Establishment Of A Virus And Viroid Elimination System For Three Tea Chrysanthemum Cultivars

Tea chrysanthemum(Chrysanthemum morifolium Ramat)is a perennial herb of the Asteraceae.It has a high medicinal value and has a long history in the field of medicinal use in China.It is a traditional Chinese medicine commonly used in traditional Chinese medicine.The chrysanthemum in Sheyang County,Jiangsu Province has a long history and many varieties.Among them,’Zaohua Hongxinju’,’Wanhua Hongxinju’ and ’Beijing Chaju’ have the widest range of cultivation and the highest medicinal value.However,with the expansion of planting area and scope,the long-term vegetative reproduction has caused the three kinds of chrysanthemum containing many virus and viroid,so the quality of chrysanthemum declines,the medicinal value is weakened,and the output is reduced year by year.It has restricted the development of its industrialization.Based on the detection of the virus and viroid of the three chrysanthemum,chemical treatment,physical treatment and genetic modification technology were used to get virus and viroid free plants,which will lay a theoretical foundation for the production and promotion of these three tea chrysanthemums.1.According to the field symptoms of ’Zaohua Hongxinju’,’Wanhua Hongxinju’ and’Beijing Chaju’,the data of nested RT-PCR showed that ’Zaohua Hongxinju’has Chrysanthemum B virus(CVB),Chrysanthemum dwarf virus(CSVd)and Tomato infertility virus(TAV).The main infections of ’Wanhua Hongxinju’ and ’Beijing Chaju’both are CVB and CSVd.2.Five different hormone ratios(6-BA:NAA)in MS medium were set up,fifteen shoot meristems ofZaohua Hongxinju’,’Wanhua Hongxinju’ and ’Beijing Chaju’were cultivated on the each medium with the different hormone ratios respectively.the optimal medium was MS with 1mg/L 6-BA and 0.1mg/L NAA.3.Fifteen shoot meristems of ’Zaohua Hongxinju’,’Wanhua Hongxinju’ and ’Beijing Chaju’were cultivated on the MS medium with 4 different concentration of ribavirin,the optimal concentration of ribavirin was 40 mg/L.4.The combination treatment of cold(4℃)and 40 mg/L ribavirin for 2,4 and 6 months respectively,6 strains of virus-free seedlings of ’Zaohua Hongxinju’,4 strains of virus-free seedlings of’Wanhua Hongxinju’,and 5 strains of virus-free seedlings of’Beijing Chaju’ were obtained respectively.5.The genetic stability of the virus-free seedlings and the conventional sterile tissue cultured seedlings obtained from three different virus elimination methods was detected with SRAP primers.The results showed that there was no difference in the genetic locus between the virus-free seedlings obtained by the three different virus elimination methods and the conventional sterile seedlings,and that the virus elimination treatment and regeneration culture has no genetic mutants and maintain the genetic stability of the three tea chrysanthemums.6.The 3D8scFv is an antibody with RNA digestion,which has a certain effect on the elimination of chrysanthemum virus.The ’Zaohua Hongxinju’ containing CSVd and the vector pORE-R4-1X35s-3D8scFv were used for the establishment of transgenic systems to eliminate viroid.The optimal regeneration medium was MS containing 2 mg/L 6-BA,0.2 mg/L NAA with 30 mg/L kanamycin.