Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.
Manjul Dutt, Zhijian T. Li, Sadanand Dhekney, and Dennis J. Gray
Sadanand A. Dhekney, Zhijian T. Li, Michael E. Compton, and Dennis J. Gray
Stamens and pistils from mature grapevines and leaves from in vitro micropropagation cultures were used to optimize parameters influencing somatic embryogenesis in Vitis. Embryogenic competence was dependent on species/variety, explant type and developmental stage, medium composition, and growth regulator concentration. Of varieties evaluated, a greater number produced embryogenic cultures from stamens and pistils (26) compared with leaves (six). Among the different stamen and pistil stages, Stage II and III explants produced the maximum embryogenic response regardless of genotype and medium composition. Of seven culture media tested, the highest embryogenic response was recorded from varieties cultured on MSI (18) and PIV (16) media. Experiments annually repeated over 3 to 10 years demonstrated reproducible results. Highly reliable protocols for somatic embryogenesis were obtained for 29 Vitis species and varieties, including 18 Vitis vinifera varieties, Vitis riparia, Vitis rupestris, Vitis champinii, and eight Vitis hybrids. Embryogenic cultures were maintained on X6 medium for a period of 6 months to 2 years depending on the variety and used in studies involving genetic transformation and transgenic plant regeneration.
Dennis J. Gray, Zhijian T. Li, Sadanand A. Dhekney, Donald L. Hopkins, and Charles A. Sims
Manjul Dutt, Dennis J. Gray, Zhijian T. Li, Sadanand Dhekney, and Marilyn M. Van Aman
A major drawback to the use of embryogenic cultures for transformation of grapevine is that their ability to undergo genetic transformation is cultivar-dependent. Also, depending on cultivar, embryogenic cultures are difficult to impossible to maintain over time, reducing their utility for use in genetic transformation. An alternative to the use of embryogenic cultures for transformation of grapevine is the use of micropropagation cultures, which are easier to initiate from a wide range of grapevine cultivars and can be maintained over time without loss of function. Vitis vinifera `Thompson Seedless' was used as a model for genetic transformation using micropropagation cultures. In vitro cultures were initiated from apical meristems of actively growing vines and maintained in C2D medium containing 4 μM of 6-benzylaminopurine (C2D4B). Shoot tips and nodes were collected from proliferating in vitro cultures for transformation studies. A variety of wounding techniques, including nicking, sonication, and fragmenting of meristematic tissues was employed in order to enable Agrobacterium infection. We used a construct containing a bidirectional 35S promoter complex with a marker gene composed of a bifunctional fusion between an enhanced green fluorescent protein (EGFP) gene and a neomycin phosphotransferase (NPTII) gene in one direction and a hybrid lytic peptide gene in the other. Transgenic shoots growing in C2D4B medium containing 200 mg·L-1 each of carbenicillin and cefotaxime and 20 mg·L-1 of kanamycin were selected based on GFP fluorescence. Transgenic shoots were rooted and transferred to a greenhouse. To date, 18 transgenic lines have been generated. Details on the transformation procedure will be discussed.