Method for inducing garlic mutant by differentiating bulbil

Abstract

The invention discloses a method for inducing a garlic mutant by differentiated bulbil, which is characterized in that a phenotypic mutation plant can be obtained by stripping the differentiated bulbil and performing EMS mutagenesis on the differentiated bulbil. Compared with the prior art, the method has the advantages that the volume of the differentiated bulbil with unclosed outer leaves is reduced by 2-3 orders of magnitude, the use of a mutagenesis reagent EMS is reduced, the cost of the mutagenesis reagent is reduced, the operation is convenient, and the potential harm of the EMS reagent to human and nature is greatly reduced. According to the method, differentiated bulbils with unclosed outer leaves are adopted, internal stored leaves are not expanded, bud points are basically exposed, EMS can fully act on growing points, and the mutation rate is high. Differentiated bulbils are cultured on an indoor culture medium after EMS treatment, the survival condition is good, and survival of harmful mutations is facilitated.

Description

Technical Field

[0001] The invention relates to a method for in vitro chemical mutagenesis and culture of differentiated bulbils and efficient acquisition of garlic mutants, belonging to the field of garlic mutation breeding. Background Art

[0002] Garlic (Allium sativum L.) is an important economic crop, widely cultivated and used worldwide. Garlic flowers are sterile and reproduce asexually. my country boasts rich garlic germplasm resources and numerous high-quality varieties, but due to the inability to perform hybrid breeding, its genetic and biological diversity is underutilized. The accumulation of viruses associated with asexual garlic reproduction has led to the serious degeneration and even extinction of local high-quality varieties. Therefore, the development of garlic germplasm resources and the efficient cultivation of new varieties are imperative. Currently, garlic breeding and germplasm development primarily rely on conventional asexual breeding, which primarily includes systematic selection and induced mutation. Systematic garlic breeding, based on somatic cell mutation, is time-consuming and inefficient. Induced mutation breeding, using radiation, chemical reagents, and spaceflight mutagenesis, accelerates somatic cell mutations, achieving high mutation rates and rapidly obtaining germplasm and varieties with superior traits. It is currently the primary method of garlic breeding.

[0003] In the garlic mutagenesis breeding, chemical mutagenesis is the main method, and induction technology adopts materials such as garlic root tip, stem tip, flower bud, stem disk, mature bulb bud and callus more, obtains mutant through mutagenic agent treatment.Root tip, stem tip and callus are as mutagenic material, and mortality rate is high after mutagenic agent treatment, and sterile operation and callus culture are needed simultaneously, and not only there is the problem that contamination rate is high, and incubation time is long, and survival rate is low, causes mutagenic efficiency extremely poor.Flower bud, stem disk and mature bulb bud are mutagenic material, and because meristem is less, or can not fully expose, the mutagenic dose that takes half lethal dose as standard is difficult to obtain higher and stable mutation rate, and in mature bulb bud mutagenesis, this drawback is particularly outstanding.CN113170728A discloses " a kind of method of inducing garlic polyploid or mutant ", and this method is to expose growing point and preserve maternal nutrition as emphasis, can directly cultivate in field (without indoor culture), shorten cycle, improve mutagenic efficiency. The aforementioned patent has several shortcomings during implementation. For example, the large size of the mutagenic material leads to high chemical mutagenic reagent consumption, which can pose environmental risks. Furthermore, the cut surface of the garlic clove is susceptible to fungal infection and rot, and the mutagenized material has a certain mortality rate, which is not conducive to the establishment of a garlic mutant library. Therefore, it is necessary to develop a new method for inducing garlic mutants to improve the efficiency of obtaining mutants. Summary of the Invention

[0004] Purpose of the invention: In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for in vitro chemical mutagenesis and culture of differentiated bulbils to efficiently obtain garlic mutants.

[0005] Technical solution: The present invention comprises the following steps:

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

[0007] (2) EMS mutagenesis of differentiated buds: Soak differentiated buds in EMS (ethyl methanesulfonate) solution, shake at low speed, filter, soak in sodium thiosulfate solution, and wash with water several times;

[0008] (3) The washed differentiated bulbils are sterilized in a sodium hypochlorite solution, rinsed with sterile water several times, and transferred to MS culture medium to obtain garlic seedlings;

[0009] (4) identifying the appearance characteristics of garlic seedlings to obtain phenotypic mutant plants;

[0010] (5) Transplanting phenotypic mutant plants into culture pots for seedling hardening;

[0011] (6) The mutant plants are transplanted into the field with soil and maintained normally.

[0012] Furthermore, in step (1), the peeling of the differentiated bulbils comprises the following steps: pulling out the garlic plant, cutting the pseudostem 2 cm above the stem disc, peeling off the enlarged part of the stem disc layer by layer, and peeling off the differentiated bulbils along the stem disc. The differentiated bulbils swell to more than 2 mm and most of the outer leaves are not closed. The differentiated bulbils with unclosed outer leaves are directly peeled off; if the outer leaves are closed, the top of the outer leaves can be cut off to ensure the opening, and then the differentiated bulbils are peeled off. The initial enlargement period of garlic is from mid-February to mid-March each year.

[0013] Furthermore, in step (2), the concentration of the EMS solution is 0.5-1.0%, the low-speed oscillation speed is 50-80 r / min, the low-speed oscillation time is 4-6 hours, the concentration of the sodium thiosulfate solution is 0.3-0.5 mol / L, the soaking time is more than 5 minutes, and the mixture is washed with water for more than 3 times, each time for 3-5 minutes.

[0014] Furthermore, in step (3), the concentration of the sodium hypochlorite solution is 15-20% (the effective chlorine concentration of the undiluted sodium hypochlorite solution is 6-10%), the sterilization time is 10-15 minutes, and the bulbils are rinsed with sterile water more than 3 times; the bulbils are cultured and differentiated in MS culture medium until they take root and germinate to the 2-3 leaf stage, the MS culture medium comprising: 38-40g MS basal culture medium (wherein the sucrose content is 2.8-3.2%) and 6.5-7.5g agar per liter of water, and the culture conditions are: temperature 23-25°C, 12-14 hours of light and 10-12 hours of dark culture, and humidity 40-60%; the MS culture medium is replaced once a month during the culture period.

[0015] Furthermore, in step (4), the appearance traits of the garlic seedling leaves are identified, including color, length, and / or width. Phenotypic mutant plants are identified by comparing them with normally differentiated garlic seedlings. If the leaf color of the leaves is significantly changed, and the EMS-induced leaves are significantly yellowed, and the difference in leaf length and width exceeds 20%, then they are identified as superior mutant bulbils.

[0016] Furthermore, in step (5), the soil matrix in the culture pot is a seedling culture matrix, comprising, by volume, imported peat: desalted coconut coir: perlite: vermiculite in a ratio of (2.5-3.5): (1-1.2): (1-1.2): (0.8-1.0), with a pH of 5.5-7.0 and organic matter >20%, and the soil matrix is ​​sterilized at 100-130° C. for more than 15 minutes; the phenotypic mutant plants are transplanted to the culture pots at the 4-leaf stage for hardening, and the hardening conditions include: a temperature of 23-25° C., 12-14 hours of light and 10-12 hours of darkness, and a humidity of 40-60%. For harmful mutations, the plants can be transplanted to the culture pots for hardening at the 5-6 leaf stage, depending on the vigor of the seedlings. Before transplanting into a culture pot, the soil matrix is ​​irrigated with a fungicide solution, wherein the fungicide is thiophanate-methyl or chlorothalonil, and the fungicide is diluted 600-800 times with water to obtain the fungicide solution; the phenotypic mutant plant is transplanted into the soil matrix and watered with a nutrient solution, wherein the nutrient solution is a urea aqueous solution (0.1% concentration) or a 1 / 3 concentration Hoagland nutrient solution. The nutrient solution is then watered once every 3-4 days, and water is applied to ensure that the matrix is ​​moist. The concentration of the nutrient solution can be gradually increased to 1 times the concentration of Hoagland nutrient solution or a concentration of 0.3% urea each time, depending on the seedling condition.

[0017] Furthermore, in step (6), the seedlings are transplanted to the field at the 6-leaf stage. Before transplanting, they need to be hardened outdoors for more than 3 days. Generally, outdoor transplanting is completed before early November in the south and before late October in the north. The later planting method is determined based on the hardening situation. If the hardened seedlings grow well, they can be transplanted to the field and maintained normally. If the growth is still weak, they can be transplanted into large flower pots and cultivated indoors.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The volume of the differentiated scale buds with unclosed outer leaves in the method of the present invention is reduced by 2-3 orders of magnitude compared with the prior art, and the amount of mutagenic agent EMS used will be greatly reduced, thereby reducing the cost of the mutagenic agent. (2) The present invention does not require the configuration of a large amount of EMS mutagenic agent, is easy to operate, and has little potential harm to humans and nature. (3) The differentiated scale buds with unclosed outer leaves used in the present invention have no swollen internal storage leaves, and the bud points are basically exposed. EMS can fully act on the growth points, resulting in a high mutation rate. (4) After the differentiated scale buds of the present invention are treated with EMS, they are cultured on an indoor culture medium with good living conditions, which is conducive to the survival of harmful mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of differentiated scale buds with unclosed outer leaves in Example 1;

[0020] Figure 2 The mutagenesis method flow chart in Example 1;

[0021] Figure 3 This is a diagram showing the variation in leaf width and length of the mutants produced in Example 1. The rightmost image is the control, and the rest are leaves of the mutants. The mutants have a wide range of leaf width and length variations.

[0022] Figure 4 This is a diagram showing the leaf color variation of the mutant produced in Example 1, wherein the rightmost leaf is the control, the middle leaf has mutated to a yellowish color, and the left leaf has mutated to a waxy and yellowish color. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below with reference to the accompanying drawings.

[0024] Example 1

[0025] (1) Preparation of early bulbils: The garlic variety "Ershui Zao" was planted on October 10, 2023. In early March 2024, 5-month-old garlic plants were taken. At the early stage of garlic bulbil expansion, the garlic plants were pulled out. The pseudostem was cut off 2 cm above the stem disc. The 2 cm expanded part of the stem disc was peeled off layer by layer with a scalpel along the stem disc until the differentiated bulbils were exposed. The differentiated bulbils with 2-3 mm outer leaves that were not closed (such as Figure 1 As shown, differentiated buds with unclosed outer leaves are approximately 3-5 mm long and 2-3 mm in diameter. Use a scalpel to slowly peel them off and place them in a Petri dish filled with room-temperature distilled water and soak for 20-30 minutes. For a small number of differentiated buds with closed outer leaves, use a scalpel to remove the top of the outer leaf to ensure an open area.

[0026] (2) EMS mutagenesis of differentiated buds: Under a fume hood, transfer the differentiated buds to a 50 ml centrifuge tube and fill it to the 30 ml mark. Then, add 0.5% EMS to the 50 ml mark, soak thoroughly, seal the tube, and place it on a shaker at 50-80 rpm for 6 hours. After soaking, filter the tube with a small mesh bag and place it in a 0.5 mol / L sodium thiosulfate solution for 5 minutes. Then, wash it with distilled water three times with shaking for 3-5 minutes each time.

[0027] (3) Sterilization and disinfection of the operating environment: Sterilize and disinfect the clean bench, hands and tweezers.

[0028] (4) The washed differentiated buds were sterilized in a 20% sodium hypochlorite solution for 10 minutes and then rinsed three times with sterile water.

[0029] (5) In a clean bench, the differentiated buds obtained in step (4) were transferred to MS culture medium (dissolve 39 g of MS powder (containing 3% sucrose) and 7 g of agar per liter of sterile water), dissolved in a high-temperature autoclave at 110-120°C, and placed in a gel state at room temperature. 5-6 differentiated buds were cultured in each culture dish.

[0030] (6) The differentiated bulbils treated in step (5) are cultured in a tissue culture room. The growth conditions in the tissue culture room are set to a temperature of 24° C., a humidity of 55%, and a photoperiod of 12 hours of light / 12 hours of darkness to allow the differentiated bulbils to root and germinate. The MS medium is replaced once a month during the culture period. After two months of culture, the differentiated bulbils take root and grow leaves. When the leaves are 2-3, garlic seedlings are obtained.

[0031] (7) Using the control seedlings as a reference, a preliminary identification is performed based on leaf color, length, width and other traits. Phenotypic mutant plants are identified and cultured separately in rotary bottles. Among them, the control seedlings are normally differentiated, that is, the peeled small scale buds are not induced by EMS, and the rest of the implementation method is consistent with the above. By comparing the phenotype of the leaf appearance traits (leaf color, length and width) with the control seedlings, the EMS-induced leaves have obvious yellowing color, and the difference in leaf length and width exceeds 20%, which is identified as an excellent mutant scale bud.

[0032] (8) When the superior mutant bulbils and the normal differentiated bulbils were about 4 leaves, the seedlings were transplanted into 5cm×5cm×7cm pots, with one seedling planted in each pot. The seedlings were cultured in a culture room at a temperature of 23-25℃, with a light intensity of 12 hours light and 12 hours dark, and humidity controlled at 40-60% for hardening. The pots were filled with soil matrix (i.e., seedling matrix: imported peat, desalted coconut coir, perlite and vermiculite (volume ratio of 3:1:1:1), pH of 5.5-7.0, organic matter >20%), and sterilized at 121℃ for more than 15 minutes. At the time of transplanting, the garlic seedlings were irrigated with 100mL of urea aqueous solution (0.1% concentration). Urea aqueous solution (0.2% concentration) was irrigated once every 5 days, and water was applied to ensure the matrix was moist.

[0033] (9) Seedlings with stunted growth and weak growth are defined as deleterious mutations. Deleterious mutations can be transplanted into culture pots at the 5-6 leaf stage according to step (8) to harden the seedlings. The whole process is as follows: Figure 2 shown.

[0034] (10) Before transplanting, 500 mL of fungicide is used to irrigate the soil matrix of each culture pot. The fungicide is a solution obtained by diluting 500-800 times of thiophanate-methyl or chlorothalonil with water. The garlic seedlings with excellent mutant bulbils and normal differentiated bulbils at the 6-leaf stage are transplanted to the field with soil, with a plant spacing of 10-12 cm and a row spacing of 25-30 cm. Normal maintenance is sufficient. The later planting method of harmful mutations is determined according to the hardening of the seedlings. If the hardening of the seedlings improves, they can be transplanted to the field. If the growth is still weak, they can be transplanted to a flower pot with an upper diameter of 15 cm and a depth of 25 cm for indoor cultivation. The soil matrix composition is the same as step (8).

[0035] After 3 months of field growth, the results of garlic plant mutagenesis are as follows Figure 3 and Figure 4 As shown, Figure 3 The figure shows the variation of leaf width and length of mutants. The rightmost one is the control seedling, and the rest are leaves of mutants. Figure 3 It can be seen that there is abundant variation in leaf width and length among the mutants;

[0036] Figure 4 The figure shows the leaf color variation of the mutants, where the rightmost one is the control seedling and the rest are leaves of the mutants. Figure 4 As can be seen, the middle leaf has a yellowish color, and the left leaf has a yellowish color with a lack of wax on the leaf surface. The mutation effects are shown in Table 1.

[0037] Comparative Example 1 Growth Point EMS Mutagenesis Method

[0038] The EMS mutagenesis method for growing points was based on the method described in CN113170728A, "A method for inducing polyploids or mutants in garlic." Mature bulbils approximately 30-50 mm in length and 10-20 mm in diameter were subjected to EMS mutagenesis using the method of this example. The results are shown in Table 1.

[0039] Comparative Example 2: Garlic stem disc cut into a "cross" shape mutagenesis method

[0040] The garlic stem discs were cut into a cross-shaped mutagenesis method. Reference was made to the "Method for in vitro mutagenesis of garlic leaf blight-resistant mutants using ethyl methanesulfonate" disclosed in CN113575416A. Approximately 5 mm stem discs were cut into four cross-shaped pieces and subjected to EMS mutagenesis using the method of this example. The mutagenesis results are shown in Table 1.

[0041] Table 1 Mutagenesis effect comparison table

[0042]

[0043] As can be seen from Table 1, the mutation rates of the growing point EMS mutagenesis method (Comparative Example 1) and the garlic stem disc cut into a "cross" shape mutagenesis method (Comparative Example 2) are both lower than those of Example 1.

[0044] In summary, Example 1 reduces the volume of mutagenic material by 2-3 orders of magnitude compared to Comparative Example 1, significantly reducing the amount of EMS mutagenic agent used and lowering mutagenic agent costs. Furthermore, due to the uncertainty of the direction of mutation and the low number of beneficial mutations in chemical mutagenesis, a large amount of material must be processed to obtain mutants with superior traits. Based on a 0.1% beneficial mutation rate for chemical mutagenesis and a median lethality, conventional mutagenesis breeding in Comparative Example 1 would require processing tens of thousands of materials. This would require preparing approximately 50 L of EMS mutagenic agent, making it inconvenient to operate in a fume hood. The toxicity and volatility of EMS, as well as the wastewater treatment and operation processes, pose potential hazards to humans and nature. Example 1 processes tens of thousands of mutagenic materials using only approximately 1 L of EMS mutagenic agent, which is convenient for shaking and soaking, allowing for operation in a fume hood, significantly reducing the harm of EMS agents to humans and nature. In Example 1, the differentiated scale buds with unclosed outer leaves and unenlarged internal storage leaves have essentially exposed bud points, allowing EMS to fully act on the growing points and achieve a high mutation rate. Example 1: After the differentiated bulbils were treated with EMS, they were cultured on a culture medium indoors, where the living conditions were good and conducive to the survival of harmful mutations.

Claims

1. A method for differentiating bulbils and inducing garlic mutants, characterized in that: The following steps are involved: (1) Early bulbil preparation: Take garlic plants, peel off differentiated bulbils at the early stage of bulbil expansion, and soak them in distilled water to obtain differentiated bulbils; (2) EMS mutagenesis of differentiated buds: Soak the differentiated buds in EMS solution, shake at low speed, filter, soak in sodium thiosulfate solution, and wash with water several times; (3) The washed differentiated bulbils are sterilized in a sodium hypochlorite solution, rinsed with sterile water several times, and transferred to MS culture medium to obtain garlic seedlings; (4) identifying the appearance characteristics of garlic seedlings to obtain phenotypic mutant plants; (5) Transplanting phenotypic mutant plants into culture pots for seedling hardening; (6) The mutant plants are transplanted into the field with soil and maintained normally.

2. The method according to claim 1, characterized in that In step (1), the differentiated bulbils peeling comprises the following steps: pulling out the garlic plant, cutting off the pseudostem on the stem disk, peeling off the enlarged part of the stem disk layer by layer, and peeling off the differentiated bulbils along the stem disk.

3. The method according to claim 1, characterized in that In step (1), the differentiated scale buds swell to more than 2 mm and most of the outer leaves are not closed. The differentiated scale buds with unclosed outer leaves are directly peeled off. If the outer leaves are closed, the top of the outer leaves are cut off to ensure the opening, and then the differentiated scale buds are peeled off.

4. The method according to claim 1, wherein In step (2), the concentration of the EMS solution is 0.5-1.0%, the low-speed oscillation speed is 50-80 r / min, the low-speed oscillation time is 4-6 hours, the concentration of the sodium thiosulfate solution is 0.3-0.5 mol / L, the soaking time is more than 5 minutes, and the mixture is washed with water for more than 3 times, each time for 3-5 minutes.

5. The method according to claim 1, characterized in that In step (3), the concentration of the sodium hypochlorite solution is 15-20%, the sterilization time is 10-15 minutes, and the bulbils are rinsed with sterile water more than three times; the bulbils are cultured and differentiated in MS culture medium to root and germinate to the 2-3 leaf stage, the MS culture medium comprising: 38-40g MS basal culture medium and 6.5-7.5g agar per liter of water, the culture conditions are: temperature 23-25°C, 12-14 hours of light and 10-12 hours of dark culture, and humidity 40-60%; the MS culture medium is replaced once a month during the culture period.

6. The method according to claim 1, wherein In step (4), the identification of phenotypic mutant plants is carried out by identifying the appearance traits of the garlic seedling leaves, which include: color, length and / or width.

7. The method according to claim 1, characterized in that In step (5), the soil matrix in the culture pot is a seedling culture matrix, which includes, by volume, imported peat: desalted coconut husk: perlite: vermiculite (2.5-3.5): (1-1.2): (1-1.2): (0.8-1.0), a pH of 5.5-7.0, organic matter>20%, and the soil matrix is ​​sterilized at a high temperature of 100-130° C. for more than 15 minutes; the conditions for hardening the seedlings include: a temperature of 23-25° C., 12-14 hours of light and 10-12 hours of darkness, and a humidity of 40-60%.

8. The method according to claim 1, characterized in that In step (5), before transplanting into the culture pot, the soil matrix is ​​irrigated with a fungicide solution, the fungicide is thiophanate-methyl or chlorothalonil, and the fungicide is diluted 600-800 times with water to obtain a fungicide solution; the phenotypic mutant plants are transplanted into the soil matrix and irrigated with a nutrient solution, the nutrient solution is a urea aqueous solution or Hoagland nutrient solution.

9. The method according to claim 1, characterized in that In step (5), nutrient solution is poured once every 3-4 days, and water is applied to ensure that the substrate is moist.

10. The method according to claim 1, characterized in that In step (6), transplanting is performed at the 6-leaf stage. Before transplanting, the seedlings need to be hardened outdoors for more than 3 days.

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