Method for obtaining garlic virus-free seedlings by in vitro culture of young sprouts

Abstract

The application discloses a method for quickly obtaining garlic virus-free seedlings through in-vitro culture of young scale buds, which comprises the following steps: firstly, taking garlic plants; in the early stage of the expansion of garlic scale buds, peeling off small scale buds and soaking them in sterile water; then sterilizing the small scale buds in a sodium hypochlorite solution; washing the small scale buds with sterile water for multiple times; culturing the small scale buds in a MS medium to make them root and germinate; when the small scale buds have 3-4 leaves, transplanting the small scale buds into a culture pot to harden the seedlings; when the small scale buds have 5-6 leaves, transplanting the garlic seedlings with soil into a field and normally maintaining the garlic seedlings. The method has the advantages of simple operation, reduced requirement for the operation skills of technical personnel, short culture period, quick acquisition of regenerated plants, improved virus-removal efficiency, satisfaction of the market demand for virus-free garlic, production of a large number of virus-free plants in a short time, improved production efficiency, suitability for large-scale production, support for the rapid growth and regeneration of plants, and expectation of becoming an innovative breakthrough in the field of garlic virus-removal culture.

Description

Technical Field

[0001] The invention relates to a method for quickly obtaining garlic virus-free seedlings through in vitro culture of young bulbils, belonging to the technical field of garlic virus-free. Background Art

[0002] Garlic (Allium sativum L.) is an important economic crop, widely cultivated and used worldwide. However, garlic sterility and long-term vegetative growth lead to significant viral accumulation in the plant. This accumulation of viruses can lead to cultivar degeneration and various disease manifestations, such as dwarf plants, low bulb yields, and poor marketability, causing significant economic losses to the garlic industry. Therefore, the development of virus-free culture technologies is crucial for addressing the problem of garlic viral diseases.

[0003] Traditional garlic virus-free culture technology mainly focuses on stem apex virus removal. This method cultivates and regenerates stem apex tissue to obtain virus-free plants. However, stem apex virus-free technology has a series of problems, including complex operation, long cycle, and low efficiency, which limit its application in large-scale production. In recent years, research on garlic virus-free culture technology has increased. Among them, in vitro culture technology, as an important biotechnology method, provides a new approach for garlic virus-free culture. In vitro culture technology can effectively remove viruses from plants by culturing tissues from different parts of the plant (such as stem apex, bulb, leaves, etc.) to obtain virus-free plants. Although garlic has great potential for virus removal, in actual operation, how to further improve regeneration rate and rooting ability remains a challenge. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for rapidly obtaining virus-free garlic seedlings by in vitro culture of young bulbils.

[0005] Technical solution: The method of the present invention for rapidly obtaining virus-free garlic seedlings by in vitro culture of young bulbils comprises the following steps:

[0006] (1) Preparation of early bulbils: Take garlic plants, peel off the small bulbils at the early stage of bulb expansion, and soak them in sterile water to obtain early small bulbils;

[0007] (2) Bulb detoxification treatment: sterilize the early bulbs in a sodium hypochlorite solution, rinse with sterile water several times, transfer to MS medium and culture the bulbs to root and germinate until the 3-4 leaf stage to obtain garlic seedlings;

[0008] (3) Transplant the garlic seedlings into culture pots and harden them until they grow to 5-6 leaves;

[0009] (4) transplant the garlic seedlings with soil obtained after step (3) to a field, and normally maintain.

[0010] Further, in step (1), the soaking time is 20 min or more, and the garlic is "space garlic".

[0011] Further, the initial bulking of garlic is generally in early March each year, and the small scale buds are bulged to 2-3 mm.

[0012] Further, in step (2), the concentration of the sodium hypochlorite solution is 15-25%.

[0013] Further, in step (2), the sterilization time is 10-15 min, and the sterilized water is rinsed for 3 times or more.

[0014] Further, in step (2), the MS medium is a commonly used medium for plant tissue culture, and 38-40 g of MS powder (containing 2.8-3.2% sucrose) and 6.5-7.5 g of agar are dissolved in 1 L of sterilized water, and the solution is dissolved at 110-120°C in a high-pressure sterilization pot, and is placed at room temperature to be in a gel state.

[0015] Further, in step (2), the rooting and germination culture conditions are: temperature 23-25°C, light 12-14 h light, 10-12 h dark culture, and humidity control 40-60%.

[0016] Further, in step (2), the MS medium is replaced every month during the culture.

[0017] Further, in step (3), the soil medium in the culture pot is a seedling culture medium, which includes imported grass charcoal, desalted coconut bran, perlite and vermiculite, and the volume ratio of the imported grass charcoal, desalted coconut bran, perlite and vermiculite is 3-4:1-1.2:1-1.2:1-1.2, the PH is 5.5-7.0, the organic matter is >20%, and the soil medium is sterilized at 100-130°C for 15 min or more.

[0018] Further, in step (3), the seedling culture is the culture of the garlic seedlings transplanted into the culture pot in a culture room with temperature 23-25°C, light 10-12 h light, 10-12 h dark, and humidity 40-60%.

[0019] Further, in step (3), before transplanting, the soil medium is irrigated with a sterilized medicine, and the sterilized medicine is a solution obtained by diluting methylthiophanate or chlorothalonil with water by 500-800 times.

[0020] Further, in step (3), the 3-4 leaf stage garlic seedlings are transplanted into the seedling substrate, and the garlic seedlings are irrigated with urea aqueous solution (1% concentration) or 1 / 2 concentration Hoagland nutrient solution for the first time.

[0021] Further, the 1 / 2 concentration Hoagland nutrient solution is used for irrigation once a week, and the concentration of the nutrient solution can be increased to 1 concentration Hoagland nutrient solution according to the seedling condition.

[0022] Further, in step (4), the seedlings need to be hardened for more than 3 days outdoors before transplanting.

[0023] Further, in step (4), the transplanting is generally completed before early November in the south and before late October in the north.

[0024] Further, in step (4), in order to ensure the detoxification effect, the 40-mesh nylon net arched shed is better for planting.

[0025] According to the above operation, the garlic variety "Taikong garlic" is treated, and the leaf blades and small scale bud growth plant leaf blades of normal garlic cloves are randomly selected for virus fluorescence quantitative PCR to detect the contents of garlic virus A, garlic virus D and garlic virus X. The results show that the relative ct value (virus gene ct value minus internal reference ct value) of garlic virus A gene in the garlic treated by the method is 15.777, and the relative ct value of the normal garlic virus gene without treatment is 3.132, and the content of garlic virus A in the small scale bud culture plant is only 0.016% of the normal garlic. It is found by similar method that the virus contents of the garlic plant of the garlic bulb are only 0.056% and 0.015% of the normal garlic, respectively, which indicates that the small scale bud culture garlic plant almost does not contain virus.

[0026] The present application provides a method for in vitro culture of garlic bulbs to obtain virus-free garlic plants. Compared with the traditional stem tip detoxification technology, the operation steps are simpler, and the requirement for the operation skill of the technical personnel is reduced. The culture period is shorter, and the regenerated plants can be quickly obtained, thereby improving the efficiency of detoxification and meeting the market demand for virus-free garlic. A large number of virus-free plants can be produced in a short time, the production efficiency is improved, and the method is suitable for large-scale production. The bulb is a storage organ of garlic, rich in nutrients, suitable for in vitro culture, and can support the rapid growth and regeneration of plants, so the method is expected to become an innovative breakthrough in the field of garlic detoxification culture.

[0027] Beneficial effects: Compared with the prior art, the present application has the following obvious advantages:

[0028] The tender scale bud of the application is cultured in vitro to quickly obtain garlic virus-free seedlings, and the operation is simple. Only the garlic scale bud is observed at a specific time every year, and when the small scale bud is 2-3 mm, the garlic plant is pulled out, and the small scale bud is gently peeled off with a scalpel into a culture dish containing sterile water. The efficiency is more than 30 times of the traditional stem tip peeling. At the same time, the peeling does not require sterile environment operation, and the peeled small scale bud is directly placed in the MS culture medium for culture. Generally, it can be transplanted to the nutrient pot within 2 months. After the seedling treatment, the indoor cultured seedlings are transplanted with soil, and healthy virus-free scale buds can be obtained within 10 months, which is 3-4 years shorter than the traditional stem tip virus elimination. The virus elimination technology has the advantages of short virus elimination period, easy operation, no need for sterile environment operation for scale bud peeling, low cost, obvious virus elimination effect, and can obviously overcome the problem of accumulation of virus in garlic asexual propagation. It is of great significance to maintain the yield and stability of garlic varieties, and effectively avoids the degradation of varieties caused by virus. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is an operation schematic diagram of scale bud virus elimination garlic,

[0030] Figure 2 is a cell-specific expression result graph of garlic virus gene. DETAILED DESCRIPTION

[0031] The technical solutions of the application will be further described below in combination with the drawings.

[0032] Example 1

[0033] (1) Early scale bud preparation: The experiment was completed in the tissue culture room, culture room and experimental field of Yangzhou University. "Space garlic" was planted on October 8, 2023, and 5-month-old "Space garlic" plants were taken in early March 2024. At the early stage of garlic scale bud (cloves) swelling, the garlic plants were pulled out, and the leaves were peeled off layer by layer until the small scale buds were exposed. The 2-3 mm small scale buds were slowly peeled off into a culture dish containing sterile water with a scalpel, and soaked in sterile water for 20 minutes (see Figure 1 ).

[0034] (2) Sterilization and disinfection of the operation environment: sterilization and disinfection of the clean bench, hands and gloves.

[0035] (3) Scale bud disinfection treatment: the early small scale buds obtained in step (1) are sterilized in 20% sodium hypochlorite solution for 10-15 minutes, and then rinsed with sterile water for 3 times.

[0036] (4) In the clean bench, the small sprouts obtained in step (3) are transferred to MS medium (40 g MS powder (containing 3% sucrose) and 7 g agar per L of sterilized water) and dissolved by a high-pressure sterilization pot at 110-120°C, and left at room temperature to become a gel state), and 5-6 small sprouts are cultured in each culture dish.

[0037] (3) The small sprouts obtained in step (4) are cultured in a tissue culture room, and the growth conditions of the tissue culture room are set as a temperature of 25°C, a humidity control of 50%, and a light cycle of 12 h light / 12 h darkness to culture the small sprouts to root and sprout. The MS medium is replaced every month during the culture, and after two months of culture, the small sprouts root and grow leaves, and the garlic seedlings are obtained at the 3-4 leaf stage.

[0038] (4) The garlic seedlings obtained in step (3) are transplanted into culture pots with a size of 5 cm x 5 cm x 7 cm, and one seedling is planted in each culture pot. The seedlings are cultured in a culture room with a temperature of 23-25°C, light for 12 h, and darkness for 12 h, and the humidity is controlled at 40-60%. The culture pots are filled with soil substrate (i.e., seedling substrate: imported grass charcoal, desalted coconut chaff, perlite, and vermiculite (volume ratio of 3:1:1:1), with a pH of 5.5-7.0 and an organic matter content of >20%). The seedling substrate is sterilized at 121°C for 15 min or more, and before transplantation, 500 ml of sterilized pesticide solution is poured into each culture pot. The sterilized pesticide solution is obtained by diluting methylthiophanate or chlorothalonil with water by 500-800 times. The 3-4 leaf stage garlic seedlings are transplanted into the seedling substrate, and 200 ml of Hoagland's nutrient solution is poured into each culture pot at the time of transplantation. The concentration of the Hoagland's nutrient solution is 1 / 2 times, and it is generally poured once a week. According to the condition of the seedlings, the concentration of the Hoagland's nutrient solution is increased to 1 time after one week, and the seedlings are grown to the 5-6 leaf stage.

[0039] (5) The garlic seedlings obtained in step (4) are transplanted into the field with soil, with a plant spacing of 6-8 cm and a row spacing of 13-15 cm. Before transplantation, the seedlings are acclimated outdoors for 3 days, and the transplantation is completed before the first week of November. In order to ensure the detoxification effect, the seedlings are planted in a 40-mesh nylon net greenhouse, and normal maintenance is performed. The next year, the virus-reduced garlic germplasm can be obtained. The operation of bulb scale detoxification is shown in Figure 1 .

[0040] In the early stage, the "space garlic" variety is selected, and samples are taken every 10 days from the early stage of bulb scale swelling to the end of bulb scale swelling. According to Livak and Schmittgen (2001) in "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 -ΔΔCTThe method proposed in the "Method" randomly detected the relative expression of three garlic virus genes at different bulbing stages. The results are shown in Figure 2

[0041] Figure 2 are the results of cell-specific expression of garlic virus genes. Figure a shows the expression of six virus genes in bulbs, and the activities of garlic virus GarVA gp6, QY65.gp6, X660.gp6, GvEgp8, GarVXgp6 and GarLVgp6 are estimated by garlic virus A, B, D, E and X and shallot latent virus, respectively. Figure b shows the expression of virus genes X660-GP6 (garlic virus D), GvEgp8 (garlic virus E), GarVXgp6 (garlic virus X) and QY65-gp6 (garlic virus B). Figure c is the relative expression level of garlic virus A (GarVAgp6), D (X660.gp6) and X (GarVXgp6) genes at six stages of bulb growth. The bulb diagram shows the state of the bulb at the beginning of bulb growth (S1) and at the end of sampling (S6). Figure d is the relative expression level of GarVAgp6 (GarA), X660.gp6 (GarD) and GarVXgp6 (GarX) virus genes in virus-accumulating (CO) and virus-free (VF) garlic. The virus-free garlic used is produced by in vitro culture of young bulb scales. The y-axis represents the relative expression level. Single-cell transcriptome sequencing and related detection show that garlic virus D accumulates gradually during bulb growth, while garlic virus A (GarVAgp6) and X (GarVXgp6) show a trend of first increasing and then decreasing Figure 2 a-c). Therefore, we selected small bulb scales at the early bulbing stage for detoxification garlic culture.

[0042] According to the above operation, garlic variety "Taikong garlic" was treated, and normal garlic scale plant leaves and small bulb scale growth plant leaves were randomly selected for virus fluorescence quantitative PCR to detect the content of garlic virus A (GarVAgp6), D (X660.gp6) and X (GarVXgp6), and six test replicates were set for each treatment. The results are shown in Table 1.

[0043] Table 1: Ct values and relative expression differences of fluorescence quantitative PCR

[0044]

[0045] ​The results in Table 1 show that the relative ct value of the garlic virus A gene treated by this method (the ct value of the virus gene minus the ct value of the internal reference) is 15.777, while the relative ct value of the normal garlic virus gene without treatment is 3.132. -ΔΔCT Method" t The method estimated that the content of garlic virus A (GarVAgp6) in garlic plants cultured with small bulbs was only 0.016% of that in normal garlic. A similar method found that the virus content of garlic virus X (GarVXgp6) and garlic virus D (X660.gp6) in garlic plants cultured with small bulbs was only 0.056% and 0.015% of that in normal garlic, respectively, indicating that garlic plants cultured with small bulbs are almost virus-free.

[0046] Comparative Example 1 Traditional stem tip detoxification method

[0047] The traditional stem tip detoxification method refers to the improved garlic stem tip breeding method disclosed in CN109220807A to cultivate "space garlic".

[0048] Comparative Example 2: Detoxification method for removing mother garlic

[0049] The method for detoxifying mother garlic refers to a method for rapidly reducing the accumulation of garlic viruses disclosed in CN117063800A to cultivate "space garlic".

[0050] Comparative Example 3: Non-detoxified garlic planting method.

[0051] Step 1: Select the "space garlic" harvested that year, and pick out plump and disease-free bulbs;

[0052] Step 2: Sterilize and disinfect the inoculation environment: sterilize and disinfect the clean bench, hands, and syringe;

[0053] Step 3: Detoxification of garlic bulbs: In a clean bench sterile environment, immerse the "space garlic" bulbs in 70% alcohol, shake for 30-60 seconds, then rinse with sterile water 1-2 times, then shake and sterilize with 10% sodium hypochlorite solution for 10 minutes, and finally wash with sterile water 5-7 times;

[0054] Step 4: Transfer the garlic bulbs disinfected in step 3 to MS culture medium in a clean bench, with three bulbs per bottle. The remaining conditions are exactly the same as steps (4) to (5) in Example 1.

[0055] The average values of the garlic viruses A (GarVAgp6), D (X660.gp6) and X (GarVXgp6) in the garlic leaves planted in different cases were detected according to the method proposed by Livak and Schmittgen (2001) in "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 -ΔΔCT The average values of the garlic viruses A (GarVAgp6), D (X660.gp6) and X (GarVXgp6) in the garlic leaves planted in different cases were detected according to the method proposed by Livak and Schmittgen (2001) in "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2

[0056] Table 2 Comparison of different detoxification methods

[0057]

[0058] As shown in Table 2, the traditional garlic cultivation methods of stem tip detoxification (Comparative Example 1) and mother garlic removal detoxification (Comparative Example 2), wherein the stem tip stripping needs to consume a large amount of manpower and time, and the detoxification effect is not significant, while the mother garlic removal detoxification cultivation method is close to the small scale bud cultivation detoxification time of Example 1 in the detoxification time, but the small scale bud cultivation detoxification effect of Example 1 is nearly 20 points higher than that of the mother garlic removal detoxification cultivation method, which shows that the method of the present application basically achieves complete detoxification.

Claims

1. A method for rapidly obtaining virus-free garlic seedlings by in vitro culture of young bulbils, characterized in that: The following steps are involved: (1) Preparation of early bulbils: Take garlic plants, peel off the small bulbils at the early stage of bulb expansion, and soak them in sterile water to obtain early small bulbils; (2) Bulb detoxification treatment: sterilize the early bulbs in a sodium hypochlorite solution, rinse with sterile water several times, transfer to MS medium and culture the bulbs to root and germinate until the 3-4 leaf stage, and obtain garlic seedlings; (3) Transplant the garlic seedlings into culture pots and harden them until they grow to 5-6 leaves; (4) The garlic seedlings obtained after the treatment in step (3) are transplanted with soil into the field and maintained normally.

2. The method according to claim 1, characterized in that In step (1), the small scale buds are peeled off by pulling out the garlic plant, peeling off the swollen parts layer by layer, and peeling off the small scale buds along the stem disk with a sterile scalpel.

3. The method according to claim 1, characterized in that In step (1), the soaking time is more than 20 minutes, and the garlic is "space garlic".

4. The method according to claim 1, wherein In step (2), the concentration of the sodium hypochlorite solution is 15-25%, the sterilization time is 10-15 minutes, and the sterile water is used to rinse more than 3 times.

5. The method according to claim 1, characterized in that In step (2), the MS culture medium is 38-40 g of MS and 6.5-7.5 g of agar dissolved in 1 L of sterile water. The conditions for rooting and germination culture are: temperature 23-25°C, light intensity 12-14 h light and 10-12 h dark culture, and humidity control at 40-60%.

6. The method according to claim 1, characterized in that In step (2), the MS culture medium was changed once a month during the culture period.

7. The method according to claim 1, characterized in that In step (3), the soil matrix in the culture pot is a seedling matrix, including imported peat, desalted coconut coir, perlite and vermiculite, with a pH of 5.5-7.0 and organic matter >20%. The soil matrix is ​​sterilized at a high temperature of 110-130°C for more than 15 minutes.

8. The method according to claim 1, characterized in that In step (3), before transplanting, the soil matrix is ​​irrigated with a sterilizing agent, which is a solution obtained by diluting thiophanate-methyl or chlorothalonil with water 500-800 times.

9. The method according to claim 1, characterized in that In step (3), the garlic seedlings at the 3-4 leaf stage are transplanted into a seedling medium, and the garlic seedlings are watered with a urea aqueous solution or Hoagland nutrient solution for the first time.

10. The method according to claim 1, characterized in that In step (4), before transplanting, harden the seedlings outdoors for more than 3 days.

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