| Wild lily species are usually characteristics with abundant genetic backgrounds and phenotypes.However,it is difficult to directly transplant wild species because most regions of China usually suffer from very high temperature and humidity in summer,which is distinctly different from the original habitat for most species and easily leading the outbreak of some disease,especially grey mould,one destructive disease for lily.Polyploids generally improve the survival rate of transplantation and stress resistance in many plant species.Many studies explored chromosome duplication protocol of wild lily species in vitro.However,the ploidy effects on wild lily,especially on growth characteristics and grey mould resistance after transplantation was seldom reported in previous studies.In this study,chromosome doubling protocol of bulblets or seeds were established in L.primulinum var.ochraceum(LPO),L.regale(LRE)and L.rosthorii(LRI).Moreover,the ploidy effects were analyzed based on the phenotypes,leaf anatomy,chloroplast ultrastructure and some photosynthetic related characteristics in three lily species and their tetraploids after transplantation.The susceptibility of grey mould was compared in diploid and tetraploid LRI,which presented more vigorous growth characteristics compared with other two species.The mechanism of this different susceptibility was compared from cytological,physiological and molecular levels.Moreover,this study firstly confirmed that oxalic acid is one of important pathogenic factor for B.elliptica,the main causal agent of grey mould in lily.The pathogenic mechanism of oxalic acid and the defense mechanism of its host was also explored in diploid and tetraploid LRI.Some main results as follow:(1)Chromosome duplication protocol in vitro of bulblets were established in LPO.The morality of scales can be decrease after preculture for 15 d.Colchicine and oryzalin were employed as antimitotic agents.The most efficient treatment of each agent was immersed in 0.100% colchicine and in 0.010%oryzalin for 24 h;the induction rate of the former(26.67%)was significantly higher than that of the latter(18.89%).In total,74 tetraploids were confirmed by FCM and chromosomal counting.For LRE,5tetraploids were obtained from 30 tissue cultured scales after treatment with 0.050% colchicine for 24 h.The stomata traits of these tetraploids were analyzed,which presented high Pearson’s coefficient with stomatal length in LPO(0.911)and LRE(0.980).(2)Chromosome duplication protocol of seeds were established in LRI.Preculture of seeds before antimitotic agents were necessary.The germinated seeds with hypocotyl 0.2-0.5 cm in length presented high survival rate in colchicine and oryzalin.The most efficient treatment of each agent was immersed in0.05% colchicine for 36 h and in 0.01% oryzalin for 24 h.The induction rate of the former(27.78%)was significantly higher than that of the latter(22.22%).In total,199 tetraploid genotypes were obtained by FCM and chromosome counting.The correlation of swollen hypocotyl phenotype and tetraploidy was analyzed.It was high in colchicine(0.989)and oryzalin(0.975),suggesting that this phenotype could serve as an early ploidy selection trait.However,the correlations were weaker between stomata length and tetraploidy(0.773),implying that stomatal characters are affected by both the ploidy level and genotype.(3)The growth characteristics of the three lily species and their tetraploids were compared in vitro and in field.Scales with slow differentiation and regenerated shoots with rough leaves were all observed in the three species after each antimitotic agent treatment.After several rounds subculture in vitro,the morphological and growth traits were not significantly different between diploids and tetraploids in LPO and LRI.However,tetraploid LRE presented more efficient rate of scale differentiation and faster weight increase of bulblets compared with diploids,although leaves still presented rough surface.In field,the phenotypes,leaf anatomical characteristics,photosynthetic pigments,chloroplast ultrastructure,leaf soluble sugar and starch,and stomata starch of mature leaves(in 5-6th month transplantation)presented different changes in the three species.Chromosome duplication not influenced photosynthetic models in the three species but the changes of photosynthetic rates were species dependent.For LPO and LRI,the photosynthetic performances were improved in different degree after chromosome duplication.However,for LRE,the maximum photosynthetic rate of light-/CO2-response curves were all significantly decreased after chromosome duplication,although net photosynthetic efficient had no significantly differences in nature conditions.After plants withered,for bulblets,the higher survival rates,larger size with more developed root systems were well observed from the three tetraploid species.(4)Tetraploids were less susceptible to grey mould in naturally and detached infected leaves.Many crystals with abnormal shapes and different sizes were observed in the infected leaves,showing similar characteristics to oxalic acid,a virulence factor for many pathogens.Healthy leaves immerse in oxalic acid solution presented same changes with infected leaves,including the disappearance of starch in guard cells,abnormal guard cell nuclei and the gradual reopening of closed stomata.These cytological results confirmed oxalic acid is a pathogenic factor for B.elliptica,which can disturb stomata function.Moreover,the results of the interaction of hyphae and leaf surface structure,the lesion points of infected leaves,and the deposition of Ca OX and callose suggested that the invasion of B.elliptica was more likely influenced by stomata density than that of stomata size.(5)To explore the physiological mechanisms of chromosome duplication influence on grey mould susceptibility,cytological and physiological traits,and transcript levels of some related genes were compared in diploid and tetraploid leaves after inoculation B.elliptica at 0-72 hours post-inoculation(hpi).The OXO activity was high in tetraploids than in diploids after 12-48 hpi,which suggested tetraploids presented well capability to defense oxalic acid and there was more degradation production(H2O2)accumulated in tetraploids than in diploids.Moreover,for ROS production system,the accumulation of O2-and H2O2 was faster after 2-24(36)hpi,and it spent less time to reach peak values in tetraploids than in diploids,although the peak ROS levels were similar in tetraploid and diploid leaves.For ROS scavenger system,the SOD activity similar in tetraploid and diploid leaves,but the POD activity was higher in tetraploids than in diploids after 2-12 hpi.After 8 hpi,the accumulation of MDA was higher in diploids than in tetraploids.Moreover,the transcript levels of some key genes related to oxalic acid degradation(Lr GLP1 and Lr GLP2),ROS production/scavenging(Lr RBOHD,Lr GST and Lr POD)and PRs synthesis(Lr CHI,Lr BGL and Lr PR10)were higher in tetraploids than in diploids after 24(12)-36(48)hpi. |
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