Study On Elimination Of Latent Viruses From In Vitro Cultured Plants Of Pear By Cryotherapy And Shoot Tip Culture

Pear cultivars are widely infected with three latent viruses, namely Apple chlorotic leaf spot virus, Apple stem grooving virus, and Apple stem pitting virus, which usually affect the growth of tree, reduce the fruit yield and quality. All these viruses can be mechanically transmitted onto other plants by grafting techniques and are naturally disseminated by infected propagating materials, and have no insect vectors. Utilization of virus-free materials is the most effective measure for the control of these viral diseases. The main methods developed for virus elimination include shoot tip culture in vitro, thermotherapy, and chemotherapy. In recent years, cryotherapy of shoot tips has been utilized widely for plant virus elimination, which is a novel technique and does not require any special equipment and only marginally add to the time and cost of the traditional procedures of shoot tip culture for pathogen eradication. In order to construct the vailable virus elimination system for different pear cultivars, we selected the in vitro cultured pear plants infected with latent viruses as the materials, and optimized the parameters of cryotherapy-based procedures and assessed the efficiency of virus elimination by cryotherapy.1. Eighteen pear cuttings were mainly collected from three regions, namely Institute of Fruit and Tea of Hubei Academy of Agricultural Science, Yantai Academy of Agricultural Sciences, and Huazhong Agricultural University. Three latent viruses were detected by RT-PCR. The result showed that all collected samples contained at least one of the three latent viruses except for the pear cultivar’Kangfolunsi’sample from Hubei province. Only four samples contained all three latent viruses, and12samples contained two latent viruses, which were ASGV and ACLSV or ASGV and ASPV.2. Shoot tips of collected samples were separated and cultured in the proliferative MS medium, and in vitro plants of14cultivars were obtained. Result showed that survival rate varied among different cultivars significantly, which was related to pear species. Among all cultivars, the survival rate of shoot tips of seven cultivars belonging to sand-pear (Pyrus pyrifolia) species was relatively higher, which varied from50%to 100%, and the survival rate of shoot tips of cultivars belonging to European pear(Pyrus communis) species was lower, which varyed from0to40%. The survival rates of shoot tips of two cultivars from White pear(Pyrus bretschneideri) species were57.4%and81.25%, respectively. The survival rate of shoot tips of cultivar’Nanguoli’belonging to Chinese pear(Pyrus ussuriensis) species was46.67%.3. The in vitro cultured plants of sand pear cultivar ’Zhenyu’ were used as the materials to optimize the important factors such as preculture time, loading time and the treatment time of PVS2solution on the survival rate of shoot tips after cryotherapy treatment. Results showed that the treatment time of PVS2solution affected survival rate of shoot tips mostly after cryotherapy. Two days of preculture treatment,20min of loading treatment and90to100minutes of PVS2solution treatment could reduce death rate of shoot tips significantly. The optimized technical system was applied to the shoot tips of four sand pear(Pyrus pyrifolia) cultivars, and virus detection was done by RT-PCR after cryotherapy treatment. Results showed that the efficiency of virus elimination varied among different sand pear cultivars. The virus elimination rate of Sand pear culivar ’Zhongli No.1’ was the highest, up to100%, while the virus from pear culivar’Huangjin’ and’Eli No.2’could not be eradicated.4. Based on the above results, virus elimination study was tried by combining in vitro thermotherapy (38℃day/32℃night,30d) and cryotherapy (Vitrification), or combining in vitro chemotherapy (20μg/mL Ribavirin) and cryotherapy. We also analyzed and compared the efficiency of two methods for virus elimination. The result showed that the growth condition of in vitro plant of different pear cultivars during heat treatment had a positive correlation with the survival rate of shoot tips after cryotherapy. The in vitro plants of cultivar ’Hongbeileisha’ and ’Yuanhuang’ were least affected during heat treatment, and the survival rate of shoot tips after cryotherapy was up to85.0%, while the in vitro plants of cultivar’Meirensu’were affected mostly during heat treatment, and the survival rate of shoot tips after cryotherapy was lowest, only40%. The virus elimination rate varied among different pear cultivars. The rate was highest in sand pear culivar ’Yuanhuang’, up to90%, followed by the pear culivars ’Fengshui’ and ’Hongbeileisha’, while pear cultivars ’Xuehua’ and ’Meirenshu’ had lowest elimination rate, only20%. The efficiency of virus elimination varied among three viruses too, and the elimination rate of ASPV was up to100%, and ASGV ranged from20%to90%.5. We selected in vitro plants of two pear cultivars (’Feishui’ and ’Meirensu’) as the materials, and analyzed the virus elimination efficiency by combining in vitro chemotherapy (20mg/L Ribavirin) with cryotherapy. The results showed that Ribavirin treatment had no obvious effect on the growth of in vitro plants. In addition, the survival rate of shoot tips after cryotherapy was higher, up to86.6%. The efficiency of virus elimination was related with the treatment time of Ribavirin treatment. When the treatment time of Ribavirin was extended from30to40days, the elimination rates of ASGV increased from84.6%to90%,60%to80%for cultivars’Fengshui’and ’Meirenshu’, respectively.