Detection Of Viruses CVB,TAV And CChMVd Infecting Chrysanthemum By RT-PCR And Plantlet Regeneration Of Procumbent Chrysanthemum

There are many kinds of viruses endangering chrysanthemum.The viruses reported are more than 20 kinds and our country has identified only 7 kinds.In our country,a part of chrysanthemum viruse diseases are quarantine disease.So,it is very important for chrysanthemum industry that the study of detecting chrysanthemum viruses.Procumbent chrysanthemum,a new chrysanthemum species,possesses good charactistics such as small plant and rapid growth.In order to use genetic transformation in the improvement of chrysanthemum,it is necessary to establish tissue culture regeneration system.In this study, the RT-PCR detection technique of chrysanthemum viruses and the regeneration of plant from procumbent chrysanthemum leaf pieces were studied.The main results were as follows:1.Three pairs of primers of CVB,TAV,CChMVd designed according to the gene sequences reported before were used to amplify by RT-PCR.Detection system was established by RT-PCR,special amplification products were cloned,and by comparing DNA sequencing results and reported homology sequences,nucleotide acid homology was analysised.Partial segment of CVB was cloned into pGEM T vector to constitute recombinant plasmid and was 633bp long.Its nucleotide acid homology with reported homology sequences was about 82%.Partial segment of TAV was cloned into pGEM T vector to constitute recombinant plasmid and was 842bp long.Its nucleotide acid homology with reported homology sequences was about 98%.CChMVd is viroid.Partial segment of CChMVd by RT-PCR was cloned into pGEM T vector to constitute recombinant plasmid and was 217bp long.Its nucleotide acid homology with reported homology sequences was above 98%.2.RT-PCR sensitivity was studied by detecting CChMVd,and the result showed only fg RNA could be detected. 3.Double RT-PCR was applied for CVB and TAV detection in virus-infected and virus-free plants and the result was similar to that of single RT-PCR.4.Prokaryotic expression was studied by TAV CP complete gene,and the result showed 28kD CP protein which could be used to make antiserum.5.This study established plantlet regeneration of procumbent chrysanthemum using MS basic culture medium with two hormones NAA and 6 BA.The culture medium of asepsis plant was MS+0.1mg/L NAA+0.5mg/L 6-BA;the medium of rooting was 1/2MS+0.1 mg/L NAA,which began rooting in a week and rooting rate was 100%;the culture of plantlet regeneration was MS+0.1 mg/L NAA+0.5 mg/L 6-BA and the rate of inducing shoot was 96%.