A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

Potassium nutrition of Cabernet Sauvignon grapevines (Vitis vinifera L.) as affected by shoot trimming

As potassium (K) requirement of grapevine (Vitis viniferaL.) berries is high and phloem translocation from mature leaves to developing organs is well established, it was posited that shoot trimming, a widely applied technique which alters the source-sink balance and the mature-to-immature foliage ratio of a canopy, may influence K deficiency. Six-year-old Cabernet Sauvignon grapevines were grown in 0.045 m³ pots (one plant per pot) filled with a soil sampled from a vineyard previously displaying K deficiency symptoms. Two levels of K supply (K₀, no K added; K_1, 25 g K per pot added in five splits from bloom to post veraison) were tested on vines that for each level were left (a) untrimmed and trimmed at ten main leaves with (b) or without (c) maintenance of lateral shoots in a split-plot design. Potassium concentration of leaf blades, berries and canes, vegetative growth and leaf gas exchange were recorded throughout the season; yield, grape quality and must characteristics were determined at fruit maturity. While adding potassium to the soil resulted in higher K concentration in blades of both main and lateral shoots and berries at harvest, trimming with removal of lateral shoots resulted in lower blade K concentration of the main leaves at harvest and more severe K deficiency symptoms regardless of K soil availability. Berry K concentration and the fraction of whole-plant K partitioned to clusters were not significantly affected by trimming. Gas-exchange, vegetative growth, yield and grape quality were not affected by the seasonal K fertilization, whereas the latter was impaired when trimming with excision of lateral shoots was applied. The results indicate that if shoot trimming is not followed by an adequate regrowth of secondary shoots an excessive depletion of potassium from the retained old basal leaves occurs during fruit maturity and increases the risk of leaf K deficiency, particularly in the K₀ treatment.     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

Warm spring temperatures induce persistent season-long changes in shoot development in grapevines

Background and Aims

The influence of temperature on the timing of budbreak in woody perennials is well known, but its effect on subsequent shoot growth and architecture has received little attention because it is understood that growth is determined by current temperature. Seasonal shoot development of grapevines (Vitis vinifera) was evaluated following differences in temperature near budbreak while minimizing the effects of other microclimatic variables.

Methods

Dormant buds and emerging shoots of field-grown grapevines were heated above or cooled below the temperature of ambient buds from before budbreak until individual flowers were visible on inflorescences, at which stage the shoots had four to eight unfolded leaves. Multiple treatments were imposed randomly on individual plants and replicated across plants. Shoot growth and development were monitored during two growing seasons.

Key Results

Higher bud temperatures advanced the date of budbreak and accelerated shoot growth and leaf area development. Differences were due to higher rates of shoot elongation, leaf appearance, leaf-area expansion and axillary-bud outgrowth. Although shoots arising from heated buds grew most vigorously, apical dominance in these shoots was reduced, as their axillary buds broke earlier and gave rise to more vigorous lateral shoots. In contrast, axillary-bud outgrowth was minimal on the slow-growing shoots emerging from buds cooled below ambient. Variation in shoot development persisted or increased during the growing season, well after temperature treatments were terminated and despite an imposed soil water deficit.

Conclusions

The data indicate that bud-level differences in budbreak temperature may lead to marked differences in shoot growth, shoot architecture and leaf-area development that are maintained or amplified during the growing season. Although growth rates commonly are understood to reflect current temperatures, these results demonstrate a persistent effect of early-season temperatures, which should be considered in future growth models.     

AFLP analysis of genetic relationships among aromatic grapevines (<Emphasis Type="Italic">Vitis vinifera</Emphasis>)

Genotypic diversity has been detected among aromatic grapevines (Vitis vinifera) by molecular markers (AFLPs). The 22 primer-pairs generated a total of 1,331 bands of which 564 (40%) were polymorphic over all the genotypes. The bootstrap analysis pointed out that a large number of polymorphic bands (200–400) has to be used for a better estimation of the genetic distances among genotypes; 383 polymorphic AFLP bands were used for the cluster and the principal coordinate analyses because they did not present missing data across all the genotypes. The cluster analysis (UPGMA), based on polymorphic AFLP markers, revealed no relationship between the Moscato and Malvasia grapevines. The Malvasias, unlike the Moscatos distinguished by their distinct muscat aroma, have to be considered a more complex group because it includes muscat and non-muscat grapevines. The principal coordinate analysis (PCO) confirmed the pattern of the cluster analysis only for those varieties which presented a low coefficient of dissimilarity, while for the other varieties there was no correspondence between the two analyses. The pattern of aggregation among aromatic grapevines in the cluster and principal coordinate analyses does not support any classification that might include an aromatic grapevine group in V. vinifera. Even though some synonyms and homonyms are present among aromatic grapevines (V. vinifera), genetic diversity exists among genotypes in AFLP markers.Communicated by H.F. Linskens     

Leaf Gas Exchange and Water Relations of Grapevines Grown in Three Different Conditions

Diurnal and seasonal changes in the leaf water potential (), stomatal conductance (g _s), net CO₂ assimilation rate (P _N), transpiration rate (E), internal CO₂ concentration (C _i), and intrinsic water use efficiency (P _N/g _s) were studied in grapevines (Vitis vinifera L. cv. Touriga Nacional) growing in low, moderate, and severe summer stress at Vila Real (VR), Pinhão (PI), and Almendra (AL) experimental sites, respectively. In VR and PI site the limitation to photosynthesis was caused more by stomatal limitations, while in AL mesophyll limitations were also responsible for the summer decline in P _N.     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.     

Somatic embryogenesis from grapevine cells. I-Improvement of embryo development by changes in culture conditions

In conventional culture conditions without auxin, somatic embryos arising from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas x Vitis berlandieri) are arrested at the heart stage of development. Starting from indications that inhibitors excreted in the culture medium could be responsible for this arrest, new culture conditions based on daily subculturing embryos in fresh medium have been successfully used to obtain full embryo development. From this technique, a microassay was devised for screening small amounts of extracellular molecules as potential inhibitors of embryonic development. Our results show that extracellular macromolecules of molecular weight higher than 10 kDa are likely involved in the inhibition of caulinary meristem initiation. However, other factors obviously cooperate to inhibit embryo development in conventional culture conditionsAbbreviations CH76 cv. Chardonnay clone 76 (Vitis vinifera) - NOA 2-naphthoxyacetic acid     

Comparative effects of salt-stress and alkali-stress on the growth,photosynthesis, solute accumulation,and ion balance of barley plants

We compared the effects of salt-stresses (SS, 1: 1 molar ratio of NaCl to Na₂SO₄) and alkali-stresses (AS, 1: 1 molar ratio of NaHCO₃ to Na₂CO₃) on the growth, photosynthesis, solute accumulation, and ion balance of barley seedlings, to elucidate the mechanism of AS (high-pH) damage to plants and the physiological adaptive mechanism of plants to AS. The effects of SS on the water content, root system activity, membrane permeability, and the content of photosynthetic pigments were much less than those of AS. However, AS damaged root function, photosynthetic pigments, and the membrane system, led to the severe reductions in water content, root system activity, content of photosynthetic pigments, and net photosynthetic rate, and a sharp increase in electrolyte leakage rate. Moreover, with salinity higher than 60 mM, Na⁺ content increased slowly under SS and sharply under AS. This indicates that high-pH caused by AS might interfere with control of Na⁺ uptake in roots and increase intracellular Na⁺ to a toxic level, which may be the main cause of some damage emerging under higher AS. Under SS, barley accumulated organic acids, Cl⁻, SO₄ ²⁻, and NO₃ ⁻ to balance the massive influx of cations, the contribution of inorganic ions to ion balance was greater than that of organic acids. However, AS might inhibit absorptions of NO₃ ⁻ and Cl⁻, enhance organic acid synthesis, and SO₄ ²⁻ absorption to maintain intracellular ion balance and stable pH.     

Leaf number and position effects on the survival and performance of grape microcuttings in vitro,and the sensitivity of the cut nodal region to the medium

The presence of leaf in microcuttings of grape cvs. Arka Neelamani and Thompson Seedless promoted rooting in vitro (MS, 1 μM IAA, 0.1 μM GA₃, 3% sucrose) but the effect varied depending on the number of leaves and position of the leaf on the cutting. Single node cuttings with a full-length lower internode and a lamina at top (LAT) showed earlier rooting and more root and shoot growth than cuttings with lamina positioned at the middle (LAM), while cuttings with a leaf at the base (LAB) of the cutting and full-length upper internode exhibited a lower percent rooting and sprouting, poor root and shoot growth, and low survival. Partial or complete removal of the upper internodal segment in LAB cuttings improved rooting and sprouting suggesting the possible operation of an inhibitory effect by the upper internode. Retaining an upper leaf in LAB cuttings (LAB+UL) resulted in necrosis of the upper leaf often followed by the lower one. The extent of necrotic damage was influenced by the leaf area and position or age of the cutting on the stock shoot. Retaining the lower internode in LAB and LAB+UL cuttings which held the node–leaf junction away from the medium, or reducing the concentration of MS medium helped significantly in improving the survival and performance of these cuttings. The difference in reaction between LAB and LAT cuttings was attributable mainly to the difference in the sensitivity of the stem part that came in contact with the medium. Removal of the leaf in LAB cuttings reduced this sensitivity. The majority of the LAB and LAB+UL cuttings, as well as non-rooting or delayed rooting LAT and LAM cuttings, exhibited high purple pigmentation of leaf, petiole and stem. Two-leafed cuttings in vitro showed poor survival, less rooting and low plantlet output compared to single-leafed cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

EBR预处理对盐胁迫下葡萄幼苗叶片抗氧化物质及酶活性的影响

以2年生葡萄(Vitis vinifera L.)酿酒品种赤霞珠扦插苗为材料,在水培条件下,分别用0、0.05、0.10和0.20mg/L 24-表油菜素内酯(EBR)预处理幼苗,然后进行50mmol/L NaCl胁迫,分别在胁迫6d和12d测定幼苗叶片中超氧阴离子(O_2~)、丙二醛(MDA)、抗氧化物质含量以及相关酶活性,探讨EBR预处理对葡萄幼苗耐盐性的影响。结果表明:与单独盐胁迫处理相比,不同浓度的EBR预处理使盐胁迫葡萄幼苗叶片O_2~和MDA含量显著降低,同时使其抗氧化物质抗坏血酸(AsA)、脱氢抗坏血酸(DHA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量以及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、超氧化物歧化酶(SOD)活性显著升高;其中,0.10mg/L EBR预处理的表现最佳,在盐胁迫12d时,其葡萄叶O_2~和MDA含量比单独盐胁迫处理分别显著降低30.5%和22.0%,其叶片相应AsA和GSH的含量较单独盐胁迫处理分别显著提高82.8%和27.9%,且GR、APX和SOD活性分别显著提高7.2%、8.5%和24.0%。研究发现,在盐胁迫条件下,适宜浓度的外源BRs预处理能够显著降低葡萄叶片中活性氧含量,提高抗氧化物质含量和抗氧化酶活性,以促进AsA-GSH循环的快速有效运转,有效减轻植株的过氧化伤害,缓解盐胁迫对葡萄幼苗的伤害,提高葡萄的耐盐性。     

PEG-tolerant cell clones of chili pepper: Growth,osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 M 2,4-d and 0.44 M BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K⁺ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - PEG polyethylene glycol     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.     

Effects of potassium iodide on the growth and metabolite accumulation of two planktonic diatoms

In this study the effects of potassium iodide on the growth and metabolite accumulation of Nitzschia closterium (Ehr.) W. Smith and Phaedactylum tricornutum Bolin were investigated to assess its possible application to the mass culture of the two diatoms in open environment, extensive systems. The results indicated that supplementation of potassium iodide at a concentration of 1000 mg L⁻¹ resulted in a reduction of the induction phase in cultures of N. closterium and P. tricornutum and led to an increase in the accumulation of biomass and extracellular polymeric substances. Conversely, the addition of potassium iodide, at all concentrations tested, showed no obvious effect on the fatty acid profiles of the two diatoms, particularly in the content of eicosapentaenoic and decosahexaenoic acid. Potassium iodide was also found to inhibit the growth of Dunaliella salina, Cryptomonas sp. and Chlorella sp. at minimum inhibitory concentrations of 356.8, 475.9 and 696.2 mg L⁻¹, respectively. It also inhibited bacteria, including species isolated from the two diatom cultures, at a minimum concentration of 400 mg L⁻¹. These results suggest that potassium iodide is an effective agent for inhibiting the proliferation of certain flagellate and non-flagellate algae, and bacteria, thus forming a favorable environment for diatoms to proliferate and consequently improving accumulation of biomass and EPS. These properties of potassium iodide provide a possible solution for preventing contamination from flagellate and non-flagellate algae in mass culture of the two diatoms without causing significant changes in their fatty acid composition.     

Characterization ofVitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme

Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5-and 3-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1-and pGS1;2-related genes.     

Solute accumulation, solute potential, germinability and seedling vigour of seeds of finger millet (Eleusine coracana Gaertn.) raised under rain-fed conditions and under irrigation

Abstract The nature of the solutes accumulated in the grains of ten finger millet genotypes grown under rain-fed conditions with moisture stress during panicle and grain development were determined. Although grain yield decreased under stress, the solute potential (SP) of seeds from stressed plants was significantly lower (more negative), probably due to the increase in sucrose content. There were no significant changes in the content of total amino acids, free proline, reducing sugars or potassium. The contribution of sucrose to total solute potential was approximately 32% in seeds obtained from stressed plants as compared with only 15% in seeds from irrigated treatment. The germinability and seedling vigour of the seeds from stressed plants was significantly higher under simulated stress with polyethylene glycol (PEG), but was similar to that of seeds of non-stressed plants under normal conditions.     

Effects of a long chain aliphatic alcohol mixture on growth and solute accumulation in water stressed wheat seedlings under laboratory conditions

Polyethylene glycol (PEG)-induced water stress adversely affected the germination and seedling growth of Sonalika and WL2265 cultivars of wheat (Triticum aestivum L.). Application of a mixture of long-chain aliphatic alcohols having the composition C-24 Tetracosanol (10%), C-26 Hexacosanol (16%), C-28 Octacosanol (15%), C-30 Tricontanol (30%), C-32 Dotriacontanol (15%) and C-34 Tetratriacontanol (14%), partially ameliorated these effects and promoted both percent germination and seedling growth. Application also stimulated the activities of the hydrolases - and -amylase and acid invertase, so increasing free sugar accumulation. A role for long chain aliphatic alcohols in the regulation of carbohydrate metabolism is suggested. The alleviation of moisture stress by application of this mixture suggest that long chain aliphatic alcohols may be most effective at low water potentials.