阅读说明：本技术 一种小滴玻璃化法草莓茎尖超低温脱毒的方法 (Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method ) 是由 赵磊 吴云锋 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种小滴玻璃化法草莓茎尖超低温脱毒的方法,属于草莓脱毒技术领域。所述的小滴玻璃化法草莓茎尖超低温脱毒的方法,具体包括以下步骤：取感染病毒的草莓外植体、切取茎尖、预培养、小滴玻璃化处理并投入液氮、解冻、恢复培养、植株再生和继代培养。本发明小滴玻璃化法草莓茎尖超低温脱毒的方法具有操作简单、耗时短、脱毒率高、再生率高的优点,具有较好的技术效果和应用前景。(The invention discloses a method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method, belonging to the technical field of strawberry detoxification. The method for ultralow-temperature detoxification of strawberry stem tips by a droplet vitrification method specifically comprises the following steps: taking a strawberry explant infected with virus, cutting a stem tip, pre-culturing, performing small droplet vitrification treatment, putting into liquid nitrogen, unfreezing, recovering and culturing, regenerating a plant and performing subculture. The method for ultralow-temperature detoxification of the stem tips of the strawberries by the droplet vitrification method has the advantages of simplicity in operation, short consumed time, high detoxification rate and high regeneration rate, and has good technical effect and application prospect.)

1. A method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method is characterized by comprising the following steps: comprises the following steps: peeling off about 2mm of strawberry stem tips, culturing on a subculture medium for 3 days, transferring to a liquid culture medium containing glycerol and sucrose, culturing for 1 day again, transferring the stem tips to PVS2, treating on ice for 40 minutes, transferring to droplets on sterilized aluminum foil strips, then immersing in liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strips and immersing in a liquid sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days^-1m^-2And (4) performing weak light culture, transferring the stem tip into a new subculture medium after regeneration for one week, and performing regeneration culture under normal illumination.

2. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the subculture medium is as follows: MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH 5.8.

3. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the culture medium containing the glycerol and the sucrose is as follows: MS +2.0M glycerol +0.8M sucrose.

4. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the liquid sucrose culture medium is as follows: MS +1.2M sucrose.

Technical Field

The invention belongs to the technical field of plant virology, and particularly relates to a method for removing strawberry viruses.

Background

Strawberries (Fragaia ananasa) are used as herbaceous plants, which are mainly propagated by means of stolon seedlings formed by mother plants as seedling. Once the strawberries are infected by virus, 100% of mother strains are directly transmitted to offspring, so that the serious variety degradation phenomenon is caused, the yield is generally reduced by 20% -80%, and the remarkable quality reduction is caused. Strawberries, once infected with virus, cannot be resolved by the necessary field management. The use of the virus-free seedlings is one of the most economical and effective means for preventing and treating the strawberry virus disease. However, the research and production of strawberry detoxification and rapid propagation in China are seriously disconnected, the conventional detoxification technology has low detoxification efficiency, and the production cost of the detoxified seedlings is high. Therefore, the method is very necessary for improving the strawberry detoxification efficiency, standardizing the high-efficiency and low-cost rapid propagation technology of the detoxified seedlings and reducing the cost of the detoxified seedlings. The strawberry stem tip ultra-low temperature therapy detoxification technology established by the research team in recent years can effectively solve the problems.

Disclosure of Invention

The research aims at the current situations that strawberry virus diseases are seriously damaged and the detoxification method is laggard in China, and provides a method for ultralow temperature detoxification of strawberry stem tips by a droplet vitrification method.

The invention can be realized by the following technical scheme: a method for ultralow-temperature detoxification of strawberry stem tips by a droplet vitrification method specifically comprises the following steps:

peeling off about 2mm of strawberry stem tips, culturing on a subculture medium for 1 day, transferring to a liquid culture medium containing glycerol and sucrose, culturing for another 1 day, transferring the stem tips to PVS2, treating on ice for 40 minutes, transferring to droplets on sterilized aluminum foil strips, then immersing in liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strips and immersing in a liquid sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days^-1m^-2Culturing under weak light, and regenerating the stem tip in a new subculture medium under normal light after one week of regeneration.

The subculture medium comprises: MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH 5.8.

The formula of the culture medium containing the glycerol and the sucrose is as follows: MS +2.0M glycerol +0.8M sucrose.

The formula of the liquid sucrose culture medium is as follows: MS +1.2M sucrose.

The method for ultralow-temperature detoxification of the stem tips of the strawberries by the droplet vitrification method has the advantages of simplicity in operation, short consumed time, high detoxification rate and high regeneration rate, and has good technical effect and application prospect.

Drawings

FIG. 1 shows the results of RT-PCR detection of 4 viruses: A-D are respectively the detection results of strawberry mottle virus (SMoV), Strawberry Vein Banding Virus (SVBV), strawberry shrunken virus (SCV) and Strawberry Mild Yellow Edge Virus (SMYEV); m: DNA molecular weight standard; 1-10 are respectively 10 strawberry seedlings regenerated after ultralow temperature detoxification of stem tips; wherein the virus was removed for 10 samples of SMoV; for SVBV, samples Nos. 1-2 and 5-10 all removed the virus. The virus was removed for 10 samples of both SCV and SMYEV.

FIG. 2 shows the regenerated tissue culture seedling of strawberry for red color after ultra-low temperature detoxification of stem tip.

Detailed Description

The present invention is further illustrated below by way of examples for the understanding of the present invention, but the following examples do not limit the present invention.

The invention provides a method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method, which comprises the following steps: peeling 50 strawberry stem tips with the diameter of about 2mm, culturing on a subculture medium (MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH5.8) for 1 day, then culturing on an MS +2.0M glycerol +0.8M sucrose culture medium for another 1 day, transferring the stem tips into PVS2, treating on ice for 40 minutes, then transferring into droplets on a sterilized aluminum foil strip, then soaking into liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strip and soaking into the MS +1.2M sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days^-1m^-2Culturing under weak light, and regenerating the stem tip in a new subculture medium under normal light after one week of regeneration. Counting the number of surviving seedlings after one week of regeneration, and calculating the survival rate. The result shows that 50 stem tips finally survive 26, and the survival rate is 52%.

And (3) continuously carrying out illumination culture on the surviving seedlings, carrying out subculture once after 3 weeks, carrying out subculture for 4 weeks after subculture, and carrying out RT-PCR detection on 4 strawberry viruses including SMoV, SVBV, SCV and SMYEV. The detection primer is synthesized by Beijing Olympic Biotechnology GmbH, and the specific primer name and sequence are shown in Table 1.

TABLE 14 detection primers for strawberry virus

The specific detection steps comprise the extraction of total RNA of a sample, RT-PCR and agarose gel electrophoresis detection. After detection, the detoxification rate of the 4 viruses is counted, and the results show that the detoxification rate of SMoV, SCV and SMYEV is 100%, and the detoxification rate of SVBV is 80%.