蝴蝶兰（Phalaenopsis）脱毒快繁关键技术及其生理基础的研究
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摘要
蝴蝶兰(Phalaenopsis)又称蝶兰,因其花形别致,色彩艳丽,具有很高的观赏价值,在观赏花卉中占有重要的地位。蝴蝶兰一般均采用设施栽培,在栽培过程中极易受病毒的侵袭,并快速蔓延造成严重损失。植株被感染后可造成植株叶斑、坏死以及花朵变色、畸形等症状,严重影响其品质。因此,研究不同脱毒技术对蝴蝶兰病毒的脱除效果无毒苗的快速繁殖具有重要的应用价值。
本试验以蝴蝶兰品系‘EG-727'试管苗为材料,在对蝴蝶兰危害较重的建兰花叶病毒(Cymbidium mosaic virus, CymMV)进行检测的基础上,采用二次茎尖培养、茎尖培养结合化学药剂处理及茎尖培养结合热处理进行脱毒,明确其脱毒效果,并对脱毒苗的获得、茎尖芽萌发和增殖等关键技术进行了研究。初步建立了蝴蝶兰品系‘EG-727’高效脱毒快繁的技术体系,为蝴蝶兰脱毒苗的快速繁殖提供科学依据和技术基础。主要研究结果如下：
1.38/32℃(昼/夜)热处理49d,蝴蝶兰试管苗叶片出现大面积黄化萎焉,甚至腐烂；可溶性蛋白含量先降低后升高；MDA含量49d内增加,49d后显著降低；相对电导率35d前变化差异较小,35d后显著增加；SOD、POD和APX活性在49d之前表现出不同程度的增加,之后均转为下降趋势；而CAT活性49d内显著降低,49d后迅速升高。该研究结果显示,38/32℃热胁迫致使蝴蝶兰试管苗发生明显氧化伤害的时间节点可能在第49d,利用38/32℃昼夜高温热处理进行脱毒的处理时间不宜超过49d。
2.应用RT-PCR技术进行CymMV检测,建立了可应用于蝴蝶兰CymMV的RT-PCR快速检测体系。剥取带病毒的蝴蝶兰试管苗茎尖,研究二次茎尖培养、茎尖培养结合化学药剂处理及茎尖培养结合热处理3种方法对蝴蝶兰CymMV脱除效果的影响。结果表明：茎尖大小对脱毒率和成苗率有显著影响,茎尖越小,成苗率越低,脱毒效果越好。一次或二次2.0mm茎尖培养均不能够有效脱除病毒。虽然茎尖培养结合化学处理及茎尖培养结合热处理降低了蝴蝶兰茎尖的成苗率,但均能显著提高脱毒率。6.0g·L-1板蓝根处理的脱毒率达到77.33%；50mg·L-1病毒唑处理的脱毒率为33.33%。38/32℃变温热处理35d,脱毒率为46.67%,脱毒率和成苗率均显著高于38℃恒温热处理。
3.选择2.0 mm的茎尖为外植体,研究了不同基本培养基、6-BA和NAA浓度配比、有机添加物和活性炭浓度对茎尖芽萌发和增殖的影响。结果表明：基本培养基ND对茎尖芽萌发最为适宜,MS培养基则有利于芽的增殖及生长,可形成大量较健壮的丛生芽；1.0mg·L-16-BA和0.1mg·L-1NAA组合时,茎尖的芽萌发效果最好,8.0mg·L-16-BA和0.5mg·L-1NAA则能有效的刺激芽的增殖；添加100m1·L-1椰子汁(CW)有利于茎尖芽的萌发,150g·L-1香蕉泥能显著促进芽的增殖。对于蝴蝶兰品系‘EG-727’试管苗茎尖芽萌发,适宜的培养基为ND+1.0mg·L-16-BA+0.1mg·L-1NAA+100 ml·L-1C W+1.0g·L-1AC+20g·L-1蔗糖+8.0g·L"1琼脂,pH5.4；对于脱毒苗芽增殖和生长,适宜的培养基为MS+8.0mg·L-16-BA+0.5mg·L-1NAA+150g·L-1香蕉泥+30g·L-1蔗糖+6.5g·L-1琼脂,pH5.7。
Phalaenopsis, is also known as butterfly orchid, because of its unique flower-shaped, colorful, has high ornamental value, and occupies an important position in ornamental flowers. Generally, Phalaenopsis is cultured in protected condition, vulnerable to viruses in cultivation process. Then the viruses are quickly spread causing severe damage. After plants were infected with viruses, it can cause leaf spots, necrosis, and flower color, deformities and other symptoms, severely affecting their quality. Therefore, it has important application value that study on the effect of different virus eliminated technology on Phalaenopsis detoxification efficiency and the rapid propagation of virus-free plantlets.
In this study, used Phalaenopsis strain'EG-727'plantlets as materials, on the basis of detection Cymbidium mosaic virus(CymMV) in Phalaenopsis, we applied secondary shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment to eliminate CymMV, to define their detoxification efficiency, and some key technologies such as virus-free plantlets obtained, buds germination and buds multiplication. We initially established a technical system for Phalaenopsis strain'EG-727' efficient detoxification and rapid propagation of virus-free plantlets, to provide a scientific evidence and technical basis for rapid propagation of Phalaenopsis virus-free plantlets. The major results are as follows:
1.49d heat treatment in 38/32℃(day/night), Phalaenopsis plantlets leaves appeared a large area yellow and wilt, even decay; soluble protein content first decreased and then increased; MDA content raised within 49d, significantly dropped after 49d; the change of relative conductivity was little difference before 35d, a significant increase after 35d; before 49d, SOD, POD and APX activity showed different degrees of increase, declined after 49d; while CAT activity reduced significantly within 49d, and raised rapidly afterwards. The results showed that:38/32℃heat stress caused significant oxidative damage, the time node may be 49dth. Therefore, Carrying out Phalaenopsis plantlets detoxification with 38/32℃(day/night) heat treatment, the treatment time should not exceed 49d.
2. We applied RT-PCR to detect CymMV, and established a RT-PCR rapid detection system, which can be used in Phalaenopsis CymMV. To study the effects on CymMV detoxification efficiency of the second shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment, the virus tips were stripped from plantlets. The results suggested that:tips size significantly affected virus-free rate and plantlets rate, the smaller the tips, the lower survival rate, the better virus-free. Stripping 2.0mm size tips, one time or secondary shoot-tip culture was not able to effectively remove the virus. Although the shoot-tip culture combined with chemical treatment and heat treatment reduced the survival rate, but could significantly increase the virus-free rate. The virus-free rate of 6.0g·L-1Banlangen treatment was 77.33%; 50mg·L-1 Ribavirin treatment was 33.33%.38/32℃(day/night) alternating temperature heat treatment 35d, virus-free rate of 46.67%, virus-free rate and survival rate were significantly higher than 38℃constant temperature heat treatment.
3. Selecting 2.0mm shoot-tip as explants, we studied the effects of different the basic medium, different 6-BA and NAA concentration ratio, organic additives and carbon concentration on the buds germination and proliferation. The results showed that:basic medium ND was the most suitable to buds germination of shoot-tip, MS medium was conducive to the proliferation and growth of buds. The buds were thick, forming a large number of clustered-like buds; the combination of 1.0mg·L-16-BA and 0.1mg·L-1NAA, was the best one of sprouting of buds, the combination of 8.0mg·L-16-BA and 0.5mg·L-1NAA was able to effectively stimulate the buds proliferation; adding coconut water (CW) 100ml·L-1 was conducive to the germination of buds, 150g·L-1banana could significantly promote the proliferation of buds. For Phalaenopsis strain'EG-727'plantlets buds germinated from shoot-tips, the suitable medium was ND+1.0mg·L-1 6-BA+0.1mg·L-1 NAA+100ml·L-1CW+1.0g·L-1AC+20g·L-1Sucrose+8.0g·L-1Agar, pH5.4; for virus-free buds multiplication and growth of plantlets, the suitable medium was MS+8.0mg·L-16-BA+0.5 mg·L-1NAA+150g·L-1Banana+30g·L-1 Sucrose+6.5 g·L-1 Agar, pH 5.7.
本试验以蝴蝶兰品系‘EG-727'试管苗为材料,在对蝴蝶兰危害较重的建兰花叶病毒(Cymbidium mosaic virus, CymMV)进行检测的基础上,采用二次茎尖培养、茎尖培养结合化学药剂处理及茎尖培养结合热处理进行脱毒,明确其脱毒效果,并对脱毒苗的获得、茎尖芽萌发和增殖等关键技术进行了研究。初步建立了蝴蝶兰品系‘EG-727’高效脱毒快繁的技术体系,为蝴蝶兰脱毒苗的快速繁殖提供科学依据和技术基础。主要研究结果如下：
1.38/32℃(昼/夜)热处理49d,蝴蝶兰试管苗叶片出现大面积黄化萎焉,甚至腐烂；可溶性蛋白含量先降低后升高；MDA含量49d内增加,49d后显著降低；相对电导率35d前变化差异较小,35d后显著增加；SOD、POD和APX活性在49d之前表现出不同程度的增加,之后均转为下降趋势；而CAT活性49d内显著降低,49d后迅速升高。该研究结果显示,38/32℃热胁迫致使蝴蝶兰试管苗发生明显氧化伤害的时间节点可能在第49d,利用38/32℃昼夜高温热处理进行脱毒的处理时间不宜超过49d。
2.应用RT-PCR技术进行CymMV检测,建立了可应用于蝴蝶兰CymMV的RT-PCR快速检测体系。剥取带病毒的蝴蝶兰试管苗茎尖,研究二次茎尖培养、茎尖培养结合化学药剂处理及茎尖培养结合热处理3种方法对蝴蝶兰CymMV脱除效果的影响。结果表明：茎尖大小对脱毒率和成苗率有显著影响,茎尖越小,成苗率越低,脱毒效果越好。一次或二次2.0mm茎尖培养均不能够有效脱除病毒。虽然茎尖培养结合化学处理及茎尖培养结合热处理降低了蝴蝶兰茎尖的成苗率,但均能显著提高脱毒率。6.0g·L-1板蓝根处理的脱毒率达到77.33%；50mg·L-1病毒唑处理的脱毒率为33.33%。38/32℃变温热处理35d,脱毒率为46.67%,脱毒率和成苗率均显著高于38℃恒温热处理。
3.选择2.0 mm的茎尖为外植体,研究了不同基本培养基、6-BA和NAA浓度配比、有机添加物和活性炭浓度对茎尖芽萌发和增殖的影响。结果表明：基本培养基ND对茎尖芽萌发最为适宜,MS培养基则有利于芽的增殖及生长,可形成大量较健壮的丛生芽；1.0mg·L-16-BA和0.1mg·L-1NAA组合时,茎尖的芽萌发效果最好,8.0mg·L-16-BA和0.5mg·L-1NAA则能有效的刺激芽的增殖；添加100m1·L-1椰子汁(CW)有利于茎尖芽的萌发,150g·L-1香蕉泥能显著促进芽的增殖。对于蝴蝶兰品系‘EG-727’试管苗茎尖芽萌发,适宜的培养基为ND+1.0mg·L-16-BA+0.1mg·L-1NAA+100 ml·L-1C W+1.0g·L-1AC+20g·L-1蔗糖+8.0g·L"1琼脂,pH5.4；对于脱毒苗芽增殖和生长,适宜的培养基为MS+8.0mg·L-16-BA+0.5mg·L-1NAA+150g·L-1香蕉泥+30g·L-1蔗糖+6.5g·L-1琼脂,pH5.7。
Phalaenopsis, is also known as butterfly orchid, because of its unique flower-shaped, colorful, has high ornamental value, and occupies an important position in ornamental flowers. Generally, Phalaenopsis is cultured in protected condition, vulnerable to viruses in cultivation process. Then the viruses are quickly spread causing severe damage. After plants were infected with viruses, it can cause leaf spots, necrosis, and flower color, deformities and other symptoms, severely affecting their quality. Therefore, it has important application value that study on the effect of different virus eliminated technology on Phalaenopsis detoxification efficiency and the rapid propagation of virus-free plantlets.
In this study, used Phalaenopsis strain'EG-727'plantlets as materials, on the basis of detection Cymbidium mosaic virus(CymMV) in Phalaenopsis, we applied secondary shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment to eliminate CymMV, to define their detoxification efficiency, and some key technologies such as virus-free plantlets obtained, buds germination and buds multiplication. We initially established a technical system for Phalaenopsis strain'EG-727' efficient detoxification and rapid propagation of virus-free plantlets, to provide a scientific evidence and technical basis for rapid propagation of Phalaenopsis virus-free plantlets. The major results are as follows:
1.49d heat treatment in 38/32℃(day/night), Phalaenopsis plantlets leaves appeared a large area yellow and wilt, even decay; soluble protein content first decreased and then increased; MDA content raised within 49d, significantly dropped after 49d; the change of relative conductivity was little difference before 35d, a significant increase after 35d; before 49d, SOD, POD and APX activity showed different degrees of increase, declined after 49d; while CAT activity reduced significantly within 49d, and raised rapidly afterwards. The results showed that:38/32℃heat stress caused significant oxidative damage, the time node may be 49dth. Therefore, Carrying out Phalaenopsis plantlets detoxification with 38/32℃(day/night) heat treatment, the treatment time should not exceed 49d.
2. We applied RT-PCR to detect CymMV, and established a RT-PCR rapid detection system, which can be used in Phalaenopsis CymMV. To study the effects on CymMV detoxification efficiency of the second shoot-tip culture, shoot-tip culture combined with chemical treatment and shoot-tip culture combined with heat treatment, the virus tips were stripped from plantlets. The results suggested that:tips size significantly affected virus-free rate and plantlets rate, the smaller the tips, the lower survival rate, the better virus-free. Stripping 2.0mm size tips, one time or secondary shoot-tip culture was not able to effectively remove the virus. Although the shoot-tip culture combined with chemical treatment and heat treatment reduced the survival rate, but could significantly increase the virus-free rate. The virus-free rate of 6.0g·L-1Banlangen treatment was 77.33%; 50mg·L-1 Ribavirin treatment was 33.33%.38/32℃(day/night) alternating temperature heat treatment 35d, virus-free rate of 46.67%, virus-free rate and survival rate were significantly higher than 38℃constant temperature heat treatment.
3. Selecting 2.0mm shoot-tip as explants, we studied the effects of different the basic medium, different 6-BA and NAA concentration ratio, organic additives and carbon concentration on the buds germination and proliferation. The results showed that:basic medium ND was the most suitable to buds germination of shoot-tip, MS medium was conducive to the proliferation and growth of buds. The buds were thick, forming a large number of clustered-like buds; the combination of 1.0mg·L-16-BA and 0.1mg·L-1NAA, was the best one of sprouting of buds, the combination of 8.0mg·L-16-BA and 0.5mg·L-1NAA was able to effectively stimulate the buds proliferation; adding coconut water (CW) 100ml·L-1 was conducive to the germination of buds, 150g·L-1banana could significantly promote the proliferation of buds. For Phalaenopsis strain'EG-727'plantlets buds germinated from shoot-tips, the suitable medium was ND+1.0mg·L-1 6-BA+0.1mg·L-1 NAA+100ml·L-1CW+1.0g·L-1AC+20g·L-1Sucrose+8.0g·L-1Agar, pH5.4; for virus-free buds multiplication and growth of plantlets, the suitable medium was MS+8.0mg·L-16-BA+0.5 mg·L-1NAA+150g·L-1Banana+30g·L-1 Sucrose+6.5 g·L-1 Agar, pH 5.7.
引文
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