Inhibitors of neomycin phosphotransferase II enzyme-linked immunosorbent assay in grapevine (Vitis vinifera L.) leaves

Grapevine tissue extracts are rich in compounds that may inhibit detection and/or extraction of protein, DNA, and RNA. One such example can be found in the use of enzyme-linked immunosorbent assays (ELISA) to detect neomycin phosphotransferase II (NPTII) in leaf tissue. The objective of this study was to identify grape leaf components that interfere with protein detection via ELISA. A series of compounds were identified, and tartaric and ellagic acids were most inhibitory to NPTII detection. Polyphenolics as well as the low pH of grape leaf extracts also reduced the effectiveness of ELISA detection

High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' ( Vitis vinifera L.) containing npt-II and antimicrobial peptide genes

A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15mg/l kanamycin; and (3) embryo induction at 27°C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for β-glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883±1,928) and stable (46±32) blue spots per plate at 2 and 95days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifer

'GR 7' Grape

'GR 7' is an early / mid-season red wine grape for use primarily in red wine blends. It is distinguished from other red wine grapes grown in cool climates by its high degree of winter hardiness, adaptation to mechanized production systems, and ability to survive in older plantings where other red wine grapes are lost due to tomato and tobacco ringspot virus infections. ?GR 7? is a highly productive, easy to manage cultivar, and is the sixth wine grape to be developed by the New York State Agricultural Experiment Station of Cornell University

Inheritance of chloroplast DNA in two full-sib Vitis populations

The mode of transmission of chloroplasts in 2 grape populations was determined using restriction fragment length polymorphisms of chloroplast DNA (cpDNA) to trace the origin of plastids in the progeny. The populations examined were formed by crossing 2 complex interspecific hybrids: NY 62.136.2 x Yates and Cayuga White x Aurore. Analysis of the restriction banding patterns of total DNA of the 4 parents probed with cpDNA of grape and petunia revealed a high level of polymorphism (63 %) between parents ot the first cross and a low level of polymorphism (15 %) between the parents of the second cross. The restriction banding patterns of the 4 parents were unique, indicating that there were 4 distinct chloroplast genotypes. Analysis of the restriction banding patterns of total DNA of the progeny probed with cpDNA showed that all progeny from both crosses exhibited the banding pattern of the maternal parent. Thus, the mode of plastid transmission in these populations of grape was strictly maternal

High frequency regeneration from grapevine petioles: Extension to new genotypes

Auxins were found to enhance shoot organogenesis when used in a high cytokinin medium at low levels. This enhancement was genotype specific. In the presence of 10.0 µM BA, Vanessa regenerated best with the inclusion of 4.0 µM IAA-dl-aspartate, while Catawba regenerated best with 2.0 µM IAA-glycine in the medium. With Ravat 51, 14-15% of explants regenerated in the presence of either 2.0 µM IAA-glycine or 4.0 µM IAA-dl-aspartate, whereas there was no regeneration in the absence of auxin. Tests with 0.0-8.0 µM TDZ as a cytokinin source indicated that petiole explants of Vanessa regenerate best (10%) at 4.0 µM TDZ. Our results clearly show that high cytokinin media can be easily extended to petiole explants of other genotypes to facilitate shoot organogenesis and that low levels of auxins, especially IAA conjugates can enhance the level of regeneration

Use of differential thermal analysis to quantify bud cold hardiness of grape selections and clones

Differential thermal analysis (DTA) was used to characterize primary bud mid-winter cold hardiness of Vitis spp. Bud hardiness reached a maximum and was rather stable during the months of January and February at Geneva, New York. Because cold tolerance increases during periods of prolonged cold, observed freezing temperature was adjusted on the basis of the freezing temperature of cv. Concord on the day of observation. DTA gives reproducible and meaningful estimates of bud freezing temperature. Such data account for at least 50 % of the among-cultivar variance in overall vine cold hardiness

Characterization of RAPD markers in Vitis

A study was initiated to investigate the possibility of using RAPD markers in related populations of Vitis. We also sought to design primers that could amplify translation initiation sites (Kozak sequence) as a mean to maximize the production of RAPD markers from single copy DNA sequences in the genome. RAPD bands were labeled and used as probes on blots with either genomic DNA or RAPD products from cvs Aurore, Cayuga White, Horizon and Illinois 547-1. Reamplification of excised RAPD products produced either several bands of smaller size, a single band of smaller size or a single band of the same size as the original band. Among 16 probes hybridized to genomic DNA blots, three probes, including one from the Kozak primer amplification, hybridized to 1-2 bands, 5 probes hybridized to 3-8 bands and 8, including two from a Kozak primer reaction, to more than 10 bands on the genomic DNA blots. Twelve RAPD bands were also probed on RAPD blots derived from the RAPD reaction that produced each probe. Three of those probes hybridized to 1-2 bands, 8 hybridized to 3-8 and one hybridized to more than 10 bands indicating the presence of probe sequences in more than one RAPD band as amplified with the same primers. This result and the observations on reamplification of RAPD bands support the hypothesis that some of the longer RAPD fragments harbor internal priming sites that are either not amplified unless the reaction mixture is saturated with longer other primers indicating amplification from the same sequence but different sized repetitive DNA. RAPD reactions were also run with 16 primers on parental DNA of 2 crosses used in genetic mapping (Cayuga White x Aurore and Horizon x Illinois 547-1). These reactions rated 140 bands; 100 bands were shared by both populations, including 47 polymorphic bands. Ten polymorphic bands in Cayuga White x Aurore and 22 in Horizon x Illinois 547-1 were population specific. The RAPD analysis as well as hybridization of RAPD markers to the genomic blots suggest that linkage analysis could be used in related segregating populations with carefully chosen markers. Tagging single copy regions with Kozak-sequence-derived primers may be possible, but the low number of probes tested and lack of DNA sequence information prevents any definite conclusions

Remaily Seedless Grape

Since the late 19th century when grape breeding began at the New York State Agricultural Experiment Station, a major goal has been to combine certain fruit attributes such as seedlessness, crisp texture, and adherent skin of Vitis vinifera L. table grapes with some of the vegetative characters such as disease resistance and cold hardiness of native American hybrid (V. labruscana, Bailey) grape cultivars

Genetic Analysis of Restriction Fragment Length Polymorphisms in Vitis

The parents and progeny from two crosses (Cayuga White x Aurore and NY62.136.2 x Yates) were examined for the presence of DNA restriction fragment length polymorphisms (RFLPs). Seventeen independent DNA sequences were used in the analysis, 15 obtained from a grape Pstl genomic library and two heterologous probes obtained from other laboratories. Most of the low copy cloned sequences hybridized to more than two restriction fragments, possibly reflecting the polyploid nature of the Vitis genome. Nine of the probes detected RFLPs between parents. Analysis of the progenies (F1generation) revealed segregation for nine distinct polymorphisms generated by seven of the probes.Thus, a relatively high level of polymorphism among parents, as well as heterozygosity within each parent, was evident. Most RFLPs gave segregation ratios close to the 1: 1 ratio predicted for a locus heterozygous in one parent. However four differences between parental phenotypes did not segregated in the progeny, and in three instances fragments present in both parents segregated in the progeny. These peculiar results may be explained by accounting for heterozygosity or homozygosity, respectively, for the DNA segment that generates the polymorphism. We conclude that RFLP studies can be performed on the first filial generation in woody perennials such as Vitis that have a relatively high level of heterozygosity in the genom

Natural infection of Run1-positive vines by naïve genotypes of Erysiphe necator

The Run1 locus for dominant resistance to powdery mildew (Erysiphe necator) has been successfully introgressed into Euvitis from Vitis rotundifolia. In the current study, Run1 vines were hybridized with breeding lines at Cornell University, and the presence of the locus was assayed using the markers GLP1-12 and VMC8g9. Signs of powdery mildew were observed on 14 of 113 Run1-positive seedlings in October 2010 in Geneva, N.Y. Severity of infection was lower for Run1-positive than for Run1-negative seedlings. Presence of mature cleistothecia suggested infection by at least two pathogen genotypes, which since V. rotundifolia is not grown within 800+ km of Geneva, N.Y., evolved from a pathogen population naïve to Run1 resistance. Therefore, caution in the deployment of the Run1 locus in new resistant cultivars is suggested so the effectiveness of Run1 does not diminish over time.