【作者】 殷朝珍；

【导师】 葛才林； 王兆龙；

【作者基本信息】 扬州大学 ， 生物物理学， 2006， 硕士

【摘要】 无融合生殖育种,是育种学科的新领域,将使杂种优势向深层次发展。由于其在农业应用上的巨大潜力,近年来吸引了很多科学家的注意。但是目前无融合生殖研究中仍然存在很多问题:例如现有的对无融合生殖遗传机制了解甚少,而植物激素可能对无融合生殖起重要的调控作用,但至今仍未有任何关于激素对无融合生殖调控的较系统的研究。针对这种情况,本课题主要进行了两方面的研究。第一部分是比较草地早熟禾无融合生殖品种、中间兼性品种与有性生殖品种在胚胎发育过程中内源植物激素水平动态变化的差异。第二部分是喷施外源激素来研究激素对多胚苗的诱导效应,从而解析植物激素对无融合生殖的调控机理。试验结果如下:(1)在不同的穗部发育阶段,无融合生殖品种(Moonlight)、中间品种和有性品种(Rite)三个品种的内源激素含量有明显的差异。孕穗时,Moonlight ABA含量明显高于Rite,差异极显著(p﹤0.01);抽穗时明显低于Rite,差异显著(p﹤0.05);抽穗2d、开花时明显高于Rite,差异极显著(p﹤0.01)。在不同发育时期内(除了抽穗50%和开花),Moonlight的t-ZR含量显著高于Rite。抽穗时Moonlight ZR/ ABA显著低于Rite;而小穗减数分裂前后(抽穗2d、开花),Moonlight ZR/ ABA比值显著高于Rite,其值最高达到2.73。中间品种的激素含量和变化趋势介于Moonlight和Rite之间,更倾向于与Rite相似。穗部内源植物激素的ELISA测定和胚囊细胞内植物激素的免疫胶体金定位研究两方面的结果表明:胚囊母细胞发育和减数分裂期是决定无融合生殖途径的关键时期,而此时胚珠或胚囊细胞内高水平的细胞分裂素和低水平的ABA或者高的ZR/ABA可能是不同生殖途径发育的重要调控因子。(2)不同激素或植物生长调节剂(除了GA3以外),对多胚苗的诱导确实存在很好的效果,但不同激素之间(除了GA3)差异不明显,而同种激素不同浓度、不同次数处理之间具有一定的规律性。6-BA、丁酰肼、2,4-D在中等浓度下随着处理次数的增加,诱导的多胚苗率呈现下降的趋势,总体趋势是处理1次、3次>5次,1次和3次之间差异不明显;同一处理次数(处理1次),中浓度6-BA、丁酰肼诱导效果明显高于低、高浓度和对照,低高浓度差异不明显,而2,4-D不同浓度之间差异不显著,同时6-BA早期喷施效果优于晚期,但丁酰肼相反,是晚期喷施效果普遍优于早期;2,4-D+6-BA联合诱导多胚苗效果较好,四个时期均能诱导出较高的多胚苗率,诱导规律与2,4-D单独诱导效应规律相似;100mg/L的GA3不同时期处理对多胚苗率诱导效果均不好,除了抽穗时期多苗率为2.00%,略高于自然发生频率之外,其它时期反而低于对照自然发生频率。就发育时期而言,抽穗100%时期的各个处理(除了GA3),均诱导出了较高的多胚苗率,其效应明显高于其它时期的处理。(3)诱导产生的后代,直接用细胞学方法鉴定其染色体倍性,因草地早熟禾的染色体数目多,染色体较小,难度较大。通过对比测定气孔密度、气孔纵径,可以间接鉴定诱导植株后代的变异。本试验中激素诱导后代的气孔密度、气孔纵径变异系数较大,不同后代之间表现出显著的差异,这可能是与染色体数目发生变异有关。(4)诱导产生的后代,POD同工酶与对照之间有显著的差异。各激素显著诱导了POD同工酶c、d两组酶带的表达,特别是c同工酶的诱导作用更为明显,表现为酶带宽、颜色较深,表明酶的活性很强。但是酶带活性在不同激素、时期之间表现出一定的差异性。表明POD同工酶可以用来鉴定无融合生殖材料。更多还原

【Abstract】 Abstract: Apomixis breeding, a new filed of breeding science, is considered to be a big push to heterosis. In recent years, more and more scientists have been paying attention to apomixis breeding due to its great potential in agriculture application. However, the actual studies on apomixis still have many problems: mechanisms of apomixis are less understood, while it is known that plant hormone may play an important part in regulating apomixis, unfortunately there is no systematic research has done about plant hormone regulation on apomixis. So our study mainly concentrates on two parts. The first is, comparing the difference between apomixis cultivars, medial cultivars and sexual cultivars of Kentucky bluegrass in term of the dynamic change of auxin level in different developing stages. The second is focused on inductive effect of auxin on poly-embryonic seedlings by spraying plant growth regulators, from which we try to demonstrate the mechanism of plant hormone regulation on apomixis. The principal results are as follows:(1)Changes of plant hormone contents were different in the fringe of three varieties at different developing stages. Content of ABA in apomixis cultivar is significantly higher than that in sexual cultivars at booting stage,two days after heading and anthesis, hower it is much lower at heading stage. Content of ZR in apomixis cultivar is significantly higher than that in sexual cultivar at different developing stages, except half of heading stage and anthesis. The content of ZR/ ABA was significant lower than that in Rite at heading period, while before or after meiosis, the number were much higher than that in Rite. The trend of changes of auxin level in.the middle cultivar is between apomixis cultivar and sexual cultivar, and more similar to the sexual. Content of auxin by ELISA in fringe and immuno-colloidal gold research both suggested: megaspore mother cell and meiosis stages are critical stages of apomixis, and higher ZR content, lower ABA content or higher ZR/ABA in ovule and megaspore may be important regulating factors of apomixis.(2) Different plant growth regulators (except Gibberellic acid (GA3)) had good effects on the inducement of poly-embryo seedlings, but effects were not obvious (except GA3) among different plant growth regulators, however spraying the same plant growth regulator, with different concentrations and times had certain regularities.①6-Benzyladenine(6-BA), daminozide(B9) 2,4-Dichlorophenoxyacetic acid (2,4-D) in medium concentration, the inducement rate of poly-embryo seedlings decreased with increasing spraying times ,in general, spraying one time or 3 times was better than 5 times, but the difference between spraying one time and 3 times was not significant;②With same spraying time, the rate of poly-embryo seedlings with medium concen- tration of 6-BA was significant higher than with lower, higher concentration and CK. 6-BA sprayed at earlier stages was better than that at later stages, but the result was opposite with B9 ; While treatment of 2,4-D was not significant different among lower, medium and higher concentration.③Combination of 6-BA and 2,4-D had good effect at four developing stages, and the regularity was similar to the inducement of single 2,4-D;④100mg/L GA3 sprayed one time were not good at four developing stages and all the treatments were lower than CK except sprayed at heading stage, which the poly-embryo seedlings was 2.00%, a little higher than CK. As for developing stages, the effects of sprayed at heading stage were better than others.(3) It is difficult to identify the induced progeny directly because there are too many chromosomes in kentucky bluegrass, so we utilized the longer diameter and density of leaf stoma to identify them directly. Our results showed: the varation of induced progeny we got were much more than CK, and different progeny were remarkable distinct, this maybe the results of variation of chromosomes.(4) POD isozymes in leaf of induced progeny were remarkable distinct from CK. Our results showed: all plant growth regulators could induced the expres– sion of c,d POD isozymes ,especially c isozymes was much more notable, indicating that activity of POD isozymes was remarkable increased. But the activities of POD isozymes with different plant growth regulators and developing stages were different. These results suggested that POD isozymes of the progeny varied to CK, so it can be used for identifying apomixis materials.更多还原