烤烟品种抗逆性的早期鉴定技术研究
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摘要
烟叶是烟草行业发展的基础,品种是烟叶生产的基础。国家烟草专卖局对烟草育种工作十分重视,将烟草育种工程列为重大战略性课题,以满足中式卷烟发展和中国烟叶生产可持续发展的需要。近年来,我国烟草育种工作虽然取得了明显的成绩,但与中国烟叶生产可持续发展的要求相比还有较大的差距。主要是烟草种质资源研究的系统性不够,对一些重要性状(如抗逆性,品质等)遗传规律的研究鉴定尚不够全面和系统。本文以通过国家审定的烤烟品种为试材,采用平板培养技术和常规技术相结合,系统研究了烤烟品种抗旱性、抗寒性等抗非生物逆境的生理基础,有关结论如下:
1、根据湿热消毒试验结果,确定烤烟成熟种子作为再生体系构建的外植体时,最佳消毒方法是50℃温水浸泡10min,再经70%酒精处理30s,然后用次氯酸钠处理10min。本消毒方法在保证较高种子发芽力的前提下,降低种子污染率,提高出愈率。另外本试验利用平板培养技术垂直竖立获得烟草无菌苗,操作即快捷简便又不受时期限制,具有环境条件易控制、影响小且周期短的特点,比大田试验节省了大量的人力、物力,而且可以准确保持完整根系,对于植物根系研究可行。另外该方法也可以运用到大量品种的抗病性、抗旱性和抗寒性等抗逆性早期筛选和综合评价研究。
2、首次利用垂直平板培养技术验证了不同烤烟品种耐低氮能力,该技术为研究烟草抗逆性的早期综合评价提供了便利。试验表明在低氮胁迫下烤烟根系的数量、总根长、干重、总吸收面积及活性均明显降低;不同烤烟基因型对低氮胁迫的敏感性不同;综合分析,红花大金元和NC82对低氮胁迫的适应性较强,属耐低氮基因型。低氮胁迫下根数、总根长、根系总吸收面积、根系活力下降幅度及根系MDA含量的上升均可用作烤烟早期筛选耐低氮品种的参考指标。
3、低钾胁迫下根系生长受到明显抑制、主根和总根长度及根冠比降低、侧根发生量减少,根直径有降低的变化趋势,但差异没有达到显著水平;低钾胁迫下根毛数量和长度明显增加,而且根毛多在根尖较近处形成。不同烤烟品种根系对低钾胁迫的响应能力具有显著性差异,在缺钾条件下,NC82根系生长受到的抑制作用相对较小。上述结果表明采用平板培养技术可较准确、快捷地对烤烟种质资源的营养特性进行研究。
4、同一烤烟品种在干旱胁迫与正常条件下相比较,其鲜重、主根长、侧根总长、根系活力、叶绿素含量及质膜透性差异显著,其中侧根总长、叶绿素含量、根系活力及质膜透性可作为烤烟抗旱性的敏感指标;不同烤烟品种的抗旱性有明显差异,5个品种抗旱性大小表现为:NC82＞RG11＞红花大金元＞G28＞K326;研究表明通过该项技术可以用于种质资源抗旱性的筛选。
5、采用8℃和5℃低温胁迫处理,研究了不同烤烟品种的耐寒性并对丙二醛含量、根系活力、叶绿素含量等相关生理指标进行了系统分析。结果表明,红花大金元的耐寒性较强,NC82、RG11次之,K326、V2的耐寒性较差。研究表明根系活力、叶绿素含量、丙二醛含量和细胞膜透性等指标对低温变化较敏感,是用于烤烟耐寒性鉴定的有效指标。综合以上试验表明,垂直平板培养技术在研究烟草抗逆性的早期评价上是可行的。
Variety is the foundation of tobacco production, which is the foundation of tobacco industry development. National Tobacco Monopoly Bureau paid great attention to tobacco breeding in order to meet the demands of Chinese-style cigarette and sustainable development of tobacco production. Nowadays, great progress has been made in the tobacco breeding, but there is still a wide gap with the sustainable development. There is a lack of comprehensive research on inheritance of several important characters (anti-adversity and quality). The seed characteristics and physiology of anti-abiotic stress (drought resistance and cold resistance) were studied through the methods of plate culture technique and regular technology with flue-cured tobacco varieties approved by the national committee as materials. The main conclusions are as follows:
1. Based on the results of wet-hot sterilization, it was proved to be the optimal method to sterilize when seeds to be soaked firstly in the aseptic water at 50℃for 10 min, secondly to be immersion in 70% ethanol for 30 s and finally to be soaked in 10% sodium hypochlorite. The method is effective to reduce the ratio of contamination and to improve the germination. The plate culture technique was convenient to get aseptic seedling without restricting of time and the environment was easy to control. So it is not only save the manpower and material resource, but also to keep the integrated roots which are feasible to conduct botanical research. Besides, it is also suitable to be applied to research of early stage screening and comprehensive evaluation on disease resistance, drought resistance and cold resistance of large number of varieties.
2. Low potassium stress restrained the root growth, decreased root/shoot ratio and total length of roots, and inhibited the occurrence of lateral root. But no significant influence was found on root diameter. Root hair number was increased and only occurred near by the root tip. The genotypic variation in response to low-potassium stress was also observed. Compared with other varieties, NC82 was affected least by low potassium stress. The results also showed that biomass of flue-cured tobacco seedling with this agar culture system can serve as an earlier selection criterion for tolerance to low soil potassium content, by which large scale screening of flue-cured tobacco germplasm can be carried out rapidly and efficiently.
3. low potassium stress restrained the root growth, decreased root/shoot ratio and total length of roots, and inhibited the occurrence of lateral root. But no significant influence was found on root diameter. Root hair number was increased and only occurred near by the root tip. The genotypic variation in response to low-potassium stress was also observed. Compared with other varieties, NC82 was affected least by low potassium stress. The results also showed that biomass of flue-cured tobacco seedling with this agar culture system can serve as an earlier selection criterion for tolerance to low soil potassium content, by which large scale screening of flue-cured tobacco germplasm can be carried out rapidly and efficiently.
4. Plant fresh weight, taproot length, total length of lateral root, root system vigor, chlorophyll content and protoplasmic membrane penetrable of a tobacco variety under normal conditions differed remarkably from those of the same variety under osmotic stress treatment. Fresh weight, total length of lateral root and permeability of plasma membrane were the sensitive indexes of drought resistance. In addition, the study indicated that drought resistance in different tobacco varieties was highly variable, and they were in order of NC82>RG11>Hongda> G28>K326. Meanwhile, this plate culture can be applied to screening and evaluation on drought resistance of tobacco germplasm resource.
5. The chilling tolerance and its related physiological indexes of several flue-cured tobacco varieties were investigated at low temperature (8℃and 5℃) stress. The results showed that Hongda had the best chilling tolerance, followed by NC82 and RG11, while K326 and V2 had the least chilling tolerance. The analysis of all correlation indexes made it clear that chilling injury indexes had highly significant correlation with the MDA content, the root activity, the chlorophyll content and the cell membrane permeability. These physiological indexes were sensitive to the change of low temperature. They were suitable parameters for evaluating chilling tolerance.
1、根据湿热消毒试验结果,确定烤烟成熟种子作为再生体系构建的外植体时,最佳消毒方法是50℃温水浸泡10min,再经70%酒精处理30s,然后用次氯酸钠处理10min。本消毒方法在保证较高种子发芽力的前提下,降低种子污染率,提高出愈率。另外本试验利用平板培养技术垂直竖立获得烟草无菌苗,操作即快捷简便又不受时期限制,具有环境条件易控制、影响小且周期短的特点,比大田试验节省了大量的人力、物力,而且可以准确保持完整根系,对于植物根系研究可行。另外该方法也可以运用到大量品种的抗病性、抗旱性和抗寒性等抗逆性早期筛选和综合评价研究。
2、首次利用垂直平板培养技术验证了不同烤烟品种耐低氮能力,该技术为研究烟草抗逆性的早期综合评价提供了便利。试验表明在低氮胁迫下烤烟根系的数量、总根长、干重、总吸收面积及活性均明显降低;不同烤烟基因型对低氮胁迫的敏感性不同;综合分析,红花大金元和NC82对低氮胁迫的适应性较强,属耐低氮基因型。低氮胁迫下根数、总根长、根系总吸收面积、根系活力下降幅度及根系MDA含量的上升均可用作烤烟早期筛选耐低氮品种的参考指标。
3、低钾胁迫下根系生长受到明显抑制、主根和总根长度及根冠比降低、侧根发生量减少,根直径有降低的变化趋势,但差异没有达到显著水平;低钾胁迫下根毛数量和长度明显增加,而且根毛多在根尖较近处形成。不同烤烟品种根系对低钾胁迫的响应能力具有显著性差异,在缺钾条件下,NC82根系生长受到的抑制作用相对较小。上述结果表明采用平板培养技术可较准确、快捷地对烤烟种质资源的营养特性进行研究。
4、同一烤烟品种在干旱胁迫与正常条件下相比较,其鲜重、主根长、侧根总长、根系活力、叶绿素含量及质膜透性差异显著,其中侧根总长、叶绿素含量、根系活力及质膜透性可作为烤烟抗旱性的敏感指标;不同烤烟品种的抗旱性有明显差异,5个品种抗旱性大小表现为:NC82＞RG11＞红花大金元＞G28＞K326;研究表明通过该项技术可以用于种质资源抗旱性的筛选。
5、采用8℃和5℃低温胁迫处理,研究了不同烤烟品种的耐寒性并对丙二醛含量、根系活力、叶绿素含量等相关生理指标进行了系统分析。结果表明,红花大金元的耐寒性较强,NC82、RG11次之,K326、V2的耐寒性较差。研究表明根系活力、叶绿素含量、丙二醛含量和细胞膜透性等指标对低温变化较敏感,是用于烤烟耐寒性鉴定的有效指标。综合以上试验表明,垂直平板培养技术在研究烟草抗逆性的早期评价上是可行的。
Variety is the foundation of tobacco production, which is the foundation of tobacco industry development. National Tobacco Monopoly Bureau paid great attention to tobacco breeding in order to meet the demands of Chinese-style cigarette and sustainable development of tobacco production. Nowadays, great progress has been made in the tobacco breeding, but there is still a wide gap with the sustainable development. There is a lack of comprehensive research on inheritance of several important characters (anti-adversity and quality). The seed characteristics and physiology of anti-abiotic stress (drought resistance and cold resistance) were studied through the methods of plate culture technique and regular technology with flue-cured tobacco varieties approved by the national committee as materials. The main conclusions are as follows:
1. Based on the results of wet-hot sterilization, it was proved to be the optimal method to sterilize when seeds to be soaked firstly in the aseptic water at 50℃for 10 min, secondly to be immersion in 70% ethanol for 30 s and finally to be soaked in 10% sodium hypochlorite. The method is effective to reduce the ratio of contamination and to improve the germination. The plate culture technique was convenient to get aseptic seedling without restricting of time and the environment was easy to control. So it is not only save the manpower and material resource, but also to keep the integrated roots which are feasible to conduct botanical research. Besides, it is also suitable to be applied to research of early stage screening and comprehensive evaluation on disease resistance, drought resistance and cold resistance of large number of varieties.
2. Low potassium stress restrained the root growth, decreased root/shoot ratio and total length of roots, and inhibited the occurrence of lateral root. But no significant influence was found on root diameter. Root hair number was increased and only occurred near by the root tip. The genotypic variation in response to low-potassium stress was also observed. Compared with other varieties, NC82 was affected least by low potassium stress. The results also showed that biomass of flue-cured tobacco seedling with this agar culture system can serve as an earlier selection criterion for tolerance to low soil potassium content, by which large scale screening of flue-cured tobacco germplasm can be carried out rapidly and efficiently.
3. low potassium stress restrained the root growth, decreased root/shoot ratio and total length of roots, and inhibited the occurrence of lateral root. But no significant influence was found on root diameter. Root hair number was increased and only occurred near by the root tip. The genotypic variation in response to low-potassium stress was also observed. Compared with other varieties, NC82 was affected least by low potassium stress. The results also showed that biomass of flue-cured tobacco seedling with this agar culture system can serve as an earlier selection criterion for tolerance to low soil potassium content, by which large scale screening of flue-cured tobacco germplasm can be carried out rapidly and efficiently.
4. Plant fresh weight, taproot length, total length of lateral root, root system vigor, chlorophyll content and protoplasmic membrane penetrable of a tobacco variety under normal conditions differed remarkably from those of the same variety under osmotic stress treatment. Fresh weight, total length of lateral root and permeability of plasma membrane were the sensitive indexes of drought resistance. In addition, the study indicated that drought resistance in different tobacco varieties was highly variable, and they were in order of NC82>RG11>Hongda> G28>K326. Meanwhile, this plate culture can be applied to screening and evaluation on drought resistance of tobacco germplasm resource.
5. The chilling tolerance and its related physiological indexes of several flue-cured tobacco varieties were investigated at low temperature (8℃and 5℃) stress. The results showed that Hongda had the best chilling tolerance, followed by NC82 and RG11, while K326 and V2 had the least chilling tolerance. The analysis of all correlation indexes made it clear that chilling injury indexes had highly significant correlation with the MDA content, the root activity, the chlorophyll content and the cell membrane permeability. These physiological indexes were sensitive to the change of low temperature. They were suitable parameters for evaluating chilling tolerance.
引文
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