【作者】 金亮；

【导师】 薛庆中；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2006， 硕士

【摘要】 花药培养技术作为产生变异和加快性状稳定的辅助手段,已在水稻育种上广泛应用。通过花药培养获得的水稻植株中,具有丰富的倍性变异,通常需在成熟期基于形态特征识别单倍体、二倍体及多倍体。花培植株中的单倍体必需进行染色体染色体加倍才能用于育种实践。本研究采用抗微管除草剂Oryzalin（3,5-二硝基N⁴,N⁴-丙基磺胺）与秋水仙碱进行了水稻单倍体加倍效果的比较实验,并探讨了使用流式细胞术（Flow Cytometry,FCM）进行倍性鉴定相关的技术及利用石蜡切片法对不同倍性的花培植株茎解剖结构进行比较研究。其主要结果如下: 1.比较Oryzalin与秋水仙碱对水稻单倍体的染色体加倍效果,设置了三组处理。（1）分别用浓度为1.25mmol/L、2.50mmol/L、5.00mmol/L的秋水仙碱和10μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L的Oryzalin对单倍体植株进行浸根24h加倍处理（以上各溶液均添加2%的二甲基亚砜）;（2）以添加2%的二甲基亚砜（DMSO）的2.50mmol/L的秋水仙碱、50μmol/L Oryzalin溶液分别设置12h、24h、48h处理;（3）在2.50 mmol/L的秋水仙碱、50μmol/L Oryzalin溶液中分别添加0%、1%、2%、4%DMSO进行浸根24h加倍处理。结果发现,对于FC311添加2%的二甲基亚砜的1.25mmol/L秋水仙碱和添加2%的二甲基亚砜的25μmol/L Oryzalin的加倍处理效率最高分别为30.6%和13.9%;而在FC322中,添加2%二甲基亚砜的2.50mmol/L秋水仙碱和添加2%二甲基亚砜的50μmol/L Oryzalin的加倍处理效率最高,分别为25.0%和22.2%。本研究表明秋水仙碱仍是水稻单倍体加倍的最有效的处理剂。 2.采用6种细胞核分离缓冲液制备用于流式细胞术分析使用的水稻细胞核悬液,发现使用Otto buffers和Marie’s nuclear isolation buffer产生的变异系数较小（CV分别为3.57%和3.41%）,其次为Lysis buffer LB01（CV为4.75%）,但Otto buffers的成分及制备的条件最简单。Tris-MgCl₂ buffer产生较多的背景碎片。作者推荐使用Otto buffers制备水稻细胞核悬液。 3.以花药培养获得的粳稻FC311单倍体（2n=x=12）、二倍体（2n=2x=24）、三倍体（2n=3x=36）,以及FC322单倍体（2n=x=12）、二倍体（2n=2x=24）、四倍体（2n=4x=48）植株为材料,利用石蜡切片法研究和分析了它们茎的解剖结构。结果表明,供试材料节间维管束数及大小及、茎壁组织面积、维管束面积基本上随着倍性的增加而增加。而表更多还原

【Abstract】 Anther culture has been demonstrated to be an effective and efficient way for rice breeding. Through rice anther culture several kinds of ploidy plants can be obtained, such as haploid, diploid, polyploid and usually based on the morphology to distinguish their ploidy during maturity. Nonetheless, the chromosome numbers of these haploids must be doubled before its application in rice breeding. The present study comparatively identified the chromosome doubling effectiveness of an antimicrotuble herbicide called Oryzalin with Colchicine. Flow Cytometry （FCM） was used for ploidy identification, and paraffin method was employed to comparatively study the stem anatomical structures in different ploidy plants. The main results are summarized as follows:1. In order to study the chromosome doubling effects of Colchicine and Oryzalin solutions on rice haploids, three groups of experiments have been designed. （1） Roots of haploid seedlings were soaked for 24h using Colchicine solutions with concentration of 1.25mmol/L, 2.50mmol/L, 5.00mmol/L, and Oryzalin with concentration of 10μmol/L, 25μmol/L, 50μmol/L, 100μmol/L, and 200μmol/L respectively（all solutions with 2% dimethyl surtoxide）;（2） Roots of haploid seedlings were soaked for 12 h, 24 h, 48 h, using 2.50mmol/L Colchicines and 50μmol/L Oryzalin sulutions with 2% dimethyl surtoxide （DMSO） respectively;（3） Roots of haploid seedlings were soaked for 24 h using 2.50mmol/L Colchicine and 50μmol/L Oryzalin solutions with 0%, 1%, 2%, and 4% DMSO respectively. The results showed that for FC311, the effectiveness of chromosome doubling with 1.25mmol/L Colchicine and 25μmol/L Oryzalin with 2% DMSO for 24 h respectively was significant compared with other treatments, their doubling rates were reached to 30.6% and 13.9% respectively;whereas for FC322, the significant effectiveness were found to soak roots for 24hrs with 2.50mmol/L Colchicine and 50μmol/L Oryzalin with 2% DMSO （25.0% and 22.2% respectively）, which indicated that Colchicine is still served as the most effective agent for rice haploids chromosome doubling.2. Six different buffers were used to prepare rice nuclei suspensions for flow cytometric analysis. And highly coefficients of variation （CV > 6%） of G_0/1 peaks were found after theanalysis of nuclei isolated by Galbraith’s buffer and Arumuganathan’s buffers;and next was the Lysis buffer LB01, its CV of Go/i peak was 4.75%;whereas the CV of Go/i peaks of Otto buffers and Marie’s nuclear isolation buffer were 3.57% and 3.41% respectively, but the composition and preparation condition of Otto buffers is the simplest of all. In addition, using Tris-MgCk buffer for preparation of rice nuclei suspension, much background debris can occur. Based on these results we introduce that the Otto buffer is better, convenient, and rapid for preparation of rice nuclei suspersions for flow cytometric analysis.3. Using anther culture-derived haploid, diploid, and triploid of FC311and haploid, diploid, and tetraploid of FC322 as materials, the anatomical structure of stems were observed and analyzed by the paraffin method. The results showed a general tendency of increase of ploidy with the increase of the number and size of vascular bundle, areas of stem wall and vascular bundle in internodes of tested materials. However, no obvious correlations were found between ploidy with the layer number and development of collenchyma cell under epiderm and in vascular bundle sheath of outer circle and inner circle, starch grains in ground tissue cells, the ratio of total area of vascular bundle and area of stem wall.4. A rice mutant termed as MTMS temporarily, having characteristics of multiple tilling numbers and male sterile was identified in the anther culture progenies from the Fi hybrid 2S258. Compared with diploid plants, this mutant shows specific characters, such as tall Culm, long leaves, multiple and big panicle, and sterile pollen. After examining its ploidy with FCM, it is found that this mutant belongs to diploid.更多还原