小茎尖培养结合热处理脱除樱桃ACLSV和PNRSV的研究
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摘要
本研究以甜樱桃(Prunus.avium L.)品系6-7、6-3和三倍体杂种樱桃Gisela5(P.cerasus×P.canescens)为试材，首先通过对影响樱桃小茎尖培养的若干因素的研究，建立了樱桃茎尖培养的快繁体系。初代培养取休眠芽和生长的茎尖，流水冲洗1～2h，经70％的乙醇表面消毒15～30S和0.1％的升汞消毒5～10min后，无菌水冲洗5次，在超净工作台上取0.5～1.0mm的小茎尖接种于WPM基本培养基并附加BA0.5mg/1、IBA0.2mg/1和GA_30.2mg/1，6-7、6-3和Gisela5的成活率分别为100％、91.6％和100％。继代培养采用MS和F_(14)培养基交替使用并附加BA1.0mg/1、IBA0.2mg/1，同时研究了蔗糖浓度、培养基，pH值对增殖的影响。试验结果表明，外植体在附加蔗糖2～3％，pH值为5.2～6.0的培养基上均能较好的生长，基本无玻璃化、茎尖枯顶和坏死现象。以F_(14)生根培养基为基本培养基，采用L_9(3~4)正交试验设计，系统地研究了影响樱桃生根和移栽的因素。结果表明，培养基附加IBA0.7mg/1和NAA1.0mg/1，18d后平均生根率达70％以上，根粗壮、二次根发达，炼苗后栽入蛭石中成活率达90％以上。
在建立樱桃小茎尖培养体系的基础上，进行了热处理结合小茎尖培养脱除ACLSV和PNRSV的研究。将试材在38℃下处理3周，取0.5～1.0mm的小茎尖培养，充分扩繁后，病毒检测结果为：热处理试管苗结合小茎尖培养(处理1)，三个品种均接种小茎尖50个，6-7、6-3和Gisela5分别成活16、24和30个，无ACLSV茎尖分别为4、7和3个，脱毒率分别为25.0％、29.2％和10.0％；无PNRSV茎尖分别为7、11和9个，脱毒率分别为43.8％、45.9％和30.0％。热处理盆栽苗结合小茎尖培养(处理2)，6-7和6-3两个品种分别接种小茎尖25和20个，分别成活24和16个，无ACLSV茎尖分别为9和5个，脱毒率分别为37.5％和31.3％；无PNRSV茎尖分别为14和10个，脱毒率分别为58.3％和62.5％。单独取小茎尖0.5～1.0mm(处理3)得到的茎尖培养物经检测极少脱除病毒。综合来说，方法2的脱毒率显著高于方法1，方法1又优于方法3，这说明单独使用小茎尖培养很难脱除核果类果树ACLSV和PNRSV病毒，而热处理与小茎尖结合则可以较好地解决这个问题。
对组织总RNA的提取方法进行了改进，使提取时间缩短至3h以内，这种方法更适合于樱桃幼嫩叶片和试管苗RNA的提取，操作简单，提取的RNA完整性好，含量和纯度达到了RT-PCR检测病毒的要求，每个样品可节约1元。以含有病毒的材料为对照，按照病毒外壳蛋白基因的一段保守序列设计引物，经RT-PCR反应后，电泳可观察到带有ACLSV和PNRSV的株系分别含有358bp和449bp的特异片段，而无病毒株系则没有特异带产生。
The sweet cherry(Prw/ms.avmm L.)varieties 6-3, 6-7 and GiselaS (P. cerasus X P.canescens) were chosen as materials in this study. After several factors including medium, genotype and hermone were studied, the micropropagation system of cherry was obtained. Dormant buds and current shoot tips were used as explants. For meristem initiation culture, explants (l~2cm ) were washed with water (l~2h) and then placed in 70% ethanol for 15-30S, 0.1%HgCl for 5~10min, then, rinsed 5-6 times with sterilled water. Meristem domes (0.5~lmm, with lesf primodia) excised from buds of cherry 6-3, 6-7 and GiselaS were cultured on basic woody plant medium supplemented with BA0.5mg/l, IBA0.2mg/l and GA30.2mg/l. For shoot multiplication, the explants were subcultured on solid medium supplemented with BA1.0mg/l, IBA0.2mg/l, 2-3% sucrose, and pH5.2-6.0. In order to avoid the explants losing vigor, we used MS and FH medium at intervals. For root development, the cultures were cultured on FH rooting medium supplemented with NAA0.7mg/l
and IBA1.0mg/l. After 18 days, high quality roots were obtained. Then, they were transferred to greenhouse. Up to 90% of plantlets were survied when the cultures were potted in vermiculite and placed in a high-humidity chamber for 3weeks.
On the basis of the micropropagation system of cherry, elimination of ACLSV and PNRSV by combining thermotherapy with meristem tip culture were studied. Materials were treated under 38癈 for 3 weeks, 0.5~lmm length meristem tips were deprived from derilled buds and cultured on WPM medium. 3-4 months later, the plants were detected for ACLSV and PNRSV by RT-PCR and virus-free plants were obtained. The results were as follows: For 6-3, 6-7 and GiselaS, in method 1, 50 meristem tips of each variety was used, 24, 16 and 30 clones of them were established in tissue culture respectively. The numbers of ACLSV free clones were 7, 4 and 3, and PNRSV free clones were 11,7 and 9 respectively. For 6-3 and 6-7 in method 2, 20 and 25 meristem tips were used and 16, 24 clones were established in tissue culture respectively. The numbers of ACLSV free were 5 and 9, and PNRSV free were 10 and 14. Viruses can be detected in almost all clones in method 3. In a word, method 2 is best and method 1 is better than method 3.
The method of RNA extraction and purification was developed. With this method, the reverse-transcritable RNA can be obtained in 3 hours. The costs accounting of RNA extraction has been reduced. The results showed that the method was suitable for RNA extraction from the tube clones and new leavies of cherry. The viruses materials were as check and primers were synthesized, the 358bp and 449bp fragments of the partial CP gene of ACLSV and PNRSV were obtained from the infected viruses materials, and no fragments be found in the virus free clones by RT-PCR.
在建立樱桃小茎尖培养体系的基础上，进行了热处理结合小茎尖培养脱除ACLSV和PNRSV的研究。将试材在38℃下处理3周，取0.5～1.0mm的小茎尖培养，充分扩繁后，病毒检测结果为：热处理试管苗结合小茎尖培养(处理1)，三个品种均接种小茎尖50个，6-7、6-3和Gisela5分别成活16、24和30个，无ACLSV茎尖分别为4、7和3个，脱毒率分别为25.0％、29.2％和10.0％；无PNRSV茎尖分别为7、11和9个，脱毒率分别为43.8％、45.9％和30.0％。热处理盆栽苗结合小茎尖培养(处理2)，6-7和6-3两个品种分别接种小茎尖25和20个，分别成活24和16个，无ACLSV茎尖分别为9和5个，脱毒率分别为37.5％和31.3％；无PNRSV茎尖分别为14和10个，脱毒率分别为58.3％和62.5％。单独取小茎尖0.5～1.0mm(处理3)得到的茎尖培养物经检测极少脱除病毒。综合来说，方法2的脱毒率显著高于方法1，方法1又优于方法3，这说明单独使用小茎尖培养很难脱除核果类果树ACLSV和PNRSV病毒，而热处理与小茎尖结合则可以较好地解决这个问题。
对组织总RNA的提取方法进行了改进，使提取时间缩短至3h以内，这种方法更适合于樱桃幼嫩叶片和试管苗RNA的提取，操作简单，提取的RNA完整性好，含量和纯度达到了RT-PCR检测病毒的要求，每个样品可节约1元。以含有病毒的材料为对照，按照病毒外壳蛋白基因的一段保守序列设计引物，经RT-PCR反应后，电泳可观察到带有ACLSV和PNRSV的株系分别含有358bp和449bp的特异片段，而无病毒株系则没有特异带产生。
The sweet cherry(Prw/ms.avmm L.)varieties 6-3, 6-7 and GiselaS (P. cerasus X P.canescens) were chosen as materials in this study. After several factors including medium, genotype and hermone were studied, the micropropagation system of cherry was obtained. Dormant buds and current shoot tips were used as explants. For meristem initiation culture, explants (l~2cm ) were washed with water (l~2h) and then placed in 70% ethanol for 15-30S, 0.1%HgCl for 5~10min, then, rinsed 5-6 times with sterilled water. Meristem domes (0.5~lmm, with lesf primodia) excised from buds of cherry 6-3, 6-7 and GiselaS were cultured on basic woody plant medium supplemented with BA0.5mg/l, IBA0.2mg/l and GA30.2mg/l. For shoot multiplication, the explants were subcultured on solid medium supplemented with BA1.0mg/l, IBA0.2mg/l, 2-3% sucrose, and pH5.2-6.0. In order to avoid the explants losing vigor, we used MS and FH medium at intervals. For root development, the cultures were cultured on FH rooting medium supplemented with NAA0.7mg/l
and IBA1.0mg/l. After 18 days, high quality roots were obtained. Then, they were transferred to greenhouse. Up to 90% of plantlets were survied when the cultures were potted in vermiculite and placed in a high-humidity chamber for 3weeks.
On the basis of the micropropagation system of cherry, elimination of ACLSV and PNRSV by combining thermotherapy with meristem tip culture were studied. Materials were treated under 38癈 for 3 weeks, 0.5~lmm length meristem tips were deprived from derilled buds and cultured on WPM medium. 3-4 months later, the plants were detected for ACLSV and PNRSV by RT-PCR and virus-free plants were obtained. The results were as follows: For 6-3, 6-7 and GiselaS, in method 1, 50 meristem tips of each variety was used, 24, 16 and 30 clones of them were established in tissue culture respectively. The numbers of ACLSV free clones were 7, 4 and 3, and PNRSV free clones were 11,7 and 9 respectively. For 6-3 and 6-7 in method 2, 20 and 25 meristem tips were used and 16, 24 clones were established in tissue culture respectively. The numbers of ACLSV free were 5 and 9, and PNRSV free were 10 and 14. Viruses can be detected in almost all clones in method 3. In a word, method 2 is best and method 1 is better than method 3.
The method of RNA extraction and purification was developed. With this method, the reverse-transcritable RNA can be obtained in 3 hours. The costs accounting of RNA extraction has been reduced. The results showed that the method was suitable for RNA extraction from the tube clones and new leavies of cherry. The viruses materials were as check and primers were synthesized, the 358bp and 449bp fragments of the partial CP gene of ACLSV and PNRSV were obtained from the infected viruses materials, and no fragments be found in the virus free clones by RT-PCR.
引文
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