Improvement Of The Cryotherapy For Elimination Of Pear Viruses And Comparison Of The Growth Characteristics Between Virus-Infected And Virus-Free In Vitro Pear Plants

Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) occurred commonly in pear (Pyrus spp.) cultivars, which result in plant decline, reduced yield and fruit quality. Cultivation of the virus-free seedlings is an important way to control the virus-inducing diseases, In this study, we used thermotherapy combined with cryotherapy method to obtain the virus-free in vitro pear plants, and evaluate the possibility of the propagation of virus-free seedlings by pear twig grafting technology. In addition, the comparison of the growth characteristics between the virus-infected and virus-free in vitro pear plants was conducted. The results provided important information for the establishment and improvement of virus elimination system on pear.1. Four pear cultivars collected from Xingcheng city in Liaoning province were used for the dormant bud differentiation culture trials. The basic MS medium and three plant growth regulators (IBA,6-BA and NAA) were used for in vitro cultivation, In order to screen out the optimal differentiation medium composition for four pear cultivars, the concentrations of three plant growth regulators were optimized with orthogonal experiment. The results showed that the optimal medium composition differentiated with the pear cultivars.6-BA was necessary for differentiation culture of all four pear species, and the concentration of that ranged from1to4mg/L, while IBA could promote the growth of three pear cultivars (P. bretschneideri cv."Jingbai", P. sinkiangensis cv."Seerkefu", P. sinkiangensis cv."Kuerlexiangli") with the concentration ranging from0.2to0.4mg/L, while NAA could only promote the growth of P. bretschneideri cv."Jingbai".2. The in vitro regenerated P. sinkiangensis cv."Kuerlexiangli" plants infected with ASPV were used for the virus elimination by thermotherapy (20,30, and40d) combined with cryotherapy. Meanwhile, the microstructure of shoot tip cells under different treatment conditions were observed by paraffin section technique. The in vitro P. pyrifolia cv."Jinshui NO.2" plants with ASGV were used for the virus elimination by chemotherapy (ribavirin processing for20,30, and40d) combined with cryotherapy. The results showed that thermotherapy could promote the growth and proliferation of P. sinkiangensis cv."Kuerlexiangli" plants, and eliminated ASPV at a certain extent (the elimination rate ranged from14%to87%). No regenerated plants were obtained caused by vitrification under cryotherapy treatment. After thermotherapy, the pear stem tip cells became larger, and stem tip cells of the plants treated with thermotherapy and cryotherapy were more seriously damaged compared with single shoot tip processing. In the virus elimination experiment of P. pyrifolia cv."Jinshui NO.2", ASGV could be completely removed from the vitro-plants with chemotherapy for40d combined with cryotherapy, while both of the elimination rates were50%with chemotherapy for20d or30d. On the other hand, single cryotherapy treatment was not appropriate for the virus elimination (the elimination rate was0%). In addition, we also found that the survival rate of shoot tips (1mm) of plants positively related to the fitness degree of original in vitro plants.3. Four physiological and biochemical indexes (SOD activity, MDA, soluble protein and chlorophyll concentrations) were measured with virus-infected and virus-free in vitro plants of P. pyrifolia cv."Yuanhuang" and P. pyrifolia cv."Xuehuali". The results showed that no significant difference in SOD activity, MDA and chlorophyll concentrations were found between virus-infected and virus-free plants. In addition, different cultivars showed opposite results in the soluble protein concentration. The results above suggested that the influence of virus may be weak to the young in vitro plants.4. P. pyrifolia cv."Fengshui", P. pyrifolia cv."Jinshui NO.2" and P. communis cv."Red Bastlett", P. communis cv."Conference" were used for virus-free seedlings propagation by twig grafting and the graft survival rate was calculated after30days. The results showed that the P. pyrifolia cv."Fengshui" had the highest survival rate (80%), and the survival rates of P. pyrifolia cv."Jinshui NO.2" and P. communis cv."Red Bastlett" ranged from40%to60%, the survival rate of P. communis cv."Conference" was low (around20%). The results showed that the twig grafting technique can be useful in the propagation of virus-free seedlings in vitro.