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Somatic embryos of grapevine (Vitis vinifera L.)`Chardonnay' were produced from liquid suspension cultures. Mature somatic embryos were blot dried briefly in the laminar flow hood and germinated directly in Magenta GA-7 Vesselse containing one of the following potting media: (1) sand, (2) commercial potting mixture (CPM), or (3) CPM overlaid with sand. Each vessel containing 20 ml of distilled water and the potting medium was sterilized by autoclaving for 30 min and cooled overnight before inoculating the somatic embryos. Five somatic embryos were placed in each vessel under aseptic conditions. The vessels were closed and incubated at 26^28CY 16 h photoperiod at 75 mmol s 21 m 22 light intensity. Results revealed that CPM overlaid with sand was best for plant development. There was more contamination of somatic embryos on pure CPM. Since direct seeding bypasses at least two subcultures in agar medium, it has implications for use of somatic embryos as`synthetic seeds' for clonal plant production. This study shows that somatic embryos of grapevine can be handled with some of the convenience of seeds, emphasizing the feasibility for further automating in vitro plant production, which might be especially useful for new varieties where propagation material is limited.

High-throughput sequencing of cDNA libraries has resulted in millions of expressed sequence tags (ESTs) from plants. To exploit such a valuable molecular resource for functional analysis of genes and genetic elements, we developed an improved thermal asymmetric interlaced (TAIL) PCR technique. We demonstrated its usefulness by recovering the complete seed-specific 2S albumin gene and promoter using a partial EST and genomic DNA of Vitis vinifera grapevine. The 2S albumin VvAlb1 (V. vinifera 2S albumin 1) gene obtained from different cultivars encompasses a coding region of 504 to 540 nucleotides corresponding to a deduced amino acid sequence of 167 to 179 residues. This deduced protein contains up to 30% glutamine residues and 8 cysteine residues arranged in a pattern highly conserved among 2S albumins for disulphide bond formation. DNA sequence alignment revealed that the same VvAlb1 gene among different grape cultivars varied greatly, including an insertion of up to 36 bp near the 3' end of the gene sequence isolated from V. vinifera 'Thompson Seedless'. DNA sequence analysis indicated that a number of highly conserved seed-specific regulatory motifs were present throughout a 2.2 kb region 5' upstream of the transcription start site of the VvAlb1 gene. Comparative analysis of promoter activity using both EGFP and GUS genes directed by various artificially truncated promoter fragments established the function of several promoter regions in regulating seed-specific gene expression in both transiently and stably transformed grape SE. In particular, a 0.4 kbp promoter fragment (-1 to-404) supported the highest level of transient expression but failed to produce any detectable level of expression in stably transformed SE. However, an opposite situation was found when a 2.2 kbp promoter fragment was used. In addition, all transgenic SE lines harboring this long promoter fragment, but not other shorter fragments, showed arrested embryo development. Plants were regenerated successfully from all transgenic SE lines and are being evaluated for temporal and spatial regulation of promoter activity. These results exemplify TAIL-PCR as a cost-effective and target-specific method to utilize the vast amount of molecular information now available.

Factors influencing Agrobacterium-mediated transformation of 15 Vitis rotundifolia, Vitis rupestris, Vitis vinifera and Vitis hybrids were explored. A green fluorescent protein/neomycin phosphotransferase II (GFP/NPT II) fusion gene was used to measure transient and stable transgene expression levels. Embryogenic cell cultures at different developmental stages were co-cultivated with Agrobacterium to determine the best stage for transformation. The effect of antioxidant treatment on recovery of transgenic embryo and plant lines was also studied. Transient and stable GFP expression varied widely among the species. For example, 1-81% of V. vinifera somatic embryos exhibited transient GFP expression, depending on the cultivar. Similarly, 0-60% of co-cultivated embryos produced stable transgenic lines. Proembryonal masses and cotyledonary stage somatic embryos were best for transformation of V. rotundifolia, V. rupestris and certain Vitis hybrids, whereas only cotyledonary stage somatic embryos were best for transformation of V. vinifera. Agrobacterium-induced browning/ necrosis was reduced by adding 1 g L-1 dithiothreitol (DTT) antioxidant to the post cocultivation wash medium. Transgenic plants have been recovered from V. rupestris 'St.