二倍体马铃薯耐盐材料筛选及其生理特性表现
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摘要
土壤盐渍化对农业生产的威胁已成为全球性的问题,严重影响到作物的生长和产量。目前马铃薯已成为世界上主要粮食作物之一,其产量仅排在小麦、玉米和水稻之后,占第四位。马铃薯是一种对盐中度敏感的作物,土壤盐渍化给马铃薯的生产造成严重的危害。解决土壤盐渍化问题最简单有效的途径就是耐盐品种选育,由于目前推广应用的马铃薯品种耐盐性较差,使马铃薯耐盐育种工作受到极大的限制,因此耐盐资源的筛选及生理特性的研究对马铃薯耐盐性育种具有非常重要的意义。
本论文针对马铃薯耐盐育种中存在着的实际问题,以适应长日照的富利亚(Solanum phureja,PHU)与窄刀薯(S.stenotomum,STN)杂种(PHU-STN)无性系试管苗为材料,选用四倍体马铃薯耐盐性强的Bintje和耐盐弱的Mainechip为对照,进行NaCl胁迫,通过芽长、根长及其鲜重和干重的测量筛选出具有耐盐性的无性系;通过对筛选出的试管苗进行同浓度不同胁迫天数和同天数不同胁迫浓度的NaCl胁迫及盐胁迫下试管薯生理指标的测定,找出与耐盐性有关的生理指标;数值在统计分析前转化成相对值。对盐胁迫下试管苗叶片细胞超微结构的观察,找到可以用于耐盐性鉴定的细胞学指标;比较同浓度NaCl和NaHCO3胁迫下试管苗的生长情况;利用RAPD技术验证耐盐性筛选的结果。为马铃薯耐盐资源的筛选和培育马铃薯耐盐新品种提供物质基础和理论依据。主要研究结果如下:
1.以芽和根的长度、鲜重、干重这六项生长参数综合评价二倍体马铃薯耐盐性,通过系统聚类分析,将45份材料分为两大类。利用聚类分析并结合平均数筛选出耐盐性比Bintje强的无性系472-1、267-1、89-2-1、188-1、566-1,感盐性比Mainechip强的感盐无性系354-1、507-1、270-2、9-3、138-1,中度耐盐无性系592-3、412-1、474-1、292-1、301-1。
2.经过生理指标的测定得出:
(1)随着盐胁迫天数的延长和盐浓度的增加,相对含水量相对值呈升高的趋势,耐盐无性系升高的幅度小于感盐无性系,在盐胁迫40、60天及20、30mmol·L-1浓度时为二倍体组间差异极显著。
(2)随着盐胁迫天数的延长和盐浓度的增加,叶绿素总含量、叶绿素a含量相对值呈降低的趋势,叶绿素b含量相对值呈没有规律的变化,耐盐无性系降低的幅度小于感盐无性系,二倍体组间叶绿素含量差异极显著。因此叶绿素含量可作为鉴定马铃薯耐盐性强弱的指标。
(3)随着盐胁迫天数的延长,SOD活性的变化呈先升高后降低的趋势,POD活性呈逐渐降低的趋势,耐盐无性系降低的幅度小于感盐无性系,二倍体组间差异极显著;随着盐胁迫浓度的增加,耐盐无性系的SOD活性呈升高的趋势,盐敏感无性系的POD活性呈降低的趋势,耐盐无性系降低的幅度小于感盐无性系,二倍体组间差异极显著;经盐胁迫后,试管薯SOD活性升高了,耐盐无性系升高的幅度高于感盐无性系,POD活性降低了,耐盐无性系降低的幅度小于感盐无性系,二倍体组间的SOD活性、POD活性均达到差异极显著水平。因此,SOD和POD活性与耐盐性有关,可以作为马铃薯耐盐性鉴定的指标。
(4)随着盐胁迫天数的延长和盐浓度的增加,试管苗的相对电导率及丙二醛含量相对值逐渐升高;试管薯经盐胁迫后相对电导率及丙二醛含量升高了,耐盐无性系升高的幅度小于感盐无性系,二倍体组间差异极显著。因此,相对电导率及丙二醛含量可以作为二倍体马铃薯耐盐性筛选的生理指标。
(5)随着盐胁迫天数的延长,脯氨酸含量相对值的变化呈先升高后降低的趋势,耐盐无性系升高的幅度高于感盐无性系,耐盐组可溶性糖含量相对值呈升高的趋势,感盐组可溶性糖含量相对值呈降低的趋势。二倍体组间差异极显著。随着盐胁迫浓度的升高,脯氨酸含量、可溶性糖含量的相对值呈逐渐升高的趋势,耐盐无性系升高的幅度高于感盐无性系。二倍体组间脯氨酸含量差异极显著,二倍体组间可溶性糖含量在10、30 mmol·L-1浓度时差异极显著。经盐胁迫后,试管薯的脯氨酸含量和可溶性糖含量均增加了,耐盐无性系升高的幅度高于感盐无性系,二倍体组间差异极显著。因此,脯氨酸的积累可以作为二倍体马铃薯耐盐性鉴定的生理指标。
(6)随着盐胁迫天数的延长和盐浓度的增加,试管苗的可溶性蛋白含量相对值降低了,然而耐盐无性系和盐敏感无性系之间变化幅度均比较小;经盐胁迫后,试管薯的可溶性蛋白含量降低了。二倍体组间差异极显著。
(7)盐胁迫后,试管薯直径、个数、最大薯重、产量均下降了,说明NaCl严重抑制了马铃薯试管薯的形成和生长,耐盐组下降的幅度小于感盐组,二倍体组间达到差异极显著水平。
(8)试管薯产量与试管薯直径、试管薯薯个数、试管薯最大薯重、SOD、POD活性、脯氨酸含量、可溶性糖含量、可溶性蛋白含量呈极显著正相关,与相对电导率、丙二醛含量呈极显著负相关
3.经盐胁迫后对试管苗叶片超微结构的观察,质膜、叶绿体、线粒体、基粒均发生变化,耐盐无性系受到的破坏小于感盐无性系。质膜、叶绿体、线粒体、基粒均可以作为耐盐性评价的细胞学指标。
4.通过对15份耐盐性不同无性系的盐碱胁迫之间的比较,在10、20、30mmol·L-1NaHCO3胁迫时芽长、芽鲜重、芽干重、根长、根鲜重、根干重的平均值均小于NaCl,说明在同浓度下NaHCO3胁迫要比NaCl严重;在碱胁迫下,耐盐组芽长、芽鲜重、芽干重、根长、根鲜重、根干重的平均值均大于感盐组,二倍体组间差异极显著。由相关分析可知,当浓度为10 mmol·L-1胁迫时,NaCl和NaHCO3在芽长、芽鲜重、芽干重之间显著正相关,说明耐盐无性系耐碱,感盐无性系对碱敏感。
5.通过对15份二倍体马铃薯的RAPD聚类分析,5份耐盐无性系分到一组,感盐无性系和中耐盐无性系之间没有被分开。说明利用生长参数进行耐盐性筛选的系统聚类结果与利用分子手段进行验证的结果基本一致。RAPD聚类分析与主坐标分析将15份二倍体马铃薯各分为4组,两种分类方法的结果基本一致。
The threat of soil salinity to agricultural production has become a global problem, which seriously affects crop growth and yield. At present, potato has become one of the world's major food crops, ranking the fourth in production only after wheat, maize and rice. Potato is a moderately salt-sensitive crop, and soil salinity causes seriously harm to the production of potatoes. Breeding salt-tolerant potatoes is the most simple and effective way to solve the problem of soil salinization. However, potato varieties being widely used are less salt-tolerance, and this makes great restriction to breeding work. So it is in urgent need to screen germplasm for salt tolerance and study their physiological response to salt stress in a potato breeding effort aimed at the development of salt tolerant potato varieties.
In this research, long-day adapted diploid Solanum phureja (PHU)– S. stenotomum (STN) hybrid clones were screened in vitro for salt (NaCI) tolerance, using tetraploid varieties, Bintje (tolerant) and Mainchipe (susceptible), as controls, and three groups of five salt tolerant, five salt susceptible and five mid-salt tolerant clones were set up based on their performance of shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, and root dry weight. Salt-tolerant physiological indicators were found by determinating physiological indices of potato plantlets in vitro at the different levels of NaCI or different treatment periods. Data were expressed in relative values prior to statistical analysis. Cell ultrastructure observation under salt stress was made to find salt-tolerant cellular indicators. Use of RAPD technology was to verify the results of salt-tolerance screening. These results could provide the material foundation and theoretical basis for screening salt-tolerant potato resources and developing new varieties of salt-tolerant potato. The main results are as follows:
1. Forty five diploid clones and two tetraploid controls were partitioned into two groups by cluster analysis based on performance of the six growth parameters, shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, and root dry weight. Five diploid clones, higher in salt tolerance than Bintje, 472-1, 267-1, 89-2-1, 188-1 and 566-1, five diploid clones, more sensitive to salt stress than Mainechip, 354-1, 507-1, 270-2, 9-3 and 138-1, and five diploid inbetween, 592-3, 412-1, 474-1, 292-1 and 301-1, were selected out based on cluster analysis combined with mean value comparison.
2. Main results after physiological index measurement:
(1) With increase in stressed period and NaCI level, the relative value of water content in these diploid potatoes tended to increase, with salt tolerant diploid potatoes being increased less compared with salt sensitive diploid potatoes. The three groups, stressed at 40 and 60 days, or at 20 and 30 mmol·L-1 NaCI, showed highly significant difference.
(2) With increase in stressed period and NaCI level, relative value of total Chlorophyll content and Chlorophyll a content tended to decrease, but the relative value of Chlorophyll b content showed no regular pattern in the. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant difference between the three groups. Therefore, Chlorophyll content can be used as the index of salt tolerance in diploid potatoes.
(3) With increase in the days of salt stress, SOD activity were increased at first and then decreased. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant differences between the three groups. With salt concentration increased, SOD activity of the salt tolerant clones was increased, while POD activity of the salt sensitive clones was reduced. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant differences between the three groups. The SOD activity of the microtuber was increased after salt stress, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. However the POD activity of the microtuber was reduced after salt stress, and the reduced range of the salt tolerant clones was less than that of the salt sensitive clones. There were highly significant differences between the three groups for SOD and POD. Therefore, the activity of SOD and POD can be used as the index of salt tolerance in diploid potatoes.
(4) With the days of salt stress and salt concentration increased, the relative conductivity and relative content of MDA in the plantlets in vitro were increased. The two parameters showed the same trend in microtuber of diploid potatoes after salt stress. However, the increased range of the salt tolerant clones was less than that of the salt sensitive clones. Therefore, the relative conductivity and relative content of MDA can be used as the indices of salt tolerance in selection for salt tolerant diploid potatoes.
(5) With the days of salt stress increased, the relative value of proline content was increased at first and then decreased, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. The relative value of soluble sugar content was increased in the salt tolerant group and decreased in the salt sensitive group. There were highly significant differences in between the three groups. With the concentration of salt stress increased, the relative value of proline content and soluble sugar content were increased, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. The proline content was highly significantly different between the groups, and soluble sugar content of diploid potatoes under stress of 10 and 30 mmol·L-1 NaCI was highly significantly different between the groups. The proline content and soluble sugar content of the microtuber were increased after salt stress. The increases in the salt tolerant clones were higher than those of the salt sensitive clones, and there were highly significant differences between the groups. Therefore, proline content can be used as the physiological index of salt tolerance in diploid potatoes.
(6) With the days and the concentration of salt stress increased, the relative value of soluble protein content in plantlets in vitro was decreased, but the change was small in both the salt tolerant and the salt sensitive clones. The soluble protein content of microtuber was decreased after salt stress. There were highly significant differences between the groups.
(7) The microtuber diameter, number, the largest sized tuber weight and microtuber yield were all decreased after salt stress. It was showed that the formation and growth of the microtuber could be seriously inhibited by NaCl. The decreased range of the salt sensitive clones was higher than that of the salt tolerant clones, and there were highly significant differences between the groups.
(8) The microtuber yield was highly significantly positively correlated to the microtuber diameter, tuber number, the largest sized tuber weight, the activities of SOD and POD, proline content, soluble sugar content and soluble protein content of the microtuber, while highly significantly negatively to relative conductivity and malondialdehyde (MAD) content.
3. The plasma membrane, chloroplast, mitochondria and grana were all changed through the ultrastructure observed of plantlet leaves after salt stress. The destructions of the salt tolerant clones were smaller than those of the salt sensitive clones. Therefore, the plasma membrane, chloroplast, mitochondria and grana could be used as the cytology index of salt tolerance evaluation.
4. The fifteen diploid clones when under stress of 10, 20, 30 mmol ? L-1 NaHCO3 were inhibited more in terms of the shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, and root dry weight than under stress of 10, 20, 30 mmol ? L-1 NaCl, suggesting that NaHCO3 would do more harm to diploid potato than NaCI at the same concentration. At the range of concentration tested, all the six parameters were higher in the salt tolerant group than in the salt sensitive group when stressed under NaHCO3, and highly significantly difference was found between the three groups. When stressed under 10 mmol·L-1 of NaCl and NaHCO3, the diploid potato showed significantly positive correlation for shoot length, shoot fresh weight, and shoot dry weight, suggesting that diploid potatoes resistant to salt are also resistant to alkali.
5. In RAPD cluster analysis, all five clones resistant to salt were assigned to one group, while salt sensitive clones and middle salt-tolerant clones were not well separated. The results demonstrated the use of growth parameters for salt tolerance screening is similar to the use of RAPD data. By RAPD cluster analysis and principal coordinates analysis, the 15 diploid potatoes were all divided into four groups, indicating that the results using the two classification methods were consistent.
本论文针对马铃薯耐盐育种中存在着的实际问题,以适应长日照的富利亚(Solanum phureja,PHU)与窄刀薯(S.stenotomum,STN)杂种(PHU-STN)无性系试管苗为材料,选用四倍体马铃薯耐盐性强的Bintje和耐盐弱的Mainechip为对照,进行NaCl胁迫,通过芽长、根长及其鲜重和干重的测量筛选出具有耐盐性的无性系;通过对筛选出的试管苗进行同浓度不同胁迫天数和同天数不同胁迫浓度的NaCl胁迫及盐胁迫下试管薯生理指标的测定,找出与耐盐性有关的生理指标;数值在统计分析前转化成相对值。对盐胁迫下试管苗叶片细胞超微结构的观察,找到可以用于耐盐性鉴定的细胞学指标;比较同浓度NaCl和NaHCO3胁迫下试管苗的生长情况;利用RAPD技术验证耐盐性筛选的结果。为马铃薯耐盐资源的筛选和培育马铃薯耐盐新品种提供物质基础和理论依据。主要研究结果如下:
1.以芽和根的长度、鲜重、干重这六项生长参数综合评价二倍体马铃薯耐盐性,通过系统聚类分析,将45份材料分为两大类。利用聚类分析并结合平均数筛选出耐盐性比Bintje强的无性系472-1、267-1、89-2-1、188-1、566-1,感盐性比Mainechip强的感盐无性系354-1、507-1、270-2、9-3、138-1,中度耐盐无性系592-3、412-1、474-1、292-1、301-1。
2.经过生理指标的测定得出:
(1)随着盐胁迫天数的延长和盐浓度的增加,相对含水量相对值呈升高的趋势,耐盐无性系升高的幅度小于感盐无性系,在盐胁迫40、60天及20、30mmol·L-1浓度时为二倍体组间差异极显著。
(2)随着盐胁迫天数的延长和盐浓度的增加,叶绿素总含量、叶绿素a含量相对值呈降低的趋势,叶绿素b含量相对值呈没有规律的变化,耐盐无性系降低的幅度小于感盐无性系,二倍体组间叶绿素含量差异极显著。因此叶绿素含量可作为鉴定马铃薯耐盐性强弱的指标。
(3)随着盐胁迫天数的延长,SOD活性的变化呈先升高后降低的趋势,POD活性呈逐渐降低的趋势,耐盐无性系降低的幅度小于感盐无性系,二倍体组间差异极显著;随着盐胁迫浓度的增加,耐盐无性系的SOD活性呈升高的趋势,盐敏感无性系的POD活性呈降低的趋势,耐盐无性系降低的幅度小于感盐无性系,二倍体组间差异极显著;经盐胁迫后,试管薯SOD活性升高了,耐盐无性系升高的幅度高于感盐无性系,POD活性降低了,耐盐无性系降低的幅度小于感盐无性系,二倍体组间的SOD活性、POD活性均达到差异极显著水平。因此,SOD和POD活性与耐盐性有关,可以作为马铃薯耐盐性鉴定的指标。
(4)随着盐胁迫天数的延长和盐浓度的增加,试管苗的相对电导率及丙二醛含量相对值逐渐升高;试管薯经盐胁迫后相对电导率及丙二醛含量升高了,耐盐无性系升高的幅度小于感盐无性系,二倍体组间差异极显著。因此,相对电导率及丙二醛含量可以作为二倍体马铃薯耐盐性筛选的生理指标。
(5)随着盐胁迫天数的延长,脯氨酸含量相对值的变化呈先升高后降低的趋势,耐盐无性系升高的幅度高于感盐无性系,耐盐组可溶性糖含量相对值呈升高的趋势,感盐组可溶性糖含量相对值呈降低的趋势。二倍体组间差异极显著。随着盐胁迫浓度的升高,脯氨酸含量、可溶性糖含量的相对值呈逐渐升高的趋势,耐盐无性系升高的幅度高于感盐无性系。二倍体组间脯氨酸含量差异极显著,二倍体组间可溶性糖含量在10、30 mmol·L-1浓度时差异极显著。经盐胁迫后,试管薯的脯氨酸含量和可溶性糖含量均增加了,耐盐无性系升高的幅度高于感盐无性系,二倍体组间差异极显著。因此,脯氨酸的积累可以作为二倍体马铃薯耐盐性鉴定的生理指标。
(6)随着盐胁迫天数的延长和盐浓度的增加,试管苗的可溶性蛋白含量相对值降低了,然而耐盐无性系和盐敏感无性系之间变化幅度均比较小;经盐胁迫后,试管薯的可溶性蛋白含量降低了。二倍体组间差异极显著。
(7)盐胁迫后,试管薯直径、个数、最大薯重、产量均下降了,说明NaCl严重抑制了马铃薯试管薯的形成和生长,耐盐组下降的幅度小于感盐组,二倍体组间达到差异极显著水平。
(8)试管薯产量与试管薯直径、试管薯薯个数、试管薯最大薯重、SOD、POD活性、脯氨酸含量、可溶性糖含量、可溶性蛋白含量呈极显著正相关,与相对电导率、丙二醛含量呈极显著负相关
3.经盐胁迫后对试管苗叶片超微结构的观察,质膜、叶绿体、线粒体、基粒均发生变化,耐盐无性系受到的破坏小于感盐无性系。质膜、叶绿体、线粒体、基粒均可以作为耐盐性评价的细胞学指标。
4.通过对15份耐盐性不同无性系的盐碱胁迫之间的比较,在10、20、30mmol·L-1NaHCO3胁迫时芽长、芽鲜重、芽干重、根长、根鲜重、根干重的平均值均小于NaCl,说明在同浓度下NaHCO3胁迫要比NaCl严重;在碱胁迫下,耐盐组芽长、芽鲜重、芽干重、根长、根鲜重、根干重的平均值均大于感盐组,二倍体组间差异极显著。由相关分析可知,当浓度为10 mmol·L-1胁迫时,NaCl和NaHCO3在芽长、芽鲜重、芽干重之间显著正相关,说明耐盐无性系耐碱,感盐无性系对碱敏感。
5.通过对15份二倍体马铃薯的RAPD聚类分析,5份耐盐无性系分到一组,感盐无性系和中耐盐无性系之间没有被分开。说明利用生长参数进行耐盐性筛选的系统聚类结果与利用分子手段进行验证的结果基本一致。RAPD聚类分析与主坐标分析将15份二倍体马铃薯各分为4组,两种分类方法的结果基本一致。
The threat of soil salinity to agricultural production has become a global problem, which seriously affects crop growth and yield. At present, potato has become one of the world's major food crops, ranking the fourth in production only after wheat, maize and rice. Potato is a moderately salt-sensitive crop, and soil salinity causes seriously harm to the production of potatoes. Breeding salt-tolerant potatoes is the most simple and effective way to solve the problem of soil salinization. However, potato varieties being widely used are less salt-tolerance, and this makes great restriction to breeding work. So it is in urgent need to screen germplasm for salt tolerance and study their physiological response to salt stress in a potato breeding effort aimed at the development of salt tolerant potato varieties.
In this research, long-day adapted diploid Solanum phureja (PHU)– S. stenotomum (STN) hybrid clones were screened in vitro for salt (NaCI) tolerance, using tetraploid varieties, Bintje (tolerant) and Mainchipe (susceptible), as controls, and three groups of five salt tolerant, five salt susceptible and five mid-salt tolerant clones were set up based on their performance of shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, and root dry weight. Salt-tolerant physiological indicators were found by determinating physiological indices of potato plantlets in vitro at the different levels of NaCI or different treatment periods. Data were expressed in relative values prior to statistical analysis. Cell ultrastructure observation under salt stress was made to find salt-tolerant cellular indicators. Use of RAPD technology was to verify the results of salt-tolerance screening. These results could provide the material foundation and theoretical basis for screening salt-tolerant potato resources and developing new varieties of salt-tolerant potato. The main results are as follows:
1. Forty five diploid clones and two tetraploid controls were partitioned into two groups by cluster analysis based on performance of the six growth parameters, shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, and root dry weight. Five diploid clones, higher in salt tolerance than Bintje, 472-1, 267-1, 89-2-1, 188-1 and 566-1, five diploid clones, more sensitive to salt stress than Mainechip, 354-1, 507-1, 270-2, 9-3 and 138-1, and five diploid inbetween, 592-3, 412-1, 474-1, 292-1 and 301-1, were selected out based on cluster analysis combined with mean value comparison.
2. Main results after physiological index measurement:
(1) With increase in stressed period and NaCI level, the relative value of water content in these diploid potatoes tended to increase, with salt tolerant diploid potatoes being increased less compared with salt sensitive diploid potatoes. The three groups, stressed at 40 and 60 days, or at 20 and 30 mmol·L-1 NaCI, showed highly significant difference.
(2) With increase in stressed period and NaCI level, relative value of total Chlorophyll content and Chlorophyll a content tended to decrease, but the relative value of Chlorophyll b content showed no regular pattern in the. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant difference between the three groups. Therefore, Chlorophyll content can be used as the index of salt tolerance in diploid potatoes.
(3) With increase in the days of salt stress, SOD activity were increased at first and then decreased. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant differences between the three groups. With salt concentration increased, SOD activity of the salt tolerant clones was increased, while POD activity of the salt sensitive clones was reduced. The reduced range of the salt tolerant clones was less than that of the salt sensitive clones, and there were highly significant differences between the three groups. The SOD activity of the microtuber was increased after salt stress, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. However the POD activity of the microtuber was reduced after salt stress, and the reduced range of the salt tolerant clones was less than that of the salt sensitive clones. There were highly significant differences between the three groups for SOD and POD. Therefore, the activity of SOD and POD can be used as the index of salt tolerance in diploid potatoes.
(4) With the days of salt stress and salt concentration increased, the relative conductivity and relative content of MDA in the plantlets in vitro were increased. The two parameters showed the same trend in microtuber of diploid potatoes after salt stress. However, the increased range of the salt tolerant clones was less than that of the salt sensitive clones. Therefore, the relative conductivity and relative content of MDA can be used as the indices of salt tolerance in selection for salt tolerant diploid potatoes.
(5) With the days of salt stress increased, the relative value of proline content was increased at first and then decreased, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. The relative value of soluble sugar content was increased in the salt tolerant group and decreased in the salt sensitive group. There were highly significant differences in between the three groups. With the concentration of salt stress increased, the relative value of proline content and soluble sugar content were increased, and the increased range of the salt tolerant clones was higher than that of the salt sensitive clones. The proline content was highly significantly different between the groups, and soluble sugar content of diploid potatoes under stress of 10 and 30 mmol·L-1 NaCI was highly significantly different between the groups. The proline content and soluble sugar content of the microtuber were increased after salt stress. The increases in the salt tolerant clones were higher than those of the salt sensitive clones, and there were highly significant differences between the groups. Therefore, proline content can be used as the physiological index of salt tolerance in diploid potatoes.
(6) With the days and the concentration of salt stress increased, the relative value of soluble protein content in plantlets in vitro was decreased, but the change was small in both the salt tolerant and the salt sensitive clones. The soluble protein content of microtuber was decreased after salt stress. There were highly significant differences between the groups.
(7) The microtuber diameter, number, the largest sized tuber weight and microtuber yield were all decreased after salt stress. It was showed that the formation and growth of the microtuber could be seriously inhibited by NaCl. The decreased range of the salt sensitive clones was higher than that of the salt tolerant clones, and there were highly significant differences between the groups.
(8) The microtuber yield was highly significantly positively correlated to the microtuber diameter, tuber number, the largest sized tuber weight, the activities of SOD and POD, proline content, soluble sugar content and soluble protein content of the microtuber, while highly significantly negatively to relative conductivity and malondialdehyde (MAD) content.
3. The plasma membrane, chloroplast, mitochondria and grana were all changed through the ultrastructure observed of plantlet leaves after salt stress. The destructions of the salt tolerant clones were smaller than those of the salt sensitive clones. Therefore, the plasma membrane, chloroplast, mitochondria and grana could be used as the cytology index of salt tolerance evaluation.
4. The fifteen diploid clones when under stress of 10, 20, 30 mmol ? L-1 NaHCO3 were inhibited more in terms of the shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, and root dry weight than under stress of 10, 20, 30 mmol ? L-1 NaCl, suggesting that NaHCO3 would do more harm to diploid potato than NaCI at the same concentration. At the range of concentration tested, all the six parameters were higher in the salt tolerant group than in the salt sensitive group when stressed under NaHCO3, and highly significantly difference was found between the three groups. When stressed under 10 mmol·L-1 of NaCl and NaHCO3, the diploid potato showed significantly positive correlation for shoot length, shoot fresh weight, and shoot dry weight, suggesting that diploid potatoes resistant to salt are also resistant to alkali.
5. In RAPD cluster analysis, all five clones resistant to salt were assigned to one group, while salt sensitive clones and middle salt-tolerant clones were not well separated. The results demonstrated the use of growth parameters for salt tolerance screening is similar to the use of RAPD data. By RAPD cluster analysis and principal coordinates analysis, the 15 diploid potatoes were all divided into four groups, indicating that the results using the two classification methods were consistent.
引文
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