黄花杓兰与菌根真菌共生关系研究

对黄花杓兰(Cypripedlium flavum P.F.Hunt et Summerh.)植株的根进行了一个生长周期的采集和切片观察,发现黄花杓兰具有相应的菌根结构,菌丝的存在状态在各物候期有所不同,可分为5个时期：(1)4月份联通菌丝时期;(2)5月份菌丝团时期Ⅰ;(3)6、7月份无菌丝团时期I;(4)8、9月份菌丝团时期Ⅱ;(5)10月份无菌丝团时期Ⅱ。可以看出,在植株生活转变期,根细胞内出现菌丝团;在植株完成正常的生命过程中,真菌营养是光合营养的补充;真菌菌丝团也作为植物营养的储存器,菌丝团消解释放营养供给植株。在此基础上探讨了菌根真菌与黄花杓兰的共生关系的一个方面：植株通过联通菌丝沟通各个细胞的物质交换,菌根真菌利用植株体内物质完成生命活动,菌丝团消解,使真菌所储藏的营养物转化,为植株所利用。     

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。     

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。     

大花杓兰根内与根际土壤真菌群落组成分析

目的：探究大花杓兰根内与根际土壤内生真菌的组成与相关性,为其种群恢复和资源可持续利用提供理论依据。方法：采用高通量测序技术对大花杓兰根内与根际土壤真菌的核基因ITS2片段进行扩增测序。结果：除极个别样本外,在OTU、属、科、目、纲和门等分类级别,大花杓兰根部内生真菌数量与根际土壤真菌数量差异较大,所有根际土壤样本内生真菌数量均大于根样本。不同样本间无共有OTUs和属。Alpha多样性结果显示,根际土壤的真菌群落丰富度大于根样本,但多样性差异不明显。在科水平上,根与根际土壤无共有优势菌。PCoA主成分分析和UPGMA聚类分析结果显示,根样本与根际土壤样本在组成上存在差异。在真菌的特异性组成方面,Rozellomycota真菌在根样本和根际土壤样本中相对丰度接近,分布较为均匀,Malassezia真菌只在植物根样本中检测到,为植物根样本的特有真菌,蜡壳耳属（Sebacina）只在根际土壤中检测到,为根际土壤的特有属。结论：大花杓兰根与其根际土壤真菌群落组成存在差异性,根际土壤真菌的丰富度大于根样本,二者真菌群落相关性小,无共有优势菌科。     

四种杓兰的菌根结构及其周年动态

对黄花杓兰、云南杓兰、西藏杓兰和紫点杓兰四种高山杓兰成年植株的根进行了一个生长周期的切片观察。发现四种杓兰的的菌根结构及其在一个生长周期中的动态变化有以下共同特征:在其成年生活期的各个时期,都有真菌入侵形成内生菌根结构,菌丝在其根部皮层细胞内有四种存在状态:(a)咖啡色或黄色、菌丝扭结缠绕而成的大菌丝结;(b)灰褐色或黄色、由片断菌丝组成的大菌丝结;(c)无色、零散的菌丝;(d)橘红色和黄色、菌丝形态模糊的小菌丝结。真菌的入侵-消解在其成年生活期中周而复始地进行,四种状态的菌丝是处于入侵-消解循环不同阶段的菌丝。并由此对高海拔环境下二者的共生关系进行了初步的探讨:真菌侵入杓兰根部以获得自身生存和繁衍所需的营养物质,杓兰利用真菌菌丝消解后的营养物质,进行混合营养。     

黄花杓兰云南中甸居群遗传结构及克隆多样性的分析

采用AFLP分子标记方法对云南中甸的6个黄花杓兰( Cypripedium flavum )居群进行了遗传多样性水平、遗传结构及克隆多样性研究,其中两个居群用样方取样法分析其克隆空间结构.POPGENE软件分析结果表明:两组引物共产生104个位点,其中86个为多态位点,多态位点百分率为82.69%.黄花杓兰具有丰富的遗传变异(物种水平上, He=0.2884, Ho=0.4312;居群水平上,P=64.59%, He=0.2449, Ho=0.3606),黄花杓兰居群的遗传分化不大( Gst=0.154),居群间基因交流较为频繁( Nm=2.7460).居群的克隆多样性水平高( D=0.9638-0.9960,G/N=0.83-0.96),同一克隆的分株相邻,克隆生长延伸范围狭窄.黄花杓兰居群之所以保持较高水平的遗传多样性,可能与其既能通过实生幼苗增加基因的杂合度,又能通过无性分株把杂合体固定下来有关.     

绿色木霉内切几丁质酶基因的克隆及其毛壳菌转化*

利用PCR方法从Trichoderma viride的基因组DNA中克隆了一个42kDa的内切几丁质酶基因,扩增的长度为1672bp,其中包含了启动子和mRNA的编码区。将该基因与来自构巢曲霉的色氨酸启动子相连后,通过原生质体转化将该基因导入球壳毛壳菌CG10。内切几丁质酶活性的测定结果表明,约1/3转化子的内切几丁质酶活性得到了明显提高。本实验为利用基因工程方法提高毛壳菌的生防能力打下了较好的基础。     

小型毛板壳虫无性分裂生殖的研究:——Ⅰ.核器和皮层纤电器的形态和形态发生

应用光学显微镜和扫描电子显微镜观察了小型毛板壳虫 (Coleps hirtus minor Kahl)的形态,描述了它的表面结构、口器、纤毛和核器在无性分裂生殖中的演化过程。     

嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性

探讨了液体发酵嗜热毛壳菌(Chaetomium thermophile)产生的内切β葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀、DEAE|Sepharose Fast Flow阴离子层析、Pheny1|Sepharose疏水层析、Sephacry1 S|100分子筛层析等步骤便可获得凝胶电泳均一的内切β|葡聚糖酶。经125%SDS|PAGE和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为67.8kD和69.8kD。该酶反应的最适温度和pH分别为60℃和40～45在pH50条件下,该酶在60℃下稳定;70℃保温1h后,仍保留30%的活性;在80℃的半衰期为25min。金属离子对内切β|葡聚糖酶的活性影响较大,其中Na+对酶有激活作用;Fe2+、Ag+、Cu2+、Ba2+、Zn2+等对酶有抑制作用。该酶对结晶纤维素没有水解能力。     

Photosynthesis in relation to reproductive success of Cypripedium flavum

* BACKGROUND AND AIMS: Cypripedium flavum is a rare, endemic alpine slipper orchid of China, which is under threat from excessive collection and habitat changes. Conservation and re-introduction of C. flavum is restricted by lack of knowledge of the plant's photosynthesis and how that affects reproductive success. The hypothesis is tested that reproductive success is determined by photosynthetic production. * METHODS: To understand the photosynthetic characteristics and adaptation of C. flavum to alpine environments, and the relation to reproductive success, measurements were made at four field sites with varying degrees of forest cover in the Hengduan Mountains, south-west China. * KEY RESULTS: Both photosynthetic capacity and reproductive traits of C. flavum are affected by light availability. Photosynthetic rate (A) is greatest around noon, following the pattern of photosynthetically active radiation (PAR) at all sites. Cypripedium flavum has highest daily mean photosynthetic rate (A(daily)) and light-saturated photosynthetic rate (A(max)) under a half to a third of full sunlight. High radiation decreased A. However, the optimum temperature for photosynthesis was similar (18-20 degrees C) at all sites. * CONCLUSIONS: The quotient of daily mean photosynthetic rate to light saturated photosynthesis (A(daily)/A(max)) is positively correlated with the ramet number m(-2) and percentage of fruiting of C. flavum. The A(daily)/A(max) ratio is a useful proxy for evaluating reproductive success of C. flavum.     

Variation of photosynthetic capacity with leaf age in an alpine orchid, Cypripedium flavum

Photosynthetic rate, chlorophyll fluorescence, leaf nitrogen and chlorophyll content of Cypripedium flavum were studied at different leaf ages. The photosynthetic capacity changed significantly with leaf age. Net photosynthesis and chlorophyll content peaked when leaf age was 60 days, decreasing at 30, 90 and 120 days. Stomatal conductance showed the highest value at 60 days, while mesophyll conductance decreased with increasing leaf age. Both leaf nitrogen content per unit area and leaf nitrogen content per unit mass decreased with increasing leaf age. The age-dependent variation in photosynthetic capacity could be linked to the changes in biochemical efficiency, leaf nitrogen content and CO₂ diffusion limitation.     

Germinability and fungal occurrence in seeds of Abies alba Mill. populations in southern Italy

Abstract

Germination tests were carried out on seeds from three Italian populations of Abies alba. Seed germinability was up to 38%. HgCl₂, NaClO and plant preservative mixture were used as seed‐sterilising agents. Despite chemical treatments, Papulaspora rubida and Chaetomium globosum were isolated. The possible role of these fungi in the physiology of silver fir seed germination process was discussed.     

珍稀植物扇脉杓兰营养器官的解剖学研究

采用石蜡切片技术对扇脉杓兰营养器官的解剖结构进行了研究。结果表明：根状茎的薄壁细胞中含丰富的淀粉粒,维管柱中分布着排列紧凑的周木维管束;根的皮层发达,有的皮层细胞中存在真菌菌丝团,木质部与韧皮部呈辐射状相间排列,根和根状茎的内皮层细胞都形成马蹄形加厚结构。茎的表面分布气孔,皮层面积较小,皮层内部的基本组织发达,外韧维管束散生分布其中,茎和叶上都附有非腺性毛;叶为等面叶,叶肉细胞排列疏松,气孔主要分布于远轴面,略外凸,保卫细胞中含有叶绿体,叶缘处的叶肉组织中含有气腔结构。扇脉杓兰营养器官的这些特征与其荫蔽湿润的生境是相适应的。     

Seed biology and in vitro seed germination of Cypripedium

Abstract

Cypripedium orchids have high horticultural value. The populations of most species are very geographically restricted and they are becoming increasingly rare due to the destruction of native habitats and illegal collection. Reduction of the commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection. Cypripedium species are commercially propagated via seed germination in vitro. This review focuses on in vitro seed germination and provides an in-depth analysis of the seed biology of this genus.     

Pollination biology of the endangered orchid Cypripedium japonicum in a fragmented forest of Japan

Pollination biology studies of the endangered orchid Cypripedium japonicum were conducted in its natural habitat using pollinator observation and hand‐pollination experiments. The observed fruit set was as follows: artificial outcross‐pollinated, 100%; artificial self‐pollinated, 100%; pollinator‐excluded, 0%; and emasculated flowers, 0%. These results show that this species, although self‐compatible, is neither autogamous nor agamospermous. The fruit set for open‐pollinated flowers was 14.9%, which suggests that the study population was subject to pollinator limitation. The nectarless flowers of C. japonicum were exclusively visited and pollinated by the queens of two bumblebee species (Bombus ardens and B. diversus diversus). It is probable that the nectarless flowers of C. japonicum attract pollinators through a generalized food deceptive system.     

三江并流区域野生杓兰属植物资源初报

对三江并流区域的野生杓兰属植物的种质资源特点及分布规律作了描述,并提出了该区域野生杓兰属资源开发利用的建议.