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 (β- (β- glucuronidase (GUS) genes. Explants were cultured on shoot-inducin g 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-protect ed 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 regener- ation system used here could produce chimerally transformed plants, but is unsuitable for the routine production of uniformly transformed plants. Transgenic grapevines have not yet been recovered by Agro- bacterium- mediated transformation, although Agrobacterium infection (Martin, 1988) and adventitious shoot regeneration (Stamp et al., 1990a) can be accomplished readily. The pro- duction of transgenic plants requires that at least some trans- formed cells also participate in regeneration. By analyzing the distribution of Agrobacterium transformation sites and the de- velopmental anatomy of adventitious shoot regeneration, we sought to identify in our system the cells that are susceptible to Agrobacterium transformation and those that participate in re- generation and to determine the extent of overlap, if any, be- tween these two sets of cells. Our overall objective is to develop a method to obtain transgenic grapevines. We have previously determined the developmental sequence of direct shoot organogenesis (Colby et al., 1991). Regenerating petiole stubs of leaf explants were fixed at intervals and sec- tioned longitudinally (Fig. 1). After several days on regenera- tion medium, three distinct regions of meristematic activity became apparent. The cut surface of the petiole underwent wound mer- istem divisions and tannin deposition, after which no further development occurred in this region (Fig. 1, stage II). New vascular bundles extended from pre-existing vascular bundles to an organogenic region in which rapid periclinal divisions resulted in nodular structures (Fig. 1, stage II), some of which later became adventitious leaves (Fig. 1, stage III). Promeri- stems also arose in the organogenic region (Fig. 1, stage II), and with the development of vascular strands and true leaf pri- mordia, many promeristems became adventitious shoot meris- tems (Fig. 1, stage III).