Study On Pokeweed Tissue Culture And Its Antiviral Effect On Tobacco Mosaic Disease

Pokeweed antiviral protein (PAP),a single chainribosome-inactivating protein (RIP) isolated from the tissues of pokeweed plants (Phytolacca americana), can remove specific adenine and guanine residues from the highly conserved, a -sarcin/ricin (S/R) loop in the large rRNA; interfere with the binding of elongation factor eEF-2 with the 60 subunit of ribosome and inhibit protein synthesis,fmally PAP makes the host cell dead.PAP is thought to be a defense protein because it is a powerful inhibitor of animal and plant viral pathogens,including tobacco mosaic virus,potato virus X,poliovirus,herpes implex virus,influenza,brome mosaic virus and human immunodeficiency virus and so on.PAP have two striking features in resistence to plant virus ie nonspecificity towards different to plant virues and strong activity at low concentration.There are two ways in using of antiviral activity of PAP: one is spraying the solution of including PAP on plants directly,the other is the transformation of PAP cDNA into the plants,transgenic plants show antiviral activity because of the PAP gene expression in plants,which the resistance to virus.This papers focus on the study of using the former method.We intended toselecting lines containing the high content of PAP from the organ of pokeweed by technology of tissue culture and the technology of hairy root transformation.The antiviral activity of callus lines to TMV was tested by machenically inoculating tobacco plant(K326) with TMV.The results is following:(1) Tissue culture. Tender leaves as ex-plants can be induced large amount of callus on MS culture Medius containing 2,4-D 0.5mg/L and 6-BA 0.2 mg/L is suitable for callus inducing.During the continuing culture procedure ,however, MS Culture Medius containing 2,4-D 2.0mg/L and 6-BA 0.5 mg/L will be better.The pokeweed calli can develop into five different colours,pink,red,white,light-yellow-green,brown.The speed of pink callus lines growing is faster in solid or liquid culture medium.The antiviral activity of five different color callus lines on TMV,pind also were proved better than the others.So in the cell liquid suspension culture,pink callus lines maybe a ideal "seed'Mn order to get the highest harveat level,suitable culture conditioning isrshaking speed: 120/RPM,volme 100ml/500ml,30%inoculation quatities,culture time 2 weeks.(2) In hairy root transformation^ have got trans-hairy root.The tender leaves as ex-plants,the concentration of inoclum was OD600 is 0.3,diluted 3 times,culture the inoculum for 2 days can get high inducing rate.(3) The pink color lines can grow well and get high amount of PAP on solid and liquid medius,Meanwhile it showed high resistance to TMV. Generally speaking,the pink color lines is a ideal candidate.