Enhancement of Stress Tolerance in Vitis vinifera
Abiotic stresses affect important aroma, flavor and color components by altering metabolite composition, improving wine quality and human health benefits. Regulated deficit irrigation has been used successfully to grow grapes with less water, an important feature in arid regions such as Nevada. As a first step toward understanding how growth is affected and wine quality improvements might arise following abiotic stress exposure, we have initiated an expressed sequence tag (EST)-based gene discovery program focused solely on stressed plants. We constructed cDNA libraries from mRNA isolated from leaf and berry tissues of Vitis vinifera cv. Chardonnay, exposed to various abiotic stress conditions. To date, we have sequenced over 3000 leaf ESTs and anticipate completing another 5000 berry sequences over the next few months before funds run out this year. Raw sequence data were processed through an automated EST analysis pipeline (ESTAP) developed at the Virginia Bioinformatics Institute (VBI; Blacksburg, VA) in collaboration with UNR and S.R. Noble Foundation (Ardmore, OK). Initial sequence analysis reveals 36%novel genes and a low redundancy of transcripts. All 1878 unique EST data generated to date have been deposited in GenBank and is freely available to the public.
In the context of genetic engineering more cold tolerant grapes, we have successfully transformed and regenerated V. vinifera in our laboratory. Embryo culture was successfully initiated using 0.5 to 1 mm immature anthers that were excised from flowers of Chardonnay plants. The excised anthers were placed on one of the following callus-initiation mediums: NB, PT or PIV medium. Tissues were subcultured to fresh plates every 6-8 weeks. Embryogenic calli suitable for transformation formed on some cultures. Single cell somatic embryos were transformed with CBF1 and CBF3 constructs including the CAMV35S promoter. The CBF/DRE transcriptional activators, CBF1 (DREB1B), CBF2 (DREB1C) and CBF3 (DREB1A) are some of the master switches for drought, salinity and cold tolerance. Eleven transgenic plants have been regenerated after selection on Kanamycin media. Three plants have been positively identified by PCR for transformation with CBF3. Confirmation of transformation by PCR and Southern analysis for the rest of the plants is underway. A second batch of transformed somatic embryos is currently going through the regeneration process. Degenerate primers designed to motifs of the CBF gene family have produced 8 distinct PCR products. These products represent putative grape CBF orthologs. Additional CBF orthologs are expected to be obtained from our on going EST sequencing program.
Must samples were obtained from well-watered and drought-stressed Chardonnay grapes. Standards were developed for gas chromatography/mass spectroscopy in preparation for must analysis.
