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.

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).

矮牵牛pMADS4基因的克隆及序列分析(英文)

利用RT-PCR方法从矮牵牛(Petunia hybrida)中克隆了MADS盒基因pMADS4,进行了全序列测定。测序结果显示,所得到的基因与发表的序列同源率为100%。该基因长度为910bp,含有一个开放阅读框,编码253个氨基酸残基组成的蛋白,具有典型的植物MADS盒基因的结构,该蛋白序列与玉米(Zeamays)ZAG3、麻竹(Dendrocalamus latiflorus)DlMADS18和拟南芥(Arabidopsis thaliana)AGL6的同源性分别为64%、64%和61%,说明pMADS4是AGL6在矮牵牛中的同源基因,推测它们具有相似的功能,都促进开花并且调节花器官形成。进化树分析显示,AP1/AGL9亚家族分为SEP、AGL6和AP1三个分枝,且AGL6分枝又进一步分为三组,分别与裸子植物、单子叶植物和双子叶植物类群相对应,预测通过分离植物的AGL6同源基因可以准确的判断植物所属类群。通过蛋白预测说明pMADS4蛋白以同源或异源二聚体的形式存在,具有结合DNA的功能。

温度对矮牵牛生长发育的影响

以矮牵牛(Petunia hybrida)重瓣豪放双瀑布、单瓣豪放梦幻、单瓣迷你地毯为研究对象,研究了温度对矮牵牛生长发育的影响。结果表明,矮牵牛生长发育最佳温度为16~25℃,且温度恒定于21~25℃区间株高、冠幅、侧枝、开花朵数增幅最大;温度处于25~35℃区间植株会发生徒长,开花朵数下降;温度高于35℃或低于15℃对矮牵牛的生长都有抑制效果,抗病性降低,叶片大面积萎蔫,甚至枯死。

低温胁迫对矮牵牛H株系花青素、游离脯氨酸及可溶性糖含量的影响

以矮牵牛[Petunia hybrida(J.D.Hooker)Vilmorin]H株系为试验材料,利用人工气候箱进行低温胁迫试验,调查低温胁迫对矮牵牛叶片中花青素和渗透调节物质游离脯氨酸、可溶性糖含量的影响。结果表明,花青素在低温处理前期2 h和中期24 h时相对含量变化较对照无显著性差异,当胁迫增至120 h,其相对含量显著提高;游离脯氨酸含量在低温处理前期2 h时含量变化较对照无明显变化,而在中期24 h达到峰值,此后表现为下降趋势;可溶性糖含量在低温处理前期2 h时含量较对照有明显提高,此后含量略微上升,直至后期120 h一直保持在这一较高水平。研究表明,矮牵牛可能通过积累花青素、渗透调节物质来提高植株的抗寒能力,从而抵御低温胁迫。

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.

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.

乳酸菌对矮牵牛生长和生理性状的影响

为进一步提高矮牵牛(Petunia hybrida)的观赏特性,以矮牵牛为试验材料,在不同浓度的乳酸菌制剂处理下,测定矮牵牛的各项生长和生理指标。结果表明,试验组较对照组在矮牵牛的生长和生理指标上均有明显提高,其中浇施1∶500乳酸菌的试验组提升效果最明显,该浓度处理下的矮牵牛冠幅、叶绿素含量、可溶性蛋白质含量和可溶性糖含量分别较对照组提高了11.69%、25.27%、32.69%、22.22%。

不同基质配比对矮牵牛生理特性的影响

以矮牵牛(Petunia hybrida)穴盘苗为供试材料,基质中分别添加不同比例的蛭石、珍珠岩、花生壳、松树壳配成栽培基质,研究不同基质配比对矮牵牛生长指标、光合作用相关指标、渗透调节物质含量、超氧化物歧化酶活等生理指标的影响。结果表明:T5、T6、T7处理下矮牵牛综合指标表现较好,矮牵牛植株抗逆性较强,三者相比,以T7最为优良。表明T5、T6和T7栽培基质的理化性质均能为矮牵牛的生长发育提供较好的条件,这与前期对矮牵牛生长形态指标的研究结果一致。

矮牵牛TPS家族基因的鉴定与PhTPS1的表达分析

研究表明海藻糖能够参与调控植物的分枝发育。本研究利用生物信息学方法,从矮牵牛全基因组数据库中鉴定出10个TPS家族成员基因。进化树分析显示,矮牵牛10个TPS蛋白家族成员分为ClassⅠ和ClassⅡ两大类。ClassⅠ中PhTPS1和PhTPS2均含有16个内含子。ClassⅡ中所有成员均含有2个内含子。TPS 10个成员中共找到6个Motif。PhTPS1、PhTPS2、PhTPS3、PhTPS4、PhTPS5、PhTPS8、PhTPS9、PhTPS10均含有Motif1~Motif6,PhTPS6和PhTPS7含有Motif1~Motif5。利用实时荧光定量PCR分析PhTPS1基因在不同组织、不同处理下的表达水平。组织特异性表达分析显示:PhTPS1在叶腋中最高,而花瓣中的表达量最低;对PhTPS1在去顶后及生长素和细胞分裂素处理后的表达水平进行了检测,结果表明:去顶6 h后PhTPS1显著上升,但随之逐渐下降;去顶后施加IAA则明显抑制了去顶引起的PhTPS1的上调;施加6-BA6 h能够引起PhTPS1表达水平的显著上升,但24 h后表达水平明显降低。该研究对于揭示矮牵牛分枝发育机理以及培育分枝特性不同的矮牵牛新品种均具有重要意义。

矮牵牛PhLcyB基因的克隆及功能分析

本研究以矮牵牛为实验材料,利用VIGS技术验证矮牵牛类胡萝卜素代谢途径关键酶基因PhLcyB的功能,为探究植物叶片中类胡萝卜素的代谢调控的分子机制及类胡萝卜素在叶片呈色方面的作用机理提供理论依据。本实验克隆获得了PhLcyB基因的完整ORF序列,通过VIGS技术构建了TRV-PhLcyB和TRV-PhCHS重组载体,通过侵染发现TRV-PhLcyB侵染的的矮牵牛植株叶片出现大面积白化现象,花瓣颜色没有明显变化。进行叶片生化检测后,发现PhLcyB基因沉默后叶片中类胡萝卜素含量明显降低,尤其是叶黄素降低明显,同时叶片中叶绿素a和叶绿素b含量明显降低。PhLcyB基因对矮牵牛叶片中类胡萝卜素(尤其是叶黄素)的代谢调节有重要作用。

矮牵牛PhUBC2-1基因响应干旱胁迫表达与功能分析

泛素/26S蛋白酶体途径(ubiquitin/26S protesome pathway,UPP)是植物适应逆境的重要方式之一,E2泛素结合酶在其中发挥着重要作用。本研究从矮牵牛(Petunia hybrida Vilm.)中克隆得到E2泛素结合酶基因PhUBC2-1。生物信息学分析表明PhUBC2-1基因在进化中高度保守,与烟草、番茄、辣椒和野草莓亲缘关系最近。实时荧光定量分析发现该基因响应干旱胁迫,在叶中表达上调、在根中呈先上升后下降趋势。酵母表达系统分析表明,PhUBC2-1基因提高酵母对干旱的耐受性。利用花序侵染法转入拟南芥发现PhUBC2-1基因可提高拟南芥成株的耐旱能力,干旱胁迫下转基因植株叶绿素含量高于野生型,丙二醛含量低于野生型。以上结果表明PhUBC2-1基因在干旱胁迫响应中起到正调控作用,为泛素结合酶在非生物胁迫下的研究提供理论支持。

芳香型矮牵牛‘Carpet Blue’高效再生体系的建立

矮牵牛(Petunia hybrida)是研究植物芳香性状的理想材料,但目前有关芳香型矮牵牛转基因技术体系的研究较少,为建立芳香型矮牵牛的高效再生体系,本研究以特异挥发苯环类物质的芳香型品种‘Carpet Blue’为材料,进行了表面消毒、愈伤组织诱导与不定芽分化、不定芽增殖、不定芽生根等研究。结果表明:先用75%酒精(C_(2)H_(5)OH)漂洗30 s,再用5%次氯酸钠(NaClO)消毒8 min,表面消毒效果最好,无菌外植体获得率可达93.75%;愈伤组织诱导与不定芽分化最适培养基为:MS+1.5 mg/L 6-苄氨基嘌呤(6-BA)+0.3 mg/L萘乙酸(NAA),诱导率可达100%,且能分化出健壮的不定芽;最适不定芽增殖培养基为MS+0.5 mg/L 6-BA+0.4 mg/L NAA,增殖系数可达14.67倍;最适生根培养基为1/2MS+0.05 mg/L吲哚丁酸(IBA),生根率为100%,平均根数达37.67条,平均根长达2.61 cm,生根指数最高,达9.82,单条不定根最长可达5.22 cm;炼苗移栽后,成活率高达92.50%。本研究初步建立了芳香型矮牵牛‘Carpet Blue’的高效再生体系,为后续通过转基因手段研究苯环类挥发物质的代谢规律奠定了坚实基础。

矮牵牛花香成分的鉴定及相关基因的表达分析

为了研究矮牵牛花香成分与相关基因之间的联系,本研究以杂交选育的3个芳香矮牵牛株系(5,6,17号)为材料,采用静态顶空进样-气相色谱-质谱联用技术(SPME-GC-MS)和实时荧光定量技术,鉴定芳香矮牵牛鲜花的香气成分,分析花香物质代谢相关基因的表达特征。结果表明:3个株系共鉴定出47种花香组分,其中萜类化合物的种类多且含量较高,株系间花香物质组成差异较大;通过比较看出,6号株系萜类化合物的含量较高,17号株系苯基/苯丙类物质的含量较高,5号株系脂肪酸类物质的含量较高;PAAS、BPBT、PAL都是调控苯基/苯丙类化合物合成的关键酶,EOBⅡ是苯丙烷类挥发性生物合成调控因子,在开花期5号株系中PAAS与PAL基因表达上调,6号株系中的BPBT基因表达上调,17号株系中的PAAS、BPBT和EOBⅡ基因表达显著上调,这些基因与香气组分中苯基/苯丙类物质的含量密切相关。研究结果可为后续培育芳香矮牵牛新品系、克隆矮牵牛花香基因、研究利用矮牵牛特有花香物质提供参考依据。

Ectopic Expression of the Pttknl Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm.

A novel knottedl-like homeobox （knox） gene, Pttknl （Populus tremulaxtremuloides knottedl）, isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on the abaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.

Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression of chsA gene in transgenic Petunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal of chsA gene, and transferred the fusion gene into Petunia hybrida via Agrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNA in situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.