Studies On Identification Of Viral Disease Pathogens And Establishment Of Virus-free Technology In Salvia Miltiorrhiza Bge.

Danshen(Salvia miltiorrhiza Bge.)is perennial herb specie of Salvia L.in Labiata,and its root as the medicinal part is a commonly used traditional Chinese medicine that has been mainly used for the prevention and treatment of vascular diseases in the clinics.Hebei province is the main producing area in China.In recent years,a disease caused by a kind of virus can cause the degeneration of Salvia miltiorrhiza Bunge and the incidence is very high and reaches up to 60-80%in the fields.The symptoms include mosaic leaves,mottling,leafrolling,yellowing,and stunting,etc.The roots of the susceptible plants are always very fine,the yield is reduced drastically,the quality becomes deterioration and the contents of medicinal ingredients decreased greatly.The continuous random sampling testing,showed that the mean value of medicianl ingredient of TanshinoneⅡA in Anguo County of Hebei is only 0.135%,less than 0.2%of the standard value set by the pharmacopoeia of China.So it is not high-quality Danshen.The virus infection and variety degeneratiaon are the main reasons what causing the bad quality,low output and serious plant disease of Salvia miltiorrhiza.And there aren't any reports about the occurrence of Salvia miltiorrhiza virus disease.Recently,there isn't any effective medicaments than control the virus disease.So the effective measures for radically improving the yielding and quality and controlling the disease of Salvia miltiorrhiza are found out the pathogens of virus diseases,removing the virus and purifing and rejuvenating,and this is also the urgent problem that must be settled in Danshen production.This study is mainly divided into three parts:the identification of virus pathogen,the study of removing virus technology and the agronomic traits comparative evaluation of virus-free Danshen.Ⅰ.A preliminary certainty about the virus pathogen of Salvia miltiorrhiza has been made by using the technology of biology assay,electron microscopy(EM)and double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA).And the molecular biology technology has also been used for the study of the virus disease,cloning the genes of the virus and analyzing the sequences so as to identifing the virus.According to specific viral gene sequence,primer was designed and then established the virus molecular detection technology based on reverse transcription polymerase chain reaction(RT-PCR)technique.Ⅱ.Virus-free methods researchs have been done,the apical buds were removed from the Salvia miltiorrhiza in the field and inoculated to the culture medium to get the tissue culture plantlets;the root segments were mechanically inoculated on the induced culture medium to get callus.By the method of the heat treatment with combing the shoot tip meristem culture,and the microcell clusters inductive regeneration technology,the virus was eliminated from Salvia miltiorrhiza plantlets.The virus was detected by DAS-ELISA and RT-PCR so as to gaining the virus-free plantlets.Based on these plantlets,we founed the systems of rapid propagation technology of virus-free Danshen plantlets and used for large-scale production of seedlings.Ⅲ.The agronomic traits and pharmacognosy characteristics comparative evaluation of virus-free plants from Salvia miltiorrhiza were conducted between virus-free and virus infected Salvia miltiorrhiza.Through the comparative analysises of growth and development characteristics, characters of seedling stage,biological characteristics,yileding characters,pharmacognosy characteristics and medical ingredients contents,the characteristics and its field performance of virus-free Salvia miltiorrhiza were made clear.The results showed that:Ⅰ.The biological inoculation asssy results showed that the inoclulated Nicotiana tabacum -White Burley showed system mosaic,chlorosis and mottle,and N.tabacum-Samsun showed partial mottling and leaf rolling,etc.This indicate that the Salvia miltiorrhiza disease can infect other species plant and it is not physiological disease but viral disease.The infection symptoms of Danshen both in field and indicator plant showed similarity to the symptoms caused by Cucumber mosaic virus(CMV)or Tobacco mosaic virus(TMV).The parlticle diameter of viruses observed by EM was 29-30nm which its size was similar to that of CMV.The DAS-ELISA detection with CMV antiserum showed that 10 samples were detected positively in all 15 samples.And the preliminary result showed that the main viral pathogen of Danshen was a isolate of CMV. Ⅱ.The CMV coat protein gene from Danshen was cloned and sequenced,the analysis showed the viral pathogen of Danshen was a new CMV strain.This coat protein gene wasdeposited by the international GenBank(the accession number is AY600989),which was the independent intelligent property right gene.According to the naming standard for the classification of International Committee on Taxonomy of Virus(ICTV),we named this new isolate as CMV-DS strain,these played a foundation on removing the virus from Danshen and the research of plant transgenetic engineering for resistance the virus.Ⅲ.In this research,we have founded "a new virus-free methods of mcirocell clusters inductive regeneration of Salvia miltiorrhiza",and it was more effective than the other methods such as heat treatment and shoot tip culture method,and its virus-free plants reached up to 75%.The method has been applied for the national invention patent in September 2006,and the patent accession mumber is 200610048342.6.This method solved the problems that Salvia miltiorrhiza cannot tolerant to heat treatment with normal virus-free methods and highly improved the virus-free efficiency.This thought and method can also be used to removing the virus from other plant species.Ⅳ.The tissue culture and quick propagation technology of Danshen was found:The best cuture medium of inducing buds from roots or stem segements was 1/3MS+BA 1.0mg/L+IBA 0.02mg/L+Cane sugar 30g/L,pH5.8;the most suitable culure medium for buds growth was MS+BA1.5 mg/L+IBA 0.2mg/L+Cane sugar 30g/L,pH5.8;the most suitable culture medium for rooting was 1/2MS+IAA 1.0mg/L+Cane sugar 20g/L,pH5.8;the optimum tempreture was 25-28℃and the optimum light intensity was 2000-3000 Lx.And the buds differentiation subculture should be one time with 25-30 days.The propagation coefficient was more than 6.0.The plantlets in vitro could root in about 20 days after inoculated on the rooting culture medium,and after 30 days,the rooting plantlets from the medium began to be cultued on the sterilization vermiculite in greenhouse where the temperature was keeping between 20-30℃,and the humidity was over 90%.After 30 days,the seedling training was controlled with water and sustained for 1-2 weeks to make the roots become older,during the seedling training,Carbendazim or thiophanate methyl are spraying once on the seedlings per 3-5 dsys in order to prevent from the infecting pathogens,and then the seedlings can be transplanted in the field.Ⅴ.The yield of virus-free Danshen was increased significantly:the biological characteristics and the growth period are similar beteen the virus-free and infecting Salvia miltiorrhiza,the virus-free seedlings that planted in the fields growing coordinately with status,the roots enlargment course are faster,the root slips are uniformed and the commodity roots pharmacognosy characteristics are better than the virus-infecint Danshen.From the roots numbers,roots diameter,roots weight per plants and the medicinal ingredients contents,the virus-free Danshen showed more superior to infecting plants,which make the production improved by 86.6%than the control.Ⅵ.The medicinal ingredient contents are improved markedly:thepharmacognosy characteristics are unifom between the the virus-free and infecting Salvia miltiorrhiza,analysis of medical compositions indicated that the average contents of tanshinoneⅡA is 0.376%in virus-free Salvia miltiorrhiza,and the control is 0.192%,which was nearly 2 times of the infecting plants and the production was improved by 88%which is regulated by Chinese Medicinal Dictionary.The extension and application of virus-free Salvia miltiorrhiza will lay a foundation for resolving radicaly the lower medicinal ingredient contents which don't accordance with the Chinese Medicinal Dictionary.