低温干旱胁迫对烟草种子发芽和幼苗生长的影响及提高其抗寒抗旱性的研究
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摘要
烟草(Nicotiana tabacum L.)起源于热带,对水分要求很高。我国大部分烟区尤其是西南山区,干旱缺水常成为制约烟草种植和品质形成的主要因素。另外,烟草是喜温作物,对低温胁迫耐受力较差。低温冷害影响烟草种子发芽、出苗和幼苗生长,最终降低烟叶的质量和产量。因此,提高烟草种子和幼苗的耐寒和耐旱性具有重要意义。本文分别对干旱和低温胁迫下20个烟草品种的耐寒和耐旱性进行了鉴定。对不同耐寒和耐旱品种生理生化特性和叶片细胞超微结构的变化进行了测定和观察。然后,尝试用浸种处理来提高烟草种子及幼苗的抗寒和抗旱性。最后,利用功能高分子材料聚N-异丙基丙烯酰胺(PNIPAm)和聚(2-丙烯酰胺-2-甲基丙磺酸)(PAMPS)来开发出新的智能型烟草丸化种子,以提高种子及幼苗的抗寒和抗旱性。主要研究结果如下：
1.对20个烟草品种发芽和苗期的耐寒性进行了鉴定。在变温(20-30℃)和低温(11℃)胁迫下测定了种子发芽特性以及幼苗素质等指标,将各性状低温与常温下测定值的比值作为耐寒性评价指标。相关性分析表明,相对发芽率、相对发芽指数、相对地上部高、相对幼苗干重之间显著相关,是较好的耐寒性评价指标；通过聚类分析,将20个品种分为耐寒型(MS云烟85、NC102、云烟97、TN86-8和红花大金元)、中等耐寒型(NC55、RGH51.巴斯玛11号、V2、NC297、云烟201和云烟202)和低温敏感型(MD-609、MS云烟87、MS K326、云烟203、云烟100、G-28、K346和TN90)3类。研究结果将为烟草抗寒性机理研究和育种提供选材依据。
2.对20个烟草品种发芽和苗期的耐旱性进行了鉴定。测定了正常供水和15%聚乙二醇(PEG-6000)溶液模拟干旱条件下烟草种子发芽特性、幼苗生长和苗期生理变化,结合各性状在正常供水与干旱胁迫下相对值,将20个品种分为耐旱型(MS云烟85、NC102、云烟97、TN86-8和红花大金元)、中等耐旱型(NC55、NC297、V2、云烟202、RGH51、巴斯玛11号、云烟201、TN90和K346)和干旱敏感型(MD-609、MS云烟87、MS K326、云烟203、云烟100和G-28)3类。相关性分析表明,相对发芽指数、相对活力指数、相对幼苗鲜重和相对幼苗干重之间显著相关,是较好的耐旱性评价指标。
3.根据聚类分析结果分别选用低温敏感和干旱敏感品种、中等耐寒性和中等耐旱性品种以及耐寒性和耐旱性品种,测定其幼苗可溶性蛋白、可溶性总糖、H2O2、O2和叶绿素含量变化,并使用透射电镜对叶片细胞进行观察。结果表明,低温和干旱胁迫下幼苗可溶性蛋白、可溶性总糖、H2O2、O2-浓度升高,而叶绿素a和b浓度降低。耐寒(旱)性品种在低温和干旱胁迫下幼苗可溶性蛋白和可溶性糖浓度高于低温(干旱)敏感品种,耐寒(旱)性品种在低温和干旱胁迫下幼苗H2O2、O2-、叶绿素a和b浓度低于低温(干旱)敏感品种。低温和干旱胁迫下幼苗叶肉细胞中出现质壁分离,叶绿体向细胞中央移动,同时伴随有嗜锇颗粒增多和淀粉颗粒减少现象,并且这种变化在低温(干旱)敏感品种中比耐寒(旱)性品种更为明显。
4.研究了不同药剂浸种处理对11℃低温逆境下不同耐寒力烟草种子发芽和幼苗生长的影响。采用不同浓度的外源脯氨酸、水杨酸、氯化钙、甘油、二甲基亚砜、乙二醇、聚乙二醇、丙二醇、多效唑、脱落酸浸种处理低温敏感品种MS K326和耐寒品种红花大金元种子,测定了低温逆境下不同烟草品种发芽及幼苗生长和生理生化指标变化。结果表明,脯氨酸、水杨酸、氯化钙、二甲基亚砜和聚乙二醇5种药剂浸种能显著促进烟草种子发芽,提高了低温逆境下幼苗根长、苗高、幼苗干鲜重以及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,其中10g/L脯氨酸、120mg/L水杨酸、15mg/L氯化钙、30ml/L二甲基亚砜和100ml/L聚乙二醇综合效果较好,可以作为提高烟草种子及幼苗抗寒性的处理方法。
5.研究了不同药剂浸种处理对干旱胁迫下不同耐旱力烟草种子发芽和幼苗生长的影响。采用不同浓度的外源多效唑、氯化钙、脯氨酸和水杨酸浸种处理干旱敏感品种MS K326和耐旱品种红花大金元种子,用15%聚乙二醇(PEG-6000)溶液模拟干旱条件,测定了干旱胁迫下不同烟草品种种子发芽及幼苗生长和生理生化指标变化。结果表明：多效唑(PP333)浸种对干旱胁迫下红花大金元和MS K326种子发芽和幼苗生长有一定抑制作用,但是能提高干旱胁迫下幼苗APX、CAT、POD和SOD活性。氯化钙、脯氨酸和水杨酸3种药剂浸种能显著促进红花大金元和MS K326种子发芽,提高干旱胁迫下幼苗根长、全苗长、幼苗干鲜重以及APX、CAT、POD和SOD活性。其中80mg/L水杨酸浸种效果最好,可以作为提高烟草种子及幼苗抗旱性的处理方法。
6.利用在种衣剂中添加温敏材料N-异丙基丙烯酰胺与甲基丙烯酸丁酯共聚物(PNIPAm-co-BMA),制备了一种智能型烟草丸化种子以提高其抗寒性。将水杨酸作为测试样品装载进PNIPAm-co-BMA,装载水杨酸的PNIPAm-co-BMA置于清水中,测定了变温条件下水杨酸从PNIPAm-co-BMA中释放速率的变化。结果显示,水杨酸从PNIPAm-co-BMA中释放速率随清水温度的变化而相应变化。最后,将装载水杨酸的PNIPAm-co-BMA粉末添加到种衣剂中用于低温敏感品种MS K326和耐寒品种红花大金元种子丸化,测定了11℃低温胁迫下丸化种子的发芽及幼苗生长和生理生化指标变化。结果表明,温敏材料丸化型种子能够显著提高两个品种种子发芽率、发芽指数、幼苗根长、幼苗干重以及POD活性,并且显著降低MDA含量。采用温敏材料控制水杨酸进行释放的智能丸化种子可以提高烟草种子和幼苗的抗寒性。
7.研究以2-丙烯酰胺-2-甲基丙磺酸(AMPS)为单体,过硫酸钾(KPS)为引发剂,N,N’-亚甲基双丙烯酰胺(Bis)为交联剂,采用溶液聚合法制备了聚(2-丙烯酰胺-2-甲基丙磺酸)高吸水树脂(PAMPS树脂),并对配方进行了优化。然后,对PAMPS树脂的红外光谱、吸水速率、保水性能以及在土壤和污泥中的降解率等特性进行了测定。最后,将干燥PAMPS粉末在水杨酸(SA)溶液中充分溶胀,干燥、粉碎后得到装载SA的PAMPS粉末,并在模拟干旱条件下,观察了装载SA的PAMPS粉末作为种衣剂抗旱成份用于干旱敏感品种MS K326和耐旱品种红花大金元种子丸化,对干旱胁迫下烟草丸化种子出苗以及幼苗生长的影响。结果表明,中和度为60%,反应条件为55℃(7h)时AMPS同KPS和Bis最佳配比为1g：0.0004g：0.001g,在此条件下合成的PAMPS高吸水树脂室温下在去离子水和0.9%NaCl溶液中吸液倍率分别为4306g/g及373g/g,吸液率远高于其他吸水树脂。在此条件下聚合的PAMPS高吸水树脂的吸液速率、保水能力以及在土壤和污泥中的降解率等特性优良。将装载SA的PAMPS粉末用于烟草种子丸化,显著提高了干旱胁迫下2个烤烟品种种子的出苗率、发芽指数、活力指数、幼苗根长和全苗长以及幼苗干重、鲜重,是一种可行的提高烟草种子及幼苗抗旱能力的种子处理方法。
Tobacco (Nicotiana tabacum L.) originated from tropical areas and very sensitive to drought stress. Tobacco seeds and seedlings often suffered from drought stress in China especially in southwestern mountain area. Meanwhile, chilling injury in tobacco is also a common problem in China, it affects seed germination and seedling growth of tobacco, finally causes the loss of tobacco quality and yield. It is important to improve drought and chilling tolerance of tobacco seeds and seedlings.
The drought and chilling tolerance of20tobacco varieties at germination and seedling stages were investigated by determining traits of germination, seedling quality and some physiological characteristics. The physiological and ultrastructural changes of tobacco varieties with different tolerances under drought and chilling stress were investigated. Then, the treatment of seed soaking was used to improve the drought and chilling tolerance of tobacco seeds and seedlings. Finally, the functional materials of PNIPAm (poly (N-isopropylacrylamide)) and PAMPS (poly (2-(acrylamido)-2-methylpropanesulfonic acid)) were used to develop new intelligent tobacco seed pellets to improve their drought and chilling tolerance. The main results were as follows:
1. The chilling tolerance of20tobacco varieties at germination and seedling growth stages was investigated by determining germination characteristics and seedling quality at normal temperature (20～30℃) and low temperature (11℃) stress. The ratios of trait values at low temperature to those at normal temperature were used to assess chilling tolerance. After the cluster analysis, tobacco varieties were clustered into three groups:chilling tolerance (MS Yunyan85, NC102, Yunyan97, TN86-8and Honghuadajinyuan), intermediate chilling tolerance (NC55, RGH51, Bashima11, V2, NC297, Yunyan201and Yunyan202) and chilling sensitivity (MD-609, MS Yunyan87, MS K326, Yunyan203, Yunyan100, G-28, K346and TN90). The correlation analyses showed that the relative values of germination percentage, germination index, vigor index and malonaldehyde content were significantly correlated with each other; they were suitable parameters for evaluating chilling tolerance. The results will provide basis for material selection during research of cold resistance mechanism and breeding in tobacco.
2. The drought tolerance of20tobacco varieties at germination and seedling growth stages was investigated by determining germination characteristics and seedling quality at normal conditions and simulated drought stress by15%polyethylene glycol (PEG-6000) solution. The ratios of trait values at low temperature to those at normal temperature were used to assess drought tolerance. Tobacco varieties were clustered into three groups:drought tolerance(MS Yunyan85, NC102, Yunyan97, TN86-8and Honghuadajinyuan), intermediate drought tolerance (NC55, NC297, V2, Yunyan202, RGH51、Bashima11, Yunyan201, TN90and K346) and drought sensitivity (MD-609, MS Yunyan87, MS K326, Yunyan203, Yunyan100and G-28). The correlation analyses showed that the relative values of germination index, vigor index, seedling fresh and dry weight were significantly correlated with each other; they were suitable parameters for evaluating drought tolerance.
3. The physiological and ultrastructural changes of tobacco varieties with different chilling/drought tolerances under chilling/drought stress were investigated. The results indicated that the concentrations of H2O2, O2-, total soluble sugar and soluble protein were increased under chilling/drought stress, while the values of chlorophyll a and chlorophyll b were decreased in all varieties. The concentrations of total soluble sugar and protein in chilling/drought tolerant variety were higher than chilling/drought sensitive variety; the concentrations of H2O2, O2-, chlorophyll a and b in chilling/drought tolerant variety were lower than those in chilling/drought sensitive variety. The separations of cell membranes and chloroplasts from cell wall along with an increase in the number of lipid droplets as well as the depletion of starch granules were observed in all tobacco varieties under the chilling/drought stress, and these ultrastructural changes in chilling/drought sensitive variety were more visible than chilling/drought tolerant and intermediate chilling/drought tolerant varieties.
4. The effects of tobacco seeds soaked in different agents on seed germination and growth characteristics were researched. Seeds of chilling sensitivity variety MS K326and chilling tolerance variety Honghuadajinyuan were soaked in different concentration solutions of exogenous proline, salicylic acid, calcium chloride, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, propylene glycol, paclobutrazol and abscisic acid. Then germination, seedling growth and physiological and biochemical changes under cold temperature stress were measured. The results showed that the germination parameters, root length, seedling length, seedling fresh and dry weight were improved, superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities of tobacco seedlings were enhanced after the treatments with proline, salicylic acid, calcium chloride, dimethyl sulfoxide and polyethylene glycol. The treatments of10g/L proline,120mg/L salicylic acid,15mg/L calcium chloride,30ml/L dimethyl sulfoxide and100ml/L polyethylene glycol had better effects, it suggests that these treatments can be used to improve chilling tolerance of tobacco seeds and seedlings.
5. The effects of tobacco seeds soaked in different agents on seed germination and growth characteristics were researched. Seeds of drought sensitivity variety MS K326and drought tolerance variety Honghuadajinyuan were soaked in different concentration solutions of paclobutrazol, calcium chloride, proline and salicylic acid. Then, the germination, seedling growth and physiological changes under drought stress (simulating with15%polyethylene glycol6000) were measured. The results showed that the germination and seeding growth were inhibited; however, the activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in seedlings were enhanced after treating with paclobutrazol (PP333). The germination characteristics, root length, seedling length, seedling fresh and dry weight were improved; the activities of APX, CAT, POD and SOD were enhanced in two tobacco varieties after treating with calcium chloride, proline and salicylic acid. Among all treatments, the treatment of80mg/L salicylic acid had the best effects, suggesting that this treatment could be used to improve drought tolerance of tobacco seeds and seedlings.
6. A kind of "intelligent" tobacco seed pellets was developed to improve their chilling tolerance by adding thermo-sensitive PNIPAm-co-BMA (poly (N-isopropylacrylamide-co-butyl methacrylate)) hydrogel. SA (salicylic acid) was used as a testing drug and was loaded into PNIPAm-co-BMA, its release rate from PNIPAAm-co-BMA undergoing temperature changes in water were measured. The results showed that the SA release rate was changed from PNIPAAm-co-BMA hydrogel in water when the temperature of the water changed. Finally, tobacco seeds of chilling sensitivity variety MS K326and chilling tolerance variety Honghuadajinyuan were pelleted by the SA-loaded PNIPAm-co-BMA, and then the germination characteristics, seedling quality and some physiological parameters of the seed pellets and seedlings were measured after a chilling stress at11℃. The results showed that the germination percentage (GP), germination index (GI), root length, seedling dry weight and the activities of POD (peroxidase) were significantly increased and the concentrations of MDA (malondialdehyde) were significantly decreased in the treatments using "intelligent" seed pellets comparing with the seeds without SA or PNIPAm-co-BMA in two tobacco varieties. It indicated that the "intelligent" seed pellets pelleted with SA-loaded PNIPAm-co-BMA could be used to improve their chilling tolerance.
7. A kind of superabsorbent polymer poly (2-(acrylamido)-2-methylpropanesulfonic acid)(PAMPS) was prepared by method of solution polymerization using2-acrylamido-2-methyl-propanesulfonic acid (AMPS) as monomer, potassium peroxydisulfate (KPS) as initiator, N,N'-methylene-bis-acrylamide (Bis) as cross-linker. The absorbency, water-keeping capacity and biodegradability of the PAMPS polymer were studied. Then the PAMPS polymer, which was loaded with SA (salicylic acid), was used for pelleting tobacco seeds of drought sensitivity variety MS K326and drought tolerance variety Honghuadajinyuan. The germination and growth experiments of these pelleting seeds were studied with two tobacco varieties. The germination characteristics and seedling quality were measured after a drought stress for16d. The optimized polymerization conditions were obtained as follows:mass concentrations of initiator and cross-linker compared to the amount of monomer are0.04%and0.1%respectively under neutralization degree was60%, and reaction temperature (time) was55℃(7hours). The absorbencies of PAMPS in deionized water and0.9NaCl aqueous solutions were4306g/g and373g/g under these conditions. The PAMPS polymer had good performance of absorbency, water-keeping capacity and biodegradability. The results of germinaton and seedling growth experiments showed that these kinds of pelleting seeds could significantly improve the drought tolerance of the tobacco seeds and seedlings.
1.对20个烟草品种发芽和苗期的耐寒性进行了鉴定。在变温(20-30℃)和低温(11℃)胁迫下测定了种子发芽特性以及幼苗素质等指标,将各性状低温与常温下测定值的比值作为耐寒性评价指标。相关性分析表明,相对发芽率、相对发芽指数、相对地上部高、相对幼苗干重之间显著相关,是较好的耐寒性评价指标；通过聚类分析,将20个品种分为耐寒型(MS云烟85、NC102、云烟97、TN86-8和红花大金元)、中等耐寒型(NC55、RGH51.巴斯玛11号、V2、NC297、云烟201和云烟202)和低温敏感型(MD-609、MS云烟87、MS K326、云烟203、云烟100、G-28、K346和TN90)3类。研究结果将为烟草抗寒性机理研究和育种提供选材依据。
2.对20个烟草品种发芽和苗期的耐旱性进行了鉴定。测定了正常供水和15%聚乙二醇(PEG-6000)溶液模拟干旱条件下烟草种子发芽特性、幼苗生长和苗期生理变化,结合各性状在正常供水与干旱胁迫下相对值,将20个品种分为耐旱型(MS云烟85、NC102、云烟97、TN86-8和红花大金元)、中等耐旱型(NC55、NC297、V2、云烟202、RGH51、巴斯玛11号、云烟201、TN90和K346)和干旱敏感型(MD-609、MS云烟87、MS K326、云烟203、云烟100和G-28)3类。相关性分析表明,相对发芽指数、相对活力指数、相对幼苗鲜重和相对幼苗干重之间显著相关,是较好的耐旱性评价指标。
3.根据聚类分析结果分别选用低温敏感和干旱敏感品种、中等耐寒性和中等耐旱性品种以及耐寒性和耐旱性品种,测定其幼苗可溶性蛋白、可溶性总糖、H2O2、O2和叶绿素含量变化,并使用透射电镜对叶片细胞进行观察。结果表明,低温和干旱胁迫下幼苗可溶性蛋白、可溶性总糖、H2O2、O2-浓度升高,而叶绿素a和b浓度降低。耐寒(旱)性品种在低温和干旱胁迫下幼苗可溶性蛋白和可溶性糖浓度高于低温(干旱)敏感品种,耐寒(旱)性品种在低温和干旱胁迫下幼苗H2O2、O2-、叶绿素a和b浓度低于低温(干旱)敏感品种。低温和干旱胁迫下幼苗叶肉细胞中出现质壁分离,叶绿体向细胞中央移动,同时伴随有嗜锇颗粒增多和淀粉颗粒减少现象,并且这种变化在低温(干旱)敏感品种中比耐寒(旱)性品种更为明显。
4.研究了不同药剂浸种处理对11℃低温逆境下不同耐寒力烟草种子发芽和幼苗生长的影响。采用不同浓度的外源脯氨酸、水杨酸、氯化钙、甘油、二甲基亚砜、乙二醇、聚乙二醇、丙二醇、多效唑、脱落酸浸种处理低温敏感品种MS K326和耐寒品种红花大金元种子,测定了低温逆境下不同烟草品种发芽及幼苗生长和生理生化指标变化。结果表明,脯氨酸、水杨酸、氯化钙、二甲基亚砜和聚乙二醇5种药剂浸种能显著促进烟草种子发芽,提高了低温逆境下幼苗根长、苗高、幼苗干鲜重以及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,其中10g/L脯氨酸、120mg/L水杨酸、15mg/L氯化钙、30ml/L二甲基亚砜和100ml/L聚乙二醇综合效果较好,可以作为提高烟草种子及幼苗抗寒性的处理方法。
5.研究了不同药剂浸种处理对干旱胁迫下不同耐旱力烟草种子发芽和幼苗生长的影响。采用不同浓度的外源多效唑、氯化钙、脯氨酸和水杨酸浸种处理干旱敏感品种MS K326和耐旱品种红花大金元种子,用15%聚乙二醇(PEG-6000)溶液模拟干旱条件,测定了干旱胁迫下不同烟草品种种子发芽及幼苗生长和生理生化指标变化。结果表明：多效唑(PP333)浸种对干旱胁迫下红花大金元和MS K326种子发芽和幼苗生长有一定抑制作用,但是能提高干旱胁迫下幼苗APX、CAT、POD和SOD活性。氯化钙、脯氨酸和水杨酸3种药剂浸种能显著促进红花大金元和MS K326种子发芽,提高干旱胁迫下幼苗根长、全苗长、幼苗干鲜重以及APX、CAT、POD和SOD活性。其中80mg/L水杨酸浸种效果最好,可以作为提高烟草种子及幼苗抗旱性的处理方法。
6.利用在种衣剂中添加温敏材料N-异丙基丙烯酰胺与甲基丙烯酸丁酯共聚物(PNIPAm-co-BMA),制备了一种智能型烟草丸化种子以提高其抗寒性。将水杨酸作为测试样品装载进PNIPAm-co-BMA,装载水杨酸的PNIPAm-co-BMA置于清水中,测定了变温条件下水杨酸从PNIPAm-co-BMA中释放速率的变化。结果显示,水杨酸从PNIPAm-co-BMA中释放速率随清水温度的变化而相应变化。最后,将装载水杨酸的PNIPAm-co-BMA粉末添加到种衣剂中用于低温敏感品种MS K326和耐寒品种红花大金元种子丸化,测定了11℃低温胁迫下丸化种子的发芽及幼苗生长和生理生化指标变化。结果表明,温敏材料丸化型种子能够显著提高两个品种种子发芽率、发芽指数、幼苗根长、幼苗干重以及POD活性,并且显著降低MDA含量。采用温敏材料控制水杨酸进行释放的智能丸化种子可以提高烟草种子和幼苗的抗寒性。
7.研究以2-丙烯酰胺-2-甲基丙磺酸(AMPS)为单体,过硫酸钾(KPS)为引发剂,N,N’-亚甲基双丙烯酰胺(Bis)为交联剂,采用溶液聚合法制备了聚(2-丙烯酰胺-2-甲基丙磺酸)高吸水树脂(PAMPS树脂),并对配方进行了优化。然后,对PAMPS树脂的红外光谱、吸水速率、保水性能以及在土壤和污泥中的降解率等特性进行了测定。最后,将干燥PAMPS粉末在水杨酸(SA)溶液中充分溶胀,干燥、粉碎后得到装载SA的PAMPS粉末,并在模拟干旱条件下,观察了装载SA的PAMPS粉末作为种衣剂抗旱成份用于干旱敏感品种MS K326和耐旱品种红花大金元种子丸化,对干旱胁迫下烟草丸化种子出苗以及幼苗生长的影响。结果表明,中和度为60%,反应条件为55℃(7h)时AMPS同KPS和Bis最佳配比为1g：0.0004g：0.001g,在此条件下合成的PAMPS高吸水树脂室温下在去离子水和0.9%NaCl溶液中吸液倍率分别为4306g/g及373g/g,吸液率远高于其他吸水树脂。在此条件下聚合的PAMPS高吸水树脂的吸液速率、保水能力以及在土壤和污泥中的降解率等特性优良。将装载SA的PAMPS粉末用于烟草种子丸化,显著提高了干旱胁迫下2个烤烟品种种子的出苗率、发芽指数、活力指数、幼苗根长和全苗长以及幼苗干重、鲜重,是一种可行的提高烟草种子及幼苗抗旱能力的种子处理方法。
Tobacco (Nicotiana tabacum L.) originated from tropical areas and very sensitive to drought stress. Tobacco seeds and seedlings often suffered from drought stress in China especially in southwestern mountain area. Meanwhile, chilling injury in tobacco is also a common problem in China, it affects seed germination and seedling growth of tobacco, finally causes the loss of tobacco quality and yield. It is important to improve drought and chilling tolerance of tobacco seeds and seedlings.
The drought and chilling tolerance of20tobacco varieties at germination and seedling stages were investigated by determining traits of germination, seedling quality and some physiological characteristics. The physiological and ultrastructural changes of tobacco varieties with different tolerances under drought and chilling stress were investigated. Then, the treatment of seed soaking was used to improve the drought and chilling tolerance of tobacco seeds and seedlings. Finally, the functional materials of PNIPAm (poly (N-isopropylacrylamide)) and PAMPS (poly (2-(acrylamido)-2-methylpropanesulfonic acid)) were used to develop new intelligent tobacco seed pellets to improve their drought and chilling tolerance. The main results were as follows:
1. The chilling tolerance of20tobacco varieties at germination and seedling growth stages was investigated by determining germination characteristics and seedling quality at normal temperature (20～30℃) and low temperature (11℃) stress. The ratios of trait values at low temperature to those at normal temperature were used to assess chilling tolerance. After the cluster analysis, tobacco varieties were clustered into three groups:chilling tolerance (MS Yunyan85, NC102, Yunyan97, TN86-8and Honghuadajinyuan), intermediate chilling tolerance (NC55, RGH51, Bashima11, V2, NC297, Yunyan201and Yunyan202) and chilling sensitivity (MD-609, MS Yunyan87, MS K326, Yunyan203, Yunyan100, G-28, K346and TN90). The correlation analyses showed that the relative values of germination percentage, germination index, vigor index and malonaldehyde content were significantly correlated with each other; they were suitable parameters for evaluating chilling tolerance. The results will provide basis for material selection during research of cold resistance mechanism and breeding in tobacco.
2. The drought tolerance of20tobacco varieties at germination and seedling growth stages was investigated by determining germination characteristics and seedling quality at normal conditions and simulated drought stress by15%polyethylene glycol (PEG-6000) solution. The ratios of trait values at low temperature to those at normal temperature were used to assess drought tolerance. Tobacco varieties were clustered into three groups:drought tolerance(MS Yunyan85, NC102, Yunyan97, TN86-8and Honghuadajinyuan), intermediate drought tolerance (NC55, NC297, V2, Yunyan202, RGH51、Bashima11, Yunyan201, TN90and K346) and drought sensitivity (MD-609, MS Yunyan87, MS K326, Yunyan203, Yunyan100and G-28). The correlation analyses showed that the relative values of germination index, vigor index, seedling fresh and dry weight were significantly correlated with each other; they were suitable parameters for evaluating drought tolerance.
3. The physiological and ultrastructural changes of tobacco varieties with different chilling/drought tolerances under chilling/drought stress were investigated. The results indicated that the concentrations of H2O2, O2-, total soluble sugar and soluble protein were increased under chilling/drought stress, while the values of chlorophyll a and chlorophyll b were decreased in all varieties. The concentrations of total soluble sugar and protein in chilling/drought tolerant variety were higher than chilling/drought sensitive variety; the concentrations of H2O2, O2-, chlorophyll a and b in chilling/drought tolerant variety were lower than those in chilling/drought sensitive variety. The separations of cell membranes and chloroplasts from cell wall along with an increase in the number of lipid droplets as well as the depletion of starch granules were observed in all tobacco varieties under the chilling/drought stress, and these ultrastructural changes in chilling/drought sensitive variety were more visible than chilling/drought tolerant and intermediate chilling/drought tolerant varieties.
4. The effects of tobacco seeds soaked in different agents on seed germination and growth characteristics were researched. Seeds of chilling sensitivity variety MS K326and chilling tolerance variety Honghuadajinyuan were soaked in different concentration solutions of exogenous proline, salicylic acid, calcium chloride, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, propylene glycol, paclobutrazol and abscisic acid. Then germination, seedling growth and physiological and biochemical changes under cold temperature stress were measured. The results showed that the germination parameters, root length, seedling length, seedling fresh and dry weight were improved, superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities of tobacco seedlings were enhanced after the treatments with proline, salicylic acid, calcium chloride, dimethyl sulfoxide and polyethylene glycol. The treatments of10g/L proline,120mg/L salicylic acid,15mg/L calcium chloride,30ml/L dimethyl sulfoxide and100ml/L polyethylene glycol had better effects, it suggests that these treatments can be used to improve chilling tolerance of tobacco seeds and seedlings.
5. The effects of tobacco seeds soaked in different agents on seed germination and growth characteristics were researched. Seeds of drought sensitivity variety MS K326and drought tolerance variety Honghuadajinyuan were soaked in different concentration solutions of paclobutrazol, calcium chloride, proline and salicylic acid. Then, the germination, seedling growth and physiological changes under drought stress (simulating with15%polyethylene glycol6000) were measured. The results showed that the germination and seeding growth were inhibited; however, the activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in seedlings were enhanced after treating with paclobutrazol (PP333). The germination characteristics, root length, seedling length, seedling fresh and dry weight were improved; the activities of APX, CAT, POD and SOD were enhanced in two tobacco varieties after treating with calcium chloride, proline and salicylic acid. Among all treatments, the treatment of80mg/L salicylic acid had the best effects, suggesting that this treatment could be used to improve drought tolerance of tobacco seeds and seedlings.
6. A kind of "intelligent" tobacco seed pellets was developed to improve their chilling tolerance by adding thermo-sensitive PNIPAm-co-BMA (poly (N-isopropylacrylamide-co-butyl methacrylate)) hydrogel. SA (salicylic acid) was used as a testing drug and was loaded into PNIPAm-co-BMA, its release rate from PNIPAAm-co-BMA undergoing temperature changes in water were measured. The results showed that the SA release rate was changed from PNIPAAm-co-BMA hydrogel in water when the temperature of the water changed. Finally, tobacco seeds of chilling sensitivity variety MS K326and chilling tolerance variety Honghuadajinyuan were pelleted by the SA-loaded PNIPAm-co-BMA, and then the germination characteristics, seedling quality and some physiological parameters of the seed pellets and seedlings were measured after a chilling stress at11℃. The results showed that the germination percentage (GP), germination index (GI), root length, seedling dry weight and the activities of POD (peroxidase) were significantly increased and the concentrations of MDA (malondialdehyde) were significantly decreased in the treatments using "intelligent" seed pellets comparing with the seeds without SA or PNIPAm-co-BMA in two tobacco varieties. It indicated that the "intelligent" seed pellets pelleted with SA-loaded PNIPAm-co-BMA could be used to improve their chilling tolerance.
7. A kind of superabsorbent polymer poly (2-(acrylamido)-2-methylpropanesulfonic acid)(PAMPS) was prepared by method of solution polymerization using2-acrylamido-2-methyl-propanesulfonic acid (AMPS) as monomer, potassium peroxydisulfate (KPS) as initiator, N,N'-methylene-bis-acrylamide (Bis) as cross-linker. The absorbency, water-keeping capacity and biodegradability of the PAMPS polymer were studied. Then the PAMPS polymer, which was loaded with SA (salicylic acid), was used for pelleting tobacco seeds of drought sensitivity variety MS K326and drought tolerance variety Honghuadajinyuan. The germination and growth experiments of these pelleting seeds were studied with two tobacco varieties. The germination characteristics and seedling quality were measured after a drought stress for16d. The optimized polymerization conditions were obtained as follows:mass concentrations of initiator and cross-linker compared to the amount of monomer are0.04%and0.1%respectively under neutralization degree was60%, and reaction temperature (time) was55℃(7hours). The absorbencies of PAMPS in deionized water and0.9NaCl aqueous solutions were4306g/g and373g/g under these conditions. The PAMPS polymer had good performance of absorbency, water-keeping capacity and biodegradability. The results of germinaton and seedling growth experiments showed that these kinds of pelleting seeds could significantly improve the drought tolerance of the tobacco seeds and seedlings.
引文
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