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Sheila M. Colby, Adrian M. Juncosa, and Carole P. Meredith

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- 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.

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James A. Stamp, Sheila M. Colby, and Carole P. Meredith

Adventitious shoots developed within 3 weeks from the petiolar stub and, less often, from wounded lamina tissues when leaves excised from nodal cultures of Vitis vinifera L. cvs. French Colombard and Thompson Seedless were cultured on solid Nitsch and Nitsch medium containing BAP at 2 mg·liter-1. The youngest leaf that could be excised, from 1 to 8 mm long, was the most responsive (90% of explants producing shoots compared to 16% for leaf 6). Removal of the lamina from the petiolar stub within the first 3 weeks of culture reduced shoot production. Increase in nodal culture age, without transfer to fresh medium, had no effect on subsequent regeneration from the youngest leaves but did reduce the regeneration frequency of leaves at the next position from 43% to 20%. In regularly subculture nodal cultures, the number of transfers had no effect on subsequent regeneration. Leaves from recently established shoot tip cultures were more responsive than leaves from nodal cultures. The frequency of shoot production was higher in laterally bisected than intact leaves (70% vs. 43%) due to additional regeneration from the distal leaf half at the sites of severed veins. Shoot outgrowth was promoted by the isolation and subculture of regenerating tissue to fresh regeneration medium. Petiolar stub removal promoted de novo shoot organogenesis from the resulting lamina wound. Shoots rooted at a high frequency on Murashige and Skoog medium with 1 mg IA-A/liter and produced morphologically normal plants. Chemical names used: 6-benzylaminopurine (BAP); indole-3-acetic acid (IAA).

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Sheila M. Colby, Adrian M. Juncosa, James A. Stamp, and Carole P. Meredith

The developmental anatomy of direct shoot organogenesis from in vitro leaves of Vitis vinifera L. cv. French Colombard was studied by light microscopy. Regenerating petiole stubs of leaf explants were fixed at intervals and were sectioned longitudinally to determine the developmental sequence of direct shoot organogenesis. After 6 days, three distinct regions of meristematic activity were apparent within expanding petiole stub: the wound-response, organogenic, and vascularization regions. In the organogenic region, divisions of vacuolate outer cortical cells formed nodular bumps that sometimes became adventitious leaves. Promeristems, which had the potential to become adventitious shoot meristems, were also initiated asynchronously in the organogenic region. Promeristem initiation occurred by two or several synchronous cell divisions occurring in the epidermal and subepidermal cell layers. Adventitious shoots and leaves developed new vascular bundles that connected to the pre-existing vascular bundles of the explant.