阅读说明：本技术 甲基磺酸乙酯离体诱变大蒜抗叶枯病突变体的方法 (Method for in vitro mutagenesis of garlic leaf blight-resistant mutant by ethyl methanesulfonate ) 是由 陈书霞 吴美骞 洪缘缘 孙银辉 于 2021-08-26 设计创作，主要内容包括：本发明提供一种甲基磺酸乙酯离体诱变大蒜抗叶枯病突变体的方法,目的是为无性繁殖的大蒜品种改良和新种质资源创制提供离体诱变定向筛选突变方法,并获得符合育种目标性状的抗病突变体。本发明以大蒜茎盘为外植体,通过对切成“十”字形的大蒜茎盘进行浓度为1%的EMS处理时长为6 h,再通过1 M硫代硫酸钠溶液终止诱变反应后,擦干,接种在附加有30%大蒜叶枯病粗毒素的MS培养基上进行离体培养,可获得抗叶枯病突变体。(The invention provides a method for in vitro mutagenesis of garlic leaf blight-resistant mutants by ethyl methanesulfonate, and aims to provide an in vitro mutagenesis directional screening mutation method for improvement of asexual propagation garlic varieties and creation of new germplasm resources and obtain disease-resistant mutants meeting breeding target characters. The invention takes a garlic stem disc as an explant, EMS treatment with the concentration of 1% is carried out on the garlic stem disc cut into a cross shape for 6 hours, then the garlic stem disc is wiped off after the mutagenesis reaction is stopped by 1M sodium thiosulfate solution, and the garlic stem disc is inoculated on an MS culture medium added with 30% garlic leaf blight crude toxin for isolated culture, so as to obtain the leaf blight resistant mutant.)

1. The method for in vitro mutagenesis of garlic leaf blight-resistant mutant by ethyl methanesulfonate is characterized by comprising the following steps: the method comprises the following steps:

1) garlic stem discs are used as a material, Ethyl Methane Sulfonate (EMS) with the concentration of 1% is used as a mutagen, the garlic stem discs are respectively treated for 2 hours, 4 hours, 6 hours, 8 hours and 12 hours, and the optimal mutagenesis time is determined through the germination rate;

2) respectively inoculating non-mutagenized and mutagenized garlic stem discs on screening culture media with the crude toxin volume numbers of 0, 5%, 10%, 15%, 20%, 25% and 30% respectively for culture, and determining the optimal crude toxin screening pressure through the germination rate;

3) EMS with the concentration of 1% is used for treating the garlic stem disk for 6 hours, the treated explant is inoculated on a culture medium with the volume fraction of crude toxin of pathogenic bacteria of leaf blight being 30%, and a regenerated plant with disease resistance is obtained by screening.

2. The method for in vitro mutagenesis of garlic leaf blight resistant mutant by ethyl methanesulfonate according to claim 1, characterized in that: the specific screening method for the optimal mutagenesis time in the step 1) comprises the following steps:

washing Bulbus Allii stem disk with tap water for 3 hr, soaking in ethanol under aseptic condition for 1min, sterilizing with 0.5% sodium hypochlorite for 15min, washing with sterile water for 3 times, cutting into stem disk with thickness of 5mm, and cutting into 4 pieces in a cross shape;

soaking the treated stem plate in 1% Ethyl Methane Sulfonate (EMS) for 2, 4, 6, 8, and 12 hr, soaking in 1M sodium thiosulfate solution for 5min, washing with distilled water, wiping, and inoculating on MS culture medium;

and (4) performing illumination culture for 28d, counting the germination number, and determining the treatment time for half of inoculated stem discs incapable of germinating as the appropriate mutagenesis time.

3. The method for in vitro mutagenesis of garlic leaf blight resistant mutant by ethyl methanesulfonate according to claim 1, characterized in that: the specific determination method of the optimal crude toxin screening pressure in the step 2) comprises the following steps:

cutting the processed stem disc into 4 blocks according to the shape of a cross, and soaking the 4 blocks in EMS phosphate buffer solution with the concentration of 2.0 percent for 8 hours;

inoculating on screening culture medium with crude toxin body integral number of 0, 5%, 10%, 15%, 20%, 25%, 30%, inoculating 40 explants per treatment, setting 3 times of repetition, and setting as control by phosphate buffer treatment without EMS;

after 30 days of illumination culture, the crude toxin screening pressure is determined by the integral number of 30 percent of crude toxin with the stem disc germination rate of 2.2 percent.

4. The method for in vitro mutagenesis of garlic leaf blight resistant mutant by ethyl methanesulfonate according to claim 1, characterized in that: the specific method of the step 3) is as follows: cutting the treated stem disc into 4 blocks according to the shape of a cross, soaking the 4 blocks in phosphate buffer of 1.0% EMS for 6h, soaking the stem disc subjected to mutagenesis treatment in 1M sodium thiosulfate solution for 5min, soaking the stem disc in distilled water for 30min, wiping the stem disc with sterilized filter paper, inoculating the stem disc on a screening culture medium with the number of crude toxin elements of 30% respectively, and screening disease-resistant plants.

Technical Field

The invention relates to the technical field of garlic disease-resistant breeding, in particular to a method for in vitro mutagenesis of garlic leaf blight-resistant mutants by using ethyl methanesulfonate.

Background

Garlic (Allium sativum L.) is a vegetable crop of Allium genus of Liliaceae family. Garlic is a favorite traditional condiment for people, has obvious food therapy effect and is popular with consumers in China. However, in recent years, with the development of garlic planting area and industrialization and the continuous change of cultivation ecological environment, the leaf blight of garlic is increasingly serious, which causes the serious loss of the yield of garlic stems and garlic bulbs. The loss due to garlic leaf blight is reported to be 20-30%, and the loss in severe years and parts of land is more than 50%. Leaf blight bacteria mainly harm leaves and pedicels of garlic, cause symptoms of withered garlic leaves, no bolting of garlic and the like in serious cases, and seriously affect the stable yield and high yield of garlic.

Although chemical medicines can be used for preventing and treating leaf blight at present, a large amount of medicines can cause increase of pesticide residue in products and reduce edible safety.

Due to the fact that the sterile characteristics of garlic and sexual breeding are almost impossible, breeding and popularization of high-quality resistant varieties and export marketable varieties in garlic production in China are seriously lagged, specific resources are seriously lacked in the breeding process of the varieties, innovation of excellent variety resources is low, and the varieties with excellent characteristics are endangered to be extinct due to sexual degeneration.

Garlic is a vegetative propagation crop, the existing garlic mainly takes local varieties as main materials, and garlic bulbs are directly adopted as sowing materials during planting; the method for breeding new species is mainly through seed selection, namely, the selection is carried out according to breeding targets through the characters such as garlic size, plant growth potential, disease resistance, bolting and the like in the field, and generally, if natural variation exists, some beneficial mutations can be selected. However, under normal conditions, the natural mutation rate is low, and the new species of garlic is difficult to obtain. Therefore, researchers generally prefer to mutate garlic by adopting a physical or chemical mutagenesis mode, and on the basis, selection is carried out, so that higher mutation frequency can be obtained, and mutants meeting the breeding target can be expected to be obtained in a relatively short time.

Therefore, the innovation of the garlic leaf blight-resistant germplasm resources and the cultivation of new varieties are taken as targets, EMS is taken as a mutagen, the garlic stem discs and the like of the garlic germplasm G024 are taken as explants for mutagenesis, and the screening of leaf blight-resistant mutant strains is carried out on a culture medium containing leaf blight crude toxin, so that theoretical and technical bases are provided for the creation of the garlic leaf blight-resistant germplasm.

Disclosure of Invention

In view of the above, the invention provides a method for in vitro mutagenesis of a leaf blight-resistant mutant of garlic by using ethyl methanesulfonate, which solves the problems that natural variation of garlic is low, screening of mutants meeting target properties is difficult, the breeding period of a conventional breeding method is long, and rapid breeding of varieties meeting the breeding target is difficult.

In order to achieve the purpose, the invention adopts the technical scheme that: the method for in vitro mutagenesis of garlic leaf blight-resistant mutant by ethyl methanesulfonate is characterized by comprising the following steps: the method comprises the following steps:

1) garlic stem discs are used as a material, Ethyl Methane Sulfonate (EMS) with the concentration of 1% is used as a mutagen, the garlic stem discs are respectively treated for 2 hours, 4 hours, 6 hours, 8 hours and 12 hours, and the optimal mutagenesis time is determined through the germination rate;

2) respectively inoculating non-mutagenized and mutagenized garlic stem discs on screening culture media with the crude toxin volume numbers of 0, 5%, 10%, 15%, 20%, 25% and 30% respectively for culture, and determining the optimal crude toxin screening pressure through the germination rate;

3) EMS with the concentration of 1% is used for treating the garlic stem disk for 6 hours, the treated explant is inoculated on a culture medium with the volume fraction of crude toxin of pathogenic bacteria of leaf blight being 30%, and a regenerated plant with disease resistance is obtained by screening.

Further, the specific screening method for the optimal mutagenesis time in the step 1) comprises the following steps:

washing Bulbus Allii stem disk with tap water for 3 hr, soaking in ethanol under aseptic condition for 1min, sterilizing with 0.5% sodium hypochlorite for 15min, washing with sterile water for 3 times, cutting into stem disk with thickness of 5mm, and cutting into 4 pieces in a cross shape;

soaking the treated stem plate in 1% Ethyl Methane Sulfonate (EMS) for 2, 4, 6, 8, and 12 hr, soaking in 1M sodium thiosulfate solution for 5min, washing with distilled water, wiping, and inoculating on MS culture medium;

and (4) performing illumination culture for 28d, counting the germination number, and determining the treatment time for half of inoculated stem discs incapable of germinating as the appropriate mutagenesis time.

Further, the specific determination method of the optimal crude toxin screening pressure in the step 2) comprises the following steps:

cutting the processed stem disc into 4 blocks according to the shape of a cross, and soaking the 4 blocks in EMS phosphate buffer solution with the concentration of 2.0 percent for 8 hours;

inoculating on screening culture medium with crude toxin body integral number of 0, 5%, 10%, 15%, 20%, 25%, 30%, inoculating 40 explants per treatment, setting 3 times of repetition, and setting as control by phosphate buffer treatment without EMS;

after 30 days of illumination culture, the crude toxin screening pressure is determined by the integral number of 30 percent of crude toxin with the stem disc germination rate of 2.2 percent.

Further, the specific method of step 3) is as follows: cutting the treated stem disc into 4 blocks according to the shape of a cross, soaking the 4 blocks in phosphate buffer of 1.0% EMS for 6h, soaking the stem disc subjected to mutagenesis treatment in 1M sodium thiosulfate solution for 5min, soaking the stem disc in distilled water for 30min, wiping the stem disc with sterilized filter paper, inoculating the stem disc on a screening culture medium with the number of crude toxin elements of 30% respectively, and screening disease-resistant plants.

Compared with the prior art, the invention has the following advantages and effects:

1) EMS with the concentration of 1 percent is taken as a mutagen, a garlic stem disc is taken as an explant, and the directional screening of the leaf blight resistant mutant is carried out in an in vitro mutagenesis mode; determining that the mutagenesis time of the garlic stem disc EMS is 6 hours, and the suitable screening pressure of the leaf blight coarse toxin is 30 percent. Under the screening system, the garlic stem discs are soaked for 6 hours by EMS with the concentration of 1 percent, and isolated culture screening is carried out by taking the semi-lethal dose of the toxin of the garlic leaf blight bacteria with the concentration of 30 percent as the directional selection pressure to obtain the mutant plants resisting the leaf blight, while the garlic stem discs which are not mutagenized by the EMS die completely on the culture medium of the leaf blight crude toxin with the screening pressure of 30 percent.

2) The invention can provide a method for in vitro mutagenesis and directional screening for variety improvement and new germplasm resource creation of the garlic of the family Liliaceae.

Drawings

FIG. 1 is a cross-shaped cut of a garlic stem disc;

FIG. 2 is a graph showing the effect of different EMS treatment times at a concentration of 1% on the germination rate of garlic stem discs;

FIG. 3 the effect of adding different concentrations of crude toxin on the germination rate of EMS-mutagenized and unmutagenized garlic stem discs;

FIG. 4 shows the growth of 4 strains selected on an additional 30% crude toxin medium.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment provides a method for mutagenizing a garlic leaf blight-resistant mutant in vitro by using ethyl methanesulfonate, which comprises the following steps:

1) washing Bulbus Allii stem disk with tap water for 3 hr, soaking in ethanol under aseptic condition for 1min, sterilizing with 0.5% sodium hypochlorite for 15min, washing with sterile water for 3 times, cutting into stem disk with thickness of 5mm, and cutting into 4 pieces in a cross shape;

soaking the treated stem plate in 1% Ethyl Methane Sulfonate (EMS) for 2, 4, 6, 8, and 12 hr, soaking in 1M sodium thiosulfate solution for 5min, washing with distilled water, wiping, and inoculating on MS culture medium;

and (4) performing illumination culture for 28d, counting the germination number, and determining the treatment time for half of the stem discs without germination as the appropriate mutagenesis time.

2) Cutting the processed stem disc into 4 blocks according to the shape of a cross, and soaking the 4 blocks in EMS phosphate buffer solution with the concentration of 2.0 percent for 8 hours;

inoculating on screening culture medium with crude toxin body integral number of 0, 5%, 10%, 15%, 20%, 25%, 30%, inoculating 40 explants per treatment, setting 3 times of repetition, and setting as control by phosphate buffer treatment without EMS;

after 30 days of illumination culture, in order to strictly and directionally screen the mutant, the final crude toxin screening pressure is determined by the 30% of the crude toxin volume of the stem disc with the germination rate of 2.2%.

3) Cutting the treated stem disc into 4 blocks according to the shape of a cross, soaking the 4 blocks in phosphate buffer of 1.0% EMS for 6h, soaking the stem disc subjected to mutagenesis treatment in 1M sodium thiosulfate solution for 5min, soaking the stem disc in distilled water for 30min, wiping the stem disc with sterilized filter paper, inoculating the stem disc on a screening culture medium with the number of crude toxin elements of 30% respectively, and screening disease-resistant plants.

In the embodiment, the influence of different EMS processing times on the garlic germination condition is tested, the garlic stem discs are used as explants to be respectively subjected to EMS processing with the concentration of 1% in mutagenesis time of 2h, 4h, 6h, 8h and 12h, and the garlic stem disc germination rate is gradually reduced along with the extension of the EMS processing time. Wherein, when EMS is used for 6 hours, the germination rate of the garlic stem discs is only 51 percent, which is the half-lethal treatment time length of the germination of the garlic stem discs. The experiment was repeated 3 times. Therefore, the invention determines that the EMS optimal mutagenesis treatment time of the garlic stem disc is 6 h.

In order to further carry out directional screening of leaf blight resistance on stem discs subjected to mutagenesis treatment, sterile crude toxins of garlic leaf blight are prepared in the experiment, and the influence of crude toxins with different concentrations on garlic germination conditions is researched. The stem discs after mutagenesis treatment were inoculated on screening media with crude toxin counts of 0, 5%, 10%, 15%, 20%, 25%, 30%, respectively. The germination rate of the garlic stem discs gradually decreases with the increase of the concentration of the crude toxin. Wherein, when the concentration of the crude toxin is 30%, the garlic stem discs which are not processed by EMS can not germinate; and the germination rate of the garlic stem discs treated by EMS on an MS culture medium with the crude toxin concentration of 30% is 2.2%, and 30% of crude toxin concentration for directionally screening the mutant is selected in order to ensure the screening effect of the crude toxin. The experiment was repeated 3 times. Therefore, the crude toxin concentration of the garlic stem disc subjected to EMS mutagenesis and then subjected to directed mutant screening is determined to be 30%.

And performing directional screening of the bacterial leaf blight resistant mutants by using 30% of crude toxins of garlic leaf blight on the garlic stem discs induced by EMS, and inoculating 200 explants together to obtain 4 bacterial leaf blight resistant mutants.

The specific steps of the method for directionally screening the leaf blight resistant mutant by in vitro mutagenesis of garlic by using ethyl methane sulfonate are as follows:

A. and (5) obtaining a garlic stem disc. Taking a G024 garlic stem disc as a material. Selecting healthy and complete garlic cloves, peeling off garlic skins, washing for 3 hours in tap water, soaking in 75% alcohol by volume fraction for 1min in an aseptic operation table, then shaking and sterilizing by 0.5% sodium hypochlorite for 15-20 min, washing for 3 times by using the aseptic water, draining, cutting off storage leaves on the upper parts of the garlic cloves, only leaving 5mm stem disks, and cutting the processed stem disks into 4 blocks according to a cross shape to be used as explants as shown in figure 1 (wherein a: garlic stem disks; b: cross-shaped explants).

B. Determination of the time of EMS mutagenesis leading to hemilethal stem-discs: and (3) soaking the cut stem-disk explants in 1.0% EMS phosphate buffer for 2, 4, 6, 8 and 12 hours. The treated stem disks were soaked in 1M sodium thiosulfate solution for 5min, in distilled water for 30min, and inoculated on MS medium, 30 explants were inoculated per treatment, 3 replicates were set, and control was performed with phosphate buffer without EMS addition. And (4) culturing for 28d in light, counting the germination number and the regeneration bud number of each explant, calculating the germination rate (the germination rate is the number of germinated explants/total number of explants multiplied by 100%), and determining that the explants are not germinated by non-differentiated adventitious roots or withered yellow within 28 d. After the EMS treatment time is 6 hours, compared with a control, the germination rate of garlic bulbels is reduced from 100% to about 51%, which is obviously lower than that of the control, as shown in figure 2 (wherein A: CK; B-F: 1.0% EMS respectively treats the germination and plantlet growth conditions of garlic stem discs after 2 hours, 4 hours, 6 hours, 8 hours and 12 hours), therefore, 1.0% of EMS treatment 6 hours are determined as the mutagenesis mode when the stem discs are explants, as shown in table 1, table 1. determination of EMS mutagenesis time for half-death of the stem discs

Note: different lower case letters indicate significant differences at the 0.05 level;

C. preparation of sterile crude toxin: culturing the bacterial leaf blight pathogenic bacteria stored on the PDA in an incubator at 25 ℃ for 7-14 days, punching out a bacterial cake with a hole puncher with the diameter of 0.5cm and uniform size, inoculating the bacterial cake to a liquid culture medium of I-Fries, continuously oscillating for 9 days in the dark, filtering by 8 layers of sterilization gauze, and filtering the filtrate by a filter membrane with the diameter of 0.45 mu m under the aseptic condition by using a vacuum air extractor to obtain crude toxin.

D. Determination of the appropriate toxin screening pressure: selecting healthy and complete garlic cloves, peeling off garlic skins, washing for 3 hours in tap water, soaking for 1 minute in 75% alcohol by volume fraction in a sterile operating table, then shaking and disinfecting for 15-20 minutes by using 0.5% sodium hypochlorite, washing for 3 times by using sterile water, draining, cutting off storage leaves on the upper parts of the stem plates, only leaving about 5mm stem plates, cutting the treated stem plates into 4 blocks according to a cross shape, and soaking for 8 hours in 2.0% EMS phosphoric acid buffer solution. Soaking the stem disc after mutagenesis treatment in 1M sodium thiosulfate solution for 5min, soaking in distilled water for 30min, wiping with sterilized filter paper, inoculating on screening culture medium with crude toxin body integral numbers of 0, 5%, 10%, 15%, 20%, 25% and 30%, inoculating 40 explants for each treatment, setting 3 times of repetition, and setting as a control by treating with phosphate buffer solution without adding EMS. After 30 days of illumination culture, the induction rate (germination rate is the number of germinated explants/total number of explants multiplied by 100%) and the development condition of the regeneration buds of each treatment are counted, and the appropriate toxin selection pressure is determined according to the statistics. The ungerminated roots were identified as being undifferentiated within 28 days or withered yellow.

When the concentration of crude toxin is 30%, the garlic stem discs which are not subjected to EMS mutagenesis can not induce bud germination on the culture medium, while the germination rate of the stem discs subjected to EMS mutagenesis is 2.2%, and a small number of garlic plantlets can be germinated, as shown in figure 3 (wherein A-G: germination and plantlet growth conditions of the garlic stem discs which are not subjected to mutagenesis on the culture medium with toxin fractions of 0%, 5%, 10%, 15%, 20%, 25% and 30%, and H-N: germination and plantlet growth conditions of the garlic stem discs which are subjected to mutagenesis on the culture medium with crude toxin fractions of 0%, 5%, 10%, 15%, 20%, 25% and 30%). For strictly directed screening of mutants, the final crude toxin screening pressure was determined by integrating 30% of crude toxin with 2.2% of stem disc germination rate, as shown in table 2;

TABLE 2 Effect of different concentrations of crude toxin on germination Rate of garlic stem discs not subjected to EMS mutagenesis and garlic stem discs subjected to EMS mutagenesis

Note: different lower case letters indicate significant differences at the 0.05 level;

E. screening of resistant plants: selecting healthy and complete garlic cloves, peeling off garlic skins, washing for 3 hours in tap water, soaking for 1 minute in 75% alcohol by volume fraction in a sterile operating table, then shaking and disinfecting for 15-20 minutes by using 0.5% sodium hypochlorite, washing for 3 times by using sterile water, draining, cutting off storage leaves on the upper parts of the stem plates, only leaving 5mm stem plates, cutting the processed stem plates into 4 blocks according to a cross shape, and soaking for 8 hours in 2.0% EMS phosphoric acid buffer solution. Soaking the stem disc after mutagenesis treatment in 1M sodium thiosulfate solution for 5min, soaking in distilled water for 30min, wiping with sterilized filter paper, inoculating on a screening culture medium with 30% of crude toxin volume fraction, then slightly separating the germinated plantlets with tweezers, further inoculating according to strains on a screening culture medium with 30% of crude toxin volume fraction, and continuing to culture disease-resistant plants, as shown in FIG. 4 (a-D: screened plantlets; A-D: expanded propagation strains obtained from disease-resistant plantlets).

The above description is only an example of the present invention, and is not intended to limit the scope of the present invention. The foregoing examples and description have been presented only to illustrate the general principles and features of the invention, and it will be apparent to those skilled in the art from this disclosure that various changes and modifications can be made in the details of the embodiments of the invention described herein, which are within the scope of the invention as hereinafter claimed.