Development Of Cryotherapy Of Shoot Tips For Virus Eradication Of Strawberry

Fragaria x ananassa Duch. is perennial herbaceous plants of Rosaceae Fragaria. The fruit that is fresh and juicy, bright color, aroma and pleasant, taste sweet and sour moderate, is one of our traditional excellent berry fruits. Its breeding mainly adopts the traditional plant breeding or stolons bury, this way of asexual improved the chances of the viruses infect plants. To avoid virus production bring huge economic loss, achieve the strawberry virus-free seedling cultivation, will certainly is the development tendency and the target of strawberry industry in our country at present. And cryogenic treatment is a new type, new technology, efficient removal of plant virus is the researchment department scholar’s favor.In this paper, Shoot tips from Fragaria xananassa’Benihoppe’were used as materials to screen and optimizate the key procedure (culture, pretreatment and thaw conditions) of cryotherapy, compared with the shoot tip culture for establishing and optimizing cryotherapy of shoot tips. In order to establish perfect multiple RT-PCR virus detection system, the parameters of RT-PCR system and process (Mg2+ concentration, primers concentration, PCR program and so on) were optimized or improved.At last, using the software of DNA to screen out of SSR-PCR primers, and analysize genetics traits of Virus-free plants of cryotherapy. The results were as follows。(1)The best technology system of cryotherapy of Fragaria×ananassa ’Benihoppe’ were in fellow procedure:preculture 7 days in the dark, with 0.3mol·L-1 sucrose,60% PVS2 loading 60 minutes, PVS2 treatment in 1 hours, liquid nitrogen treated 1h and and throwing in 40℃ water for 120 s, using liquid MS culture medium containing sucrose 1.2 mol·L-1 washing twice, each time 10 min.The system can simultaneously removal four kind of virus (strawberry vein banding virus, strawberry mild yellow edge virus, strawberry crinkle virus, strawberry mottle virus) and the survival rate was 69.03%. Then this system were used in Fragaria^ananassa’shufeng’, Fragaria xananassa’toyonoka’ and Fragaria×ananassa’zoji’. Their survival rates were 68.15%,71.00% and 69.78%. The results achieved the target of the desired test.(2)The best primer pairs for multiple RT-PCR were SC1/SC2, SM1/SM2, SY1/SY2, SV1/SV2 and A1/A2. The size of the target fragments were 345 bp,394 bp,861 bp,271 bp and 295 bp respectively. The best multiple RT-PCR system used to diagnose strawberry virus as followed,1μl cDNA,2 μ110×PCR buffer,2.0 mmol·L-1MgCl2,0.5 U of Taq enzyme,0.2 mmol·L-1 dNTPs, primer concentration 0.2 μmol·L-1(SCV 0.25 μmol·L-1), then pure water was added to the 20μl system; RT-PCR procedure:initial denaturation at 94℃ for 3 min, denaturation at 94℃ for 1 min,55℃ annealing for 40s,68℃ extension for 40 s,35 cycles,72℃ extension of 5min.