【作者】 王晓云；

【导师】 曹翠玲； 张锡梅； 杜胜利；

【作者基本信息】 西北农林科技大学 ， 植物学， 2004， 硕士

【摘要】 离体雌核发育是单倍体诱导的主要途径之一，有关离体雌核发育方面的生理生化机制的研究报道尚少。天津科润黄瓜研究所在国内首次通过黄瓜未受精子房培养获得单倍体植株，建立了高效、稳定的黄瓜单倍体诱导体系。本试验在前期有关离体黄瓜雌核发育形态学和生理生化研究的基础上，以诱导离体雌核发育的 M99 培养基上和非雌核发育的 W5 培养基上培养的黄瓜未受精子房为试材，采用生物化学技术和组织化学技术，深入研究了离体雌核发育早期外植体内的标志酶——过氧化物酶及其同工酶的活性及分布的变化、可溶性蛋白质的含量和总蛋白质分布的变化及核酸和多糖（淀粉）分布的变化，为揭示离体雌核发育早期的细胞分化机制提供科学依据。试验结果如下： 1. 研究了过氧化物酶同工酶在两种培养基上的外植体内的变化，结果发现，在培养的 1-9 天，在可发生离体雌核发育的 M99 培养基上的黄瓜未受精子房内，总的 POD和 NADHPOD、GSH-Px 两种同工酶活性均比 W5 上的外植体内酶活性高，COX，ASP活性较 W5 上的低。培养在 M99 上的外植体内 IAAO 活性一直保持较高的水平，而 W5上的 IAAO 活性在培养前 5 天略高于 M99，培养 5 天后则急剧下降。 2. 培养在 M99 上的黄瓜未受精子房内，在培养的第 0~2 天过氧化物酶主要分布于珠被和珠心组织的细胞质中。子房的薄壁组织随着培养天数的增加染色加深，POD 主要分布于其细胞壁及邻近的区域。培养 4 天后 POD 在胚珠内整个细胞都有分布，而薄壁组织的细胞壁中 POD 分布较多；在 W5 上的外植体内，在培养的 0~4 天一直以胚珠，尤其珠被细胞较周围的薄壁细胞分布多，发现培养 4 天时珠被细胞开始增生，增生细胞的细胞壁及邻近区域染色。 3. 在培养的前 4 天，M99 和 W5 两种培养基上的外植体内可溶性蛋白的含量变化略有上升，培养 4 天后，M99 上的外植体内可溶性蛋白含量呈下降趋势，而 W5 上的则呈上升趋势；总蛋白质在 M99 上以胚珠内分布一直较多而薄壁组织中分布减少，W5 上的胚珠与薄壁组织中蛋白质分布都较多，随着培养天数的增加，胚珠与薄壁组织的细胞染色都加深，细胞壁着色明显。 4. M99 上的外植体，胚珠内尤其是珠心组织中 DNA、RNA 含量增加，薄壁细胞内DNA 含量逐渐减少；胚珠细胞内有很小的淀粉颗粒出现，且细胞壁很薄，而薄壁组织的细胞内没有淀粉粒出现。在 W5 上的外植体内胚珠和薄壁组织细胞的 DNA、RNA 都增加，第 6-9 天，增生的细胞 DNA 含量减少；薄壁组织中多糖含量一直增加，并且发现增生的细胞内布满了大的淀粉粒。更多还原

【Abstract】 In vitro gynogenesis is one of the major methods for haploid production. There isfew report about physiological and biochemical changes of cucumber in vitro gynogenesis.An efficient and stable system for cucumber haploid production via in vitro gynogenesis wasestablished in Tianjin Keneil Cucumber Research Institute. On the basis of this system andpreviously morphological, physiological and biochemical studies on cucumber in vitrogynogenesis. Materials from both typical gynogenesis using M99 as induction medium andnon gynogenesis using W5 as induction medium were collected. The activities anddistribution of peroxidase and its isoenzymes, content of soluble protein and distribution of allprotein during early stage of cucumber in vitro gynogenesis and non gynogenesis wereinvestigated by biochemistry and histochemistry technology. Nucleic acid and amylose werealso studied by histochemistry. These researches aimed to give a probative value for celldifferentiation mechanism during the early stage of cucumber in vitro gynogenesis. The results showed that total peroxidase, NADH peroxidase, and glutathione peroxidaseactivities of explants in mediums M99 were all increased more and earlier than that in W5.While cytochrome peroxidase and ascorbate peroxidase of explants in mediums M99 were alwayslower than that in W5. The Indole-3-acetic acid oxidase activity of explants in medium M99increased markedly and remained high level, while that in W5 increased in the first 5 days anddecreased markedly after 5-day culture. Peroxidase localization in explants of medium M99 showed that total peroxidase wasfound mainly in cytoplasm of outer integument and nucellar tissue during 0-2 days of culture,peroxidases activity in parenchyma tissue of ovary increased gradually which showedgradient staining following culture days and peroxidases mainly localized in cell wall and theextracellular gaps. Whole ovule cells were completely stained. While in medium W5peroxidases mostly localized in ovule cells especially in integument during 2-4 days afterculture. Peroxidase located in cell wall and in the extracellular gaps of proligerous cells inintegument at day 4. Soluble proteins in explants of both M99 and W5 were all increased slightly in the first 4days of culture. But it decreased in explants of mediums M99, while it increased in that of W5after 4 days culture. Total protein in ovules increased, while that in parenchyma of mediumsM99 decreased. But total protein in both ovules and parenchyma of mediums W5 were allincreased. In explants of M99, DNA and RNA content increased in ovules, especially in nucellus.Small starch grains appeared in ovule cells, while no starch grains appeared in parenchyma.<WP=7>In explants of W5, DNA and RNA content increased in both ovules and parenchyma. DNAcontent in proligerous cells decreased after 6 days of culture. Amylose increased inparenchyma gradually and big starch grains appeared in proligerous cells.更多还原