Method for screening drought-resistant mutants after EMS mutagenesis of bamboo reed cluster buds

Abstract

The invention relates to the technical field of plant stress resistance screening, in particular to a method for screening drought-resistant mutants after EMS mutagenesis of bamboo reed cluster buds, which comprises the following steps: S1, performing hydroponic seedling strengthening on wild tissue culture seedlings and EMS-treated rooting tissue culture seedlings to obtain wild bamboo reed seedlings and EMS-treated bamboo reed seedlings; s2, screening suitable concentration of PEG-6000: screening wild type bamboo reed seedlings by using a series of PEG-6000 solutions with different concentrations, comparing survival rates of the bamboo reed seedlings under different treatments, and screening suitable concentration for simulating drought stress by a PEG-6000 water culture method; s3, simulating drought stress conditions by using the screened PEG-6000 solution with proper concentration as a permeable medium, and screening the EMS treated bamboo reed seedlings to obtain drought-resistant plants. The screening method provided by the invention is not limited by seasons, has high accuracy, and is a simple, convenient and rapid method for screening drought-resistant bamboo reed plants.

Description

Technical Field

[0001] The present invention relates to the technical field of plant stress resistance screening, and particularly relates to a method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax cluster buds. Background Art

[0002] Arundo donax is a plant of the genus Arundo in the family Poaceae. It resembles reeds and bamboo but belongs to different species. Arundo donax grows fast, has a high biological yield, and is easy to manage in the field, and is planted all over the world. Young Arundo donax has a high protein content and good nutritional value, and can be used for edible mushroom cultivation and high-quality feed production. Arundo donax is an excellent fiber plant, with fast reproduction, high yield, and a short income period. Its fiber is long, has high tensile strength, and a high beating rate, making it a good raw material for papermaking and rayon, and can also be used for making boards and biomass energy. Arundo donax has strong stress resistance, with characteristics such as salt tolerance, barren tolerance, low temperature tolerance, strong adaptability, and high photosynthetic efficiency. It can be planted in barren soils or marginal lands without competing with food crops for land, and can be applied to soil remediation, sewage purification, wind prevention and sand fixation, and protection of the water-level-fluctuation zone. By screening drought-tolerant Arundo donax varieties, it can better survive and grow under drought conditions, thereby improving its adaptability and yield. Summary of the Invention

[0003] To solve the above problems, the present invention provides a method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax cluster buds. It is not restricted by seasons, has a high accuracy rate, and is a simple and rapid method for screening drought-tolerant Arundo donax plants.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax cluster buds, comprising the following steps:

[0006] S1. Hydroponically strengthen the wild-type tissue culture seedlings that have taken root and the root-taking tissue culture seedlings treated with EMS with a nutrient solution to obtain wild-type Arundo donax seedlings and EMS-treated Arundo donax seedlings;

[0007] S2. Screen the appropriate concentration of PEG-6000: Screen the wild-type Arundo donax seedlings with a series of different concentrations of PEG-6000 solutions, and screen the appropriate concentration for simulating drought stress by PEG-6000 hydroponics method by comparing the survival rates of Arundo donax seedlings under different treatments. Preferably, when screening the wild-type Arundo donax seedlings, the concentrations of the PEG-6000 solution are 0, 10%, 20%, and 30%;

[0008] S3. Use the screened appropriate concentration of PEG-6000 solution as an osmotic medium to simulate drought stress conditions, and screen the EMS-treated Arundo donax seedlings to obtain drought-tolerant plants.

[0009] In the method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax L. cluster buds of the present invention, the rooted tissue culture seedlings after EMS treatment are obtained through the following steps:

[0010] S1.1: Take the stem segments with axillary buds of Arundo donax L. in the current year without pests and diseases and with plump buds. After disinfection, transfer them to an MS medium supplemented with 0.7% agar powder, 3% sucrose, 4 mg / L 6-BA, and 1 mg / L IBA for cluster bud induction. After multiple subcultures, well-grown tissue culture seedlings are obtained.

[0011] S1.2: After treating the well-grown tissue culture seedlings with 0.1 mol / L EMS for 6 h, select the surviving tissue culture seedlings and subculture them for rooting using a 1 / 2MS rooting medium supplemented with 0.7% agar powder, 3% sucrose, 1 mg / L 6-BA, 1 mg / L IBA, and 0.1 mg / L NAA to obtain the rooted tissue culture seedlings after EMS treatment.

[0012] In the method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax L. cluster buds of the present invention, in step S1, the formula of the nutrient solution is: prepared by mixing Huaduoduo 01 and water at a ratio of 1:1000 for hydroponic seedling strengthening.

[0013] In the method for screening drought-tolerant mutants after EMS mutagenesis of Arundo donax L. cluster buds of the present invention, in step S3, a 20% PEG-6000 solution is used as the osmotic medium to simulate drought stress conditions.

[0014] The present invention has established a technical system for screening drought-tolerant Arundo donax L. plants using PEG-6000 indoors, reducing outdoor operations, enabling artificial control of experimental conditions, being simple to operate, having a short cycle, and being able to accurately screen out drought-tolerant plants, providing technical support for drought-tolerant Arundo donax L. screening and Arundo donax L. drought resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:

[0016] Figure 1 Shows the effects of wild-type Arundo donax L. tissue culture seedlings after treatment with different PEG concentrations.

[0017] Figure 2 Shows the effects of Arundo donax L. tissue culture seedlings after mutagenesis with a 20% PEG concentration. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made. These all belong to the protection scope of the present invention.

[0019] Example 1

[0020] 1) Obtaining Arundo donax tissue culture seedlings

[0021] Take the axillary bud-bearing stem segments of Arundo donax in the current year without pests and diseases and with plump buds, disinfect them, and transfer them to a subculture medium (MS medium with 0.7% agar powder, 3% sucrose, 6-BA concentration of 4 mg / L, and IBA concentration of 1 mg / L) for adventitious bud induction. After subculturing 3 - 5 times, well-grown tissue culture seedlings are obtained.

[0022] 2) Obtaining experimental materials

[0023] Treat the tissue culture seedlings in step 1) with 0.1 mol / L EMS for 6 h, then select the surviving tissue culture seedlings and transplant them to a rooting medium (1 / 2MS rooting medium with 0.7% agar powder, 3% sucrose, 6-BA concentration of 1 mg / L, IBA concentration of 1 mg / L, and NAA concentration of 0.1 mg / L) for subculture rooting to obtain rooted tissue culture seedlings after EMS treatment;

[0024] Hydroponically strengthen the rooted wild-type tissue culture seedlings and the rooted tissue culture seedlings after EMS treatment with a nutrient solution (prepared by mixing Huaduoduo 01 and water at a ratio of 1:1000). When the seedlings grow to 8 - 10 cm, select healthy wild-type Arundo donax seedlings and EMS-treated Arundo donax seedlings with consistent plant height growth for PEG-6000 drought stress experiments.

[0025] 3) Screening for the appropriate PEG-6000 concentration

[0026] Transfer the wild-type Arundo donax seedlings into containers pre-prepared with PEG-6000 solutions of four different mass fractions: 0, 10%, 20%, and 30%. Place 20 plants in each hydroponic box and repeat 3 times. The laboratory temperature range is (25 ± 2) °C and the humidity is 60%. Start timing after all the plants are transferred into the solution. Take digital photos of the treated seedlings every 3 - 4 days, observe the external morphological changes of the leaves using the direct observation method, and calculate the survival rate of Arundo donax seedlings under different treatments. Select the PEG-6000 solution concentration with a lower survival rate of Arundo donax seedlings as the appropriate concentration for simulating drought stress by the PEG-6000 hydroponic method;

[0027] 4. Obtaining drought-tolerant materials

[0028] Using the screened PEG-6000 solution with appropriate concentration as the osmotic medium to simulate drought stress conditions, hydroponic screening was carried out on EMS-treated Arundo donax seedlings to obtain drought-tolerant plants.

[0029] Results:

[0030] 1. The external morphological changes of plants are the most intuitive manifestation of their suffering from drought stress and also the most basic criterion for judging whether they can survive in their environment. Drought stress was imposed on wild-type Arundo donax seedlings with PEG-6000 solutions at concentrations of 0, 10%, 20%, and 30%. The results showed that when the PEG-6000 concentration was 0, the Arundo donax seedlings grew normally; when the concentration was 10%, the leaves of the plants showed shrinkage at 11 days, and all shrank and grew slowly but still survived at 14 days; when the concentration was 20%, the leaves of the plants began to show shrinkage at 7 days, all shrank and the leaves began to turn yellow at 11 days, the plants began to wither at 14 days, and the survival rate was 20%; when the concentration was 30%, the leaves of the plants began to show shrinkage, withering, and yellowing at 4 days, almost all the plants died at 7 days, and all had died at 11 days. Therefore, the appropriate PEG-6000 concentration is 20%.

[0031] 2. By treating the EMS-mutagenized Arundo donax seedlings, i.e., EMS-treated Arundo donax seedlings, with 20% PEG-6000, the results showed that: at 7 days, the plants could still grow normally, with the leaf tips shrinking and turning yellow; at 11 days, the leaves of the plants shrank but most of the seedlings still survived; at 14 days, some Arundo donax seedlings began to wither and die as a whole, and only about 30% of the Arundo donax seedlings survived. These seedlings are the initially screened drought-tolerant plants, and further physiological, biochemical, and molecular tests will be carried out on them to identify the screened mutants.

[0032] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots, characterized in that: The steps include: S1, hydroponically strengthening the wild-type tissue culture seedlings and the rooted tissue culture seedlings treated with EMS to obtain wild-type Arundo donax seedlings and EMS-treated Arundo donax seedlings; S2. Screening the appropriate PEG-6000 concentration: A series of PEG-6000 solutions with different concentrations were used to screen wild-type Phyllostachys australis seedlings, and the appropriate concentration of PEG-6000 for simulating drought stress in hydroponics was screened by comparing the survival rates of Phyllostachys australis seedlings under different treatments; S3. Using the selected PEG-6000 solution of appropriate concentration as the infiltration medium to simulate drought stress conditions, the EMS-treated Phragmites australis seedlings were screened to obtain drought-resistant plants.

2. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clump shoots as claimed in claim 1, characterized in that: The rooted tissue culture seedlings after EMS treatment are obtained by the following steps: S1.

1. Take the current year's disease- and insect-free stem segments of Phragmites australis with full buds and axillary buds, sterilize them, transfer them to subculture medium for cluster bud induction, and obtain well-growing tissue culture seedlings after 3-5 subcultures; S1.

2. After treating the well-growing tissue culture seedlings with 0.1 mol / L EMS for 6 h, the surviving tissue culture seedlings were selected for subculture and rooting to obtain rooted tissue culture seedlings treated with EMS.

3. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots as claimed in claim 1, characterized in that: In the step S1, the rooted wild-type tissue culture seedlings and the rooted tissue culture seedlings treated with EMS are hydroponically cultured with a nutrient solution to strengthen the seedlings, wherein the nutrient solution is prepared with Huaduoduo 01 and water at a ratio of 1:1000.

4. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots as claimed in claim 2, characterized in that: In the step S1.1, the subculture medium is MS medium supplemented with 0.7% agar powder, 3% sucrose, 4 mg / L 6-BA and 1 mg / L IBA.

5. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots as claimed in claim 2, characterized in that: In the step S1.2, the subculture rooting adopts 1 / 2MS rooting medium supplemented with 0.7% agar powder, 3% sucrose, 1 mg / L 6-BA, 1 mg / L IBA, and 0.1 mg / L NAA.

6. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots as claimed in claim 1, characterized in that: In the step S2, when the wild-type Phragmites australis seedlings are screened, the concentration of the PEG-6000 solution is 0%, 10%, 20%, and 30%.

7. The method for screening drought-tolerant mutants after EMS mutagenesis of Phragmites australis clustered shoots as claimed in claim 1, characterized in that: In step S3, a PEG-6000 solution with a concentration of 20% is used as an infiltration medium to simulate drought stress conditions.

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