藏红花细胞和组织培养的过程调控
文献类型：学位论文
| 作者 | 陈书安 |
| 学位类别 | 博士 |
| 答辩日期 | 2004 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 王玉春 |
| 关键词 | 藏红花 藏红花素 愈伤组织 过程调控 脱毒 快繁 |
| 其他题名 | Process Regulation in Crocus sativus L. Cell and Tissue Culture |
| 学位专业 | 化学工艺 |
| 中文摘要 | 为解决藏红花资源短缺和藏红花病毒感染的问题，本论文较系统地研究了高产藏红花素细胞系的诱导、筛选和培养的过程调控，并利用生物反应器规模化培养细胞直接生产藏红花的主要活性成分藏红花素；同时对筛选到的细胞系进行脱毒，调控胚性愈伤的发生、增殖和分化过程，建立了稳定高效的藏红花芽再生体系，为藏红花的人工种植提供大量、无毒、优质种苗。首先对藏红花球茎、芽、叶子和花愈伤组织以及桅子叶子和种子愈伤组织进行了系统诱导，获得了大量的藏红花和桅子愈伤组织细胞系。首次建立了一简便的筛选高产藏红花细胞的方法即目视法和HPLC相结合的方法。采用该方法从诱导的229株藏红花细胞系和15株桅子细胞系中快速筛选出一株藏红花素含量较高、生长快速、不易褐化的藏红花球茎1细胞系。建立了藏红花细胞的悬浮培养体系。悬浮培养时细胞的生长周期约为20d，在20d时生物量达到最大，为12.3 g DW／L，但是藏红花素的合成周期大约为28d，在28d时藏红花素的含量和产量达到最大，分别为95.8mg／g和0.92g／L。藏红花素的积累与细胞的生长之间的关系为半生长偶联型。首次采用二步培养法生产藏红花素。在接种量、培养温度、光照条件和培养时间都相同的条件下，同样的藏红花细胞系采用该二步法生产的藏红花素是一步法的3.04倍。首次进行了稀土元素对藏红花细胞生长和藏红花素合成影响的研究，结果表明0.06mM La3+对藏红花细胞生长的促进效果最好，与不添加任何稀土元素的对照处理相比较，细胞生物量提高了58％。0.06mM MRE对藏红花素合成促进的效果最好，与不添加任何稀土元素的对照处理相比较，藏红花素产量提高了4倍。与导流筒气升式和筛网导流筒气升式生物反应器相比，采用鼓泡塔式反应器培养，藏红花细胞的生长速度快，藏红花素的产量也比较高，因此更有利于规模化生产藏红花素。藏红花细胞在2.5L鼓泡式生物反应器中悬浮培养 28d后，细胞生物量为9.6gDW／L，增长3.2倍，藏红花素的产量为0.459／L。在愈伤组织再分化成苗的过程中对藏红花材料进行了脱毒处理，并首次采用间接 ELISA和RT-PCR方法检测了国内外报道的藏红花五种病毒。经过ELISA和RFPCR检测，在本研究筛选的藏红花愈伤组织和再分化芽中没发现有TuMV、TRV、CMV和Potyvirus病毒。通过优化一些物理和化学因素，提高藏红花胚性愈伤的增殖系数和分化能力，首次建立了高效稳定的再生体系。在优化的条件下藏红花胚性愈伤的繁殖系数为12g／g，生物量为13.3 g DW／L；出芽率高达84.5％，远远高于国内报道的1％和国外报道的20％。 |
| 英文摘要 | The induction and screening of Crocus sativus L. callus, the regulation and optimization of the cell growth and crocin biosynthesis, and the cell culture in 2.5L bioreactors were investigated. In addition, the induction of virus-free shoot and callus as well as the regulation of the initiation, growth and differentiation of embryogenic callus of Crocus sativus L. were also studied. Those investigations were fundament to solve the problems of limited availability and virus infection of Crocus sativus L. The callus inductions of the corm, shoot, leaf and flower of Crocus sativus L. as well as the leaf and seed of Gardenia jasminoides Ellis were systematically investigated. One simple method was first established to screen the cell line with high crocin biosynthesis ability by the line color and HPLC assay. One cell line (corml) having characteristics of high croin biosynthesis ability, fast growth and not easy browning was screened from 229 cell lines of Crocus sativus L. and 15 cell lines of Gardenia jasminoides Ellis. The suspension culture system was established. The highest biomass reached 12.3g DW7L on 20*1 d. The highest crocin content and production were obtained on 28th d, they were 95.8mg/g and 0.92g /L respectively. The process of croin biosynthesis was semi-associated with the cell growth of Crocus sativus L. Two-stage culture method for producing crocin was first established on the basis of the optimization of the culture medium and conditions of Crocus sativus L.callus. The amount of crocin obtained by two-stage culture method was 3.04 folds of that produced by one-stage method under the same cell line, inoculation size and culture conditions. The effect of rare earth elements on the cell growth of Crocus sativus L. and crocin biosynthesis was investigated. La3+ and MRE could markedly enhance the callus growth and crocin biosynthesis separately. When 0.06mM La3+was added to solidified B5 medium supplemented with 2mg/L NAA, 1 mg/L 6-BA and 300mg/L CH, the callus biomass of 16g DW/L was obtained. When 0.06mM MRE was added to solidified B5 medium supplemented with 2mg/L NAA, 1 mg/L 6-BA and 300mg/L CH, the crocin production reached 64.5mg/L. Comparing with the internal loop airlift bioreactor and internal loop airlift bioreactor with sifter riser, the bubble column bioreactor was more suitable for Crocus sativus L. cell culture and crocin production. When 5% inoculation size cells were cultured in 2.5 L bubble column bioreactor for 28d, 9.6g DW/L biomass and 0.45g/L crocin production were obtained. The methods of indirect ELIS A and RT-PCR were first used for the detection of all the virus which could infect Crocus sativus L. No TuMV and potyvirus were found in the callus and differentiation shoot of Crocus sativus L. by the method of indirect ELIS A. No TuMV, potyvirus, CMV and TRV were found in the callus and differentiation shoot of Crocus sativus L. This result indicated that the virus-free Crocus sativus L. callus and shoot induced in this study were obtained. The conditions for the proliferation of Crocus sativus L. embryogenic callus and the shoot germination of the callus were optimized. The highest biomass and proliferation factor reached 13.3 g DW/L and 12 g/g respectively. The germination capacity of the callus reached 84.5%, while the highest germination capacity of Crocus sativus L. callus was reported only about 20% abroad. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 173 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1421]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 陈书安. 藏红花细胞和组织培养的过程调控[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004. |
入库方式： OAI收割
来源：过程工程研究所
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