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John E. Bowers and Carole P. Meredith

RFLP data were used to assess genetic similarity among 33 Vitis vinifera L. cultivars and one interspecific cultivar. A similarity matrix was constructed on the basis of the presence or absence of 49 bands generated by eight RFLP probes and cluster analysis was performed. The mean similarity index for all pairwise comparisons was 0.696 and ranged from 0.444 between `St. Emilion' and the interspecific hybrid `Salvador' to 0.952 between `Chenin blanc' and `Semillon'. Mean similarity among all V. vinifera cultivars was 0.705. Several groupings of similar cultivars are consistent with historical reports and presumed geographic origins: `Chardonnay' and `Melon', `Colombard' and `Folle blanche', `Gewürztraminer' and `Trousseau', `Cabernet franc' and `Cabernet Sauvignon', `Mission' and `Palomino'. The similarity between `Mission' and `Palomino' is the first genetic evidence to support the putative Spanish origin of `Mission'. Some groupings are unexpected (`Sauvignon blanc' and `Gewürztraminer', `Chenin blanc' and `Semillon') because the cultivars are not thought to have originated in the same regions. While some relationships suggested by this study may be artifacts of RFLP analysis or of the statistical method, they raise questions for further genetic inquiry into the origins of grape cultivars.

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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, 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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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.

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Summaira Riaz, Keith E. Garrison, Gerald S. Dangl, Jean-Michel Boursiquot, and Carole P. Meredith

In total, 25 clones of Vitis vinifera `Pinot noir' and 22 clones of `Chardonnay' were analyzed with 100 microsatellite markers, selected from an initial screening of 228 markers. Of the 100 markers, 17 detected polymorphism within one or both of the cultivars. In `Pinot noir', 15 polymorphic markers detected 15 different genotypes, uniquely distinguished 12 clones out of the 25 and separated the remaining 13 clones into 3 groups. In `Chardonnay', 9 polymorphic markers detected 9 genotypes and uniquely distinguished 6 clones out of the 22. The remaining 16 clones were separated into 3 groups. For markers that were polymorphic in `Pinot noir' and `Chardonnay', none of the variant alleles were common to both cultivars. It is inferred from this result that the natural cross that produced `Chardonnay' probably occurred when `Pinot' was still relatively young. Many of the variant genotypes were expressed as three alleles. Further analysis revealed the presence of chimeras in which the third allele was present in leaf but not root or wood tissues, confirming that the grape apical meristem is functionally two-layered. Some clones that share the same microsatellite genotype are documented to have originated in the same locality, suggesting that the origins of undocumented clones may be traced by comparing their microsatellite genotypes with those of well-documented clones. Because clones of `Pinot noir' and `Chardonnay' are often visually indistinguishable, microsatellite genotyping may also be useful to detect identification errors in collections and nurseries.