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题名: 兜兰属植物离体繁殖和系统学研究
作者: 龙波
学位类别: 博士
答辩日期: 2009-05-20
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 龙春林
关键词: 兰科:兜兰属:离体繁殖:离体花粉萌发:离体保存:系统学:ITS:RAPD
学位专业: 植物学
中文摘要: 全世界兜兰属(Paphiopedilum)植物总数为85种,中国是世界上兜兰属植物种类最丰富的国家之一,占了全世界的1/3,共29种。由于兜兰属中许多种类具有很高的观赏价值,导致资源破坏十分严重,一些种类已濒临灭绝。2004年出版的《中国物种红色名录》第一卷里,18种兜兰属植物中有5种被列为极危种,12种被列为濒危种。早在1997年制定的《华盛顿公约》(即《濒危野生动植物种国际贸易公约》,Convention on International Trade in Endangered Species of Wild Fauna and Flora,简称为CITES),所有兜兰被列入附录I,属于绝对禁止国际贸易的物种。本文从蒴果成熟度、培养基无机盐浓度和有机添加物、激素浓度入手,来探讨对兜兰属植物离体繁殖的最佳途径;研究兜兰花粉的寿命,外源的钙和硼对兜兰花粉萌发的影响;通过对兜兰属离体保存技术的研究,探索兜兰属植物离保存技术的最佳手段和方法,制定兜兰属植物保存规范;从形态学、分子生物学方面对兜兰属的系统学进行初步研究,对其亚属和组的划分、种间关系以及一些种的归属问题进行探讨。主要研究结果如下: 1. 中国兜兰属植物离体繁殖技术的研究对20种兜兰属植物进行离体繁殖技术的研究。以密毛兜兰为代表,实验结果证实了种子成熟度、培养基、碳源、天然添加剂对密毛兜兰种子萌发、原球茎和植株生长有明显影响:120 d、130 d、140 d种子不能萌发;随着种子每推迟10 d成熟,种子萌发率随之提高;200 d的种子达到萌发最高点;在KC培养基接种40 d达到最高萌发率,而VW培养基的原球茎直径最长;葡萄糖有利于种子萌发和叶的生长,其次是蔗糖、麦芽糖和甘露醇;10%椰子汁是最好的种子萌发天然添加剂,而土豆泥、苹果泥有利于叶的生长,相对差的是佛手瓜泥和LH。以密毛兜兰、巨瓣兜兰、波瓣兜兰、杏黄兜兰为例,证明了激素浓度对兜兰的芽繁殖率有显著影响:密毛、波瓣兜兰在BA 5.0 mg l-1 + NAA 0.5 mg l-1,BA 1 mg l-1 + NAA 0.5 mg l-1水平能达到增殖率100%;BA 5.5 mg l-1 + NAA 0.5 mg l-1 组合能使巨瓣兜兰达到增殖率为33%;每种激素水平都能使杏黄兜兰得到增殖,最高增殖率发生在BA 4 mg l-1 + NAA 0.1 mg l-1为200% (2.0芽/节间)。通过我们的实验方法,同样地成功离体繁殖和保存了另外15种兜兰属植物,以及1个自主杂交品种♀P. delicatum × ♂P. coccineum:卷萼兜兰、硬叶兜兰、巨瓣兜兰、同色兜兰、紫毛兜兰、紫纹兜兰、白花兜兰、长瓣兜兰、带叶兜兰、亨利兜兰、麻栗坡兜兰、包氏兜兰、浅斑兜兰、根茎兜兰、红旗兜兰。而且,在3个月内,1/4 MS medium + BA 2 mg + NAA 0.2 mg l-1, KT 2 mg + NAA 0.2 mg l-1分别使麻栗坡兜兰和长瓣兜兰增殖3倍和4倍。 2. 中国兜兰属植物的花粉离体萌发研究5种兜兰(格丽兜兰、波瓣兜兰、紫毛兜兰、长瓣兜兰、红旗兜兰)花粉的寿命,外源的钙和硼对兜兰花粉萌发的影响。在培养基缺糖、缺钙条件下,花粉都不萌发。虽然缺硼的培养基下,花粉仍会有极微量的萌发,但几乎可以忽略不计。在液体布氏培养基上,2个月、3个月成熟度的花粉依然可以萌发,与新鲜的20天的花粉萌发率没有多大区别。兜兰植物花粉管伸长需要的时间较长,一般要等到第4天、第5天。少数花粉在24小时以后才开始萌发,完全没有在24小时前萌发的花粉。所以在人工授粉后,五天内尽量不要浇水,以免柱头和花药聚集水分,导致霉菌容易生长,不利于花粉的萌发。 3. 兜兰属植物离体保存技术的研究在4 ℃保存兜兰幼苗,1个月以内取出复壮,恢复的状况比较好,超过1个月,植株就容易受到冻害。15 ℃的条件下,植株长势的缓慢,平均18个月继代1次,可以起到延缓继代周期的目的。通过对兜兰属15个种(含自主杂交品种1种)的离体保存技术的研究,我们制定了兜兰属植物离体保存规范。 4. 分子系统学研究基于ITS序列的数据对兜兰属70个种的研究,结合RAPD对于中国兜兰30个种的研究结果进行分析,支持唐沢耕司(1982)所提出的将兜兰属分为6个亚属的观点,其中产于中国的兜兰属植物可以划分为5个亚属:Sigmatopetalum、Paphiopedilum、Parvisepalum、Brachypetalum和Polyantha。①杏黄兜兰、硬叶兜兰、麻栗坡兜兰、白花兜兰和德氏兜兰为亚属Parvisepalum的成员;②长瓣兜兰和飘带兜兰所在的Pardalopetalum组升为亚属Polyantha;③同色兜兰、文山兜兰和巨瓣兜兰所在的Concolaria组升为亚属Brachypetalum;④Barbata组(即彩云兜兰、紫纹兜兰、胼胝兜兰、卷萼兜兰所在的组)升为亚属Sigmatopetalum;⑤Paphiopedilum亚属包括紫毛兜兰以及其变种包氏兜兰和密毛兜兰、根茎兜兰、波瓣兜兰、小叶兜兰和带叶兜兰等。 9个RAPD引物在中国兜兰属32个个体中总共扩增出产生99条可区分的DNA条带,根据种间的遗传相似系数和遗传距离的结果分析,巧花兜兰与海伦兜兰的遗传距离为0.3466,相似系数为0.7071。RAPD聚类图和ITS树状图显示,这两个种并没有聚成一支,而是远远的分开,并不是一个单系。因此,我们认为巧花兜兰不能并入海伦兜兰中,最多可以作为其变种处理,或者独立成种,隶属于兜兰亚属的Paphiopedilum兜兰组。 与海伦兜兰同样,将文山兜兰归并到巨瓣兜兰或同色兜兰中,也是不合理的。RAPD遗传相似度表明:文山兜兰与同色兜兰的相似系数为0.2384,与巨瓣兜兰的为0.3324,亲缘关系较近。但不至于为同一个种。因此,不支持将文山兜兰并入巨瓣兜兰或者同色兜兰中,可以作为是其变种处理,隶属于Concoloria组。海伦兜兰、根茎兜兰和陈莲兜兰在亚属等级的归并还没有文献报道,在我们基于ITS序列的邻接树和严格一致树以及RAPD聚类图的结果,这几个种都嵌套在兜兰亚属的分支中,因此我们支持将这些种划分入兜兰亚属。
英文摘要: There are about 85 species of Paphiopedilum (Orchidaceae) in the world, while China is one of the countries which harbors the richest Paphiopedilum species, with 29 species. A few species are regarded as very endangered or even extinct in the wild as a result of over-collecting from natural habitats and a large scale of illegal trade. Five species of Paphiopedilum were regarded as Critically Endangered, twelve species as Endangered in Volume 1 of the China Species Red List (2004). The whole genus have been listed in Appendix I by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, Appendices 2008). In the present study, we conducted a series of experiments to establish an efficient and effective protocol for propagating native Chinese Paphiopedilum species in vitro through seed maturity, medium composition, sugar type and organic nutrient additives, the combination of cytokinins and auxin. This work is a start to develop a propagation protocol for mass production. The pollen longevity, and the effect of sugar, calcium and boron on pollen germination of Paphiopedilum species were studied in the present paper. Throughout the study on preservation in vitro of Paphiopedilum species, the Standardization of Paphiopedilum Germplasm Preservation has been developed. We also have tentatively studied and discussed the phylogeny of Paphiopedilum based on the morphological and molecular evidences. The results are summarized as follows: 1. In vitro propagation of Chinese Paphiopedilum species The present study focuses on in vitro propagation of Paphiopedilum, taking four species, P. villosum var. dentissimum, P. bellatulum, P. insigne and P. armeniacum as experiment materials. The P. dentissimum seed germination, protocorm and plant growth was affected by different seed age, media and carbon source and natural complexes addenda: 1) 120 d, 130 d, or 140 d seeds could not germinate; the germination rate increased when maturity period was postponed 10 d intervals and the 200 d seeds reached the highest rate. 2) Germination rate was the highest for KC medium (Knudson C) after 40 d inoculation; protocorm size was the biggest at 100 d for VW medium (Vauin and Went). 3) Glucose was the most effective for germinating; Leaf width was relatively high for glucose, sucrose, and maltose and low for mannitol. 4) Best seed germination was observed on the 10% coconut juice treatment while leaf width was the highest for potato, apple mash treatments and the lowest for chayote mash and LH. Shoot multiplication of four species in Paphiopedilum was influenced by medium with hormone concentration: The combination of BA (6-benzyladenine) 5.0 mg l-1 with NAA (a-Naphthalene acetic acid) 0.5 mg l-1 and BA 1 mg l -1 with NAA 0.5 mg l-1 doubled the number of P. dentissimum and P. insigne plants. For P. bellatulum, BA 5.5 mg l-1 and NAA 0.5 mg l-1 resulted in maximum shoot formation of 0.33 per plant. Shoot formation occurred at all hormone concentration in P. armeniacum, the highest frequency of shoot multiplication occurred in BA 4 mg l-1 and NAA 0.1 mg l-1 treatment (2.0 shoots / node). We also made ♀P. delicatum × ♂P. coccineum, P. micranthum, P. purpuratum, P.concolor, P. villosum, P. villosum var. boxllii, P. appletonianum, P. emersonii, P. dianthum, P. hirsutissimum, P. charlesworthii, P. rhizomatosum, P. henryanum, P. malipoense and P. malipoense var. jackii propagated in vitro successfully though our method. Moreover, 1/4 MS medium + BA 2 mg + NAA 0.2 mg l-1, KT 2 mg + NAA 0.2 mg l-1 made P. malipoense and P. dianthum three times and four times multiplicated in three months respectively. 2. In vitro pollen germination of Chinese Paphiopedilum species The pollen longevity of five Paphiopedilum species was studied: P. gratrixianum, P. villousum, P. dianthum, P. charlesworthii and P. insigne. The pollens which are two-month and three-month old can still germinate on BM media without agar. The germination rate of two months, three months maturity pollens were not different from the rate of 20 days maturity pollens. The pollens could not germinate on the media without sugar, calcium, boron. The tube of pollen needs four or five days to elongate, a few pollen began to germinate after 24 hours, no pollen germinate before 24 hours. Therefore, after artificial-pollination, the stigma and pollen would not be watered, lest become mildewed. 3. Study on in vitro preservation of Chinese Paphiopedilum species It is good for the recovery of Paphiopedilum plantlets when they were preserved less than one month under 4 ℃ preservation. They would hurt at 4 ℃ for more than one month. Under 15 ℃ preservation, the plantlets only need one subgeneration after 18 months resulting from the slowly growing. The plantlets at 15 ℃ may slow down the cycle of subgeneration. Though studying the preservation of 15 species of Paphiopedilum (including one hybrid species we bred by ourselves), the Standardization of Paphiopedilum Germplasm Conservation has been established. 4. Molecular Phylogeny of Paphiopedilum With 39 species nrDNA ITS sequence from Genbank, and by using nrDNA ITS sequence information of our study 31 species (from China), a phylogenetic analysis was carried out for 70 Paphiopedilum species. The results have been analyzed with the results from RAPD molecular marker of 30 Chinese species. It indicated that, we supported Kirasawa’s Paphiopedilum System proposed in 1982 that this genus can be divided into six subgenera. The species from China belong to 5 subgenera: Sigmatopetalum, Paphiopedilum, Parvisepalum, Brachypetalum and Polyantha. These are some re-treatments: 1) P. malipoense and P. armeniacum are included in Subg. Parvisepalum, 2) Sect. Pardalopetalum (P. dianthum and P. parishii) is promoted to be Subg. Polyantha, 3) Subg. Brachypetalum includes P. concolor and P. bellatulum, 4) Sect. Barbata (including P. appletonianum, P. wardii, P. callosum, P. purpuratum) is promoted to be Subg. Sigmatopetalum, and 5) Subg. Paphiopedilum includes P. villosum, P. insigne, P.hirsutissimum, P. henryanum and so on. Nine primers produced 99 polymorphic marker bands in 31 samples from 30 Chinese Paphiopedilum species. According to recent taxonomic treatment, P. delicatum should be included in P. halenae, and P. wenshanense should be in P. conclor. Our results of RAPD genetic identity and genetic distance revealed that the genetic distance between P. delicatum and P. halenae was 0.3466, the genetic identity was 0.7071; and the genetic distance between P. wenshanense and P. conclor was 0.2384. And the dendrogram from RAPD and phylogeny tree from ITS showed P. delicatum and P. halenae was not monophyletic. We therefore suggested P. delicatum should not be included in P. halenae, but an independent species in this genus. And P. wenshanense should not be included in P. concolor, but as a variety within P. concolor. The belongingness of P. helenae, P. rhizomatosum or P. tranlienianum have not been reported yet. Based on our results derived from ITS and RAPD, these species should belong to the subgenus Paphiopedilum for being nested in the branch of the subgenus.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/476
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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