| Sugarcane（Saccharum spp.）is one of the most important cash crop in China,which is mainly produced in southern China.With global warming,some major sugarcane planting areas have experienced reduced precipitation or even severe drought,resulting in sugarcane production reduction.More and more scientific researchers have begun to attach importance to the research of sugarcane drought resistant varieties.In this study,Yuetang 93-159 was used as experimental material to establish sugarcane regeneration system through exploring the effects of different kinds and concentrations of hormones and activated carbon.Sugarcane callus was mutagenized by EMS and screened for drought resistant strains under PEG6000 stress.Agronomic traits and physiological indexes of the mutant were measured before and after drought stress.Principal component analysis and cluster analysis were used to evaluate the drought-resistant performance of the mutant.Transcriptome analysis was performed on the drought-resistant mutant 1-17.The main research results are as follows:1.Establishment of sugarcane regeneration system of Yuetang 93-159The young leaf sheaths of Yuetang 93-159 were used as explants to culture regenerated seedings in the MS medium with the different concentrations of 2,4-D,6-BA,NAA and activated carbon at callus induction and differentiation stage.The best medium for callus induction was MS+2 mg·L-12,4-D+30 g·L-1sucrose+7 g·L-1agar,and the induction rate was 95.37%.The best medium for differentiation was MS+1 mg·L-16-BA+0.2 mg·L-1NAA+30 g·L-1sucrose+7 g·L-1agar,and the differentiation rate was 42.22%.The best medium for rooting was 1/2MS+3 mg·L-1NAA0.5 mg·L-1 activated carbon+30 g·L-1sucrose+7 g·L-1agar.2.Establishment of EMS mutagenesis system and screening criteria for mutantsThe sugarcane callus was treated by EMS at 0,0.1%,0.2%and 0.3%for 4 h and 6 h,respectively.According to the differentiation rate of the callus,the combination of EMS mutagenesis concentration and time was determined to be 0.3%EMS for 4 h.When 0,5%,10%,15%and 20%PEG6000 were added into the root medium to simulate drought stress,the survival rate of tissue culture seedlings was 100%,76.67%,45.56%,27.78%and 14.44%,respectively.The survival rate of sugarcane tissue culture seedlings treated with different concentration PEG6000 was compared.Finally,20%PEG6000 was selected as the threshold for drought resistance screening,and the survival rate of tissue culture seedlings under this concentration was 14.44%.3.Identification of drought-resistant mutantsThe mutant was treated with drought stress,and 7 morphological indexes including plant height,total root length,root volume,root mean diameter,root surface area,root fresh weight and root dry weight were determined.The 7 indexes were converted into 3 comprehensive indexes by principal component analysis.The D-value was used for cluster analysis,and 20 strains were divided into 5 categories according to the criterion of Euclidean distance greater than 5.The best drought-resistant mutant 1-17was selected.By measuring the physiological indexes of different drought-resistant mutant lines,it was found that the POD,SOD activities and MDA,CAT content were increased in different amplitude before and after drought stress.4.Transcriptome analysis of mutants 1-17 under drought stressTranscriptome analysis of the mutant 1-17 and Yuetang 93-159responding to drought tress showed that the mutant had 2,168 differentially expressed genes（DEGs）before and after drought stress.6,244 and 12,754DEGs were identified between the mutant and control plants before and after drought,respectively.GO enrichment analysis revealed that a total of237 GO entries were enriched in the two strains under normal condition,including jasmonic acid mediated signal pathway regulation,intracellular signal transduction,glutathione metabolism,heme binding,calcium binding,and iron binding.A total of 412 GO entries were enriched in the mutant and control after drought stress,including the reaction to unfolded proteins,lignin biosynthesis processes,anionic transmembrane transport,amino acid transmembrane transport activity,CTP synthase activity,and protein serine/threonine phosphatase activity.KEGG enrichment analysis revealed that the DEGs of mutants and controls before and after drought stress were mainly enriched in signal transduction,biosynthesis of secondary metabolites,carbohydrate metabolism,lipid metabolism,energy metabolism,and other pathways,indicating that these pathways played an important role under drought stress. |
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