CN108450325B

CN108450325B - Method for creating red kidney bean mutant by adopting EMS mutagen and application

Info

Publication number: CN108450325B
Application number: CN201810117101.5A
Authority: CN
Inventors: 陈喜明; 韩云丽; 邵林生; 郭贵青; 李晓峰
Original assignee: Corn Research Institute Shanxi Academy Of Agricultural Sciences
Current assignee: Corn Research Institute Shanxi Academy Of Agricultural Sciences
Priority date: 2018-02-06
Filing date: 2018-02-06
Publication date: 2021-08-03
Anticipated expiration: 2038-02-06
Also published as: CN108450325A

Abstract

The invention belongs to the technical field of kidney bean breeding, and particularly relates to a method for creating a red kidney bean mutant by using an EMS mutagen and application of the method. Comprises the steps of seed pretreatment, mutagenesis treatment and sowing cultivation, wherein the mutagenesis treatment comprises the following steps: alternately soaking the sterile seeds in a plant growth regulator and EMS solution with the volume fraction of 0.01-0.05%, wherein the soaking time of the plant growth regulator is 5-10min, and the soaking time of the EMS solution is twice that of the plant growth regulator; the alternation times are 2-5 times; drying the surface moisture of the seeds to obtain first-stage mutagenized seeds; radiating the first-stage mutagenized seeds by rays to obtain second-stage mutagenized seeds; sowing and cultivating, and screening out the red kidney bean mutant. The method is a special efficient mutagenesis method for red kidney beans, improves mutagenesis efficiency, reduces the using amount of carcinogens such as EMS and the like, and reduces health threats to breeding workers.

Description

Method for creating red kidney bean mutant by adopting EMS mutagen and application

Technical Field

The invention belongs to the technical field of kidney bean breeding, and particularly relates to a method for creating a red kidney bean mutant by using an EMS mutagen and application of the method.

Background

The red kidney bean is of angiosperm of Leguminosae, and has large particle size, bright color, high nutritive and medicinal value, and good health. The red kidney bean is rich in anthocyanin and saponin, can reduce the content of local inflammatory tissues of joints, has obvious anti-inflammatory effect, and has the effects of diminishing inflammation and easing pain for arthritis patients. The red kidney bean is a special local product of Shanxi in China, the planting area of the red kidney bean in the whole county of Goulan county reaches 13 ten thousand mu, and 350 ten thousand dollars can be exported, so that the income of farmers is greatly increased. The quality of the existing red kidney beans is maintained, and the development of new red kidney bean varieties and strains is an important target of agricultural scientists.

The traditional red kidney bean breeding method adopts a crossbreeding mode, namely, a male parent and a female parent of the red kidney bean with excellent characters are selected for crossbreeding, and a variety with excellent characters is finally selected after generations. Therefore, a method of mutagenizing breeding using a mutagen is gradually used.

EMS is a commonly used plant mutagen, belongs to one of alkylating agents, and has high mutagenesis efficiency, in the prior art, the commonly used volume fraction of the EMS mutagen is 0.1-1.0%, but the substance has strong carcinogenicity and volatility, and strict protection work must be carried out during use, otherwise, the EMS has serious threat to the physical health of breeding workers. Therefore, the development of the EMS mutagen special for the red kidney beans has important significance in the breeding of the red kidney beans, and the use amount of EMS is reduced while the high-efficiency mutagenesis efficiency is ensured.

Disclosure of Invention

In order to solve the problems, the method for creating the red kidney bean mutant by using the EMS mutagen and the application thereof provided by the invention have the advantages that the EMS mutagen special for the red kidney beans is used for carrying out mutagenesis on red kidney bean seeds, the use amount of the EMS is reduced while the high-efficiency mutagenesis efficiency is ensured, the red kidney bean mutant is created, and a foundation is laid for the breeding of red kidney bean varieties.

The first purpose of the invention is to provide a method for creating a red kidney bean mutant by using an EMS mutagen, which comprises the following steps:

s1, pretreatment of seeds

Selecting red kidney bean seeds for disinfection treatment, and then soaking the red kidney bean seeds in a gibberellin solution of 20-30mg/L for 2-3h to obtain sterile seeds;

s2, mutagenesis treatment

Alternately soaking the sterile seeds in a plant growth regulator and EMS solution with the volume fraction of 0.01-0.05%, wherein the soaking time of the plant growth regulator is 5-10min, and the soaking time of the EMS solution is twice that of the plant growth regulator; the alternation times are 2-5 times; drying the surface moisture of the seeds to obtain first-stage mutagenized seeds;

radiating the first-stage mutagenic seeds by rays to obtain second-stage mutagenic seeds M0;

s3, sowing and cultivating

Soaking the second-level mutagenic seeds M0 generation in a rooting agent for 1-2h, sucking the moisture on the surfaces of the seeds, sowing and cultivating in the field, screening out single plants with mutated characters, and harvesting seeds of M1 generation; respectively planting seeds of M1 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening out mutant strains with different characters from the red kidney bean seeds which are not mutagenized to obtain seeds of M2 generation, and taking the seeds of M2 generation as the red kidney bean mutant.

Preferably, in the method for creating the red kidney bean mutant by using the EMS mutagen, in S2, the plant growth regulator is any one or a combination of gibberellin, cytokinin, naphthylacetic acid and 6-benzylaminopurine.

Preferably, in the method for creating the red kidney bean mutant by using the EMS mutagen, in S2, the concentration of the plant growth regulator is 80-150 mg/L.

Preferably, in the method for creating the red kidney bean mutant by using the EMS mutagen, S2, the radiation is⁶⁰Co gamma ray or¹³⁷And the radiation dose rate of the Cs gamma rays is 1Gy/min, and the radiation dose is 30 Gy.

Preferably, in the method for creating the red kidney bean mutant by using the EMS mutagen, in S3, the rooting agent is gibberellin, and the concentration of the gibberellin is 5-10 mg/L.

Preferably, in the method for creating the red kidney bean mutant by using the EMS mutagen, the sterilization treatment in S1 is as follows: soaking red kidney bean seeds in 70% ethanol for 50-60s, soaking in 0.1g/100mL mercuric chloride solution for 10-15min, washing with sterile water, and draining.

The second purpose of the invention is to provide an application of the method for creating the red kidney bean mutant by using the EMS mutagen in screening the red kidney bean mutant strain with stable characters.

Preferably, the above application comprises the steps of:

(1) respectively planting seeds of M2 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening mutant strains with phenotypic characters different from those of the red kidney bean seeds which are not mutagenized, and harvesting seeds of M3 generation;

(2) and (3) repeating the step (1) until M6 generation red kidney bean seeds with stable mutation characters are obtained, and thus, screening out red kidney bean mutants with stable characters.

Compared with the prior art, the method for creating the red kidney bean mutant by adopting the EMS mutagen and the application have the following beneficial effects:

1. the method can efficiently create the red kidney bean mutant, improve the mutagenesis efficiency, reduce the using amount of carcinogens such as EMS and the like, and reduce the health threat to breeding workers. After the sterilization treatment in S1, other bacteria are prevented from damaging the seeds and the generation of seed diseases is avoided, the gibberellin solution in S1 can break the dormancy of the seeds, so that the cell metabolism in the seeds is in an active state, and the generation of mutants is facilitated, and experiments show that the distortion rate of the seeds subjected to the dormancy treatment is 35.6% higher than that of the seeds not subjected to the dormancy treatment; s2, adopting plant growth regulator and EMS to mutate alternately, then carrying out ray radiation mutagenesis, combining multiple mutagenesis means to improve mutagenesis efficiency, and finding that if the plant growth regulator and the EMS mutagen are used alternately, the effect is better; s3, the rooting agent is soaked before the seeds are sown, so that the seeds can root and sprout as soon as possible after being sown, and the growth time is shortened.

2. Through comparison experiments, the method is a special efficient mutagenesis method for red kidney beans, has low aberration rate on cotton seeds, wheat seeds and corn seeds, and has poor effect.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

The invention provides a method for creating a kidney bean mutant by adopting an EMS mutagen, which specifically comprises the following steps:

s1, pretreatment of seeds

s2, mutagenesis treatment

s3, sowing and cultivating

The method specifically includes the following embodiments, and the method for the sterilization processing in S1 in the following embodiments is as follows: soaking red kidney bean seeds in 70% ethanol for 50s (disinfection can be realized within 50-60 s), soaking in 0.1g/100mL mercuric chloride solution for 10min (disinfection can be realized within 10-15 min), washing with sterile water, and draining.

Example 1

A method for creating a red kidney bean mutant by adopting an EMS mutagen comprises the following steps:

s1, pretreatment of seeds

Selecting red kidney bean seeds with consistent color, plump seeds and no worm damage, sterilizing, and soaking in 20mg/L gibberellin solution for 3h to obtain sterile seeds;

s2, mutagenesis treatment

Alternately soaking the sterile seeds in a plant growth regulator and EMS (ethyl methane sulfonate) solution with the volume fraction of 0.01 percent, wherein the soaking time of the plant growth regulator is 10min, and the soaking time of the EMS solution is twice that of the plant growth regulator; the alternation times are 2 times (namely 10min of the plant growth regulator-20 min of EMS solution-10 min of the plant growth regulator-20 min of EMS solution); drying the surface moisture of the seeds to obtain first-stage mutagenized seeds;

the plant growth regulator is cytokinin (N6-isopentenyl adenine), and the concentration of the plant growth regulator is 80 mg/L;

by using⁶⁰Co gamma-rays are used for irradiating the first-stage mutagenized seeds, the radiation dose rate is 1Gy/min, the radiation dose is 30Gy, and second-stage mutagenized seeds M0 are obtained;

s3, sowing and cultivating

Soaking the second-level mutagenized seeds M0 generation in a rooting agent gibberellin for 1h, wherein the concentration of the gibberellin is 10mg/L, absorbing water on the surfaces of the seeds, sowing and cultivating in the field, screening out single plants with mutant characters, and harvesting seeds of M1 generation; respectively planting seeds of M1 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening out mutant strains with different characters from the red kidney bean seeds which are not mutagenized to obtain seeds of M2 generation, and taking the seeds of M2 generation as the red kidney bean mutant.

Example 2

s1, pretreatment of seeds

Selecting red kidney bean seeds with consistent color, plump seeds and no worm damage, sterilizing, and soaking in 30mg/L gibberellin solution for 2h to obtain sterile seeds;

s2, mutagenesis treatment

Alternately soaking the sterile seeds in a plant growth regulator and EMS (ethyl methane sulfonate) solution with the volume fraction of 0.05 percent, wherein the soaking time of the plant growth regulator is 5min, and the soaking time of the EMS solution is twice that of the plant growth regulator; the alternation frequency is 5 times (namely 5min of the plant growth regulator-10 min of EMS solution-5 min of the plant growth regulator-10 min of the EMS solution); drying the surface moisture of the seeds to obtain first-stage mutagenized seeds;

the plant growth regulator is naphthylacetic acid, and the concentration of the plant growth regulator is 100 mg/L;

by using¹³⁷The Cs gamma rays irradiate the first-stage mutagenized seeds, the radiation dose rate is 1Gy/min, the radiation dose is 30Gy, and second-stage mutagenized seeds M0 are obtained;

s3, sowing and cultivating

Soaking the second-level mutagenized seeds M0 generation in a rooting agent gibberellin for 2h, wherein the concentration of the gibberellin is 5mg/L, absorbing the surface moisture of the seeds, sowing and cultivating in the field, screening out single plants with mutant characters, and harvesting seeds of M1 generation; respectively planting seeds of M1 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening out mutant strains with different characters from the red kidney bean seeds which are not mutagenized to obtain seeds of M2 generation, and taking the seeds of M2 generation as the red kidney bean mutant.

Example 3

s1, pretreatment of seeds

Selecting red kidney bean seeds with consistent color, plump seeds and no worm damage, sterilizing, and soaking in 25mg/L gibberellin solution for 2.5h to obtain sterile seeds;

s2, mutagenesis treatment

Alternately soaking the sterile seeds in a plant growth regulator and EMS (ethyl methane sulfonate) solution with the volume fraction of 0.02%, wherein the soaking time of the plant growth regulator is 8min, and the soaking time of the EMS solution is twice of that of the plant growth regulator; the alternation times are 3 times (namely 8min of the plant growth regulator-16 min of EMS solution-8 min of the plant growth regulator-16 min of EMS solution); drying the surface moisture of the seeds to obtain first-stage mutagenized seeds;

the plant growth regulator is 6-benzylaminopurine, and the concentration of the plant growth regulator is 150 mg/L;

s3, sowing and cultivating

Soaking the second-level mutagenized seeds M0 generation in a rooting agent gibberellin for 1.5h, wherein the concentration of the gibberellin is 8mg/L, sucking water on the surfaces of the seeds, sowing and cultivating in the field, screening out single plants with mutated characters, and harvesting seeds of M1 generation; respectively planting seeds of M1 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening out mutant strains with different characters from the red kidney bean seeds which are not mutagenized to obtain seeds of M2 generation, and taking the seeds of M2 generation as the red kidney bean mutant.

Example 4

A method for creating a red kidney bean mutant by using an EMS mutagen is basically the same as the operation steps of example 1, except that the plant growth regulator is formed by mixing gibberellin and cytokinin according to the mass ratio of 1: 1.

Example 5

A method for creating a red kidney bean mutant by using an EMS mutagen is basically the same as the operation steps in the example 2, and the difference is that the plant growth regulator is formed by mixing gibberellin, cytokinin and naphthylacetic acid according to the mass ratio of 1:1: 2.

Example 6

A method for creating a red kidney bean mutant by using an EMS mutagen is basically the same as the operation steps in the example 1, and the difference is that the plant growth regulator is formed by mixing gibberellin, cytokinin, naphthylacetic acid and 6-benzylaminopurine according to the mass ratio of 1:1:2: 1.

Example 7

A method for creating a red kidney bean mutant by using an EMS mutagen is basically the same as the operation steps in the example 1, except that the plant growth regulator is formed by mixing naphthylacetic acid and 6-benzylaminopurine according to the mass ratio of 2: 1.

Example 8

A method for creating a red kidney bean mutant by using an EMS mutagen is basically the same as the operation steps in the example 1, and the difference is that the plant growth regulator is formed by mixing cytokinin and naphthylacetic acid according to the mass ratio of 5: 1.

The effects of the process of the present invention will be described below by taking examples 1 to 8 as examples.

First, evaluation of mutation Effect

(1) The mutagenized seeds obtained in S2 of examples 1 to 8 were wrapped in 3 layers of medical gauze, germinated in a climatic chamber, and the germination rate (the number of seeds germinated in 200 seeds) of the seeds was measured, and 3 parallel tests were performed for each comparative example, and 200 seeds were selected for each test.

(2) Investigation of seed distortion: planting the normally germinated seeds in a pot culture mode, observing the height and tillering number of seedlings, counting the number of deformed plants such as weak growth or spot leaves and the like, and calculating the distortion rate (the number of deformed plants in each 100 seedlings).

TABLE 1 statistics of seed germination and distortion rates for different examples

	Seed germination rate	Rate of distortion
			Example 1	85％	22.5％
Example 2	88％	16.5％
			Example 3	85％	23.9％
Example 4	88％	19.3％
			Example 5	86％	20.0％
Example 6	86％	21.4％
			Example 7	90％	20.1％
Example 8	87％	21.5％

Table 1 shows that although the data in examples 1 to 8 are slightly different and the plant growth regulators selected are slightly different, the seed germination rate and the distortion rate are high. The distortion rate is 16.5-22.5%, and the mutation effect of the method is good. It is to be noted that the methods of examples 1 to 8 are applicable to all red kidney bean varieties.

Table 2 shows the influence of simple seed soaking with different volume concentrations of EMS mutagen on the seed germination rate, and the results in Table 2 show that the seed germination rate gradually decreases with the increase of seed soaking time and the increase of the concentration of EMS mutagen, and even if the seeds are soaked with pure clear water, the seeds have only a germination rate of less than 75 percent, and the red kidney bean seeds have lower germination rate compared with the seeds of examples 1-8.

TABLE 2 Germination rates after seed soaking of EMS mutagens at different volume concentrations

Second, different mutagenesis methods, comparative tests of plant seeds

Comparative example 1

The treatment method was the same as the steps S1-S3 of example 1, except that the red kidney bean seeds were replaced with cotton seeds to prepare secondary mutagenized seeds.

Comparative example 2

The treatment procedure was the same as in steps S1-S3 of example 1, except that the red kidney bean seeds were replaced with corn seeds to make secondary mutagenized seeds.

Comparative example 3

The treatment method was the same as the steps S1-S3 of example 1, except that the red kidney bean seeds were replaced with wheat seeds to prepare secondary mutagenized seeds.

Comparative example 4

The treatment method was the same as the S1 and S3 steps of example 1, and the S2 mutagenesis treatment step was changed to:

soaking the sterile seeds in EMS solution with the volume fraction of 0.01% for 1 h; and (5) absorbing the water on the surface of the seeds to obtain the first-stage mutagenized seeds.

Comparative example 5

soaking the sterile seeds in EMS solution with the volume fraction of 0.5% for 1 h; drying the surface moisture of the seeds to obtain first-stage mutagenized seeds; by using⁶⁰And (3) irradiating the first-stage mutagenic seeds by Co gamma rays, wherein the radiation dose rate is 1Gy/min, and the radiation dose is 30Gy, so as to obtain second-stage mutagenic seeds.

Comparative example 6

The treatment method was the same as that of S1 in example 1, except that the S2 mutagenesis treatment step was changed to:

soaking the sterile seeds in EMS solution with the volume fraction of 0.5% for 1 h; and (5) absorbing the water on the surface of the seeds to obtain the first-stage mutagenized seeds.

The mutagenized seeds obtained in the above comparative examples were subjected to recording of seed germination rate and distortion rate in the manner described in "one, evaluation of mutation Effect" and the results are shown in Table 3.

TABLE 3 statistics of seed germination and distortion rates for different protocols

	Seed germination rate	Rate of distortion
			Example 1	85％	18.5％
Comparative example 1	86％	6.2％
			Comparative example 2	83％	5.1％
Comparative example 3	80％	6.3％
			Comparative example 4	80％	2.3％
Comparative example 5	81％	10.6％
			Comparative example 6	82％	6.1％

Table 3 results show that although the same mutagenesis treatment method is used in example 1 and comparative examples 1 to 3, the distortion rates of cotton seeds, wheat seeds and corn seeds are significantly smaller than those of red kidney bean seeds, which indicates that the method of the present invention is limited to the preparation of red kidney bean mutants and has no significant effect on other plants. Comparing different treatment modes of example 1 and comparative examples 4-6, the distortion rate is obviously improved because the plant growth regulator is added in example 1, which shows that the mutation efficiency can be improved because the plant growth regulator and the EMS mutagen are alternately used for red kidney beans; in comparative example 5, EMS solution with a high concentration volume fraction of 0.5% was treated with radiation, and the seed distortion rate reached 10.6%; however, in comparative example 4, the EMS solution of low concentration of 0.005mol/L was treated with radiation, the seed distortion rate was only 2.3%, and the mutagenesis efficiency was poor due to the low concentration of the mutagen; comparative example 6 increased the concentration of EMS but was not combined with radiation, so the mutagenesis efficiency was lower than comparative example 5.

In addition, we found that if the plant growth regulator and EMS mutagen were not used interchangeably, but the plant growth regulator was used first and then the EMS mutagen, the mutagenesis effect was also inferior to that of example 1, indicating that the "alternate use" procedure had a significant effect on promoting the rate of red kidney bean seed aberration.

In conclusion, the method provided by the embodiment of the invention can efficiently create the red kidney bean mutant, improve the mutagenesis efficiency, reduce the using amount of carcinogens such as EMS (EMS), and reduce the health threat to breeding workers.

Based on the reasons, the method disclosed by the invention is applied to screening of the red kidney bean mutant strain with stable properties, so that the production rate of a new variety can be improved, and the breeding efficiency can be improved. Taking example 1 as an example, the method provided by the invention is applied to screening of red kidney bean mutant strains with stable properties, and comprises the following steps:

(2) repeating the step (1) until M6 generation red kidney bean seeds with stable mutation characters are obtained, so as to screen out red kidney bean mutants with stable characters, which specifically comprise the following steps: respectively planting seeds of M3 generation and red kidney bean seeds which are not mutagenized in a field, observing the phenotypic characters of the plants in the field, screening mutant strains which have different phenotypic characters from the red kidney bean seeds which are not mutagenized, and harvesting seeds of M4 generation;

respectively planting seeds of M4 generation and red kidney bean seeds which are not mutagenized in a field, observing the phenotypic characters of the plants in the field, screening mutant strains which have different phenotypic characters from the red kidney bean seeds which are not mutagenized, and harvesting seeds of M5 generation;

respectively planting seeds of M5 generation and red kidney bean seeds which are not mutagenized in a field, observing the phenotypic characters of the plants in the field, screening out mutant strains with the phenotypic characters different from those of the red kidney bean seeds which are not mutagenized, and harvesting the seeds of M6 generation with stable mutational characters, thereby screening out the red kidney bean mutant strains with stable characters.

It should be noted that the preferred embodiments and effects of the present invention have been described for the purpose of preventing redundancy, and although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for creating a red kidney bean mutant by adopting an EMS mutagen is characterized by comprising the following steps:

s1, pretreatment of seeds

s2, mutagenesis treatment

Alternately soaking the sterile seeds in a plant growth regulator and EMS solution with the volume fraction of 0.01-0.05%, wherein the soaking time of the plant growth regulator is 5-10min, and the soaking time of the EMS solution is twice that of the plant growth regulator; the alternation times are 2-5 times; drying the surface moisture of the seeds to obtain first-stage mutagenized seeds; the plant growth regulator is any one of N6-isopentenyl adenine, naphthylacetic acid and 6-benzylaminopurine, and the concentration of the plant growth regulator is 80-150 mg/L;

radiating the first-stage mutagenic seeds by rays to obtain second-stage mutagenic seeds M0; the ray is⁶⁰Co gamma ray or¹³⁷Cs gamma rays, the radiation dose rate is 1Gy/min, and the radiation dose is 30 Gy;

s3, sowing and cultivating

2. The method for creating the red kidney bean mutant by using the EMS mutagen according to claim 1, wherein in S3, the rooting agent is gibberellin, and the concentration of the gibberellin is 5-10 mg/L.

3. The method for creating the kidney bean mutant by using EMS mutagen according to claim 1, wherein the sterilization treatment in S1 is as follows: soaking red kidney bean seeds in 70% ethanol for 50-60s, soaking in 0.1g/100mL mercuric chloride solution for 10-15min, washing with sterile water, and draining.

4. Use of the method of creating a mutant of red kidney bean according to claim 1 using EMS mutagen for screening of a mutant of red kidney bean with stable traits.

5. Use according to claim 4, characterized in that it comprises the following steps:

(1) respectively planting seeds of M2 generation and red kidney bean seeds which are not mutagenized in a field, observing plant characters in the field, screening mutant strains with different characters from the red kidney bean seeds which are not mutagenized, and harvesting seeds of M3 generation;