【作者】 贺佳玉；

【导师】 李云；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2008， 硕士

【摘要】 以四倍体刺槐胚珠和成熟种胚为材料,探讨了胚胎发育、败育与内源激素含量变化的关系,以及成熟种胚的变异和生理生化特性,并通过离体组织培养技术进行了挽救。结果表明:(1)四倍体刺槐幼胚在授粉后10d和100d时败育现象明显,败育率可达60%～70%,30d时败育率最低,仅为27%,败育主要发生在前期和后期。由胚珠横径和重量生长曲线看出:生长旺盛期出现在花后15d到50d,直至55d基本趋缓。(2)整个发育过程中,正常胚珠中ABA、IAA、GA3含量5d时达到峰值,随后呈下降趋势,但IAA和GA3含量在30～70d有所回升,败育胚珠中ABA含量在胚胎发育后期持较高水平,IAA和GA3含量则降到最低点。败育胚珠中ABA/(IAA+GA3)始终高于正常胚珠。(3)对不同胚龄的幼胚进行离体培养挽救试验,结果表明:荚果用70%乙醇浸泡60s,灭菌效果最佳,污染率降至9.4%;胚珠的适宜离体培养时期为授粉后30d,即败育率最低和胚珠生长最旺盛时期,萌动率为47.7%;适宜萌动培养基为Nitsch+6-BA 0.5mg/L+ IBA 2mg/L +GA3 0.5mg/L,萌动率最高达63.6%;幼胚诱导愈伤组织的最佳培养基为MS+ 2,4-D 3.0mg/L+6-BA 0.5mg/L+NAA 0.2mg/L,诱导率为38.6%;较好的分化培养基为MS+BA 3.0mg/L+NAA 0.5mg/L+KT 3.0mg/L,初步诱导出了不定芽,分化率最高为16%;愈伤组织切片结果表明,四倍体刺槐幼胚愈伤组织的增殖生长以内起源为主。(4)四倍体刺槐种子有胚率在37.77%,归纳为6类畸形胚,畸形种胚率92.31%,;四倍体和二倍体刺槐种胚在可溶性蛋白含量、丙二醛含量、过氧化物酶活性及种胚活力方面存在明显差异,而且种胚萌发的条件与其密切相关。通过倍性鉴定发现有活力的实生胚仍然是四倍体。(5)对成熟种子和种胚进行了离体培养挽救试验,结果表明:经10%H2O2 24h、5mg/L GA3 6h、7mg/L NAA 6h,处理后接种到MS或1/2MS培养基上,平均萌发率为5.5%、最高达6.0%。成熟种胚接种到培养基上,7d后有小部分种子胚萌发。更多还原

【Abstract】 In the studies, both the ovule and mature embryo of Tetraploid Robinia pseudoacacia were employed to investigate and discuss the relationship between embryonic development and changes of endogenous hormones in ovules; the variation and the physiology and biochemical mechanism of mature embryo; the rescuing technique by culture in vitro. The results indicates that:(1) At both 10d and 100d after anthesis, abortion was more serious than other points with a frequency of 60%～70%. At 30d after anthesis the abortion was the lowest with a frequency 27%. So the abortion mainly happened during younger and older stage of embryo development. From the growth curve of embryo’s diameter and weight, the peak period of growth happened during 15d to 50d, and tended to be flat till 55d.(2) During the whole development, the contents of ABA、IAA、GA3 in normal ovules reached their highest level on the 5th day after anthesis, and then decreased, but the contents of IAA and GA3 increased again during 30～70d, ABA content in aborted ovules maintained a relatively high level, but the contents of IAA and GA3 dropped to the lowest level. In the aborted ovule, ABA/(IAA+GA3) was always higher than that of normal ovule.(3) The results of saving young embryos in different development stages by culture in vitro indicate that preferable sterilization was to dip the young legumen in 70% alcohol for 60s, the frequency of pollution drops to 9.4%; the suitable stage for ovule culture is 30d after anthesis and the embryo’s culture-response-rate is of 47.7%; the suitable development medium for ovule is Nitsch+6-BA0.5mg/L+ IBA2 mg/L +GA30.5mg/L and the ovules’s culture-response-rate is of 63.6%; the efficient medium for inducing callus from young embryo is MS+ 2,4-D3.0mg/L+6-BA0.5mg/L+NAA 0.2mg/L and the callus-inducing rate is 38.6%; the suitable medium for differentiation is 6-BA3.0mg/L+NAA0.5mg/L+ KT3.0mg/L and on which some buds are induced from callus, the differentiation rate reached 16%; the callus anatomy showed that the proliferation growth of young embryo callus mainly originates from inner.(4) Within the seeds, the frequency of embryo is 37.77%, yet the abnormal embryo were classified into 6 groups, he frequency accounts for 92.31%.. From the physiological and biochemical properties, and studies from ploidy showed that between Diploid and Tetraploid Robinia pseudoacacia, the content of soluble protein and MDA, the activity of POD, and the vitality of embryo, which are associated with embryo’s germination, were different. By ploidy identification, most of the embryos of vitality were tetraploid. (5) The results of rescuing the mature seeds and embryos by culture in vitro indicate: after the treatment of 10%H2O2 24h、5mg/l GA3 6h, 7mg/l NAA 6h, the average frequency of germination is 5.5%, and the peak value is 6.0%. Meanwhile, a small proportion of the mature embryos can germinate after 7d.更多还原