【作者】 陈智慧；

【导师】 姚家玲；

【作者基本信息】 华中农业大学 ， 植物学， 2004， 硕士

【摘要】 禾本科植物龙须草(Eulaliopsis binata)具有无融合生殖现象，本研究以两种不同生态型龙须草窄叶型和红杆型为材料，利用去雄、杂交等田间遗传学实验方法，通过花粉活力检测，传粉后花粉粒和柱头的荧光染色观察等，并利用流式细胞仪检测成熟种子内胚和胚乳细胞的相对倍性，采用种子萌发的根尖进行常规染色体压片，观察龙须草细胞染色体数目和倍性水平，进一步从遗传学和细胞学角度研究龙须草的无融合生殖特性，评价供试龙须草的无融合生殖程度，为研究龙须草无融合生殖遗传规律打下基础。 主要研究结果如下： 1．窄叶型龙须草去雄套袋平均结实率为12.2％，杂交(窄叶型龙须草为母本，红杆型龙须草为父本)平均结实率为11.23％，去柱头平均结实率为10.78％，去雄对照平均结实率为12.38％；红杆型龙须草去雄套袋平均结实率为9.38％，杂交(红杆型龙须草为母本，窄叶型龙须草为父本)平均结实率为8.88％，去柱头平均结实率为9.29％，去雄对照平均结实率为9.13％。经过差异显著性方差分析表明：窄叶型龙须草各种处理的结实率之间没有显著差异，花粉对龙须草的结实率没有显著影响；红杆型龙须草各种处理的结实率之间也没有显著差异，花粉对龙须草的结实率没有显著影响； 2．对龙须草散粉当天的花粉生活力的测定表明(用蔗糖溶液对花粉进行离体萌发)：窄叶型龙须草花粉萌发率达到28.1％，红杆型龙须草花粉萌发率达到34.13％，两者均具有正常的活性；红杆型龙须草花粉经干燥后低温保存(4℃)2天的花粉萌发率为24.17％，四天后萌发率为15.31％，保存6天后其萌发率仍有10.98％。 3．传粉后数小时(1-5)的雌蕊整体装片，经过苯胺蓝染色处理，分别在荧光显微镜和普通显微镜上观察，结果表明：花粉在柱头上的附着率不高，柱头上可以观察到少量正常萌发的花粉和生长的花粉管，荧光显微镜下，花粉管发出较强的荧龙须草无融合生殖特性的研究光;传粉后5小时的花粉管在柱头表面伸长缠绕，多数不进入柱头和花柱;但观察到(5%的花粉管能进入柱头毛状细胞。 4.采用Partec流式细胞仪检测两种生态型龙须草的成熟种子，以种子萌发苗的叶片做对照，设定其细胞内的C值为ZC，检测结果表明:窄叶型龙须草种子(每次被检样品中含15一20颗磨碎的种子，混合检测，每个样品重复三次)出现3个峰值，其比例为卜1.48:1.9，其种子细胞内的相对C值约等于ZC:3C:4C;红杆型龙须草种子出现2个峰值，其比例为1:1.9，即种子内细胞的相对C值约等于ZC:4C。流式细胞仪检测结果显示，红杆型和窄叶型龙须草种子中胚和胚乳细胞的相对C值具有与有性生殖植物不同的水平:两种生态型龙须草的无融合生殖程度有差异。 5.建立了适宜于龙须草根尖压片的实验技术:龙须草种子萌发的适宜基质是河沙，根尖细胞分裂旺盛期是上午9点半至n点，下午4一6点;用对二氯苯饱和溶液+Q一嗅代蔡前处理4h;盐酸解离时间以15一25min为宜;改良的卡宝品红染色10一15min。龙须草种子细胞染色体数目为36条。 本课题从遗传学、细胞学角度进一步研究证实了龙须草具有无融合生殖特性，两种生态型龙须草的种子形成过程不依赖于花粉，种子中胚和胚乳都可以自发形成:两种生态型龙须草具有不同的无融合生殖程度，其中红杆型龙须草具有高度无融合生殖特性，其种子的形成没有雄性配子的参与，胚和胚乳细胞的相对C值为ZC:4C，窄叶型龙须草为兼性无融合生殖类型，其胚和胚乳细胞的相对C值为ZC:3C:4C;龙须草花粉具有活力和正常萌发率，可以在无融合生殖工程中作为花粉供体;龙须草种子根尖细胞的染色体数目为36条.更多还原

【Abstract】 Eulaliopsis binata is a valuable apomictic resource in Graminea. Two ecotypes of E. binata with different leaf shape were artificially or open pollinated after emasculation to investigate their apomixis degree and genetic pathway. Pollen vigor was tested by germination in vitro; In vivo germination on stigma were observed by fluorescence staining method. The relative DNA content and ploidy of the embryo and endosperm cell in ripe seed were examined by Flow Cytometry. Chromosome number of root tips were calculated. The results are as follows:1 In slender-leaf type of E. binata, the seed setting in treatment of no pollination (bagging after emasculation) reached 12.2%, while that in treatment of cross-pollination was 11.23%, and in another treatment of no pollination (removal of stigma) was 10.78%. In treatment of open pollination after emasculation the seed setting was 12.38%; In red type E. binata, the seed setting of emasculation by bagging was 9.38%, the seed setting of cross-pollination was 8.88%, the seed setting of removal of stigma was 9.29%, the seed setting of emasculation comparison was 9.13%; Variances analysis suggest that there are no significant difference among the seed setting of emasculation by bagging, cross-pollination and removal of stigma, respectively as compared with control in both ecotypes.2 Fresh pollen grains were germinated in sugar solution. The pollen germination rate of slender leaf type E. binata is up to 28.1%, the pollen germination rate of red pole type E. binata is up to 34.13%, the both have normal activity; Vigor of pollen of red-haulm type E. binata that was kept at low temperature (4癈) after dryness is 24.17% after two days, the vigor is 15.31% after four days, the vigor is 10.98% after.keeping for six days.3 Pistil was stained with aniline blue at l-5h after pollinating, then wholly observed on,fluorescence microscope and ordinary microscope respectively. The result indicated the pollens of E. binata had poor adherences to stigma. Though pollen grains could germinate on stigma but the pollen tube could not stretch into stigma further more. After 5h, the pollen tubes prolonged and twisted on the stigma surface and rare pollen tubes (<5%) could enter stigma and style.4 Using leaves of seedling as control, C values of seeds in two ecotypes E. binata were screened by Partec Flow Cytometry. The observed three peaks in seeds of slender leaf type revealed relative three types of C value as 2C:3C:4C. In red-haulm type, only two peaks were screened indicated two types of C value in 2C:4C. These results were quite different from normal sexual plant in which the ripe seeds produce two relative C value as 2C:3C, standing for zygote (2n) and endosperm (3n)..5 The methods of chromosome identify of root tip of E. binata: the suitable medium of seed germination is river sand, the best time for root tip cell division are am 9-11 and pm 4-6; the pre-treatment need 4hrs in p-Dichlorobenzene saturated solution + a -Bromonaphthalene, it is good with 15-25min that using the hydrochloric acid to solve roots; The dyeing time of improved fuchsin need 10-15 min. The numbers of chromosome of E. binata seed is 36.Above results provides evidences in genetics> cytology and seed ploidy to support the opinion that E. binata is an apomictic plant in Graminea. The seed could be produced without pollination and the embryo and endosperm could develop autonomously. Two kinds of ecotype E. binata have different apomixis degree. The red-haulm type had highly apomixis characteristic, its seed formed without male gamete participation, and its embryo and endosperm’s C value is 2C:4C. The slender leaf type should be facultative apomixis.Its embryo and endosperm’s relative C value is 2C:3C:4C. E. binata possess vigorous pollen which could germinate normally and might act as a potential valuable donor for apomixis gene. The chromosome number of E. binata is 36.更多还原