【作者】 许矛；

【导师】 张美萍；

【作者基本信息】 吉林农业大学 ， 作物遗传育种， 2006， 硕士

【摘要】 本实验以平贝母为供试材料，筛选出最优外植体和培养基质，建立了平贝母的高效再生体系，探讨了激素自养型培养物生理生化指标与形态发生的关系，还探讨了不同离体保存方法与细胞存活率和长势的关系，并进行了细胞形态学观察，确定了细胞分化的类型，为进一步研究形态发生控制和工厂化生产鳞茎奠定了基础。 平贝母鳞茎和茎段诱导不定芽丛的最佳基质为MS+NAA0.5mg/L+BA1.5mg/L，二者诱导愈伤组织的最佳基质分别为MS+2.4-D2.0mg/L和MS+2.4-D1.5mg/L，pH=6.0，光培养。对休眠鳞茎进行GA500mg/L的处理可打破休眠，再生植株的周期为40-50天。将平贝母培养物先减半原激素浓度，再以无激素MS基本基质继代培养10余月，可获得激素自养型培养物。器官发生过程中从愈伤组织到不定芽再到再生植株，游离氨基酸的含量先升高再下降，而可溶性糖含量是先下降再升高，其中不定芽丛中游离氨基酸含量最高为10.90％，愈伤组织中可溶性总糖的含量最高为6.49％；不定芽丛中的淀粉酶活力较高，并且几种培养物中以α-淀粉酶变化趋势与总淀粉酶变化趋势相似，其变化幅度也高于β-淀粉酶；过氧化物同工酶以不定芽的酶带带数最多，这说明不定芽中酶的种类最多，含量也较高。在培养物发育时期各种酶的活性和功能均不相同，可见发育是一个复杂的各种功能元件相互协调配合的过程。 平贝母培养物有着和生药相同的生物碱斑点。其中不定根中的总生物碱含量高于愈伤组织和不定芽丛。 通过超低温的方法可以达到中长期保存平贝母种质资源的目的。保存程序为：加入5％DMSO+5％甘油冷冻保护剂→-20℃停留2h→超低温冰箱保存→38℃水浴迅速解冻→MS培养基冲洗→接于新鲜培养基再培养。另外本实验中加入甘露醇、PP₃₃₃也成功保存了平贝母培养物。更多还原

【Abstract】 Taking bulbs of fritillaria ussuriensis maxim as explants, sieve the optimal explant and medium . and set up high performance regeneration system,discuss the relations between different method in vitro conservation and the rate of survivorship, determine the type of differentiation in cells by paraffin section,analysis the regulations between physiological and biochemistry aspects and morphogensis of hormone autotrophy culture , In order to control the secondary metabolism and ground the foundation for mass production.The optimal basic medium of bud inducement from bulbuds and little stem is MS+NAA0.5mg/L+BA1.5mg/L, the optimal mediums of calli inducement are MS+2.4-D2.0mg/L and MS+2.4-D1.5mg/L,pH=6.0,light,Immerse the bulbuds with GA500mg/L to break dormancy ,and the regeneration circle is 40-50 days,we gain the hormone autotrophy type culture by changing the medium with half reducing the concentration of hormone and then culture with MS free of hormone, during the organogenesis course,the content of free amino acid is to go up and then go down,but the opposite condition is in the content of soluble sugar.The buds have the highest content of free amino acid is 10.90% and the calli has the highest contents of soluble sugar is 6.49% , the amylase activity is higher in buds, the change trend of α-amylase activity is similarly with the whole amylase activity’s and it may play an important role in starch degradation. The zymogram of buds is complex which has many line and deep dying , it indicate that many enzyme have different activities and function during the culture period.Fritillaria culture and the crude drug have the same alkaloids spots. And the contents of total alkaloid in adventitious roots is higher than that in calli and buds.Using cryopreservation to conserve, the course is : add 5%DMSO+5%glycerol—>stay 2h in -20°C→conserve in ultra colder→defrosting in 38°C water bath→ wash with MS medium→subculture in fresh MS medium. Also add mannitol and PP333 can conserve fritillaria ussuriensis maxim culture in short period.更多还原