变温热处理结合茎尖培养脱除沙梨潜隐病毒研究
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摘要
苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus, ACLSV)、苹果茎沟病毒(Apple stem grooving virus, ASGV)和苹果茎痘病毒(Apple stem pitting virus, ASPV)是世界仁果类果树上普遍发生的三种潜隐病毒,我国主栽的梨品种的平均带病毒率为86.3%。根据果树病毒病通过嫁接传染等特点,目前主要通过栽培无病毒苗木减轻病毒病的危害。本研究以同时带有这三种病毒的沙梨离体植株为材料,采用变温热处理结合茎尖培养方法进行病毒脱除试验。
1.对采集的21个沙梨品种的枝条材料进行病毒检测,结果显示所有材料均带至少一种潜隐病毒,其中ACLSV的带毒率为90.48%, ASGV 85.71%, ASPV 42.86%。通过茎尖培养(诱导分化培养基为MS+0.2mg/L IBA+1.0mg/L 6-BA,琼脂5.4g/L,蔗糖30g/L, pH5.8)方法处理21个沙梨品种251个茎尖,共获得20个沙梨品种366株离体植株。结果显示,不同沙梨品种的诱导分化成活率和茎尖分化系数有异,安农一号、清香、桂冠和丰水等5个品种的成活率最高,达到100%,而金水一号未成活。中梨一号的茎尖分化系数最高,新高其次,桂冠和翠冠最低。对15个品种离体植株79个芽系进行病毒检测的结果显示,ACLSV检出率为67.09%(53/79), ASGV 86.08%(68/79), ASPV 32.91%(26/79).
2.采用变温热处理(34℃/40℃,8h/16h交替)结合茎尖(大小约1 mm)培养对8个沙梨品种的1014株带毒离体植株进行病毒脱除处理,共获得8个沙梨品种的69株脱毒材料。结果显示不同品种处理后的成活率和增殖系数有异,成活率中梨一号为14.7%,清香和金秋为9.0%,黄金为2.11%。增殖系数圆黄为2.7,新高为2,丰水、爱甘水和黄金等3个品种没有增殖。通过RT-PCR等方法对8个沙梨品种的65株脱毒材料进行病毒检测的结果显示,有5个沙梨品种的39株离体植株不带病毒,且不同病毒的脱除率有差异,ASGV为70.77%(46/65), ACLSV 90.77%(59/65), ASPV 95.38%(62/65)。
Apple chlortotic leaf spot virus (ACLSV),apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are three latent viruses in pome fruit trees,which are distributed worldwide. About 86.3% of main pear tree varieties in China are infected by the three latent viruses.Based on viral disease infection by grafting techniques on pear for these virus,the major efficient way of mitigating them is via the planting of virus-free germplasm.In this study,with the in vitro sand pears infected three latent viruses was used as source for viral materials.Heat treatment under 34℃/40℃combined shoot tip culture were used as techniques for virus elimination.
Branches were collected from 21 varieties sand pear plants and virus detection was done by RT-PCR.Detections showed that all the materials collected contain at least one of three latent viruses. The infected rate was as follow:90.48% ACLSV,85.71% ASGV and 42.86%ASPV.For the 21 varieties containing 251 buds,shoot tip culture was done in a proliferative MS (MS+0.2 mg/L, IBA+1.0 mg/L 6-BA, Agar 5.4 g/L, sucrose 30 g/L, pH5.8),366 in vitro plants of 20 cultivars were obtained.Result showed that survival rate and differentiation coefficient varied between cultivars. The highest survival rate of 100% was reached for Anlongyihao, Qingxiang, Guiguan and Fengshui,the lowest of 0% reached of Jingshuiyihao. For tip differentiation coefficient, Zhongliyihao was the highest followed by Xingao.Guiguan and Cuiguan recorded the lowest becquse they show no tip polarization.15 cultivars of in vitro sand pear have been dectected,including 79 buds.Results showed that the detection rate of ACLSV was 67.09%(53/79), the detection rate of ASGV was 86.08%(68/79), the detection of ASPV was 32.91%(26/69).
8 varicties of sand pear were treated under 34℃/4℃(8h/16h alteration) coupled with shoot tips culture,for1014 buds.For the same cultivars 69 strains processing materials were obtained. It resulted that cultivars'survival rate and multiplication coefficient were different.The survival rate was 14.7%,9% and 2.11% or Zhongliyihao Qingxiang and Jingqiu and Huangjing.The proliferation coefficient of Yuanhuang was 2.7,that of Xinggao was 2; Aiganshui,Fengshui and Huangjing were not appear proliferation.8 pear cultivars of 65 regenerated plans were detected by RT-PCR.Results showed that 5 sand pear clutivars were obtained,including 39 in vitro plants.The virus eliminate rate of different cultivars were different.The eliminate rate of ACLSV stands at 90.77%, that of ASGV at 70.77%,and 95.38% for ASPV.
1.对采集的21个沙梨品种的枝条材料进行病毒检测,结果显示所有材料均带至少一种潜隐病毒,其中ACLSV的带毒率为90.48%, ASGV 85.71%, ASPV 42.86%。通过茎尖培养(诱导分化培养基为MS+0.2mg/L IBA+1.0mg/L 6-BA,琼脂5.4g/L,蔗糖30g/L, pH5.8)方法处理21个沙梨品种251个茎尖,共获得20个沙梨品种366株离体植株。结果显示,不同沙梨品种的诱导分化成活率和茎尖分化系数有异,安农一号、清香、桂冠和丰水等5个品种的成活率最高,达到100%,而金水一号未成活。中梨一号的茎尖分化系数最高,新高其次,桂冠和翠冠最低。对15个品种离体植株79个芽系进行病毒检测的结果显示,ACLSV检出率为67.09%(53/79), ASGV 86.08%(68/79), ASPV 32.91%(26/79).
2.采用变温热处理(34℃/40℃,8h/16h交替)结合茎尖(大小约1 mm)培养对8个沙梨品种的1014株带毒离体植株进行病毒脱除处理,共获得8个沙梨品种的69株脱毒材料。结果显示不同品种处理后的成活率和增殖系数有异,成活率中梨一号为14.7%,清香和金秋为9.0%,黄金为2.11%。增殖系数圆黄为2.7,新高为2,丰水、爱甘水和黄金等3个品种没有增殖。通过RT-PCR等方法对8个沙梨品种的65株脱毒材料进行病毒检测的结果显示,有5个沙梨品种的39株离体植株不带病毒,且不同病毒的脱除率有差异,ASGV为70.77%(46/65), ACLSV 90.77%(59/65), ASPV 95.38%(62/65)。
Apple chlortotic leaf spot virus (ACLSV),apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are three latent viruses in pome fruit trees,which are distributed worldwide. About 86.3% of main pear tree varieties in China are infected by the three latent viruses.Based on viral disease infection by grafting techniques on pear for these virus,the major efficient way of mitigating them is via the planting of virus-free germplasm.In this study,with the in vitro sand pears infected three latent viruses was used as source for viral materials.Heat treatment under 34℃/40℃combined shoot tip culture were used as techniques for virus elimination.
Branches were collected from 21 varieties sand pear plants and virus detection was done by RT-PCR.Detections showed that all the materials collected contain at least one of three latent viruses. The infected rate was as follow:90.48% ACLSV,85.71% ASGV and 42.86%ASPV.For the 21 varieties containing 251 buds,shoot tip culture was done in a proliferative MS (MS+0.2 mg/L, IBA+1.0 mg/L 6-BA, Agar 5.4 g/L, sucrose 30 g/L, pH5.8),366 in vitro plants of 20 cultivars were obtained.Result showed that survival rate and differentiation coefficient varied between cultivars. The highest survival rate of 100% was reached for Anlongyihao, Qingxiang, Guiguan and Fengshui,the lowest of 0% reached of Jingshuiyihao. For tip differentiation coefficient, Zhongliyihao was the highest followed by Xingao.Guiguan and Cuiguan recorded the lowest becquse they show no tip polarization.15 cultivars of in vitro sand pear have been dectected,including 79 buds.Results showed that the detection rate of ACLSV was 67.09%(53/79), the detection rate of ASGV was 86.08%(68/79), the detection of ASPV was 32.91%(26/69).
8 varicties of sand pear were treated under 34℃/4℃(8h/16h alteration) coupled with shoot tips culture,for1014 buds.For the same cultivars 69 strains processing materials were obtained. It resulted that cultivars'survival rate and multiplication coefficient were different.The survival rate was 14.7%,9% and 2.11% or Zhongliyihao Qingxiang and Jingqiu and Huangjing.The proliferation coefficient of Yuanhuang was 2.7,that of Xinggao was 2; Aiganshui,Fengshui and Huangjing were not appear proliferation.8 pear cultivars of 65 regenerated plans were detected by RT-PCR.Results showed that 5 sand pear clutivars were obtained,including 39 in vitro plants.The virus eliminate rate of different cultivars were different.The eliminate rate of ACLSV stands at 90.77%, that of ASGV at 70.77%,and 95.38% for ASPV.
引文
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