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凤眼莲等植物组织和原生质体培养及遗传转化的研究
李学宝
Subtype博士
Thesis Advisor俞敏娟 ; 刘永定
1996
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Keyword植物细胞学 细胞遗传 植物生物化学 植物组织 厚生质体 豆子斗 紫云英 苜蓉
Abstract凤眼莲植株在低温(2-4 ℃)下培养4-48小时,叶片SOD等多种酶活性减弱,细胞呼吸减慢,酶活性及呼吸减弱程度与低温持续的时间有关。凤眼莲茎段培养在含BA3mg/L的MS培养基础上,可直接分化出芽,分化频率为46.7%。凤眼莲植株必须在高温度条件下培养,这显然与其水生特性有关。绿豆未成熟子叶、紫云英和南苜蓿子和下胚轴培养均获得再生植株。紫云英下胚轴分化频率为62.5%,南苜蓿下胚轴为48.1%,绿豆未成熟子叶为22.5%。从豇豆和绿豆未成熟子叶、紫云英和南苜蓿成熟子叶、凤眼莲叶片和根尖分离原生质体,纯化后的产量豇豆为1.5 - 2 * 10~6/gFW,绿豆为0.5 - 1 * 10~6/gFW,紫云英为1.5 - 2 * 10~6/gFW,南苜蓿为2 - 3 * 10~6/gFW,凤眼莲为0.5 - 1.5 * 10~6/gFW。比较了MS、B_5和KM8P三种原生质体培养基,发现豇豆和凤眼莲原生质体以MS为优,绿豆等则以B_5较佳。豇豆原生质体培养在附加2,4-D 0.2mg/1,BA 0.5mg/l和NAA lmg/l的MS培养基中,10天后的细胞分裂频率可达25.1-32.7%。培养20-30天后,形成肉眼可见的小愈伤组织。凤眼莲、绿豆、紫云英和南苜蓿原生质体也能在15-30天内形成小愈伤组织,其中以南苜蓿原生质体分裂最快,频率最高。豇豆原生质体来源的愈伤组织继代培养在含2,4-D2mg/L,BA0.5mg/L的MSB培养基上,获得胚性愈伤组织。首次从豇豆未成熟子原生质体来源的胚性愈伤组织诱导出大量体细胞胚,进而萌发生长成完整植株。其余几种植物原生质体培养获得根或芽分化。研究了凤眼莲等几种植物细胞遗传转化的条件,建立了农杆菌介导的和PEG介导的基因转移系统。根癌农杆菌经含乙酰丁香酮(AS)等诱导物质以及低pH、低磷酸的培养基诱导培养后,用来感染凤眼莲、紫云英、豇豆和绿豆等原生质体,随后的细胞GUS瞬间表达活性显著提高。这间接证明了上述诱导培养活化了细菌Vir区基因,促进了T-DNA向植物细胞转移。在PEG介导的DNA直接转移中,较高的pH和Ca~(2+)浓度能够提高细胞GUS活性。质粒DNA浓度及启动子类型对外源基因在植物细胞内表达也有一定的影响。首次建立了紫云英等植物的转化系统。采用原生质体-农杆菌共培养法,成功地将外源基因导入凤眼莲、豇豆等细胞中,获得稳定表达gus等外源基因的转化愈伤组织。采用农杆菌感染法,成功地将外源基因导入紫云英、南苜蓿、油菜和芥菜等植物,筛选标记为npt II基因。所用外植体为带有子叶柄的完整子叶或下胚轴切段,与农杆菌共培养2-3天后,转移到含500mg/l头孢霉素(或羧苄青霉素)的杀菌培养基上培养7-10天,启动细胞分裂和分化。然后,再将外植体移入含20-50mg/l卡那霉素的选择培养基上筛选转化体。把在选择培养基上分化出的茎芽切下,插入生根培养基中,诱导生根。将完整的卡那霉素抗生植株移入盆栽,生长发育至成熟,收获到T_1和T_2代种子。GUS和NPT II酶活性分析表明,外源基因在紫云英等植物细胞内得到稳定表达。用B.t.Cry IA基因或T-DNA左右边界作探针,进行Southern杂交。结果表明,部分抗性植株含有Cry IA基因或T-DNA边界的同源顺序。害虫饲喂试验证明,外源Cry IA基因导入油菜和芥菜获得的转Cry IA基因植物具有明显的抗虫性。在渗透胁迫下,紫云英转proB基因植株叶片游离脯氨酸大量积累,一些转化植株较对照组高2倍,其耐盐能力也有所提高。对油菜转基因植株后代株系所做的遗传分析表明,Km抗性性状遗传符合孟德尔分离规律。部分后代植株PCR试验为阳性,并具杀虫活性,表明Cry IA基因已遗传传递到T_1和T_2代植株基因组中。
Other AbstractLow temperature culture caused a decline in superoxide dismutase, peroxidase and glucose-6-phosphate dehydrogenase activity and cell respiration of water hyacinth leaves. This effect became more pronounced as the culture time was prolonged. When stem explants of water hyacinth were cultured on MS medium with BA 3 mg/L for 2-4 weeks, many adventitious shoots were induced from the explants, with shoot induction frequency reaching 46.7%. Plants of water hyacinth were cultured in the condition of high humidity and grew well, suggesting that is related with the water-growth feature of the hydrophyte. Regenerated plants were obtained from hypocotyl and cotyledon explants of Astragalus sinicus and Medicalgo hispida and immature cotyledon explants of mung bean, with shoot induction frequency reaching 62.5%, 48.1% and 22.5% respectively. Immature cotyledons of Vigna sinensis (cowpea) and phaseolus aureus (mung bean), and cotyledons of Astragalus sinicus and Medicago hispida, leaves and roots of water hyacinth were used for protoplast isolation. The purified protoplasts, with protoplast yield reaching 1.5 - 2 * 10~6/gFW in cowpea, 0.5 - 1 * 10~6/g FW in mung bean, 1.5 - 2 * 10~6/g FW in Astragalus sinicus, 2 - 3 * 10~6/g FW in Medicago hispida, and 0.5 - 1.5 * 10~6/g FW in water hyacinth, were cultured in MS, B_5 or KM8p liquid medium in dark(25 ℃) at a density of 1 - 5 * 10~5/ml. The protoplasts started cell division in 3-5 days. Sustained cell division resulted in formation of cell clusters and small calli. A comparison among MS, B_5 and KM8p media shows MS medium suitable for water hyacinth and cowpea protoplast culture and B_5 medium suitable for protoplast culture of mung bean, Astragalus sinicus and Medicago hispida. The cell division frequency of cowpea reached 25.1 - 32.7% in MS medium after 10 days of culture, and Medicago hispida protoplasts had a highest cell division frequency in B_5 medium. When protoplasts-derived calli of cowpea were transferred onto MSB (MS salts and B_5 vitamins) agar medium containing 2,4-D 2mg/l, BA 0.5mg/l for further growth, the embryogenic calli were selected and subcultured on the same medium. A lot of somatic embryos formed from the embryogenic calli on MSB medium containing IAA 0.1 mg/l, KT 0.5mg/l and 5% mannitol under light (2000 lux, 12 hrs/day). Some somatic embryos germinated and developed into plants. However, only roots or shoots formed from the protoplasts-derived calli of mung bean, Astragalus sinicus and Medicago hispida. Conditions of cell genetic transformation were studied, and Agrobacterium-mediated and PEG-mediated gene transfer systems were established for water hyacinth, cowpea, mung bean, Astragalus sinicus, Medicago hispida, Brassica napus, B. juncea and B. naphbrassica. Gene transfer efficiency was influenced by different, factors. The experimental results indicated that low pH, phosphate starvation and phenolic compounds (such as acetosyringone, etc.) could activate Agrobacterium tumefaciens vir genes and facilitate T-DNA transfer into plant cells, and therefore transient expression of gus gene markedly enhanced in the protoplasts transformed by Agrobacterium tumefaciens strain C_(58) (pKIWI 105). In PEG-mediated DNA direct transfer, transient expression of gus gene was promoted by higher pH and higher Ca~(2+) concentration of fusion medium. In the same experimental condition, expression of gus gene under the control of MAS-CaMV 35S promoter was more effective than that under the control of CaMV 35S promoter, and intensity of gus gene expression was positively correlated with the amount of foreign plasmid DNA in range of 10-100 ug. 48 hours after DNA uptake was the optimal by flurometric assay. The transformed calli from water hyacinth, cowpea, mung bean, etc. protoplasts were selected on MS medium containing 20 mg/l kanamycin or 50 mg/l chloramphenicol after the protoplasts were cocultivated with Agrobacterium tumefacients (strains PGV2260 (pBI121) or C_(58)C_1 (pBZ 6111), and confirmed by analysis of neomycin phosphotransferase II (NPT II), glucuronidase (GUS) and nopaline synthetase (NOS) activity. Adventitious shoots were induced from cotyledon and hypocotyl explants of Astragalus sinicus, Medicago hispida, rape and Brassica naphbrassica, etc. treated with Agrobacterium tumefaciens (strains PGV 2260(pBI121), PGV 2260 (proB_4), ASE367 (pBS21), LBA4404 (BY2 471) or C_5 _8C_1 (pBZ 6111), and subcultured on MS medium containing 20-50mg/l kanamycin or 50mg/l chloramphenicol to select transformants. Then the transformed shoots were rooted. Assay of NPT II, GUS and NOS activity and Southern blot analysis confirmed that foreign genes integrated into plant genomes and expressed in the plants. Transgenic plants of rape and Brassica naphbrassinca exhibited tolerance to insects such as pierce rapae (cabbage caterpilar) in leaf feeding experiments. Transgenic plants of Astragalus sinicus exhibited tolerance to NaCl on MS medium containing 0.5-1% NaCl and proline over-production and accumulation at salt-stress. Seeds were obtained from self-pollinated transgenic plants. Kanamycin-resistance and insect-resistance were maintained in the rape progenies.
Pages96
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12500
Collection学位论文
Recommended Citation
GB/T 7714
李学宝. 凤眼莲等植物组织和原生质体培养及遗传转化的研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1996.
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