马铃薯（Solanum tuberosum L.）愈伤组织培养和耐盐突变体的离体筛选
摘要
本试验以费乌瑞它、东农303、下波帝、鄂1号四个马铃薯品种的茎段、叶片为外植体，以MS+蔗糖3％+琼脂0.8％为基本培养基，进行了愈伤组织培养再生与耐盐突变体筛选的研究。建立了马铃薯叶片、茎段的高效再生体系，研究了高盐直接胁迫与系列盐胁迫两种筛选方法、外植体类型、继代次数、盐的胁迫浓度等因素与耐盐突变体诱导关系的规律，获得了部分耐盐植株；并对耐盐植株进行了耐盐性鉴定。试验结果表明：
马铃薯茎段的愈伤组织发生频率高于叶片的愈伤组织发生频率。从诱导效果和生长状态来看，由叶片诱导愈伤组织的最佳培养基：东农303、鄂1号两品种为MS+6-BA 2.5mg·L~(-1)+NAA 0.2mg·L~(-1)；费乌瑞它为MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1)；夏波帝为MS+6-BA 2.0mg·L~(-1)+NAA 0.2mg·L~(-1)。由茎段诱导愈伤组织的最佳培养基：东农303、夏波帝、鄂1号为MS+6-BA 3.0mg·L~(-1)+NAA 0.01mg·L~(-1)；费乌瑞它为MS+6-BA 3.0mg·L~(-1)+NAA 0.1mg·L~(-1)。
马铃薯不定芽分化对培养基的要求较为严格，不同品种的不同外植体对分化培养基要求不同。从叶片愈伤诱导的出芽天数、诱导率和生长状态来看：费乌瑞它的最佳分化培养基为MS+6-BA 2.5mg·L~(-1)+GA_3 5.0mg·L~(-1)；东农303为MS+6-BA 1.0mg·L~(-1)+IAA 0.1mg·L~(-1)+GA_3 2.5mg·L~(-1)；夏波帝为MS+6-BA 2.5mg·L~(-1)+IAA 0.5mg·L~(-1)+GA_3 2.5mg·L~(-1)；鄂1号为MS+6-BA 2.5mg·L~(-1)+IAA 0.1mg·L~(-1)+GA_3 1.0mg·L~(-1)。由茎段愈伤组织诱导不定芽的最佳分化培养基：费乌瑞它、鄂1号为MS+6-BA 2.0mg·L~(-1)+NAA 0.2mg·L~(-1)；东农303为MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1)；夏波帝为MS+6-BA 2.0mg·L~(-1)+NAA 0.3mg·L~(-1)。
分别以叶片和茎段为外植体，以叶片和茎段诱导的愈伤组织为原始材料，进行耐盐突变体筛选，实验结果表明：不同品种的愈伤组织的耐盐性是有差异的：东农303、鄂1号两品种在细胞水平对NaCl的最大耐受浓度为2.0％；费乌瑞它为1.5％；夏波帝为1.0％。通过系列盐胁迫诱导产生的耐盐愈伤组织，易产生生理适应性，获得的耐盐愈伤组织在无盐的培养基上培养过程中，其耐盐性会减弱或消失。而通过高浓度盐胁迫下产生的耐盐愈伤组织没有此现象，其耐盐性一般不会减弱或消失。
叶片的耐盐愈伤组织，只有继代次数低于10次的在无盐胁迫诱导分化培养基
上有进一步诱导再生植株发生的能力，而在盐胁迫诱导分化培养基中，愈伤组织则
继代次数超过8次即很难继续分化再生;茎段的耐盐愈伤组织，在有盐胁迫与无盐
胁迫的诱导分化培养基上能继续分化的愈伤组织，最高继代次数分别是8代与12
代。因此，减少继代次数，而采用高盐直接胁迫筛选的方法，较为恰当。
经盐水灌溉试验鉴定，再生植株的耐盐性与愈伤组织的耐盐性有一定的差异，
东农303、鄂1号两品种的再生植株的耐盐性与愈伤组织的耐盐性水平相当，而费
乌瑞它和夏波帝两品种的再生植株没有达到愈伤组织的耐盐水平。
In this research, the callus culture, regeneration and salt-tolerant mutants screening were studied by use of four potato varieties, Favorite, Dongnong 303, Sharpdi and E No.1, with stem and leaf as the explants and MS+sucrose 3%+arg 0.8% as the basic medium. The high efficient regeneration system was established, and the effect of different treatments, different explant type, the subculture times and the salt concentration on screen of salt-tolerant mutant were investigated. Some salt-tolerant mutants were abtained and the salt-tolerant characters were identified. The results showed:
The optimal medium for callus induction from leaf discs was found to be MS+6-BA 2.5 mg/L+NAA 0.2 mg/L for "Dongnong 303" and "E No.l", MS+6-BA 2.0 mg/L+NAA 0.1 mg/L for "Favorite" and MS+6-BA 2.0 mg/L+NAA 0.2 mg/L for "Sharpdi", and for callus induction from stem explants was found to be MS+6-BA 3.0 mg/L+NAA 0.01 mg/L for "Dongnong 303", "Sharpdi" and "E No.1", MS+6-BA 3.0 mg/L+NAA 0.1 mg/L for "Favorite".
The optimal medium for adventitious bud differentiation from leaf callus was found to be MS+6-BA 2.5 mg/l+GA3 5.0 mg/L for "Favorite", MS+6-BA 1.0 mg/L+IAA 0.1 mg/L+GA3 2.5 mg/L for "Dongnong 303", MS+6-BA 2.5 mg/L+IAA 0.5 mg/L+GA3 2.5 mg/L for "Sharodi" and MS+6-BA 2.5mg/L+IAA 0.1 mg/L+GA3 1.0 mg/L for "E No.1", and for adventitious bud differentiation from stem callus was found to be MS+6-BA 2.0 mg/L+NAA 0.2 mg/L for "Favorite" and "E No.l", MS+6-BA 2.0mg/L+NAA 0.1 mg/L for "Dongnong 303" and MS+6-BA 2.0 mg/L+NAA 0.3 mg/L for "Sharpdi".
Two methods were tried to select salt tolerant callus. One was screen of salt tolerant callus by directly inducing from explants of leaf discs and stem segments on medium with salt (NaCl). The second was by culture of callus firstly from explants on medium with no salt (NaCl), and then inducing salt tolerant callus from normal callus on medium with different level of salt step by step (progressively). There were differences in
salt-tolerant charaters among callus of different vareities. "Dongnong 303" and "E No.1" were tolerant to 2.0 % NaCl, "Favorite" were tolerant to 1.5% NaCl but "Sharpdi" was inferior in salt tolerane and only tolerant to 1.0% NaCl. The salt tolerance of mutants derived by progressive method was found to be unstable. Only the plantlets regenerated from high level NaCl directly induced salt-tolerant calli showed high tolerance under NaCl stress.
Adventitious buds could not be induced from salt-tolerant leaf discs callus on differentiation medium without salt if the calli were subcultured more than 10 times on medium with salt, or on differentiation medium with salt if the calli were subcultured more than 8 times. However, the adventitious buds could be induced from stem segment calli even the calli were subcultured more than 8 or 12 times on medium with or without salt. Therefore, the optimal mehord to got salt-tolerant mutants were to reduce subculture times and enhance directly salt stress.
There were differences in salt-tolerant characters between the regenerated plantlets and the salt tolerant callus. The salt-tolerant ability of regenerated plantlets was on the same level as the salt tolerant callus only in varieties of "Dongnong 303" and "E No. 1". The salt-tolerant ability of regenerated plantlet was found lower than that of the salt tolerant callus.
马铃薯茎段的愈伤组织发生频率高于叶片的愈伤组织发生频率。从诱导效果和生长状态来看，由叶片诱导愈伤组织的最佳培养基：东农303、鄂1号两品种为MS+6-BA 2.5mg·L~(-1)+NAA 0.2mg·L~(-1)；费乌瑞它为MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1)；夏波帝为MS+6-BA 2.0mg·L~(-1)+NAA 0.2mg·L~(-1)。由茎段诱导愈伤组织的最佳培养基：东农303、夏波帝、鄂1号为MS+6-BA 3.0mg·L~(-1)+NAA 0.01mg·L~(-1)；费乌瑞它为MS+6-BA 3.0mg·L~(-1)+NAA 0.1mg·L~(-1)。
马铃薯不定芽分化对培养基的要求较为严格，不同品种的不同外植体对分化培养基要求不同。从叶片愈伤诱导的出芽天数、诱导率和生长状态来看：费乌瑞它的最佳分化培养基为MS+6-BA 2.5mg·L~(-1)+GA_3 5.0mg·L~(-1)；东农303为MS+6-BA 1.0mg·L~(-1)+IAA 0.1mg·L~(-1)+GA_3 2.5mg·L~(-1)；夏波帝为MS+6-BA 2.5mg·L~(-1)+IAA 0.5mg·L~(-1)+GA_3 2.5mg·L~(-1)；鄂1号为MS+6-BA 2.5mg·L~(-1)+IAA 0.1mg·L~(-1)+GA_3 1.0mg·L~(-1)。由茎段愈伤组织诱导不定芽的最佳分化培养基：费乌瑞它、鄂1号为MS+6-BA 2.0mg·L~(-1)+NAA 0.2mg·L~(-1)；东农303为MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1)；夏波帝为MS+6-BA 2.0mg·L~(-1)+NAA 0.3mg·L~(-1)。
分别以叶片和茎段为外植体，以叶片和茎段诱导的愈伤组织为原始材料，进行耐盐突变体筛选，实验结果表明：不同品种的愈伤组织的耐盐性是有差异的：东农303、鄂1号两品种在细胞水平对NaCl的最大耐受浓度为2.0％；费乌瑞它为1.5％；夏波帝为1.0％。通过系列盐胁迫诱导产生的耐盐愈伤组织，易产生生理适应性，获得的耐盐愈伤组织在无盐的培养基上培养过程中，其耐盐性会减弱或消失。而通过高浓度盐胁迫下产生的耐盐愈伤组织没有此现象，其耐盐性一般不会减弱或消失。
叶片的耐盐愈伤组织，只有继代次数低于10次的在无盐胁迫诱导分化培养基
上有进一步诱导再生植株发生的能力，而在盐胁迫诱导分化培养基中，愈伤组织则
继代次数超过8次即很难继续分化再生;茎段的耐盐愈伤组织，在有盐胁迫与无盐
胁迫的诱导分化培养基上能继续分化的愈伤组织，最高继代次数分别是8代与12
代。因此，减少继代次数，而采用高盐直接胁迫筛选的方法，较为恰当。
经盐水灌溉试验鉴定，再生植株的耐盐性与愈伤组织的耐盐性有一定的差异，
东农303、鄂1号两品种的再生植株的耐盐性与愈伤组织的耐盐性水平相当，而费
乌瑞它和夏波帝两品种的再生植株没有达到愈伤组织的耐盐水平。
In this research, the callus culture, regeneration and salt-tolerant mutants screening were studied by use of four potato varieties, Favorite, Dongnong 303, Sharpdi and E No.1, with stem and leaf as the explants and MS+sucrose 3%+arg 0.8% as the basic medium. The high efficient regeneration system was established, and the effect of different treatments, different explant type, the subculture times and the salt concentration on screen of salt-tolerant mutant were investigated. Some salt-tolerant mutants were abtained and the salt-tolerant characters were identified. The results showed:
The optimal medium for callus induction from leaf discs was found to be MS+6-BA 2.5 mg/L+NAA 0.2 mg/L for "Dongnong 303" and "E No.l", MS+6-BA 2.0 mg/L+NAA 0.1 mg/L for "Favorite" and MS+6-BA 2.0 mg/L+NAA 0.2 mg/L for "Sharpdi", and for callus induction from stem explants was found to be MS+6-BA 3.0 mg/L+NAA 0.01 mg/L for "Dongnong 303", "Sharpdi" and "E No.1", MS+6-BA 3.0 mg/L+NAA 0.1 mg/L for "Favorite".
The optimal medium for adventitious bud differentiation from leaf callus was found to be MS+6-BA 2.5 mg/l+GA3 5.0 mg/L for "Favorite", MS+6-BA 1.0 mg/L+IAA 0.1 mg/L+GA3 2.5 mg/L for "Dongnong 303", MS+6-BA 2.5 mg/L+IAA 0.5 mg/L+GA3 2.5 mg/L for "Sharodi" and MS+6-BA 2.5mg/L+IAA 0.1 mg/L+GA3 1.0 mg/L for "E No.1", and for adventitious bud differentiation from stem callus was found to be MS+6-BA 2.0 mg/L+NAA 0.2 mg/L for "Favorite" and "E No.l", MS+6-BA 2.0mg/L+NAA 0.1 mg/L for "Dongnong 303" and MS+6-BA 2.0 mg/L+NAA 0.3 mg/L for "Sharpdi".
Two methods were tried to select salt tolerant callus. One was screen of salt tolerant callus by directly inducing from explants of leaf discs and stem segments on medium with salt (NaCl). The second was by culture of callus firstly from explants on medium with no salt (NaCl), and then inducing salt tolerant callus from normal callus on medium with different level of salt step by step (progressively). There were differences in
salt-tolerant charaters among callus of different vareities. "Dongnong 303" and "E No.1" were tolerant to 2.0 % NaCl, "Favorite" were tolerant to 1.5% NaCl but "Sharpdi" was inferior in salt tolerane and only tolerant to 1.0% NaCl. The salt tolerance of mutants derived by progressive method was found to be unstable. Only the plantlets regenerated from high level NaCl directly induced salt-tolerant calli showed high tolerance under NaCl stress.
Adventitious buds could not be induced from salt-tolerant leaf discs callus on differentiation medium without salt if the calli were subcultured more than 10 times on medium with salt, or on differentiation medium with salt if the calli were subcultured more than 8 times. However, the adventitious buds could be induced from stem segment calli even the calli were subcultured more than 8 or 12 times on medium with or without salt. Therefore, the optimal mehord to got salt-tolerant mutants were to reduce subculture times and enhance directly salt stress.
There were differences in salt-tolerant characters between the regenerated plantlets and the salt tolerant callus. The salt-tolerant ability of regenerated plantlets was on the same level as the salt tolerant callus only in varieties of "Dongnong 303" and "E No. 1". The salt-tolerant ability of regenerated plantlet was found lower than that of the salt tolerant callus.
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