CELLULAR DIFFERENCES IN AGROBACTERIUM SUSCEPTIBILITY AND REGENERATIVE CAPACITY RESTRICT THE DEVELOPMENT OF TRANSGENIC GRAPEVINES

Although grape is easily infected with Agrobacterium and plants can be regenerated routinely, it has proven recalcitrant to the recovery of transgenic plants. Anatomical and histochemical analyses of cocultivated regenerating leaf explants were used to investigate the compatibility of direct shoot organogenesis with Agrobacterium-mediated transformation. Leaves of Vitis vinifera L. cvs. French Colombard and Thompson Seedless were cocultivated with Agrobacterium tumefaciens containing a binary vector carrying kanamycin resistance (APH(3')II) and beta-glucuronidase (GUS) genes. Explants were cultured on shoot-inducing medium containing levels of kanamycin inhibitory to the formation of untransformed shoots and assayed for GUS expression after 2 or 4 weeks. Cells expressing GUS were most frequently observed either at the cut surface, in vascular bundles, or in inner cortical cells of the petiole, but none of these regions produce adventitious shoots. GUS expression was also frequently found on leaf laminae, where it marked the center of a zone of cross-protected cells, but unwounded lamina cells never participated in shoot regeneration. Cells expressing GUS were found less frequently in the epidermal and subepidermal locations where exogenous, multicellular promeristem initiation occurred. These observations suggest that the direct shoot regeneration system used here could produce chimerally transformed plants, but is unsuitable for the routine production of uniformly transformed plants.