| Pears are one of the most important fruits in our country.Pears fruit not only delicious and juicy,but also have rich nutrition and health benefits.Pear industry is the third largest fruit industry in our country,but pear trees generally carry virues,which causes the pear trees vigor to weaken,the growth decline,the fruit quality and the output decline,seriously affects the development of pear industry in China.The main method for solving the harm of virus diseases is to cultivate virus-free seedlings.This study used‘Yuluxiang’,‘Zaosu’,‘Korla’pear,‘Dangshansu’and bud mutation of‘Dangshansu’as materials to study the main latent viruses apple stem grooving virus,apple stem pitting virus and apple chlorotic leaf spot virus in pear.The main findings are as follows:1.Cultivation of sterile seedlingsIn spring,the buds of‘Yuluxiang’,‘Zaosu’,‘Korla’pear,‘Dangshansu’and bud mutation of‘Dangshansu’were collected from the field,and sterilized in clean bench before being inoculated into a culture medium(MS+1.0 mg·L-16-BA+0.3 mg·L-1IBA).The survival rates of‘Yuluxiang’,‘Zaosu’,‘Korla’pear,‘Dangshansu’and bud mutation of‘Dangshansu’were 46.4%,37.5%,52.5%,59.7%and 53.6%,respectively.2.Subculture cuiture and rooting culture‘Korla’pear subculture medium was MS+1.0 mg·L-16-BA+0.3 mg·L-1IBA,the multiplication coefficient was 7.5;the rooting medium adopted two-step rooting method,the first step of culture medium was 1/2MS+0.5 mg·L-1NAA+0.5 mg·L-1IBA,and the second step medium is 1/2MS,the rooting rate was 56%and the average root length was 3.43 cm.Bud mutation of‘Dangshansu’subculture medium was DKW+0.8 mg·L-16-BA+0.3 mg·L-1IBA,the multiplication coefficient is 4.7;the rooting medium was 1/2MS+1.5 mg·L-1IAA+0.5 mg·L-1IBA,the rooting rate was51%and the average root length was 2 cm.‘Zaosu’subculture medium was MS+0.8mg·L-16-BA+0.3 mg·L-1IBA,the multiplication coefficient is 5.4;the rooting medium adopted two-step rooting method,the rooting rate was 49%and the average root length was 3 cm.‘Yuluxiang’subculture medium was DKW+1.0 mg·L-16-BA+0.3 mg·L-1IBA,the multiplication coefficient was 6.2;the rooting medium adopted two-step rooting method,the rooting rate was 38%and the average root length was 2.71 cm.‘Dangshansu’subculture medium was DKW+1.0 mg·L-16-BA+0.3 mg·L-1IBA,the multiplication coefficient is 5.7;the rooting medium was1/2MS+1.5 mg·L-1IAA+0.5 mg·L-1IBA,the rooting rate was 57%and the average root length was 2.86 cm.The above subculture media were all supplemented with agar 7 g·L-1and sucrose 30 g·L-1,while the rooting medium was supplemented with agar 7 g·L-1and sucrose 20 g·L-1.3.Virues eliminationIn the process of virus elimination by shoot tip culture,the survival rate was22%when the length of shoot tip was 0.5 mm,the virus-free rate of ASPV and ACLSV was 79%and 48%respectively,and the virus-free rate of ASGV was 15%.The survival rate was 46%when the length of shoot tip was 1 mm.The elimination rate of ASGV,ASPV and ACLSV was 7%,14%and 8%,respectively.To explore the effect of heat treatment temperature on survival rate of plants after heat treatment,four temperature gradients of 32/38°C（16/8 h）,34/40°C（16/8 h）,38°C and 40°C were set,the survival rates of’Dangshansu’were 15%,0%,7%and0%at four temperatures,while those of‘Korla’pear were 45%,8%,22%and 1%at four temperatures.The survival rates of’Zaosu’at four temperatures were 24%,1%,25%and 0%,and those of’Yuluxiang’at four temperatures were 27%,2%,21%and 5%,respectively.The survival rates of bud mutation of’Dangshansu’were26%,3%,22%and 2%at four temperatures,respectively.The suitable heat treatment temperature was 32/38℃（16/8 h）,and it was found that‘Korla’pear is more heat-resistant,with a significantly higher survival rate than other varieties,reaching 45%.Exploring the effect of heat treatment time on plant survival rate at a temperature of 32/38℃（16/8 h）,four gradients of 25 d,30 d,35 d,and 40 d were set.Except for‘Korla’pear,the survival rate of other varieties significantly decreased after 30 d,so a treatment time of about 30 d is more reasonable.The virus-free rates of ASPV from’Dangshansu’,‘Korla’pear,’Zaosu’,’Yuluxiang’and bud mutation of’Dangshansu’were 65%,72%,77%,80%and 93%,respectively.The virus-free rates of ACLSV were 88%,84%,100%,89%and 96%,but the virus-free rates of ASGV were 40%,31%,28%,56%and 39%.The detoxification effect of heat treatment on ASPV and ACLSV is good,and‘Korla’pear was the most suitable for heat treatment.Explored the precultivation sucrose concentration and PVS2 treatment time for ultra-low temperature detoxification.It was found that the regeneration rate was 89%when the concentration of sucrose was 0.6 mol·L-1and the treatment time of PVS2was 60 min.The virus-free rate of ASGV by ultra-low temperature treatment was higher than that by heat treatment.The method of ultra-low temperature combined with heat treatment is the best for the removal of all three viruses,and can reach about80%for ASGV which is difficult to be removed by other methods,but the disadvantage is that the survival rate is very low,ranging from 12%to 20%.Through the above methods,detoxified seedlings of‘Yuluxiang’,‘Zaosu’,‘Korla’pear,‘Dangshansu’and bud mutation of‘Dangshansu’were obtained.4.Transplantation of virus-free seedlingsThe combination of nutrient soil:vermiculite:perlite=7:2:1 has a better effect of transplanting,and the survival rate is 94.8%... |
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