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     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.     

Somatic embryogenesis from stigmas and styles of grapevine

Summary An in vitro protocol has been developed for callus indiction, somatic embryogenesis, and plant regeneration from stigma-style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.: cvs. ‘Bombino Nero’, ‘Greco di Tufo’, ‘Merlot’, and ‘Sangiovese’) were tested. Exlants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA: 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot’, which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma-style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA). 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.     

Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K⁺ representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca²⁺ from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K⁺. Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca²⁺ uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca²⁺ uptake which results in callose deposition by a direct activition of the Ca²⁺-dependent and plasma-membane-located 1,3--glucan synthase.     

Tracheary-element differentiation in suspension-cultured cells ofZinnia requires uptake of extracellular Ca2+

Tracheary-element (TE) differentiation in suspension cultures ofZinnia elegans L. mesophyll cells was inhibited by blocking calcium uptake in three ways: 1) reducing the [Ca²⁺] of the culture medium, 2) blocking calcium channels with the non-permeant cation La³⁺, and 3) blocking calcium channels with permeant dihydropyridine calcium-channel blockers. Calcium-channel blockers were effective when added at any time between 0 and 48 h after culture initiation; after 48h, calcium sequestration and secondary cell-wall deposition began. In contrast, calmodulin antagonists inhibited TE differentiation when added at the beginning of culture, but not when added after 24h. These results indicate that TE differentiation involves at least two calcium-regulated events: one calmodulin-dependent and occurring shortly after exposure to inductive conditions, and the other calmodulin-independent and occurring just prior to secondary cell-wall deposition.     

Study of genetic relationships between wild and domesticated grapevine distributed from Middle East Regions to European countries

Archaeobotanical-archaeological, cultural and historical data indicate that grapevine domestication can be dated back from 6000 to 7000 years ago and that it took place in the Caucasian and Middle East Regions. However, events leading to the domestication of this crop species are still an open issue. In this paper, 6 chloroplast microsatellites have been used to assess genetic similarities among, and within, domesticated and wild grapevine accessions representative of 7 distinct geographical regions from the Middle-East to Western Europe. Results show that 2 out of the 6 analyzed chloroplast loci are polymorphic within the 193 domesticated individuals and the 387 samples of 69 wild populations. Allele variants of the Cp-SSR loci combine in a total of 6 different haplotypes. The data show that the haplotype distribution is not homogeneous: the 6 haplotypes are present in the domesticated varieties, but only 5 (haplotype VI is absent) are observed in wild populations. The analysis of haplotype distribution allows discussion of the relationships between the two grape subspecies. The contribution of the wild grape germplasm to the domesticated gene pool still growing in different geographical regions can be, in cases, made evident, suggesting that beside domestication, gene introgression has also played a role in shaping the current varietal landscape of the European viticulture.

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Partitioning and mobilization of starch and N reserves in grapevine (Vitis vinifera L.)

We followed C and N reserves of grapevines grown in trenches under semi-controlled conditions over a 3-year period after planting. Temporal mobilization of stored C and N and subsequent distribution of reserve materials within the vines were described in parallel with 15N uptake, particularly during the third growing season. Storage C in the perennial tissues (roots, trunk, canes) was mainly made of starch, which accumulated in the ray parenchyma of the wood. In the permanent tissues, starch and total nitrogen contents were found to decrease early in the development (bleeding sap, budbreak) whereas, on a concentration basis, they decreased only after stage 7 (first leaf fully expanded). Starch started to accumulate again in the perennial tissues during flowering. The same observation was made with total nitrogen, although N levels were much lower than those of starch. The 15N study showed that N uptake by the roots started at budbreak and increased with vine development, becoming predominant over reserve mobilization only after the onset of flowering. Taken together, these results indicate that the spring growth period can be divided into three main phases: In the first (dormancy to budbreak), significant losses of C and N proceed mainly via root necrosis. In the second period (first leaf to the onset of bloom), a strong mobilization of starch (and, to a lower extent, of N) occurred for supporting vegetative and reproductive growth. At that point, most of the C and N reserves used on the spring flush were those of the roots, rather than those of the old wood (trunk, canes). In the third period (bloom and early berry development), the mobilization process became low and was relieved by N uptake (and CO2 assimilation) supplying nutrients to the sink structures.     

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Humid incubation period and plantlet age influence acclimatization and establishment of micropropagated grapes

Summary In vitro rooted grape (Vitis vinifera L.) plantlets (4 or 8 wk from culturing microcuttings) were plantedex vitro in polythene sachets (24×12 cm) filled to one-third their height with planting mixture. The sachets were misted, closed, and incubated at ambient temperature (25–30°C) under 16 h photoperiod (40–50 μ E·m^−2.·sec⁻¹) for 1,2, or 3 wk before opening. Maximum establishment with no or minimum damage toin vitro formed leaves was obtained with 3 wk of closed sachet incubation in both age groups. Opening the sachet at 2 wk from planting resulted in marginal scorching of lower leaves and some reduction in establishment and vigor. Opening at 1 wk led to severe leaf scorching and significant reduction in establisment particularly in 4-wk-oldin vitro plantlets while growth was more affected in 8-wk-old ones. Relative humidity ofin vitro culture vessel was 68–75% while closed sachets had RH values of 45–77% depending on length of incubation and ambient RH. Diurnal variation in RH of sachet in relation to ambient RH was the major factor that facilitated acclimatization rather than the overall fall in RH during the period of closed incubation. Satisfactory acclimatization of plantlets to withstand the open sachet RH (50–55%) by 3 wk and the ambient RH (30–40%) by 4 wk was achieved. Monitoring water loss from detached leaves of plantlets showed a significant reduction between the date of planting and Week 3, and again between Weeks 3 and 4. Comparison of growthin vitro andex vitro suggested that shifting toex vitro earlier was more beneficial. This observation was confirmed by transferring 3-, 4-, and 5-wk-old plantlets fromin vitro rooting medium toex vitro and recording the growth at 8 wk fromin vitro culturing when 3 wkin vitro plus 5 wkex vitro combination showed maximum vigor. The leftover stumps after subculturing of 1–4-mo.-old stock cultures could also be effectively used forex vitro establishment.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H(2)O(2) in grapevine leaves

Biosynthesis of both ascorbic acid (AsA) and peroxidase activity were induced by light in cv. Sultana grapevine leaves. Induced peroxidase activity mainly involved basic isoenzymes of pI 9.8 and 9.6 and catalyzed the oxidation of flavonoids like quercetin and kaempferol and derivatives of hydroxycinnamic acids such as ferulic and p-coumaric acids, but not AsA. However, the peroxidase-dependent oxidation of ferulic acid and quercetin was temporarily suppressed by AsA as long as it remained in the reaction medium. Kinetics and spectroscopic results indicated that AsA was oxidized to dehydroascorbic acid only in the presence of phenols or flavonoids, and did not interfere with the catalytic activity of the peroxidase. Ascorbate peroxidase isoenzymes (APx), whose activities are widely considered central for detoxification of H(2)O(2) in most plant cells, were not detected in grape leaves extracts. The significance of light stimulus on peroxidase activity and leaf AsA content is discussed in terms of a flavonoid-redox cycle proposed as an alternative system to detoxify H(2)O(2) in grapevine leaves.     

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF,is not only expressed in flowers but also throughout berry development

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.     

Leaf area estimation by simple measurements and evaluation of leaf area prediction models in Cabernet-Sauvignon grapevine leaves

For two growing seasons (2005 and 2006), leaves of grapevine cv. Cabernet-Sauvignon were collected at three growth stages (bunch closure, veraison, and ripeness) from 10-year-old vines grafted on 1103 Paulsen and SO4 rootstocks and subjected to three watering regimes in a commercial vineyard in central Greece. Leaf shape parameters (leaf area-LA, perimeter-Per, maximum midvein length-L, maximum width-W, and average radial-AR) were determined using an image analysis system. Leaf morphology was affected by sampling time but not by year, rootstock, or irrigation treatment. The rootstock×irrigation×sampling time interaction was significant for all the leaf shape parameters (LA, Per, L, W, and AR) and the means of the interaction were used to establish relationships between them. A highly significant linear function between L and LA could be used as a non-destructive LA prediction model for Cabernet-Sauvignon. Eleven models proposed for the non-destructive LA estimation in various grapevine cultivars were evaluated for their accuracy in predicting LA in this cultivar. For all the models, highly significant linear functions were found between calculated and measured LA. Based on r ² and the mean square deviation (MSD), the model proposed for LA estimation in cv. Cencibel [LA = 0.587(L×W)] was the most appropriate.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+ -transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca2+ + Mg2+)-ATPase activity and in Ca2+ efflux at 37 degrees C, depending on the side of the membrane at which the monovalent cations K+ and Na+ were placed. Under the conditions used, (Ca2+ + Mg2+)-ATPase activity and Ca2+ efflux was highest when K+ (35 +/- 0.5 mM (+/- S.E.), mean of four experiments) was at the inside and Na+ (130 mM) at the outside of the ghost membrane.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Characterization of VvSERK1, VvSERK2, VvSERK3 and VvL1L genes and their expression during somatic embryogenesis of grapevine (Vitis vinifera L.)

Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Paul Schellenbaum and Alban Jacques contributed equally to this work.