The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Effects of Temperature Acclimation Pretreatment on the Ultrastructure of Mesophyll Cells in Young Grape Plants （Vitis vinifera L. cv. Jingxiu） Under Cross-Temperature Stresses

Leaves from annual young grape plants （Vitis vinifera L. cv. Jingxiu） were used as experimental materials. The ultrastructural characteristics of mesophyll cells in chilling-treated plants after heat acclimation （HA） and in heat-treated plants after cold acclimation （CA） were observed and compared using transmission electron microscopy. The results showed that slight injury appeared in the ultrastructure of mesophyll cells after either HA （38℃ for 10 h） or CA （8℃ for 2.5 d）, but the tolerance to subsequent extreme temperature stress was remarkably improved by HA or CA pretreatment. The increases in membrane permeability and malondialdehyde concentration under chilling （0℃） or heat （45℃） stress were markedly inhibited by HA or CA pretreatment. The mesophyll cells of plants not pretreated with HA were markedly damaged following chilling stress. The chloroplasts appeared irregular in shape, the arrangement of the stroma lamellae was disordered, and no starch granules were present. The cristae of the mitochondria were disrupted and became empty. The nucleus became irregular in shape and the nuclear membrane was digested. In contrast, the mesophyll cells of HA-pretreated plants maintained an intact ultrastructure under chilling stress. The mesophyll cells of control plants were also severely damaged under heat stress. The chloroplast became round in shape, the stroma lamellae became swollen, and the contents of vacuoles formed clumps. In the case of mitochondria of control plants subjected to heat stress, the outer envelope was digested and the cristae were disrupted and became many small vesicles. Compared with cellular organelles in control plants, those in CA plant cells always maintained an integrated state during whole heat stress, except for the chloroplasts, which became round in shape after 10 h heat stress. From these data, we suggest that the stability of mesophyll cells under chilling stress can be increased by HA pretreatment. Similarly, CA pretreatment can protect chloroplasts, mitochondria, and the nucleus against subsequent heat stress; thus, the thermoresistance of grape seedlings was improved. The results obtained in the present study are the first, to our knowledge, to offered cytological evidence of cross-adaptation to temperature stresses in grape plants.

Anthocyanin profi les and color properties of red wines made from Vitis davidii and Vitis vinifera grapes

Spine grape(Vitis davidii Foex.)is an important wild grape species native to China.Fifteen red spine grape clones and three red Vitis vinifera grape varieties were used to evaluate the differences in the anthocyanin profiles and color properties of wines made from V.davidii and V.vinifera grapes.Among spine wines,‘Junzi#2’wine had the highest total phenolic and total anthocyanin,‘Xiangzhenzhu’wine had the highest total flavonoids,and‘Junzi#1’wine had the highest total tannin.The anthocyanin compositions of all of the spine wines were dominated by Mv-3,5-diglucoside.The total individual anthocyanin contents in spine wines,except‘Gaoshan#5’,‘Junzi#5055’,‘Junzi#5061’,and‘Junzi#5044’,were signifi cantly higher than in V.vinifera wines.Most of the spine wines had a stronger red intensity and a brighter chroma with a bluer hue than V.vinifera wines.Correlation analysis revealed that the color properties were closely related to the anthocyanin composition.These results suggest that wines made from spine grapes may be useful for the wine industry for their color properties and high individual anthocyanin contents.

Development of a mobile application for identification of grapevine(Vitis vinifera L.)cultivars via deep learning

Traditional vine variety identification methods usually rely on the sampling of vine leaves followed by physical,physiological,biochemical and molecular measurement,which are destructive,time-consuming,labor-intensive and require experienced grape phenotype analysts.To mitigate these problems,this study aimed to develop an application(App)running on Android client to identify the wine grape automatically and in real-time,which can help the growers to quickly obtain the variety information.Experimental results showed that all Convolutional Neural Network(CNN)classification algorithms could achieve an accuracy of over 94%for twenty-one categories on validation data,which proves the feasibility of using transfer deep learning to identify grape species in field environments.In particular,the classification model with the highest average accuracy was GoogLeNet(99.91%)with a learning rate of 0.001,mini-batch size of 32,and maximum number of epochs in 80.Testing results of the App on Android devices also confirmed these results.

Effects of mesoclimate and microclimate variations mediated by high altitude and row orientation on sucrose metabolism and anthocyanin synthesis in grape berries

Climate change and extreme weather pose significant challenges to the traditional viticulture regions.Emerging high-altitude grape-producing regions with diverse orientations have shown great potential in coping with this challenge.Stable,high-quality wine grape production may be achieved by synchronizing the meso-and microclimate.To clarify the role of high altitude and row orientation in meso-and microclimate and the response of berries to it,we evaluated seven years(2012-2018)of climate data,two years of basic grape(Cabernet Sauvignon,Vitis vinifera L.)quality,and one-year microclimate from veraison to harvest.By comparing two locations(Sidon 2047 m,Sinon 2208 m)in Yunnan Province,China,we found that the average temperature has been stable at approximately 15℃ for seven years,with no extreme weather or,noticeable global warming.The light intensity(LI)in the north-south(NS)was more balanced than the east-west(EW)direction,and the east-west to the south(EW-S)canopy side was almost higher than the other sides.High LI was associated with high photosynthetically active radiation(PAR),ultraviolet(UV),and infrared(IR)light and vice versa.The north-south to the east(NS-E)and east-west to the north(EWN)sides were characterized by lower LI and higher UV and IR light,and higher total anthocyanin content.Most anthocyanin synthesis-related genes,for example,VvF3'H and VvF3'5'H,were highly expressed in NS-E from veraison to maturity.Perhaps UV and IR light induced their expression.This study provides new insights on the role of differently orientated rows in controlling grape quality due to varied light quality.The findings are globally significant,particularly in the context of climate change,and offer fresh insights into berry physiological responses and decision-making for the management of existing vineyards.

基于改进BiSeNet的葡萄黑麻疹病害程度分级预测

为了准确对葡萄(Vitis vinifera L.)黑麻疹病害程度进行分级预测,通过语义分割模型将叶片部分和病斑部分分割出来,以同一叶片上病斑面积与总叶面积的比值作为疾病严重程度分级的依据,对葡萄黑麻疹病害程度进行分级预测。精确标注了PlantVillage公开数据库中的419张葡萄疾病图像,细分为背景、叶片和病斑3个类别,并应用了数据增强技术增加样本多样性。以BiSeNet作为基准模型,引入GhostNet作为上下文路径的主干提取网络,不仅保持了较小的模型参数量,而且在精度上实现了明显提升,满足病害程度分级预测的需求。提出了累加空洞空间金字塔池化(CASPP)模块,用来替换BiSeNet模型中单一的上下文嵌入模块,以增强BiSeNet模型的多尺度上下文信息提取能力,提高了模型的分割精度。经过测试,本研究模型在测试集中的平均交并比为94.11%,在对葡萄黑麻疹病害程度进行分级预测时,准确率达98.21%,能够精确地对葡萄黑麻疹病害程度进行分级预测。

硫华菊混种葡萄对两种植物镉积累的影响

为降低果树镉积累,采用盆栽试验,将观赏型镉富集植物硫华菊(Cosmos sulphureus)和葡萄(Vitis vinifera L.)混种在镉污染的土壤上,研究了硫华菊混种葡萄对2种植物镉积累的影响。结果表明,硫华菊混种葡萄提高了硫华菊植株的生物量和镉含量,分别较硫华菊单种提高8.63%和22.40%;降低了葡萄植株的生物量和镉含量,分别较葡萄单种降低6.74%和34.84%。硫华菊混种葡萄提高了硫华菊植株的镉积累量,降低了葡萄植株的镉积累量,其中,混种的硫华菊植株镉积累量较其单种提高33.00%,而混种的葡萄植株镉积累量较其单种降低39.21%。此外,2种植物的整株生物量与整株镉含量、整株镉积累量、富集系数均呈负相关,整株镉含量与整株镉积累量、富集系数呈正相关,整株镉积累量与富集系数也呈正相关。因此,硫华菊混种葡萄能促进硫华菊对镉的积累、降低葡萄对镉的积累。

木醋液对葡萄灰霉病菌的抑制作用

为挖掘更多葡萄(Vitis vinifera L.)灰霉病的生防资源,分析不同浓度木醋液对葡萄灰霉病菌菌丝生长、孢子量、孢子萌发的抑制作用以及对葡萄离体叶片灰霉病菌的防治效果。结果表明,木醋液浓度为0.500 0%,灰霉病菌菌株HM22-5菌丝生长和产孢受到明显抑制,菌丝生长抑制率为55.93%,分生孢子量显著降低,分生孢子萌发抑制率为97.08%;当木醋液浓度为1.000 0%时,葡萄灰霉病菌菌丝生长抑制率可达90.00%以上,分生孢子萌发抑制率为100.00%,对葡萄离体叶片的防效达77.32%;木醋液浓度为2.000 0%时,葡萄灰霉病菌菌丝生长抑制率为100.00%,对葡萄离体叶片的防效达89.69%。

Ectopic expression of VvFUS3,B3-domain transcription factor,in tomato influences seed development via affecting endoreduplication and hormones

FUSCA3(FUS3)is a member of B3-domain transcription factor family and master regulator of seed development.It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle,especially in seed germination,dormancy,embryo formation,seed and fruit development,and maturation.However,there is limited information about its functions in seed and fruit development of grapevine.In this study,we expressed VvFUS3 in tomato for its functional characterization.Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size.Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected.However,there were no obvious differences in pollen size,shape,and viability,suggesting that VvFUS3 affects seed development but not the pollen grains.Moreover,the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3.These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size.In conclusion,our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

Grapevine Phenological Quantitative Trait SSR Genotyping Using High-Throughput HRM-PCR Analysis

Discrimination among grapevine varieties based on quantitative traits,such as flowering,veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs.These traits are under complex genetic control for which 6 linked SSR loci(VVS2,VVIn16,VMC7G3,VrZAG29,VMC5G7,and VVIB23)have been identified.Using these markers in HRM-PCR analysis,we assessed genetic diversity among a large collection of 192 grapevine varieties.The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties,3 prominent foreign varieties and 11 varieties of Palestinian origin.The SSR markers used were highly polymorphic,displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis.This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism.Hence,HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis.The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties,signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.

Effect of Proline Pretreatment on Grapevine Shoot-Tip Response to a Droplet-Vitrification Protocol

Proline has been shown to accumulate in plants in response to biotic and abiotic stresses. Exogenous proline has thus been used for improving some plant cryopreservation protocols. Further enhancement of cryopreservation efficiency for in vitro grapevines could be expected if stresses linked to cryopreservation procedures could be reduced. We therefore studied the possible beneficial effect of proline in grapevine cryopreservation. Single-node explants from in vitro grown grapevine plantlets (Vitis vinifera L. cv Portan) were cultured on shooting media (half-strength MS + 1 μM BAP) containing no proline (control) or 50, 500, or 2000 μM filter-sterilized L-proline. Shoot tips excised from these microshoots were subjected to a PVS2-based droplet-vitrification procedure. Control and rewarmed explants were grown on a recovery medium containing 1 μM BAP. Shoot development on control medium and lower proline concentrations did not notably differ whereas the highest concentration of proline inhibited shoot development. Carry-over effects were observed since lower survival and regrowth were obtained both for non-frozen or LN-treated explants excised from micro-shoots obtained on the 2000 μM proline medium. No significant differences in survival and regrowth were observed for non-frozen explants subjected to pretreatment without LN exposure. A slightly enhancing effect (although non-significant) on post-cryopreservation survival was observed for explants derived from shoots developed on 50 or 500 μM proline, but no significant improvement of regrowth percentage was observed for these two conditions. Although a slight increase in survival could be observed, no significant beneficial effect of proline pretreatment on post-cryoconservation regrowth could be evidenced in our conditions. However, the 2-week period before explant excision could have allowed at least partial metabolism and catabolism of exogenous proline;the results observed could thus have been the consequence of complex interactions. Shorter proline treatments applied closer to the actual LN exposure step might produce different results and allow for clearer interpretation.

Phenology and Vegetative Growth in a New Production Region of Grapevines:Case Study in Sao Joaquim,Santa Catarina,Southern Brazil

In Brazil winemaking is expanding not only in the renewal of vineyards in traditional production areas, but also in the introduction of new crops in the newer winemaking regions. Promising characteristics were observed in the quality of the grapes and wines produced in altitude regions of Santa Catarina. The aim of this study is to characterize the geographic and climatic conditions which affect the phenology and vegetative growth of grapevine in S?o Joaquim, in the mountains region of Santa Catarina. The vineyards are located in areas with undulated and strongly undulated relief, predominantly in soils originating from basaltic rocks, in climate conditions classified as Cfb according to the K?ppen, as Region I according to Winkler classification. The phenological stage of the grapevines in the S?o Joaquim region begins later and lasts longer in comparison to other winegrowing regions in Brazil. Decrease in photoperiod and low temperatures (<10?C) were the determining factors for the induction of growth cessation of the shoots in S?o Joaquim. The geographic location in a lower latitude and its high altitude results in a distinct growing climate compared to the other winegrowing regions in Brazil.

壳聚糖对葡萄幼苗养分吸收的影响

为筛选出有利于葡萄(Vitis vinifera L.)幼苗养分吸收的壳聚糖浓度,为葡萄生产提供参考,试验以葡萄幼苗为材料,对其喷施不同浓度的壳聚糖溶液,研究壳聚糖对葡萄幼苗养分吸收的影响。结果表明,喷施壳聚糖溶液提高了葡萄幼苗整株氮、磷、钾含量,在壳聚糖浓度为4 g/L时达最大值,分别较对照增加25.53%、18.67%、12.63%。壳聚糖处理也提高了土壤碱解氮、速效磷、速效钾和有机质含量,其中,壳聚糖浓度4 g/L时,土壤有机质含量较对照增加42.58%。葡萄幼苗的中微量元素钠和钙含量随壳聚糖浓度的增大呈先降后升再降的变化趋势,而镁含量呈先升后降的变化趋势。就土壤酶活性而言,壳聚糖提高了土壤蔗糖酶、脲酶、过氧化氢酶和磷酸酶活性。4 g/L壳聚糖最有利于葡萄幼苗养分的吸收。

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virusinduced oxidative stress

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics.Currently,neuraminidase(NA)inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration(FDA)for the prevention and treatment of influenza.Here,we show that vitisin B(VB)inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells.Reactive oxygen species(ROS),which frequently occur during viral infection,increase virus replication by activating the NF-κB signaling pathway,downmodulating glucose-6-phosphate dehydrogenase(G6PD)expression,and decreasing the expression of nuclear factor erythroid2-related factor 2(Nrf2)antioxidant response activity.VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity,and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation.In addition,VB reduced body weight loss,increased survival,decreased viral replication and the inflammatory response in the lungs of influenza A virus(IAV)-infected mice.Taken together,our results indicate that VB is a promising therapeutic candidate against IAV infection,complements existing drug limitations targeting viral NA.It modulated the intracellular ROS by G6PD,Nrf2 antioxidant response pathway,and NF-κB signaling pathway.These results demonstrate the feasibility of a multi-targeting drug strategy,providing new approaches for drug discovery against IAV infection.

不同设施栽培模式对葡萄生长和产量的影响

以武汉都市圈项目合作为契机,在湖北省天门市九真镇开展葡萄(Vitis vinifera L.)高产高效标准化技术示范,并对不同设施栽培模式(简易避雨敞棚、单膜覆盖、双膜覆盖)下的阳光玫瑰、夏黑生育性状进行比较研究。结果表明,在相同的田间种植管理模式下,双膜栽培改善1—3月低温波动较大的气温环境,使棚内日平均温度维持在葡萄萌芽生长的生物学温度(10.0℃)以上,提早打破休眠期,促进葡萄早萌芽、早开花、早成熟。双膜覆盖栽培经济效益明显提升,产投比平均为3.45∶1.00,双膜覆盖栽培的阳光玫瑰、夏黑产值分别比简易避雨敞棚增加68.01%、55.47%。合理运用3种设施栽培模式,可以延长葡萄销售期,降低集中上市的市场风险。

TRV介导的葡萄叶片VvANR基因瞬时表达分析

为建立葡萄叶片TRV-VIGS系统,分析验证VvANR基因功能,本研究以烟草脆裂病毒(Tobacco rattle virus,TRV)介导基因沉默表达载体pTRV2-ANR,采用真空侵染和主叶脉针孔注射法分别侵染葡萄幼嫩、成熟叶片,观察表型并测定原花青素含量,实时荧光定量PCR测定被侵染叶片中VvANR及其相关基因表达。结果表明,真空侵染的幼嫩、成熟叶片分别于第6、第7天出现漂白,侵染15 d后,幼嫩叶片几乎全部漂白,成熟叶片大面积漂白。主叶脉针孔注射侵染的幼嫩、成熟叶片分别于第17、第19天出现漂白;侵染25 d后,幼嫩和成熟叶片发生全叶漂白,且漂白均沿主叶脉逐渐扩展至整个叶面;对照均未发生漂白现象。pTRV2-ANR侵染后叶片中原花青素含量极显著降低,VvANR基因表达量极显著降低,且原花青素生物合成相关基因VvLAR1、VvLAR2、VvMYBPA1、VvMYBPA2、VvUFGT表达量显著降低,除VvUFGT表达量呈显著差异外,其余均呈极显著差异;而VvANS、VvDFR表达量轻微上调,且与对照差异不显著。本研究建立了葡萄叶片VIGS体系,为基因功能快速验证提供了新方法;同时,揭示了葡萄叶片中原花青素含量、VvANR表达及与相关基因的关系,本研究为完善葡萄原花青素积累机制奠定了一定的理论基础。

不同营养液对限根栽培葡萄生长及其栽培基质的影响

以一年生葡萄(Vitis vinifera L.)阳光玫瑰和甬优一号为试验材料,在限根栽培模式下研究不同营养液对葡萄生长及栽培基质的影响。结果表明,对于阳光玫瑰品种来说,30 mg/L N处理显著促进植株生长,对于甬优一号品种来说,180和120 mg/L N处理显著促进植株生长。阳光玫瑰为营养高效类型,在低营养液处理下,氮素的利用效率更高,栽培基质中积累的氮较少,而甬优一号为营养低效型,在高营养液条件下,栽培基质中积累的氮较少。

基于SSR分子标记技术的葡萄种间杂交F1代鉴定

SSR分子标记技术以其多态性高、重复性好和检测方便快捷等优点,被广泛应用于植物F1代真假杂种的鉴定。为了长期有效的开展葡萄杂交育种工作,本研究以葡萄杂交组合‘北冰红’伊‘贵人香’F1代159个单株及‘双红’伊‘贵人香’F1代121个单株为试验材料,利用SSR分子标记技术结合田间形态学性状对葡萄杂交后代的真伪进行鉴定。在12对SSR引物中筛选出同时在亲本间具有特异性位点的引物,对F1代进行扩增检测,结果表明:‘北冰红’伊‘贵人香’及‘双红’伊‘贵人香’2个杂交组合中分别有63和94个单株具有双亲特异性条带,结合田间形态学鉴定,认为其为真杂种,故2个杂交群体的杂种率分别为40.38%和77.69%,该结果可为今后开展葡萄遗传连锁图铺构建及数量性状的QTL定位提供依据。

葡萄GRF基因家族的生物信息学分析

GRF(Growth-regulating factor)基因家族是一种广泛分布于种子植物的转录因子,在调节植物的生长、发育中发挥重要的作用。本研究从葡萄基因组鉴定出8个VvGRF基因,命名为VvGRF1~VvGRF8。采用生物信息学的方法,系统地对葡萄Vv GRF家族的蛋白理化性质、进化关系、保守基序及在不同发育时期的差异性表达进行预测和分析。结果表明,VvGRF基因内含子与外显子数量差异较小,在所有预测蛋白的N端含有两个典型的保守结构域QLQ与WRC;系统进化分析将GRF家族成员分为6组,其中葡萄与甜瓜的亲缘关系较拟南芥、番茄两种双子叶植物近;顺式作用元件表明,调控分生组织、胚、种子发育及生长素、赤霉素等信号传导和逆境胁迫响应元件,在VvGRFs启动子序列中大量存在;基因特异性表达分析显示,多数GRF基因在葡萄发育的花后2周、10周时期的表达丰富度高,在接近成熟时期的表达丰富度较低,表明GRF在葡萄生长、转色早期起着重要的作用,参与组织、器官的生长调节。本研究为进一步探究葡萄GRF基因家族的结构及生长发育时期的功能调节提供了参考依据。