Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

Effect of water deficit and rewatering on leaf gas exchange and transpiration decline of excised leaves of four grapevine (Vitis vinifera L.) cultivars

Grapevine cultivars are known to differ in their drought adaptation mechanisms, but there is little knowledge on how they behave when recovering after a drought event. The effects of increasing water deficit and recovery after rewatering were evaluated on four widely grown red grapevine cultivars native from different climates (Cabernet Sauvignon, Cs; Garnacha, syn. Grenache, Ga; Merlot, Me; and Tempranillo, Te) through the study of gas exchange (GE) measurements and transpiration decline curves (TDC). As a whole, Ga has proved to be the cultivar best adapted to water deficit, since it showed the highest water use efficiency (WUE) and the greatest water saving ability after leaf excision. Te, on the contrary showed the lowest values for those parameters under increasing stress, although when rewatered showed greater acclimation ability than Cs and Me, remarkably improving its behaviour. The two methodological approaches (GE and TDC) used at different water deficit levels and after rewatering, have complemented each other, allowing a better cultivar characterization than each method would had allowed itself.     

The influence of preplanting dips and postplanting temperatures on root growth and development of nonprecooled tulips,daffodils and hyacinths

The effects of preplanting dips and postplanting temperatures were studied, using nonprecooled bulbs of Tulipa gesneriana L. ‘Paul Richter’, Narcissus pseudonarcissus L. ‘Explorer’ and Hyacinthus orientalis L. ‘Pink Pearl’, for 2 consecutive years. Preplanting treatments consisted of a non-dip control, and 30-min dips of either tap water or benomyl—ethazol. After planting, the tulips and daffodils received temperatures of 5, 9, 13, 17 or 21° C for a 5-week period. Hyacinths received temperatures of 9, 13, 17, 21, 25, 29 or 33° C for 5 weeks in the first year and for 25 days in the second year.It was determined that 17° C for 3 weeks and 17° C for 3–4 weeks were the optimal temperatures and periods of time for root development of tulips and daffodils, respectively. For hyacinths, a range of from 17 to 25° C for 10–14 days was optimal. Under these conditions, a minimum root length of 70 mm was obtained and all bulbs of each species had rooted.The benomyl-ethazol and water preplanting dips stimulated root growth for the first 2 weeks for tulips. This effect was not observed for daffodils and hyacinths.     

Antifungal effects of iron sulfate on grapevine fungal pathogens

The present study aimed to determine the most efficient experimental conditions of iron sulfate use leading to optimal inhibition in the development of fungal pathogens. Assays have been focused on fungal species inducing severe grapevine diseases. FeSO₄ directly inhibited the in vitro mycelial growth of Botrytis cinerea, Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Diplodia seriata, and Neofusicoccum parvum with variable efficiency in the range of 0.5–10 mM. The development was always completely inhibited at 20 mM. This inhibitory effect was greatly increased at acidic pH values. The anionic moiety of the molecule was of importance since bromide, chloride and sulfate were highly active, whereas acetate and oxalate showed a small effect. Electron microscope observations on E. lata and B. cinerea showed that a treatment with FeSO₄ induced dramatic changes in the hyphal organization leading to cell death. No toxicity was observed on grapevine leaves following repeated FeSO₄ sprays in the antifungal concentration range. Therefore, FeSO₄ may be proposed to effectively replace the long-term pollutant use of CuSO₄ as an antifungal agent, with the additional advantage of iron being an important plant micronutrient.     

贺兰山东麓葡萄毛毡病消长动态以及新型防控药剂的研究

以赤霞珠为试验品种,40%与50%仿生胶和10%浏阳霉素乳油为试验药剂,40%毒死蜱乳油、1.8%阿维菌素乳油和20%哒螨灵可湿性粉剂为对照药剂,研究毛毡病的消长动态和防治毛毡病的新型农药,探讨贺兰山东麓葡萄毛毡病消长动态以及2种防治药剂在不同时期对其的影响。结果表明:引起葡萄毛毡病的葡萄缺节瘿螨1a发生3代。在整个葡萄生育期中的危害有2次低谷时期:5月中旬和7月中旬。40%与50%仿生胶对葡萄毛毡病有很好的防控效果,药后21 d防效可以达到87.00%以上,而且对葡萄生长没有影响,是防治葡萄毛毡病的新型农药。在葡萄膨大期,10%浏阳霉素乳油可以有效控制葡萄毛毡病的发生。毛毡病的防治时期以葡萄刚出土时预防为最佳时期。葡萄毛毡病在贺兰山东麓发生普遍且危害重,在葡萄刚未展叶以前施用仿生胶可以有效地控制毛毡病的发生及危害。     

Molecular cloning and characterization of a novel gene encoding an EF-hand calcium-binding protein related to fruit seedlessness of grapevine

EF-hand calcium-binding protein (CBP) gene plays a critical role during embryonic development in animals, but it remains unclear whether this gene possesses similar function in plants. In this study, a novel CBP gene was isolated from Vitis vinifera cv. Thompson Seedless and designated as VvCBP1. Using a combination of sequence structural investigation, expression pattern analysis of different embryo development in grapevine, and silencing assay of VvCBP1 homolog in tomato, the functional role of VvCBP gene in embryo development of seedless grapevine was studied. Structural investigations showed that VvCBP1 is 907 bp in length, and contains two EF-hand motifs. Expression pattern of this gene is different in embryo between seedless and seeded grapevine species. Moreover, the silencing of homology gene led to the seeds number significantly decreased in tomato. These data demonstrate clearly that VvCBP1 plays an important role in embryo development of seedless grapevine.     

秋季叶面喷施多胺和尿素对酿酒葡萄贮藏营养和抗冻性的影响

为提高酿酒葡萄休眠期贮藏营养水平和抗性,以6a生、长势一致的“赤霞珠”葡萄为试材,研究了秋季叶面喷施不同浓度的多胺、尿素对赤霞珠葡萄叶片光合作用、树体内碳水化合物含量、活性氧代谢、主芽及花序质量的影响.结果表明:在秋季叶面喷施4mM的精胺、1mM的亚精胺或3％的尿素,可显著的提高叶片气孔导度、叶绿素含量、叶片光合速率.这些处理可显著提高休眠期树体内碳水化合物含量和主芽质量,降低枝蔓相对电导率和丙二醛含量,提高SOD、CAT活性,提高了花序的质量.因此,在秋季,叶面喷施适当浓度的多胺或尿素可通过提高叶片光合速率来显著提高酿酒葡萄贮藏营养水平和抗冻性,提高主芽和花序质量,这也有助于酿酒葡萄品质或产量的提高.     

Sustainable Mitigation of Alkaline Stress in Grapevine Rootstocks (<Emphasis Type="Italic">Vitis</Emphasis> spp.) by Plant Growth-Promoting Rhizobacteria

Practical use of plant growth-promoting rhizobacteria (PGPR) on plants under stress conditions remains elusive because most of the studies focused on merely evaluating the plant growth-promoting effects on non-stressed plants. This study focused on the effect of root inoculation of different PGPRs on the growth and physiology of grapevine rootstocks 41 B, 99?R and 140 Ru grown in soilless culture with elevated pH. The rootstocks in pots under glasshouse condition were inoculated with Agrobacterium rubi A18 and Bacillus subtilis OSU 142 bacteria in early spring. To increase the pH of growth medium, the plants were watered with 250?mL plant^?1 bicarbonate solution (840?g L^?1 NaHCO₃) four times (beginning at 3–4?cm shoot growth) with one-month interval during the vegetation. Along with the bicarbonate supplementation to growth medium, root rhizosphere pH increased from an initial value 7.76 to the final values between 8.10 and 8.26. Although the bacteria population decreased progressively, they were able to alleviate the negative effects of high pH by improving vegetative growth, leaf physiology and nutrient acquisition in many cases. The bacteria strains employed in this study can be recommended to support grapevine growth and physiology under alkaline conditions for a sustainable and environment-friendly viticulture.     

In vitro regeneration from alginate-encapsulated microcuttings of Quercus sp.

Microcuttings with apical and nodal buds from common oak (Quercus robur L.) and Turkey oak (Quercus cerris L.) in vitro clones were encapsulated into an alginate matrix. Apical segments of common oak showed significantly greater in vitro regeneration potential as compared with the nodal ones. Encapsulated Turkey oak nodal segments demonstrated successful regeneration after different periods (2–6 weeks) of cold storage (4 °C), showing no significant deviation from the regenerative frequency of non-cold-treated segments.     

Stenting of roses; a method for quick propagation by simultaneously cutting and grafting

A method was developed for quick propagation of roses under conditions prevailing in The Netherlands, based on cutting and grafting in one action and called “stenting” (to stent), being a contraction of the dutch words “stekken” (to strike a cutting) and “enten” (to graft). A piece of stem of the cultivar with one leaf and a dormant bud is grafted on a single internode of the rootstock. Formation of the graft union and of adventitious roots occur simultaneously, resulting in a complete plant in 3 weeks.Absence of buds on the rootstock is essential to diminish wild suckering. Factors influencing the percentage of successful stentlings were investigated. With R. chinensis ‘Indica Major’ as a rootstock, the best results were obtained with mature internodes 6–8 mm in diameter after application of 5000 mg 1^?1 IBA. Pre-treatment of the rootstock internodes by application of IBA and storage at 4° C for 3 weeks improved rooting. Comparison of grafting by hand and by machine showed that machine-grafting offers good possibilities.The influence of the cultivar, after cutting or stenting, on root-formation and root-growth was studied. Grafting on seedling rootstocks (bench-grafting) and stenting proved to give similar results. In the first year after stenting, R. chinensis ‘Indica Major’ was a better rootstock for the cultivar ‘Cocktail 80’ than R. canina ‘Inermis’, while ‘Inermis’ was better for ‘Motrea’.Rose culture can be improved by using cultivars stented on a convenient rootstock. Stenting can be applied around the year, is ideal for screening new scion/stock combinations and to investigate the interaction between shoots and roots.     

Shedding of the upper parts of grapevine shoots

Shedding of the upper parts of unlignified grapevine (Vitis vinifera L.) canes is frequent during autumn. Stress conditions during the growing-season lead to similar shedding of shoot tips. The separation process occurs above a node in the boundary area between the distal part of the diaphragm and the pith.Ethephon (2-chloroethyl-phosphonic acid) sprays, at 750 or 1500 mg l^?1, induced separation of the shoot tips of ‘Perlette’ and ‘Queen of the Vineyards’. Ethylene (C₂H₄) 200 vpm and ethephon at 10, 100 and 1000 mg l^?1 enhanced separation in shoot explants, whereas 2,4,5-TP (2,4,5-trichlorophenoxy-propionic acid) at concentrations above 1 mg l^?1 arrested it.This phenomenon may be regarded as a shoot abscission process which may represent adaptation of self-pruning of the grapevine.     

Effects of high-humidity storage on quality,decay and storage life of cherry,lemon and peach fruits

Sweet cherries (Prunus avium (L.) ‘Lambert’ and ‘Blackboy’), lemons (Citrus limon (L.) Burm. f. ‘Lisbon’) and peaches (Prunus persica (L.) Batsch, ‘Summerset’) were stored at 77–83, 90–94 and 95–99% RH (high humidity) at near-optimal storage temperatures after harvest and treatment with fungicides. High-humidity storage did not increase the storage life of peaches held at 0°C, but the life expectancy of cherries (both cultivars) was extended by 7–10 days at 0°C, and of lemons by at least 4 weeks at 10°C when fruit were stored at 95–99% RH compared with levels below 95%. The beneficial effects of high humidity were attributed to retardation of peel desiccation and associated reductions in fruit deformation, peel de-greening, chilling-injury and decay in lemons and to the maintenance of a fresher stalk and a firmer, less shrivelled fruit in cherries. High humidity had no effect on decay in cherries or peaches, but it significantly reduced weight loss and delayed the appearance of shrivel in peaches stored at 0°C. However, after storage at high humidity for 4 weeks, peaches ripened with low rates of C₂H₄ evolution and showed severe low-temperature injury, slight peeling-injury and a poor flavour ex-store.     

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus (Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.     

Effect of the Period of Maceration on the Content of Antioxidant Substances in Grape Juice

The study deals with the monitoring of the content of antioxidant components in grape juice in the course of cold maceration of grapevine varieties ‘Italian Riesling’ and ‘Cabernet Sauvignon’, vintages 2011 and 2012. For analyses, grape juice was sampled after the maceration intervals of 0; 2; 4; 6; 8; 12 and 24?h. Spectrophotometry was used to estimate antioxidant activity (by the DPPH test), total flavanols, contents of anthocyans and contents of hydroxycinnamic acids. The content of total acidity and pH were estimated as well. Obtained results indicated that contents of aforementioned compounds increased with the increasing time of mush maceration.     

Effects of high vineyard temperatures on the grapevine leafroll associated virus elimination from Vitis vinifera L. cv. Napoleon tissue cultures

In vitro culture of shoot tips and axillary buds was used for virus elimination from the Spanish autochthonous table grapevine cultivar Napoleon which was infected by Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine fanleaf virus (GFLV). High percentages of GLRaV-3-free plants (91–100%) were obtained by establishing shoot tip cultures from infected mother plants of the 29-228, 74-16 and 77-266 clones. Lower percentages of virus-free plants (71–87%) were obtained by in vitro culture of the first, the second and the third axillary buds of the growing distal shoots. Percentages of virus-free plants obtained with both shoot tips and axillary buds varied according to the time of the year when the explants were collected and the bud position on the shoot. A increased efficiency of in vitro tissue culture methods was observed when cultures were established in summer and it was due in part to the high vineyard temperatures reached in the southeast of Spain during the hot season. GFLV- and GLRaV-3-free plants were only obtained by thermotherapy in combination with tissue culture methods from co-infected plants of the clone 39-29.     

Discrimination of the grapevine cultivars ‘Picolit’ and ‘Kéknyelű’ with molecular markers

The name of the grapevine cultivar ‘Kéknyel?’ has become inseparable from the name of the Badacsony vine region, whose fame is well known beyond our frontier as well. In the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/) ‘Kéknyel?’ is reported, as the synonym of the Italian grapevine cultivar ‘Picolit’. Vertical poliacrylamide-gel electrophoresis was used for the investigation isoenzymes of catechol-oxidase (CO) and acid phosphatase (AcP). Microsatellite analyses were carried out at 6 loci (VVS2, VVS16, VrZag79, VVMD7, VMC4A1, VMC4G6). The results of the isoenzymatic and microsatellite analyses confirmed, that this two cultivars are different.     

Origin and genetic diversity of Tunisian grapes as revealed by microsatellite markers

Ten SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability, cultivar relatedness, and parentage in a collection of 61 autochthonous Vitis vinifera cultivars from Tunisia.The number of alleles per locus ranged from 6 to 11, while the number of genotype patterns varied between 10 and 21. The expected heterozygosity varied between 0.621 and 0.855 and the observed heterozygosity was higher than 0.9 at 4 loci (VVMD28, VVMD5, VVIP31 and VVS2) indicating that the SSRs were highly informative.Cluster analysis using unweighted pair group method with arithmetic averaging (UPGMA) suggested 14 groups among studied cultivars and 53 grapevine denominations out of 61 were unequivocally distinguished, with all accessions showing at least one-specific combination of alleles.On the other hand, in order to overcome the existing confusion in Tunisian grapevine nomenclature, of the analyzed homonymous pairs of cultivars, only ‘Balta 2’ and ‘Balta 3’ have shown identical allelic profiles, consistent with their being the same genotype. Hence, nomenclature distinction is meaningless and only one denomination should be retained.Due to the high overall power of exclusion (Q) (greater than 99.99%) and to the absence of null alleles, the set of microsatellite loci used is appropriate to determine parentage in Tunisian grapevines beyond any reasonable doubt. The analysis of fingerprints indicated that the Tunisian grape vines have evolved through out crossing between five possible parents: Balta 1, Beldi Baddar, Beldi Rafraf, Beldi Local Rafraf and Khedhiri 3.     

Effects of Fertigation on Pigment Pattern and Fruit Quality of cv. ‘Gala’ Apples

Fruit pigmentation in ripening cv. ‘Gala, Schniga’ apples was monitored under the influence of fertigation weekly during 21 July–31 August 2009, i.e. 34 days before and 7 days after the optimal harvest date in Slovenia. The soil was a heavy loam clay with high humus content (2.8 %) and high acidity (low pH 4.9). The spectral indices NDVI (Normalized Difference Vegetation Index) and NAI (Normalized Anthocyanin Index) were obtained by a PA 1101 on attached fruit on the transition between the ground and blush colour of the fruit surface. NAI increased slowly from 0.25 to 0.6 two weeks prior to optimum maturity. NDVI values fell from 0.9 to 0.2 before and during ripening, with significant changes starting ca. 3 weeks before and during the week after the optimum harvest date. Fertigation delayed fruit ripening viz harvest date by ca. 4 days. Fertigation delayed changes in the intensity of the fruit’s epidermal pigments, expressed as NDVI for 1 week and NAI for 4 weeks, respectively, but had no significant effect on fruit colour or NDVI and NAI values at the optimal harvest date. At this time, those fruits from the fertigated trees were firmer with a lower starch index compared with the un-fertigated control. Statistically, highly significant correlations were found (p?≤?0.01) (r?=?0.35 to r?=?0.91, R²?=?0.11–0.83) between all the studied spectral and standard parameters i.e. fruit fresh firmness, soluble solids content and starch index.     

Rapid vegetative propagation of Hyacinthus orientalis L. in vitro

A simple in vitro system was developed to increase the vegetative propagation rate of hyacinth, Hyacinthus orientalis L. Excised bulb scale segments were induced to regenerate bulblets on the basal sides of the inverted explants. Within 12 weeks, 240–300 bulblets were obtained from a single 17–18 cim bulb when cut into basal scale segments, 3–4 cm long and 0.5 cm wide.     

‘品丽珠’葡萄不同营养系酚类物质差异分析

筛选山东蓬莱产区酿酒葡萄‘品丽珠’黄烷醇、花色素苷含量较高的优良营养系，以期为发挥其酿酒性能提供理论依据。以蓬莱产区品丽珠母本及3个芽变营养系（214、327、623）为试材，采用分光光度计法分析葡萄果皮和种子中总酚、总单宁、总花色素含量，利用高效液相色谱法分析酚酸类、黄酮醇类、黄烷醇类、花色苷单体及白藜芦醇含量。研究结果表明，黄烷醇是种子和果皮中主要的单体酚，其中儿茶素含量最高；二甲花翠素葡萄糖苷是果皮中主要的花色苷。在相同的栽培管理条件和自然条件下，品丽珠不同营养系间的酚类物质差异较明显，相同营养系不同年份间也有显著差异。其中营养系623果皮和种子的总酚、种子的单宁和黄烷醇以及果皮和种子中的酚酸类、单体花色苷含量显著高于其他营养系和母本。