Study On Selection System Of Somaclonal Variation Resistant To White Rot In Garlic

Garlic white rot,a damaging disease of garlic,mainly harms the roots,bulbs,and leavesof garlic.It may cause death of young plants and influence garlic production in fields,withgreat subsequent losses or even no harvest during severe incidence.At present,chemicalcontrol is the major method to control white rot but it is not so effective,and overuse offungicides may also result in chemical residuals in garlic products which may be a health risk.To solve this problem,the usual way is to develop a resistant variety.However,culturedgarlic is sterile,and it is very difficult or even impossible to achieve sexual hybridization,which is necessary for traditional plant breeding.The selection of somatic mutants is one ofthe important alternative breeding strategy for garlic.The biological characteristics of white rot pathogen,culture conditions for theproduction of crude toxin,components of curde toxin,selection of cell mutant lines resistantto white rot,physiological and biochemical characterization of garlic somatic mutants weresystematically studieed in this thesis.It is important for opening up a new breeding waytaking garlic as the representative of the asexual reproduction crops.White rot,caused by Sclerotium cepivorum Berk.is a ruined disease of garlic in thecourse of the production.The research on biological characteristics of Sclerotium cepivorumBerk.may provide a theoretical basis for the prevention and control of the disease.The effectsof temperature,pH,light,atmospheric relative humidity(RH)on mycelium growth andsclerotia germination were studied in this experiment.The results showed that mycelium grewwell when cultured at 18.0℃and pH 4.0～5.0.Light was beneficial to mycelium growth.Conditions such as 15.0～18.0℃,pH 4.0～5.0,12 hour light/12 hour dark were beneficial tosclerotia production.The sclerotia germinated well when cultured at 12.0～24.0℃,pH 4.0～9.0,and under RH 100%.The lethal temperature ofmycelial growth and sclerotia germinationwere 45.0℃and 50.0℃,respectively.The changes of defensive enzymes after inoculation by Sclerotium cepivorum Berk.intwo different resistant cultivars were studied.After obvious symptom appearing,the changesof defensive enzymes were determined in health and sick plants.The inoculation of pathologycould activated the activities of defensive enzymes in garlic.The results suggested thatactivities of PAL,SOD,PPO and POD were increased after inoculating regardless of in resistant cultivars or susceptible cultivars,and the enzyme activities in resistant cultivarHanzhong Red Skin were higher than those in susceptible cultivar Gailiang.However,theactivities of CAT was lower after inoculating regardless of in resistant cultivars or susceptiblecultivars,and the activities of CAT in susceptible cultivar dropped more than that in resistantcultivar.Resistance of the different garlic cultivars was significantly correlated with thedefensive enzymes.The activity of defensive enzymes is thought closely related to the diseaseresistance of cultivars.The relation between crude toxin production and culture conditions was examined bytesting the toxicity of crude toxin with garlic root growth inhibition rate.The results showedthat the optimum conditions for toxin production were Fries liquid medium at pH 5 and at18℃,with successive dark periods and shaking during culturing.Production of crude toxinreached the highest level on the 6th day of culturing.The dynamic changes of defensive enzyme activities,content of superoxide radical(O₂^-)and malonaldehyde(MDA)in leaf of cultivars Hanzhong Red Skin and Gailiang treated bycrude toxin of Sclerotium cepivorum Berk.were investigated at seedling stage.The resultssuggested that the activities of superoxide dismutase(SOD)and peroxidase(POD),werestimulated and increased treating by crude toxin,and the activities of catalase(CAT)werelower after treating.The enzyme activities in resistant cultivar Hanzhong Red Skin werehigher than that in susceptible cultivar Gailiang.The peaks of the activities of SOD and PODin the resistant cultivar appeared earlier than that of the susceptible cultivar and POD wasmost sensitive to the toxin coercion.The results also showed that the content of O₂^-inresistant cultivar was lower than that in susceptible cultivar,after treating for 24 hours.Thedynamic changes of the content of MDA were basically in conformity with that of O₂^-.Theseresults indicated that the content of reactive oxygen species(ROS)and activities of protectionenzymes in leaf had a close relationship with disease resistance in garlic.GC-MS was used to analyse the components of curde toxin produced by Sclerotiumcepivorum Berk.The results suggested that the major components of curde toxin were organicacids,alcohols,ester,steroids and heterocyclic compounds.Dodecanoic acid and dibutylphthalate were active components of pathogenic toxin;toxicity of dodecanoic acid wasstronger than dibutyl phthalate.The somatic variant clones of disease resistance were screened by in vitro culture ofgarlic stem base explants in the medium with pathogen crude toxin added.The resultsshowed that the crude toxin had clearly negative effect on callus induction and growth.It alsoextremely inhibited differentiation of adventitious buds.The inhibitory effects becameintensified with the toxin concentration increased.Some somaclonal variation lines and plantlets resistant to Sclerotium cepivorum Berk.were obtained at crude toxin concentrationof 50%.Changes of activities of phenylalanine ammonia-lyase(PAL)and polyphenol oxidase(PPO)and contents of lignin and hydroxyproline-rich glycoprotein(HRGP)in the selectedmutant and the original callus were investigated 72 h after crude toxin treatment.The resultsshowed that the activities of PAL and PPO were stimulated and increased by crude toxintreatment.The enzyme activities in mutant callus were higher than that of original callus.Thepeaks of the activities of PAL and PPO in mutant callus appeared earlier than that in theoriginal callus and PAL was most sensitive to the toxin coercion.The results also showed thatthe content of lignin in original callus was lower than that in mutant callus.The dynamicchanges of the content of HRGP were basically in conformity with that of lignin.Theseresults indicated that the accumulation of HRGP and lignin deposition in cell wall had a closerelationship with disease resistance in garlic.These results suggested that in vitro screenedgarlic somatic mutants resistant to white rot had the similar resistant mechanism as thegeneral resistant varieties.