甘肃省杓兰属植物资源调查及2新记录种

通过对甘肃省杓兰属植物进行系统的调查和资料整理,共发现甘肃省杓兰属植物15种,占中国杓兰属植物种类的39.47%,其中9种为中国特有种。调查发现2种甘肃省杓兰属植物分布新记录种——巴郎山杓兰(Cypripedium palangshanense T.Tang et F.T.Wang)和离萼杓兰(Cypripedium plectrochilum Franch.)。通过整理重新编制了甘肃省杓兰属植物的分种检索表。     

大花杓兰亲环素基因的克隆及生物信息学分析

亲环素是一个多基因家族,在植物生命活动中发挥着重要的作用。该研究以大花杓兰(Cypripedium macranthum)为材料,采用RT-PCR技术克隆到1个亲环素基因(CyP),并对其进行生物信息学分析。结果表明:大花杓兰CyP基因的开放阅读框序列为525 bp,命名为CmCyP(GenBank登录号为MH411125),编码174个氨基酸。预测CmCyP蛋白是一个位于细胞质、相对分子量约为18 kD、理论pI为8.73、无信号肽、跨膜结构域的亲水性蛋白质。磷酸化和糖基化位点预测分析发现,CmCyP蛋白存在18个潜在的磷酸化位点和2个潜在的糖基化位点。蛋白保守结构域预测分析发现,CmCyP蛋白包含一个高度保守的肽脯氨酰顺反异构酶结构域,属于单结构域亲环素。对二级结构进行预测分析发现,CmCyP蛋白中存在无规卷曲70个、延伸链56个、α-螺旋23个、β-折叠25个,这4种结构元件在三级结构中也有体现。系统进化树结果显示,大花杓兰CmCyP蛋白与铁皮石斛(Dendrobium catenatum)和万带兰(Vanda hybrid cultivar)的CyP蛋白的亲缘关系较近。该研究首次克隆了大花杓兰亲环素基因(CmCyP),为进一步探讨CmCyP基因的生物学功能奠定了基础。     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

浙江扇脉杓兰野生居群的表型性状变异及其与地理-土壤养分因子的相关性

为揭示扇脉杓兰(Cypripedium japonicum Thunb.)表型性状的变异规律,以浙江临安6个野生居群(老殿、宝剑石、红蛇洞、清凉峰、大明山和里横塘)为研究对象,分析了叶片、萼片、子房及唇瓣等18个表型性状的变异幅度,并讨论了表型性状变异与地理-土壤养分因子间的相关性以及居群间和居群内的表型多样性.结果显示:6个居群18个表型性状(地径、下叶长、下叶宽、下叶长/宽比、上叶长、上叶宽、上叶长/宽比、中萼片长、中萼片宽、苞片长、苞片宽、子房长、花梗直径、花梗长、唇瓣长、唇瓣宽、植株展度和株高)测量值的F值为1.240 ～ 18.666,除花梗直径和株高外,其他16个性状均有极显著差异.各性状的变异系数为5.937％ ～ 26.349％,其中,唇瓣长的平均变异系数最小(6.119％)、苞片宽的平均变异系数最大(24.124％);居群间各性状的变异系数为8.139％～ 13.663％,平均变异系数为11.143％,其中,里横塘居群的平均变异系数最大、宝剑石居群的最小.各性状的多样性指数平均为1.977,其中地径的多样性指数最大(2.045)、苞片宽的最小(1.663).通过UPGMA聚类分析,可将6个居群划分为3组:清凉峰、大明山、老殿和宝剑石居群为1组;红蛇洞和里横塘居群分别各自成组,其中清凉峰和大明山居群的遗传距离最近.各表型性状间存在显著或极显著的相关关系;但仅地径与速效钾含量显著正相关(r=0.854),子房长、唇瓣长和宽与土壤全磷含量显著正相关(r值分别为0.916、0.854和0.914),植株展度与土壤pH显著正相关(r=0.810),其余性状与地理因子(包括经度、纬度和海拔)及土壤养分因子均无显著相关性.研究结果表明:扇脉杓兰野生居群表型性状存在丰富的变异和多样性,且与生境土壤中速效钾及全磷含量及土壤pH有关.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

黄花杓兰种子无菌萌发的培养条件研究

授粉１５周后的黄花杓兰（Cypripedium flavum P.F.Hunt et Summerh.)种子经０．５％ＮaClO溶液处理后,无菌下播于培养基因表面,２０周后种子最高萌发率达到９０％。ＫＴ和ＢＡ促进兰花种子萌发的机理是作为一种萌发诱导物质直接起作用。而不仅仅是拮抗ＡＢＡ的抑制作用而促进种子的萌发,培养基,激素和种子预处理萌发率高低的关键,种皮是阻碍黄花杓兰种子萌发的主要原因之一。     

黄花杓兰的花芽发育

对黄花杓兰（Cypripedium flavum P.F.Hunt et Summerh.）成年植株做了一个生长季的研究,提出了一年芽、二年芽和多年休眠芽的概念。指出由芽形成到植株开花需两年时间,其具体发育路线是：第一年6－7月份,根状茎顶端二年芽基部外侧有两个新的小芽产生,即“一年芽”,至9－10月份发育出7－9片幼叶,然后随气温下降停止生长;第2年4月份复苏,即为“二年芽”,二年芽在本生长季内发育成混合芽,但一般情况下只有一个充分发育,另一个未能充分发育并且一般将来也不再有发育的机会,被称为“多年休眠芽”;第3年5月份充分发育的二年芽长出地面,形成植株,迅速开花、结果,至9月底植株枯萎。本文还讨论了黄花杓兰发育过程与环境的关系。     

Stimulatory effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of Cypripedium macranthos seed in vitro

Cypripedium macranthos is a wild orchid that is becoming endangered. Efficient methods for its propagation from seed, which is indispensable for conservation, production and breeding, have not been reported. The effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of seeds of Cypripedium macranthos Swartz were examined. The duration of treatment with a solution of hypochlorite prior to sowing was one of the critical factors that affected germination. Approximately 70% of seeds that had been treated with either a solution of NaClO that contained 0.5% available chlorine for 60 min or with one of Ca(ClO)₂ with 3.2% available chlorine for 7 h, germinated after 3 months of culture at 20°C, subsequent to 2 months chilling at 4°C. Chilling seeds at 4°C prior to culture at 20°C was another factor that stimulated germination. Even chilling for 2 weeks had a promotive effect on germination, and chilling for 2 months enhanced it most effectively: the frequency of germination was 67% after 3 months of culture at 20°C. However, the promotive effects of chilling on germination were reduced by holding seeds at 20°C for 3 and 6 weeks prior to chilling treatment. Germination of 58‐70% was achieved by the addition of 1 µ M cytokinin to the medium, while the frequency was only 17% in cytokinin‐free medium. We report a reproducible and efficient method for enhancing seed germination of C. macranthos , which involves treatment with hypochlorite prior to sowing, and the combination of chilling at 4°C prior to germination and exposure to a cytokinin.     

杓兰属(兰科)中一个国产种类的新等级(英文)

丽江杓兰大围山变种(Cypripedium lichiangensem var.daweishanense)被提升为种的等级:大围山杓兰(Cypripedium daweishanense)。此种的特征是:中萼片浅黄绿色,上面疏被栗色斑点;花瓣较短,近等长于或略长于唇瓣。而丽江杓兰(Cypripedium lichiangense)的中萼片为紫肝色;花瓣要长得多,大约为唇瓣长的2倍。它们均属于三组(Cypripediumsect.Trigonopedium)。该组共含11种,均为中国特有种。文中提供了一个该组的分种检索表。