Study On The Physiological Response To Low Temperature Stress And Micropropagation In Vitro Of Jasminum Sambac L.

In this study, the physiological and biochemical characteristics of chilling stress under artificial low temperature and natural low temperature were investigated in2-year-old cuttings of two cultivars of Jasminum sambac L., single petal jasmine (SPJ) and double petal jasmine (DPJ). In order to unravel mechanism underlying the chilling tolerance of jasmine and to facilitate chilling tolerance breeding, the effect of exogenous salicylic acid (SA) and abscisic acid (ABA) on physiological and biochemical response of these two cultivars were examined. Also, the relationship between delta-1-Pyrroline-5-Carboxylate Synthetase(P5CS) gene and the proline content was studied through semi quantitative reverse transcription and polymerase chain reaction (Sq RT-PCR) under low temperature stress. Besides, the efficient in vitro micropropagation system of jasmine was developed by nodal segments with axillary buds.From this research, the main results were as follows:1. During the artificial temperature falling experiment, the nine kinds of physiological and biochemical indexes of chilling tolerance during the low temperature stress were investigated in2-year-old cuttings of two cultivars of J. sambac, SPJ and DPJ. The results showed that the effect of low temperature stress on two cultivars intensified gradually with temperature falling and elongation of treating time. The several physiological and biochemical indexes of jasmines changed severely while they were treated under3℃for8days. The relative electrolyte leakage (REL), peroxidase (POD) activity, the content of malondialdehyde (MDA), proline and soluble protein increased gradually; while the root activity and chlorophyll content decreased by degrees; and the other physiological and biochemical indexes changed complicatedly under different low temperature and treating time. The superoxide dismutase (SOD) activity of SPJ increased firstly and then decreased when it was treated under different temperature for2days and8days. The soluble protein content of DPJ was not different remarkably under different temperature treating for2days and4days but increased significantly under different temperature treating for8days. Through comprehensive analysis of these indexes, the chilling tolerance of DPJ was betterr than that of SPJ. 2. The chilling injury parameter and eight physiological and biochemical indexes of chilling-tolerance were investigated in2-year-old cuttings of SPJ and DPJ under the condition of natural temperature falling in2009year, Nanjing City. The results showed that the REL and the content of MDA and proline of these two cultivars increased significantly, the content of soluble protein increased at first and decreased afterwards, while the tendencies of antioxidant enzymes activity of the two cultivars were different and their root activity decreased gradually. Comprehensive assessment of those physiological indexes showed that the DPJ was a little bit more chilling-resistant than SPJ, especially the change of antioxidant enzymes activity of DPJ.3. These two cultivars which were pretreated with SA solution (50,100,175and250mg·L-1) or ABA solution (0.5,5,10and20mg·L-1) respectively for4days were used for detecting the physiological and biochemical responses to6℃treating for3days. The REL, the content of MDA, proline and the soluble sugar, the activity of SOD and POD of two jasmines were investigated. Comprehensively comparation of above indexes of these two cultivars, both of50mg·L-1SA solution and5mg·L-1ABA solution were the suitable pretreatment to enhance the chilling resistance of these two jasmines under6℃treating for3days.4. The P5CS gene segment which was concerned with proline synthesis was obtained from leaves of DPJ by method of homologous cloning. The gene segment had1205nucleotides and coded401amino acids. In comparison with the published sequences of P5CS gene by BLAST program, the P5CS gene segment of DPJ showed more than89%identity to that gene from Aegiceras corniculatum, Actinidia deliciosa, Nicotiana tabacum and Solanum lycopersicum. The P5CS gene expression during3℃was investigated through Sq RT-PCR method with the18S gene as reference gene. The results showed that the expression of P5CS was first up-regulated and then down-regulated during low temperature stress. The expressional fastigium of P5CS gene was at the fourth day of3℃treating, while the fastigium of proline content was at the sixth day of3℃treating. Although there was hysteresis phenomenon between the P5CS gene expression and proline synthesis, the trend of them was consilient. But further studies were needed to elucidate the relationship between them.5. The micropropagation in vitro was investigated in8-year-old cuttings of DPJ by stem segment. Several experiments were carried out to detecte the disinfection of explant, germination culture of axillary buds, the optimum formula of proliferation and induction of multiple shoots and rooting. The results were as follows:(1) April to September (avoiding the mould rains season) was the best period to pick the explants. The one-year-old and semi-lignification twigs with axillary buds were most suitable to be explants. The treatment with75%ethanol for1min,0.1%mercuric chloride for52mins then washing3times in aseptic water sequentially was the best disinfection method. The optimum basic medium of germination for axillary buds was WPM medium.(2) In order to find out the optimum formula for axillary buds elongation, the experiments with different plant growth regulators alone or in combination were carried out. The best formula for axillary buds elongation was WPM+2mg·L-1IBA+0.5mg·L-16-BA+0.7mg·L-1GA3. The optimum combination of plant growth regulators for induction of multiple shoots, with generation of11axillary shoots, was1mg·L-1ZT supplemented with0.5mg·L-1IBA,(3)The best rooting method was that shoots were first dipped in450mg·L-1NAA solution for10mins and cultured in the basal1/2WPM sequentially. The rooting rate was up to86.7%. In this study, the plantlets were acclimatized with78%survival rate under the suitable humidity and illumination during the initial period of acclimation.