Articles
TRANSFER OF GRAPE FANLEAF VIRUS COAT PROTEIN GENE THROUGH HYBRIDIZATION WITH XIPHINEMA INDEX RESISTANT GENOTYPES TO OBTAIN ROOTSTOCKS RESISTANT TO VIRUS SPREAD
Article number
603_42
Pages
325 – 334
Language
English
Abstract
Fanleaf degeneration, caused by grapevine fanleaf virus (GFLV) and vectored by the nematode Xiphinema index, is one of the worlds most serious grape diseases.
Strong resistance to X. index feeding and multiplication has been reported in Muscadinia rotundifolia and introduced by hybridization in Vitis genotypes potentially usable as rootstocks resistant to virus spread.
In order to increase the level of resistance of these genotypes to the virus itself, attempts were done to introduce the coat protein (CP) gene of GFLV by hybridization with genetically engineered rootstock cultivars.
Two genotypes were used as female parents : One F1 hybrid V. vinifera x M. rotundifolia, with high resistance to X. index but low gametic fertility, and one BC1 hybrid (F1 x V. vinifera), with moderate resistance to X. index but high gametic fertility.
Eight transformed lines of the rootstock cultivar 110 Richter (V. rupestris x V. Berlandieri), and four transformed lines of V. rupestris cv du Lot were used as male parents.
The seedlings were grown in vitro and screened for the expression of the uidA (GUS) gene on roots and leaves.
In 5 progenies of the BC1 female crossed by the transgenic lines of 110 R, GUS expression was observed with a ratio not significantly different from 1:1. In all progenies, presence of the CP-GFLV gene was detected by PCR in the plants GUS-positive.
The expression of nptII gene was studied on the GUS-positive plants, using axillary micropropagation on medium added with 25 µg/µ ;l kanamycine.
In 4 progenies, all the plants GUS-positive proved to be kanamycin resistant.
In one progeny, all the plants proved to be susceptible to the antibiotic, and absence of the nptII gene was confirmed by PCR analysis.
Production of the coat protein was tested by ELISA, but was observed with a very low level in only few plants.
Resistance of the transformed plants to GFLV was tested by micrografting.
First results reveal considerable differences between genotypes in their reaction to micrografting and virus inoculation.
Plants have been acclimatized in a confinement greenhouse and tests for resistance to GFLV inoculated by X. index are in progress.
Strong resistance to X. index feeding and multiplication has been reported in Muscadinia rotundifolia and introduced by hybridization in Vitis genotypes potentially usable as rootstocks resistant to virus spread.
In order to increase the level of resistance of these genotypes to the virus itself, attempts were done to introduce the coat protein (CP) gene of GFLV by hybridization with genetically engineered rootstock cultivars.
Two genotypes were used as female parents : One F1 hybrid V. vinifera x M. rotundifolia, with high resistance to X. index but low gametic fertility, and one BC1 hybrid (F1 x V. vinifera), with moderate resistance to X. index but high gametic fertility.
Eight transformed lines of the rootstock cultivar 110 Richter (V. rupestris x V. Berlandieri), and four transformed lines of V. rupestris cv du Lot were used as male parents.
The seedlings were grown in vitro and screened for the expression of the uidA (GUS) gene on roots and leaves.
In 5 progenies of the BC1 female crossed by the transgenic lines of 110 R, GUS expression was observed with a ratio not significantly different from 1:1. In all progenies, presence of the CP-GFLV gene was detected by PCR in the plants GUS-positive.
The expression of nptII gene was studied on the GUS-positive plants, using axillary micropropagation on medium added with 25 µg/µ ;l kanamycine.
In 4 progenies, all the plants GUS-positive proved to be kanamycin resistant.
In one progeny, all the plants proved to be susceptible to the antibiotic, and absence of the nptII gene was confirmed by PCR analysis.
Production of the coat protein was tested by ELISA, but was observed with a very low level in only few plants.
Resistance of the transformed plants to GFLV was tested by micrografting.
First results reveal considerable differences between genotypes in their reaction to micrografting and virus inoculation.
Plants have been acclimatized in a confinement greenhouse and tests for resistance to GFLV inoculated by X. index are in progress.
Authors
A. Bouquet, G. Marck, D. Pistagna, L. Torregrosa
Keywords
Grapevine, rootstock, genetic resistance, grape fanleaf virus, nematode, Muscadinia rotundifolia, hybridization, genetic transformation.
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