A method for rapidly reducing the accumulation of garlic viruses

Abstract

The present invention relates to a method for rapidly reducing the accumulation of garlic viruses, comprising the following steps: (1) Preparation of mother garlic cloves; the garlic cloves are treated at a low temperature of 10-12°C for 15-20 days, then the garlic cloves are soaked in water for 20 minutes and placed in a humid environment for 1-2 days for root culture. The number of roots should be less than 5 to obtain mother garlic cloves; (2) Virus detoxification treatment of garlic cloves; the storage cloves of the mother garlic cloves obtained in step (1) are cut off with a knife, only the bulbils are retained, sterilized, and immediately transplanted into a sterilized soil substrate to obtain virus-detoxified garlic cloves; (3) The virus-detoxified garlic cloves obtained in step (2) are cultured indoors. After one week of culture, nutrient elements are added to strengthen the seedlings until the 3-4 leaf stage; (4) The garlic cloves obtained in step (3) are transplanted into the field with soil. Garlic germplasm with a significant reduction in viruses can be obtained in the coming year. Through the present invention, the virus detoxification cycle is short, easy to operate, and does not require aseptic environment operation, and can significantly overcome the problem of virus accumulation in the asexual reproduction of garlic.

Description

Technical Field

[0001] The present invention relates to a method for rapidly reducing the accumulation of garlic viruses, belonging to the technical field of garlic detoxification. Background Art

[0002] Garlic belongs to the species of Allium in the family Amaryllidaceae and is an important vegetable crop. Garlic is rich in sulfur-containing compounds, which give garlic a unique flavor, so garlic is also an important condiment crop. The sulfur-containing compounds in garlic also have a broad-spectrum bactericidal and anti-inflammatory effect and have a significant curative effect on diseases such as cardiovascular diseases, so garlic is also widely used in the pharmaceutical industry. China is a major garlic-growing country, with an annual sown area of more than 10 million mu and a production accounting for about 75% of the world's total output. Garlic is also one of the crops with the largest single agricultural product export value in China, with an annual export value of up to 3 billion US dollars.

[0003] Garlic is sterile, and long-term vegetative propagation has led to serious accumulation of viruses in garlic. The accumulation of viruses will cause the degeneration of garlic varieties and various disease manifestations, such as short plants, low garlic bulb yields, and poor commercial quality. At present, the main method for garlic detoxification is the shoot tip detoxification technology. However, this technology has some deficiencies, mainly manifested in: (1) The extraction of shoot tips is time-consuming and laborious. Generally, only 20-30 shoot tips can be extracted by one person per day; (2) The culture of shoot tips takes a long time. Generally, it takes 9 months for a single shoot tip to regenerate into a garlic seedling; (3) The shoot tip detoxification has high requirements for platform conditions, requires operation in a sterile environment, and requires a good light culture room. This process is extremely prone to contamination, resulting in the failure of the detoxification experiment. (4) The detoxification cycle is long. The transplanted detoxified seedlings in the field generally form single-clove garlic and need to be reproduced for another year to form normal garlic bulbs. Coupled with the shoot tip culture, the whole process takes 3-4 years. It can be seen that the garlic shoot tip detoxification work platform has high requirements, large input costs, and a long effective cycle, which limits the large-scale application of garlic shoot tip detoxification. At present, there are few detoxified garlic seeds in the garlic seed market. Therefore, it is imperative to develop a technical method that does not rely on a high-standard platform, has low costs, and can rapidly reduce the virus content. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for rapidly reducing the accumulation of garlic viruses in view of the problems existing in the above-mentioned prior art.

[0005] In order to achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows: A method for rapidly reducing the accumulation of garlic viruses, characterized by including the following steps:

[0006] (1) Preparation of maternal garlic cloves;

[0007] Treat the garlic cloves at a low temperature of 10-12°C for 15-20 days, then soak the garlic cloves in water for 20 minutes, and place them in a humid environment for 1-2 days for rooting culture. The number of roots should be less than 5 to obtain the maternal garlic cloves;

[0008] (2) Detoxification treatment of garlic cloves;

[0009] Cut off the storage cloves of the mother garlic cloves obtained in step (1) with a knife, only keep the scale buds, sterilize them, and immediately transplant them into the sterilized soil substrate to obtain detoxified garlic cloves;

[0010] (3) Cultivate the detoxified garlic cloves obtained in step (2) indoors. After one week of cultivation, add nutrient elements to strengthen the seedlings until the 3-4 leaf stage;

[0011] (4) Transplant the garlic cloves obtained in step (3) with soil to the field, and virus-reduced garlic germplasm can be obtained the following year.

[0012] In step (1), the preparation of the mother garlic cloves is generally completed before the end of August.

[0013] In step (1), the rooting culture temperature is 23-25 °C, the light culture condition is 14 hours of light and 10 hours of dark culture, and the humidity is controlled between 40-60%.

[0014] In step (2), the method for cutting off the storage cloves is as follows: Peel the garlic cloves, make a circular cut along 1 cm above the stem disc with a sterile scalpel, do not cut through to prevent damage to the base of the germinating leaves; then make a longitudinal cut of the garlic cloves at 1 cm on the left and right from the middle position, protect the germinating leaves from being cut off, and use sharp tweezers to remove the excess garlic cloves around the germinating leaves.

[0015] In step (2), after cutting off the storage cloves, soak them in the sterilizing medicine for 30-60 seconds for sterilization.

[0016] In step (2), the soil substrate is a seedling-raising substrate, including peat, desalted coconut coir, perlite and vermiculite, the substrate pH = 5.5-7.0, and the organic matter > 20%;

[0017] The soil substrate should be sterilized at 121 °C for 15 minutes and placed at room temperature to cool for later use.

[0018] In step (2), when transplanting, pour the sterilizing medicine through the soil substrate; the sterilizing medicine is a solution obtained by diluting thiophanate-methyl or chlorothalonil 500-800 times.

[0019] In step (3), for the first time, 0.1% urea water or MS culture solution can be used to strengthen the seedlings;

[0020] Use urea water to strengthen the seedlings once a week. Each time, according to the situation of the seedlings, increase the urea concentration and fertilization amount, but the maximum concentration should not exceed 0.2%; for the MS culture solution, strengthen the seedlings twice a week.

[0021] In step (4), transplanting is completed before the first ten days of November in the south and before the last ten days of October in the north.

[0022] In step (4), before transplanting, the seedlings should be hardened off outdoors for more than 3 days; in order to ensure the virus elimination effect, they should be planted in a 40-mesh net house.

[0023] The method of the present invention is advanced and scientific. Through the present invention, the method includes: (1) Preparation of mother garlic cloves; the garlic cloves are treated at a low temperature of 10-12 °C for 15-20 days, then the garlic cloves are soaked in water for 20 minutes, and then placed in a humid environment for 1-2 days for root culture, but the time should not be too long. Generally, it is more appropriate that the number of roots is less than 5. (2) Virus elimination treatment of garlic cloves; the storage cloves of the mother garlic cloves are cut off with a knife, and only the scale buds are retained (see Figure 1 ), and sterilization is carried out, and then immediately transplanted into a sterilized soil substrate. (3) The treated virus-eliminated garlic cloves are cultured indoors. After one week of culture, nutrient elements can be added to strengthen the seedlings until the 3-4 leaf stage. (4) The treated garlic cloves are transplanted into the field with soil, and garlic germplasm with a significantly reduced virus content can be obtained in the coming year.

[0024] According to the above operations, the garlic variety "Ershuizao" is treated. Then, the plant leaves are taken at the bolting stage, and virus fluorescence quantitative PCR is carried out to detect the contents of garlic virus B and garlic virus D. Three experimental replicates are set for each treatment. It is found that in the garlic treated by this method, the relative ct value of the garlic virus B gene (the virus gene ct value minus the internal reference ct value) is 3.173, while the relative ct value of the normal garlic virus gene without treatment is 0.2825. According to the 2 -ΔΔct method proposed by Livak and Schmittgen in the paper "Analysis of Relative Gene Expression Data Using Real-TimeQuantitative PCR and the 2 -ΔΔct Method" published in the international journal "METHODS" in 2001, the content of garlic virus B in the garlic plants without mother garlic is only 13.5% of that of normal garlic. Through a similar method, it is found that the virus content of garlic virus D in the garlic plants without mother garlic is only 20.7% of that of normal garlic.

[0025] Beneficial effects:

[0026] The virus-free treatment of garlic cloves has the advantages that the removal of storage cloves is simple. Generally, one person can peel 30 cloves per hour, and the efficiency is more than 10 times that of shoot tip dissection. At the same time, this dissection does not require operation in a sterile environment. The dissected storage cloves are directly cultured in a sterilized soil substrate and can generally be transplanted to the field within 2 months. After the seedlings cultured indoors are treated, they can be transplanted with soil and healthy virus-free bulbs can be obtained within 10 months, which is much shorter than the 3-4 years required for shoot tip virus elimination, greatly shortening the time. The virus-eliminating technology of the present invention has a short virus-eliminating cycle, is easy to operate, does not require operation in a sterile environment, has low cost, and has obvious virus-eliminating effect. It can significantly overcome the problem of virus accumulation in the asexual reproduction of garlic, which is of great significance for maintaining the high and stable yield characteristics of garlic and effectively avoiding the degeneration of varieties caused by viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram for resection of storage cloves; where the left is the mother garlic and the right is the bulb bud with the storage clove resected.

[0028] Figure 2 Distribution diagram of RNA-seq reads in storage cloves and bulb buds; where the left two diagrams show the proportions of RNA-seq reads mapped to the garlic genome and the virus genome, and the right diagram shows the top 10 viruses in terms of RNA-read proportion.

[0029] Figure 3-1 Comparison diagram of the sizes of the mother plant of Chaling purple-skinned garlic and virus-free garlic; where the left in the figure is the mother plant of Chaling purple-skinned garlic and the right is the virus-free garlic.

[0030] Figure 3-2 Comparison diagram of the numbers of cloves of the mother plant of Chaling purple-skinned garlic and virus-free garlic; where the left cloves in the figure are the cloves of the mother plant of Chaling purple-skinned garlic and the right are the cloves of the virus-free garlic. DETAILED DESCRIPTION OF THE INVENTION

[0031] The following further elaborates on the present invention in detail in conjunction with the specific embodiments, but it shall not be used as a basis for limiting the present invention.

[0032] A method for rapidly reducing virus accumulation in garlic includes the following steps:

[0033] (1) Preparation of mother garlic cloves;

[0034] The garlic cloves are treated at a low temperature of 10-12°C for 15-20 days, then soaked in water for 20 minutes, and placed in a humid environment for 1-2 days for root culture. The number of roots should be less than 5 to obtain the mother garlic cloves;

[0035] (2) Virus-free treatment of garlic cloves;

[0036] The mother garlic cloves obtained in step (1) are cut with a knife to remove the storage cloves, and only the scale buds are retained. After sterilization, they are immediately transplanted into a sterilized soil substrate to obtain virus-free garlic cloves.

[0037] (3) The virus-free garlic cloves obtained by the treatment in step (2) are cultured indoors. After one week of culture, nutrient elements are added to strengthen the seedlings until the 3-4 leaf stage.

[0038] (4) The garlic cloves obtained after the treatment in step (3) are transplanted into the field with soil, and garlic germplasm with a significant reduction in virus can be obtained the following year.

[0039] According to the above operations on the garlic variety "Ershuizao", we took plant leaves at the bolting stage, performed virus fluorescence quantitative PCR, and detected the contents of garlic virus B and garlic virus D. Three experimental replicates were set for each treatment. The results showed that the relative ct value (virus gene ct value minus internal reference ct value) of the garlic virus B gene in the garlic treated by this method was 3.173, while the relative ct value of the virus gene in the untreated normal garlic was 0.2825. According to the method proposed by Livak and Schmittgen (2001), the content of garlic virus B in the garlic plants without mother garlic was only 13.5% of that in the normal garlic. By a similar method, it was found that the virus content of garlic virus D in the garlic plants without mother garlic was only 20.7% of that in the normal garlic. -ΔΔct The content of garlic virus B in the garlic plants without mother garlic was only 13.5% of that in the normal garlic estimated by the method proposed by Livak and Schmittgen (2001). By a similar method, it was found that the virus content of garlic virus D in the garlic plants without mother garlic was only 20.7% of that in the normal garlic.

[0040] Table 1 Ct values of fluorescence quantitative PCR and relative expression differences

[0041]

[0042] Chaling purple-skinned garlic belongs to a local garlic variety and has accumulated a large amount of virus. We took plant leaves at the bolting stage and treated the mother garlic cloves of "Chaling purple-skinned garlic" according to the above operation steps (1). The storage cloves and scale bud parts of the garlic cloves were separated and RNA sequencing (RNA-seq) was performed separately. Three sample replicates were carried out for each part, and finally 81.4 and 79.8 million sequences / sample were obtained in the storage cloves and scale buds respectively. These sequences were aligned to the garlic genome, and it was found that 27.6% of the RNA-seq sequences in the storage cloves could be aligned to the garlic genome, while 72.4% of the sequences could not be aligned to the garlic genome. Further aligning these sequences to the NCBI Nr database, it was found that 72.4% of these sequences were mainly aligned to garlic viruses ( Figure 1 ). By a similar method, it was found that 78.9% of the RNA-seq sequences in the scale buds, and the sequence aligned to the virus genome was 21.1% ( Figure 2) It can be seen that the virus in the storage cloves mainly aggregates in the garlic cloves, and the virus activity in the scale buds is only 29.1% of that in the storage cloves. Compared with the storage cloves, we found that the virus reduction of Garlic virus A and Garlic virus D in the scale buds is the most significant, especially Garlic virus D( Figure 1 )。

[0043] Based on the research finding that the virus types and contents in the scale buds are significantly lower than those in the storage cloves, the present invention cultivates the scale buds by removing the storage cloves at the seedling stage, preventing the infection of the scale buds by the virus in the storage cloves before the garlic mother bulb withers, and greatly reducing the virus accumulation in the asexual reproduction process of garlic. The present invention can significantly reduce the garlic virus content, and the contents of Garlic virus B and Garlic virus D can be reduced by more than 80%.

[0044] For the above method, preferably, in step (1), the preparation of the mother garlic cloves is generally completed before the end of August,

[0045] Preferably: the rooting culture temperature is 23 - 25 °C, the light culture condition is 14 h of light and 10 h of darkness, and the humidity is controlled between 40 - 60%.

[0046] Preferably, in step (2), the storage clove excision technique is: peel the garlic cloves, use a sterile scalpel to make a circular cut along 1 cm above the stem disc without cutting through to prevent damage to the base of the germinating leaf. Then make a longitudinal cut on the garlic cloves at 1 cm on both sides of the middle position, protecting the germinating leaf from being excised, and use sharp tweezers to remove the excess garlic cloves around the germinating leaf.

[0047] Preferably, after the storage cloves are excised, soak them in a sterilizing medicine for 30 - 60 seconds for sterilization.

[0048] Preferably, the soil substrate is a seedling raising substrate, including imported peat, desalted coconut coir, perlite and vermiculite, the substrate pH = 5.5 - 7.0, and the organic matter > 20%.

[0049] Preferably, the soil substrate should be sterilized at 121 °C for 15 min and placed at room temperature to cool for standby.

[0050] Preferably, when transplanting, water the soil substrate thoroughly with a sterilizing medicine

[0051] Preferably: the sterilizing medicine is a solution obtained by diluting thiophanate-methyl or chlorothalonil 500 - 800 times.

[0052] Preferably, in step (3), for the first time, 0.1% urea water or MS culture solution can be used to strengthen the seedlings.

[0053] Preferably, the urea water is used to strengthen the seedlings once a week. Each time, according to the seedling situation, the urea concentration and fertilization amount can be increased, but the maximum concentration should not exceed 0.2%:

[0054] Preferably, the MS culture medium is used to strengthen the seedlings twice a week.

[0055] Preferably, in the step (4), the transplanting is generally completed before the first ten days of November in the south and before the last ten days of October in the north.

[0056] Preferably, before transplanting, the seedlings should be acclimatized outdoors for more than 3 days.

[0057] Preferably, in order to ensure the virus-free effect, it is better to plant in a 40-mesh net room.

[0058] For the virus-free treatment of garlic cloves in the present invention, the removal of storage cloves is simple. Generally, one person can peel 30 cloves per hour, and the efficiency is more than 10 times that of shoot tip peeling. At the same time, the peeling does not require operation in a sterile environment. The peeled storage cloves are directly cultured in a sterilized soil matrix and can generally be transplanted to the field within 2 months. After the indoor-cultured strong seedlings are treated, they are transplanted with soil and strong virus-free bulbs can be obtained within 10 months, which is much shorter than 3-4 years for shoot tip virus-free treatment. The virus-free technology of the present invention has a short virus-free cycle, is easy to operate, does not require operation in a sterile environment, has low cost, and has obvious virus-free effect. It can significantly overcome the problem of virus accumulation in the asexual reproduction of garlic, is of great significance for maintaining the high and stable yield of garlic, and effectively avoids the degeneration of varieties caused by viruses.

[0059] Case:

[0060] The local variety 'Chaling Purple Skin Garlic' is a local variety and has never carried out virus-free work. The virus accumulation is large and the varietal characteristics degenerate strongly. Using it as the material to carry out the virus-free work of removing mother cloves, the obtained virus and agronomic trait performance data of virus-free garlic are as follows.

[0061] (1) Virus content

[0062] We took the plant leaves during the bolting period of Chaling Purple Skin Garlic and randomly detected the content of four viruses. The relevant detection results showed that compared with the virus-free garlic (female parent), the virus rates detected in the leaves after shoot tip virus-free treatment and the virus-free treatment of removing mother cloves in this patent were lower, and the virus-free effect was obvious. The virus-free effect of shoot tip virus-free treatment was slightly better than that of removing mother cloves, but the difference was not significant.

[0063] Table 2 Virus detection results of Chaling Purple Skin Garlic leaves before and after virus-free treatment

[0064]

[0065]

[0066] Note: The data in the column are the relative expression levels of virus genes. A high expression level represents a high virus content, and undetected means that the virus was not detected. Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0067] (2) Trait performance of mother-removed virus-free garlic;

[0068] Bulb traits of mother-removed virus-free garlic were investigated (see Table 3). The bulbs of mother-removed virus-free garlic were larger in size and had more scale buds ( Figure 3-1 、 Figure 3-2 ), and the economic traits were significantly improved. Among them, the bulb weight and bulb width increased by 171.0% and 52.1% respectively, and the scale bud weight and scale bud number increased by 105.3% and 40.9%.

[0069] Table 3 Investigation form of bulb traits of mother-removed virus-free "Chaling Purple Skin" garlic

[0070]

Claims

1. A method for rapidly reducing the accumulation of garlic viruses, characterized in that, It includes the following steps: (1) Preparation of mother garlic cloves; Treat the garlic cloves at a low temperature of 10 - 12°C for 15 - 20 days, then soak the garlic cloves in water for 20 minutes, and place them in a humid environment for 1 - 2 days for root culture. The number of roots should be less than 5 to obtain mother garlic cloves; (2) Virus detoxification treatment of garlic cloves; Cut off the storage cloves of the mother garlic cloves obtained in step (1) with a knife, only keep the scaly buds, sterilize them, and immediately transplant them into a sterilized soil substrate to obtain virus - detoxified garlic cloves; (3) Cultivate the virus - detoxified garlic cloves obtained in step (2) indoors. After one week of cultivation, add nutrient elements to strengthen the seedlings until the 3 - 4 leaf stage; (4) Transplant the garlic cloves with soil obtained in step (3) to the field, and virus - greatly reduced garlic germplasm can be obtained the next year.

2. The method for rapidly reducing the accumulation of garlic viruses according to claim 1, wherein In step (1), the preparation of mother garlic cloves should be completed before the end of August.

3. A method for rapidly reducing the accumulation of garlic viruses according to claim 1 or 2, characterized in that, In step (1), the temperature for root culture is 23 - 25°C, the light culture conditions are 14 hours of light and 10 hours of darkness, and the humidity is controlled between 40 - 60%.

4. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (2), the method for cutting off the storage cloves is as follows: Peel the garlic cloves, use a sterile scalpel to make a circular cut 1 cm above the stem disc without cutting through to prevent damage to the base of the germinating leaves; then make a longitudinal cut 1 cm to the left and right from the middle position of the garlic cloves, protecting the germinating leaves from being cut off, and use sharp tweezers to remove the excess garlic cloves around the germinating leaves.

5. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (2), after cutting off the storage cloves, soak them in a sterilizing medicine for 30 - 60 seconds for sterilization.

6. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (2), the soil substrate is a seedling - raising substrate, including peat, desalted coconut coir, perlite and vermiculite. The substrate pH = 5.5 - 7.0, and the organic matter > 20%; The soil substrate should be sterilized at 121°C for 15 minutes and placed at room temperature to cool for standby.

7. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (2), when transplanting, pour the sterilizing medicine thoroughly into the soil substrate; the sterilizing medicine is a solution obtained by diluting thiophanate - methyl or chlorothalonil 500 - 800 times.

8. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (3), use 0.1% urea water or MS culture solution to strengthen the seedlings for the first time; Use urea water to strengthen the seedlings once a week. Each time, according to the situation of the seedlings, increase the urea concentration and fertilization amount, but the maximum concentration should not exceed 0.2%; for the MS culture solution, strengthen the seedlings twice a week.

9. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that In step (4), the transplanting should be completed before the first ten - day period of November in the south and before the last ten - day period of October in the north.

10. A method for rapidly reducing the accumulation of garlic viruses according to claim 1, characterized in that, In step (4), before transplanting, the seedlings should be hardened off outdoors for more than 3 days; in order to ensure the virus - detoxification effect, plant them in a 40 - mesh net room.

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