EMS处理诱发大白菜变异的研究
摘要
作物种质资源是品种改良的物质基础。种质资源匮乏是目前大白菜品种改良中面临的最大问题,创造具有丰富遗传基础的育种材料是大白菜育种工作者研究的重点之一。人工诱变可使自然突变率提高千倍以上,从而使人们定向地创造和筛选变异成为可能。但人工诱变存在着方向不确定性和多为隐性突变,将人工诱变与小孢子培养相结合,可达到基因迅速纯合和缩短突变鉴定时间的目的。
本研究以6份不同基因型大白菜为试验材料,采用EMS为诱变剂,分别进行了种子诱变、花蕾诱变结合游离小孢子培养、游离小孢子诱变与培养以及花粉诱变处理方法的研究,确定了不同处理方法适宜的EMS浓度和处理时间,探讨了种子处理后种子和幼苗防御系统相关生理生化指标的变化,并对蕾期授粉自交后代和游离小孢子培养后代进行了田间形态学鉴定及分子辅助鉴定,初步获得了一定类型的变异材料。主要研究结果如下:
1.以‘99-2’和‘津育80’大白菜种子为材料,研究了不同EMS浓度浸泡种子诱变4h后种子的萌发情况、电导率变化及种子与幼苗的SOD、POD活性和MDA的含量变化。结果表明,EMS浓度范围为0~1.0%,随着EMS浓度的增大,种子的发芽势、发芽率逐渐降低,幼苗根尖和叶缘出现褐化并逐渐加剧,成活率下降;种子浸出液电导率及种子和幼苗的MDA含量均逐渐增大;EMS对种子及幼苗SOD、POD活性影响表现为低浓度促进、高浓度抑制。初步确定大白菜种子诱变处理的适宜EMS浓度为0.4%~0.6%。
2.以大白菜‘85-1’的花蕾为处理对象,研究了不同浓度的EMS浸泡花蕾不同时间小孢子膨大以及产胚成苗情况的影响,结果表明随着EMS浓度增大或处理时间延长,小孢子膨大率和产胚率逐渐降低,畸形胚率增加;以0.1%的EMS处理花蕾15min和30min可得到一定数量的活性胚并能够正常发育成植株,变异率较高,1%浓度处理15min也可以得到少量的活性胚胎但是再生植株能力差,未发育成苗。
3.对大白菜‘85-1’‘A01’‘A06’3个自交系的小孢子进行了EMS诱变处理并培养,对小孢子培养过程的细胞学观察结果表明,随着培养基中EMS含量的增加和诱变培养时间的延长,小孢子膨大率出现了明显的下降,各浓度时间下均未形成成熟的胚状体。
4.用EMS-石蜡油处理‘85-1’‘A12’的花粉,研究花粉离体萌发半致死的EMS浓度和处理时间。结果表明:常温下存于液体石蜡油中的大白菜花粉在1h内萌发率不受影响,之后随储存时间延长而降低;随着EMS浓度的增大或时间的延长,花粉萌发率降低;适宜大白菜花粉诱变的EMS诱变剂量为0.15%,处理时为45~60min。
5..对诱变后代进行了常规和分子鉴定,从中筛选了不同类型的突变体。其中种子诱变M1代无显著性状差异,M2代出现能够直接观察到的不同差异类型。花粉诱变、花蕾浸泡诱变后获得的小孢子植株差异较明显,且变异类型较多。
Germplasm resources are the foundation of crop varietal improvement. The scarcity of germplasm resources is the most serious problem in Chinese cabbage breeding, the creation of breeding materials with rich genetic basis is the emphasis of research for breeding scientists. Natural mutation ratio can be improved more than a thousand times by artificially inducing variation, which makes it possible to create and screen orientation variation. The direction of artificial mutagenesis is uncertainty, and most of mutants are recessive. Combined artificial mutagenesis and microspore culture together, we can get homozygous genes rapidly and reduce the time used for mutations identification.
In this experiment, different genotypes of Chinese cabbage were used as materials , the methods of seed mutagenesis,bud mutation combined with microspore culture, isolated microspore mutation and culture and pollen mutagenesis were investigated by EMS inducing. The suitable EMS concentration and processing time of different treatment methods were determined. The variation in related physiological and biochemical characteristics on the defense system in seeds and seedlings after dealing with seeds were discussed. Furthermore, The morphological identification and molecular profiling were conducted in offsprings of buds pollination selfing and free microspore culturing, resulting in some types of mutation materials. The main research results are as follows:
1. Using Chinese cabbage genotypes’99-2’and‘Jinyu 80’, the effects of 4h seed-treatment with different EMS concentration solutions on several major characteristics such as seeds germination, seeds conductivity and the activity of SOD, POD and MDA in seeds and seedlings of Chinese cabbage were studied in the present paper. The results showed that EMS concentrations ranged from 0-0.1%, with EMS concentration increasing, seed germination potential, seed germination rate and seedling surviving rate gradually decreased, the root tip and cotyledon edge of seedlings became brown gradually. The conductivity of seeds and the MDA content of seeds and seedlings were both increased with EMS concentration increasing. The SOD, POD activity of seeds and seedlings was promoted by low EMS concentration solution treatment and inhibited by high EMS concentration solution treatment. The optimal EMS concentrations inducing Chinese cabbage seeds mutations ranged from 0.4% to 0.6%.
2. Using buds of Chinese cabbage '85-1' as materials,the effects of different EMS concentrations on the rate of microspore swollen and the condition of embryos alive and regeneration were studied. The results showed that with EMS concentration and treatment time increasing, both the rate of microspore swollen and embryonic generation reduced gradually, and the number of deformity embryos increased. Treating buds by 0.1% EMS for 15min or 30min, we can get quite some energetic embryos which can be regenerated into seedlings as normal, with a high variation ratio. But if the EMS concentration was reached 1%, with treatment time of 15min, only a few activity embryos were produced with a low regeneration ratio and without seedlings at all.
3. mutagenesis microspore of three inbred lines in vitro induced by EMS then cultibated , In the process of the microspore culture , though cytology observation, we can get the result that the rate of microspore expands is significantly reduced along with the increase of concentration and the extension of mutagenesis time, nothing embryos was got at last。
4. Pollens of two different varieties of Chinese cabbage were treated with EMS-paraffin oil solution, half lethal EMS concentration and treatment time of pollen germination in vitro were studied. The result showed that the germination rate of pollen stored in liquid paraffin oil was not affected within 1h, and then was decreased with prolonged storage. With the increasing EMS concentration or the mutagenesis time gradually, the rate of pollen germination reduced. The suitable EMS concentration induced for pollen mutagenesis was 0.015%,with treatment time of 45~60min.
5. By routine identification and molecular appraisal, sdifferent types of mutants from mutagenesis offsprings were screened.After the seeds mutagenesis, there was no significant differences in traits variation in M1 generation, but some mutants can be observed in M2 generation. There were large differences in phenotypic variation among the plants obtained from microspore culture of pollens mutagenesis and buds mutagenesis, with multiple mutant types.
本研究以6份不同基因型大白菜为试验材料,采用EMS为诱变剂,分别进行了种子诱变、花蕾诱变结合游离小孢子培养、游离小孢子诱变与培养以及花粉诱变处理方法的研究,确定了不同处理方法适宜的EMS浓度和处理时间,探讨了种子处理后种子和幼苗防御系统相关生理生化指标的变化,并对蕾期授粉自交后代和游离小孢子培养后代进行了田间形态学鉴定及分子辅助鉴定,初步获得了一定类型的变异材料。主要研究结果如下:
1.以‘99-2’和‘津育80’大白菜种子为材料,研究了不同EMS浓度浸泡种子诱变4h后种子的萌发情况、电导率变化及种子与幼苗的SOD、POD活性和MDA的含量变化。结果表明,EMS浓度范围为0~1.0%,随着EMS浓度的增大,种子的发芽势、发芽率逐渐降低,幼苗根尖和叶缘出现褐化并逐渐加剧,成活率下降;种子浸出液电导率及种子和幼苗的MDA含量均逐渐增大;EMS对种子及幼苗SOD、POD活性影响表现为低浓度促进、高浓度抑制。初步确定大白菜种子诱变处理的适宜EMS浓度为0.4%~0.6%。
2.以大白菜‘85-1’的花蕾为处理对象,研究了不同浓度的EMS浸泡花蕾不同时间小孢子膨大以及产胚成苗情况的影响,结果表明随着EMS浓度增大或处理时间延长,小孢子膨大率和产胚率逐渐降低,畸形胚率增加;以0.1%的EMS处理花蕾15min和30min可得到一定数量的活性胚并能够正常发育成植株,变异率较高,1%浓度处理15min也可以得到少量的活性胚胎但是再生植株能力差,未发育成苗。
3.对大白菜‘85-1’‘A01’‘A06’3个自交系的小孢子进行了EMS诱变处理并培养,对小孢子培养过程的细胞学观察结果表明,随着培养基中EMS含量的增加和诱变培养时间的延长,小孢子膨大率出现了明显的下降,各浓度时间下均未形成成熟的胚状体。
4.用EMS-石蜡油处理‘85-1’‘A12’的花粉,研究花粉离体萌发半致死的EMS浓度和处理时间。结果表明:常温下存于液体石蜡油中的大白菜花粉在1h内萌发率不受影响,之后随储存时间延长而降低;随着EMS浓度的增大或时间的延长,花粉萌发率降低;适宜大白菜花粉诱变的EMS诱变剂量为0.15%,处理时为45~60min。
5..对诱变后代进行了常规和分子鉴定,从中筛选了不同类型的突变体。其中种子诱变M1代无显著性状差异,M2代出现能够直接观察到的不同差异类型。花粉诱变、花蕾浸泡诱变后获得的小孢子植株差异较明显,且变异类型较多。
Germplasm resources are the foundation of crop varietal improvement. The scarcity of germplasm resources is the most serious problem in Chinese cabbage breeding, the creation of breeding materials with rich genetic basis is the emphasis of research for breeding scientists. Natural mutation ratio can be improved more than a thousand times by artificially inducing variation, which makes it possible to create and screen orientation variation. The direction of artificial mutagenesis is uncertainty, and most of mutants are recessive. Combined artificial mutagenesis and microspore culture together, we can get homozygous genes rapidly and reduce the time used for mutations identification.
In this experiment, different genotypes of Chinese cabbage were used as materials , the methods of seed mutagenesis,bud mutation combined with microspore culture, isolated microspore mutation and culture and pollen mutagenesis were investigated by EMS inducing. The suitable EMS concentration and processing time of different treatment methods were determined. The variation in related physiological and biochemical characteristics on the defense system in seeds and seedlings after dealing with seeds were discussed. Furthermore, The morphological identification and molecular profiling were conducted in offsprings of buds pollination selfing and free microspore culturing, resulting in some types of mutation materials. The main research results are as follows:
1. Using Chinese cabbage genotypes’99-2’and‘Jinyu 80’, the effects of 4h seed-treatment with different EMS concentration solutions on several major characteristics such as seeds germination, seeds conductivity and the activity of SOD, POD and MDA in seeds and seedlings of Chinese cabbage were studied in the present paper. The results showed that EMS concentrations ranged from 0-0.1%, with EMS concentration increasing, seed germination potential, seed germination rate and seedling surviving rate gradually decreased, the root tip and cotyledon edge of seedlings became brown gradually. The conductivity of seeds and the MDA content of seeds and seedlings were both increased with EMS concentration increasing. The SOD, POD activity of seeds and seedlings was promoted by low EMS concentration solution treatment and inhibited by high EMS concentration solution treatment. The optimal EMS concentrations inducing Chinese cabbage seeds mutations ranged from 0.4% to 0.6%.
2. Using buds of Chinese cabbage '85-1' as materials,the effects of different EMS concentrations on the rate of microspore swollen and the condition of embryos alive and regeneration were studied. The results showed that with EMS concentration and treatment time increasing, both the rate of microspore swollen and embryonic generation reduced gradually, and the number of deformity embryos increased. Treating buds by 0.1% EMS for 15min or 30min, we can get quite some energetic embryos which can be regenerated into seedlings as normal, with a high variation ratio. But if the EMS concentration was reached 1%, with treatment time of 15min, only a few activity embryos were produced with a low regeneration ratio and without seedlings at all.
3. mutagenesis microspore of three inbred lines in vitro induced by EMS then cultibated , In the process of the microspore culture , though cytology observation, we can get the result that the rate of microspore expands is significantly reduced along with the increase of concentration and the extension of mutagenesis time, nothing embryos was got at last。
4. Pollens of two different varieties of Chinese cabbage were treated with EMS-paraffin oil solution, half lethal EMS concentration and treatment time of pollen germination in vitro were studied. The result showed that the germination rate of pollen stored in liquid paraffin oil was not affected within 1h, and then was decreased with prolonged storage. With the increasing EMS concentration or the mutagenesis time gradually, the rate of pollen germination reduced. The suitable EMS concentration induced for pollen mutagenesis was 0.015%,with treatment time of 45~60min.
5. By routine identification and molecular appraisal, sdifferent types of mutants from mutagenesis offsprings were screened.After the seeds mutagenesis, there was no significant differences in traits variation in M1 generation, but some mutants can be observed in M2 generation. There were large differences in phenotypic variation among the plants obtained from microspore culture of pollens mutagenesis and buds mutagenesis, with multiple mutant types.
引文
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