S. A. Dhekney

OPTIMIZING AGROBACTERIUM-MEDIATED TRANSFORMATION OF GRAPEVINE

SummaryA translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.

Temporal and spatial control of gene expression in horticultural crops

Biotechnology provides plant breeders an additional tool to improve various traits desired by growers and consumers of horticultural crops. It also provides genetic solutions to major problems affecting horticultural crops and can be a means for rapid improvement of a cultivar. With the availability of a number of horticultural genome sequences, it has become relatively easier to utilize these resources to identify DNA sequences for both basic and applied research. Promoters play a key role in plant gene expression and the regulation of gene expression. In recent years, rapid progress has been made on the isolation and evaluation of plant-derived promoters and their use in horticultural crops, as more and more species become amenable to genetic transformation. Our understanding of the tools and techniques of horticultural plant biotechnology has now evolved from a discovery phase to an implementation phase. The availability of a large number of promoters derived from horticultural plants opens up the field for utilization of native sequences and improving crops using precision breeding. In this review, we look at the temporal and spatial control of gene expression in horticultural crops and the usage of a variety of promoters either isolated from horticultural crops or used in horticultural crop improvement.

Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco.

We report the development of a convenient plant-based reporter system to analyze promoters and facilitate selection of genetically engineered plants. The VvMybA1 gene of grapevine (Vitis vinifera L.) regulates the last metabolic step of anthocyanin biosynthesis and its ectopic expression leads to anthocyanin production in otherwise non-pigmented cells. To develop an anthocyanin-based quantitative reporter system, the VvMybA1 gene was isolated from V. vinifera ‘Merlot’ and placed under control of three promoters to test its ability to distinguish different activity levels. Promoters included a double enhanced CaMV35S (d35S) promoter, a double enhanced CsVMV (dCsVMV) promoter or a bi-directional dual promoter (BDDP), resulting in transformation vectors DAT, CAT and DEAT, respectively. These vectors were introduced into grapevine and tobacco via Agrobacterium-mediated transformation for transient and stable expression analysis. A linear relationship between the mean red brightness (MRB) and optical density (OD) values with a 0.99 regression coefficient was identified in a dilution series of anthocyanin, thus allowing the use of histogram data for non-destructive and real-time assessment of transcriptional activity. Results of histogram-based analysis of color images from transformed grapevine somatic embryos (SE) and various tissues of transgenic tobacco showed a consistent six to sevenfold promoter activity increase of DEAT over DAT. This expression increase was verified by spectroscopic measurement of anthocyanin concentrations in sepal tissue of transgenic tobacco plants. These results were congruent with previously findings of promoter activity derived from GUS fluorometric assay, thus demonstrating for the first time that the VvMybA1 gene could offer a simple, versatile and reliable plant-based alternative for quantitative promoter analysis in plants.

Initiation and transformation of grapevine embryogenic cultures.

Protocols for the production and transformation of grapevine embryogenic cultures are described. Embryogenic cultures are initiated from leaves or stamens and pistils and transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Cultures are transferred to induction medium in the dark for callus formation and proliferation. Resulting cultures are transferred to somatic embryo development medium to induce secondary embryogenesis and formation of transgenic somatic embryos. Transgenic embryos identified on the basis on GFP fluorescence and kanamycin resistance are transferred to germination medium to regenerate transgenic plants. The presence of transgenes in independent plant lines is confirmed by PCR.

An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis

Plant regeneration from grapevine (Vitis spp.) via somatic embryogenesis typically is poor. Recovery of plants from Vitis rotundifolia Michx. (muscadine grape) is particularly problematic due to extremely low efficiency, including extended culture durations required for embryo–plant conversion. Poor plant recovery is an obstacle to the selection of improved genetically modified lines. Somatic embryos (SEs) of V. rotundifolia cultivar Delicious (Del-HS) and Vitis vinifera L cultivar Thompson Seedless (TS) were used to identify culture media and conditions that promoted embryo differentiation and plant conversion; this resulted in a two-step culture system. In comparative culture experiments, C2D medium containing 6% sucrose was the most effective, among four distinct formulae tested, for inducing precocious SE germination and cell differentiation. This medium, further supplemented with 4 µM 6-benzylaminopurine (C2D4B), was subsequently determined to enhance post-germinative growth of SE. MS medium supplemented with 0.5 µM 1-naphthaleneacetic acid (MSN) was then utilized to stimulate root and shoot growth of germinated SE. An average of 35% and 80% ‘Del-HS’ and ‘TS’ SE, respectively, developed into plants. All plants developed robust root and shoot systems and exhibited excellent survival following transfer to soil. Over 150 plants of ‘Del-HS’ were regenerated and established within 2.5 months, which is a dramatic reduction from the 6- to 12-month time period previously required. Similarly, 88 ‘TS’ plant lines were obtained within the same time period. Subsequently, seven out of eight Vitis cultivars exhibited significantly increased plant conversion percentages, demonstrating broad application of the two-step culture system to produce the large numbers of independent plant lines needed for selection of desired traits.