated.
Here we report the regeneration of transgenic grapevine plants (V. vinifera ‘Nebbiolo’) in which the GFLV Coat Protein gene has been inserted through an Agrobacterium-mediated transformation system, and the first results in the analysis of the transgene expression.
Immature anthers and ovaries of the cv.
Nebbiolo were cultivated on media inducing embryogenic callus formation; calli were then transferred to an embryo differentiation medium which, for long term maintenance of embryogenic cultures, was periodically alternated with a callus propagation medium.
A binary vector was used for transformation experiments.
The plasmid pGA-CP+ carries the full-length GFLV-CP genes with an introduced start codon.
The binary vector also contains the nptII coding region conferring kanamycin resistance.
Embryogenic calli of ‘Nebbiolo’ were co-cultivated with a diluted Agrobacterium suspension.
Putatively transformed tissues were selected by continued culture on kanamycin-containing media.
Thirty-nine lines of putatively transformed ‘Nebbiolo’ grape were obtained, each line deriving from a single somatic embryo.
Twenty four lines were so far tested by Southern blot analysis.
In all the lines the GFLV-CP gene could be detected, and the number of T-DNA insertions in the genome ranged from 1 to 3. In order to study the expression of the GFLV-CP gene, RNA extraction and RT-PCR analyses were performed on 20 lines (all tested positively for T-DNA presence). Fifteen lines showed the presence of the specific mRNA, while 5 lines did not.
Although preliminary, the results indicate that the adopted transformation protocol is suitable for the cv Nebbiolo, and the DNA analyses confirm that the applied selection procedure ensures the recovery of transformed plants only.
Not all the transformed lines showed accumulation of the specific mRNA
As new truly resistant grapes has not been developed yet by traditional breeding methods, genetic engineered virus-resistant grape plants have been proposed as an alternative approach to control this important disease.
The effectiveness of the adopted strategy in conferring virus resistance has to be carefully investigated.
Here we report the regeneration of transgenic grapevine plants (V. vinifera ‘Nebbiolo’) in which the GFLV Coat Protein gene has been inserted through an Agrobacterium-mediated transformation system, and the first results in the analysis of the transgene expression.
Immature anthers and ovaries of the cv.
Nebbiolo were cultivated on media inducing embryogenic callus formation; calli were then transferred to an embryo differentiation medium which, for long term maintenance of embryogenic cultures, was periodically alternated with a callus propagation medium.
A binary vector was used for transformation experiments.
The plasmid pGA-CP+ carries the full-length GFLV-CP genes with an introduced start codon.
The binary vector also contains the nptII coding region conferring kanamycin resistance.
Embryogenic calli of ‘Nebbiolo’ were co-cultivated with a diluted Agrobacterium suspension.
Putatively transformed tissues were selected by continued culture on kanamycin-containing media.
Thirty-nine lines of putatively transformed ‘Nebbiolo’ grape were obtained, each line deriving from a single somatic embryo.
Twenty four lines were so far tested by Southern blot analysis.
In all the lines the GFLV-CP gene could be detected, and the number of T-DNA insertions in the genome ranged from 1 to 3. In order to study the expression of the GFLV-CP gene, RNA extraction and RT-PCR analyses were performed on 20 lines (all tested positively for T-DNA presence). Fifteen lines showed the presence of the specific mRNA, while 5 lines did not.
Although preliminary, the results indicate that the adopted transformation protocol is suitable for the cv Nebbiolo, and the DNA analyses confirm that the applied selection procedure ensures the recovery of transformed plants only.
Not all the transformed lines showed accumulation of the specific mRNA
Authors
I. Gribaudo, V. Scariot, G. Gambino, A. Schubert, R. Göller, M. Laimer
Keywords
nepovirus, pathogen-derived resistance, somatic embryogenesis, grapevine.
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