Method for rapidly obtaining virus-free garlic seedlings through in-vitro culture of tender bulbils

Abstract

The invention discloses a method for rapidly obtaining virus-free garlic seedlings by in-vitro culture of young and tender bulbil, which comprises the following steps: firstly, taking a garlic plant, stripping small bulbil to soak in sterile water at the initial expansion stage of the garlic bulbil, then sterilizing in a sodium hypochlorite solution, washing with sterile water for multiple times, culturing the small bulbil by using an MS culture medium to root and germinate, and after the 3-4 leaf stage, culturing the small bulbil to obtain the virus-free garlic seedlings. Transplanting the garlic seedlings into a culture bowl for seedling hardening, transplanting the obtained garlic seedlings with soil into a field after the garlic seedlings grow to a 5-6-leaf period, and performing normal maintenance. The method is easy to operate, and the requirement for the operation skill of technicians is reduced; the culture period is short, and regenerated plants can be rapidly obtained, so that the detoxification efficiency is improved, and the demand of the market on virus-free garlic is met; a large number of virus-free plants can be generated in a short time, the production efficiency is improved, and the method is suitable for large-scale production; rapid growth and regeneration of plants can be supported, and the method is expected to become an innovative breakthrough in the field of garlic detoxification 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, and belongs to the technical field of garlic virus-free. Background Art

[0002] Garlic (Allium sativum L.) is an important economic crop and is widely cultivated and used worldwide. Garlic is sterile and long-term vegetative reproduction leads to serious accumulation of viruses in garlic, which can lead to the degeneration of garlic varieties and the emergence of various diseases, such as short plants, low garlic yield, and poor commercial quality, causing huge economic losses to the garlic industry. Therefore, the development of virus-free culture technology is of great significance to solving the problem of garlic virus diseases.

[0003] Traditional garlic virus-free culture technology is mainly based on stem tip virus-free. This method cultivates and regenerates stem tip tissue to obtain virus-free plants. However, there are a series of problems with stem tip virus-free technology, including complex operation, long cycle, low efficiency, etc., 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 idea for garlic virus-free culture. In vitro culture technology can effectively remove viruses in plants and obtain virus-free plants by culturing tissues from different parts of plants (such as stem tips, bulbs, leaves, etc.). Although garlic has great potential for virus-free, in actual operation, how to further improve the regeneration rate and rooting ability is still 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 for rapidly obtaining garlic virus-free seedlings by in vitro culture of young bulbils of the present invention comprises the following steps:

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

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

[0008] (3) transplanting the garlic seedlings into culture pots and hardening them until they grow to the 5-6 leaf stage;

[0009] (4) The garlic seedlings obtained after the treatment in step (3) are transplanted to the field with soil and maintained normally. Furthermore, in step (1), the small scale buds are peeled off by pulling out the garlic plants, peeling off the swollen parts layer by layer, and peeling off the small scale buds along the stem disk with a sterile scalpel.

[0010] Furthermore, in step (1), the soaking time is more than 20 minutes, and the garlic is "space garlic".

[0011] Furthermore, the initial stage of garlic enlargement is generally in early March each year, when the small bulbs enlarge to 2-3mm.

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

[0013] Furthermore, in step (2), the sterilization time is 10-15 minutes, and the sterile water is used for more than 3 times.

[0014] Furthermore, in step (2), MS medium is a commonly used medium for plant tissue culture. 38-40 g MS powder (containing 2.8-3.2% sucrose) and 6.5-7.5 g agar are dissolved in every 1 L of sterile water, and dissolved in an autoclave at 110-120° C. and left at room temperature to form a gel state.

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

[0016] Furthermore, in step (2), the MS culture medium is replaced once a month during the culture period.

[0017] Furthermore, in step (3), the soil matrix in the culture pot is a seedling matrix, including imported peat, desalted coconut bran, perlite and vermiculite, the volume ratio of imported peat, 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 matrix is ​​sterilized at a high temperature of 100-130°C for more than 15 minutes.

[0018] Furthermore, in step (3), the seedling hardening is that after the garlic seedlings are transplanted into the culture pots, they are cultured in a culture room with a temperature of 23-25° C., a light intensity of 10-12 h light, 10-12 h dark, and a humidity of 40-60%.

[0019] Furthermore, in step (3), before transplanting, the soil matrix is ​​irrigated with a sterilizing agent, wherein the sterilizing agent is a solution obtained by diluting thiophanate-methyl or thiophanate-methyl with water 500-800 times.

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

[0021] Furthermore, use 1 / 2 concentration Hoagland nutrient solution for watering once a week, and each time you can increase the concentration of the nutrient solution to 1 concentration Hoagland nutrient solution depending on the condition of the seedlings.

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

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

[0024] Furthermore, in step (4), in order to ensure the detoxification effect, it is better to use a 40-mesh nylon mesh arch shed for planting.

[0025] The garlic variety "Space Garlic" was processed according to the above operation, and leaves of normal garlic clove plants and leaves of plants grown with small scale buds were randomly selected for virus fluorescence quantitative PCR to detect the contents of garlic virus A, garlic virus D and garlic virus X. The results showed that the relative ct value of garlic virus A gene (the ct value of the virus gene minus the ct value of the internal reference) of the garlic processed by the method of the present invention was 15.777, while the relative ct value of the virus gene of normal garlic without treatment was 3.132, and the content of garlic virus A in the plants cultured with small scale buds was only 0.016% of that in normal garlic. By a similar method, it was found that the virus contents of garlic virus X and garlic virus D in the garlic plants without mother garlic were only 0.056% and 0.015% of that in normal garlic, respectively, which indicated that the garlic plants cultured with small scale buds contained almost no virus.

[0026] The present invention proposes a method for in vitro culture using garlic bulbs to obtain virus-free garlic plants. Compared with the traditional stem tip detoxification technology, the operation steps are simpler, reducing the requirements for the technical staff's operating skills; the culture cycle is short, and 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, improving production efficiency, and being suitable for large-scale production; the bulb, as a storage organ of garlic, is rich in nutrients, suitable for in vitro culture, and can support the rapid growth and regeneration of the plant. Therefore, 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 invention has the following significant advantages:

[0028] The young and tender bulb buds of the present invention are cultured in vitro to quickly obtain garlic virus-free seedlings, and the operation is simple. It is only necessary to observe the expansion of garlic bulb buds at a specific time each year, and the garlic plant is extracted when the small bulb buds expand to 2-3mm. The small bulb buds are gently peeled off in a culture dish equipped with sterile water with a scalpel, and the effect is more than 30 times that of traditional stem tip peeling. At the same time, peeling does not require aseptic environment operation, and the peeling small bulb buds are directly placed in MS culture medium for cultivation, and can generally be transplanted to a nutrient pot within 2 months. After the seedlings of indoor culture are treated, they are transplanted with soil, and robust virus-free bulbs can be obtained within 10 months, which is 3-4 years less than the traditional stem tip detoxification, greatly shortening the time. The detoxification technology of the present invention has a short detoxification cycle, is easy to operate, and bulb bud peeling does not require aseptic environment operation, is low in cost, and has obvious detoxification effects. It can obviously overcome the problem of asexual reproduction virus accumulation of garlic, and is of great significance to the species of keeping garlic high and stable yield, and effectively avoids the degradation caused by virus reasons. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the operation of garlic bulb detoxification.

[0030] Figure 2 This is a diagram showing the cell-specific expression results of garlic virus genes. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0032] Example 1

[0033] (1) Early bulbil preparation: This experiment was completed in the tissue culture room, culture room and experimental field of Yangzhou University. The "space garlic" was planted on October 8, 2023. In early March 2024, the "space garlic" plants that had been planted for 5 months were taken. At the beginning of the expansion of garlic bulbils (garlic cloves), the garlic plants were pulled out and the leaves were peeled off layer by layer until the small bulbils were exposed. The small bulbils of 2-3 mm were selected and slowly peeled off with a scalpel into a culture dish filled with sterile water and soaked in sterile water for 20 minutes (see Figure 1 ).

[0034] (2) Sterilization and disinfection of the operating environment: Sterilize and disinfect the clean workbench, hands and tweezers.

[0035] (3) Disinfection of scale buds: sterilize the early scale buds obtained in step (1) in a 20% sodium hypochlorite solution for 10-15 minutes, and then rinse with sterile water for 3 times.

[0036] (4) In a clean bench, transfer the small scale buds obtained in step (3) to MS culture medium (dissolve 40 g MS powder (containing 3% sucrose) and 7 g agar per liter of sterile water, dissolve at high temperature of 110-120°C in an autoclave, and leave at room temperature to form a gel state), and culture 5-6 small scale buds in each culture dish.

[0037] (3) The small scale buds treated in step (4) are cultured in a tissue culture room, and the growth conditions in the tissue culture room are set to a temperature of 25° C., a humidity of 50%, and a photoperiod of 12 h light / 12 h dark to culture the small scale buds for rooting and germination. During the culture period, the MS medium is replaced once a month. After two months of culture, the small scale buds take root and grow leaves, and when the 3-4 leaf stage is reached, garlic seedlings are obtained.

[0038] (4) transplanting the garlic seedlings obtained after the treatment in step (3) into a culture pot of 5 cm×5 cm×7 cm, planting one seedling in each culture pot, and culturing in a culture room at a temperature of 23-25° C., a light intensity of 12 h light and 12 h dark, and hardening the seedlings under the condition of humidity controlled at 40-60%, wherein the culture pot is filled with a soil matrix (i.e., a seedling matrix: imported peat, desalted coconut bran, perlite and vermiculite (volume ratio of 3:1:1:1), pH of 5.5-7.0, organic matter>20%), the seedling matrix is ​​sterilized at 121° C. for more than 15 min, and before transplanting, each culture pot is irrigated with 500 ml of a sterilizing agent, the sterilizing agent is a solution obtained by diluting methyl thiophanate or thiophanate-methyl with water 500-800 times, and the garlic seedlings at the 3-4 leaf stage are transplanted into the seedling matrix, and each culture pot is irrigated with 200 ml of Hoagland nutrient solution at the time of transplanting. The concentration of Hoagland nutrient solution is 1 / 2 times. Generally, water it once a week. Depending on the condition of the seedlings, increase the concentration of 1 / 2 times Hoagland nutrient solution to 1 times after one week and water it again until it grows to the 5-6 leaf stage.

[0039] (5) The garlic seedlings obtained after the treatment in step (4) are transplanted with soil into the field, with a plant spacing of 6-8 cm and a row spacing of 13-15 cm. Before transplanting, the seedlings are hardened outdoors for 3 days and the transplanting is completed before early November. In order to ensure the virus-free effect, the garlic seedlings are planted in a 40-mesh nylon net arch shed and maintained normally. In the next year, garlic germplasm with a large reduction in viruses can be obtained. Figure 1 shown.

[0040] In the early stage, the "space garlic" variety was selected and samples were taken every 10 days from the early stage of bulbil expansion to the end of bulbil expansion. According to Livak and Schmittgen (2001) in "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 -ΔΔCTThe relative expression levels of three garlic virus genes at different expansion stages were randomly detected by the method proposed in the “Method”. Figure 2 shown.

[0041] Figure 2 It is the result diagram of cell-specific expression of garlic virus genes. Among them, Figure a shows the expression of six virus genes in bulbs, and the activities of garlic viruses GarVAgp6, QY65.gp6, X660.gp6, GvEgp8, GarVXgp6 and GarLVgp6 are estimated by garlic viruses A, B, D, E and X and Shallot latent virus. Figure b shows the expression of viral genes X660-GP6 (garlic virus D), GvEgp8 (garlic virus E), GarVXgp6 (garlic virus X) and QY65-gp6 (garlic virus B). Figure c shows the relative expression levels of garlic virus A (GarVAgp6), D (X660.gp6) and X (GarVXgp6) genes in six stages of bulb bud expansion growth. The bulb diagram shows the status at the early stage of bulb bud growth (S1) and at the end of sampling (S6). Figure d shows the relative expression levels of GarVAgp6 (GarA), X660.gp6 (GarD) and GarVXgp6 (GarX) viral genes in virus-accumulating (CO) and virus-free (VF) garlic. The virus-free garlic used was produced by in vitro culture of young petals. The y-axis represents the relative expression level. Single-cell transcriptome sequencing and related tests showed that garlic virus D gradually accumulated during the bulb bud expansion period, while garlic virus A (GarVAgp6) and X (GarVXgp6) showed a trend of first increasing and then decreasing ( Figure 2 ac). Therefore, we selected small bulbs in the early stage of expansion for virus-free garlic culture.

[0042] The garlic variety "Space Garlic" was treated according to the above operation, and leaves of normal garlic clove plants and leaves of plants with small bulbs were randomly selected for virus fluorescence quantitative PCR to detect the content of garlic virus A (GarVAgp6), D (X660.gp6) and X (GarVXgp6). Six experimental 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 was used to find 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 contain almost no viruses.

[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: Method for detoxifying mother garlic

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

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

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

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

[0053] Step 3: Detoxification of garlic bulbs: In the sterile environment of a clean bench, first 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 bulbils disinfected in step 3 to MS culture medium in a clean bench, with three bulbils per bottle. The remaining conditions are exactly the same as steps (4) to (5) in Example 1.

[0055] According to Livak and Schmittgen (2001) in "Analysis of Relative Gene ExpressionData Using Real-Time Quantitative PCR and the 2 -ΔΔCT The method proposed in “Method” was used to detect the average values ​​of garlic virus A (GarVAgp6), D (X660.gp6) and X (GarVXgp6) in garlic leaves grown in different cases, and the detoxification efficiency was calculated. The results are shown in Table 2.

[0056] Table 2 Comparison of different detoxification methods

[0057]

[0058] As can be seen from Table 2, the traditional detoxification garlic culture method stem tip detoxification (Comparative Example 1) and the mother garlic detoxification (Comparative Example 2), wherein the stem tip peeling requires a lot of manpower and time, and the detoxification effect is not significant, and the mother garlic detoxification culture method is close to the detoxification time of the small scale bud culture of Example 1, but the detoxification effect of the small scale bud culture of Example 1 is nearly 20 points higher than that of the mother garlic detoxification culture method, indicating that the method of the present invention basically achieves complete detoxification.

Claims

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

2. The culture 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 culture 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 culture method according to claim 1, characterized in that 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 for more than 3 times.

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

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

7. The culture 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 husk, perlite and vermiculite, with a pH of 5.5-7.0, organic matter>20%, and the soil matrix is ​​sterilized at 110-130°C for more than 15 minutes.

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

9. The culture 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 culture method according to claim 1, characterized in that: In step (4), before transplanting, the seedlings are hardened outdoors for more than 3 days.

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