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新疆雪莲体胚诱导及成苗方法研究
贾莉芳
学位类型硕士
导师王晓军
2007-06
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物有机化学
关键词新疆雪莲 体细胞胚胎发生 形态建成 同工酶 可溶性蛋白
摘要

本研究以新疆特色药用植物-新疆雪莲的子叶为外植体,通过固体培养和液体培
养相结合的方法,对影响体细胞胚胎发生的多种影响因子进行筛选,建立了一个稳定的新疆雪莲体细胞胚胎发生的培养体系。并从组织细胞学、同工酶酶谱和特异蛋白质三个方面,对新疆雪莲体细胞胚胎形态建成的机理进行了初步探索,其主要结果如下:1、新疆雪莲可通过以下途径实现体细胞胚胎发生:以生长15d 左右无菌苗的子叶为外植体,在附加0.1mg/L2,4-D、0.5mg/LBA 的MS 固体培养基上诱导愈伤组织。随后挑选胚性愈伤组织转入MS+0.05mg/L 2,4-D 的培养基中进行液体培养。20d 后,培养基中除去2,4-D,并在继代培养过程中先后加入5g/LPEG、2mg/LGA3,以实现体细胞胚胎的诱导分化及成熟。最后将成熟体胚转入琼脂固化培养基1/2MS 中,实现植株再生。2、对新疆雪莲体细胞胚诱导分化及成熟的条件进行了优化,实现了新疆雪莲体细胞胚胎的萌发与成苗。并在体胚发育后期引入干化法处理成熟体胚,有效地提高了新疆雪莲体细胞胚的萌发率和成苗率,成苗率可达50%。3、 新疆雪莲体细胞胚多发生于胚性愈伤组织表面,少数发生于胚性愈伤组织的内部。体细胞胚胎形态发生经历了与合子胚相似的阶段,即球形胚-心形胚-鱼雷胚-子叶胚,最后发育成再生植株。体胚发育是不同步的,在同一块胚性愈伤组织上有处于不同发育阶段的体细胞胚。4、 新疆雪莲体细胞胚胎形态建成过程中同工酶酶谱的研究表明,POD 和EST同工酶的谱带都随着体细胞胚的发育而增多,并在不同发育阶段有特征谱带出现,可作为新疆雪莲体细胞胚胎发生的生化标记。5、 新疆雪莲体细胞胚胎形态建成过程中SDS-PAGE 图谱表明,非胚性愈伤组织的蛋白质含量较少,蛋白表达较弱;分子量为25KD、45KD 和91KD 的蛋白是球形胚期所产生的特征蛋白;而14.3KD 的蛋白条带是器官发生所特有的。

其他摘要
With cotyledons of Saussurea involucrata – a distinctive medicinal plant in Xinjiang as explant, various culture factors suitable for the somatic embryogenesis were selected by using the method of solid culture combining with liquid culture. A stable somatic embryo culture system for S. involucrata was established. Then we discussed the mechanisms of morphogenesis of somatic embryos of S. involucrata from the following three aspects: histological observation, isozyme spectra and specific proteins. The results are the follows: 1 The somatic embryogenesis of S. involucrata could be obtained through the following pathway: the embryogenic callus(EC) was induced by culture the cotyledons of sterile seedlings, which had grown for 15 days, on the MS medium added with 0.1mg/L 2,4-D and 0.5mg/LBA. Then EC was transferred into the medium of MS+0.05mg/L2,4-D for liquid culture. During the process of subculture, 5g/LPEG and 2mg/LGAwere added in turn for the realization of the differentiation and maturation of somatic embryo. Finally the mature embryos were transferred on the 1/2MS solid medium and regenerative plants were developed. 2 For S. involucrata, it is realized the germination and regeneration of somatic embryos and the differentiation and maturation of somatic embryos were optimized. Moreover, we treated the mature embryos by desiccation. It raised the rate of germination and plantlet formation of somatic embryos effectively. The frequency of seedling formation reached 50%. 3 Somatic embryos of S. involucrata mainly rooted from the epidermic cells of embryonic callus, minority rooted from the internal cells. The development of somatic embryo was similar as zygotic embryo, i.e. the process included globular embryo, heart embryo, torpedo embryo and cotyledon embryo, finally regenerative plantlet. However, the developmental process was asynchronous, and there were different stages of somatic embryo on the same embryonic callus. 4 The experiment results on isozyme spectra during the morphogenesis of somatic embryos of S. involucrata showed that the spectrum bands of POD and EST increased with the development of somatic embryos, and some special bands at the different stages were found, which could act as the embryogenesis of S. involucrata. 5 The protein analysis by SDS-PAGE during the morphogenesis of somatic embryos of S. involucrata indicated that non-embryonic callus (NEC) had a lower amount and expression of proteins. Some proteins, such as the proteins of 25KD, 45KD and 91KD, could be found only at globular stage, while organogenesis had specific proteins of 14.3KD.
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/3499
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
推荐引用方式
GB/T 7714
贾莉芳. 新疆雪莲体胚诱导及成苗方法研究[D]. 北京. 中国科学院研究生院,2007.
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