【作者】 汤飞宇；

【导师】 王国英；

【作者基本信息】 中国农业大学 ， 遗传学， 2004， 博士

【摘要】 玉米是重要的粮食和饲料作物之一。玉米育种的核心是自交系的选育，常规条件下选育自交系需要4-6年时间，消耗大量的人力物力。通过离体诱导雄核或雌核发育则可以快速获得纯系，大大缩短了育种时间，提高了育种效率。本研究的目的之一是建立一套有效的诱导玉米离体雌核发育的技术体系，探讨离体雌核发育的机理。研究了不同授粉处理的玉米子房离体诱导单倍体的效果，比较了不同染色体加倍方法的加倍效果，利用SSR分子标记对子房培养诱导的植株进行了遗传分析。所获结果如下： 1．玉米子房培养胚诱导培养基为N₆+2.4-D 3.0mg/L+CH 500mg/L+Pro 690mg/L+AgNO₃10mg／L+Sugar3％-12％；胚分化培养基为MS+KT 0-1mg／L+CH 500mg／L+Pro 690mg／L+Sugar3％-6％；再生植株壮根培养基为1／2MS+NAA0.5mg/L+Sugar1.5％-2％。 2．低温预处理（5℃）有助于玉米子房胚胎的发生。当授粉时间为15、17小时，未经低温处理的子房不会产生胚胎，反之则产生胚胎，当授粉时间为15小时，5℃冷藏60小时出胚率为3.33％，当授粉时间为17小时，5℃冷藏24小时，出胚率为1.55％。 3．授粉处理的时间长度是影响诱导效果的重要因素。当授粉时间为15-22小时均可以诱导获得单倍体，其中诱导率最高的是19小时，诱导率为0.2％。但对于温室材料，授粉时间应延长至26-30小时，其中诱导率最高是29小时，单倍体诱导率为0.18％。 4．白花授粉处理的效果好于异花授粉．在异花授粉条件下，诱导单倍体所需的授粉时间宜短，在自花授粉条件下，诱导单倍体所需的时间宜长。 5．不同季节授粉处理的效果存在差异。春秋两季授粉处理的效果好于夏季。春季授粉处理的单倍体的平均诱导率为0.109％，秋季为0.105％，夏季为0.058％。 6．在两年时间内共获得玉米子房再生植株281棵，经根尖压片检查染色体数目，有24棵为单倍体植株，其中2棵被加倍成功，另有一棵单倍体植株自发散粉。 7．利用20对在两亲本（178，黄C）之间表现多态性的SSR引物（每条染色体两对）对部分非单倍体再生植株进行了遗传分析，其中一个株系在大田生长条件下，株高、株型、穗位等诸多农艺性状表现出整齐一致，SSR检测结果表明：该株系在所检测的20个位点上均表现纯合，其中11个位点与黄C一致，其他9个位点与178一致。从而断定该株系应当是一个自发加倍的双单倍体。 8．对一个自发散粉的单倍体株系也进行了SSR检测，在所检测的20个位点上同样表现纯合，其中15个位点与黄C一致，其他5个位点与178一致。 以上结果证明了利用玉米授粉子房来离体诱导孤雌生殖是可行的。更多还原

【Abstract】 Maize (Zea mays L.) is one of the most important cereal crops, being used both as human food and animal feed. Usually it may take 4-6 years to breed a pure line by using traditional breeding method. But, haploids production via in vitro androgenesis or in vitro gygogenesis can accelerate the process and enhance breeding efficiency. In maize, in vitro-produced haploids can save 2-3 years. The investigations were aimed at establishing an effective protocol for in vitro production of maize haploids, and understanding the mechanism of in vitro gygogenesis. The effect of various pollination treatment for maize ovaries on haploid production was investigated, including duration of pollination treatment, mode of pollination, season of pollination, et al. Two different chromosome doubling techniques was compared. The plants derived from maize ovaries were subjected to genetic analysis using SSR molecular marker. The results were as follows:1. Embryo induction medium in maize ovary culture was N6 basal medium, supplemented with 3.0mg/L 2.4-D, 500mg/L casein hydrolysate, 690mg/L L-proline, 30g/L-120g/L sucrose , 10mg/L silver nitrate. Embryo differentiation medium was MS basal medium, contented with 0-1mg/L KT, 500mg/L, 690mg/L L-proline, 30g/L-60g/L sucrose. Medium for rooting was 1:1 dilute MS salts, MS organic compounds, enriched with 0.5mg/L NAA, 15-20g/L sucrose.2. Cold pre-treatment led to increased embryogenesis in maize. Ovaries without cold pre-treatment failed to produce embryo. On the contrary embryogenesis occurred. The embryo induction frequency was 3.33% in ovaries with 15 hpp after cold pre-treatment for 60 hours at 5C. The embryo production ratio was 1.55% in ovaries with 17 hpp after cold pre-treatment for 24 hours at 5C.3. Hpp was an important factor affecting induction result. Haploid plants can be obtained from pollinated ovaries with hpp ranged from 15 hours to 22 hours . The highest induction frequency was 02.%, occurred in ovaries with 19 hpp. However, for ovaries growing in the greenhouse, the hpp should be prolonged to 26-30 hours for haploid induction. The highest was 0.18%, occurred in ovaries with 29 hpp.4. Better result achieved in self- pollination than cross-pollination. The hpp for cross-pollination was found to be shorter than the one for self-pollination for in vitro gynogenesis.5. Gygogenesis efficiency related to pollination season. The ovaries collected in spring (February, March) and autumn (September, October) were more responsive to in vitro gygogenesis than ones in summer (July). The best result occurred in spring (0.109%). The second in autumn (0.105%). The poor result obtained in summer (0.058%).6. A total of 281 plantlets was obtained in two years. Among of them, 24 were determined to be haploids after chromosome counting. Two haploids were diploidized successfully after applying colchicines. One haploid shed pollen spontaneously and set seed.7. Twenty different SSR primers showing polymorphism between 178 and Huang C were used to genetic analysis for .diploid plants from maize ovaries. A R1 generation plant , whose progenyrevealed a high level of homogeneity for many agromorphological traits such as : plant height, leaf shape, the location of ear, etc, was homozygous at 20 loci ( 2 loci per chromosome), showing paternal ( Huang C ) bandings at 11 loci and maternal (178 ) bandings at the other 9 loci. It was concluded that the R1 diploid plant originated from an unfertilized haploid cell.8. The haploid shed pollen spontaneously was also subject to be genetic analysis using SSR molecular markers. SSR analysis revealed that it was homozygous at 20 loci, showing paternal (Huang C ) bandings at 15 loci and maternal (178) bandings at the other 5 loci.The above results demonstrated the it is feasible to induce maize gygogenesis by in vitro culture of pollinated ovaries.更多还原