Construction And Screening Of Mutant Library In Chrysanthemum Indicum Var.aromaticum

Chrysanthemum is a perennial herbaceous species that is widely used in different ways.such as ornamental flowers in gardens,herbal medicine,and making perfumes in cosmetic industry in China.Among them,C.indicum var.aromaticum has been used as Chinese traditional herbal medicine,because its flowers,leaves,and roots are rich in aroma.Even though C.indicum var.aromaticum is a very important plant,its distribution range is restricted to a few areas.Moreover,the breeding for improvement of various traits was hindered not only because of limited genetic diversity but also paucity of studies.Induced mutation is one of possible alternatives to conventional breeding for plant improvement program.Among the common chemical mutagens,Ethyl Methanesulfonate(EMS)is the most effective and powerful mutagen to induce functional variations in plants.However,inducing mutants on C.indicum var.aromaticum with EMS has not been reported so far.In this study,EMS was used as an exogenous inducer on C.indicum var.aromaticum,using different doses to observe its effect on plant germination,potential mutants and attempted to construct and screen mutant library of C.indicum var.aromaticum.During the experiment,five types of mutants were observed,including early flower,large flower,large leaf shape,bending stem and salt tolerance.Morphological,anatomical,physiological traits and assessed molecular changes in C.indicum var.aromaticum induced by EMS in response to salt stress using RNA sequencing.The main results are as follows:1.To determine the optimum EMS condition of the dosage for C.indicum var.aromaticum,four concentration levels of 0%,0.1%,0.2%,and 0.5%v/v of EMS were used for the treatments of the seeds.The study revealed that both germination index and survival percentage of the seedlings decreased significantly with increase in EMS dose.Approximately 86%and 73%of the seeds planted germinated when treated with 0.1%v/v and 0.2%v/v of EMS,respectively.Conversely,only 32%of the seeds treated with 0.5%v/v of EMS germinated.This clearly shows that high dosage of EMS reduces the seeds germination.The survival rate of seedling when exposed for 6 hours with 0.1%v/v,0.2%v/v and 0.5%v/v EMS dosage were 75%,56%and 25%,respectively.This implied that 44%of the seeds treated with 0.2%v/v EMS would also not survive,so the LD50 of C.indicum var.aromaticum was found at 0.2%v/v EMS dosage.2.The seedlings of C.indicum var.aromaticum were treated with 0.1%,0.2%,and 0.5%v/v EMS solution for 8 h,1200 mutant seedlings were gained.The flowering period of the mutant plants(ET3-16 and ET3-21)occurred much earlier than that of CK plant.Moreover,the net photosynthetic rate(Pn).stomatal conductance(Gs)and transpiration rate(Tr)reduced significantly in the mutant plants than CK plant.No significant difference was observed in the intercellular CO2 concentration(Ci)of all plants.The average flower diameter of ET1-4 and ET3-9 were 25.06 mm and 22.82 mm,respectively,while the average flower diameter of CK plant was 15.69 mm,which was significantly smaller than those of the mutant.3.This study also revealed that the morphology of leaf size included leaf width(6.36 cm);leaf length(8.46 cm)and leaf area(32.69 cm2)of mutant plants(ET2-138)were larger than that of CK plants(3.64 cm,4.22 cm,and 8.89 cm2).Plant height of mutant plant of 48.10 cm was significantly higher than that of CK plants 26.2 cm.The anatomical characters of the leaves of mutants plants exhibited obvious changes.The average leaf thickness(27.80 ?m),upper epidermis thickness(2.63 ?m)and lower epidermis thickness(1.57?m)of mutant plants were thicker than those of CK plants(24.29 ?m,1.53 ?m,1.24 ?m).Analysis of variance revealed significant difference between the stem length of mutant plants and CK plants.4.The average stem length of ET1-55,ET1-56 and ET2-61 were 37.16 cm,31.93 cm and 32.40 cm respectively;while that of CK plants was only 26.44 cm.Moreover,the stem growth morphology of the plants was quite different.The stems of CK plants were erect and grew straight,whereas the stems of mutant plants bent transversely bend and were creeping.The total lignin and cellulose contents in the stem of the mutant plants were lower compared with that of CK plant.These shows that lignin and cellulose contents were related to plant stem strength,and EMS treatment could affect cell metabolism,lignin and cellulose contents in mutant plants.5.In order to study the effects of EMS induced salt-tolerance mutant on plant growth parameters of C.indicum var.aromaticum,salt-tolerance was compared between EMS treatment and CK treated plant.This study revealed that salty stress significantly reduced the chlorophyll contents in both ET1-312 and CK treated.A comparison of the chlorophyll content from day 0 to day 15 of after salinity treatment revealed a reduction of 34%and 32%of the chlorophyll a content in CK and ET1-312 plants,respectively.Chlorophyll b content in CK treated and ET1-312 plants reduced by 77%and 76%,respectively.The decrease in chlorophyll was attributed to the salt stress which increases the activity of chlorophyll enzymes and reduces the degradation of chlorophyll.Net photosynthetic rate(Pn),stomatal conductance(Gs)and transpiration rate(Tr)decreased under salt stress in both ET1-312 and CK treated.On the contrary,carotenoids content and intercellular CO2 concentration(Ci)accumulation increased slightly in ET1-312 compared to CK treated.In addition,the activities of antioxidant enzymes,peroxidase(POD),catalase(CAT),and superoxide dismutase(SOD)of the mutant plants,were on average higher than that of the CK treated.Expression level of antioxidant genes(POD42,CAT1,CAT2,SOD-Cu-Zn,SOD-Mn1 and SOD-Mn2)was increased in the ET1-312,compared with their expression in CK treated,this was consistent with its physiological manifestations.The mutant ET1-312 has strong salt resistance and with fertility,which can improve the adaptability of C.indicum var.aromaticum,in saline-alkali land.6.The transcriptome of leaves and roots of ET1-312 and CK under salt stress were sequenced.A total of 60,595 Unigenes were obtained,of which 62.46%of unigenes could be annotated in at least one database.Approximately 3,956 and 3,123 differential genes(DEGs)were obtained in the comparison group of ET312vsCK leaf and ET312vsCK root,respectively.Differential gene expression analysis of plant hormone signal transduction pathways,plant salt stress-related transporters,antioxidant enzymes,phenylpropane biosynthesis pathway and flavonoid biosynthesis pathway were carried out.The gene expression of these pathways was enhanced in the ET1-312 mutant.and the DEGs in roots had more up-regulated genes in phenylpropane biosynthesis pathway,flavonoid biosynthesis pathway and transcription factors than DEGs in leaves.The qRT-PCR test results were basically consistent with the transcriptome sequencing results which implied that the gene expression profile data of transcriptome data sequencing was credible.