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天山雪莲新种质创制方法的研究
王卉
学位类型硕士
导师王晓军 ; 宁慧霞
2012-05
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业有机化学
关键词天山雪莲 花药培养 植株再生 莴苣 原生质体融合
摘要以天山雪莲花药为材料,系统研究了其愈伤诱导与再生体系建立的过程,对花药培养阶段的影响因素进行了探讨,并对天山雪莲和莴苣原生质体分离的条件进行了优化,在此基础上对天山雪莲与莴苣原生质体进行融合,为天山雪莲新种质选育奠定了基础,实验结果如下 1. 天山雪莲花药培养最适宜的花期是花蕾期,比盛花期诱导率高,且诱导出的愈伤组织质量好。在进行低温预处理7天后的诱导率较好,褐变率较低,综合效果最好。天山雪莲花药培养MS培养基附加激素组合2,4-D 0.5mg/L+6-BA 1mg/L诱导天山雪莲花药愈伤的效果最好,诱导率达到34%,显著高于其他组合。 2. 天山雪莲种子最佳消毒处理为75% C2H5OH处理50s ,0.5% HgC12处理6min, 15%H2O2处理30min,无菌水冲洗6次,发芽率和污染率分别为46.15%和45%。 3. 天山雪莲原生质体分离最优的方案为1.5%纤维素酶,1.0%果胶酶,0.2%离析酶,9%甘露醇,在25℃黑暗条件下振荡酶解12h的原生质体产量最高为9.073×105个•g-1 ,原生质体活力为87.95%,有活力的原生质体数目为7.98×105个•g-1。 4. 天山雪莲在继代培养10到12天左右进行酶解分离原生质体是最佳的。原生质体产量为8.89×105个•g-1 ,原生质体活力为87.1%。 5. 以莴苣无菌苗的幼叶为材料,研究了不同的分离材料、质壁分离时间、苗龄、酶液组合、酶解时间及酶液渗透压等对原生质体分离效果的影响。结果表明:幼叶在含有13%甘露醇的CPW溶液里质壁分离2h,获得的原生质体产量存活率均高于其他质壁分离时间,为5.04×105个•g-1;苗龄为20d的幼叶分离得到的原生质体质量高,原生质体呈圆形、内含物多、活力强;酶液组合为1.0%纤维素酶 + 0.2%离析酶,25℃黑暗条件下酶解12h可获得大量有较高活力的原生质体;酶解液中添加浓度为9%的甘露醇较适合原生质体分离。 6. PGE4000诱导天山雪莲与莴苣进行原生质体融合的最适浓度为30%,诱导融合率为8.3%。
其他摘要The tissue culture and regeneration system were systematically studied by using anther of Saussurea involucrate Kar. et Kir. as meterial. And the influence of the whole process were researched detailedly. Meanwhile, primary study on studies on intergeneric somatic hybridization between Saussurea involucrate Kar. et Kir and Lactuca sativa L. By protoplast fusion.The results are as follow: 1. The most suitable period of culture is bud stage,its mutation rate is higher than that in flowering,and its induced callus has good quality;The induction rate is better after 7 days low-temperature pretreatment ,browning rate lower; MS medium supplemented with hormones contain 2,4-D 0.5mg/L+6-BA 1mg/L to induces callus were best. The induction rate reached 34%, and significantly higher than other combinations 2. The most suitable disinfection method for the seeds of Saussurea involucrate is 75% C2H5OH treating 50s, 0.5% HgCl2 treating 6min, 15%H2O2 treating 30min, and washing by germfree water 6 times. The rate of shoot is 46.15%, the rate of pollution is 45%. 3. The experiment indicated that the best enzyme solution for isolating protoplasts was 1.5%Cellulase+1%Pectinase+0.2%Macerozyme, pH5.85.The digestion condition was incubated for 12 hours on a rotary shaker at 40 rpm in dark condition at 25℃.The field and viability of protoplasts were9.073×105 cells /g and 87.95%,dynamic number of protoplast were7.98×105个•g-1 . 4. Enzymatically isolated protoplasts is best after Saussurea involucrate in the subculture around 10-12 days,The field and viability of protoplasts were 8.89×105 cells /g and 87.1%. 5. Lactuca sativa L. was used to study the effections of material resources,plasmolysis time, seedling age, enzyme combination, enzymolysis time and ostimum pressure on protoplast isolation. The result showed that when young leaves were plasmolysed in CPW solution which contained 13% mannitol for 2h, the yield of protoplast and its survival rate were higher than those of other plasmolysis time.protoplasts quality is higher using the yong leaves that ege is 20d than the oher eges . The protoplasm takes the shape of circular, with many inclusion in it and have strong vitality; The enzyme concent ration was 1.0% cellulase + 0.2% macerozyme, isolated ezyme. A large amount of high-active protoplast could be obtained by enzymolysis for 12h at 25℃ under dark conditions. Minstra with 9% of manitol added was uitable for protoplast dissociation 6. Hybrid cells of Saussurea involucrate Kar. et Kir and Lactuca sativa L. Were obtained using protoplast fusion by PEG method,The best fusion condition 30% PEG(4000) can obtain the hybrid cell,the fusion rate is 8.3%.
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4389
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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王卉. 天山雪莲新种质创制方法的研究[D]. 北京. 中国科学院研究生院,2012.
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