Exogenous calcium enhances the physiological status and photosynthetic capacity of rose under drought stress

Drought(water shortage)can substantially limit the yield and economic value of rose plants(Rosa spp.).Here,we characterized the effect of exogenous calcium(Ca^(2+))on the antioxidant system and photosynthesis-related properties of rose under polyethylene glycol 6000(PEG6000)-induced drought stress.Chlorophyll levels,as well as leaf and root biomass,were significantly reduced by drought;drought also had a major effect on the enzymatic antioxidant system and increased concentrations of reactive oxygen species.Application of exogenous Ca^(2+)increased the net photosynthetic rate and stomatal conductance of leaves,enhanced water-use efficiency,and increased the length and width of stomata following exposure to drought.Organ-specific physiological responses were observed under different concentrations of Ca^(2+).Application of 5 mmol·L^(-1)Ca^(2+)promoted photosynthesis and antioxidant activity in the leaves,and application of 10 mmol·L^(-1)Ca^(2+)promoted antioxidant activity in the roots.Application of exogenous Ca^(2+)greatly enhanced the phenotype and photosynthetic capacity of potted rose plants following exposure to drought stress.Overall,our findings indicate that the application of exogenous Ca^(2+)enhances the drought resistance of roses by promoting physiological adaptation and that it could be used to aid the cultivation of rose plants.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Direct Adventitious Bud Induction and Plant Regeneration of Rosa hybrida Samantha

Effect of explant, site of leaflet, induction period in the dark and combinations of plant growth regulators on direct adventitious bud induction and plant regeneration of Rosa hybrida Samantha was investigated. The results showed that after an induction period of 8 d on MS medium with 1.5 mg L-1 TDZ and 0.05 mg L-1 NAA in the dark and a subculture on MS medium with 0.5 mg L-1 BA and 0.01 mg L-1 NAA under light, the best plant regeneration was obtained and the regeneration frequencies of leaflets and petioles were 51.8 and 10% respectively. There was no significant difference in regeneration ability between leaflets at different sites of the compound leaves, longer time of induction in the dark or high concentration of auxin would cause callus formation, which was disadvantageous for shoot regeneration, and the regeneration frequency was significantly reduced. This regeneration system could be applied for genetic transformation of this cultivar in the future.

Antimicrobial Effectiveness on Selected Bacterial Species and Alkaloid and Saponin Content of <i>Rosa nutkana</i>C. Presl (Nootka Rose) and <i>Urtica dioica</i>L. (Stinging Nettle) Extracts

Nootka rose (Rosa nutkana C. Presl) and stinging nettle (Urtica dioica L.) have been traditionally used in the treatment of skin infection by Indigenous peoples of Vancouver Island, British Columbia, Canada. The main objective of this study was to examine the antibacterial efficacy of extracts of Nootka rose and stinging nettle against the common pathogenic skin bacteria Staphylococcus aureus, Micrococcus luteus, and Pseudomonas aeruginosa using Indigenous science and standard methods of analysis. The Indigenous science method of plant extraction by steeping as advised by the Traditional Knowledge keeper was performed to examine minimum inhibitory concentration (MIC) values and minimum bactericidal concentrations (MBC) by serial dilution and bacterial population counts. Soxhlet extractions and Kirby Bauer disc sensitivity testing showed that Nootka rose extracts possessed antibacterial effectiveness against all three bacterial species while stinging nettle extracts were effective against M. luteus. Results for MIC and MBC indicated antibacterial activity against M. luteus and S. aureus for the Nootka rose when using full-strength solutions;all three bacterial species exhibited growth when undiluted stinging nettle treatments were used. When considering bacterial population counts for S. aureus, results indicated that only the Nootka rose treatment offered effective inhibition. Chemical analysis showed that alkaloid percentage was greater in the stinging nettle (0.17%) than Nootka rose (0.07%), while saponin percentage was greater in the Nootka rose (0.87%) than stinging nettle (0.17%). Overall, Nootka rose showed a greater level of antibacterial effectiveness than stinging nettle by Indigenous and Western scientific methods of plant extract preparation.

Study on the Introduction and Screening of Cut Roses in the Tropical Coastal Area of China

[Objective] This study aimed to select suitable cultivars of cut roses with prominent comprehensive performance which is suitable for the commercial promotion in the tropical region of South Hainan Province,including Sanya and the low-altitude area.[Method] Introduction experiment and production test were designed to observe the agronomic and economic traits of cultivars in a rose resources nursery established in Sanya.[Result] Through observation in the resources garden,18 varieties were chosen to conduct the introduction experiment,of which 8 varieties were selected to accomplish the production test.The results of the production test showed that ＇Vendela＇,＇Golden Emblem＇,＇Samantha＇ and ＇Tineke＇ showed the best adaptability;the cut flower quality of ＇Carola＇,＇Black Magic＇ and ＇Golden Emblem＇ belonged to the first grade;the yield of ＇Movie Star＇,＇Golden Emblem＇ and ＇Black Magic＇ was more than one million per hm2;except ＇Golden Emblem＇,the vase life of the other seven cultivars was more than 10 days;＇Carola＇,＇My Choice＇ and ＇Perfume White＇ had better disease-and insect-resistance;＇Carola＇,＇Vendela＇,＇Rouge Meilland＇,＇Tineke＇,＇Golden Emblem＇,＇Samantha＇,＇Perfume White＇,＇Saiun＇,＇Asagumo＇ and ＇Double Delight＇ were more resistant to blackspot,while ＇Black Magic＇,＇Versilla＇,＇Alec＇s Red＇ and ＇Movie Star＇ were less resistant to blackspot.[Conclusion] Cultivars of cut roses were able to grow and develop normally in the tropical coastal area of Hainan Province,including South Hainan and Sanya,where it was appropriate for the production of cut roses.However,it is necessary to pay attention to the prevention and control of thrips and blackspot.＇Black Magic＇,＇Carola＇,＇Vendela＇,＇Rouge Meilland＇,＇Golden Emblem＇,＇Tineke＇,＇Movie Star＇ and ＇Samantha＇ showed best comprehensive performance,which were thus worthy of large-scale popularization in Hainan Province.

月季RcLEA基因表达提高大肠杆菌对非生物胁迫耐性

[目的]研究RcLEA基因在月季品种中热诱导表达模式及RcLEA基因对多种非生物胁迫耐性。[方法]将耐热和不耐热月季品种进行38℃/3h热激处理,研究RcLEA基因在月季品种的热诱导表达模式;为验证月季RcLEA基因功能,将其转化E.coliBL21,将重组菌株BL21分别置于4、50℃及LiCl、NaCl、Na2CO3、CdCl2、H2O2胁迫下,研究重组菌株对高温、低温及非生物胁迫的响应。[结果]38℃/3h热激处理后,该基因在耐热月季品种‘曼海姆宫殿’(Schloss mannieim,SM)、‘赌城’(Lasvegas,LV)中强表达,而在不耐热品种‘新十全’(Kordes Perfecta,KP)弱表达或不表达,表明该基因与月季耐热性关系密切。重组菌株提高了对高温、低温、重金属、高盐、高pH值、氧化等非生物胁迫的耐性,表明RcLEA参与了上述非生物胁迫的响应。[结论]该研究为后续该基因导入不耐热月季品种提高月季耐热品质及其机理研究提供了思路,也为月季等园林观赏植物的耐热品种筛选提供了理论支持。

The Expression of RcLEA Gene Improves Tolerance of E. coli Cells to Abiotic Stress

[Objective] This study was to reveal the heat induced expression model of RcLEA gene and its tolerance to various abiotic stresses.[Method] Heat resistant and heat sensitive varieties of Rosa hybrida L.were subjected to heat shock treatment at 38 ℃ for 3 h;then RcLEA gene from both varieties treated was cloned and transformed into Escherichia coli strain BL21;finally recombinant colonies were separately cultured at 4 ℃ and 50 ℃ under the stresses of LiCl,NaCl,Na2CO3,CdCl2 and H2O2 to study the responses of recombinant E.coli strains to high temperature,low temperature and some other abiotic stresses.[Result] After heat shock treatment at 38 ℃ for 3 h,RcLEA gene expressed highly in ＇Schloss mannieim＇（SM）and ＇Las vegas＇（LV）variety,but weakly or even not expressed in ＇Kordes＇ Perfecta＇（KP）,indicating that this gene is closely related with heat resistance of R.hybrida.Compared with WT strains,recombinant clones showed higher tolerance to abiotic stresses including high temperature,low temperature,heavy metal,high salt,high pH value and oxidation,suggesting that RcLEA is concerned with the response of R.hybrida to abiotic stresses mentioned above.[Conclusion] These results provide thoughts for increasing heat resistance by introducing RcLEA into heat sensitive R.hybrida varieties and studying the heat-resistant mechanism of R.hybrida,and also provide theoretical support for selecting heat resistant variety of landscape and ornamental plants like R.hybrida.

Exogenous ethylene influences flower opening of cut roses (Rosa hybrida) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1- aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh- ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-in- duced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhib-ited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.

三类切花月季品种花器乙烯生成量和ACC含量的时空变化

以切花月季品种‘天使’、‘唐娜小姐’和‘红成功’为试材,测定了花朵开放过程中整朵花以及花瓣、雄蕊、花柱、花托和子房、花萼5部分的乙烯生成量和ACC含量。结果表明,花朵的乙烯生成量变化类型主要取决于花瓣,品种间乙烯变化类型的区别也取决于花瓣。3个品种花朵不同部位乙烯生成量的变化趋势相近,都在花朵开放前期出现明显的峰值。花蕾发育早期,3个品种花瓣ACC含量占整朵花ACC总量的比例远低于乙烯生成量所占比例,而花器其它部位则相反,尤其是雄蕊的ACC含量极高而乙烯生成量却相对很低;在花朵开放后期,3个品种表现不同。推测,在花朵开放进程中花器其它部位可能向花瓣运输ACC,不同乙烯变化类型的品种可能在花器不同部位间ACC的运输分配上存在差异。

两个切花月季品种花朵开放和衰老对乙烯的反应及其与内肽酶的关联

以切花月季(Rosa hybrida)品种 Samantha 和 Kardinal 为试材,探讨了乙烯和乙烯作用抑制剂 1-MCP处理对不同品种花朵开放和衰老以及内肽酶活性和种类的影响。结果显示,两供试品种都对外源乙烯敏感,但花朵在乙烯处理后形态上呈现不同的变化趋势。Samantha 表现为乙烯处理显著促进花朵开放和衰老进程,导致花瓣脱落;而 Kardinal 则明显抑制花朵开放进程,出现僵花,直接衰老萎蔫。两品种在乙烯引起花瓣总可溶性蛋白含量下降和内肽酶活性的升高方面,以及 1-MCP 处理获得与乙烯处理相反的效果等方面呈现相同的变化趋势,但是Kardinal 的变化远比 Samantha 剧烈。同时,乙烯处理引起花瓣丝氨酸蛋白酶所占比例的增加方面依然是 Kardinal比 Samantha 显著。上述结果表明,本试验供试的两个切花月季品种开花和衰老对外源乙烯反应的不同可能与相应花瓣中内肽酶的变化程度有关。

切花月季Samantha失水胁迫耐性与其超氧化物歧化酶之间的关联

以探讨月季切花失水胁迫耐性与其超氧化物歧化酶(SOD)之间的关联为目的,选用耐失水胁迫品种Samantha,用DDTC(SOD专一性抑制剂)进行12h预处理,之后在20℃、RH35%～40%、80μEm-2·s-1光照(12h/d)条件下进行12～48h的失水胁迫处理,然后进行瓶插观察。结果表明,20mmol·L-1DDTC预处理在显著抑制花瓣SOD活性的同时,明显减弱切花失水胁迫耐性,表现在切花弯颈率明显提高,复水率和水势值明显降低,瓶插寿命显著缩短。在花朵自然开放初期,花瓣MnSOD、FeSOD和Cu/ZnSOD这3种类型都存在。其中,MnSOD有1种,FeSOD有3种,Cu/ZnSOD有2种;迁移率大小依次为Cu/ZnSOD>FeSOD>MnSOD;谱带强弱依次为FeSOD>Cu/ZnSOD>MnSOD。经失水胁迫处理花瓣中诱导产生了3条新的FeSOD(FeSOD4、FeSOD5和FeSOD6)条带,与此同时MnSOD条带消失。DDTC预处理抑制了任何与上述失水胁迫诱导有关的新的条带产生。由此推测,切花月季Samantha失水胁迫耐性与其SOD活性相关,与FeSOD4～6的诱导产生相关联。

表油菜素内酯对月季切花衰老的影响

0.5mg·L^(-1)表油菜素内酯明显缓解水分对月季切花的胁迫,促进切花体内水分平衡的改善和还原糖含量的增加,抑制花瓣溶质外渗,从而延缓切花衰老。

一氧化氮对瓶插月季呼吸作用及相关酶活性的影响

以硝普钠(SNP)为一氧化氮供体,研究了NO及其清除剂PTIO(2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide)处理对切花月季瓶插过程中生理变化及呼吸作用相关酶活性变化的影响。结果表明:0.1mmol.L-1SNP释放的NO可以降低月季切花的萎蔫率,延长瓶插寿命,增加花枝鲜样质量,抑制COX(细胞色素氧化酶)、AO(抗坏血酸氧化酶)、PPO(多酚氧化酶)活性,抑制月季呼吸强度和乙烯释放,延缓二者跃变高峰的出现,延缓MDA含量和膜相对透性的升高,PTIO的处理提高了上述SNP处理下COX、AO、PPO的活性,提高了呼吸速率,促进MDA含量和膜相对透性的上升,但抑制了乙烯的释放,初步说明NO可能参与衰老过程中呼吸作用的末端氧化酶的活性调节。

月季组织培养和遗传转化体系的研究进展

月季通过器官和体细胞胚发生途径都可以获得再生植株,在遗传转化中主要是利用体细胞胚作为转化受体。目前,利用农杆菌介导法和基因枪法已成功将外源基因如报告基因、抗病基因和改变花色的基因等导入月季基因组中。本文对近年来月季组织培养和转基因研究进展进行了综述,为建立月季高效遗传转化体系奠定了理论基础。

月季MYB基因cDNA全长克隆和表达分析

【目的】克隆月季花色代谢相关新基因的全长cDNA序列,并分析其表达功能。【方法】根据月季花色突变体SSH文库分析得到的差异表达EST序列设计引物,采用RACE技术进行全长cDNA克隆。【结果】克隆到1 125 bp的cDNA全长,GenBank登录号为Eu082130。这一cDNA序列编码1个包含294个氨基酸的前体蛋白,它与小金海棠MxMYB1和大豆的GmMYB176蛋白的保守区同源性分别为70%和58%,都是只有1个MYB结构;而与具有两个MYB结构域的棉花GhMYB26、拟蓝芥AtMYB305和金鱼草AmMYB340同源性很低。该蛋白的分子量为32.33 kD,等电点为9.65,分子式为C1387H2201N435O440S10。半定量PCR分析证实了该RhMYB1基因与CHS基因在红花突变体中比在黄花亲本中表达量高。【结论】推测该基因是一个转录因子,参与调控花青苷的合成。

月季丁香酚合成酶基因RhEGS1的克隆及表达分析

【目的】克隆月季丁香酚合成酶基因RhEGS1的全长cDNA序列,分析其序列及表达特征,并对该基因编码的蛋白进行原核表达分析,为深入探讨丁香酚合成酶的生化特性奠定基础。【方法】根据已发表的其它植物EGS基因序列的保守结构域设计简并引物,结合RACE技术,获得RhEGS1的全长cDNA序列,并进行生物信息学分析;利用半定量RT-PCR对不同组织及不同花发育时期RhEGS1进行表达分析。采用Gateway克隆技术,构建原核表达载体,并进行原核蛋白表达。【结果】月季RhEGS1的cDNA全长为1 207 bp,包含一个927 bp的ORF,编码309个氨基酸。同源序列比对发现RhEGS1与仙女扇的CbEGS2有83.87%的同源性,与矮牵牛PhEGS1具有81.55%同源性。表达谱分析表明,RhEGS1主要在雄蕊中表达,且在花盛开期表达最强,而在花蕾期及凋谢期表达较弱。原核表达分析发现,在37℃、0.5 mmol.L-1IPTG诱导4 h后,携带RhEGS1 ORF的原核表达载体在大肠杆菌中生成了大量的分子量约为35 kD的蛋白质,其分子量大小与预测的理论值相一致。【结论】从月季雄蕊中克隆到丁香酚合成酶基因RhEGS1,具有EGS基因的结构特征和完整的编码框,且在盛开期雄蕊中表达量最高。

现代月季45S rDNA的荧光原位杂交分析

以45S rDNA为探针对和平、粉和平、奇异玫瑰、红双喜、蜜糖等5个现代月季品种进行了荧光原位杂交研究。结果表明,5个品种的45S rDNA杂交位点数量均为3个,杂交位点的大小和荧光强度比较一致。在细胞分裂间期的核中,5个品种的45S rD-NA杂交位点都位于核仁内,均表现为较小且荧光较弱的杂交点,呈圆形,相互间在大小和荧光强度上比较接近。基于研究结果对现代月季品种45S rDNA的特点及演化过程进行了初步探讨。

月季萨蔓莎不定芽的直接诱导和植株再生的研究

以月季品种萨蔓莎为试材研究不同外植体、叶片位置、暗培养时间和激素组合对月季不定芽诱导和植株再生的影响。结果表明,月季萨蔓莎在含1.5 mg·L-1TDZ 和0.05 mg·L-1NAA 的MS 培养基上黑暗培养8 d 后转入含0.5 mg·L-1 BA 和0.01 mg·L-1NAA 的MS 培养基光下培养,再生效果最好,小叶外植体和复叶柄的再生率分别为51.8%和10%。小叶的叶位对植株再生无显著影响,暗培养时间过长,外植体易形成愈伤组织而不利于不定芽的分化,植物生长调节剂中生长素浓度升高易导致愈伤组织形成而影响植株再生。本研究所建立的不定芽直接再生系统可用于该品种的转基因遗传改良研究。

杂交茶香月季演化与遗传背景分析

通过文献收集、整理,融合最新研究成果,以时间为轴,大量分析包括图象资料在内的中外文献材料,明确了杂交茶香月季演化历程,解析其遗传背景,为杂交茶香月季育种提供参考。绘制了更为详细的杂交茶香月季历史演化图,通过对比发现切花月季最主要花型"中高型或高芯型"品种特性可能源自于中国古老月季资源月月粉,总结出杂交茶香月季的遗传背景。杂交茶香月季遗传背景较为复杂,主要来源于中国的Rosa chinensis(月月红、月月粉)、R.ordorata(淡黄香水月季、彩晕香水月季)、巨花蔷薇(R.gigantea)以及欧洲的法国蔷薇、大马士革蔷薇、麝香革蔷薇、异味蔷薇等9个原种及其变种,甚至包括一些人为的外源基因。

45S rDNA在中国古老月季品种染色体上的荧光原位杂交分析

采用荧光原位杂交(FISH)技术研究45S rDNA在10个中国古老月季品种染色体上的差异。结果表明:(1)中国古老月季品种的45S rDNA杂交位点数与染色体倍性变化基本一致,中国古老月季的起源可能比较简单:二倍体的‘月月红’、‘月月粉’和‘绿萼’均有2个45S rDNA杂交位点,三倍体的‘湖中月’、‘青莲学士’和‘春水绿波’有3个45S rDNA杂交位点,四倍体品种除‘大富贵’有3个杂交位点外其他都有4个45S rDNA杂交位点;(2)中国古老月季品种之间在45S rDNA杂交信号的强弱上存在差异,说明中国古老月季品种之间在染色体结构及45S rDNA拷贝数上存在多样性。