【作者】 姚娜；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2010， 博士

【摘要】 竹类是全球重要的森林资源之一,具有较高的经济价值和生态价值,在生产、生活中起着不可或缺的作用。近年来,植物组织培养和细胞培养已成为细胞诱变、细胞融合、遗传转化途径培育新品种的基础技术。建立竹类植物从种苗微繁、离体再生到细胞、原生质体培养、遗传转化一套完整的组织培养技术体系,将为竹类植物生理学、分子生物学乃至遗传育种研究提供良好平台。目前,竹类植物离体再生和细胞培养存在褐化、再生困难等问题;其中,散生竹离体再生成功的报道较少,毛竹未见离体再生成功报道。本研究以毛竹、空竹和孝顺竹为材料进行了愈伤组织培养;并对筛选出的具有分化潜力的毛竹愈伤组织进行了基因表达分析,研究结果如下:1.毛竹高度脱分化愈伤组织(黄色松散型愈伤组织)在MS大量+B5微量+B5有机物+MS铁盐+500 mg/L脯氨酸+500 mg/L谷氨酰胺+300 mg/L胰蛋白胨+0.5-1.0 mg/L 2,4-D+30 g/L蔗糖+6 g/L琼脂上可大量增殖;在未添加2,4-D的培养基中会发生褐化、死亡。2.对毛竹愈伤组织的分化特性进行了观察和分类。从毛竹愈伤组织中筛选出紫色致密、紫色松散、黄色致密、绿色致密4类有分化潜力的愈伤组织。其中,紫色致密、紫色松散和黄色致密愈伤组织可在未添加2,4-D的培养基上继代和分化出不定芽、不定根或组织、器官原基;绿色致密愈伤组织继代培养易发生褐化、死亡。3.分别将毛竹紫色致密、黄色致密和绿色致密愈伤组织与高度脱分化愈伤组织cDNA组合,进行双色荧光标记后与水稻cDNA芯片杂交。芯片分析结果表明,与高度脱分化愈伤组织比较,紫色致密愈伤组织共检测到513个差异表达基因;黄色致密愈伤组织检测到412个差异表达基因;绿色致密愈伤组织检测到567个差异表达基因。对差异表达基因中的unigene进行GO分析表明,与高度脱分化愈伤组织比较,以上3类有分化潜力的毛竹愈伤组织中编码与细胞分化、发育、胚胎发生、植物激素相关基因以及编码信号转导相关的激酶基因均有不同程度的差异表达。在绿色致密愈伤组织中检测到促进细胞死亡基因的差异表达。4.通过与水稻cDNA芯片杂交,比较了在不添加2,4-D培养基上培养15 d的毛竹愈伤组织与高度脱分化愈伤组织间基因表达的差异。其中,编码乙烯合成、茉莉酸合成、胁迫应答、DNA修复和抗坏血酸过氧化物酶等基因存在差异表达。5.孝顺竹愈伤组织增殖培养基为MS大量+B5微量+B5有机物+MS铁盐+500 mg/L脯氨酸+500 mg/L谷氨酰胺+300 mg/L胰蛋白胨+ 1.0 mg/L 2,4-D+30 g/L蔗糖+6 g/L琼脂。黄色致密型愈伤组织可分化不定芽,其中部分为白化苗。白化苗和正常苗在未添加植物生长调节剂的培养基中均可正常增殖和生根。6.毛竹和空竹愈伤组织在MS+500 mg/L脯氨酸+500 mg/L谷氨酰胺+300 mg/L胰蛋白胨+1.5 mg/L 2,4-D+30 g/L蔗糖液体培养基中振荡培养后可获得质地均一、分散性良好的悬浮细胞。悬浮细胞继代周期为9 d。选择20 g/L接种密度,将悬浮细胞进行平板培养,可获得愈伤组织,用于悬浮细胞遗传转化后阳性单克隆的筛选。更多还原

【Abstract】 Bamboo is one of the most important forest resources, which has a high commercial value and ecological function. Bamboo is indispensable in manufacture industry and our daily life. However, genetic improvement researches on bamboo are lag now. In recent years, plant tissue culture and suspension cell culture have been the basic technologies on breeding new varieties by the approaches of somatic cells mutation, cell fusion and genetic transformation. The construction of a tissue culture technology system of bamboo on in vitro micropropogation, regeneation, culture of suspension cell and protoplast, and genetic transformation will provide a research platform for the researches on physiology, molecular biology, and genetics and breeding of bamboo. Browning and hard to regenerate are huge problems on tissue culture of bamboo. The scattered bamboos such as Phyllostachy edulis, are especially hard to regenerate.In this study, Phyllostachys edulis, Cephalostachyum fuchsianum and Bambusa multiplex were sued as materials to establish the culture system of callus or suspension cell lines. The gene expressions of Phyllostachys edulis calli with differentiation potentials were detected by cDNA microarray. The research contents and results are as follows:1. The calii of Phyllostachys edulis can proliferation on the medium of MS macro salt +B5 micro salt +B5 organic +MS ferric salt +500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone +0.5-1.0 mg/L 2,4-D+30 g/L sucrose +6 g/L agar. The highly dedifferentiation calli can not survive on the medium without 2,4-D.2. Four types of calli were seemed as calli with differentiation potential. They were purple campact calli, purple friable calli, yellow compact calli and green compact calli. The purple campact calli, purple friable calli and yellow compact calli can survive on the medium without 2,4-D and differentiate adventitious buds or some tissues wiith structure. However, the green compact calli would brown during subculture.3. The purple campact calli, yellow compact calli and green compact calli were made a pair with yellow friable calli respectively to hybridize to the probes in cDNA microarraies. There were 412 differentially expressed genes detected in the microarray of purple compcat calli VS yellow friable calli. There were 412 differentially expressed genes detected in the microarray of yellow compcat calli VS yellow friable calli. And 567 differentially expressed genes were detected in the microarray of green compcat calli VS yellow friable calli. The unigenes of the differentially expressed genes were analyzed by GO. Genes related to cell differentiation, development, somatic embryogenesis, plant hormones and kinases in signal transduction of differentiation were differentially expressed respectively in 3 types of calli comparing to yellow friable calli. And genes stimulated cell death were upregulated in green compact calli.4. Genes related to ethylene synthesis, JA synthesis, stress response, DNA repair and Ascorbate peroxidase were detected differently expressed in the calli cultured on the medium without 2,4-D for 15 d.5. The calii of Bambusa multiplex could proliferation on the medium of MS macro salt +B5 micro salt +B5 organic +MS ferric salt +500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone + 1.0 mg/L 2,4-D+30 g/L sucrose +6 g/L agar. Adventitious buds could differentiate from yellow compact calli. And amonts of albino buds were acquired. Both normal buds and albino buds could proliferation and rooting.6. Fine suspension cell line could be acquired respectively by inoculation the calli of Phyllostachys edulis and Cephalostachyum fuchsianum into the liquid medium of MS+500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone +1.5 mg/L 2,4-D +30 g/L sucrose. The subculture cycle were 9 d. Calli could be acquired after the cells being cultured in the solid medium by the inoculation density of 20 g/L.更多还原