【作者】 哀建国；

【导师】 丁炳扬；

【作者基本信息】 浙江大学 ， 植物学， 2005， 硕士

【摘要】 百山祖冷杉(Abies beshanzuensis M.H.Wu)是百山祖自然保护区的特有植物,为全球最濒危的12种植物之一,属国家一级重点保护野生植物,在植物学、地理学、气候学等学科研究上具有十分重要的意义。本研究采用ISSR分子标记技术对百山祖冷杉种群内个体的遗传多样性进行了研究,用染色法和培养基法作了花粉活力测定和萌发试验,用石蜡切片法进行了胚胎发育过程的观察,用TTC法作了种子活力测定,主要获得以下结果。 1.利用优化反应体系从100个ISSR引物中,筛选出了13个稳定性高、重复性好的引物,对40个百山祖冷杉个体材料的DNA进行正式扩增,共扩增得到91条带,其中39条为多态性条带,多态位点百分率(PPB)为42.86%。百山祖冷杉的3个群体遗传多样性比较分析表明,经人工授粉的实生苗群体(PPB=37.36%)遗传多样性明显高于老树群体(PPB=16.48%)和嫁接树群体(PPB=20.88)。 2.用联苯胺染色法和氯化三苯基四氮唑(TTC)染色法测定百山祖冷杉花粉活力,均未取得满意的结果,改用培养基培养法能得到较为满意的结果。百山祖冷杉花粉在5%蔗糖、20mg/L 硼酸培养基上萌发率最高,萌发率达到78.5%,为其花粉萌发的最适培养基。 3.通过对石蜡切片的观察,表明百山祖冷杉的最佳授粉期在5月中下旬,而雄球花开花是在4月下旬至5月上旬之间。因此百山祖冷杉的确存在雌雄花期不遇现象,在传粉机制上存在生殖障碍。大量的百山祖冷杉胚珠在功能大孢子经有丝分裂形成多细胞结构的过程中,存在败育现象,多细胞结构在进一步发育形成雌配子体的过程中也存在一定量的败育现象。 4.百山祖冷杉人工授粉种子的千粒重为44.73g,自然授粉种子的千粒重为39.38g。t 检验表明,经人工辅助授粉产生的种子与自然授粉产生的种子在种子饱满度方面存在显著差异。经人工授粉有生活力的种子占20.2%,自然授粉有生活力的种子占18.7%。 5.百山祖冷杉对环境变化的适应能力弱,遗传变异低,比其他大种群的物种具有更大的脆弱性。建议采取以老树为主,嫁接树和幼树辅助的取样策略,保存百山祖冷杉的核心种质,以确保当前和未来有足够利用的种质材料得到及时有更多还原

【Abstract】 Abies beshanzuensis M.H.Wu which is only distributed in Baisanzhu natural reserve, Zhejiang province, is one of the twelve most endangered species in the world and it is also a wild plant of national first class focused protection. To study the plant has very important meanings in such disciplines as botany, geography, climatology, etc. Based on ISSR molecule mark technology, the genetic diversity and genetic variation of different individuals within population were determined, and pollen vitalities, embryo development processes and seed vitality were also studied in this paper. The main results were showed as follows:1. Based on optimized reacting system, 13 primers were screened from 100 ISSR primers, with high stability and repeatability. By using these primers, DNA from 40 individual plants were reporduced, as a result, 91 discernible DNA fragments were generated, of which 39(42.86%) was polymorphic bands, indicating low genetic variations within the species. The level of genetic variation in population originated from seedings (PPB=37.36%) was higher than that in population of old trees (PPB= 16.48%) and population of graft-originated (PPB=20.88%).2. The pollen vitality could not be mensured by benzidine staining method and TTC staining method. But it could be mensured by the method of culture medium. The percentage of pollen bourgeon was 78.5% on the culture medium which consisted of sucrose (5%) and boric acid (20mg/L).3. By observing the paraffin sections, it was indicated that the best time of pollination was the middle and last ten days of May. But the blossom of male cones was from the last ten-day of April to the first ten days of May. So there was reproductive disorder in pollination mechanism of male cones and famle cones. A great deal of ovules were abortive during the function megaspores formed the multicellular formations and the multicellular formations formed macrogametophytes.4. The 1000-seed weight of artificial pollination seeds and of the spontaneouspollination seeds of Abies beshanzuensis was 44.73g and 39.38g. The results of t-test showed that there was significant difference of the seed plumpness between the artificial pollination seeds and the spontaneous pollination.The vitality of the seeds through artificial pollination was 20.2%, while the seeds through spontaneous pollination wasl8.7%.5. The adaptablity of Abies beshanzuensis was poor and the genetic variation was low, so it had higher frangibility than other species of great number population. The sampling action strategies that the old trees were main and the graft trees and seedling trees were assistant was advised to conserve the main germplasm . Adequate germplasm should be conserved timely and effectively for study nowdays and in the future. The genetic diversity of seeding trees was high, the technology system for in vitro reproducing of the megasporophylls, seeds and macrogametophytes of seedling trees should be established. The artificial suplementary pollination should be done between old tree and old tree, old tree and graft tree, graft tree and graft tree. Enforcing studies on breeding and raising seedlings, observing environment continuously were advised. The ture reasons that the macrogametophytes aborted should be further studied.更多还原