Optimization Of The Method Of Removing Several Common Strawberry Viruses By Meristem Culture And The System Of Virus Detection By Polymerase Chain Reaction (PCR)

In this paper, strawberry (Fragaria×ananassa Duch.) was taken as test material; shoot tips of creeping stems were used as explants to establish fast propagation system, followed by virus detection and virus-free culture. The main purpose of the study is to improve and optimize the method of shoot tip culture removing several common strawberry viruses (Strawberry crinkle virus SCV, Strawberry mottle virus SMoV, Strawberry mild yellow edge virus SMYEV, and Strawberry vein banding virus SVBV) and PCR virus detection system. The main study content including (1) to choose the best way to extract strawberry total RNA, and it is suitable for more repeated extractions of RNA;(2) to screen out of the primers of four virus-specific fragments and plant mRNA internal control of the subject, which will be used to virus detection and multiplex RT-PCR system optimization;(3) to eliminate DNA from extracted RNA, whose reverse transcription product cDNA was used as template to filter out the primers, then specific fragment was amplified, the parameters of PCR system and process were optimized or improved;(4) to remove viruses from strawberry through shoot tip culture combined with the constant temperature water bath heat treatment (35℃,40℃) and variable temperature water bath heat treatment (35℃and40℃were changed). Through different combinations of the processing temperature, the time of water bath (2h,4h,6h) and length of the shoot tip (0.5mm,0.8mm,1.0mm), compared with the shoot tip culture without heat treatment, detected the corresponding effect on virus-free of strawberry, filter out a bigger shoot tip length under the most suitable way for removing viruses and facilitate the production and application. The main findings of this study are (1) the modified CTAB method can be regarded as the best method of extraction high-quality total RNA in strawberry leaves compared with the plant RNAOUT kit and plant column RNAOUT kit, in terms of the quality and purity or its cost of the extracted RNA.(2) primer pairs for PCR detection finalized SD1/SD2, D1/D3, Y1/Y2, SV1/SV2, Nla/N1b, N1a/N2b, the latter two pairs’sequence data from mitochondrial NADH dehydrogenase ND2subunit (ndhB gene) of the complete genome sequence of Atropa belladonna L. used as internal control, and the size of the target fragments were345bp,219bp,271bp,422bp,571bp and188bp respectively.(3) the optimization of the PCR system including the20μl reaction system and PCR program design. The best RT-PCR system used to diagnose strawberry virus as followed,1μl cDNA,10×PCR buffer,1.5mmol/L MgCl2,0.2mmol/L dNTPs,0.5U of Taq enzyme, primer concentration0.1μmol/L, pure water was added to the20μl system; PCR procedure:94℃initial denaturation for5min, denaturation at94℃for1min,54℃annealing for40s,68℃extension for1.5min,35cycles,68℃extension of10min.(4) in this experiment, shoot tip culture combined water bath treatment at35℃for2h and then at40℃for2h is the best, the length of shoot tip is1.00mm。Four viruses are removed at the same time, and the rate of it is up to90%. In addition, a relatively higher seeding rate and survival rate are71.11%and88.89%. This brings convenience to the production of the shoot tip detoxification of strawberry to a large extent.