Study On The Application Of Three Chemical Mutagens In Breeding Of Chinese Fir

Chinese fir （Cunninghamia lanceolata） as an important arbor tree mainly growth in SouthChina with many characteristics including fast-growing, good material, functional diversities, andso on. In order to abundant the category of Chinese fir genetic variations, provide more originalmaterials for breeding new cultivars. The Chinese fir seedling mutants induce experiments werecarried out by using three chemical mutagens, colchicine,ethyl methane sulfonate and sodiumazide, the screened excellent families of Chinese fir were treated with these chemical mutagens atdifferent time and concentration. To select the optimum mutagenic treatment combinations andscreening out mutant seedlings, some biology effects including the mortality rate, morphologicalvariations, and Chlorophyll Fluorescence parameters were investigated. The results of this studywill lay a foundation for further study on mutation breeding by chemical mutagen in Chinese firseedlings. The main results as follows：1、In the colchicine mutagenic experiment,the obviously inflated roots of seedlings wereobserved during the treatment, and the aneuploid were found in the swollen root cells through thedetection of the content of DNA with Flow Cytometry. And the seedlings without showing a largenumber of death at the end of treatment, however, death became more evident due to inabilitygrowth of bud and leaves after a period of transplant, and the number of survival seedlingsmaintained at a relative steady level after45days of transplant. In the experiment of EMStreatment, with the increase of EMS concentration and treatment time, more seedlings becomehypocotyl softening and browning,and died ultimately. And the number of survival seedlingsmaintained at a relative steady level after20days of transplant. While, in the experiment ofsodium azide treatment, many seedlings became died and brown within12h after the end ofsoaking treatment.The number of survival seedlings also maintained at a relative steady level aftertransplant for20days, which showed a similar pattern as the treatment of EMS.2、The regression equation of the mortality rate and the colchicine concentration, treatmenttime, light and root length of fir seedlings in the sixth family isy6=0.031+0.83c+0.145t+0.126g0.055m（R²=867**）， the results showed that theoptimum mutagenic treatment combinations of colchicine concentration and treatment time of this family is0.05%+4d,under light and the root length of seedling is3cm, when the semi-lethal doseselected as a criterion. while, the regression equation of the seventh family of Chinese firseedlings isy₇=0.065+0.93c+0.113t+0.172g0.168m（R²=0.85**）,and the optimummutagenic treatment combinations of colchicine concentration, treatment time,light,root lengthfor them are0.05%+3d+no light+1.5cm and0.1%+2d+light+1.5cm. However, the regressionequation of the eighth family of fir seedlings isy₈=-0.046+0.98c+0.127t+0.236g0.166m（R²=0.858**）,and the optimum mutagenic treatment combinations of colchicine concentration,treatment time,light,root length for Chinese fir seedlings are0.3%+4d+light+3cm.3、The regression equation of the mortality rate and the EMS concentration, treatment time inthe sixth family of fir seedlings is y₆=0.604+0.556c+0.23t（R²=0.87**）,the resultsshowed that the optimum mutagenic treatment combinations of EMS concentration and treatmenttime of the family are1.16%+2h and0.95%+2.5h. The regression equation of the seventh familyfir seedlings is y₇=0.606+0.598c+0.237t（R²=0.959**）, the suitable Mutagenic treatmentcombinations of EMS concentration and treatment time for the fir seedlings are1.06%+2h and0.86%+2.5h. The regression equation of the eighth family fir seedlingsis y₈=0.562+0.685c+0.181t（R²=0.927**）, and the suitable Mutagenic treatmentcombinations of EMS concentration and treatment time of this family are1.15%+1.5h and0.89%+2.5h.4、The regression equation of the mortality rate and the sodium azide concentration，treatment time in the sixth family of Chinese fir seedlings isy₆=0.356+7.865c+0.295t（R²=0.969**）,the results showed that the optimum mutagenic treatment combinations of sodiumazide concentrations and treatment time of this family are0.053%+1.5h and0.034%+2h. And theregression equation of the seventh family of fir seedlings isy₇=0.492+9.879c+0.343t（R²=0.973**）,the optimum mutagenic treatment combinations of sodium azide concentrationsand treatment time for the fir seedlings are0.048%+1.5h and0.031%+2h. The regression equationof the sixth family of Chinese fir seedlings isy₈=0.501+9.326c+0.364t（R²=0.940**）,the optimum mutagenic treatment combinations of sodium azide concentrations and treatmenttime for the fir seedlings are0.05%+1.5h and0.03%+2h.5、During the cultivation of the mutants inducted by colchicine, three kinds of mutants werefound compared with control, namely the type of thick short cotyledon with normal crown, thickshort cotyledon with dead crown type, three pieces of cotyledon type. the seedling height and the shoot length of different mutant types of fir seedlings among three families were significantlydifferences compared with the corresponding control with the exception of the three pieces ofcotyledon type, which did not showed any significantly differences, In addition, the former mutanttypes also displayed many dwarf seedlings.The results of chlorophyll fluorescence detectionshowed that the permanently damages of leaves of thick short cotyledon with dead crown typemutants caused by chemical mutagens was bigger than that of control.however, the maximalfluorescence （Fm） is lower in these mutants compared with control.The variable fluorescence（Fv）, the changes of PSâ…¡ potential activity （Fv/Fo）and the photosynthetic efficiency （Fv/Fm）were significantly lower than the other mutant types.The initial fluorescence （Fo） in the mutants ofthick short cotyledon with normal crown type and the three pieces of cotyledon type were lowerthan that of control, which might due to the developing of resistances in these mutants in responseto unfavorable environment stress after mutation. furthermore, the Fv/Fm and Fv/Fo were higherin these two mutants compared with control, which indicated that these two mutants possesshigher vitality.6、During the cultivation of the mutants by different EMS treatments, the effects of EMS onthe morphology of fir seedlings mainly showed in cotyledon and true leaf compared withcontrol,,,and there were three kinds of mutants were found during the cultivation processes, that iscotyledon curl type, leaf brown type and albino type. There were no significantly differences incotyledon curl type and seedling height of fir seedlings treated with EMS compared with control,while EMS treatment significantly increased the frequency of the appearance of leaf brown andalbino, and both of these mutants display dwarf as well, which may due to the blocked synthesisof chlorophyll by mutagens, further studies is needed to explain this phenomenon.. The originalfluorescence of seedlings of different mutants in three families upon EMS treatment did notshowed significantly differences compared with corresponding control, which suggested that thepermanently damages of leaf PSâ…¡ i n seedlings upon EMS treatment was not obviously after3months cultivation. The mutant types of cotyledon curl and leaf brown seedlings in three familiesall showed a similar trends at. Fm, which did not showed significantly differences compared withcorresponding control. As the case for Fv,Fv/Fo and the Fv/Fm,, the mutant seedlings ofcotyledon curl among three families did not varied significantly, however, the corresponding indexof brown type mutant seedlings were significantly lower compared with the control.7、During the cultivation of the mutants by different sodium azide treatments,the effects of odium azide on the morphology of fir seedlings mainly showed in cotyledon and true leafcompared with control, the seedlings displayed different types of variations as the concentrationand treatment time of odium azide changed, namely completely albino type and half leaf albinotype. There is no significantly differences in the seedlings height and length of bud in the half leafalbino type of the three families compared with control. However, the obviously dwarf seedlingsin completely albino mutant type was observed, this may result from severe loss of chlorophylltriggered by treatment of chemical mutagens, which in turn lead to the dysfunction ofphotosynthesis, and ultimately result in the retarded growth of seedlings and bud or even stopgrowing..The results of chlorophyll fluorescence showed that the completely albino type seedlingscouldn’t detect values due to the loss of chlorophyll. The Fo, Fm, Fv and other chlorophyllfluorescence parameters of half leaf albino type in the three families did not varied significantly,compared with the control, which indicated that although half of the leaves showed albinophenotype, the rest ones still can function normally, thus such kind of leaf did not disturbance thenormal growth of seedlings.