Study On Technology Of Virus-free, Rapid Propagation And Virus-free Plantlet’s Field Adaptation Of Huaiqing Chrysanthemum

Xiaohuangju, which is a excellent variety of Huai chrysanthemum, was used as experimental materialsin this study. The technology system of virus-free and its rapid propagation of Huai chrysanthemum wasestablished, and it had been systematically studied that physio-biochemical characteristics, genetic stabilityand the field adaptation of virus-free test-tube plantlets. The results as followings:1. Established the technology system of virus-free and its rapid propagation of Huaichrysanthemum(1) The best virus-free method was screened out. Firstly, apical buds were heat treated for sixweeks(day,38℃,12h; night,30℃,12h), then, we cut shoot tips with leaf primordium(0.3～0.5mm),cultured on the optimum shoot tips medium(MS+KT0.03mg/L); last, when the shoot tip became seedling,we cut shoot tips with leaf primordium twice. The plant regeneration ratio was54%; the virus-free ratiowas80%.(2) The optimum propagation medium had been confirmed, it was MS+NAA0.05mg/L+6-BA0.01mg/L, the best rooting and strength medium of vires-free plantlets was1/2MS+PP3330.2mg/L, apicalbuds were cultured on the rooting medium, The rooting percentage was higher and test-tube plantlets wasstrong. When the test-tube plantlets were transplanted, they had developed roots, green leaves and strongstems; the survival rate of transplanted plantlets was98%. 2. Identified the effect of virus-free on physiology and biochemistry and genetic stability of test-tube plantlets of Huai chrysanthemum(1) Virus-free treatment improved photosynthetic capacity and N metabolism level. With the growthperiod increased, the chlorophyll a contents, chlorophyll b contents and total chlorophyll contents of theleaves of virus-free test-tube plantlets of Huai chrysanthemum increased gradually compared to the controlplantlets. And the chlorophyll contents of virus-free test-tube plantlets were significantly higher thancontrol seedlings. Otherwise, the NR activity, Proline and soluble protein contents were significantly higherthan control seedlings.(2) Virus-free treatment reduced membrane lipid peroxidation, enhanced the anti-membrane lipidperoxide ability and improved the activity of relevant protective enzymes. Compared with control plantlets,the MDA and H2O2contents, and O2·-produce rate of the leaves were significantly lower during the growthstage. The activities of five protective enzymes (SOD, POD, CAT, APX, GR) in enzymatic system forremoving were improved, the contents of two protective enzymes (ASA, GSH) were increased, andcompared with control seedlings, the difference had reached the significantly level (P=0.05).(3) The evidences of isozyme level and molecular level showed that virus-free treatment didn’t changegenetic stability of Huai chrysanthemum. Number of bands of SOD isozyme and POD isozyme were samebetween virus-free test-tube plantlets and control seedlings, but the band brightness of virus-free test-tubeplantlets were brighter than the latter, that proved the SOD and POD specific activities of virus-freetest-tube plantlets were higher than control seedlings. On DNA molecular level, it also proved thatvirus-free treatment didn’t change genetic stability of virus-free Huai chrysanthemum. ISSR primer05thatwas selected was used to determine the genetic stability of virus-free test-tube plantlets. There were nodifference bands among9ISSR fragments detected. 3. Defined the advantages of virus-free, the field adaption of Huai chrysanthemum was enhanced, the yield was increased, the quality was improved, on the base, we screened virus-freeexcellent strain.(1) Virus-free enhanced the field growth of Huai chrysanthemum. The plant height and crown width ofvirus-free Huai chrysanthemum were higher than control seedlings, and the virus-free strain YD5J6was themost prominent. Compared with the control strain YD5, plant height showed significant difference inAugust、September and October, crown width showed significant difference in the four months (P=0.05).(2) Virus-free didn’t change flowering period of Huai chrysanthemum, but increased the quality.Compared with the control strains, the fresh weight per plant, dry weight per plant, branch number perplant, tiller number per plant, flower capita per plant, the length and width of ray florets all increased atdifferent levels. There was significantly difference between virus-free strain YD5J6and control seedlingabout the flower capita per plant, dry weight per plant, the fresh weight per plant and yield per mu (P=0.05), it could be1170.12kg FW per mu, the increasing range was16.4%.(3) Virus-free promoted the activity of some enzymes that related to synthesis of chlorogenic acidand luteoloside, improved quality. The activities of PAL, C4H,4CL of virus-free strains were higher thancontrol seedlings, reached the highest at the period of revealing the colour of buds, there had significantlydifference between virus-free strain YD5J6and control seedlings YD5(P=0.05); the contents ofchlorogenic acid and luteoloside of virus-free strains were higher than control seedlings, and there hadsignificantly difference between virus-free strain YD5J6and control seedling YD5(P=0.05). The contentsof chlorogenic acid and luteoloside of virus-free strain YD5J6was0.79%,0.22%respectively, compared tocontrol seedling YD5, and increasing range was39.7%,62.6%respectively.Taken together, this study no only established an efficient virus-free and rapid propagation technologysystem of Huai chrysanthemum, but also screened a virus-free strain showed a good peiformance, provideda theoretical references for extensive application of virus-free Huai chrysanthemum.