Carole P. Meredith

Genetic structure and differentiation in cultivated grape, Vitis vinifera L.

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.

Kanamycin sensitivity of cultured tissues of Vitis

SummaryThe kanamycin sensitivity of callus growth and adventitious shoot and root formation was studied in several cultivars of Vitis vinifera L. and in V. rupestris Scheele cv. St. George to investigate the suitability of kanamycin resistance as a selectable marker for grape transformation. Kanamycin concentrations ranged from 0 to 30 mg/l. Carbenicillin was added to the medium in all experiments at concentrations of 500 or 250 mg/l, as normally used in cocultivation experiments with Agrobacterium. Callus formation, root initiation, and adventitious shoot formation were completely inhibited by 20, 10, and 7 mg/l kanamycin, respectively; suggesting that these are the minimum concentrations that should be necessary to select transformed plants. Carbenicillin produced inhibitory effects that sometimes resembled those of growth regulators. The high kanamycin sensitivity of adventitious shoot formation in grape exceeds that reported for any other plant species and is likely to hinder the recovery of transformed plants.

Selection and characterization of aluminum-resistant variants from tomato cell cultures

Abstract Aluminum-resistant variants were obtained from cultured tomato cells by challenging callus and plated suspension cultures with 200 μM aluminum for several months. The frequencies of occurrence of the variants are estimated at 2.03 × 10 −7 per cell in callus and 8.32 × 10 −6 in suspension cultures, and are in agreement with those reported for spontaneous mutation. The variants generally grew vigorously in the presence of aluminum, although not as well as they did in the absence of aluminum. Although several variants lost some of their tolerance in the absence of aluminum, for the most part they were stable. All variants analyzed took up aluminum to the same extent as did the control. High levels of polyploidy were detected in the cell cultures. The behavior of the resistant variants suggests that they are the result of mutation, but the possibility, of an epigenetic basis for the resistance cannot be ruled out.

Direct shoot organogenesis and plant regeneration from leaves of grape ( Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl-1 than with 1 or 4 mgl-1. On media containing 2 mgl-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of ‘Cabernet Sauvignon’, ‘French Colombard’, ‘Thompson Seedless’, and ‘White Riesling’ produced adventitious shoots compared to 14, 14, and 29%, respectively, for ‘Grenache’, ‘St. George’, and ‘Ganzin 1’. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.

Genetic characterization and relationships of traditional grape cultivars from Transcaucasia and Anatolia

We present here the first large-scale genetic characterization of grape cultivars from Transcaucasia and Anatolia. These regions where wild grapes still grow in nature have been cultivating wine and table grapes for thousands of years and are considered the cradles of viticulture. Using 12 nuclear microsatellite markers, we genotyped 116 accessions of traditional grape cultivars from Armenia, Georgia and Turkey and we detected 17 identical genotypes and six homonymy cases, mainly within each national germplasm. Neighbour-joining analysis of genetic distance showed that each germplasm could have multiple origins and although they are now separated, they might have some common ancestors. In addition, four varieties from Western Europe included as outgroups turned out to be more related to Georgian cultivars than other germplasms, suggesting a possible ancient origin in Georgia. This work represents a first step towards germplasm management of this rich ampelographic heritage.

An improved polyurethane support system for monitoring growth in plant cell cultures

An improved culture system for plant cells that employs filter paper resting on polyurethane saturated with liquid medium is described. It combines a simplified version of the system outlined by Weber and Lark [1979, Theor Appl Genet 55: 81–86] with the method of growth estimation described by Horsch et al. [1980, Can J Bot 58: 2402–2406]. The growth of plated cells or callus can be conveniently monitored through repeated non-destructive fresh weight measurements of the filter paper and adhering cells, thereby allowing the construction of a complete growth curve over the course of an experiment. Experiments with 3 Nicotiana genotypes (N. plumbaginifolia Viv., N. tabacum L. ‘SC 58’ and N. tabacum ‘WI 38’) and 3 Vitis vinifera L. genotypes (‘Chenin Blanc’, ‘Dogridge’ and ‘White Riesling’) clearly demonstrate higher growth rates of plated cells on polyurethane supports compared with agar. Further experiments with N. plumbaginifolia illustrate the use of polyurethane supports for culturing cells at low pH (4.0) and the recovery of spent medium for monitoring changes in pH. These features will greatly facilitate quantitative studies of mineral nutrition and metal toxicity in cultured cells. Polyurethane supports also allow the incorporation of conditioned medium or feeder cells to support the growth of cells at low densities and facilitate the rapid recovery of variant cells.

Response of cultured tomato cells to aluminum

Abstract Callus cultures of tomato cultivars “Marglobe” and “VFNT Cherry” were subjected to 200 and 400 μM aluminum added to the culture medium. The growth of the “Marglobe” callus was completely inhibited after two passages on aluminum-containing medium, while the “VFNT Cherry” callus was only partly inhibited. The 200 and 400 μM aluminum treatments did not differ significantly in toxicity with either cultivar. Both cultivars took up similar amounts of aluminum, apparently reaching an equilibrium with the culture medium. These experiments demonstrate that aluminum toxicity in tomato is not exclusively a whole-plant phenomenon, and that genotypic differences in aluminum tolerance may be expressed in cell cultures.