Method for performing EMS efficient mutagenesis by applying dendrobium seeds

Abstract

The invention relates to a method for performing EMS efficient mutagenesis by applying dendrobium seeds, and relates to the technical field of botany. The method comprises the following steps: (1) disinfecting dendrobium seeds; (2) sowing: sowing the seeds on a germination culture medium with sterile glass paper laid on the surface; (3) seed culture: culturing the sown seeds until the seeds just germinate; (4) mutagenesis of the seeds: soaking the newly germinated seeds in an EMS buffer solution with the volume concentration of 0.4%, and mutagenizing for 4 hours at 25 + / -2 DEG C under the oscillation condition; and then terminating mutagenesis. Compared with the prior art in which dendrobium protocorm and protocorm-like body are used as the semi-lethal dose of the mutagenesis material, the method has the advantages that the dose of EMS which reaches the semi-lethal dose by using the newly germinated seeds as the mutagenesis material is obviously reduced.

Description

Technical Field

[0001] The present application relates to the field of botanical technology, and in particular to a method for using Dendrobium officinale seeds to carry out EMS efficient mutagenesis. Background Art

[0002] Dendrobium Dendrobium ) is an important ornamental flower and medicinal plant of the Orchidaceae family, and is deeply loved by the Chinese people. At present, the breeding of Dendrobium is mainly based on hybridization, but because the seeds of Dendrobium themselves have special biological characteristics, they have no endosperm and are very small. Under natural conditions, their reproduction rate is low and their growth cycle is long. Therefore, traditional breeding methods have disadvantages such as long growth cycle and low efficiency. Compared with traditional breeding methods, chemical mutagenesis breeding is a fast and effective breeding method, which is of particular significance for plant variety improvement. Ethyl methane sulfonate (EMS) is an alkylating agent that can cause gene mutations and induce plant phenotypic variations by changing base pairs in DNA. It is currently the most widely used and most effective chemical mutagen in crop mutation breeding. Median lethal dose (LD 50 ) is the optimal dose for mutation breeding.

[0003] Dendrobium plants have no endosperm and their seeds are as small as dust. During the aseptic germination process, the seeds need to attach to the culture medium to obtain nutrients, but it is difficult to collect them. At present, the mutagenic materials for EMS mutagenesis breeding of Dendrobium are mainly protocorms and protocorm-like bodies. In the prior art, there are no reports on the direct EMS mutagenesis of Dendrobium seeds. When using protocorms and protocorm-like bodies as mutagenic materials, due to their large tissue volume, it is difficult for mutagens to penetrate into their cells, so the mutagenesis efficiency is often low and chimeras are easily produced. When using protocorms-like bodies as mutagenic materials for EMS mutagenesis disclosed in the prior art, LD 50 0.8 % EMS for 90 min or LD 50 1.8% EMS treatment for 4h; when EMS mutagenesis was performed using protocorms as mutagenic material, LD 50 The samples were treated with 1.0% EMS for 6 h. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides a method for using Dendrobium seeds for EMS efficient mutagenesis, directly using Dendrobium seeds as EMS mutagenesis materials, and performing EMS mutagenesis when the seeds just germinate. The results show that LD 50The volume concentration of EMS was 0.4% and the treatment time was 4 h. Compared with the semi-lethal dose of Dendrobium protocorms and protocorm-like bodies used as mutagenic materials in the prior art, the EMS dose reaching the semi-lethal dose using the newly germinated seeds as mutagenic materials was significantly reduced.

[0005] The present application discloses a method for using Dendrobium officinale seeds to carry out EMS efficient mutagenesis, comprising the following steps: (1) Disinfection of Dendrobium seeds Disinfect the Dendrobium fruit pods; (2) Sowing Cut the sterilized Dendrobium fruit pods and sow the seeds on a germination medium covered with sterile cellophane paper; (3) Seed cultivation Cultivate the sown seeds until they just germinate; (4) Seed mutagenesis The newly germinated seeds were immersed in an EMS solution with a volume concentration of 0.4%, and induced for 4 hours at 25±2℃ and shaking conditions; then, a sodium thiosulfate solution with a mass concentration of 25% was added to the EMS solution to terminate the mutagenesis, wherein the volume ratio of the 25% mass concentration sodium thiosulfate solution to the EMS solution was 1:4; after mixing, the mixture was allowed to stand for 10 minutes, the supernatant was removed, and the seeds after the mutagenesis was terminated were washed with sterile distilled water to obtain the induced Dendrobium seeds.

[0006] Furthermore, the specific operation of disinfecting the Dendrobium pods in step (1) is: soaking the Dendrobium pods in 75% alcohol for 3 minutes, washing with sterile distilled water twice; then soaking and sterilizing with 0.1% mercuric chloride for 8 minutes, washing with sterile distilled water 3 times; then soaking and sterilizing with 1% sodium hypochlorite for 5 minutes, washing with sterile distilled water 10 times.

[0007] Furthermore, the specific operation of washing the seeds after the termination of the reaction with sterile distilled water is as follows: 40 mL of distilled water is added to the centrifuge tube each time, inverted and mixed 10 times, and then allowed to stand, and after the seeds sink to the bottom of the tube, the supernatant is absorbed; and this operation is repeated 10 times.

[0008] Furthermore, the method also includes the steps of sowing and culturing the induced dendrobium seeds. The specific sowing operation is: after resuspending the induced dendrobium seeds with sterile distilled water, the resuspended liquid is spread in a culture bottle filled with solid sowing culture medium; the specific culturing operation is: placing the culture bottle under the conditions of temperature 25±2°C, light 12 h / d, and light intensity of 2000-3000 lx for culturing.

[0009] Furthermore, the formula of the germination medium is: 1 / 2 MS, 1 mg / L 6-BA, 0.1 mg / L NAA, 20 g / L sucrose, 0.3% activated carbon, 7 g / L agar, pH 5.8.

[0010] The beneficial effects of this application are: This application firstly uses Dendrobium seeds as EMS mutagenesis materials, and uses the method in this application to carry out EMS mutagenesis on Dendrobium seeds. The results show that LD 50 The volume concentration of EMS was 0.4%, and the treatment time was 4 h. This result shows that the method in this application only needs a lower EMS dose to achieve LD 50 , that is, the mutagenesis method in this application is more efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0012] Figure 1 This is a schematic diagram of the process after seeds are sown on a germination medium in Example 1 of the present application; Figure 2 A schematic diagram of the germination process of Dendrobium officinale from sowing to before mutagenesis in Application Example 1; Figure 3 This is a schematic diagram of the phenotype of Dendrobium induced by different EMS concentrations and time in Example 1 of the present application; Figure 4 This is a statistical diagram of the seedling rate of Dendrobium officinale induced by different EMS concentrations and time in Application Example 1; Figure 5 Schematic diagram of the comparison between the seedlings obtained by not subjecting the Dendrobium seeds to EMS mutagenesis in Example 2 and the seedlings obtained by subjecting the Dendrobium seeds to EMS mutagenesis in Example 1. DETAILED DESCRIPTION

[0013] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0014] Example 1 In this embodiment, the method of inducing mutagenesis of Dendrobium officinale seeds by using EMS includes the following operations: (1) Disinfection of Dendrobium seeds In a sterile clean bench, soak the Dendrobium fruit pods in 75% alcohol for 3 min, wash with sterile distilled water twice; then soak and sterilize with 0.1% mercuric chloride for 8 min, wash with sterile distilled water 3 times; then soak and sterilize with 1% sodium hypochlorite for 5 min, wash with sterile distilled water 10 times; after taking out the fruit pods, dry the surface moisture with sterile absorbent paper and set aside; (2) Sowing Use a sterile blade to cut the pods open, sow the seeds on a germination medium covered with sterile cellophane, add a small amount of sterile distilled water, and slightly tilt and shake the culture dish to evenly distribute the seeds on the cellophane. Figure 1 The diagram shows the seeds after sowing on the germination medium. Each pod is sown on 5 petri dishes.

[0015] The germination medium formula is: 1 / 2 MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 20 g / L sucrose + 0.3% activated carbon + 7 g / L agar, and the pH is adjusted to 5.8; specifically, the prepared medium is sterilized by high pressure at 121°C for 15 min, cooled to 55°C, and then poured into a culture dish with a diameter of 90 mm and a height of 20 mm. After the culture medium solidifies, sterilized glass paper with a diameter of 95 mm is spread on the culture medium for use.

[0016] (3) Seed cultivation The culture dish with the seeds sown was placed in a dark culture room at 25℃, taken out every Monday, and observed for germination under a microscope. The seeds began to germinate on the 14th day and sprouted on the 21st day. Figure 2 Shown is a schematic diagram of the germination process of Dendrobium seeds from sowing to before mutagenesis.

[0017] (4) Mutation of newly germinated Dendrobium seeds: ① Prepare 100 mM phosphate buffer, pH 7.5.

[0018] Prepare 100 mL of 1M K2HPO4 solution and 100 mL of 1M KH2PO4 solution; take 70 mL of 1M K2HPO4 solution and 20 mL of 1M KH2PO4 solution respectively; adjust the pH to 7.5 with 1M KH2PO4 solution; dilute the 1 M phosphate buffer to 100 mM concentration by adding water.

[0019] ② Preparation of EMS at different concentrations.

[0020] Take 40 mL of phosphate buffer in a 50 mL sterile centrifuge tube, add EMS solution sterilized by filtration with a 0.22 um filter to the buffer in a fume hood, so that the final concentration (v / V) is 0%, 0.2%, 0.4%, and 0.6%, respectively, and mix them upside down for use. The 0% treatment group is the control group. Prepare 3 copies of each concentration and set 3 replicates.

[0021] ③ Mutagenesis conditions: In a sterile clean bench, use a sterile medicine spoon to take the newly germinated seeds on the cellophane, divide them into 12 parts roughly evenly, and place them in centrifuge tubes containing different concentrations of EMS. Each treatment group contains about 800 seeds. Place the centrifuge tubes in an oscillating incubator at 25°C and 50 rpm for mutagenesis reaction.

[0022] ④ Termination of reaction: Prepare 25% sodium thiosulfate solution, filter and sterilize with a 0.22 um filter, and set aside. Take 13 mL of sample solution into a new 50 mL sterile centrifuge tube at 4 h, 8 h, and 12 h of mutagenesis, add 3.2 mL of 25% sodium thiosulfate, 25 ℃, 50 rpm, terminate the reaction, let it stand for 10 min, wait for the seeds to sink to the bottom of the tube, and then drain the supernatant, and transfer the supernatant to the waste bottle containing 50% sodium thiosulfate solution.

[0023] ⑤ Wash with water to remove residual EMS: Wash the seeds that have terminated the reaction with sterile distilled water, add 40 mL of distilled water each time, invert and mix 10 times, and let it stand. After the seeds sink to the bottom of the tube, absorb the supernatant and transfer the supernatant to a waste liquid bottle containing 50% sodium thiosulfate solution. Repeat 10 times; the induced Dendrobium seeds are obtained.

[0024] (5) Sowing of Dendrobium officinale seeds after mutation Add 15 mL of sterile distilled water to each centrifuge tube, resuspend the mutagenized Dendrobium seeds, mix well, divide the seed resuspension into 5 parts, and spread them in culture bottles containing 100 mL of solid sowing medium. Considering that some seeds are lost during the operation, 150 seeds are calculated in each culture bottle. The culture bottles are placed at a temperature of (25±2) °C, a light intensity of 12 h / d, and a light intensity of 2000-3000 lx. After 60 days of cultivation, the seedling rate is statistically observed.

[0025] Figure 3 Shown is a schematic diagram of the phenotypes of Dendrobium officinale induced by different EMS concentrations and time; Figure 4 This is a statistical diagram of the seedling rate of Dendrobium officinale induced by different EMS concentrations and time.

[0026] Example 2 In Example 2, the Dendrobium seeds were not subjected to EMS mutagenesis, which was a control group compared to the mutagenesis in Example 1.

[0027] Comparison of the seedlings obtained without EMS mutagenesis in Example 2 and after EMS mutagenesis in Example 1 shows that Figure 5 As shown. Figure 5 It can be seen that after the EMS mutagenesis treatment in Example 1, some plants showed leaf width variation, and some plants turned yellow and did not grow leaves.

[0028] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for using Dendrobium seeds for EMS efficient mutagenesis, characterized in that: The following steps are involved: (1) Disinfection of Dendrobium seeds Disinfect the Dendrobium fruit pods; (2) Sowing Cut the sterilized Dendrobium fruit pods and sow the seeds on a germination medium covered with sterile cellophane paper; (3) Seed cultivation Cultivate the sown seeds until they just germinate; (4) Seed mutagenesis The newly germinated seeds were immersed in an EMS solution with a volume concentration of 0.4%, and induced for 4 hours at 25±2℃ and shaking conditions; then, a sodium thiosulfate solution with a mass concentration of 25% was added to the EMS solution to terminate the mutagenesis, wherein the volume ratio of the 25% mass concentration sodium thiosulfate solution to the EMS solution was 1:4; after mixing, the mixture was allowed to stand for 10 minutes, the supernatant was removed, and the seeds after the mutagenesis was terminated were washed with sterile distilled water to obtain the induced Dendrobium seeds.

2. The method of using Dendrobium seeds for EMS efficient mutagenesis according to claim 1, characterized in that: The specific operation of disinfecting the Dendrobium fruit pods in step (1) is as follows: soak the Dendrobium fruit pods in 75% alcohol for 3 minutes, and wash them twice with sterile distilled water; then soak and sterilize them in 0.1% mercuric chloride for 8 minutes, and wash them three times with sterile distilled water; then soak and sterilize them in 1% sodium hypochlorite for 5 minutes, and wash them 10 times with sterile distilled water.

3. The method of using Dendrobium seeds for EMS efficient mutagenesis according to claim 1, characterized in that: The specific operation of washing the seeds after the termination reaction with sterile distilled water is as follows: add 40 mL of distilled water to the centrifuge tube each time, invert and mix 10 times, and then let it stand, wait for the seeds to sink to the bottom of the tube, and then absorb the supernatant; repeat the above operation 10 times.

4. The method of claim 1 for high-efficiency mutagenesis using EMS on Dendrobium officinale seeds, characterized in that: The method also includes the steps of sowing and culturing the induced dendrobium seeds. The specific sowing operation is: after resuspending the induced dendrobium seeds with sterile distilled water, the resuspended liquid is spread in a culture bottle filled with solid sowing culture medium; the specific culturing operation is: placing the culture bottle under the conditions of temperature of 25±2°C, light of 12 h / d, and light intensity of 2000-3000 lx for culturing.

5. The method of using Dendrobium seeds for EMS efficient mutagenesis according to claim 1, characterized in that: The formula of the germination medium is: 1 / 2 MS, 1 mg / L 6-BA, 0.1 mg / L NAA, 20 g / L sucrose, 0.3% activated carbon, 7 g / L agar, pH 5.8.

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