建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)的脱除研究

doi:10.11924/j.issn.1000-6850.casb2020-0342

摘要/Abstract

摘要：

本研究的主要目的是通过茎尖培养的技术手段来探索蝴蝶兰病毒的脱除问题,为建立工厂化无毒苗培养体系提供技术支撑。长期的工厂化生产,使蝴蝶兰经常感染各种病毒,导致叶片产生枯斑、褪绿、坏死等现象,使花朵畸形变色。其中建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)是最常见的2种病毒。实验取已检测出含有CymMV和ORSV病毒的蝴蝶兰植株花梗,进行诱导丛生芽的培养,对丛生芽进行继代增殖形成组培苗用于脱毒处理。切取组培苗的茎尖顶端分生组织进行培养,茎尖长度在1~6 mm之间,随着茎尖长度的增加,成活率上升,脱毒率明显降低。取2~3 mm组培苗茎尖,经0、10、20、30、40 mg/L的三氮唑核苷浸泡15 min处理和在含有相应浓度的三氮唑核苷培养基中培养30天后,继代培养形成再生植株。实验采用ELISA法对脱毒前的蝴蝶兰植株以及脱毒后的再生植株进行2种病毒检测,通过显色反应判断是否含有病毒病原。实验结果表明,蝴蝶兰脱毒苗的成活率随着三氮唑核苷浓度的增加而降低,当其浓度达到40 mg/L时,茎尖顶端分生组织出现严重的透明和褐变现象,未获得再生植株。而试管再生苗的脱毒率则随着三氮唑核苷浓度的升高而增加。在添加30 mg/L的三氮唑核苷进行化学抑制病毒的处理后,可以比单纯使用茎尖培养的方式脱毒率提高22.3%。

关键词: 蝴蝶兰, 建兰花叶病毒, 齿兰环斑病毒, 化学处理, 茎尖培养

Abstract:

The paper aims to explore the virus removal of Phalaenopsis with shoot tip culture and provide technical support for the industrialized virus-free seedling culture. Due to long-term industrial production, Phalaenopsis is often infected with various viruses, which have resulted in withered leaves, chlorosis, necrosis and so on, causing abnormal discoloration of flowers. The peduncles of plants detected with CymMV and ORSV were cultured to induce cespitose buds. The cespitose buds were multiplicated by sub-culture and test-tube plantlets were taken for virus eradicated treatment. Meristem-tips of vitro plantlets were cut for culturing. When the stem-tips were 1-6 mm in length, with the tip length increase, the survival rate of tips increased, and the virus-free rate of regenerated plantlets was significantly reduced. 2-3 mm length of meristem-tips were cut and immersed separately with 0, 10, 20, 30 and 40 mg/L of antiviral agent ribavirin for 15 min, and then cultured for 30 d in the medium with ribavirin of the corresponding concentration, followed by sub-culture, and the regenerated plantlets were obtained. The plants before virus eradicating and the regenerated plantlets after virus eradicating were detected for CymMV and ORSV by the way of ELISA, and whether there were viruses in the samples were judged by chromogenic reaction. The experiment results showed that the survival rate of the regenerated plantlets reduced as the concentration of ribavirin increased, under the treatment of 40 mg/L of ribavirin, the meristem of vitro plantlets was transparent and browned seriously, which could not achieve regenerated plantlets. Oppositely the virus-free rate of the regenerated plantlets rose with the increase of the ribavirin concentration, by adding 30 mg/L ribavirin to the culture medium, the virus-free rate increased by 22.3% compared with that of the purely shoot tip culture.

Key words: Phalaenopsis aphrodita, Cymbidium mosaic virus (CymMV), Odontogssum ringspot virus (ORSV), chemical treatment, shoot tip culture

中图分类号:

S682.31

靖晶, 李军, 刘凤军, 徐君, 姜红卫, 李青. 建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)的脱除研究[J]. 中国农学通报, 2021, 37(28): 115-120.

Jing Jing, Li Jun, Liu Fengjun, Xu Jun, Jiang Hongwei, Li Qing. Eradication of CymMV and ORSV from Tissue Culture of Phalaenopsis aphrodita with Chemotherapy[J]. Chinese Agricultural Science Bulletin, 2021, 37(28): 115-120.

图/表 4

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茎尖大小/mm	茎尖成活数	茎尖总数	茎尖成活率/%
1~2	10	50	20Ee
2~3	38	50	76Dd
3~4	43	50	86Cc
4~5	46	50	92Bb
5~6	50	50	100Aa

茎尖大小/mm	茎尖成活数	茎尖总数	茎尖成活率/%
1~2	10	50	20Ee
2~3	38	50	76Dd
3~4	43	50	86Cc
4~5	46	50	92Bb
5~6	50	50	100Aa

茎尖大小/mm	ELISA		茎尖脱毒率/%
茎尖大小/mm	阴性反应的样品数	被检样品数	茎尖脱毒率/%
1~2	10	10	100Aa
2~3	26	38	68Bb
3~4	23	43	54Cc
4~5	12	46	26Dd
5~6	6	50	12Ee
阳性对照	8	8	0
阴性对照	0	8	100

茎尖大小/mm	ELISA		茎尖脱毒率/%
茎尖大小/mm	阴性反应的样品数	被检样品数	茎尖脱毒率/%
1~2	10	10	100Aa
2~3	26	38	68Bb
3~4	23	43	54Cc
4~5	12	46	26Dd
5~6	6	50	12Ee
阳性对照	8	8	0
阴性对照	0	8	100

处理浓度/(mg/L)	ELISA		茎尖脱毒率/%
处理浓度/(mg/L)	阴性反应样品数	被检样品数	茎尖脱毒率/%
0	26	38	68Dd
10	28	35	80Cc
20	30	33	91Bb
30	31	31	100Aa
40	—	—	褐变,未出苗
阳性对照	8	8	0
阴性对照	0	8	100