Study On Mutation Breeding Of Docosahexaenoic Acid High Yield Crypthecodinium Cohnii

In this thesis，we used Crypthecodinium cohnii ATCC30772as the original strain inthe test of mutation breeding of DHA high yield mutant by⁶⁰Co γ-ray irradiation，withdifferent irradiation dose．We screened and re-screened the mutants according to thoseindexes such as observation of mutants＇growth and development、the result of Sudan blackB staining、biomass、oil content、DHA content，etc．At last，the optimum mutant waselected．The main findings are as follows：1. Pre-experiment of⁶⁰Co-γ ray irradiationWe made a⁶⁰Co γ-ray irradiation on C.Cohnii6#that we used as the original strain inthis pre-experiment，the cell density、irradiation dose、incubation time and other factorswere explored，different death rate and growth results of C.Cohnii6#under the irradiationdose of1.2kGy、1.8kGy、2.4kGy、3.0kGy、4.2kGy、5.4kGy were studied．The experimentalresults showed that：the death rate of cells is proportional to the radiation dose．Althoughthe growth rate of those algal cells which were irradiated was restricted，the mutants whichwas irradiated with irradiation dose around1.8kGy reached maximum growth rate earlierthan the original strain．All treatment groups had higher biomass than the original strainexcept for4.2kGy and5.4kGy treatment group．In this testing condition，we decided that：it’s more appropriate to mutate C.Cohnii by⁶⁰Co γ-ray irradiation with low irradiation dose（under2.4kGy）after synthetic analysis．2. Mutant preliminary screening of C.Cohnii ATCC30772As the original strain，C.Cohnii ATCC30772was irradiated by⁶⁰Co-γ ray withirradiation does of0.8kGy、1.6kGy、2.4kGy，mutants were screened according to syntheticindexes for breeding DHA high yield mutants．Results showed that：death rate of cellsincreased with increasing irradiation dose．The results of the measurement of algalcolonies’ diameters and the observation of colonies’ shape and microscopic smearindicated that：algal cells in irradiated treatment groups grew and reproduced fastersignificantly，the ability of cell division was strengthened notably，the size of cell gotbigger，as well．The results of the observation of mutants’ liquid-culture test tube andSudan black B staining suggested that：the growth rate of mutant strains was faster than theoriginal strain，the fat accumulation of mutants was more than the original strain．3. Rescreening of mutant strains of C.Cohnii ATCC30772We rescreened the27mutants which were selected in preliminary screening byshaking-flask fermentation and continuous fermentation test．The experimental results revealed that：the optimal DHA high yield mutant of C.Cohnii ATCC30772（2.4k-2A2-5）was obtained by⁶⁰Co γ-ray irradiation with the radiation dose of2.4kGy．The oil weightand oil content of this mutant was51.66%and66.37%higher than the original strainrespectively，the average DHA content of this mutant was39.04%，increased by3.39percent than the original strain，the DHA yield（measured in g/L fermented liquid）increased by56.92%than the original strain．The mutant of C.Cohnii ATCC30772（2.4k-2A2-5）was the desired positive mutation algae species in this experimentalcondition．...