Study On Mutation And Identification Of Hibiscus Syriacus Seeds By Colchicine And EMS

Hibiscus syriacus Linn.,a deciduous shrub of Malvaceae,is an excellent ornamental species with edible and medicinal value.Aiming at the late start of Hibiscus germplasm innovation and breeding in China,using Hibiscus seeds as materials,colchicine and EMS impregnation method were used to explore the best mutagenesis combination under different factors.The mutant plants were identified by mor:phology,cytology and molecular analysis,then the mutant plants were screened out.The physiological indexes were determined to predict the resistance change direction of the mutants.It provides innovative materials for Hibiscus landscape application,and lays a foundation for Hibiscus genetic analysis,gene mapping and map cloning from the molecular point of view.The main results are as follows:In Hibiscus mutant breeding with colchicine,the highest germination rate of Hibiscus seeds was 65%when soaked in clear water for Oh.Combining the lethal rate,seedling rate and variation rate of Hibiscus seeds,the optimum combination of colchicine-induced variation of Hibiscus seeds was determined as 2 day germination of seed and soaking for 12h with 0.2%colchicine.The mutant plants showed variations of plant height dwarfing,stem segment coarsening,shortening internode spacing,basal angle and ratio of length to width of leavesincreasing,and leaves yellowing and shrinking.The stomatal morphology of leaves was significantly different,and the cells showed polyploid giant.The stomatal number and stomatal density decreased by 55.90%.Flow cytometry showed that nuclear DNA content increased with the number of chromosomes doubling.Effective variation can be quickly and accurately identified by morphological and cytological identification.The chloroplast pigments content of the mutant plants increased significantly with the chromosome number,and the leaves showed dark green.The electrolyte leakage rate of the mutant cells decreased while the ploidy increase.It is speculated that the resistance of the mutant plants increased after the mutagenesis of colchicine.In Hibiscus mutant breeding with EMS,the best combination of EMS-induced seed variation was 2 day germination of seed and 0.2%EMS soaking for 12h.The mutants showed dwarfing,thickening,shortening internodes,yellowing,curling and obvious veins of leaves.Molecularly,the optimal reaction system of Hibiscus ISSR-PCR(25?l)was established:10×buffer(Mg2+ free)2.5?l,Taq enzyme 0.75U,Mg2+ 2mmol/L,dNTPs 0.1mmol/L,template DNA 100ng,primer 0.5?mol/L,and annealing temperature 50.0?.The percentage of polymorphic bands of six primers was 95.08%.In ISSR-PCR amplification,the bands of mutants showed deletion sites,new sites,and both deletion and addition.The average genetic similarity(SM)was 0,885 and genetic distance(GD)was 0.142.When the genetic distance coefficient was 0.85,UPGMA clustering results showed that the mutants were divided into three categories,which were consistent with the three categories of morphological preliminary classification.This indicating that ISSR-PCR molecular identification method was a reliable method for identification of EMS mutagenesis in Hibiscus.The content of chloroplast pigments in the mutant plants corresponds to the change of leaf color,and the electrolyte leakage rate of the mutant plants mostly increases.Which speculated that the resistance of the mutant plants decreases after EMS mutation.