WO2021189544A1 - 一种高效的百合脱毒方法 - Google Patents

一种高效的百合脱毒方法 Download PDF

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WO2021189544A1
WO2021189544A1 PCT/CN2020/084135 CN2020084135W WO2021189544A1 WO 2021189544 A1 WO2021189544 A1 WO 2021189544A1 CN 2020084135 W CN2020084135 W CN 2020084135W WO 2021189544 A1 WO2021189544 A1 WO 2021189544A1
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lily
virus
scales
callus
seedlings
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PCT/CN2020/084135
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French (fr)
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滕年军
吴慧君
吴泽
张德花
蓝令
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南京农业大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

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  • the invention belongs to the technical field of plant tissue culture and detoxification, and relates to a lily detoxification method, in particular to a lily detoxification method for detoxification by inducing callus.
  • viruses that have been reported to infect lilies, three are the most common and most harmful viruses: Cucumber mosaic virus (CMV) and Lily symptomless virus (LSV) And lily mottle virus (LMoV), they often appear in the form of compound infection, causing the stems, leaves, and flowers of plants to become mottled or deformed, which seriously leads to short plants, degeneration and even death, and seriously affects the yield and quality of lilies. . These viruses are mainly spread through insect vectors such as aphids, which spread quickly and are difficult to directly and effectively control with chemical or biological agents.
  • CMV Cucumber mosaic virus
  • LSV Lily symptomless virus
  • LoV lily mottle virus
  • the problems of the detoxification method are: the demand for detoxification materials is extremely large, the stem tip is not easy to strip, the survival rate is low, the process is complicated, the time-consuming, the technical requirements are high, and it is difficult to apply to factory production.
  • the Chinese patent "A comprehensive detoxification method for lily bulbs" with the patent number CN201410586353.4 firstly conducts heat treatment and detoxification under greenhouse environmental conditions of 36-38°C and humidity of 70-86%, and then conducts external detoxification. Disinfection of the plant body and the collection and induction culture of the growth point.
  • the problems of this detoxification method are: high equipment cost, difficult extraction of the growth point, technical difficulty, and serious degradation of the formed detoxification bulb, although it can remove part of the detoxification method.
  • the existing patents basically do not perform virus detection on the lily bulbs before treatment to confirm whether the raw materials are infected with the virus, so the statistics of the detoxification rate are not scientific.
  • the purpose of the present invention is to address the shortcomings of the existing lily detoxification technology, to perform virus detection before the detoxification treatment, and to provide a detoxification method that is highly sustainable, easy to operate, and has a short cycle and can be applied to large-scale production of lilies.
  • This method can effectively improve the resistance of seedlings, and has the advantages of simple operation, fewer procedures, and short detoxification time.
  • An efficient detoxification method for lily includes the following steps:
  • the callus is induced to become seedlings after differentiation culture
  • step 5) Cut the leaves in step 4) for virus detection, and the lily seedlings that pass the test are virus-free seedlings.
  • step 1) The detailed process of virus detection in step 1) is as follows: select non-rotted and healthy lily scales and freeze them in liquid nitrogen, and extract RNA by the Trizol method; then use takara reverse transcription kit for reverse transcription; design cucumber mosaic virus
  • the primers of lily symptomless virus and lily mottle virus are amplified by PCR to detect whether they contain these three viruses; the important reagent for RNA extraction is Trizol. Random 6mers, an important reagent in reverse transcription.
  • Step 2 The detailed process of cultivating and obtaining sterile seedlings is: select bulbs with fewer lesions, peel off healthy scales in the middle and inner layer, rinse them under running water, and then sterilize them with 75% ethanol and 1% sodium hypochlorite, and then sterilize them. Rinse with ddH 2 O several times, put it upside down on filter paper to dry; cut the scales into squares, place the concave surface up in the bud induction medium, and place it in the tissue culture room to grow;
  • Step 3 Cut the small scales of sterile seedlings, and the detailed process of inducing dense callus is as follows: cut off the leaves of plants that grow to 3-8cm high, peel off the small scales and place them in the callus induction medium with the concave side up; Place it in the tissue culture room to grow;
  • Step 4) The detailed process of callus induction into seedlings after differentiation culture is as follows: the dense callus is cut out of the mother, after 1-2 subcultures, it is transferred to the differentiation medium; placed in the tissue culture Grow under room conditions;
  • Step 5) The detailed process of cutting the leaves in step 4) for virus detection is: randomly extract 10-20% of the lily bulblets that have been detoxified in step 4) for RT-PCR retrovirus detection. It is confirmed that it does not carry cucumber mosaic virus, lily symptomless virus and lily mottle virus, which is a virus-free lily seedling.
  • step 2) the healthy scales in the middle inner layer are peeled and rinsed under running water for 2 hours, 75% ethanol for 30 seconds, 1% sodium hypochlorite for 15-20 minutes, and sterilized ddH 2 O for 5-8 Then, place it upside down on filter paper and let it dry.
  • the budding medium described in step 2) is: MS+2.0mg/L6-BA+0.2mg/LNAA+7g/L agar+15g/L sucrose.
  • the callus induction medium described in step 3) is: MS+1.0mg/LTDZ+2.0mg/L PIC+7g/L agar+15g/L sucrose.
  • Step 3 The light culture condition for medium and small scales to induce callus is dark and 20°C.
  • the differentiation medium described in step 4) is: MS+0.4g/L activated carbon+0.2mg/L NAA+9g/L agar+40g/L sucrose.
  • step 4 the transfer interval for the callus succession is 10-15 days.
  • step 4 the culture conditions for callus induction and seedling formation are as follows: light intensity is 1500 Lx and temperature is 20°C.
  • the detoxification principle of the high-efficiency lily detoxification method of the present invention is:
  • Step 2) in the process of establishing sterile seedlings with field seedlings Part of the virus may be removed: using lily scales as explants, after inducing buds, cut out the buds for proliferation and growth. The growth rate of the bud is faster than the amplification rate of the virus. If the mother body containing the virus is discarded, a part of the virus may be removed.
  • a part of the virus is removed again: using the scales of sterile seedlings as explants to induce callus, the dense callus After cutting out the mother body, put it in the differentiation medium.
  • the growth rate of dense callus is much faster than the amplification rate of the virus, and the maternal part containing the virus is discarded, so the virus is removed.
  • the research of the present invention shows that only the process of establishing and cultivating sterile seedlings with field seedlings cannot completely remove the three viruses at the same time, and only the combination of two steps can remove the three viruses at the same time.
  • the callus induction medium used in this study is the first one, MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L sucrose+7g/L agar.
  • PIC or 2,4-D is often added.
  • the present invention uses TDZ and PIC in combination.
  • Using callus induction for detoxification the state of the callus is the key to the success of detoxification. Under the induction of this medium, we get dense and fast-growing embryogenic callus (as shown in Figure 1).
  • the method of this study can be used to obtain virus-free seedlings in about 14 weeks: it takes 5 weeks to establish a sterile seedling system, and the sterile seedling scales are used as explants to induce callus and callus succession. The time is 5 weeks, and the time for callus to induce seedlings is 4 weeks.
  • the efficient detoxification method for lily described in the present invention is the result obtained by the inventor on the basis of a large number of experiments after a long period of exploration and research.
  • the beneficial effects of the present invention are as follows:
  • the present invention effectively overcomes the problems of many detoxification materials, lengthy detoxification steps, long detoxification time, and high required technology and equipment conditions, and can efficiently and quickly obtain detoxification tissue culture plants.
  • the sterile seedling system was established by first using the field bulbs, which greatly reduced the pollution rate and the required materials, thereby speeding up the detoxification efficiency. .
  • the invention adopts a generation of sterile seedling scales to induce callus, which can remove cucumber mosaic virus, lily symptomless virus and lily mottle virus at one time, and obtain virus-free seedlings in 14 weeks. If sterile seedlings are directly used as experimental materials, the detoxification time only takes 9 weeks.
  • the present invention After obtaining the virus-free seedlings, the present invention adds an appropriate amount of activated carbon to the conventional culture medium, and uses tissue culture to rapidly obtain a large number of virus-free seedlings at a very high propagation rate, and can be widely used in industrialized production.
  • Figure 1 shows the state of lily callus under a stereo microscope.
  • A initial callus
  • B late callus
  • Figure 2 shows the results of the original seed ball virus detection.
  • M DNA Marker DL2000; 1: CMV; 2: LSV; 3: LMoV).
  • Figure 3 shows the results of virus detection after callus induction and detoxification treatment.
  • M 2000marker; 1: positive control; 2-4: treated seedlings; 5: negative control.
  • the callus is induced to become seedlings after differentiation culture: the callus that grows into a dense state is cut out of the mother, and subcultured 1-2 times. Among them, the transfer interval of callus subculture is 10-15 days, and it is transferred to differentiation medium, which is: MS+0.4g/L activated charcoal+0.2mg/L NAA+9g/L agar+40g/ L sucrose; grow under the condition of tissue culture room, light intensity is 1500Lx, temperature is 20°C;
  • step 5) Cut the leaves in step 4) for virus detection. If the test is qualified, they are detoxified lily seedlings: 10-20% of the lily bulblets that have been detoxified in step 4) are randomly selected for RT-PCR reverse transcription Virus testing, after testing confirmed that it does not carry cucumber mosaic virus, lily symptomless virus and lily mottle virus, it is a lily virus-free vaccine.
  • Callus is a mass of undifferentiated tissue formed from the inside of the explant or the surface of the incision. This tissue has the ability to redifferentiate.
  • the present invention uses the characteristics of callus to obtain virus-free vaccines, and does not include other meanings.
  • cDNA synthesis refers to the instructions of takara kit.
  • the sample addition system is:
  • the sample addition system is:
  • the synthesized 5'end primer and 3'end primer are used for PCR amplification.
  • the PCR amplification system is:
  • the reaction was carried out for 36 cycles on a PCR amplification machine; extension was carried out at 72°C for 2 min, and the reaction was terminated at 4°C. Take 10 ⁇ L of the amplified product for 1% agarose gel electrophoresis, and use the BioRad gel imaging system to observe the results.
  • Example 1 An efficient detoxification method for lily.
  • the detailed steps of the detoxification method for lily are as follows:
  • the callus induction medium is :MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L sucrose+7g/L agar; put it in the dark and grow at 20°C;
  • the callus is induced to become seedlings after differentiation culture; the callus that grows into a dense state is cut out of the mother, and subcultured twice.
  • the transfer interval of callus subculture is 10 days, and it is transferred to differentiation medium, which is: MS+0.4g/L activated charcoal+0.2mg/L NAA+9g/L agar+40g/L sucrose ;Grow under the condition of tissue culture room, the light intensity is 1500Lx, the temperature is 20°C;
  • step 5) Cut the leaves in step 4) for virus detection. If the test is qualified, they are detoxified lily seedlings: 10-20% of the lily bulblets that have been detoxified in step 4) are randomly selected for RT-PCR reverse transcription Virus detection (the specific steps are as described above), and it is confirmed that it does not carry cucumber mosaic virus, lily symptomless virus and lily mottle virus, which is a virus-free lily vaccine.
  • Example 2 An efficient detoxification method for lily.
  • the detailed steps of the detoxification method for lily are as follows:
  • the callus induction medium is :MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L sucrose+7g/L agar; put it in the dark and grow at 20°C;
  • the callus is induced to become seedlings after differentiation culture; the dense callus is cut out of the mother body, and subcultured once. Among them, the transfer interval of callus subculture is 13 days, and it is transferred to differentiation medium, which is: MS+0.4g/L activated charcoal+0.2mg/L NAA+9g/L agar+40g/L sucrose ;Grow under the condition of tissue culture room, the light intensity is 1500Lx, the temperature is 20°C;
  • step 5) Cut the leaves in step 4) for virus detection. If the test is qualified, they are detoxified lily seedlings: 10-20% of the lily bulblets that have been detoxified in step 4) are randomly selected for RT-PCR reverse transcription Virus detection (the specific steps are as described above), and it is confirmed that it does not carry cucumber mosaic virus, lily symptomless virus and lily mottle virus, which is a virus-free lily vaccine.
  • Example 3 An efficient detoxification method for lily.
  • the detailed steps of the detoxification method for lily are as follows:
  • the callus induction medium is :MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L sucrose+7g/L agar; put it in the dark and grow at 20°C;
  • the callus is induced to become seedlings after differentiation culture; the densely grown callus is cut out of the mother body, and subcultured twice. Among them, the transfer interval for callus subculture is 15 days, and it is transferred to the differentiation medium, which is: MS+0.4g/L activated charcoal+0.2mg/L NAA+9g/L agar+40g/L sucrose ;Grow under the condition of tissue culture room, the light intensity is 1500Lx, the temperature is 20°C;
  • step 5) Cut the leaves in step 4) for virus detection. If the test is qualified, they are detoxified lily seedlings: 10-20% of the lily bulblets that have been detoxified in step 4) are randomly selected for RT-PCR reverse transcription Virus detection (the specific steps are as described above), and it is confirmed that it does not carry cucumber mosaic virus, lily symptomless virus and lily mottle virus, which is a virus-free lily vaccine.
  • the present invention effectively overcomes the problems of many detoxification materials, lengthy detoxification steps, long detoxification time, and high required technology and equipment conditions, and can efficiently and quickly obtain detoxification tissue culture plants.
  • the use of a first generation of sterile seedling scales to induce callus can remove cucumber mosaic virus, lily symptomless virus and lily mottle virus at one time, and obtain a virus-free seedling in 14 weeks.
  • an appropriate amount of activated carbon is added to the conventional medium (activated carbon is added to the differentiation medium to assist in the growth of the tissue culture seedlings), and the tissue culture method is used to quickly obtain a large amount at a very high reproduction rate.
  • Virus-free vaccines can be widely used in industrial production.
  • the present invention establishes a generation of sterile seedling system, takes its small scales for callus induction, uses the extremely strong redifferentiation ability of callus to induce seedlings, and effectively overcomes the cumbersome and complicated detoxification steps in the past lily detoxification technology.
  • a first generation of aseptic seedling system is established, and then its small scales are cut for callus induction, and tissue culture seedlings free of cucumber mosaic virus, lily symptomless virus and lily mottle virus are obtained at one time, which has high detoxification efficiency,
  • the advantages of simple operation, short time, environmental requirements and low cost are suitable for large-scale industrial production.

Abstract

本发明公开了一种高效的百合脱毒方法，该方法包括以下步骤：1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测；2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗；3)切取无菌苗小鳞片，诱导获得致密愈伤组织；4)愈伤组织经过分化培养诱导成苗；5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗。本发明的百合脱毒方法通过诱导愈伤组织快速获得无毒苗，能够满足产业化生产的需求。

Description

一种高效的百合脱毒方法 技术领域
本发明属于植物组织培养及脱毒技术领域，涉及一种百合脱毒方法，具体涉及一种通过诱导愈伤进行脱毒的百合脱毒方法。
背景技术
百合作为球根花卉，在花卉家族中占有非常重要的地位。但其长期采用以鳞茎、鳞片、珠芽等营养体进行无性繁殖，极易发生病毒感染，且常伴有球根退化，严重影响百合的品质和产量。其中，病毒病是近年来世界范围内发生的主要病害之一。随着我国百合的进口、引种、种植面积的迅速增加，以及种球的不规范种植和自繁，病毒病开始在我国百合种植区蔓延。一般发病率在40％-50％，二代种球的带病率在90％以上。在已报道的十余种侵染百合的病毒中，发生最为普遍、危害最为严重的病毒有3种：黄瓜花叶病毒(Cucumber mosaic virus，CMV)、百合无症病毒(Lily symptomless virus，LSV)和百合斑驳病毒(Lily mottle virus，LMoV)，它们经常以复合感染的形式出现，使植株的茎、叶、花发生斑驳或变形，严重导致植株矮小、退化甚至死亡，严重影响百合的产量和品质。这些病毒主要通过蚜虫等昆虫媒介传播，传播速度快，难以用化学药剂或生物药剂直接有效控制。
目前关于百合脱毒技术的研究主要集中在剥茎尖、热处理和化学处理等技术的综合应用，这些方法虽然能有效脱除百合病毒。但存在所需脱毒材料较多、污染率高、技术难度操作较大、工序复杂、周期长等问题，难以满足规模化生产的要求。例如，专利号为CN201410420844.1的中国专利“一种基于胚性愈伤特异诱导的百合脱毒方法”，它将大田百合鳞片经消毒后诱导出愈伤组织，诱导成苗后进行热处理，剥离茎尖后再次诱导愈伤组织，再将其诱导成苗。该脱毒方法存在的问题是：脱毒材料需求量极大，茎尖剥取不易且成活率低，工序复杂，耗时长，技术要求高，难以应用于工厂化生产。此外，专利号为CN201410586353.4的中国专利“一种百合种球综合性脱毒方法”，它先以36-38℃，湿度为70-86％的大棚环境条件进行热处理脱毒，后进行外植体的消毒和生长点采集诱导培养，该脱毒方法存在的问题是：设备成本高，生长点剥取不易，技术难度大，形成的脱毒鳞茎种球退化严重，虽然能够脱除一部分的病毒，但易二次感染病毒，故实际应用价值低。此外，已有专利基本未对处理前的百合种球进行病毒检测以确认原材料是否感染病毒，因此其脱毒率的统计有失一定的科学性。
发明内容
本发明的目的是针对现有百合脱毒技术的不足，在脱毒处理前就进行病毒检测，提供一种可持续化高、易操作、周期短且能够应用于规模化生产百合的脱毒方法，该方法能有效提高种苗抗性，具有操作简单、工序少、脱毒时间短等优势。
本发明的目的是通过以下技术方案实现的：
一种高效的百合脱毒方法，该方法包括以下步骤：
1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测；
2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗；
3)切取无菌苗小鳞片，诱导获得致密愈伤组织；
4)愈伤组织经过分化培养诱导成苗；
5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗。
作为一种优选技术方案：
步骤1)中进行病毒检测的详细过程为：选取无腐烂、健康的百合鳞片冻入液氮中，采用Trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物进行PCR扩增，检测是否含有此三种病毒；RNA提取的重要试剂是Trizol。反转录中重要的试剂的Random 6mers。
步骤2)培养获得无菌苗的详细过程为：选取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗后依次经75％乙醇和1％次氯酸钠消毒，再经灭菌ddH 2O冲洗多次，倒置于滤纸上晾干；将鳞片切成方块，凹面向上置于诱芽培养基中，放至组培室条件下生长；
步骤3)切取无菌苗小鳞片，诱导获得致密愈伤组织的详细过程为：将长至3-8cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中；放至组培室条件下生长；
步骤4)愈伤组织经过分化培养诱导成苗的详细过程为：将长成致密状态的愈伤组织切离出母体，继代1-2次后，转至分化培养基中；放至组培室条件下生长；
步骤5)切取步骤4)中的叶进行病毒检测的详细过程为：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。
进一步优选的：步骤2)中剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒15-20分钟，经灭菌ddH 2O冲洗5-8次，倒置于滤纸上晾干。
步骤2)中所述的诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/LNAA+7g/L琼脂+15g/L蔗糖。
步骤3)中所述的诱导愈伤培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+7g/L琼脂+15g/L蔗糖。
步骤3)中小鳞片诱导愈伤的光照培养条件为黑暗，20℃。
步骤4)中所述的分化培养基为：MS+0.4g/L活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖。
步骤4)中愈伤继代的转接间隔时间为10-15天。步骤4)中愈伤诱导成苗的培养条件为：光照强度为1500Lx，温度为20℃。
本发明高效的百合脱毒方法的脱毒原理为：
1)、以大田苗建立无菌苗过程中的步骤2)中可能脱去一部分病毒：以百合鳞片作为外植体，诱导出芽后，切取芽的部分进行增殖生长。芽的生长速度快于病毒的扩增速度，舍弃了含有病毒的母体，可能脱去一部分病毒。
2)、以无菌苗的鳞片作为外植体诱导愈伤的步骤3)中又脱去了一部分病毒：以无菌苗的鳞片作为外植体，诱导愈伤组织，将致密的愈伤组织切离出母体后放至分化培养基中。致密愈伤组织的生长速度远远大于病毒的扩增速度，并舍弃了含有病毒的母体部分，因此脱去了病毒。
本发明研究表明，只是以大田苗建立培养无菌苗的过程是没办法彻底同时脱掉三种病毒，只有两个步骤结合才能同时脱去三种病毒。
本发明技术方案的创新点：
1)、与现有发明内容相比，大多数的发明采取的方法大多以茎尖作为外植体，经热处理后再一次切取茎尖等，操作难度大且步骤冗杂，本研究中利用了无菌苗的鳞片，经一次愈伤组织的培养，就成功获得了脱毒苗，实际操作容易，步骤少。鳞片作为愈伤诱导的外植体，愈伤诱导率高、获得的愈伤组织状态好。因此本研究具明显的创新。
2)、本研究中应用的愈伤组织诱导的培养基是首创的，MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L蔗糖+7g/L琼脂。常规的愈伤诱导培养基内，常添加是PIC或是2，4-D，本发明将TDZ和PIC结合一起使用。利用愈伤诱导进行脱毒，愈伤组织的状态是脱毒成功的关键，在该培养基的诱导下，我们得到的是致密、生长快速的胚性愈伤组织(如图1所示)。
3)、在经历近两年多的摸索后，本研究发现经鳞片诱导的愈伤组织在转接10-15天后诱导分化的脱毒效果是最好的，在此培养时间内，愈伤组织的生长速度远远快于病毒的扩繁速度。并在10-15天后诱导其分化，快速成苗。
4)、本研究先对原种球进行了病毒检测，确定脱毒材料确实是感染病毒的(如图2所示的脱毒前的病毒检测胶图所示)，最后对完成脱毒处理的材料进行病毒检测(如图3所示经愈伤诱导脱毒处理后病毒检测结果)，更加充分说明脱毒方法的有效性。其他的发明中只是检测最后完成脱毒处理的材料。
5)、以本研究的方法进行操作可在14周左右获得脱毒苗：建立无菌苗体系需要5周，以无菌苗鳞片为外植体诱导出愈伤组织与愈伤组织继代的时间是5周，愈伤组织诱导成苗的时间是4周。
因此，本研究在脱毒方法的结合上具有创新性，对愈伤组织诱导的培养基探究上具有创新性，对脱毒方法的有效性的考量上有创新性，且能在较短时间内就获得脱毒苗。
本发明所述的一种高效的百合脱毒方法，是发明人经过长时间的摸索和研究，在大量实验的基础上获得的结果，相比现有的技术，本发明的有益效果为：
本发明通过采取愈伤诱导的方法，有效的克服了脱毒材料多、脱毒步骤冗长、脱毒时间长、所需技术与设备条件高的问题，能高效快速的获得脱毒组培苗。
与其他与众多的研究直接以大田苗的各个部位进行脱毒不同，本研究中采用先利用大田种球建立了无菌苗体系，大大降低了污染率及所需材料，从而加快了脱毒效率。
本发明采用一代无菌苗鳞片诱导愈伤组织可一次性去除黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒，并在14周获得脱毒苗。如直接以无菌苗为实验材料，脱毒时间只需要9周。
本发明在获得脱毒苗后，在常规培养基中添加适量的活性炭，用组培的方式以极高的繁殖速率快速获得大量脱毒苗，可广泛适用于产业化生产。
附图说明
图1为体视显微镜下百合愈伤组织状态。
其中，A：初期愈伤；B：后期愈伤。
图2为原种球病毒检测结果。
其中，M：DNA Marker DL2000；1：CMV；2：LSV；3：LMoV)。
图3为经愈伤诱导脱毒处理后病毒检测结果。
其中，a):CMV病毒检测情况；b):LSV病毒检测情况；c):LMoV病毒检测情况；
M：2000marker；1:阳性对照；2-4：处理苗；5：阴性对照。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
一种高效的百合脱毒方法，该方法的详细步骤如下：
1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测：选取无腐烂、健康的百合鳞片0.5g，冻入液氮中，采用trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物(见表1)，采用20微升体系进行PCR扩增，检测是否含有此三种病毒；
表1 RT-PCR特异引物
Figure PCTCN2020084135-appb-000001
2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗：选取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒15-20分钟，经灭菌ddH 2O冲洗5-8次，倒置于滤纸上晾干；将鳞片切至1×1cm大小的方块，凹面向上置于诱芽培养基中，该诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/L NAA+15g/L蔗糖+7g/L琼脂，该培养基的pH＝5.8，每个培养基放至2-3块；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
3)切取无菌苗小鳞片，诱导获得致密愈伤组织：将长至3-8cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中，该愈伤诱导培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L蔗糖+7g/L琼脂；放至黑暗，20℃条件下生长；
4)愈伤组织经过分化培养诱导成苗：将长成致密状态的愈伤组织切离出母体，继代1-2次。其中，愈伤继代的转接间隔为10-15天，转至分化培养基中，该分化培养基为：MS+0.4g/L活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。愈伤组织是从外植体的内部或切口表面形成的一团没有分化的组织，这种组织具有再分化的能力。本发明借助于愈伤组织的特性得到脱毒苗，而不包括其他含义。
RT-PCR反转录病毒检测具体步骤：
1)反转录
cDNA的合成参照takara试剂盒说明。
加样体系为：
Figure PCTCN2020084135-appb-000002
65℃保温5min后，冰上迅速冷却，继续加样。
加样体系为：
Figure PCTCN2020084135-appb-000003
缓慢混匀，按以下条件进行反转录：
30℃10min；42℃30～60min；95℃5min；冰上冷却。
2)以合成的cDNA第一链为模板，用合成的5’端引物和3’端引物进行PCR扩增。
PCR扩增体系为：
Figure PCTCN2020084135-appb-000004
反应程序为：
Figure PCTCN2020084135-appb-000005
反应在PCR扩增仪上进行36个循环；72℃延伸2min，4℃终止反应。取10μL扩增产物进行1％琼脂糖凝胶电泳，利用BioRad凝胶成像系统观察结果。
实施例1一种高效的百合脱毒方法，该百合脱毒方法的详细步骤如下：
1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测：选取无腐烂、健康的百合鳞片0.5g，冻入液氮中，采用Trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物(见表1)，采用20微升体系进行PCR扩增，检测是否含有此三种病毒；
2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗：选取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒15分钟，经灭菌ddH 2O冲洗5次，倒置于滤纸上晾干；将鳞片切至1×1cm大小的方块，凹面向上置于诱芽培养基中，该诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/L NAA+15g/L蔗糖+7g/L琼脂，该培养基的pH＝5.8，每个培养基放至3块；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
3)切取无菌苗小鳞片，诱导获得致密愈伤组织；将长至3cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中，该愈伤诱导培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L蔗糖+7g/L琼脂；放至黑暗，20℃条件下生长；
4)愈伤组织经过分化培养诱导成苗；将长成致密状态的愈伤组织切离出母体，继代2次。其中，愈伤继代的转接间隔为10天，转至分化培养基中，该分化培养基为：MS+0.4g/L活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测(具体步骤如前述)，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。
实施例2一种高效的百合脱毒方法，该百合脱毒方法的详细步骤如下：
1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测：选取无腐烂、健康的百合鳞片0.5g，冻入液氮中，采用Trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物(见表1)，采用20微升体系进行PCR扩增，检测是否含有此三种病毒；
2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗：选 取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒15分钟，经灭菌ddH 2O冲洗5次，倒置于滤纸上晾干；将鳞片切至1×1cm大小的方块，凹面向上置于诱芽培养基中，该诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/LNAA+15g/L蔗糖+7g/L琼脂，该培养基的pH＝5.8，每个培养基放至3块；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
3)切取无菌苗小鳞片，诱导获得致密愈伤组织；将长至5cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中，该愈伤诱导培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L蔗糖+7g/L琼脂；放至黑暗，20℃条件下生长；
4)愈伤组织经过分化培养诱导成苗；将长成致密状态的愈伤组织切离出母体，继代1次。其中，愈伤继代的转接间隔为13天，转至分化培养基中，该分化培养基为：MS+0.4g/L活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测(具体步骤如前述)，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。
实施例3一种高效的百合脱毒方法，该百合脱毒方法的详细步骤如下：
1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测：选取无腐烂、健康的百合鳞片0.5g，冻入液氮中，采用Trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物(见表1)，采用20微升体系进行PCR扩增，检测是否含有此三种病毒；
2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗：选取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒20分钟，经灭菌ddH 2O冲洗8次，倒置于滤纸上晾干；将鳞片切至1×1cm大小的方块，凹面向上置于诱芽培养基中，该诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/L NAA+15g/L蔗糖+7g/L琼脂，该培养基的pH＝5.8，每个培养基放至3块；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
3)切取无菌苗小鳞片，诱导获得致密愈伤组织；将长至8cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中，该愈伤诱导培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+15g/L蔗糖+7g/L琼脂；放至黑暗，20℃条件下生长；
4)愈伤组织经过分化培养诱导成苗；将长成致密状态的愈伤组织切离出母体，继代2 次。其中，愈伤继代的转接间隔为15天，转至分化培养基中，该分化培养基为：MS+0.4g/L活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖；放至组培室条件下生长，光照强度为1500Lx，温度为20℃；
5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测(具体步骤如前述)，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。
本发明通过采取愈伤诱导的方法，有效的克服了脱毒材料多、脱毒步骤冗长、脱毒时间长、所需技术与设备条件高的问题，能高效快速的获得脱毒组培苗。上述实施例采用一代无菌苗鳞片诱导愈伤组织可一次性去除黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒，并在14周获得脱毒苗。本发明在获得脱毒苗后，在常规培养基中添加适量的活性炭(活性炭添加在分化培养基中，作用是辅助组培苗生长)，用组培的方式以极高的繁殖速率快速获得大量脱毒苗，可广泛适用于产业化生产。
本发明通过建立一代无菌苗体系，取其小鳞片进行愈伤诱导，利用愈伤组织极强的再分化能力诱导成苗，有效地克服了过去百合脱毒技术中存在的脱毒步骤繁琐、环境条件要求高、脱毒成本高、时间长的问题。本发明中先建立一代无菌苗体系，再切取其小鳞片进行愈伤诱导，一次性得到脱去黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的组培苗，具有脱毒效率高、操作简单、时间短、环境要求及成本低等优点，适用于规模化产业化生产。
本发明未涉及部分均与现有技术相同或可采用现有技术加以实现。以上所述仅为本发明的优选实施例而已，并不用与限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种高效的百合脱毒方法，其特征在于：该方法包括以下步骤：
    1)选取健康无腐烂的百合鳞片提取RNA，经反转录进行病毒检测；
    2)选取中内层健康的百合鳞片进行外植体消毒，培养获得大量长势良好的无菌苗；
    3)切取无菌苗小鳞片，诱导获得致密愈伤组织；
    4)愈伤组织经过分化培养诱导成苗；
    5)切取步骤4)中的叶进行病毒检测，检测合格即为百合脱毒种苗。
  2. 根据权利要求1所述的方法，其特征在于：
    步骤1)中进行病毒检测的详细过程为：选取无腐烂、健康的百合鳞片冻入液氮中，采用Trizol法提取RNA；后用takara反转录试剂盒进行反转录；设计黄瓜花叶病毒、百合无症病毒以及百合斑驳病毒的引物进行PCR扩增，检测是否含有此三种病毒；
    步骤2)培养获得无菌苗的详细过程为：选取较少病斑的种球，剥取中内层健康的鳞片在流水下冲洗后依次经75％乙醇和1％次氯酸钠消毒，再经灭菌ddH 2O冲洗多次，倒置于滤纸上晾干；将鳞片切成方块，凹面向上置于诱芽培养基中，放至组培室条件下生长；
    步骤3)切取无菌苗小鳞片，诱导获得致密愈伤组织的详细过程为：将长至3-8cm高的植株切去叶片，剥其小鳞片凹面朝上放至诱导愈伤培养基中；放至组培室条件下生长；
    步骤4)愈伤组织经过分化培养诱导成苗的详细过程为：将长成致密状态的愈伤组织切离出母体，继代1-2次后，转至分化培养基中；放至组培室条件下生长；
    步骤5)切取步骤4)中的叶进行病毒检测的详细过程为：对步骤4)中完成脱毒处理的百合小鳞茎随机抽取10-20％，进行RT-PCR反转录病毒检测，经检测确认不带黄瓜花叶病毒、百合无症病毒和百合斑驳病毒，即为百合脱毒苗。
  3. 根据权利要求2所述的方法，其特征在于：步骤2)中剥取中内层健康的鳞片在流水下冲洗2个小时，75％乙醇消毒30秒，1％次氯酸钠消毒15-20分钟，经灭菌ddH 2O冲洗5-8次，倒置于滤纸上晾干。
  4. 根据权利要求2所述的方法，其特征在于：步骤2)中所述的诱芽培养基为：MS+2.0mg/L6-BA+0.2mg/L NAA+7g/L琼脂+15g/L蔗糖。
  5. 根据权利要求2所述的方法，其特征在于：步骤3)中所述的诱导愈伤培养基为：MS+1.0mg/LTDZ+2.0mg/L PIC+7g/L琼脂+15g/L蔗糖。
  6. 根据权利要求2所述的方法，其特征在于：步骤3)中小鳞片诱导愈伤的光照培养条件为黑暗。
  7. 根据权利要求2所述的方法，其特征在于：步骤4)中所述的分化培养基为：MS+0.4g/L 活性炭+0.2mg/L NAA+9g/L琼脂+40g/L蔗糖。
  8. 根据权利要求2所述的方法，其特征在于：步骤4)中愈伤继代的转接间隔时间为10-15天。
  9. 根据权利要求1或2所述的方法，其特征在于：步骤4)中愈伤诱导成苗的培养条件为：光照强度为1500Lx，温度为20℃。
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