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植物受体再生及遗传转化体系的建立是对植物进行转基因操作时至关重要的一个技术环节。以菊花优良种质怀黄菊(Chrysanthemum morifolium cv.‘Huaihuang’)为试材,探讨胚状体诱导所需的最佳培养基及芽分化和根生长的最适抗生素选择压。在此基础上,将抗病基因Cm TGA1通过同源转化的方法转入怀黄菊,获得再生植株。结果表明,在附加1.5 mg·L-1IAA、0.5 mg·L-1 6-BA和1.0 mg·L-1 2,4-D的MS培养基上诱导培养15天后,再去除2,4-D进行分生培养,并进一步诱导芽再生,最终86%的供试外植体通过胚状体途径获得再生芽;在芽分化时所需潮霉素选择压为5.0 mg·L-1,生根时潮霉素选择压为4.5 mg·L-1。对转化植株进行半定量PCR(semi-quantitative PCR)和实时荧光定量PCR(real-time quantitative PCR)检测,结果表明,Cm TGA1基因成功整合到转化植株基因组中,从而建立了怀黄菊间接体细胞胚途径转基因受体再生体系。该技术的建立为通过转基因手段解决生产中存在的怀黄菊病害感染严重和种质退化等问题奠定了基础。
The establishment of plant receptor regeneration and genetic transformation system is one of the most important technical aspects of transgenic plants. Chrysanthemum morifolium cv.’Huaihuang ’was used as material to investigate the optimum medium for embryoid induction and the optimum antibiotic selection pressure for bud differentiation and root growth. On this basis, the resistance gene Cm TGA1 was transferred into the Huaihuang Ju by homologous transformation to obtain regenerated plants. The results showed that on the MS medium supplemented with 1.5 mg · L-1 IAA, 0.5 mg · L-1 6-BA and 1.0 mg · L-1 2,4-D for 15 days, 2,4-D And then regenerated buds were induced further. At the final 86% of explants, regenerated shoots were obtained through embryoid body pathway. The selection pressure of hygromycin was 5.0 mg · L-1 at bud differentiation, Hygromycin selection pressure of 4.5 mg · L-1. Semi-quantitative PCR and real-time quantitative PCR analysis of the transformed plants showed that the Cm TGA1 gene was successfully integrated into the genome of the transformed plant, thereby establishing an indirect somatic cell cycle Pathway Transgene Regeneration System. The establishment of this technology laid the foundation for solving the problems of the serious infection of Huismania and the degeneration of germplasm in the production by means of transgenic means.