【作者】 周群；

【导师】 陈家春；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 湖北麦冬Liriope spicata（Thunb．）Lour．var．prolifera Y．T．Ma是中药“山麦冬”的主要基原植物，药用其块根，具有养阴生津，润肺清心之功效，它是常用中药麦冬的主流品种和湖北省的道地药材。湖北麦冬开花不结实，在种植生产中只能行无性繁殖。由于长期无性繁殖，种质呈明显的退化趋势，严重影响其产量和质量。本文利用本草学、植物形态学、细胞生物学、植物胚胎学、生药学和分子生物学等多学科的方法和技术对湖北麦冬的资源品质与不育机理进行研究，为优化种质及品种改良提供理论依据和应用基础，对于提高湖北麦冬的产量和质量，提升湖北麦冬产业，具有重要的理论和实际意义。主要的研究成果如下：1湖北麦冬的本草学、生药学及RAPD分子标记鉴定研究。本草学考证结果表明，麦冬药用历史悠久，而宋代《图经本草》及《重修政和经史证类备用本草》收载的“随州麦门冬”似山麦冬（Liriope）属植物，其形态、产地与现今药用的湖北麦冬基本一致。对湖北襄樊GAP基地生产的湖北麦冬进行生药学研究，补充和完善了湖北麦冬的品种鉴别标准。结果表明，湖北麦冬与其它麦冬来源植物在形态学及细胞学上均存在一定差异。可以通过花丝是否明显，花药锐头或钝头，子房的位置，地下走茎的有无，花葶长度及花序在花后是否长叶簇或小苗等形态学特征将麦冬、湖北麦冬、山麦冬和短葶山麦冬区别开来。根据块根横切面韧皮部束数目的多少易于区别麦冬和湖北麦冬。采用随机扩增多态DNA（RAPD）技术首次从DNA水平检测和分析了不同栽培措施处理的湖北麦冬、山麦冬、恩施产麦冬、杭麦冬和川麦冬的亲缘关系和遗传多样性，用SPSS软件对扩增片断的相关性进行聚类分析。从100个随机引物中筛选出10个扩增条带清晰、稳定、重现性好和多态性明显的引物，10个引物共扩增出75个条带，其中多态性条带58个，占总扩增条带的77.3％。湖北麦冬不同栽培条件和组织培养植株的相关系数均接近1。湖北麦冬与山麦冬的相关系数在0.74～0.88之间。麦冬（杭麦冬和川麦冬）与湖北麦冬和与山麦冬之间的相关系数分别为0.41～0.66和0.48～0.69。杭麦冬与川麦冬的相关系数为0.74。不同的山麦冬样品相关系数均在0.8以上。结果表明，22种麦冬类药用植物之间的亲缘关系与形态分类结果基本一致，组织培养和栽培措施对遗传关系影响不大。但产于不同地区（四川、杭州、湖北恩施）的麦冬间具有明显的遗传差异。2湖北麦冬的传粉生物学及交配系统与其不育的关系研究。考察了湖北麦冬花的形态特征、花期、花粉与胚珠比、花粉活力、柱头可授性及传粉媒介，结果表明湖北麦冬花期为6月下旬至8月中下旬，单花花期1 d，09：20左右开花，16：30左右关闭。自然状态下不存在风媒花粉流，为虫媒传粉。单花花期内柱头都具有过氧化氢酶活性，开花后1～2 h为可授期。花粉计数为25 778±9 682，花期内散播出的花粉没有活力。湖北麦冬花粉的畸形率为95.55％（2 066个）。湖北麦冬花粉与胚珠比Np／No为4 296±1 614，为专性异交（Obligate xenogamy）的交配系统。结论：花粉畸形率高无活力且花粉数相对稀少，为湖北麦冬不育的生殖生物学原因。3湖北麦冬染色体核型研究。研究结果表明，湖北麦冬为三倍体植物，其核型公式为2n=3x=54=30m+24sm（1SAT），遗传进化上属2B型。一般认为，三倍体具有自然不育性，提示湖北麦冬“花而不实”可能是其三倍体所致。湖北麦冬核型为首次报道。我们的研究结果支持湖北麦冬不应为山麦冬的变种，而应为两个种的观点。另外，本研究推测三倍体湖北麦冬的来源可能是二倍体与四倍体自然杂交后形成的。结论：染色体核型为三倍体是湖北麦冬不育的细胞遗传学原因。4湖北麦冬花粉发育与小孢子母细胞减数分裂行为的观察。采用石蜡制片显微镜下观察湖北麦冬花药和花粉发育过程，常规压片显微镜下观察小孢子母细胞减数分裂行为。湖北麦冬小孢子母细胞减数分裂过程中染色体异常行为多表现为：中期出现单个染色体游离在纺锤体外面，在后期出现染色体桥和落后染色体，在末期出现单个染色体游离在细胞核外形成微核的现象。花粉发育异常，产生大量畸形花粉。花药绒毡层在减数分裂早期已大部分解体退化，绒毡层细胞液泡化膨大，单核小孢子败育，花粉粒畸形。少数成熟花粉为2-细胞型。结论：减数分裂过程中高频率的染色体异常行为是引起小孢子的败育，从而导致湖北麦冬不育的原因。这些异常现象也支持湖北麦冬为三倍体植物的观点。绒毡层发育异常和提前退化是湖北麦冬花粉败育的重要原因。5采用湖北麦冬的花药为外植体离体培养再生植株。以湖北麦冬花药为外植体，采用MS培养基，附加不同的植物激素进行实验。常规压片通过显微镜进行再生植株染色体的计数分析。结果表明，MS+2，4-D 1.0mg·L^-1+KT 2.0 mg·L^-1诱导胚性愈伤组织效果最好，愈伤组织诱导率可达41.07％。MS+6-BA 1.5～2.0 mg·L^-1+NAA 0.1～0.3 mg·L^-1适于不定芽的诱导，不定芽转入附加NAA 0.1～0.3 mg·L^-1的1／2 MS的生根培养基上，生根后获得完整的再生植株。再生植株为体细胞起源。4℃低温预处理对湖北麦冬花药培养的影响不显著。湖北麦冬种子和芽经该体系培养，愈伤组织诱导率高，并可获得再生植株。结论：本研究建立了湖北麦冬花药培养体系和快速繁殖途径。为进一步采用细胞和基因工程技术改良湖北麦冬奠定了良好基础。更多还原

【Abstract】 Liriope spicata （Thunb.） Lour. var. prolifera Y. T. Ma is one main original plants ofthe traditional Chinese materia medica "Shanmaidong". Its tuberous roots are usedmedicinally, with effects of nourishing Yin to promote the production of body fluid andmoistening the lung to clear away heart-fire. L. spicata var. prolifera is one geoherb fromHubei province and the main variety of common traditional Chinese materia medica"Maidong". It has no seeds after efflorescence and reproduces only by vegetativepropagation. Because of long-term vegetative propagation, its germplasm degenerated andproduction and quality were influenced severely. In this paper the resource-quality and themechanism of sterility were studied by using the method of bencaology, plant morphology,cell biology, plant embryology, pharmacognosy and molecular biology. It providesavailable data and theoretical foundation for breeding. Our findings have importantpractical significance for increasing production and quality and raising industry of L.spicata var. prolifera. The results were as following.1 Studies on bencaology, pharmacognosy and RAPD analysis of L. spicata var.prolifera.The research on bencaology indicated that Chinese materia medica "Maidong" wasused medicinally for long history. Suizhou Maidong was recorded in Classified MateriaMedica for Emergency in Song Dynasty, which was similar with plants of the genusLiriope. Its shapes and place of produce were consistent with L. spicata var. prolifera.In order to provide the scientific evidence for identification and estimation, L. spicatavar. prolifera from Xiangfan city, Hubei province was studied in pharmacognosy. Theresults show that there exists certain difference in morphology and cytology between L.spicata var. prolifera and other original plants of "Maidong". L. spicata var. prolifera, L.spicata, Ophiopogon japonicus and L. muscari Bailey can be distinguished by lengths offilaments of flowers, place of ovary, blunt-end or sharp-end anthers, lenth of scape and littlebuds after efflorescency. On the other hand, L. spicata var. prolifera and O. japonicus canbe distinguished by the number of phloem bunch in cross sections of root tubers.To analyze the genetic relationships and diversity, random amplified polymorphic DNA （RAPD） was applied to germplasm accessions in 22 cultivated and wild medicinalbotanic samples of L. spicata var. prolifera, L. spicata and O. japonicus. 10 random primersof high stable quality were screened from 100 primers. 10 arbitrary primers produced 75distinctive bands in total and 58 of them were found to be polymorphic, which accountedfor 77.3%. Cluster analysis showed that histological culture and cultivated methods couldn’tobviously affect the genetic relationships and there are obvious genetic differences amongOphiopogon japonicus in the different areas of zhejiang, Sichuan and Hubei provinces. Thephylogenetic relationships of them are basically accordant with the results of classical taxology.2 The relation between pollination biology and sterility of L. spicata var. prolifera was studied.The morphological character, florescence, pollen viability, stigma receptivity,pollen-ovule ratio and pollinating agent of flower of L. spicata var. prolifera were estimated.L. spicata var. prolifera bloomed from late June to early August. The anthesis was only oneday. It bloomed at around 09:20 and finished at around 16:30. Wind pollination did notexist in this area. The flowers of this species were pollinated by insects. The stigma of L.spicata var. prolifera could only be receptive 1 to 2 hours after blooming, although stigmahad catalase activity in the whole anthesis. Pollen in the anthesis had no viability and theiraberration rate was 95.55%. The pollen-ovule ratio, ranging from 4 296±1 614, reflected asan obligate xenogamy system. Conclusion: Its high aberration rate, pollen without viabilityand low pollen number were sterility of pollination biology.3 The karyotype of L. spicata var. prolifera was examined.The results are as follow: L. spicata var. prolifera is a triploid plant. The karyotypeformula is 2n=3x=54=30m+24sm（1SAT）, which belongs to symmetrical 2B type. Becauseof triploid plants being naturally sterile, the sterility of L. spicata var. prolifera was inducedby its triploid. Karyotype of L. spicata var. prolifera was reported for the first time. Theresults agree with the views treating them as separate species. In addition, our findingsindicate that L. spicata var. prolifera might originate from hybridization between tetraploidand diploid species. Conclusion: the discovery of triploid L. spicata var. prolifera revealedthe cytogenetic mechanism of its sterility.4 Meiotic behavior and pollen development of L. spicata var. prolifera were describedin detail for the first time. A study on anther and pollen development process was carried out in L. spicata var.prolifera by means of paraffin section under a microscope. Meiotic behavior was alsoobserved. During the meiosis of microspore mother cells （MMC）, most of cells displayedabnormally. The abnormity was showed that some chromosomes or their fragments movedout of the spindle of the cell at metaphase. Some chromosome bridges, fragments andlagging ones were formed at anaphase, and many microspores displayed the micronucleusat the telophase. The pollen development was abnormal and with high aberration rate ofpollen. The degradation of the tapetum was taken place at the early time of meiosis stageand tapetum cells inflated like vacuolization. The mononuclear pollens were abortive,which eventually formed abnormal pollens.Conclusion: These results indicate that someabnormalities of meiotic behavior and pollen development are significant reasons inducingmicrospore abortion during its development. It also supports the viewpoint that L. spicatavar. prolifera belongs to a triploid plant. The pollen abortion of L. spicata var. prolifera isassociated with tapetum abnormalities and degradation in advance.5 To study the technique of the callus induction from anther and plant regeneration ofL. spicata var. prolifera.Callus was induced from anther of L. spicata var. prolifera on a MS mediumsupplemented with different hormones. The squash methods combined with a microscopewere used to analyse chromosomes of regenerated plantlets. Result: MS+2, 4-D 1.0mg·L^-1+KT 2.0 mg·L^-1 gave the highest induction ratio which was 41.07%. MS+6-BA1.5-2.0 mg·L^-1 +NAA 0.1-0.3 mg·L^-1 was suitable for the induction and proliferation ofindefinite buds. The buds were transferred to 1 / 2 MS medium supplemented with NAA0.1-0.3 mg·L^-1 for rooting. The shoots produced roots of culture and formed completeplantlets. The regenerated plantlets originated from somatic cells. At the same time, theeffects of pretreatment of low temperature at 4℃on the callus induction were studied anddiscussed. Conclusion: This paper sets up the method of tissue culture of anthersomatic-cells and intermediate propagation of L. spicata var. prolifera.更多还原