桑（Morus）离体再生体系的建立及生理生化研究

【作者】 杨金富；

【导师】 余茂德； 鲁成；

【作者基本信息】 西南农业大学 ， 细胞生物学， 2002， 硕士

【摘要】 本研究以桐乡青冬芽叶片、沙二×伦109种子子叶和25个桑品种冬芽为材料，比较了不同处理条件下的植株再生和愈伤组织诱导的差异，获得再生植株，建立了离体再生体系，并对桑离体培养过程中内源激素、SOD和POD变化与形态发生的相关性进行了探讨，为有效地调控桑离体形态发生和遗传转化奠定基础。主要的工作和结果如下： 1．离体再生体系的建立 1．1外植体对桑离体培养的影响 在MS+NAA0．5 mg／L+BA0．5 mg／L培养基上，25个材料的试管苗得率为0～87％，表现出较大的差异；桑枝条基部的培养效果较好。1月份采样的冬芽试管苗得率高于12月和2月采样的冬芽。 1．2愈伤组织和不定芽诱导 正交试验表明，在MS+BA0．5mg／L+NAA0．5mg／L，附加30％果糖，8p维生素，500mg／L酪蛋白水解物，叶片愈伤组织诱导率达68％，叶片不同部位的愈伤组织诱导率差异较大。在MS+BA3．0mg／L+IAA0．3 mg／L的培养基上，子叶不定芽诱导率为71．6％，附加0．5mg／L的硝酸银能提高子叶不定芽诱导率(75％)，并降低褐化率。 1．3试管苗增殖和生根 在MS+BA3．0 mg／L+IAA0．5mg／L的培养基上培养10天，试管苗获得较大的增殖系数(4．67)。1／4MS+IBA0．5，附加浓度300～600mg／L的肌醇是桑试管苗生根的较适培养基，2000Lx的光照强度有利于桑试管苗生根。 2．生理生化研究 2．1内源激素变化 桑子叶内源激素的浓度随培养时间而改变，ABA随培养时间的增加而增加，在培养16天时有一个高峰；iPA、ZR、DHZR在培养的第8天有一个峰值，此后下降；IAA在培养的第6天和第16天各有一个峰值。 2．2SOD和POD分析 在离体培养过程中桑SOD和POD同工酶和酶活性成一定的规律性变化。SOD同工酶的谱带表现上，在不定芽的发生时，一些谱带消失，不定根发生时，出现二条新的同工酶谱带；POD同工酶谱带在不定芽发生时，一条同工酶带消失，POD和SOD在培养8天和16天各出现两个活性峰。 2．3硝酸银对离体生理变化的影响 添加一定浓度的硝酸银能够降低ABA、IAA、iPA的浓度，提高细胞分裂素和生长素的比值，促进芽的分化而抑制根的发生；外植体的SOD和POD同工酶谱带也有变化，同时提高芽分化时两种酶的活性，降低根原基发生时SOD和POD的活性更多还原

【Abstract】 Leaves, cotyledon, and winter buds of 25 mulberry cultivars were employed as explants in this experiment. Under different treatment, difference of plant regeneration and callus initiation between them was compared. In this paper, regeneration system was established and variation of endogenous hormone, SOD and POD was studied during in vitro culture. It may be usefull in efficiently controlling in vitro morphopoiesis of mulberry and lay foundation for its genetic transformation. The main resuls were as follows: 1. Establishment of in vitro regeneration system.1.1 The effects of explants on in vitro culture.Cultured on medium containing 0.5mg/L NAA and 0.5mg/L BA, seedlings of the 25 materials were obtained with rate range from 0% to 87%. Basal portion of mulberry branch gave the best results. The seedling rate of winter buds taken in January are higher than that of taken in Feberuary and December.1.2 Induction of callus and advertious shootOrthnnormal test showed that 68% induction rate of leaves was observed with medium containing 0.5mg/L NAA, 0.5mg/L BA, 30% fructose, 8p vitamins and 500mg/L CH .Induction rate between different parts of leaves was significantly different.1.3 Propagation and rooting of seedlingsMaxium propagation coefficient (4.67) of seedlings was reached after cultured on medium containing 3.0mg/LBA and 0.5mg/L IAA for 10 days. Quarter strength MS medium supplemented with 0.5mg/L IBA and 300-600mg/L inose was better used for rooting.with light intensity of 2000Lx. 2 Studies of physiology and biochemistry2.1 Variation of endogeious hormoneConcentration of endgenous hormone in mulberry leaves varied with the culture time. Concentration of ABA increases till to reach peak value after 16 days culture. Concentration of iPA, ZR and DHZR reach peak value in the eighth day respectively and decrease thereafter.Two peaks was observed with IAA in the sixth and sixteenth day respectively.2.2 Analysis of SOD and PODConcentration of SOD and POD and their enzyme activity varied rhythmical during in vitro culture. Some bands of SOD isoenzyme disappear when adventitous shoots were initiated and two novel bands appeared when adventitous roots were initiated. For POD, one band disappeared when shoots were initiated. Two enzyme activity peak of SOD and POD were observed in the eighth and sixteenth cuture day respectively.2.3 Effects of AgNO3 on physiological variation of mulberry in vitro.Concentration of ABA, IAA and iPA was decreased when AgNO3 was added to the culture medium. Increasing the ratio of cytokinin and auxin, differentiation of bud was promoted and rooting was inhibited. Enzy activity of isoenzyme SOD and POD were increased when differentiation of bud was promoted and were reduced during the period of root morphoresis.更多还原