明叶藓组织培养体系的建立及多倍体育种

	明叶藓组织培养体系的建立及多倍体育种; Establishment of tissue culture system and polyploid breeding of Vesicularia montagnei
	李青
导师	刘莉
摘要	Vesicularia montagnei is a kind of bryophyte widely used in ornamental landscaping because of its high aesthetic and economic values. However there is no efficient tissue culture system for V. montagnei, which leads to over-excavation of wild V. montagnei for its commercial use and thus great reduction in wild moss resources, and also limits the large-scale application of V. montagnei. To preserve the wild moss resources and exploit their potential for industrialized production, it is an urgent need to breed varieties for V. montagnei and to develop efficient methods for in vitro propagation. In this study, V. montagnei was propagated by tissue culture and then colchicine was applied to induce polyploid V. montagnei in vitro, ploidy level of these mutants was confirmed by flow cytometry, and the phenotype of mutants with stable inheritance was observed. We found that some mutants with doubled chromosomes exhibited an enhanced rhizoid density phenotype that could be stably inherited. To further explore the molecular mechanisms underlying increased rhizoid, RNA-Seq was performed with these mutant and wild-type V. montagnei. The major results obtained in this study are as follow: (1) The tissue culture system and regeneration system of V. montagnei were established successfully: 1) The sterilization of V. montagnei capsula : immerse the V. montagnei capsula into 10% sodium hypochlorite for 7 min, finally the success rate of spore sterilization could reach 100%; 2) Spore germination: inoculate the disinfected spore suspension after disinfection in a modified BCD medium. The petri dishes were placed in a 25℃ incubator for cultivation. Spores started to germinate 3 days after inoculation, and a large number of protonemata were obtained 14 days after inoculation, and protonemata differentiated into gametophytes 40 days after culture; 3) Regeneration of V. montagnei: On the modified BCD medium without hormones, protonemata were regenerated from the original protonemata, leaves, rhizoids, stems and other tissues. A large number of protonemata of V. montagnei could be obtained by culturing the moss suspensions on the modified BCD, which were made by crushing the protonemata or gametophyte with a homogenizer. (2) Induction and identification of polyploid V. montagnei: 1) Induction of polyploid V. montagnei: immerse the V. montagnei protonema into colchicine solution (concentration gradients are 0%, 0.1% and 0.3%)for different durations (2 h, 6 h). The results showed that high concentration of colchicine led to a higher mutagenesis efficiency; especially treatments with high concentration of colchicine for 6 hours could result in much more polyploidy mutants; 2) Mutant identification: 600 samples were screened by flow cytometry; 10 homozygous mutants with doubled genome and 16 chimeras were identified; 6 mutants with doubled genome and stable inheritance were eventually obtained after long-term subcultures and validations with flow cytometry for 3 more tim
	2020-05
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74145
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	李青. 明叶藓组织培养体系的建立及多倍体育种, Establishment of tissue culture system and polyploid breeding of Vesicularia montagnei[D],2020.

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