Induction of somatic embryogenesis and plantlets in tendrils of Vitis vinifera L.

Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium. Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed. Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997     

The effect of fruit-removal on cytokinins and gibberellin-like substances in grape leaves

Removal of fruit from potted cuttings of Vitis vinifera L. increased the concentration of a cytokininglucoside in leaf tissue extracts and decreased the level of extractable gibberellin-like substances. The glucoside (of zeatin riboside) is not present in xylem exudate of V. vinifera L., and appears to be synthesized in the leaves. Berry extracts contain zeatin-riboside and smaller amounts of cytokinin-glucoside. The changes in the level of these hormones are discussed in relation to previous results on abscisic acid and phaseic acid levels in grape leaves.Abbreviations ABA abscisic acid - PA phaseic acid - GA gibberellin     

Sterols of male and female compound inflorescences of Zea mays L.

A comparison has been made between the sterols of male and female inflorescences and of pollen from Zea mays. The female inflorescence was shown to contain cholesterol, 24-methylcholesterol, 24-ethyl-5,22-cholestadien-3-ol, 24-ethylcholesterol and (28Z)-24-ethylidenecholesterol. Themale inflorescence contained the same five compounds together with 24-methylenecholesterol. Pollen contained 24-methylenecholesterol as its main sterol together with lesser amounts of cholesterol, 24-ethylcholesterol, (28Z)-24-ethylidenecholesterol, 24-methylene-5-cholest-7-en-3-ol and 4-methyl-24-methylene-5-cholest-7-en-3-ol.     

Variation in recombination rates across Vitis species

Recombination rate data are presented for three populations of grape based on framework genetic linkage maps developed with simple-sequence repeat markers. These linkage maps were constructed from different Vitis species and represent three genetic backgrounds. The first population is pure Vitis vinifera, derived from a cross of the European cultivars Riesling and Cabernet Sauvignon. The second is an interspecific cross between two commercially used rootstock cultivars of different North American Vitis species parentage, Ramsey (Vitis champinii) and Riparia Gloire (Vitis riparia). The third population, D8909-15 (Vitis rupestris × (Vitis arizonica/Vitis girdiana form)) × F8909-17 (V. rupestris × (V. arizonica/Vitis candicans form)), is an F1 from two half-sibs. Genome-wide and chromosome-wide recombination rates varied across the three populations and among the six Vitis parents. Global recombination rates in the parents of the third F1 population, with a complex Vitis background, were significantly reduced. In the first and third populations, the recombination rate was significantly greater in the male parent. Specific genome locations with frequent heterogeneity in recombination were identified, suggesting that recombination rates are not equal across the Vitis genome. The identification of regions with suppressed or high recombination will aid grape breeders and geneticists who rely on recombination events to introgress disease resistance genes from the genomes of wild Vitis species, develop fine-scale genetic maps, and clone disease resistance genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro

Two Vitis species were cultured in vitro under photoautrophic (sucrose-free culture medium) and photomixotrophic (sucrose 15 g l^-1) conditions during the period following microcutting rooting (day 34 to day 120). Several parameters were measured at the end of the culture: growth, plant dry weight, carbohydrate uptake from the medium and rates of photosynthesis and dark respiration. The two species behaved very differently. Under photoautotrophic conditions, dark respiration, net photosynthesis and daily CO₂ fixation were higher in Vitis vinifera than in Vitis rupestris. Culture under mixotrophic conditions caused increase in growth, respiration and photosynthesis in Vitis rupestris. In contrast, photosynthesis decreased in Vitis vinifera under the same conditions.     

Factors affecting isolation of protoplasts from leaves of grape (Vitis vinifera)

A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 E m^-2 s^-1) for 24 hours. Over 30×10⁶ protoplasts per cm² of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species.     

The inflorescences structure of Cyperus L. section Luzuloidei Kunth (Cyperaceae)

In the present work the inflorescences of 12 species of Cyperus sect. Luzuloidei Kunth sensu Kükenthal were analyzed using the methodology and terminology of Troll's school. All the inflorescences studied are polytelic (indeterminate). The inflorescences are anthela-like or capitate, and can be terminal or pseudolateral. Below the main florescence a paracladial zone is present. In this zone three types of branching were observed, i.e. normal, accessory-axillar and prophyllar branching, whose position vary among the different species. In the paracladial zone a subzone of long paracladia and a subzone of short paracladia were observed. In the short paracladial subzone the spikelets are clustered in fascicles, which can be serial, prophyllar or mixed. Variation in length and position of pherophylls, length of prophyll, number and degree of branching were noticed. The systematic value of the inflorescences in the section Luzuloidei is discussed.     

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml^-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10⁶ viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10⁵ protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [¹⁴C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN₃, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.     

Abundance of phytoseiid mites on Vitis species: effects of leaf hairs,domatia, prey abundance and plant phylogeny

We observed the number of predatory mites (Phytoseiidae:Typhlodromus caudiglans) on the foliage of 20 North American species of grapes (Vitis spp) plus the domesticated EuropeanVitis vinifera, all grown in a common garden. We found relatively few phytophagous mites. The numbers of phytophagous mites were not correlated with the plant characteristics that we measured. We found approximately five times as many predatory mites as phytophagous mites and the numbers of these phytoseiid predators were not affected by the availability of prey. Similarly, numbers of phytoseiids were unaffected by plant gender and, hence, the availability of pollen, another source of food. The numbers of phytoseiids were not clustered according to the taxonomic grouping of the tested plant species. Leaf surface characteristics explained over 25% of the variance in the numbers of phytoseiids. Numbers of phytoseiids were positively associated with the density of vein hairs, the density of bristles in leaf axils, and the presence of leaf domatia. These results suggest that sheltered habitats rather than food availability may limit the numbers of phytoseiid mites on grapevines.     

Development of inflorescences inArabidopsis thaliana

Those mutants were studied whose defects resulted in the morphological changes of inflorescences inArabidopsis thaliana. We characterized newly isolatedcorymbosa mutants andacaulis5 mutants. Thecorymbosa1-1 mutation was caused by the defects in the elongation of pedicels and the previously identifiederecta mutation belonged to this class. Thecorymbosa2-1 mutation was caused mainly by the increase of the number of the floral buds in the inflorescence. The expression of theERECTA gene whose defect resulted to the corymbose inflorescence was analyzed. TheERECTA gene was expressed in subsets of cells in both the peripheral zone and central zone and was thought to have important role for the development of inflorescences. The phenotypes of theacaulis5 mutation was pronounced just after the transition from the vegetative to reproductive growth phase. We found that the expressions of the genes for EXGT-A1 and γ-TIP were drastically reduced in theacaulis5 mutants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Indications for post-translational regulation of Vitis vinifera L. arginine decarboxylase

In order to study arginine decarboxylase regulation, we produced an antiserum against a hybrid of a 615 amino acid residue fragment of grapevine arginine decarboxylase cDNA with maltose-binding protein. The antiserum generated recognized mainly a protein band of ca. 80 kDa in extracts from grapevine tissues. Extracts from leaves and internodes in different developmental stages showed differences in the quantity of the 80 kDa band recognized by the antiserum. However, these differences did not correspond with changes in arginine decarboxylase specific activity. Furthermore, western blot analysis of extracts from cell cultures, where enzyme-specific activity was induced or repressed, did not reveal respective changes in the quantity of the 80 kDa protein band. Digestion of the hybrid by the specific protease factor Xa resulted in a polypeptide of 90 kDa instead of the expected two polypeptides of 43 and 66 kDa. Finally, western blot analysis of shoot extract incubated with factor Xa or the hybrid protein previously digested by factor Xa revealed that factor Xa-digested hybrid protein cleaved the 80 kDa band, resulting in two bands of ca. 38 and 40 kDa, whereas factor Xa alone did not affect it. These results suggest that arginine decarboxylase protein levels and/or activity is post-translationally regulated, as has been shown for other enzymes of polyamine biosynthesis.     

Retranslocation of carbon reserves from the woody storage tissues into the fruit as a response to defoliation stress during the ripening period in Vitis vinifera L.

A technique for reliable labeling of the carbon reserves of the trunk and roots without labeling the current year's growth of grapevines was developed in order to study retranslocation of carbon from the perennial storage tissues into the fruit in response to defoliation stress during the ripening period. A special training system with two shoots was used: the lower one (feeding shoot) was cut back and defoliated to one single leaf (¹⁴CO₂-feeding leaf) while the other (main shoot) was topped to 12 leaves. The potted plants were placed in a water bath at 30 °C to increase root temperature and therefore their sink activity. Additionally, a cold barrier (2–4 °C) was installed at the base of the main shoot to inhibit acropetal ¹⁴C translocation. Using this method, we were able to direct labeled assimilates to trunk and roots in preference to the current year's growth. On vines with root and shoot at ambient temperature, 44% of the ¹⁴C activity was found in the main shoot 16 h after feeding whereas only 2% was found in the temperature-treated vines. At the onset of fruit ripening, and at three-week intervals thereafter until harvest, potted grapevines were fed with ¹⁴CO₂ using the temperature treatment described above. Sixteen hours after feeding, half of the vines of each group were defoliated by removing all except the two uppermost main leaves. Three weeks after each treatment, vines were destructively harvested and the dry weight and ¹⁴C incorporation determined for all plant parts. Under non-stressing conditions, there was no retranslocation of carbon reserves to support fruit maturation. Vines responded to defoliation stress by altering the natural translocation pattern and directing carbon stored in the lower parts to the fruit. In the three weeks following veraison (the inception of ripening in the grape berry), 12% of the labeled carbon reserves was translocated to the fruit of defoliated plants compared to 1.6% found in the clusters of control vines. Retranslocation from trunk and roots was highest during the middle of the ripening period, when 32% of the labeled carbon was found in the fruit compared to 0.7% in control plants. Defoliation during this period also caused major changes in dry-matter partitioning: the fruit represented 31% of total plant biomass compared to 21% measured in the control vines. Root growth was reduced by defoliation at veraison and during the ripening period. Defoliation three weeks before harvest did not affect dry matter or ¹⁴C partitioning.     

In vitro culture of Vitis: the effects of light spectrum,manganese sulfate and potassium iodide on morphogenesis

The effects of blue and red light, manganese sulfate concentration (100 and 5 M), and potassium iodide (5 and 0 M) on shoot and root production from subcultured shoots of the Vitis hybrid Remaily Seedless were studied.Shoot production was greater in blue light. It was increased by lowering the manganese sulfate concentration. The response to manganese in blue light was greatest when there was no potassium iodide addition.Root production was decreased by red light and lower manganese concentration.The effects of manganese, iodide and light spectrum on morphogenesis are discussed in relation to their known effects on IAA metabolism.     

Xylem and Phloem Derived Polyamines during Flowering in Two Diverse Rose Species

Polyamine contents in xylem (root) and phloem (leaf) exudates in two diverse species of rose, viz. Rosa damascena Mill and Rosa bourboniana Desport, were analyzed before, during, and after flowering in the main flowering season, that is, April–May. Only free putrescine (Put) was detected in the xylem and phloem exudates at these time points, and it was high during the peak flowering period. In phloem, Put content was significantly higher in R. bourboniana than in R. damascena at all three stages; whereas in the xylem exudate it was relatively higher in R. damascena at the peak flowering period. A spray of α-difluoromethylornithine (DFMO), an irreversible inhibitor of the putrescine biosynthetic inhibitor ornithine decarboxylase (ODC), markedly decreased the flowering. This effect was reversed by application of Put alone or in combination with DFMO. The significance of this finding is discussed in light of polyamine translocation during flowering. *IHBT Communication: 0354     

Observation of Flowering Process of Grape (<i>Vitis vinifera</i> L.)—Insight into the Starting of Pollination of Flower Hiding in Calyptras (Cap)

It is generally agreed that many Vitis vinifera L. cultivars are self-fertile, where self-pollination often occurs before capfall in a process called cleistogamy. Therefore, it is difficult to identify the right time to remove stamens before self-pollination during the cross-breeding of grape. For this paper, we observed the process of grape flowering and measured the pollen viability and stigma receptivity of grape flowers of ‘Shine Muscat’ in order to identify the starting time of self-pollination before capfall and to provide useful information for improving the efficiency of cross-breeding. The results demonstrate that the anther is not cracked during the visible clusters and separated clusters stages. Meanwhile, in the separated floral buds, flowering begins, and full bloom stages, the pollen viability is 60.7%, 73.2% and 80.3%, respectively; however, at the berry set stage, pollen viability drops to zero. The top of the mature stigma is composed of a layer of nearly cylindrical papillary cells, and the stigma receptivity for pollen changes with the development of flowers: in particular, no reaction was observed in the visible clusters stage; weak positive reaction at the separated clusters stage; strong positive reaction at the separated floral buds, flowering begins, and full bloom stages; and no reaction at the berry set stage. In the separated floral buds stage, pollen tubes were seen germinating in the style. In the flowering begins stage, more pollen tubes were observed at the entry of the ovary. During the full bloom stage, most pollen tubes elongated into the ovary base and some entered the pearl hole. At the berry set stage, newborn endosperm nucleus could be seen in the ovule. From the above, we can conclude that the initiation time of closed fertilization for ‘Shine Muscat’ grape can be judged as the separated floral buds stage, and it is best to discard the stamen before the separated floral buds stage when conducting cross-breeding.     

Benzylaminopurine induces phenocopies of floral meristem and organ identity mutants in wild-typeArabidopsis plants

Arabidopsis thaliana (L.) Heynh. has been used as a model system to investigate the regulatory genes that control and coordinate the determination, differentiation and morphogenesis of the floral meristem and floral organs. We show here that benzylaminopurine (BAP), a cytokinin, influences flower development inArabidopsis and induces partial phenocopies of known floral homeotic mutants. Application of BAP to wild-type inflorescences at three developmental stages results in: (i) increase in floral organ number; (ii) formation of abnormal floral organs and (iii) induction of secondary floral buds in the axils of sepals. These abnormalities resemble the phenotypes of mutants,clv1 (increase in organ number),ap1,ap2,ap3 (abnormal floral organs) andap1 (secondary floral buds in the axils of first-whorl organs). In addition, BAP induces secondary floral buds in the axils of perianth members ofapt2-6, ap3-1 andag mutants, and accentuates the phenotype of theapt2-1 mutant to resemble theapt2-6 mutant. These observations suggest that exogenous BAP suppresses the normal functioning of the genes for floral meristem identity and thereby affects flower development and the later stages of floral organ differentiation.Abbreviations BAP N6-benzylaminopurine - CK cytokinin     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

Tissue origins,cell lineages and patterns of cell division in the developing dermal system of the frut of Vitis vinifera L.

Cell division during development of the dermal system of fruit of the grape cv. Gordo is confined to the first growth period. The epidermis is conserved with anticlinal proliferative cell divisions providing for increase in cell number. The hypodermis is the layer of origin of the collenchymatous dermal system. Six or seven layers are differentiated by periclinal cell divisions early in the first growth period, and later increase in size is obtained by proliferative anticlinal cell divisions. These observations are related to developmental and genetic control of fruit shape and volume.