Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus |
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Authors: | Torregrosa L Bouquet A |
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Institution: | (1) Unité de Formation et de Recherche en Viticulture, Ecole Nationale Supérieure Agronomique de Montpellier, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France;(2) Institut National de la Recherche Agronomique, Domaine du Chapítre, 34751 Villeneuve lès Maguelone Cedex, France |
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Abstract: | Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and
disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and
gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar
‘Gravesac’, 72% of the excised root tips initiated hairy root cultures on growth regulator-free media. According to the nature
of the strains used in co-inoculation, co-transformation frequencies of the hairy root clones ranged from 4 to 16%. Co-transformed
roots showed resistance to kanamycin and hygromycin but responses varied from clone to clone. Fluorometric GUS expression
and GCMV coat protein production showed a large variability among hairy root clones co-transformed by pKHVG2+. Though the
presence of gus, nptII and GCMV coat protein genes was checked by polymerase chain reaction and Southern blotting, it was
difficult to establish a clear relationship between expression of the different transgenes. The regeneration of plants was
not achieved, but the possibility to graft in vitro transgenic roots to non transformed shoot systems could permit rapid testing
of the resistance induced by nepovirus coat protein in roots of cultivars that are recalcitrant to A. tumefaciens-mediated
transformation.
This revised version was published online in June 2006 with corrections to the Cover Date. |
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Keywords: | Agrobacterium coat protein grapevine hairy root nepovirus transformation |
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