Radiation mutation breeding method for dormant buds of arundo donax

Abstract

The invention discloses a bamboo reed dormant bud radiation mutation breeding method which comprises the following steps: selecting robust bamboo reed stalks with high lignification degree and dormant buds, sawing the stalks into stalk sections section by section, performing 60Co-gamma ray radiation treatment on a part of the stalks, performing no radiation treatment on the rest of the stalks, and taking the stalks as a control group; putting the stalk sections with the dormant buds and the stalk sections with the dormant buds of the control group, which are subjected to radiation treatment, into an illumination culture room for rooting culture, performing water culture on the obtained seedlings in the illumination culture room, and performing cadmium-tolerant screening on the obtained water-cultured seedlings to obtain cadmium-tolerant seedlings. Observing and selecting survival transplanted seedlings with characters different from those of the control group in the whole growth process as primary radiation mutants; and carrying out asexual propagation on the obtained primary radiation mutant single plant, and carrying out secondary cadmium-resistant screening on the primary radiation mutant single plant to obtain the cadmium-resistant mutant. According to the method, the bamboo reed mutagenesis material can be subjected to early screening, the stably inherited mutant can be rapidly obtained, the operation is convenient, and the method has important significance on breeding of a new bamboo reed variety.

Description

Technical Field

[0001] The invention belongs to the technical field of mutation breeding, and in particular relates to a radiation mutation breeding method for dormant buds of Phragmites australis. Background Art

[0002] Phragmites australis, also known as "reed bamboo", belongs to the genus Phragmites of the Poaceae family and is a perennial tall clump of herbaceous plants. At present, the main functions of Phragmites australis are reflected in the three aspects of economy, energy and environmental protection. In terms of economy, the comprehensive cellulose content of Phragmites australis stems is as high as 57.71%, which can be used as a source of cellulose and lignin. At the same time, Phragmites australis leaves can be made into high-quality green fodder. In terms of energy, Phragmites australis is one of the most promising bioenergy crops. It has high biomass and low irrigation and nitrogen input requirements. At the same time, Phragmites australis has a huge root system and grows rapidly, which can play a role in soil consolidation, embankment protection and soil and water conservation. In terms of environmental protection, Phragmites australis can be used to repair polluted soil and wetlands, which can not only achieve the repair effect, but also bring certain economic value while repairing.

[0003] Phragmites australis has a high tolerance to abiotic stress conditions (including herbicides, salinity and heavy metals). In heavy metal polluted environments, Phragmites australis shows good adaptability and also shows significant advantages in the process of plant self-repair. Studies have found that under cadmium stress, many morphological and physiological characteristics of Phragmites australis are not greatly affected. It has a strong tolerance to cadmium stress and can be used to repair cadmium-contaminated soil. However, Phragmites australis cannot be propagated by seeds and can only be propagated through asexual reproduction. 60 Co-γ ray irradiation treatment of dormant buds of Phragmites australis can induce gene mutations, produce various types of mutants, and promote the innovation of Phragmites australis germplasm resources and the breeding of new varieties. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a radiation-induced mutation breeding method for dormant buds of Phragmites australis, which is simple to operate and mature and stable.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A radiation-induced mutagenesis breeding method for dormant buds of Phragmites australis, characterized by comprising the following steps:

[0007] S1. Select robust, highly lignified Phragmites australis stalks with dormant buds, and saw them into stalk segments with one dormant bud section by section. The stalk segments are 9 cm long, with the upper end of the stalk being 6 cm away from the dormant bud and the lower end being 3 cm away from the dormant bud;

[0008] S2, taking a portion of the stem segments with dormant buds obtained in step S1 and performing 60Co-γ ray radiation treatment, the radiation intensity is 2Gy / min, the radiation dose is 80Gy~120Gy, the radiation time is 40min~60min, preferably, the radiation dose can be 80Gy, 100Gy, 120Gy, the radiation time is 40min, 50min, 60min respectively; the remaining stem segments with dormant buds are not treated 60 Co-γ-ray irradiation treatment, as the control group;

[0009] S3, will be completed 60 The stem segments with dormant buds treated with Co-γ-ray radiation and the stem segments with dormant buds in the control group were placed in a light culture room for rooting for 24 h;

[0010] S4, hydroponically growing the rooted seedlings in a light culture room for 21 days to obtain seedlings with roots longer than 3 cm and buds longer than 10 cm;

[0011] S5, the hydroponic seedlings obtained in step S4 are placed in a 0.5 mmol / L Cd 2+ The mutants were cultured in 1 / 2 Hogland and the mutants with obvious differences in leaf color and plant height from the control group were observed and selected as radiation primary mutants during the whole growth process.

[0012] S6, the radiation primary mutant single plant obtained in step S5 is cultured and propagated to obtain clustered buds, and the clustered buds are transferred to a medium containing 0.5 mmol / L Cd 2+ The target mutants were cultured in subculture medium for one week and those that grew and developed normally were the target mutants.

[0013] Furthermore, in the step S3, 100 mg / L ABT No. 1 rooting powder solution is used to immerse the dormant buds of the stems for rooting treatment.

[0014] Furthermore, in the step S4, the seedlings are hydroponically cultured in a hydroponic box to which 1 / 2 Hogland nutrient solution is added.

[0015] Furthermore, in the step S4, hydroponics is carried out under the conditions of a temperature of 25° C., a daily illumination time of 16 h, a light intensity of 18,000 to 20,000 lux, and a humidity of 60%.

[0016] Furthermore, in step S6, the subculture medium is: MS medium, 0.7% agar powder and 3% sucrose, 6-BA, IBA.

[0017] The present invention utilizes 60Co-γ irradiation of dormant buds of Phragmites australis increased its mutation rate. Tissue culture of radiation-induced mutant plants can obtain mutants with stable genetic traits in a relatively short period of time, thereby effectively shortening the breeding cycle of Phragmites australis and overcoming the defects of long natural mutation breeding cycle, low breeding efficiency and few mutations of Phragmites australis. This is of great significance for the selection and breeding of new Phragmites australis varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0019] Figure 1 for 60 The results of hydroponics after 21 days of Co-γ-ray radiation.

[0020] Figure 2 The phenotype of the radiation mutants was selected initially;

[0021] Figure: A shows the growth of the control strain in 500mmol / L Cd2+ medium; B shows the growth of the cadmium-resistant mutant in 500mmol / L Cd 2+ Growth conditions of the culture medium. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0023] Example 1

[0024] A radiation-induced mutagenesis breeding method for dormant buds of Phragmites australis comprises the following steps:

[0025] S1. Select robust, highly lignified Phragmites australis stalks with dormant buds, and saw them into stalk segments with one dormant bud section by section. The stalk segments are 9 cm long, with the upper end of the stalk being 6 cm away from the dormant bud and the lower end being 3 cm away from the dormant bud;

[0026] S2, 138 Phragmites australis stems with dormant buds were selected for 60 Co-γ-ray radiation treatment, the radiation intensity is 2Gy / min, the radiation dose is 80Gy, and the radiation time is 40min;

[0027] S3, use 100mg / L ABT No. 1 rooting powder solution to complete 60 The stems of Phragmites australis with dormant buds treated with Co-γ-rays were subjected to rooting treatment for 24 h;

[0028] S4, placing the rooted Phragmites australis stems with dormant buds in a hydroponic box with 1 / 2 Hogland nutrient solution for hydroponics, the culture conditions are: temperature 25°C, light duration 16h, light intensity 18000-20000 lux, humidity 60%, changing the nutrient solution every 3 days, and culturing for 21 days;

[0029] S5, the hydroponic seedlings obtained in step S4 are placed in a 0.5 mmol / L Cd 2+ The plants were cultured in 1 / 2 Hogland nutrient solution, and the mutant phenotypes with obvious differences in leaf color and plant height from the control group (Comparative Example 1) were observed and selected during the entire growth process as radiation primary mutants;

[0030] Example 2

[0031] 132 Phragmites australis stems with dormant buds were selected for 60 The Co-γ ray radiation treatment has a radiation dose of 100 Gy, and the rest is exactly the same as in Example 1.

[0032] Example 3

[0033] 125 Phragmites australis stems with dormant buds were selected for 60 The radiation dose in the method for radiation-induced mutation breeding of dormant buds of Phyllostachys australis was 120 Gy, and the others were exactly the same as in Example 1.

[0034] Comparative Example 1

[0035] The radiation dose in the radiation-induced mutagenesis breeding method for dormant buds of Phragmites australis is 0 Gy, and the rest is exactly the same as in Example 1.

[0036] 1) Observe and record the initial radiation mutants of each group. The specific results are as follows:

[0037] like Figure 1 As shown, in Example 1, among the 138 transplanted seedlings after 80 Gy irradiation, 8 primary mutant transplanted seedlings were found, and the leaf color of 5 of them changed from green to light yellow compared with the control group after transplantation and culture; and the plant height of 3 of them became shorter compared with the control group after transplantation and culture;

[0038] In Example 2, among the 125 transplanted seedlings treated with 100 Gy irradiation, 12 were primary mutant transplanted seedlings, of which 8 had leaf color changed from green to light yellow compared with the control group after transplantation and culture; and 4 had shorter plant heights compared with the control group after transplantation and culture;

[0039] In Example 3, 9 primary mutant seedlings were transplanted among the 132 seedlings after 120GY irradiation treatment, of which 5 had leaf color changed from green to light yellow compared with the control group after transplantation and culture; 4 had shorter plant heights compared with the control group after transplantation and culture;

[0040] 2) The radiation primary mutant transplanted seedlings obtained in each group and the control group were subjected to tissue culture and propagation by taking their axillary buds, with each tissue culture growth time being 25 to 30 days, to obtain primary mutant cluster seedlings and control group cluster seedlings; then the plant traits of the primary mutant cluster seedlings and the control group cluster seedlings were compared, and the transplanted seedlings that survived from the primary mutant cluster seedlings and whose leaf color and plant height were different from those of the control group cluster seedlings after tissue culture were selected, which were the cadmium-tolerant mutants.

[0041] 3) The seedling formation rate and mutation rate of dormant buds of Phyllostachys australis in each group were counted, wherein the mutation rate (%) = the number of transplanted mutant seedlings from a certain radiation dose / the total number of plants in the test group. The results are shown in Table 1.

[0042] Table 1 60 Effects of different Co-γ-ray radiation doses on the seedling rate and mutation rate of dormant buds of Phragmites australis (21 days of hydroponic culture)

[0043] Group Radiation dose / Gy Seedling rate / % Mutation rate / % Comparative Example 1 0 58.89 0 Example 1 80 40.58 5.79 Example 2 100 28.99 9.60 Example 3 120 14.49 6.81

[0044] Application Example 1

[0045] A radiation-induced mutagenesis breeding method for dormant buds of Phragmites australis comprises the following steps:

[0046] 1) Select robust, highly lignified Phragmites australis stalks with dormant buds, and saw them into stalk segments with one dormant bud section by section. The stalk segments are 9 cm long, with the upper end of the stalk being 6 cm away from the dormant bud and the lower end being 3 cm away from the dormant bud;

[0047] 2) 138 Phragmites australis stems with dormant buds were tested. 60 Co-γ radiation treatment, the radiation intensity is 2Gy / min, the radiation dose is 80Gy, and the radiation time is 40min;

[0048] 3) Use 100mg / L ABT No. 1 rooting powder solution to complete 60 The Co-γ-irradiated Phragmites australis culms with dormant buds were subjected to rooting treatment for 24 h;

[0049] 4) placing the rooted Phragmites australis stalks with dormant buds in a hydroponic box with 1 / 2 Hogland nutrient solution for hydroponics, the culture conditions being: temperature 25° C., daily light duration 16 h, light intensity 18,000-20,000 lux, humidity 60%, replacing the nutrient solution every 3 days, culturing for 21 days, and obtaining seedlings with roots longer than 3 cm and buds longer than 10 cm;

[0050] 5) The obtained hydroponic seedlings were placed in a solution containing 0.5 mmol / L Cd 2+ The plants were cultured in 1 / 2 Hogland nutrient solution. During the whole growth process, the mutant phenotypes with obvious differences in leaf color and plant height from the control group were observed and selected as radiation primary mutants. A total of 8 radiation primary mutants were obtained.

[0051] 6) The radiation primary mutant single plant was cultured and propagated to obtain clustered shoots, and the clustered shoots were transferred to a medium containing 0.5 mmol / L Cd 2+ The mutants were cultured in subculture medium for one week. Those with normal growth and development were cadmium-resistant mutants. A total of 6 cadmium-resistant mutants were obtained.

[0052] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A radiation-induced mutagenesis breeding method for dormant buds of Phragmites australis, characterized in that: The following steps are involved: S1. Select robust, highly lignified Phragmites australis stalks with dormant buds, and saw them into stalk segments with one dormant bud section by section. The stalk segments are 9 cm long, with the upper end of the stalk being 6 cm away from the dormant bud and the lower end being 3 cm away from the dormant bud; S2, taking a portion of the stem segments with dormant buds obtained in step S1 and performing 60 Co-γ-ray radiation treatment, the radiation intensity is 2Gy / min, the radiation dose is 80Gy~120Gy, and the radiation time is 40min~60min; the remaining stem segments with dormant buds are not treated. 60 Co-γ-ray irradiation treatment, as the control group; S3, will be completed 60 The stem segments with dormant buds treated with Co-γ-ray radiation and the stem segments with dormant buds in the control group were placed in a light culture room for rooting for 24 h; S4, hydroponically growing the rooted seedlings in a light culture room for 21 days to obtain seedlings with roots longer than 3 cm and buds longer than 10 cm; S5, the hydroponic seedlings obtained in step S4 are placed in a 0.5 mmol / L Cd 2+ The plants were cultured in 1 / 2 Hogland nutrient solution, and the mutant phenotypes with obvious differences in leaf color and plant height from the control group were observed and selected as radiation primary mutants during the entire growth process; S6, the radiation primary mutant single plant obtained in step S5 is cultured and propagated to obtain clustered buds, and the clustered buds are transferred to a medium containing 0.5 mmol / L Cd 2+ The mutants that grow and develop normally are cultured in the subculture medium for one week and are cadmium-resistant mutants.

2. The radiation-induced mutagenesis breeding method according to claim 1, characterized in that: In the step S3, the dormant buds of the stems are immersed in a 100 mg / L AB T No. 1 rooting powder solution for rooting for 24 hours.

3. The radiation-induced mutation breeding method according to claim 1, characterized in that: In the step S4, the seedlings are hydroponically cultured in a hydroponic box into which 1 / 2 Hogland nutrient solution is added.

4. The radiation-induced mutation breeding method according to claim 1, characterized in that: In the step S4, hydroponics is carried out under the conditions of a temperature of 25° C., a daily illumination time of 16 h, a light intensity of 18,000 to 20,000 lux, and a humidity of 60%.

5. The radiation-induced mutation breeding method according to claim 1, characterized in that: In step S6, the subculture medium is: MS medium, 0.7% agar powder and 3% sucrose, 6-BA, IBA.

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