马铃薯的组织培养及耐盐诱变育种研究
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摘要
马铃薯(Solanum tuberosum L.)是我国重要的粮食及经济作物,在农业生产和人民生活中占有非常重要的地位,盐渍土壤严重影响了马铃薯的生长和产量。因此,选育耐盐的马铃薯品种,具有重要的经济价值。
本研究以马铃薯中1、中2、中3、中4为实验材料,通过茎尖脱毒作用,用分化培养基(MS+BA 2mg/L+NAA 0.1mg/L+GA30.1 mg/L+3%蔗糖+6%琼脂),建立了四个马铃薯品种的组织培养再生体系。
实验以马铃薯脱毒试管苗茎段为材料,1.0%NaCl为筛选压力,通过60Coγ射线辐照、EMS浸泡及60Coγ射线和EMS复合的方法诱导马铃薯耐盐突变株,并获得了耐1.0%NaCl的马铃薯突变株WL-1。同时本实验研究了60Coγ射线、EMS处理对马铃薯茎段生长及突变率的影响。研究结果表明:小剂量(10Gy)的60Coγ射线能促进马铃薯茎段的生长,随着辐照剂量的增大,对马铃薯试管苗的抑制作用逐渐增强。顶芽的辐射敏感性低于侧芽;60Coγ射线辐照马铃薯茎段的半致死剂量和突变高峰为20Gy,致死剂量为40Gy;小剂量(0.1%)的EMS对茎段损伤极小,而高剂量长时间的EMS处理会导致茎尖或茎段白化,失去生长能力,EMS处理马铃薯茎段的半致死剂量为0.7%+4h,致死剂量为1.0%+3h。
通过试管苗外部指标分析、生化分析及盆栽盐胁迫的方法对耐盐突变株WL-1进行了分析,结果表明:当NaCl浓度高于0.6%时,突变株WL-1的株高、脯氨酸含量、叶绿素含量显著高于对照,而丙二醛含量较低。通过分析WL-1和对照的SDS-PAGE图谱显示:WL-1的一条蛋白表达量明显高于对照。盆栽盐胁迫中,当盐浓度为0.4%时对照株开始出现叶色变黄萎蔫,两者长势差别不大,而当盐浓度大于0.8%时耐盐突变株长势明显好于对照。
用RAPD分子标记方法进一步对突变株WL-1进行DNA水平鉴定,得出了突变株WL-1与中3马铃薯的遗传相似系数为0.703,因此从基因的水平上证明了该株系为突变株。
Potato (Solanum tuberosum L.), which is one of most important food crops in agricultural production plays a very important role in people's life, and the saline soil could seriously affect potatoes’s growth and productivity. Therefore, breeding the salt-tolerant varieties of potatoes does not only have a very important economic value but also an important social value.
In our study, we built up four culture systems of potatoes tissue using potato 1, 2, and 3, in 4 as the material, through meristem tip, and differentiation medium was MS + BA 2mg / L + NAA 0.1mg / L + GA30.1 mg / L +3% +6% sucrose agar.
We use the potatoes stems as the material, 1.0% NaCl as the selection pressure, through ~(60)Coγ-ray radiation、EMS treatment and composite method to induce salt tolerant mutant, and obtained the salt tolerant mutant of 1.0% NaCl named WL-1. At the same time, we studied the effection of ~(60)Coγ-ray radiation、EMS to the growth of potato stems and the mutation rate. Low dose (10Gy) of ~(60)Coγ-ray radiation could promote the stems’growth, with the increase of the radiation dose; the inhibition to the plantlet is also increased. Sensitivity of terminal bud is lower than lateral bud, the suitable doses for potato stems were 20Gy, and lethal dose were 40 Gy. The damage of low dose (0.1%) EMS treatment is minimal, and the high dose or long time EMS treatment could cause stem tip and stems whitening, lose the ability of growth, the suitable doses and treatment time for potato stems were 0.7%+4h, and lethal dose were 1.0%+3h.
Through outside indexes analysis、biochemical indexes analysis and salt stress of pot cultivation, evaluated the salt tolerance of WL-1. The results showed that: as the NaCl concentration higher than 0.6%, the plant height、proline content、chlorophyll content of salt tolerant mutant WL-1 is significantly higher than control group, MDA content is lower. Results of SDS-PAGE showed that: One of WL-1 expression content is higher than control group.
The RAPD text revealed that the gentic similarity between WL-1 and Zhong 3 was 0.707; so all the evidence mentioned above proved that WL-1 is salt tolerant mutant.
本研究以马铃薯中1、中2、中3、中4为实验材料,通过茎尖脱毒作用,用分化培养基(MS+BA 2mg/L+NAA 0.1mg/L+GA30.1 mg/L+3%蔗糖+6%琼脂),建立了四个马铃薯品种的组织培养再生体系。
实验以马铃薯脱毒试管苗茎段为材料,1.0%NaCl为筛选压力,通过60Coγ射线辐照、EMS浸泡及60Coγ射线和EMS复合的方法诱导马铃薯耐盐突变株,并获得了耐1.0%NaCl的马铃薯突变株WL-1。同时本实验研究了60Coγ射线、EMS处理对马铃薯茎段生长及突变率的影响。研究结果表明:小剂量(10Gy)的60Coγ射线能促进马铃薯茎段的生长,随着辐照剂量的增大,对马铃薯试管苗的抑制作用逐渐增强。顶芽的辐射敏感性低于侧芽;60Coγ射线辐照马铃薯茎段的半致死剂量和突变高峰为20Gy,致死剂量为40Gy;小剂量(0.1%)的EMS对茎段损伤极小,而高剂量长时间的EMS处理会导致茎尖或茎段白化,失去生长能力,EMS处理马铃薯茎段的半致死剂量为0.7%+4h,致死剂量为1.0%+3h。
通过试管苗外部指标分析、生化分析及盆栽盐胁迫的方法对耐盐突变株WL-1进行了分析,结果表明:当NaCl浓度高于0.6%时,突变株WL-1的株高、脯氨酸含量、叶绿素含量显著高于对照,而丙二醛含量较低。通过分析WL-1和对照的SDS-PAGE图谱显示:WL-1的一条蛋白表达量明显高于对照。盆栽盐胁迫中,当盐浓度为0.4%时对照株开始出现叶色变黄萎蔫,两者长势差别不大,而当盐浓度大于0.8%时耐盐突变株长势明显好于对照。
用RAPD分子标记方法进一步对突变株WL-1进行DNA水平鉴定,得出了突变株WL-1与中3马铃薯的遗传相似系数为0.703,因此从基因的水平上证明了该株系为突变株。
Potato (Solanum tuberosum L.), which is one of most important food crops in agricultural production plays a very important role in people's life, and the saline soil could seriously affect potatoes’s growth and productivity. Therefore, breeding the salt-tolerant varieties of potatoes does not only have a very important economic value but also an important social value.
In our study, we built up four culture systems of potatoes tissue using potato 1, 2, and 3, in 4 as the material, through meristem tip, and differentiation medium was MS + BA 2mg / L + NAA 0.1mg / L + GA30.1 mg / L +3% +6% sucrose agar.
We use the potatoes stems as the material, 1.0% NaCl as the selection pressure, through ~(60)Coγ-ray radiation、EMS treatment and composite method to induce salt tolerant mutant, and obtained the salt tolerant mutant of 1.0% NaCl named WL-1. At the same time, we studied the effection of ~(60)Coγ-ray radiation、EMS to the growth of potato stems and the mutation rate. Low dose (10Gy) of ~(60)Coγ-ray radiation could promote the stems’growth, with the increase of the radiation dose; the inhibition to the plantlet is also increased. Sensitivity of terminal bud is lower than lateral bud, the suitable doses for potato stems were 20Gy, and lethal dose were 40 Gy. The damage of low dose (0.1%) EMS treatment is minimal, and the high dose or long time EMS treatment could cause stem tip and stems whitening, lose the ability of growth, the suitable doses and treatment time for potato stems were 0.7%+4h, and lethal dose were 1.0%+3h.
Through outside indexes analysis、biochemical indexes analysis and salt stress of pot cultivation, evaluated the salt tolerance of WL-1. The results showed that: as the NaCl concentration higher than 0.6%, the plant height、proline content、chlorophyll content of salt tolerant mutant WL-1 is significantly higher than control group, MDA content is lower. Results of SDS-PAGE showed that: One of WL-1 expression content is higher than control group.
The RAPD text revealed that the gentic similarity between WL-1 and Zhong 3 was 0.707; so all the evidence mentioned above proved that WL-1 is salt tolerant mutant.
引文
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