Differential gene expression for isozymes in somatic mutants of Vitis vinifera L. (Vitaceae)

Isozyme electrophoresis was used as a method to provide a measure of relationship among Italia, Rubi, Benitaka, and Brasil cv of Vitis vinifera traditionally grown in Marialva, a town in the northwestern region of the state of Paraná, southern Brazil. No allelic variation was observed for esterase (EST), malate dehydrogenase (MDH), peroxidase (POD), glutamate dehydrogenase (GTDH), alkaline phosphatase (AKP), acid phosphatase (ACP), and aspartate amino transferase (AAT). Tissue specific and variation in staining intensity of EST, MDH, POD, and GTDH isozymes indicate differential gene expression in colour grape varieties. Regulatory genes may be operative in determining the number of molecules of enzymes in a cell and determining the berry skin polymorphism in four cultivars. Change frequency for berry skin colour suggest the occurrence of somatic crossing-over in naturally cultivated plants and a periclinal chimerism in Brasil cv. The four grape colour cultivars seem to be clones of the same cultivar.     

Pruning-induced tylose development in stems of current-year shoots of Vitis vinifera (Vitaceae)

Tyloses form in xylem vessels in response to various environmental stimuli, but little is known of the kinetics or regulation of their development. Preliminary investigations indicated that wounds seal quickly with tyloses after pruning of grapevine shoots. In this study, tylose development was analyzed qualitatively and quantitatively at different depths and times from pruning cuts along current-year shoots of grapevines at basal, middle, and apical stem regions. Tyloses developed simultaneously within a single vessel but much separated in time among vessels. Pruning caused prodigious tylosis in vessels of grape stems, extending to approximately 1 cm deep and to 7 d after wounding, but about half of the vessels did not become completely occluded. The fraction of vessels forming tyloses was greatest in basal (85%) and least in apical (50%) regions. The depth of maximum density of tyloses was 4 mm from the cut in the basal region and 2 mm from the cut in the middle and apical regions. Tylose development was faster in the basal and middle than in the apical region. The pattern of tylose development is discussed in the context of wound repair and pathogen movement in grapevines.     

Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants

Summary Transgenic grape plants were regenerated from somatic embryos derived from immature zygotic embryos of seedless grape (Vitis vinifera L.) selections. Somatic embryos were bombarded twice with 1 m gold particles using the Biolistic PDS-1000/He device (Bio-Rad Laboratories) and then exposed to Agrobacterium tumefaciens strain C58/Z707 containing the binary plasmid pGA482GG or pCGN7314. Following cocultivation, secondary embryos were allowed to proliferate on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 20–40 g/ml kanamycin (kan). Transgenic embryos were identified after 3–5 months under selection and allowed to germinate and develop into rooted plants on Woody Plant Medium containing 1 M 6-benzylaminopurine (BAP), 1.5% sucrose, 0.3% activated charcoal and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kan, positive GUS and PCR assays, and Southern analysis.     

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Wound-induced vascular occlusions in Vitis vinifera (Vitaceae): Tyloses in summer and gels in winter1

Vascular occlusion in xylem conduits is a common response to environmental stresses, and plant species are recognized as primarily tylose-forming or gel-forming. These stresses occur throughout the year, but there is little information on the wound responses throughout the year and in growing and dormant tissues. Wound-induced vascular occlusions were evaluated by type (tylose or gel), temporal progress, and spatial distribution for grape stems pruned in four seasons through an entire year. Tyloses were formed predominantly in summer and gels in winter. Cytohistological analyses indicated that wound-induced gels were pectin-rich. Both gel formation and tylose development were complete within 7 d and 10 mm from the cut regardless of the season of the wounding. Most vessels were affected by wounding, but a higher fraction of vessels developed occlusions in summer and autumn (over 80%) than in winter and spring (about 60%). The study is the first to show a single species is capable of producing primarily either tyloses or gels and that the type of wound-induced occlusion is dependent upon the season in which wounding occurs. Winter conditions limit the wound response to reversible gel formation that may contribute to refilling of embolized vessels in the spring.     

Microsatellite genotyping of cultivars of the Holy Land grapevine, Vitis vinifera ssp. sativa (Vitaceae)

Analysis of DNA microsatellites was used to assign genomic identity to the local grapevines ( Vitis vinifera ssp. sativa ) of the Holy Land. Most of the 24 analysed cultivars were sampled from the Indigenous Fruit Trees Rescue Gardens (Sataf collection) near Jerusalem. To determine the genotype identity of these cultivars, primers of the following microsatellites were used: VrZAG47, VrZAG62, VrZAG79, VVS2, VVMD5 and VVMD7. The amplicon sizes of the various microsatellites were measured by genotyping. The genetic similarity between cultivars within the local collection was computed and presented as a dendrogram. Three vines showed identical allele sizes for all six microsatellites. Two of these cultivars are likely to be genetically identical, whereas one, although potentially closely related, showed phenotypic difference at least in the colour of the berries. In comparing the allelic frequencies of the various microsatellite sizes with those of European cultivars (available in accessible web databases), it was found that the cultivar group most similar to the Holy Land grapevines is the Greek vine population. Historical and archaeological information indicates that the Sataf collection may represent only part of the expected diversity of local vines. It is thus possible that many of the missing vines still occur as unattended feral plants.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 513–521.     

Polyamines and somatic embryogenesis in two Vitis vinifera cultivars

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.     

Structural and Cytochemical Characteristics of the Stigma and Style in Vitis vinifera L. var. Sangiovese (Vitaceae)

Studies were carried out on structural and cytochemical aspectsof the stigma and style ofVitis vinifera . The stigma is ofthe wet papillate type with a continuous cuticle and pellicle.During the development of the papillae, the cell walls increasein thickness and produce a secretion product constituted oflipids that pass through the wall forming the exudate. The styleis solid with a central core of transmitting tissue which hasconspicuous intercellular spaces that increase remarkably fromthe periphery to the centre where the cuticle is present. Theintercellular spaces, where the pollen tubes grow, contain amatrix that includes polysaccharides, pectic substances andscattered areas of lipidic nature. Cytochemistry; stigma; style; ultrastructure; Vitis vinifera     

Biochemical and Genetic Polymorphisms for Carboxylesterase and Acetylesterase in Grape Clones of Vitis vinifera L. (Vitaceae) Cultivars

Native polyacrylamide gel electrophoresis (PAGE) was employed to show the highest number of esterase loci and to detect alpha- and beta-esterase polymorphisms in leaf buds of Vitis vinifera cultivars. A total of 16 esterase isozymes were detected in leaf buds from 235 plants including Italia, Rubi, Benitaka, and Brasil cultivars. Biochemical characterization of the grape esterases using ester substrates revealed alpha-, beta-, and alpha/beta-esterases with inhibitor tests distinguishing both carboxylesterases (EST-2, EST-3, EST-5, EST-6, EST-7, EST-8, EST-9, EST-10, and EST-16 isozymes) and acetylesterases (EST-4, EST-11, EST-12, EST-13, EST-14, EST-15 isozymes). No allele variation for alpha-, beta-, and alpha/beta-esterases was detected; however, EST-3 alpha-carboxylesterase was absent in 61.7% of vines, and EST-4 alpha/beta-acetylesterase was absent in one vine of Rubi cv. Null EST-3 carboxylesterase phenotype (61.7%) cannot be explained in this article, but the high genetic polymorphism in four V. vinifera clones is a positive aspect for genetic selection and development of new clones with different characteristics.     

Genetic variation and biogeography of the disjunct Vitis subg. Vitis (Vitaceae)

Aim Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location Asia, Europe, North America. Methods We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra‐specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH–psbA–trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re‐examine certain aspects of the current subgeneric classification.     

葡萄两性花花蜜腺的发育解剖学研究

葡萄两性花的花蜜腺位于子房基部的花盘上,共5枚,呈椭圆形,与雄蕊相间排列,属于花盘蜜腺,蜜腺由表皮和泌蜜组织组成,缺乏维管束,表皮具薄的角质层,无气孔器,蜜腺原基由子房基部表层细胞恢复分裂能力形成,在蜜腺发育过程中,泌蜜组织的液泡规律 性变化和多糖动态变化均不明显,原蜜汁由子房维管束的韧皮部提供,蜜汁通过表皮细胞排出。     

Precociously germinating somatic embryos of Vitis vinifera have lower ABA and IAA levels than their germinating zygotic counterparts

Somatic embryos of Vitis vinifera (cv. Grenache noir) develop normally up to the torpedo stage, but they germinate precociously and form viable plantlets with very low frequency. Because a peak in abscisic acid (ABA) in mid‐embryogenesis could be one factor preventing precocious germination during normal seed development, we followed the development of ABA content concurrent with that of the somatic embryos. Additionally, we measured changes in indoleacetic acid (IAA) levels. We also compared the levels of both hormones during precocious germination of somatic embryos and during normal germination of their zygotic counterparts. Somatic embryos were able to accumulate ABA and IAA throughout their development but no peak in ABA concentration was detected during embryogenesis. This suggests that the switch from mid‐ to late‐embryogenesis is not triggered. Furthermore, during precocious germination, i.e. from the torpedo stage onwards, the concentrations of ABA and IAA in somatic embryos were much lower than during normal germination of zygotic embryos. Thus, it is likely that when precocious germination occurs, grape somatic embryos do not accumulate ABA and/or IAA in sufficient concentrations to support normal plantlet development. Therefore, for grape somatic embryos we propose that prevention of precocious germination, i.e. triggering late‐embryogenesis, is attainable by an ABA treatment followed by slow desiccation, as already shown for conifer somatic embryos. Our results also suggest that the role of ABA and IAA for improving normal germination after imposed quiescence should be investigated.     

Three species of Phymatodes Mulsant (Coleoptera: Cerambycidae) new to South Korea that hosted on Vitis vinifera Linnaeus (Vitaceae)

Two unrecorded Phymatodes species, P. (Phymatodellus) jiangi Wang and Zheng, P. (P.) zemlinae Plavilstshikov and Anufrieva, and a known species from North Korea, P. (P.) murzini Danilevsky, are newly recognized in South Korea, as insect pests of Vitis vinifera Linnaeus (Vitaceae). In the present paper, male genitalia of P. (P.) jiangi Wang and Zheng is presented for the first time. We also provide the diagnoses and illustrations of habitus of three Phymatodes species with a revised key to Korean species.     

Somatic embryogenesis and plant development from immature zygotic embryos of seedless grapes (Vitis vinifera L.)

Summary Somatic embryo formation occurred from immature zygotic embryos within ovules of stenospermocarpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to Emershad/Ramming medium supplemented with 1 M benzylaminopurine and 0.65% TC agar. Plant development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage (torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium supplemented with 1.5% sucrose + 1 M benzylaminopurine + 0.3% activated charcoal and 0.65% TC agar.Abbreviations ABA abscisic acid - BAP benzylaminopurine - ER Emershad/Ramming - GLM general linear model - MGLH multivariate general linear hypothesis - MS Murashige/Skoog - NaClO sodium hypochlorite - TC tissue culture - WP woody plant     

The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA,ABA and polyamines

In somatic embryogenesis (SE) of conifers, the inability of many embryogenic cell lines to form well-developed somatic embryos may results from failure and constraints during the transition of proembryogenic masses (PEMs) to early somatic embryos. In the present work, we propose the inclusion of a preculture and prematuration steps looking at enhancing PEM III-to-early somatic embryos transition. It was further hypothesized that these results would correlate with the contents of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamines (PA). To test these hypotheses, the embryogenic culture was subjected to preculture with fluridone (FLD) and prematuration treatments with different combinations of carbon source and polyethylene glycol (PEG). The frequency of PEM III was increased after FLD preculture and the contents of IAA and ABA decreased, while the contents of PA increased. Putrescine (Put) was the most abundant PA present at this stage, followed by spermidine (Spd) and spermine (Spm). In early embryogenesis, prematuration treatments supplemented with maltose or lactose plus PEG enhanced the PEM III-to-early somatic embryos transition. IAA and ABA contents increased at this stage, while a decrease of the total free PA levels was observed. Put was the most abundant PA, followed by Spd and Spm, mainly in the treatment supplemented with PEG. This resulted in a decrease of PA ratio (Put/Spd + Spm) and, hence, PEM III-to-early somatic embryos transition. It was concluded that the preculture with FLD and prematuration treatments promote the PEM III-to-early somatic embryos transition throughout the whole early developmental process in Araucaria angustifolia.     

Berry morphology and composition in irrigated and non-irrigated grapevine (Vitis vinifera L.)

The present study was carried out in a 5-year-old vineyard (Vitis vinifera L., cv. Aglianico) located in Southern Italy. Half of the plants (IRR) were fully irrigated, whereas the other half were not irrigated (NIRR). In both of the treatments, plant water status, gas exchange, photosynthetic efficiency and productive performance were determined. The arid conditions resulted in significant decreases in stem water potential in NIRR (minimum values of -1.34 and -1.52 MPa in IRR and NIRR, respectively). The values of yield per plant, cluster weight and total berry weight were significantly higher in IRR. Grape berries were separated into four weight classes, and morphometric and microscopic analyses were carried out to measure and calculate berry skin characteristics. Irrigation determined a marked shift toward heavier (+23% in the class ≥ 1.25 g) and bigger (336.35 mm3 vs 299.15 mm3) berries, and induced significant changes in other morphometric berry parameters. No differences among berry weight classes and irrigation treatments were observed for berry skin thickness. In all of the berry weight classes, total anthocyanins extracted from berry skins were significantly higher in NIRR than in IRR (12301.53 and 9585.52 mg kg?1 fresh berry skin, respectively), and appeared to be positively related to berry weight, whereas total flavonols were not significantly different between the two treatments. Qualitative changes in the levels of single anthocyanin and flavonol compounds were detected between IRR and NIRR. In addition, iron, copper and zinc, whose high concentration can negatively affect wine quality, were significantly higher in the IRR treatment. The results highlighted that the absence of irrigation did not determine decreases in grape quality. Such data can be of primary importance in environments where water availability is by far the most important limiting factor for plant growth.