EMS和秋水仙素对寒兰根状茎化学诱变的研究
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摘要
以甲基磺酸乙酯(EMS)和秋水仙素(colchicine)为诱变剂,以寒兰(Cymbidium kanran Makino)根状茎为材料,探讨2种诱变剂对寒兰根状茎生长的影响,筛选出半致死剂量,并分析了2种诱变剂对根状茎的生理、分子水平上的诱变效应,结果表明：
1、2种化学诱变剂对寒兰根状茎的生长均有一定的抑制作用,随着浓度升高或时间的延长,根状茎的褐化率和死亡率升高,褐化率始终小于死亡率。总体表现为根状茎早期生长缓慢,先出现褐化现象,后逐渐导致根状茎死亡。
2、计算了2种诱变剂对寒兰根状茎的半致死剂量,它与浓度和时间都有关系,可作为寒兰离体化学诱变的参考剂量。秋水仙素1d、2d、3d、4d的半致死剂量分别为1.05%、0.81%、0.59%和0.38%；EMS1d、2d、3d、4d的半致死剂量分别为0.607%、0.237%、0.145%和0.119%。可见,EMS对根状茎的致死率影响高于秋水仙素。
3、经2种诱变剂处理后,根状茎可溶性糖含量与对照组相比均有不同程度的增加,且达到显著性差异,随着2种诱变剂浓度的增加和时间延长而升高。随着EMS浓度的增加和时间延长,可溶性蛋白含量呈现出先升高后降低的趋势,与对照组具有显著性差异；随着秋水仙素浓度的增加和时间延长,可溶性蛋白含量都有降低,但变化不明显,仅比对照减少了7.84%-20.2%,没有统计学意义上的差异。
4、经2种诱变剂处理后,根状茎MDA含量与对照组相比均有不同程度的增加,呈现出先升高后降低并趋于稳定的趋势。经过不同浓度的EMS和秋水仙素诱变处理后,根状茎的H202含量与对照相比均有不同程度的增加,总体上是随着浓度的增加而增大。经过不同时间的EMS和秋水仙素诱变处理后,随着时间的延长,H202含量出现先上升后降低趋势。
5、经2种诱变剂处理后,根状茎的SOD、POD活性与对照组相比均有不同程度的增加。随着EMS浓度的增加、时间的延长,SOD、POD活性出现先上升后降低的趋势。随着秋水仙素浓度的增加、时间的延长,SOD、POD、CAT活性出现逐渐上升的趋势。
6、诱变后的根状茎和对照之间的基因组ISSR扩增图谱条带均有差异性,EMS多态性条带比例为25%,秋水仙素无多态性条带。
Using EMS, colchicine as mutagenic agents, and using the rhizomes of Cymbidium kanran Makino as material, we studied the effects on growth of rhizomes treated with mutagenic agents, chosed the semi-lethal dose (LD50), analyzed the physiological and gene changes of rhizomes treated with mutagenic agents. The results were as follows:
1. Two mutagenic agents could resrain the growth of rhizomes, with the increase of concentration and the prolonged time, the brown ratio and death ratio rised, the brown ratio were always less than death ratio.
2. Calculating the LD50on rhizomes of mutagenic agents, it related to concentration and time, the results could be the referenced dosage for chemical mutation of Cymbidium kanran Makino.The LD50of1,2,3,4d, respectively,1.05%,0.81%,0.59%,0.38%for colchicine. The LD50of1,2,3,4d, respectively,0.607%,0.237%,0.145%and0.119%for EMS.
3. After two mutagenic agents treatment,the soluble sugar contents of rhizomes all increased in various degrees compared with the control group.The soluble sugar contents of rhizomes were significantly different from the control group. With the increase of concentration and the prolonged time of EMS, the soluble protein contents rised frist, and then decreased. The soluble protein contents of rhizomes were significantly different from the control group. With the increase of concentration and the prolonged time of colchicine, the soluble protein contents decreased by7.84%-20.2%,the change was not significant.
4. After two mutagenic agents treatment,the MDA contents of rhizomes all increased in various degrees compared with the control,but rised frist and then decreased. With the increase of concentration of EMS and colchicine, the H2O2contents of rhizomes rised. The H2O2contents of rhizomes were significantly different from the control. With the increase of treating time of EMS and colchicine, the H2O2contents of rhizomes rised frist and then decreased.
5. After two mutagenic agents treatment,the SOD、POD activity of rhizomes all increased in various degrees compared with the control. With the increase of concentration and the prolonged time of EMS, the SOD、POD activity of rhizomes rised frist and then decreased. With the increase of concentration and the prolonged time of colchicine, the SOD、POD、CAT activity of rhizomes rised gradually.
6. The ISSR-PCR amplified genome bands of rhizomes treated with two mutagenic agents were different from normal rhizomes. The polymorphic rate was25%between rhizomes from EMS treatment and control.There were no polymorphism bands between rhizomes from colchicine treatment and control.
1、2种化学诱变剂对寒兰根状茎的生长均有一定的抑制作用,随着浓度升高或时间的延长,根状茎的褐化率和死亡率升高,褐化率始终小于死亡率。总体表现为根状茎早期生长缓慢,先出现褐化现象,后逐渐导致根状茎死亡。
2、计算了2种诱变剂对寒兰根状茎的半致死剂量,它与浓度和时间都有关系,可作为寒兰离体化学诱变的参考剂量。秋水仙素1d、2d、3d、4d的半致死剂量分别为1.05%、0.81%、0.59%和0.38%；EMS1d、2d、3d、4d的半致死剂量分别为0.607%、0.237%、0.145%和0.119%。可见,EMS对根状茎的致死率影响高于秋水仙素。
3、经2种诱变剂处理后,根状茎可溶性糖含量与对照组相比均有不同程度的增加,且达到显著性差异,随着2种诱变剂浓度的增加和时间延长而升高。随着EMS浓度的增加和时间延长,可溶性蛋白含量呈现出先升高后降低的趋势,与对照组具有显著性差异；随着秋水仙素浓度的增加和时间延长,可溶性蛋白含量都有降低,但变化不明显,仅比对照减少了7.84%-20.2%,没有统计学意义上的差异。
4、经2种诱变剂处理后,根状茎MDA含量与对照组相比均有不同程度的增加,呈现出先升高后降低并趋于稳定的趋势。经过不同浓度的EMS和秋水仙素诱变处理后,根状茎的H202含量与对照相比均有不同程度的增加,总体上是随着浓度的增加而增大。经过不同时间的EMS和秋水仙素诱变处理后,随着时间的延长,H202含量出现先上升后降低趋势。
5、经2种诱变剂处理后,根状茎的SOD、POD活性与对照组相比均有不同程度的增加。随着EMS浓度的增加、时间的延长,SOD、POD活性出现先上升后降低的趋势。随着秋水仙素浓度的增加、时间的延长,SOD、POD、CAT活性出现逐渐上升的趋势。
6、诱变后的根状茎和对照之间的基因组ISSR扩增图谱条带均有差异性,EMS多态性条带比例为25%,秋水仙素无多态性条带。
Using EMS, colchicine as mutagenic agents, and using the rhizomes of Cymbidium kanran Makino as material, we studied the effects on growth of rhizomes treated with mutagenic agents, chosed the semi-lethal dose (LD50), analyzed the physiological and gene changes of rhizomes treated with mutagenic agents. The results were as follows:
1. Two mutagenic agents could resrain the growth of rhizomes, with the increase of concentration and the prolonged time, the brown ratio and death ratio rised, the brown ratio were always less than death ratio.
2. Calculating the LD50on rhizomes of mutagenic agents, it related to concentration and time, the results could be the referenced dosage for chemical mutation of Cymbidium kanran Makino.The LD50of1,2,3,4d, respectively,1.05%,0.81%,0.59%,0.38%for colchicine. The LD50of1,2,3,4d, respectively,0.607%,0.237%,0.145%and0.119%for EMS.
3. After two mutagenic agents treatment,the soluble sugar contents of rhizomes all increased in various degrees compared with the control group.The soluble sugar contents of rhizomes were significantly different from the control group. With the increase of concentration and the prolonged time of EMS, the soluble protein contents rised frist, and then decreased. The soluble protein contents of rhizomes were significantly different from the control group. With the increase of concentration and the prolonged time of colchicine, the soluble protein contents decreased by7.84%-20.2%,the change was not significant.
4. After two mutagenic agents treatment,the MDA contents of rhizomes all increased in various degrees compared with the control,but rised frist and then decreased. With the increase of concentration of EMS and colchicine, the H2O2contents of rhizomes rised. The H2O2contents of rhizomes were significantly different from the control. With the increase of treating time of EMS and colchicine, the H2O2contents of rhizomes rised frist and then decreased.
5. After two mutagenic agents treatment,the SOD、POD activity of rhizomes all increased in various degrees compared with the control. With the increase of concentration and the prolonged time of EMS, the SOD、POD activity of rhizomes rised frist and then decreased. With the increase of concentration and the prolonged time of colchicine, the SOD、POD、CAT activity of rhizomes rised gradually.
6. The ISSR-PCR amplified genome bands of rhizomes treated with two mutagenic agents were different from normal rhizomes. The polymorphic rate was25%between rhizomes from EMS treatment and control.There were no polymorphism bands between rhizomes from colchicine treatment and control.
引文
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