Molecular characterization of a SCAR marker purportedly linked to seedlessness in grapevine (Vitis)

Molecular markers associated with seedlessness in grapevine (Vitis vinifera) could be used for early trait selection in breeding programs and provide useful tools for elucidating the molecular mechanisms underlying seed abortion. In this study, the sequence fidelity of a previously reported seedlessness-linked Sequence-Characterized Amplified Region (SCAR) marker (GenBank accession #AY327513, Yang et al. in Chinese J Agri Biotech 3:13–17, 2006) was determined by comparative sequence analysis. Using available genomics resources, the SCAR marker was mapped to an overlapping region between exon-1 and intron-1 of a putative histidine triad protein (HIT) gene corresponding to an ORF of 588 bp for a peptide of 195 residues and a gene structure with 8 exons in a 14.7 kbp genomic region. However, PCR and DNA sequence analyses revealed that both seeded and seedless phenotypes of Vitis species contained highly homologous sequences in an EcoRI-delimited region of up to 5 kbp surrounding the SCAR marker. The absence of linkage between the marker and the seedlessness trait does not support a role for the HIT gene in seedlessness development, or the potential for the use of this SCAR marker in the identification/selection of seedless progeny.     

PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco

A functional contribution of pathogenesis-related 1 (PR-1) proteins to host defense has been established. However, systematic investigation of the PR-1 gene family in grapevine (Vitis spp.) has not been conducted previously. Through mining genomic databases, we identified 21 PR-1 genes from the Vitis vinifera genome. Polypeptides encoded by putative PR-1 genes had a signal sequence of about 25 residues and a mature protein of 10.9–29 kDa in size. PR-1 mature proteins contained a highly conserved six-cysteine motif and pI values ranging from 4.6 to 9. A major cluster with 14 PR-1 genes was mapped to a 280-kb region on chromosome 3. One particular PR-1 gene within the cluster encoding a basic-type isoform (pI 7.77), herein named VvPR1b1, was isolated from various genotypes of grapevine (Vitis spp.) for functional studies. Sequence analysis of PCR-amplified DNA revealed that all genotypes contained a single VvPR1b1 gene except for a broad-spectrum bacterial and fungal disease resistant Florida bunch grape hybrid, ‘BN5-4’, from which seven different homologues were identified. Duplication of VvPR1b1-related genes encoding acidic-type PR-1 isoforms was also observed among several genotypes. However, transgenic expression analysis of grapevine PR-1 genes under strong constitutive promoters in transgenic tobacco revealed that only the basic-type VvPR1b1 gene duplicated in ‘BN5-4’ was capable of conferring high level resistance to bacterial disease caused by Pseudomonas syringae pv. tabaci.     

Temporal and spatial patterns of gene expression in skeletal muscles in response to swim training in adult zebrafish (Danio rerio)

In adult zebrafish, 4 weeks of exercise training is known to induce an increase in mitochondrial enzymes such as citrate synthase (CS) when determined in mixed (red and white) muscle. However, this remodeling is not accompanied by changes in PGC-1α mRNA, a potent inducer of mitochondrial biogenesis in mammals. To further understand this response, we examined absolute and relative changes in red muscle area by histochemistry after 4 weeks of swim training. We also examined fiber-type specific responses in the expression of metabolic genes and putative regulators in red and white muscle of adult zebrafish at 1 and 8 weeks of training and in recovery from a single bout of exercise. Total red muscle area was unaltered after 4 weeks of training. The mRNA expression of CS was unaffected in red muscle, while it was increased in white muscle after 1 week of training and remained elevated at 8 weeks of training, suggesting an increase in oxidative capacity of this fiber type. In contrast, PGC-1α mRNA was elevated in both muscles only after 1 week of training. In both muscles, an acute bout of exercise rapidly (within 0–2 h post-exercise) induced PGC-1α mRNA and a delayed (24 h) increase in CS mRNA post-exercise. These results suggest complex temporal and spatial adaptive molecular responses to exercise in the skeletal muscles of zebrafish.     

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

Summary A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of Parafilm^TM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.     

Transgenic plants from shoot apical meristems of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. “Thompson Seedless” via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Shoot apical meristem explants of Vitis vinifera “Thompson Seedless” were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L⁻¹ in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L⁻¹ kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.     

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

     

Correction to: Improving micropropagation of Mentha × piperita L. using a liquid culture system

In Vitro Cellular & Developmental Biology - Plant - This correction reflects Sadanand A. Dhekney’s updated e-mail address and affiliation.     

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A 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.     

Factors influencing induction and maintenance of <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. embryogenic cultures

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of ‘Supreme’ and unopened leaves and petioles of ‘Delicious’. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of ‘Alachua’ and ‘Carlos’ cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in ‘Carlos’ (379.3) followed by ‘Alachua’ (350.0) and ‘Delicious’ (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 μM Benzyladenine (BA). Although high embryo germination rates (80–100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6–47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.