大百合无性系突变体诱导及其鉴定研究
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摘要
本研究主要以大百合组织培养苗为实验材料,在前期工作的基础上,将材料在30W的紫外线30cm处进行0h,3h,6h,12h照射以及在0、1.0、2.0、3.0、4.0mmol/L的叠氮化钠(pH=3)浸泡3h诱变。继代培养60d后,对诱导材料进行无性系苗的外观形态观察,生理生化测定以及RAPD分析以期寻找发生变异的株系,同时筛选优化处理浓度和建立大百合无性系突变体鉴定方法,为大百合的引种驯化和后期诱变育种研究作前期准备。得到如下结果:
1.继代培养60d后观察发现,在紫外线照射处理3h、6h的大百合组织培养苗的形态变化不明显,与对照相比几乎没有差别,但是12h照射处理发现大百合组织培养苗开始失绿并且变紫,苗体也开始大量的死亡。继代培养60d后在叠氮化钠处理后观察发现,3.0mmol/L处理的大百合有一半小苗逐渐的萎蔫死亡,4.0mmol/L处理的大百合小苗几乎全部都死亡,因此3.0mmol/L为半致死浓度。
2.未经处理的大百合组培苗,叶片呈长形,叶片较薄;紫外线照射后的材料,叶片比较宽大,呈卵圆形,叶片较厚,同时叶片的颜色较浅,可以看见叶脉分布,苗体矮小,气孔变大,随着照射时间的增加现象更加明显;经过不同NaN_3浓度处理后的大百合组织培养苗的叶片长度以及气孔大小没有发生明显的变化。
3.随着紫外线辐照的时间逐渐变长,大百合组织培养苗中CAT、POD的活性呈上升趋势,增长变化明显,与对照呈显著关系。其中SOD活性在辐照6h、12h后含量有所下降;NaN_3处理后测定发现POD的活性随着浓度的增加而增加,而SOD的活性随着浓度的增加反而呈下降趋势,CAT的活性变化未呈规律性变化。
4.本实验对大百合进行RAPD体系建立与优化发现较为理想的各因子用量和扩增程序为:Mg~(2+)(2.0mmol/L)1.0μl、dNIPs(0.2mmol/L)1.0μl、primer(0.6μmol/L)1.3μl、DNA(30ng/μl)1.0μl、Taq酶取1U、10×buffer取2μl,总反应体系为20μl;扩增程序为:在94℃下预变性,然后进行35个循环(94℃变性30s、37℃退火50s、72℃延伸1min),最后在72℃延伸10min下。电泳电压为50V,时间为1h。
5.通过对紫外线和叠氮化钠处理后大百合组织培养苗进行RAPD分析,发现紫外线处理的材料6-23、12-14在RAPD条带上和其他不同,叠氮化钠处理的材料D-3-11组织培养苗在RAPD条带上也出现差异。由此可以推断这三株材料已发生了变异。
6.通过对大百合外观形态观察、生理生化测定和RAPD检测的表现,建立了大百合组织培养无性系突变体鉴定方法。
The seedlings were used as subjects in this investigation.Based on the early-days work,some seedlings were exposed by the UV(30W) in the distance of 30 cm for 0,3,6, 12 hours respectively,and the others were soaked in the solution of sodium azide(pH=3) with the concentration of 0,1.0,2.0,3.0,4.0mmol/L for 3 hours.The seedlings were investigated for the morphous and physiological functions.And the RAPD system of Cardiocrinum giganteum was established and optimized,and the mutations of Cardiocrinum giganteum were identified.The purpose of this investigation was to gaining the seedlings of Cardiocrinum giganteum,and also making preparation for the introduction and mutation breeding of Cardiocrinum giganteum.The results are as follows,
1.Under the treatment of ultraviolet irradiation,the changes of the seedlings were obvious in the comparison with the check group.But considerable seedlings were dead under the treatment of ultraviolet irradiation for 12 hours.
After soaked in the solution of sodium azide,almost 1/3 of the seedlings that soaked in the concentration of 3.0mmol/L were dead,and almost all of the seedlings that soaked in the concentration of 4.0mmol/L were dead.These results are very obvious.
2.The Cardiocrinum giganteum was untreated,the leaves were strip and thin,and the seeding was tall.While Cardiocrinum giganteum was under the treatment of ultraviolet irradiation,the leaves were orbicular-ovate,the color was light,the leaf vein could be seen easily,the seeding was short,and the stoma was bigger.The longer the time of the treatment was,the more manifest all the changes were.The leaf lengths and the stoma sizes of the he seedlings of Cardiocrinum giganteum were not changed so much under different concentrations of sodium azide.
3.The activities of the CAT,POD were increasing in accordance with the accumulation time of the ultraviolet irradiation.The increasing of POD was sharp,and there was significant difference between this group and the CK group.The increasing of SOD,CAT was obvious,and the activity of SOD was decreasing 6h,12 h after the ultraviolet irradiation.
The activities of POD increased in accompany with the increase in concentrations of sodium azide,while the activities of SOD decreased.And the changes in the activities of CAT had no regularity.
4.The RAPD system of Cardiocrinum giganteum was established and optimized,and the ideal factors are as follows,Mg~(2+)(2.0mmol/L)1.0μl、dNTPs(0.2mmol/L)1.0μl、primer(0.6μmol/L)1.3μl、DNA(30ng/μl)1.0μl、Taq(1U)、10×buffer(2μl).The overall reaction system is 20μl.The amplification procedure is as follows,94℃-5 min,than 35 cycles:94℃-30s,37℃-50s,72℃-1 min,final elongation72℃-1 min.The voltage of electrophoresis was 50V,the time consuming is 1 hour.
5.According to the RAPD analysis of treated seedlings of Cardiocrinum giganteum,the PCR bands of two seedlings were different from the others.Based on these results, Inductions have occurred already.
6.According to the changes of the seedlings,the changes of the enzymatic activity and the RAPD analysis,building a method of identification of Mutations in Cloned Line of Cardiocrinum giganteum
1.继代培养60d后观察发现,在紫外线照射处理3h、6h的大百合组织培养苗的形态变化不明显,与对照相比几乎没有差别,但是12h照射处理发现大百合组织培养苗开始失绿并且变紫,苗体也开始大量的死亡。继代培养60d后在叠氮化钠处理后观察发现,3.0mmol/L处理的大百合有一半小苗逐渐的萎蔫死亡,4.0mmol/L处理的大百合小苗几乎全部都死亡,因此3.0mmol/L为半致死浓度。
2.未经处理的大百合组培苗,叶片呈长形,叶片较薄;紫外线照射后的材料,叶片比较宽大,呈卵圆形,叶片较厚,同时叶片的颜色较浅,可以看见叶脉分布,苗体矮小,气孔变大,随着照射时间的增加现象更加明显;经过不同NaN_3浓度处理后的大百合组织培养苗的叶片长度以及气孔大小没有发生明显的变化。
3.随着紫外线辐照的时间逐渐变长,大百合组织培养苗中CAT、POD的活性呈上升趋势,增长变化明显,与对照呈显著关系。其中SOD活性在辐照6h、12h后含量有所下降;NaN_3处理后测定发现POD的活性随着浓度的增加而增加,而SOD的活性随着浓度的增加反而呈下降趋势,CAT的活性变化未呈规律性变化。
4.本实验对大百合进行RAPD体系建立与优化发现较为理想的各因子用量和扩增程序为:Mg~(2+)(2.0mmol/L)1.0μl、dNIPs(0.2mmol/L)1.0μl、primer(0.6μmol/L)1.3μl、DNA(30ng/μl)1.0μl、Taq酶取1U、10×buffer取2μl,总反应体系为20μl;扩增程序为:在94℃下预变性,然后进行35个循环(94℃变性30s、37℃退火50s、72℃延伸1min),最后在72℃延伸10min下。电泳电压为50V,时间为1h。
5.通过对紫外线和叠氮化钠处理后大百合组织培养苗进行RAPD分析,发现紫外线处理的材料6-23、12-14在RAPD条带上和其他不同,叠氮化钠处理的材料D-3-11组织培养苗在RAPD条带上也出现差异。由此可以推断这三株材料已发生了变异。
6.通过对大百合外观形态观察、生理生化测定和RAPD检测的表现,建立了大百合组织培养无性系突变体鉴定方法。
The seedlings were used as subjects in this investigation.Based on the early-days work,some seedlings were exposed by the UV(30W) in the distance of 30 cm for 0,3,6, 12 hours respectively,and the others were soaked in the solution of sodium azide(pH=3) with the concentration of 0,1.0,2.0,3.0,4.0mmol/L for 3 hours.The seedlings were investigated for the morphous and physiological functions.And the RAPD system of Cardiocrinum giganteum was established and optimized,and the mutations of Cardiocrinum giganteum were identified.The purpose of this investigation was to gaining the seedlings of Cardiocrinum giganteum,and also making preparation for the introduction and mutation breeding of Cardiocrinum giganteum.The results are as follows,
1.Under the treatment of ultraviolet irradiation,the changes of the seedlings were obvious in the comparison with the check group.But considerable seedlings were dead under the treatment of ultraviolet irradiation for 12 hours.
After soaked in the solution of sodium azide,almost 1/3 of the seedlings that soaked in the concentration of 3.0mmol/L were dead,and almost all of the seedlings that soaked in the concentration of 4.0mmol/L were dead.These results are very obvious.
2.The Cardiocrinum giganteum was untreated,the leaves were strip and thin,and the seeding was tall.While Cardiocrinum giganteum was under the treatment of ultraviolet irradiation,the leaves were orbicular-ovate,the color was light,the leaf vein could be seen easily,the seeding was short,and the stoma was bigger.The longer the time of the treatment was,the more manifest all the changes were.The leaf lengths and the stoma sizes of the he seedlings of Cardiocrinum giganteum were not changed so much under different concentrations of sodium azide.
3.The activities of the CAT,POD were increasing in accordance with the accumulation time of the ultraviolet irradiation.The increasing of POD was sharp,and there was significant difference between this group and the CK group.The increasing of SOD,CAT was obvious,and the activity of SOD was decreasing 6h,12 h after the ultraviolet irradiation.
The activities of POD increased in accompany with the increase in concentrations of sodium azide,while the activities of SOD decreased.And the changes in the activities of CAT had no regularity.
4.The RAPD system of Cardiocrinum giganteum was established and optimized,and the ideal factors are as follows,Mg~(2+)(2.0mmol/L)1.0μl、dNTPs(0.2mmol/L)1.0μl、primer(0.6μmol/L)1.3μl、DNA(30ng/μl)1.0μl、Taq(1U)、10×buffer(2μl).The overall reaction system is 20μl.The amplification procedure is as follows,94℃-5 min,than 35 cycles:94℃-30s,37℃-50s,72℃-1 min,final elongation72℃-1 min.The voltage of electrophoresis was 50V,the time consuming is 1 hour.
5.According to the RAPD analysis of treated seedlings of Cardiocrinum giganteum,the PCR bands of two seedlings were different from the others.Based on these results, Inductions have occurred already.
6.According to the changes of the seedlings,the changes of the enzymatic activity and the RAPD analysis,building a method of identification of Mutations in Cloned Line of Cardiocrinum giganteum
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