Prunes necrotic ring spot virus (PNRSV) is one of important virus disease in sweet cherry. Because having a wide-ranging host, it had been researched as one of the main virus-free object in the world. Especially, it is belong to second -class quarantine object in China, Shoot tips tissue virus-free culture, leaves regeneration and the cryopreservation of sweet cherry in vitro has been studied according to practical production. PNRSV was detected in some of sweet cherry plantlets in vitro with RT-PCR technique. Buds or tender stem of sweet cherry as the explants,the micropropagation has been studied,the culture medium of MS+ 6-BA1.0 mg/L+NAA0.2mg/L+AgNO35.0~10.0mg/L +sucrose 30 g/L+agar 7g/L was screened out used to differentiate and multiply and differentiating quantities of buds up to more 6. Meanwhile,the inducing rate on culture medium of 1/2MS+IBA 0.7mg/L+NAA0.2mg/L+sucrose20g/L+agar 7g/L being responsible for root growth up to 75.1% and the survival rate by transplanting up to more than 80.5% were accounted for the optimum one. Studies with leaf discs from micropropagated axillary shoots in sweat cherry showed that different type,concentrations and combinations of hormones in the medium could influence rcgeneration of shoots. 6-BA and NAA was more efficient than the others, 3 subcultures of leaf discs from adventitious shoots on the medium amended with MS+6-BA3mg/L+NAA0.2mg/L GA30.5mg/L+AgNO35.0~10.0mg/L gave 6.4% of regeneration frequency.The experiment demonstrated that the first condition of sweet leaves in vitro is choosing the different type, concentrations and combinations of hormones. The shoot tips of in vitro cultured sweet cherry were successfully cryopreserved. The shoot tips were excised from the plantlets cold-hardened at 5℃for 30 days, precultured on MS medium with 0.3,0.5,0.7mol/L sucrose for 1 day every concentration, cryopreserved using encapsulation-dehydration technique and pretreated in PVS2(30%glycerol+15%glycol +15%DMSO+0.4mol/L sucrose) for 60 min before immersion into liquid nitrogen directly. After thawed at 0℃bath of ice water for 2 hours, shoot tips were transferred onto standard medium for regrowth. The survival rate of shoot tips reached 28.7%, after 21 days regrowth. Total RNA, isolated from 3 sweet cherry cultivars, was used as template to amplify PNRSV by polymerase chain reaction (PCR). The results showed the virus-free rate of PNRSV is 78.5% on 0.5~0.8mm shoot tip; The virus-free rate of PNRSV is 93.3% less than 0.5mm shoot tip, it was proved that PNRSV could be eliminated effectively from sweet cherry plantlets in vitro by culture of shoot tip less than 0.5mm. Studies in sweat cherry showed that different type, concentrations and combinations of hormones in the medium could influence regeneration of shoots, were commentated by our research, demonstrated the potential of cryopreservation in vitro shoot tip tissue virus-free culture in sweet Cherry. The results not only help to apprehend the knowledge of cryopreservation, but also provide the new way, used for shoot tips virus-free of sweet cherry. Some condition of leaf regeneration in vitro were found, as well as the medium. All of above can help us to conduct cherry culture technique.
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