矮牵牛(Petunia hybrida Vilm)组织培养技术研究

实验以矮牵牛(Petunia hybrida Vilm)的花瓣为培养材料,在MS培养基中附加不同浓度的细胞分裂素(6-BA)和生长素(NAA),进行矮牵牛快速繁殖技术研究。结果表明,MS+6-BA(2.0 mg.L-1)+NAA(0.1mg.L-1)培养基愈伤组织发生的较早,数量多,培养基为最佳分化培养基,不定芽发生早,粗壮,数量多,玻璃化程度很小;诱导不定根时,1/2 MS+NAA(0.5 mg.L-1)培养基诱导生根的时间较早,不定根发生率达100%。

矮牵牛(Petunia hybrida)开花过程中的可溶性蛋白质分析(英文)

在进行矮牵牛(Petunia bybrida)光周期诱导开花的过程中,对叶子中的可溶性蛋白质进行了分析,其中有4条与开花有关的特异蛋白,分子量分别为49.45 kD(a)、35.45 kD(b)、17.98 kD(c)和11.74 kD(d)。在不开放的花苞中不含有蛋白质a和d,只有这4种蛋白质全出现时,才能形成花苞并且开放。花开了以后,蛋白质c和d就消失。即使在开花的植株中,各组织中的蛋白质 也是不同的。茎中完全不含有蛋白质c和d,叶子和花中的蛋白质组成也是不同的。

矮壮素对矮牵牛(Petunia hybrida Vilm)试管内调整株型的作用

在培养基中加入矮壮素可以使试管矮牵牛矮化,枝叶紧凑,叶片颜色加深,在使用的浓度范围内(0,0.05,0.1,0.5mg/L),呈现浓度越高,矮化作用越强.在其他诱导措施相同的情况下,矮壮素对矮牵牛的开花有利,当矮壮素在0.1mg/L的浓度时对花苞的形成最有效.

Improvement of the Hydroponic Growth and Waterlogging Tolerance of Petunias by the Introduction of vhb Gene

The coding sequence of Vitreoscilla hemoglobin (vhb) was cloned with PCR technique from Vitreoscilla stercoraria Pringsheim. The plant expression vector with vhb gene under the control of CaMV 35S promoter was constructed and used in the transformation of Petunia hybrida Vilm by the Agrobacterium mediated procedure. The results of PCR amplification and Southern hybridization indicated that the vhb gene had been integrated into the petunia genome and the vhb gene expression had been detected by RT-PCR amplification. In hydroponic culture the transgenic petunias grew much better than non-transgenic controls. For further analysis of hypoxia tolerance of transgenic petunia, the petunia plants with vhb gene were submerged into liquid MS medium. The transgenic plants survived in hypoxic condition and grew out of the liquid surface in a few weeks, while non-transgenic plants were still submerged and suffocated in culture solution without ability to grow out of liquid medium in submersed culture for four to five weeks. The vhb gene transformed petunia plants had been planted and tested in a simulated flooding condition, and showed obvious tolerance to water-logging. It seen is that hemoglobin gene from Vitreoscilla might have the potential use in molecular breeding for the improvement of plant resistance to hypoxia and flooding.

上海矮牵牛(Petunia hybrida)病毒病的毒源鉴定

从花叶状矮牵牛植株上分离到两类病毒分离物:(1)P-14;(2)P-33和P-a_1。根据鉴别寄主反应和寄主范围、病毒粒体形态和大小、传毒方式、体外稳定性、内含体的类型以及血清学反应,P-14分离物被鉴定为黄瓜花叶病毒(CMV);P-a_1和P-33被鉴定为芜菁花叶病毒(TuMV)。这两类分离物在少数寄主上的症状有所差异。经鉴别寄主反应和血清学检测,表明黄瓜花叶病毒是危害矮牵牛的主要病毒。

上海矮牵牛（Petunia hybrida）病毒病的脱毒技术

用黄瓜花叶病毒接种矮牵牛，取发病植株的叶片、花芽和茎尖进行培养，用间接ＥＬＩＳＡ和离体蚕豆叶片进行检测，结果表明：用叶片、花芽以及大于０．３～０．５ｍｍ的茎尖培养，不能获得无毒苗，从病株上剥取０．１５～０．２５ｍｍ的茎尖培养可获得６．６％的无毒苗；连续高温处理病株８ｄ结合茎尖（０．３～０．５ｍｍ）培养，可获１０％的无毒苗；分别变温处理（３８℃８ｈ＋２２℃１６ｈ）１６ｄ和３０ｄ结合茎尖（０．３～０．ｓｍｍ）培养，均可获得１１．１％的无毒苗。

Systematic Identification of the Light-quality Responding Anthocyanin Synthesis-related Transcripts in Petunia Petals

Previous studies have shown that high light intensity can induce anthocyanin synthesis(AS)in petunia plants.To identifywhich kind of light quality plays a role in inducing such metabolic process,and what transcripts participate in controlling it,we carried out whole-transcriptome sequencing and analysis of petunia petals treated with different light-quality conditions.Among the red and white light treatments,a total of 2205 differentially expressed genes and 15,22,and 20 differentially expressed circRNAs,miRNAs,and lncRNAs,were identified respectively.The AS-related genes,including the structural genes CHSj,F3H,F35H,DFR,and ANS,and the regulatory genes AN4,DPL,PHZ and MYBx were found to be downregulated under red light condition compared with their levels under white light condition.Furthermore,the light photoreceptor Cryptochrome 3(CRY3)and a series of light-dependent genes,such as PIF,HY5,andBBXs,were also determined to respond to the light treatments.The anthocyanin contents in early petunia petals under red light were significantly lower than that under white and blue light.The results of qRT-PCR further confirmed the expression pattern of some AS-related and light-response genes in response to different light quality.Yeast two-hybrid results showed that the key elements in the light signal pathway,HY5 can interact with BBX19,BBX24 and BBX25.And PHZ,the important AS regulator can induce anthocyanin synthesis in response to blue light quality fromtransient expression analysis in petunia petals.These findings presented here not only deepen our understanding of how light quality controls anthocyanin synthesis,but also allow us to explore potential target genes for improving pigment production in petunia flower petals.

Effect of Different Plant Growth Regulators on Callus Induction and in vitro Rapid Propagation of Wild Petunia Juss.

In this study,the seeds of wild Petunia Juss.were used as explants to investigate the optimal condition for tissue culture.Several different kinds and concentrations of growth regulators were adopted to produce more multiple bud clumps,callus or roots in this study.The experiments may provide experimental foundation for the rapid propagation technology and establishment of tissue culture system for wild Petunia Juss.

Influences of ascorbic acid and gibberellic acid in alleviating effects of salinity in Petunia under in vitro

Salinity is one of the abiotic stresses that limits the growth and productivity of many crops.A possible survival strategy for plant under saline conditions is to use compounds that could minimize the harmful effects of salt stress on the plant development.The objective of the presented study was to investigate the effect of exogenous ascorbic acid(ASA)with or without gibberellic acid(GA3)on key growth and biochemical parameters in two petunia cultivars‘Prism Rose’and‘Prism White’under saline(150 mM NaCl)and non-saline in vitro condition.Nodal cutting with an axillary buds were used as explants.Application of 1 mM ascorbic acid with or without 0.05 mM gibberellic acid into the MS medium stimulated the length of shoots and the number of new shoots of‘Prism Rose’;whereas,it decreased the root length and the number of roots of both‘Prism Rose’and‘Prism White’under non-saline condition.The addition of ascorbic acid with or without gibberellic acid into the MS medium under saline condition,increased the length of plants and the number of new shoots,but did not affect their root number and length.NaCl treatments increased the proline content and lipid peroxidation which was indicated by the accumulation of malondialdehyde(MDA).The study revealed a correlation between chlorophylls a and b content and the leaf pigmentation intensity–parameter a*.Addition of 1 mM ascorbic acid with 0.05 mM gibberellic acid into the MS medium plays a protective role in salinity tolerance by improving the shoot growth and the development as well as increasing the activities of the antioxidant enzymes and other antioxidant substances.

Transgenic Petunia hybrida with Silicon Transporter Protein OsLsi1 and OsLsi2 Genes and Its Drought Resistance Analysis

As one of the important materials in landscaping for flower terrace and border, Petunia hybrida needs high environmental conditions and its growth is seriously influenced by the drought. Silicon is considered to be a necessary element for plant growth, and soluble silicon can improve plant resilience. To improve the drought resilience of Petunia hybrida, the silicon transporter protein OsLsi1 and OsLsi2 genes cloned from rice（Oryza sative） were transferred into Petunia hybrida by Agrobacterium-mediated method, and finally got 26 and 32 positive plants, respectively by PCR and RT-PCR detections. With a control of non-transgenic plants, the obtained transgenic plants were taken by drought treatment stress for 0, 4, 7, 10 and 14 days, then re-watered and measured physiological indexes as malondialdehyde（MDA） content, free proline（Pro） content, superoxide dismutase（SOD） activity and peroxidase（POD） activity to study the effect of Petunia＇s drought resistance. All the results proved that the silicon transporter protein OsLsi1 and OsLsi2 genes were normally transcripted and expressed in transgenic Petunia hybrida; OsLsi1 gene could improve the abilities of plants＇ drought resistance and recover after drought stress, while OsLsi2 gene could reduce the above abilities. The order of the drought resistance ability of the three strains from strong to weak was OsLsi1〉CK〉OsLsi2; and silicon indeed improved the ability of drought resistance as well. All these results provided a new way to improve the drought resistance of Petunia, and laid a foundation to improve the ability of garden plants＇ drought resistance and water saving.

Types of Irrigation Water and Soil Amendment Affect the Growth and Flowering of Petunia x alkinsiana ‘Bravo Pinc’

Water insufficiency is the hampering feature of crop sustainability,especially in arid and semi-arid regions.So,the effectual usage of all water resources especially underground brackish water represents the core priority in Saudi Arabia.The present study aimed to recognize the influence of different types of water irrigation(tap water as a control,salinized well water,and magnetized salinized well water)with or without soil amendments(soil without any amendment as a control,peat-moss,ferrous sulfate,and peat-moss plus ferrous sulfate)on petunia plant growth and flowering as well as ion content.Irrigating Petunia plants with saline well water adversely affected growth and flowering as compared to tap water and magnetized saline well water.Additionally,plants irrigated with magnetized water showed a significant enhancement in all the studied vegetative and flowering growth parameters as compared to those irrigated with salinized well water.Furthermore,mineral contents and survival of Petunia plants irrigated with magnetized well water were higher than those irrigated with tap water.Irrigation with magnetized well water significantly reduced levels of Na+and Cl−ions in leaves of Petunia plants indicating the role of magnetization in alleviating harmful effects of salinity.In conclusion,we recommend the utilization of magnetized saline well water for irrigating Petunia plants either alone or in combination with soil amendments(peat moss plus ferrous sulfate).

Influence of Foliar Fertilizers on Growth and Development of Petunia hybrida in Winter-Spring 2015-2016 in Thua Thien Hue

The experiment was conducted in winter-spring 2015-2016 in Thua Thien Hue to identify different foliar fertilizer for Petunia hybrida having good growth and development,beautiful colors and long lifetime under local conditions.The experiment included four treatments with three kinds of forliar fertilizers—Dau Trau MK 30-10-5,gibberellin 25-10-10 and abscisic acid.The control treatment(T0)used sterilized water without foliar fertilizer.The results showed that all the foliar fertilizers influenced well on growth and development of Petunia hybrida.Dau Trau MK 30-10-5 helped Petunia hybrida have high quality and high value/cost ratio than the others.

Regulation of Petunia Pollen Tube Growth by Phytohormones： Identification of Their Potential Targets

It is known that cytoskeleton-dependent trafficking of cell wall and membrane components to apical plasma membrane （PM） coupled with ion transport across pollen PM is crucial for maintaining polar pollen tube growth. To elucidate whether plant hormones are involved in these processes, the effects of exogenous phytohormones, indole-3-acetic acid （IAA）, abscisic acid （ABA）, gibberellin A3 （GA3） and cytokinin （kinetin） on the growth, PM polarization, actin cytoskeleton （AC） organization and cytoplasmic pH （pile） of in vitro 4 h-growing petunia pollen tubes were investigated. IAA, ABA and GA3 displayed the growth-stimulating effects and these were accompanied by orthovanadate-sensitive hyperpolarization of the PM. Fluorescent labeling the enzyme with H＋-ATPase antibodies exhibited IAA- and ABA-induced lateral PM redistribution of it into the subapical zone of pollen tube PM. Pollen cultivation on the medium with latrunculin B, the inhibitor of actin polymerization, resulted in inhibition of pollen tube growth and simultaneously in the drop of endogenous IAA content. The IAA-growth stimulating effect was correlated with increased content of actin filaments （AF） in both apical and subapical zones of tubes, while ABA and GA3 exerted the same effect but it was accompanied by redistributing F-actin only to apical zone. In contrast, kinetin decreased the total F-actin content and inhibited pollen tube growth. It has been shown that the pHe of growing pollen tubes is sensitive to the plant hormones. In the case of male gametophyte growing for 1, 2 and 4 h, IAA induced alkalinization of the cytosol, while ABA and GA3 exerted qualitatively similar effect only after its growth for 1 h and 4 h, respectively. Kinetin, in contrast, resulted in acidification of the cytosol. All these results, taken together, indicate, for the first time, potential targets of the phytohormone action in pollen tubes.

矮牵牛花瓣衰老和逆境胁迫响应相关NAC基因的鉴定与分析

【目的】NAC(NAM, ATAF and CUC)参与植物生长发育和多种逆境胁迫响应过程的调控。本文旨在鉴定和研究对矮牵牛生长发育和逆境胁迫响应的关键NAC成员,为优质抗逆矮牵牛育种提供基因资源。【方法】以腋生矮牵牛(Petunia axillaris)基因组为参考基因组,利用矮牵牛花器官衰老过程、烟草脆裂病毒(Tobacco rattle virus,TRV)侵染、低磷、低温、NaCl、铜离子和干旱胁迫处理后的转录组数据,分析矮牵牛NAC基因(PaNACs)差异表达情况,并对差异表达PaNACs的启动子顺式作用元件及转录因子结合位点进行分析。利用实时荧光定量PCR验证了部分差异表达PaNACs在矮牵牛花衰老过程中的表达情况,并预测了差异表达PaNACs编码蛋白的潜在靶基因。【结果】鉴定的131个PaNAC基因中,59个(45.04%)被鉴定为花器官衰老和逆境胁迫响应过程中的差异表达基因。PaNAC72、 PaNAC22、 PaNAC29、 PaNAC40、 PaNAC2、 PaNAC90、 PaNAC83、 PaNAC56、 PaNAC36和PaNAC35在至少3个生物学过程响应中差异表达显著,其中拟南芥衰老关键基因AtNAP的直系同源基因PaNAC29在花器官衰老过程和低温、低磷、铜离子胁迫逆境处理中显著上调表达;PaNAC72在除受铜离子胁迫外的所有6种处理中表达差异显著;PaNAC22在花器官衰老过程和低温和低磷胁迫中上调表达,在铜离子和干旱逆境下调表达。启动子分析结果显示这10个PaNAC启动子区域存在多种逆境胁迫响应相关元件,且大量响应衰老和逆境胁迫的差异表达基因的启动子区域存在NAC的结合位点。【结论】PaNACs广泛参与矮牵牛生长发育及逆境胁迫响应,其中PaNAC29可能是花衰老关键的正调控因子,PaNAC72广泛响应多种逆境胁迫。

芯片行业再生水灌溉矮牵牛的植物毒理效应及环境风险评价

芯片行业废水的再生利用对减少水环境污染、缓解水资源短缺、推动行业绿色发展具有重要意义。为明确芯片行业再生水灌溉对植物的毒理效应,采用矮牵牛作为模型生物,分析2种再生水灌溉期间其生长形态及对总蛋白、叶绿素、抗氧化系统和能量系统标志物的响应特征,基于第二代综合生物标志物响应指数(IBRv2)法评估再生水灌溉的环境风险。结果表明:2种再生水灌溉期间未对矮牵牛的生长形态产生明显不良影响;灌溉中期(9 d)矮牵牛总蛋白、叶绿素均被显著诱导,随后诱导作用减弱,抑制作用开始显现;灌溉中长期(9~15 d)矮牵牛的抗氧化标志物以抑制效应为主,但机体未出现明显的氧化损伤;灌溉期间矮牵牛能量系统标志物的响应规律无明显的一致性。芯片行业再生水灌溉矮牵牛的平均IBRv2为0.85~1.72,环境风险水平较低,但因再生水仍含有高浓度的氯离子和溶解性总固体(TDS),可选择敏感的生物标志物组合谷氨酸脱氢酶、丙酮酸激酶、丙二醛(MDA)或超氧化物歧化酶、MDA对环境风险进行监测和管控。建议进一步研发可有效降低芯片行业再生水中氯离子和TDS浓度的处理工艺,以保障芯片行业再生水回用于绿化灌溉的生态环境安全。

矮牵牛PhSPL9b基因的克隆及功能分析

为研究SPL(SQUAMOSA-promoter binding protein-like)转录因子在矮牵牛成花转换中的作用,克隆矮牵牛PhSPL9b基因,并将该基因对应的miR156/157靶位点进行点突变获得rPhSPL9b,将PhSPL9b和rPhSPL9b分别构建超量表达载体,转化矮牵牛和拟南芥,最终获得拟南芥35S∶∶PhSPL9b与35S∶∶rPhSPL9b转基因植株以及矮牵牛35S∶∶PhSPL9b转基因植株。研究结果显示,过表达PhSPL9b和rPhSPL9b导致拟南芥莲座叶显著减少,花期明显提前,其中35S∶∶rPhSPL9b转基因表型更为明显;过表达PhSPL9b促进矮牵牛提前开花。RT-PCR和qRT-PCR分析结果显示,表型明显的转基因株系中,PhSPL9b基因表达量均显著高于对照。转录激活实验结果表明,PhSPL9b是一个具有转录激活活性的转录因子。以上结果表明,矮牵牛PhSPL9b基因对开花时间具有重要调控作用,其功能具有保守性,同时,它可能是通过转录激活下游基因的表达而影响植物开花。

黑果枸杞花青素单体PrG对H_(2)O_(2)诱导的小鼠颗粒细胞氧化损伤的保护机制研究

目的探讨黑果枸杞花青素单体矮牵牛花色素-3-O-(6-O-对香豆酰)芸香糖苷-5-O-葡萄糖苷(PrG)对H_(2)O_(2)诱导的小鼠颗粒细胞(GCs)氧化损伤的保护机制。方法将20只ICR雌性小鼠的GCs进行原代培养。将GCs随机分为对照组(Control组)、过氧化氢处理组(H_(2)O_(2)组)、PrG处理组(H_(2)O_(2)+PrG组)。首先,构建了由不同浓度(0μmol/mL、25μmol/mL、50μmol/mL、100μmol/mL、200μmol/mL、400μmol/mL)、不同作用时间(30 min、60 min、120 min、180 min)H_(2)O_(2)诱导的GCs氧化应激损伤模型。随后应用PrG不同浓度(20μg/mL、40μg/mL、80μg/mL)、不同作用时间(12 h、24 h、48 h)处理氧化损伤后的GCs。采用CCK8法筛选H_(2)O_(2)与PrG的最佳作用浓度与作用时间,采用EdU法检测H_(2)O_(2)诱导的GCs增殖情况,使用DCFH-DA活性氧荧光探针检测GCs内的ROS水平,使用JC-1探针和Mito-tracker Red CMXRos活细胞染料检测线粒体膜电位和线粒体分布。结果H_(2)O_(2)在作用浓度为200μmol/mL、作用时间为2 h时小鼠GCs存活率下降约60%。GCs活力随着PrG浓度的升高而显著升高(P<0.01),且在浓度为80μg/mL时对活性影响最佳。PrG能显著促进GCs增殖能力,降低活性氧水平(P<0.01),增加线粒体分布数量(P<0.01),恢复线粒体膜电位(P<0.01)。结论黑果枸杞花青素单体PrG通过清除ROS自由基、增强线粒体功能对H_(2)O_(2)诱导的小鼠GCs氧化损伤发挥保护作用。

矮牵牛花朵大小及相关性状遗传分析

为了探究矮牵牛花朵大小的遗传规律,以大花型和小花型矮牵牛高代自交系为亲本构建四世代遗传群体(P_1、P_2、F_1、F_2),对花朵大小遗传特征进行主基因+多基因混合遗传模型分析,并将F_1植株与中花型矮牵牛W115株系进行杂交,验证遗传规律。同时以F_2群体为材料,对花径、萼片长、叶片长等23个表型性状进行测定,并研究其相关性。结果表明,矮牵牛大花对小花性状符合2MG-A模型,即由2对加性主基因控制,主基因遗传率为95.38%;大、小花杂交F_1与中花W115进一步杂交,后代出现大花与中花性状分离(1∶1),且中花植株的叶片和苞片叶绿素含量显著高于大花植株(P<0.01)。大花×小花F_2群体的表型性状变异丰富,变异系数在7.67%~59.93%,平均22.38%。相关性分析结果表明,花部性状、叶部性状以及两者之间均存在一定的相关性,其中花径与其他器官大小均呈显著正相关,与部分植株性状呈显著负相关。

不同供磷水平对矮牵牛生长的影响

为提升矮牵牛的育苗和养护技术,本研究以矮牵牛品种‘梦幻玫红’为试验材料,设置5个不同磷浓度的营养液处理矮牵牛幼苗,研究不同磷供应水平对矮牵牛生长相关指标的影响。结果表明,不同浓度磷供应对矮牵牛植株的株高随处理时间不同有所差异,至第5周,磷浓度为7.5 mg·L^(-1)处理组的平均株高最大(达到14.25 cm)。随着处理时间延长,磷浓度高的处理组和磷浓度低的处理组的冠幅差异逐渐增大,至第5周,磷浓度越高冠幅越大,磷浓度为45.0 mg·L^(-1)处理组冠幅达到22.90 cm。叶片数、分枝数、花芽数随磷浓度升高呈上升趋势,但各处理组间叶片SPAD值基本无显著差异。破坏性取样后发现,植株地上和地下部分磷积累量和干重也随磷浓度升高而增加,但根冠比随着磷浓度升高而降低。因此,不同供磷水平对矮牵牛的营养生长有明显的影响,磷供应不足会降低矮牵牛的冠幅、叶片数、分枝数和花芽数量等重要观赏指标。

城市绿地土壤解磷菌的筛选及促生效果分析

为了从城市绿地土壤环境中筛选解磷菌,明确其菌属,并从中获得具有促生效果的优势解磷菌株。以园林绿地土壤为对象,通过解磷圈观察从筛选平板上获取解磷菌株,分析16S rDNA片段序列同源性并构建系统发育树。利用钼锑抗显色法检测菌株的解磷能力,同时测定其培养基pH。挑选3个解磷能力强的菌株,对应T1、T2和T3处理组,设置未接菌对照(CK),通过矮牵牛栽培试验检验其促生效果。结果表明,从绿地土壤中筛选出的29株解磷菌分属Kluyvera、Pantoea、Enterobacter、Burkholderia、Paraburkholderia和Ochrobactrum属。菌株的解磷能力分布在8.67~632.62 mg/L之间,对应培养基的pH分布在6.38~4.42之间,二者存在明显的负相关(R^(2)=0.8775)。3株优势解磷菌能显著提升矮牵牛的冠幅、叶片数、花芽数和干重,对株高和SPAD无明显作用。另外,T3菌株能显著提升根体积,T2和T3菌株均能提升植株地上和地下部分磷含量,T1菌株则对上述生长指标无明显促进作用。综合来看,绿地土壤中获得的29株解磷菌酸化培养基的能力在一定程度上能反映其解磷能力。挑选的3株优势菌均对矮牵牛有促生效果,解磷能力最强的T3菌株表现最佳,具备进一步开发为菌剂的潜力。