滇楸体细胞胚胎发生及其机理研究
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摘要
本研究综合运用固体培养和液体培养相结合的方法,建立了滇楸(Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour)体细胞胚胎发生培养体系,筛选出了较合适的培养基和培养条件,培养出了滇楸体细胞胚胎再生植株;通过体细胞发育过程的解剖学观察,初步掌握了滇楸体细胞胚胎的起源与发育过程;通过体细胞胚胎不同发育阶段主要生理生化特性及蛋白质代谢动态的分析进一步探讨了滇楸体细胞发生的生理及分子机理。主要研究结论如下:
1.以滇楸无菌实生苗子叶为外植体,接种于1/2MS+6-BA2.00mg·L~(-1) +NAA 0.10 mg·L~(-1)培养基中诱导体胚,建立了滇楸体细胞胚胎直接发生体系;以滇楸无菌实生苗子叶、下胚轴为外植体,接种于N6+NAA0.10mg·L~(-1)+ 6-BA2.00 mg·L~(-1)或1/2MS+TDZ0.50mg·L~(-1)上诱导愈伤,将胚性愈伤接种于1/2MS+1.00mg·L~(-1)6-BA +0.01mg·L~(-1)NAA +500mg·L~(-1)肌醇上诱导体胚,将萌发后正常体胚苗移栽驯化,建立了滇楸体细胞胚胎间接发生体系。
2.滇楸体胚发生存在内部发生和外部发生两种方式,起源于单细胞,依次经历二细胞原胚,多细胞原胚、球形胚、心形胚、鱼雷胚、子叶胚时期;体细胞胚胎与周围愈伤组织存在着明显的生殖隔离。滇楸体细胞胚胎发生不同材料和不同发育时期的胚状体生理生化特性变异明显,生理生化特性的差异与玻璃化体胚的发生以及畸形苗的形成有关。
3.愈伤组织蛋白质代谢较弱,中期体胚、成熟体胚?和体胚萌发幼苗蛋白质代谢旺盛。胚性愈伤组织与早期体胚混合物、中期体胚、成熟体胚?和成熟体胚Ⅱ四种材料的蛋白质双向电泳结果差异很大,4个时期共表达341个蛋白点,相对分子量范围在15.1~85.0kD之间,等电点范围为4.47~6.92之间,四个时期分别有79、200、102和191蛋白质点,分别有40、65、30和36个特异表达的蛋白质点,其余蛋白质点随着体细胞胚胎的发育呈现不同的表达趋势。
The system of somatic embryogenesis in Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour was established with comprehensive application of solid and liquid culturing methods.The best medium and conditions were chosen for somatic embryogenesis and plantlets originated from somatic embryos were cultured; the origins and processes of somatic embryogenesis and development had been primarily acknowledged during the anatomical observation. Physiological and biochemical characteristics and the dynamic of protein metabolism during the processes of the development of SE were analyzed. Furthermore, the physiological and molecular mechanism was evaluated further. The main results as follows :
1. The cotyledons of seedlings in C. fargesii f. duclouxi was cultured on the medium of 1/2MS+6-BA2.00mg·L~(-1) +NAA 0.10 mg·L~(-1) to induce somatic embryos directly.The system of somatic embryogenesis was directly established. The cotyledons and the axis of seedlings in C. fargesii f. duclouxi was cultured on the medium of N6+NAA0.10mg·L~(-1)+ 6-BA2.00 mg·L~(-1) or 1/2MS+TDZ0.50mg·L~(-1) to induce callus. Then the embryogenic callus was cultured on the medium of 1/2MS+1.00mg·L~(-1)6-BA +0.01mg·L~(-1)NAA + Inositol 500mg·L~(-1) to induce somatic embryos indirectly. The plantlets developed from embryoid were transplanted and domesticated, thus the syetem of somatic embryogenesis was established indirectly.
2. Somatic embryos can be induced from the internal and external of callus. Somatic embryo was originated from single cell, and then developed to multicellular proembryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo, cotyledon embryo in sequence. There is obvious reproductive isolation between the embryos and the peripheral tissue. There were obvious physical and chemical variations between different types of callus and somatic embryos, and the variations of physical and chemical properties were related to the vitrification of somatic embryos and the development of deformed plantlet.
3. The metabolism of protein in callus was relatively weak, while that in the midstage embryos, the matured embryos ? and the matured embryosⅡwere vigorous. The results of two-dimensional electrophoresis of protein showed great variance between 4 kinds of material which included the mixture of little callus and the early-stage embryos, the midstage embryos, matured embryos ? and matured embryosⅡ. There were 341 proteins expressed in the 4 stages in total. The relative molecular weight was in the range of 15.1kD to 85.0kD. The isoelectric point was in the range of 4.47 to 6.92. There were 79、200、102 and 191 proteins expressed in the 4 stages respectively, and 40、65、30 and 36 proteins specially expressed in the 4 kinds of material respectively. The other proteins expression changed along with the development of somatic embryos.
1.以滇楸无菌实生苗子叶为外植体,接种于1/2MS+6-BA2.00mg·L~(-1) +NAA 0.10 mg·L~(-1)培养基中诱导体胚,建立了滇楸体细胞胚胎直接发生体系;以滇楸无菌实生苗子叶、下胚轴为外植体,接种于N6+NAA0.10mg·L~(-1)+ 6-BA2.00 mg·L~(-1)或1/2MS+TDZ0.50mg·L~(-1)上诱导愈伤,将胚性愈伤接种于1/2MS+1.00mg·L~(-1)6-BA +0.01mg·L~(-1)NAA +500mg·L~(-1)肌醇上诱导体胚,将萌发后正常体胚苗移栽驯化,建立了滇楸体细胞胚胎间接发生体系。
2.滇楸体胚发生存在内部发生和外部发生两种方式,起源于单细胞,依次经历二细胞原胚,多细胞原胚、球形胚、心形胚、鱼雷胚、子叶胚时期;体细胞胚胎与周围愈伤组织存在着明显的生殖隔离。滇楸体细胞胚胎发生不同材料和不同发育时期的胚状体生理生化特性变异明显,生理生化特性的差异与玻璃化体胚的发生以及畸形苗的形成有关。
3.愈伤组织蛋白质代谢较弱,中期体胚、成熟体胚?和体胚萌发幼苗蛋白质代谢旺盛。胚性愈伤组织与早期体胚混合物、中期体胚、成熟体胚?和成熟体胚Ⅱ四种材料的蛋白质双向电泳结果差异很大,4个时期共表达341个蛋白点,相对分子量范围在15.1~85.0kD之间,等电点范围为4.47~6.92之间,四个时期分别有79、200、102和191蛋白质点,分别有40、65、30和36个特异表达的蛋白质点,其余蛋白质点随着体细胞胚胎的发育呈现不同的表达趋势。
The system of somatic embryogenesis in Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour was established with comprehensive application of solid and liquid culturing methods.The best medium and conditions were chosen for somatic embryogenesis and plantlets originated from somatic embryos were cultured; the origins and processes of somatic embryogenesis and development had been primarily acknowledged during the anatomical observation. Physiological and biochemical characteristics and the dynamic of protein metabolism during the processes of the development of SE were analyzed. Furthermore, the physiological and molecular mechanism was evaluated further. The main results as follows :
1. The cotyledons of seedlings in C. fargesii f. duclouxi was cultured on the medium of 1/2MS+6-BA2.00mg·L~(-1) +NAA 0.10 mg·L~(-1) to induce somatic embryos directly.The system of somatic embryogenesis was directly established. The cotyledons and the axis of seedlings in C. fargesii f. duclouxi was cultured on the medium of N6+NAA0.10mg·L~(-1)+ 6-BA2.00 mg·L~(-1) or 1/2MS+TDZ0.50mg·L~(-1) to induce callus. Then the embryogenic callus was cultured on the medium of 1/2MS+1.00mg·L~(-1)6-BA +0.01mg·L~(-1)NAA + Inositol 500mg·L~(-1) to induce somatic embryos indirectly. The plantlets developed from embryoid were transplanted and domesticated, thus the syetem of somatic embryogenesis was established indirectly.
2. Somatic embryos can be induced from the internal and external of callus. Somatic embryo was originated from single cell, and then developed to multicellular proembryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo, cotyledon embryo in sequence. There is obvious reproductive isolation between the embryos and the peripheral tissue. There were obvious physical and chemical variations between different types of callus and somatic embryos, and the variations of physical and chemical properties were related to the vitrification of somatic embryos and the development of deformed plantlet.
3. The metabolism of protein in callus was relatively weak, while that in the midstage embryos, the matured embryos ? and the matured embryosⅡwere vigorous. The results of two-dimensional electrophoresis of protein showed great variance between 4 kinds of material which included the mixture of little callus and the early-stage embryos, the midstage embryos, matured embryos ? and matured embryosⅡ. There were 341 proteins expressed in the 4 stages in total. The relative molecular weight was in the range of 15.1kD to 85.0kD. The isoelectric point was in the range of 4.47 to 6.92. There were 79、200、102 and 191 proteins expressed in the 4 stages respectively, and 40、65、30 and 36 proteins specially expressed in the 4 kinds of material respectively. The other proteins expression changed along with the development of somatic embryos.
引文
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