| 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. |
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