Zhijian T. Li

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.

Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco.

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.

Constitutive expression of Vitis vinifera thaumatin-like protein after in vitro selection and its role in anthracnose resistance

Anthracnose-resistant grapevine (Vitis vinifera L. cv. Chardonnay) plants were regenerated from embryogenic cultures that had been subjected to in vitro selection with culture filtrate of Elsinoe ampelina (de Bary) Shear. Three secreted proteins differentially expressed by in vitro-selected embryogenic cultures and regenerated plants were identified. An 8-kDa protein was identified as a lipid-transfer protein (LTP) by N-terminal amino acid sequence comparison. Two other differentially expressed proteins, with estimated molecular weights of 21.6 and 22kDa, immuno-reacted with antiserum raised against a thaumatin-like protein (TLP) protein from pinto bean (Phaseolus vulgaris L.). N-terminal amino acid sequencing of the 21.6-kDa protein showed a high degree of homology to V. vinifera thaumatin-like protein 2 (VVTL-2 = grapevine osmotin; Acc no. CAA71883), and that of the 22-kDa protein was homologous to V. vinifera thaumatin-like protein 1 (VVTL-1; AAB61590). Interestingly, both VVTL-1 and VVTL-2 are pathogenesis-related (PR) proteins, belonging to the PR-5 group. Protein produced from the cloned grapevine VVTL-1 gene significantly inhibited E. ampelina spore germination and hyphal growth in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in vivo. Enhanced expression of antifungal VVTL-1 in anthracnose resistant grapevine strongly suggests that it plays an important role, either alone or in conjunction with other PR proteins, by suppressing pathogen growth.

An improved enzyme-linked immunoabsorbent assay protocol for the detection of small lytic peptides in transgenic grapevines (Vitis vinifera)

An enzyme-linked immunoabsorbent assay (ELISA) protocol was developed for the detection of small lytic peptides in transgenic grapevines (V. vinifera). The protocol requires a high concentration of protease inhibitor in the extraction buffer; the use of antiserum cross-absorbed with control tissue, an increased concentration of blocking reagents in the antiserum buffer, and performing all coating and/or binding processes at 37°C while reducing the time period for each step to 1 h. The procedure greatly reduced protein degradation, increased the signal-to-noise ratio, and it allowed the effective detection of the Shiva-1 lytic peptide (5 kDa) at concentrations as low as 0.1 μM. This procedure made it possible for routine analysis of transgene expression in Shiva-1 gene-containing transgenic grape plants.

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.

Direct seeding of grapevine somatic embryos and regeneration of plants

SummarySomatic 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 Vessels™ 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±2°C, 16 h photoperiod at 75 μmol s−1 m−2 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.

Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.

Somatic Embryogenesis and Genetic Modification of Vitis

Grapevine embryogenic cultures are ideal target tissues for inserting desired traits of interest and improving existing cultivars via precision breeding (PB). PB is a new approach that, like conventional breeding, utilizes only DNA fragments obtained from sexually compatible grapevine plants. Embryogenic culture induction occurs by placing leaves or stamens and pistils on induction medium with a dark/light photoperiod cycle for 12-16 weeks. Resulting cultures produce sectors of embryogenic and non-embryogenic callus, which can be identified on the basis of callus morphology and color. Somatic embryo development occurs following transfer of embryogenic callus to development medium and cultures can be maintained for extended periods of time by transfer of the proliferating proembryonic masses to fresh medium at 4-6-week intervals. To demonstrate plant recovery via PB, somatic embryos at the mid-cotyledonary stage are cocultivated with Agrobacterium containing the desired gene of interest along with a, non-PB, enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Modified cultures are grown on proliferation and development medium to produce uniformly modified somatic embryos via secondary embryogenesis. Modified embryos identified on the basis of green fluorescence and kanamycin resistance are transferred to germination medium for plant development. The resulting plants are considered to prototype examples of the PB approach, since they contain egfp/nptII, a non-grapevine-derived fusion gene. Uniform green fluorescent protein (GFP) fluorescence can be observed in all tissues of regenerated 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.