论文标题:兰州百合外源DNA花粉管通道法导入的初步研究
The Frist Stage Research of the Exogenous DNA Pollen-tube Pathway Transferring in Lilium Davidii Var. Unicolor (hong) Cotton
论文作者 杜捷
论文导师 幸亨泰,论文学位 硕士,论文专业 植物学
论文单位 西北师范大学,点击次数 741,论文页数 46页File Size2179k
2003-05-01论文网 http://www.lw23.com/lunwen_123242/ 兰州百合,外源DNA,花粉管通道导入法
Lilium davidii var.Unicolor(hong),exogenous,DNA pollen-tube pathway
兰州百合是百合科百合属百合的一个变种，其鳞片丰满白嫩，质地细腻，清香可口，毫无苦味，食用品质优良，是食用百合中的上品，在兰州及周边地区广为栽种，其栽培历史已有四百多年，由于多年的栽培，在一些地方的栽培品种出现了品质的退化。我国近代的百合育种工作开始于上世纪80年代，利用百合的远缘杂交培育获得了一些百合种间杂种，其育种方法基本采用柱头切割技术及杂种胚的培养进行。本论文对一些百合的杂交育种和基因的导入方法进行了文献综述，认为花粉管通道法导入外源基因(DNA)的方法在百合育种工作中有利用的前景。 本实验利用兰州百合、甘肃产野生百合及一些观赏百合品种，对其性状、物候期进行了较为系统的观察和记录，对其进行了有性杂交。结果表明，兰州百合与一些野生百合及观赏百合在性状上有较大的差异，且在其优良性状上具有互补性，但进行互相间的杂交较为困难，杂交后不易结实和形成有胚种子。在对这些百合进行了自交后表明，大多数品种自交的结实率不高，兰州百合在进行自交时，不同的授粉方式后有不同的结实率，其中异株异花授粉时的结实率略高。 在进行了有性杂交的基础上，选取具有较高结实率的兰州百合作为受体，利用花粉管通道导入法，对一些百合品种的总DNA在在兰州百合中进行了初步研究，基本掌握了在兰州百合中进行外源DNA导入的基本操作方法，提供了一组以结实率和有胚种子率为标准的优化参数。 在导入时，通过对授粉后4个时间段滴加外源DNA的子房膨大、结实和有胚种子情况的研究后得出结论，选择外源DNA滴加的时机在授粉后3h左右较为适宜，此时的子房膨大率和有胚种子率均较高。 通过对授粉后在柱头或花柱断面滴加后的结实与有胚种子的分析，表明滴加的部位选择在柱头滴加为宜，采用切割柱头的花柱断面滴加时，子房膨大和结实率会受到影响而降低。 在实验中设置了滴加的DNA溶液的3个浓度，结果表明较高浓度的DNA溶液在授粉后滴加会对结实造成影响，在兰州百合中进行外源DNA的花粉管通道法导入时，50u留nil的浓度的结实率较高，而滴加时DNA溶液选择中性为宜。 对导入后代部分植株进行了蛋白质凝胶电泳，但在所做电泳中未能出现有蛋白条带的差异，同时对部分的导入后代植株和未进行导入后代植株根尖染色体进行了观察，在两种植株中均有染色体数目的变异，变异的频率基本相同。
Lilium davidii var.Unicolor(hong) is an variety of lily family with its characteristics as full-grown tender and white squamae; delicate texture; scent and bitterless taste;superior edible quality. Owing to its 400 years cultivation history in Lanzhou and its peripherical areas, the quality of some cultivated species of Lilium davidii var. Unicolor (hong) comes to retrogress. The Chinese research of lily"s breeding can be fraced back in 1980"s by using distant hybridization to get some lily"s distant hytrids. Its breeding technique to improve lily"s quality was stigma cutting and hybridized embryo cultivating. This thesis presets the literature review of lily"s breeding and its genetic breeding transferring technique. In this thesis, it is stated that the technique of transferring the pollen-tube pathway to exogenous gene has available and bright prospect.In this experiment, the anthor uses Lilium davidii var.Unicolor(hong). Gansu wild lily and some decorative lily varieties as the different materials and makes the respectively systematic observation and concerned records on their traits, phenological periods.Through the hybridization, it is concluded that Lilium davidii var.Unicolor(hong) gretly differ from the wild lily varieties and decorative lily in traits; some of their traits can be mutually complementary, but it is difficult to hybridize them each other. It isn"t subject to seeds and embryo forming under inter-hybridization condition. Their autogamy is also tested and the result show that most of lily varieties have low rata self-fruitfulness. Lilium davidii var. Unicolor(hong) in autogamy shows different the seed-setting percentage in different pollinating methods, with the result as the higher rate of self-fruitfulness through xenogamy. Based on hybridization, genetransferring through pollen-tube pathway to Lilium davidii van Unicolor(hong) is researched. The master of basic technique of exogenous DNA transferring to Lilium davidii var.Unicolor(hong) provides us a set of improved parameters based on the rata of fruit and embryo seeds.After pollinating the conclusion of the items of ovary enlargement, fruitfulness, embryo seeds of transferring exogenous DNA should be better transferred to Lilium davidii var.Unicolor(hong) after 3 hours of pollinating in that the rate of ovary enlargement and embryo forming in this period is respectively high.While, the form of transferring is also tested, which shows that the position and percentrite of DNA are of great effect on Lilium davidii var.Unicolor(hong). The drip DNA to plane of stigma and style or drip high concentration DNA will make the rate of ovary enlargement and embryo seeds decrease.In this experiment, 3 concentrations of DNA transferring solution is set up . The results show that the higher concentration DNA solution after pollinating will effect the embryo seeds. And it also show that when Lilium davidii var. Unicolor(hong) is accepting the exogenous DNA pollen-tube pathway transferring, the embryo seeds rate would be higher with 50 ug/ml concentration solution and the neutral drip DNA solution could be advisable.The gel electrophoresis of protein of the variation of the number of chromosome don"t show any difference between transgene plant and their parental generation.
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