百合的快速繁殖与多倍体新种质的培育

【作者】 王丽艳；

【导师】 梁国鲁；

【作者基本信息】 西南农业大学 ， 细胞生物学， 2004， 硕士

【摘要】 百合属多年生草本植物，具有较高的经济价值，是一种集观赏、食用、药用于一体的花卉，并且是我国卫生部审批通过的首批药食两用植物。本试验在前人研究的基础上，对兰州百合和川百合这两个品种进行了系统的组织培养研究，以期找到最佳的快速繁殖方式，为百合大规模的工厂化生产及国外百合优良品种的引种快繁和品种改良打下基础；同时还在组织培养的基础上结合染色体工程技术对选育百合多倍体新种质的技术和方法进行了研究，以期找到最佳的选育方法，为百合的倍性育种奠定基础，并且获得大量高产优质的同质多倍体新种质可用于生产。 以取自陕西省杨陵的兰州百合、川百合作为试验材料，建立了百合高效的离体再生体系，对外植体不同消毒方式的筛选、外植体类型的选择、培养基的筛选、组培苗生根、移栽等方面作了系统的研究。并成功的在试管内诱导出了百合小鳞茎，弥补了百合组织培养的不足之处，为百合的快速繁殖提供了一条很有价值的新途径。 两种百合离体再生体系培养条件的探索结果表明：获得无菌的外植体是植物组织培养中最基础的一环，因此外植体的灭菌效果和芽诱导率直接影响着试验的进程。筛选出的最佳消毒方式，即以洗涤剂浸泡10min，自来水冲洗30min，75％的乙醇消毒120s，0.1％的升汞消毒12min(升汞中滴加1～2滴2％的吐温—20)，无菌水冲洗4～6次。可以获得90％的芽诱导率，污染率也仅为2％，出芽所需时间为20～27d；诱导芽分化的最适培养基川百合为MS+1.0mg/L6-BA+0.1mg/LNAA+3％蔗糖，兰州百合为MS+1.0mg/L6-BA+0.2mg/LNAA+3％的蔗糖；芽继代增殖培养基川百合宜使用1.0mg/L6-BA+0.5mg/LNAb+5％蔗糖，兰州百合宜使用0.5mg/L-BA+0.1mg/LNAb+5％蔗糖；百合以鳞片为外植体效果较好，鳞片以鳞茎外层鳞片的基部培养效果最好；试管内诱导形成鳞茎的最适培养基兰州百合为1/2MS+0.5mg/LIBA-3％蔗糖，川百合为1/2MS+1.0mg/LIAA+3％蔗糖。两种百合所结鳞茎的部位不同，兰州百合是在基部产生的平均多达3.5个，而川百合是在组培苗顶端产生的，因而每株只能诱导形成1个小鳞茎；百合的生根相对来说较容易，最适生根培养基川百合的为1/2MS+0.5mg/LIhh+2％蔗糖，兰州百合为1/2MS+0.2mg/LIAA+2％蔗糖。生根率均可达到100.0％，平均生根条数川百合多达8.2，兰州百合多达8.0。 在建立百合离体再生体系的基础上，利用秋水仙素溶液处理对兰州百合进行多倍体的诱导，诱导方法采用了浸泡法和混培法，浸泡法又分浸泡无菌试管苗的方式和浸泡鳞瓣的方式。结果表明：两种方法中以浸泡法最佳，浸泡法中以浸泡无菌试管苗的方式效果最好。以0.05％的秋水仙素溶液浸泡处理25h为处理的最佳组合，诱变产生的植株经染色体鉴定，其染色体数目为2n=4X=48，属四倍体植株类型，诱导率最高可达40.5％。 对获得的兰州百合同质四倍体与二倍体进行了外部形态和细胞学的比较观察，结果表明：在同一生长阶段和环境条件下，在叶片形态上，四倍体植株充分体现了器官的巨大性。叶片 西南农业人学映l_学位论文旦旦旦旦旦鱼鱼旦旦鱼旦旦鱼鱼里口的宽度和厚度均人T-一二倍体，厚度为二倍体的l了l%，宽度为二倍体的182%，叶形指数变小，为一倍体的49%，方差分析差异达到极显著水平:川片表面略显粗糙，有时还略呈扭曲状;植株较二倍体粗壮，根系较二倍体的肥大:对获得的四倍体与二倍体进行了气孔特性的比较，结果表明:四倍体保卫细胞明显增人，纵径约为一倍体的162%，横径约为二倍体的151%。保 只细胞内叶绿体数日明显增多，约为二倍体的204%。气孔增人，长度约为二倍体的15既，宽度约为二倍体的138%，而密度卜降，约为二倍体的62%，方差分析差异均达到极显著水平。 对诱导形成的兰州百合同质四倍体与二倍体进行了核型分析，染色体类型均由中部着丝点(m)、近端部着丝点(st)及端部着丝点(t)染色体组成，其核型公式四倍体为Zn二4X二48二12m+4m(SAT)+ZOst+12t，二倍体为 Zn二ZX=24二6m+Zm(SAT)+10st+6t。最长染色体与最短染色体的比值二倍体为2.07、四倍体为2.35，均在2一4之间;平均臂比值二倍体为5.51、四倍体为5.34:臂比大于2的染色体均占染色体组的66.6%，均属“3B”核型。从二倍体与四倍体的核型上看，百合经秋水仙素溶液诱导所产生的四倍体可能是由二倍体直接加倍产生的同源四倍体。 在进行染色体数目检测的同时，我们观察到二倍体与四倍体百合的染色体有的有最高一级结构一螺旋结构出现，其螺旋方向没有规律性，是完全随机的。我们的观察结果符合Rattner提出的有丝分裂中期染色体的最高一级结构一螺旋结构模型。更多还原

【Abstract】 Lily posses the better economical value, is a kind of flower, acting on ornamental, edible and officinal function. And it is the both ornamental and edible plant examined and approved by department of sanitation firstly. Accordingly, In the study, based on the previous research, the systemic tissue culture for the best rapid mode propagation is studied, which can be used for industrial production of the lily on a great scale and fine lily genus abroad rapid propagation and genus improvement; Meanwhile, combining with chromosome engineering, the method and technology on selectively breeding lily germplasm is done for obtaining the optional breeding method.The effective system in vitro regeneration of the lilium davidii var. Unicolor (hoog)Cotton and Lilium davidii Duchautre from yangling of Shanxi is built. And It is done systematically that filtration of sterilizing explants , selecting the type of the explants, sieving the culture medium, and the transplanting and rooting of the tissue plantlets. And the bulb of the lily in the tube is induced successfully, further to compensate the deficiency of tissue culture, and it provides a new method for rapid propagation of lily.The result in the culture in vitro of the two kinds of lily shows that obtained sterile explants is the critical step in plant tissue culture. As a result, the explants sterilizing and the inducing rate of bud affect directly the experiment process. The best sterile method is that 10 min immersing by scour, 30 min scouring by water, 120 min sterilizing by 75% ethanol, 12 min sterilizing by 0.1% hydrargyrum, rinsing 4-6 times by sterilized water. The induced rate is 90%, and the polluted rate is only 2%. The time of shoot formation is 20-27 days, and the result show that the appropriate medium for inducing the differentiation on bud of the Lilium davidii Duchautre is MS+6-BA 1 .0mg/1 +NAA0.5mg/L + sucrose 3%; The appropriate medium for inducing the differentiation on bud of the Lilium davidii var. Unicolor(hoog)Cotton is MS + 6-BA 1 .0mg/1 + MAA 0.2mg/L + sucrose 3%. As for the Lilium davidii Duchautre, the appropriate medium on differentiation and propagation of bud is 6-BA1.0mg/l +NAA0.5mg/L+ sucrose 5%; As for the Lilium davidii var.Unicolor(hoog)Cotton . the appropriate medium on step-generation and propagation of bud is 6-BA0.5mg/l +NAA0.1mg/L+sucrose5%. The result shows that it is the best that lily explants is squama and the base of squama is the better. As for the Lilium davidii var. Unicolor(hoog)Cotton , the optimal medium induced to form the bulb of lily is l/2MS+IBA0.5mg/L; As for Lilium davidii Duchautre, the optimal medium induced to form the bulb of lily is 1/2MS+IAA1.0mg/L the position formed the bulb of the both Lily differed, Lilium davidii var .Unicolor(hoog)Cotton form in the base for more than 3.5 on the average, the Lilium davidii Duchautre form in the top of the cultured plant and the every plant only form one bulb. It is easy to root comparatively, the both optimal medium for rooting is also 1/2MS+IBA0.5mg/L, the rate of rooting is 100%, the number of roots produced by everyplant is more than 8.2. Lilium davidii vor.Unicolor(hoog)Cotton is 8.0.On the base of building the system of regeneration in vitro, The inducing tetraploids of the Lilium davidii var. Unicolor(hoog)Cotton is done by the solution of colchicines. Two methods were adapted: marinating and composite culture. The marinating individe further the marinating of tube seeding and the marinating of bulb. The result show: the marinating is a better method and the marinating of the tube seeding is the best. The autoployploid could be induced by immersed the stems with buds in 0.05% colchicines solution for 25 hours that is prior to culture. The induced seeding is identified by the number of the polyploidy. The number of the chromosome is 2n=4x=48, along to tetraploids plaats. The highest rate of inducing is 40.5%.Plant morphology and cytology observation on two ploidy of Lillian davidii vor.Unicolor . (hoog)Cotton with similar growth stage and en更多还原