Composition for Co-60 γ-ray irradiation mutagenesis breeding of Dendrocalamus brandisii and its application

Abstract

The present invention relates to the technical field of crop breeding, and specifically discloses a composition for Co-60γ ray irradiation mutagenesis breeding of Dendrocalamus brandisii, which is composed of an irradiation pretreatment solution and an irradiation post-treatment solution. The present invention also provides a method for breeding Dendrocalamus brandisii by Co-60γ ray irradiation mutagenesis, which includes soaking the seeds of Dendrocalamus brandisii in an irradiation pretreatment solution containing L-hydroxyproline to fully imbibe them, irradiating them with Co-60γ rays at a semi-lethal dose, and then treating them with an irradiation post-treatment solution containing 2-hydroxyglutaric acid, glutathione and vitamin C. The method of the present invention effectively improves the mutagenesis efficiency of Dendrocalamus brandisii by adding L-hydroxyproline as an inducer for specific pretreatment before irradiation, adding 2-HG as an irradiation sensitizer, and adding GSH and Vc as radiation protectors for post-treatment after irradiation. Mutant plants with excellent traits are screened and can be used as new germplasms or new varieties of bamboo.

Description

Technical Field

[0001] The present invention relates to the technical field of crop breeding, and particularly relates to a composition for Co-60 γ-ray irradiation mutagenesis breeding of Dendrocalamus brandisii. Background Art

[0002] Co-60 γ-rays are the most commonly used radiation mutagenic sources in forestry, horticulture and crop breeding. They have a high induced mutation frequency and a wide mutation spectrum. Researchers can screen out excellent mutant strains that are beneficial to the cultivation of new varieties from a large number of random mutations generated after radiation, increasing the economic value of plants. Co-60 γ-rays acting on the body mainly cause DNA damage, and its types include: changes or deletions of bases, DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs). Among them, DSBs are considered the most serious DNA damage. Ineffective repair or incorrect repair of DSBs can trigger genomic disorders, ultimately leading to the occurrence of tumors and other related diseases. However, organisms, especially plants, can repair DNA damage through epigenetic regulation or activation of regulatory factors such as STEMIN (Yoshiyama et al., DNA damage response in plants: conserved and variable response compared to animals. Biology 2, 1338–1356 (2013)). As Gu et al. (2020) found in their research, after the leaf cells of Physcomitrella patens were soaked in a DNA damage-inducing reagent for 6 hours, the genomic DNA strands were broken; subsequently, the damaged DNA was repaired to its original state in about one day (Gu et al., DNA damage triggers reprogramming of differentiated cells into stem cells in Physcomitrella, Nature Plants, 2020). These research results indicate that DNA damage has a positive inducing effect in cell reprogramming and is a new strategy for plants to adapt to stress environments.

[0003] Dendrocalamus brandisii (Munro) Kurz is an indigenous bamboo species in Yunnan and is an excellent bamboo species that can be used for both bamboo shoots and timber, with high economic value. Due to the complexity of the genetic background and the biological particularity of bamboos, that is, the flowering of bamboo plants is uncertain and they die after flowering, it is very difficult to improve their varieties through traditional breeding methods. Mutagenesis breeding is an effective means. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to breed more new varieties of Dendrocalamus brandisii.

[0005] To solve the above technical problem, the present invention provides a composition for Co-60γ ray irradiation mutagenesis breeding of Dendrocalamus brandisii, which is composed of an irradiation pretreatment solution and an irradiation post-treatment solution:

[0006] The irradiation pretreatment solution is composed of a solute and a solvent. The solvent is 10 mM MES buffer solution with a pH value of 6.5, and the solute and its content are 5 mM magnesium chloride (MgCl2) and 5 mM L-hydroxyproline (Hyp).

[0007] The irradiation post-treatment solution is composed of a solute and a solvent. The solvent is 10 mM MES buffer solution with a pH value of 6.5, and the solute and its content are 10-20 μm 2-hydroxyglutaric acid (2-Hydroxyglutarate, 2-HG), 100 mM glutathione (glutathione, GSH) and 200 mM vitamin C (Vc).

[0008] In the above composition, the concentration of 2-hydroxyglutaric acid in the irradiation post-treatment solution is preferably 10 μm.

[0009] The present invention also provides another composition for Co-60γ ray irradiation mutagenesis breeding of Dendrocalamus brandisii, which is the above-mentioned irradiation pretreatment solution.

[0010] The present invention also provides a third composition for Co-60γ ray irradiation mutagenesis breeding of Dendrocalamus brandisii, which is the above-mentioned irradiation post-treatment solution.

[0011] The present invention also provides a method for Co-60γ ray irradiation mutagenesis breeding of Dendrocalamus brandisii, including soaking the seeds of Dendrocalamus brandisii with the above-mentioned irradiation pretreatment solution to make them fully imbibed, irradiating them with Co-60γ rays at a semi-lethal dose, and then treating them with the above-mentioned irradiation post-treatment solution.

[0012] In the above method, the treatment with the irradiation post-treatment solution is soaking in the dark at 25°C for 3-6 hours, preferably soaking for 6 hours.

[0013] In the above method, the semi-lethal dose is 96 Gy.

[0014] In the above method, the Dendrocalamus brandisii is Dendrocalamus brandisii (Munro) Kurz.

[0015] The breeding method of Dendrocalamus brandisii by Co-60 γ-ray irradiation mutagenesis effectively improves the mutagenesis efficiency of Dendrocalamus brandisii through specific pre-irradiation pretreatment (adding L-hydroxyproline as an inducer) and post-irradiation treatment (adding 2-HG as an irradiation sensitizer, adding GSH and Vc as radiation protectants). Mutant plants with excellent traits are screened and can be used as new germplasms or new varieties of bamboo. Brief Description of the Drawings

[0016] Figure 1 Seeds of Dendrocalamus brandisii in Example 1 of the present invention.

[0017] Figure 2 Plants of two-year-old Dendrocalamus brandisii in Example 2 of the present invention. Among them, CK is the unirradiated control; M0-7 and M0-19 are plants obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M0 (without 2-HG) in the dark for 3 hours; M10-3-12 and M10-3-25 are plants obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 3 hours; M10-6-12, M10-6-22, and M10-6-35 are plants obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 6 hours; M20-3-29 and M20-3-33 are plants obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M20 (containing 20 μm 2-HG) in the dark for 3 hours; M20-6-25 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M20 (containing 20 μm 2-HG) in the dark for 6 hours.

[0018] Figure 3 Polymorphic bands of mutant plant DNA amplified by primer P-21 in Example 2 of the present invention. Among them, CK is the unirradiated control; M0 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M0 (without 2-HG) in the dark for 3 hours; M10-3 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 3 hours; M10-6 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 6 hours; M20-3 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M20 (containing 20 μm 2-HG) in the dark for 3 hours; M20-6 is a plant obtained by planting seeds that were irradiated and then treated with post-irradiation treatment solution M20.

[0019] Figure 4This is the phylogenetic tree of cluster analysis of EST-SSR molecular markers of mutagenized plants in Example 2 of the present invention. Among them, CK is the unirradiated control; plants numbered starting with M0, such as M0-3, M0-7, M0-9, M0-17, M0-19, and M0-32, are obtained from seeds treated with irradiation and then treated with the post-irradiation treatment solution M0 (without 2-HG) in the dark for 3 hours; plants numbered starting with M10-3, such as M10-3-2 and M10-3-12, are obtained from seeds treated with irradiation and then treated with the post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 3 hours; plants numbered starting with M10-6, such as M10-6-22 and M10-6-37, are obtained from seeds treated with irradiation and then treated with the post-irradiation treatment solution M10 (containing 10 μm 2-HG) in the dark for 6 hours; plants numbered starting with M20-3, such as M20-3-29 and M20-3-33, are obtained from seeds treated with irradiation and then treated with the post-irradiation treatment solution M20 (containing 20 μm 2-HG) in the dark for 3 hours; plants numbered starting with M20-6, such as M20-6-2 and M20-6-25, are obtained from seeds treated with irradiation and then treated with the post-irradiation treatment solution M20 (containing 20 μm 2-HG) in the dark for 6 hours. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0021] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0022] The seeds of Dendrocalamus brandisii described in the following embodiments can be obtained commercially.

[0023] 2-Hydroxyglutarate (2-HG) is an abnormal metabolite in organisms. In 2020, Sulkowski et al. reported that 2-HG inhibits α-ketoglutarate (α-KG)-dependent dioxygenases and demethylases, hindering DNA repair (Sulkowski et al., Oncometabolites suppress DNA repair by disrupting local chromatin signaling, Nature, 2020). The latest research by the inventors shows that adding a certain concentration of 2-HG during the post-treatment of Co-60 γ-ray irradiation, as a "radiosensitizer", reduces DNA repair and can activate the response pathway to a certain extent, promote gene expression, and improve the mutation efficiency. At the same time, Vc is added as an antioxidant during the post-treatment to protect the cell membrane from oxidative damage. Meanwhile, glutathione (GSH) is added as a low-molecular-weight peptide containing sulfhydryl groups to scavenge free radicals caused by ionizing radiation and reduce radiation damage.

[0024] The method of the present invention uses Co-60 γ-ray irradiation to mutagenize the imbibed seeds of Dendrocalamus brandisii, and through specific pre-irradiation pretreatment (adding L-hydroxyproline as an inducer) and post-irradiation treatment (adding 2-HG as a radiosensitizer, adding GSH and Vc as radiation protectors), effectively improves the mutagenesis efficiency of Dendrocalamus brandisii. Mutant plants with excellent traits are screened and can be used as new germplasms or new varieties of bamboo.

[0025] Example 1. Semi-lethal dose (LD50) of Co-60 γ-ray mutagenesis of imbibed seeds of Phyllostachys edulis

[0026] Dendrocalamus brandisii (Munro) Kurz belongs to Dendrocalamus.

[0027] The tested seeds of Dendrocalamus brandisii (see Figure 1 ) were obtained commercially (the direct provider is Yang Tingfu, contact information: phone: 13890356248), which were new seeds harvested in September 2020, collected in Yunnan Province, with a thousand-grain weight of about 28 g, and stored in a 4°C refrigerator for later use after collection.

[0028] Set 2 kinds of irradiation pretreatment solutions for treatment:

[0029] 1. Irradiation pretreatment solution with L-hydroxyproline added: Magnesium chloride (MgCl2) and L-hydroxyproline (Hyp) were added to 10 mM MES buffer to prepare irradiation pretreatment solution 1 with a pH value of 6.5. The concentration of magnesium chloride in irradiation pretreatment solution 1 was 5 mM, and the concentration of L-hydroxyproline was 5 mM. That is, irradiation pretreatment solution 1 consisted of a solute and a solvent. The solvent was 10 mM MES buffer with a pH value of 6.5, and the solute and its content were 5 mM magnesium chloride and 5 mM L-hydroxyproline.

[0030] 2. Irradiation pretreatment solution without L-hydroxyproline added: Magnesium chloride (MgCl2) was added to 10 mM MES buffer to prepare irradiation pretreatment solution 2 with a pH value of 6.5. The concentration of magnesium chloride in irradiation pretreatment solution 2 was 5 mM. That is, the irradiation pretreatment solution consisted of a solute and a solvent. The solvent was 10 mM MES buffer with a pH value of 6.5, and the solute and its content were 5 mM magnesium chloride.

[0031] The seeds of Dendrocalamus brandisii were soaked in irradiation pretreatment solution 1 and irradiation pretreatment solution 2 respectively for 12 h to allow them to fully imbibe water, and the seeds imbibed with the two irradiation pretreatment solutions were obtained for Co-60 γ-ray gradient dose irradiation.

[0032] For Co-60 γ-ray gradient dose irradiation, dose treatments of 0 Gy (CK), 50 Gy, 80 Gy, 100 Gy, 120 Gy, and 150 Gy were set. For each dose treatment, 200 seeds of the seeds imbibed with each irradiation pretreatment solution were used, with 3 replicates.

[0033] To determine the effects of different radiation doses and pretreatment methods on the semi-lethal dose, the treated seeds were directly placed in a germination box containing filter paper (since this experiment was to determine the semi-lethal dose of irradiated and imbibed seeds of Phyllostachys edulis, no post-irradiation treatment solution was added), and the filter paper was kept moist. The germination rate was counted after 30 d.

[0034] Germination rate (%) = (Number of germinated seeds / Number of treated seeds) × 100.

[0035] The SPSS software was used to perform a regression analysis on the germination rates of different dose treatments. The semi-lethal dose (LD50) was calculated. The results are shown in Table 1:

[0036] Table 1 Radiation dose and germination rate of Co-60 γ-ray mutagenized seeds of Dendrocalamus brandisii (t test, p < 0.01)

[0037]

[0038] As can be seen from the statistical results in Table 1, with the increase of radiation dose, the germination rate of Dendrocalamus brandisii seeds generally shows a downward trend. At the same radiation dose, except for the treatments of 0 and 150 Gy, adding 5 mM Hyp (L-hydroxyproline) during the seed imbibition process has no significant effect on the seed germination rate.

[0039] Taking the radiation dose as X and the relative germination rate at different doses as Y, a regression equation was established and the semi-lethal dose LD50 when the germination rate was 50% was calculated. The germination rate regression equation for the imbibed seeds without adding Hyp was y = -0.4555X + 96.17, while the germination rate regression equation under the treatment of 5 mM Hyp was y = -0.4522X + 93.50, and the corresponding LD50 were 101.4 Gy and 96.2 Gy respectively. Based on this, the suitable radiation dose for the subsequent experiments of Dendrocalamus brandisii seeds treated with 5 mM Hyp imbibition was set at 96 Gy.

[0040] Example 2. Phenotypic mutation frequencies after different post-irradiation treatments

[0041] Soak the Dendrocalamus brandisii seeds in the irradiation pretreatment solution 1 containing L-hydroxyproline (the irradiation pretreatment solution 1 consists of a solute and a solvent. The solvent is 10 mM MES buffer with a pH value of 6.5, and the solute and its content are 5 mM magnesium chloride and 5 mM L-hydroxyproline) for 12 h to make them fully imbibed, and obtain the imbibed seeds, and conduct Co-60 γ-ray irradiation with a radiation dose of 96 Gy.

[0042] After the irradiation treatment, the seeds were transferred to the post-irradiation treatment solution 1, the post-irradiation treatment solution 2, and the post-irradiation treatment solution 3 for treatment respectively.

[0043] Post-irradiation treatment solution M0 (without 2-HG): Add glutathione (GSH) and vitamin C (Vc) to 10 mM MES buffer to prepare the post-irradiation treatment solution M0 with a pH value of 6.5. The concentration of glutathione in the post-irradiation treatment solution M0 is 100 mM, and the concentration of vitamin C is 200 mM. That is, the post-irradiation treatment solution M0 consists of a solute and a solvent. The solvent is 10 mM MES buffer with a pH value of 6.5, and the solute and its content are 100 mM glutathione and 200 mM vitamin C.

[0044] Post-irradiation treatment solution M10 (containing 10 μm 2-HG): 2-Hydroxyglutarate (2-HG), glutathione (GSH), and vitamin C (Vc) were added to 10 mM MES buffer to prepare the post-irradiation treatment solution M10 with a pH value of 6.5. The concentration of 2-hydroxyglutarate in the post-irradiation treatment solution M10 was 10 μm, the concentration of glutathione was 100 mM, and the concentration of vitamin C was 200 mM. That is, the post-irradiation treatment solution M10 consisted of a solute and a solvent. The solvent was 10 mM MES buffer with a pH value of 6.5, and the solute and its content were 10 μm 2-hydroxyglutarate, 100 mM glutathione, and 200 mM vitamin C.

[0045] Post-irradiation treatment solution M20 (containing 20 μm 2-HG): 2-Hydroxyglutarate (2-HG), glutathione (GSH), and vitamin C (Vc) were added to 10 mM MES buffer to prepare the post-irradiation treatment solution M20 with a pH value of 6.5. The concentration of 2-hydroxyglutarate in the post-irradiation treatment solution M20 was 20 μm, the concentration of glutathione was 100 mM, and the concentration of vitamin C was 200 mM. That is, the post-irradiation treatment solution M20 consisted of a solute and a solvent. The solvent was 10 mM MES buffer with a pH value of 6.5, and the solute and its content were 20 μm 2-hydroxyglutarate, 100 mM glutathione, and 200 mM vitamin C.

[0046] A total of 7 groups of treatments were set:

[0047] CK: Control of seeds without irradiation.

[0048] M0-3: After irradiation treatment, the seeds were soaked in the post-irradiation treatment solution M0 (without 2-HG) at 25 °C in the dark for 3 hours.

[0049] M0-6: After irradiation treatment, the seeds were soaked in the post-irradiation treatment solution M0 (without 2-HG) at 25 °C in the dark for 6 hours.

[0050] M10-3: After irradiation treatment, the seeds were soaked in the post-irradiation treatment solution M10 (containing 10 μm 2-HG) at 25 °C in the dark for 3 hours.

[0051] M10-6: After irradiation treatment, the seeds were soaked in the post-irradiation treatment solution M10 (containing 10 μm 2-HG) at 25 °C in the dark for 6 hours.

[0052] M20-3: After irradiation treatment, the seeds were soaked in the post-irradiation treatment solution M20 (containing 20 μm 2-HG) at 25 °C in the dark for 3 hours.

[0053] M20-6: Seeds after irradiation were soaked in the post-irradiation treatment solution M20 (containing 20 μm 2-HG) at 25 °C in the dark for 6 hours.

[0054] For each treatment, 200 seeds were used and repeated 3 times.

[0055] The treated seeds were planted in the substrate (field soil: peat = 5:1) for cultivation, and the surviving plants were planted in the field. The phenotypic traits such as lateral buds, stems, sheaths, internode lengths, and plant heights of the mutagenized plants in the second year of growth were observed and recorded, and the mutation frequency was calculated.

[0056] The mutation frequency was calculated as = number of phenotypic variations / number of surviving plants × 100%.

[0057] The results are shown in Table 2, and the photos of some plants are shown in Figure 2 .

[0058] Table 2 Phenotypic mutation frequencies of plants under different post-irradiation treatments

[0059]

[0060]

[0061] In Table 2, * indicates significant difference (P < 0.05), and ** indicates extremely significant difference (P < 0.01).

[0062] The results showed that radiation mutagenesis caused phenotypic trait variations in Dendrocalamus brandisii plants, and the variable traits were diverse (see Figure 2 ). Compared with the M0 treatment, adding 10 - 20 μm 2-HG (DNA repair inhibitor) in the post-irradiation treatment had a certain effect on the emergence rate, but the phenotypic mutation frequency of the surviving mutagenized plants increased by 19% - 44% (Table 2). Among them, when the treatment time of 10 μm 2-HG (M10) increased from 3 h to 6 h, the total phenotypic variation rate increased by 17.4%, of which the growth-type variation frequency increased by 9.4% and the reduction-type variation frequency increased by 7.7%. When the treatment time of 10 μm 2-HG (M10) was 6 h, the mutagenesis rate was the highest, reaching 72.5%, which was higher than the variation frequency of 20 μm 2-HG treatment (M20) (47.8% - 52.8%).

[0063] Therefore, the treatment effect was the best when the seeds after irradiation were treated with the post-irradiation treatment solution M10.

[0064] The control (CK, non-irradiated plants), and irradiated plants with obvious phenotypic variations obtained under the treatments of M0, M10-3 (M10 treatment for 3 hours), and M10-6 (M10 treatment for 6 hours) were selected respectively, and genomic DNA was extracted by the CTAB method. Eight pairs of primers with good polymorphism were screened out from 122 EST-SSRs to analyze the effectiveness of radiation mutagenesis of Dendrocalamus brandisii.

[0065] The EST-SSR molecular marker technology is as follows:

[0066] (1) Extraction of genomic DNA from leaves. The specific method is as follows: Take 0.5 g of fresh leaves and pre-cool them in liquid nitrogen, then put them into a 1.5 mL centrifuge tube, add a small amount of liquid nitrogen, and quickly and evenly grind them into powder. Add 500 μL of CTAB buffer and mix it with the ground material. Incubate in a water bath at 65 °C for 30 min, gently shake and mix every 5 min in the middle. Centrifuge at 15 °C and 12,000 rpm for 10 min. Take the supernatant into a new 1.5 mL centrifuge tube, add 500 μl of chloroform-isoamyl alcohol mixture (chloroform:isoamyl alcohol = 24:1), mix well, and centrifuge at 15 °C and 12,000 rpm for 10 min. Add an equal volume of isopropanol to the supernatant and mix well, then precipitate at low temperature for 20 min. Pour out all the liquid, add 500 μl of 70% ethanol to wash the precipitate for 2 min, pour out the supernatant, and continue to add 100 μl of 70% ethanol solution to wash, repeat twice. Dry in a sterile fume hood, add an appropriate amount of 40 μl of TE to dissolve the precipitate, and store the sample at -20 °C.

[0067] (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification with EST-SSR primers;

[0068] Eight pairs of EST-SSR primers in Table 3 were used respectively.

[0069] Table 3 EST-ISSR primer sequences and amplification results

[0070]

[0071] Reaction system (20 μL): 0.9 μL of dNTP mixture solution (2.5 mM each), 5.0 μL of 2×GC buffer (2.5 mM), 0.2 μL of rTaq DNA polymerase solution (5 U / μL), 2.0 μL of genomic DNA, 0.5 μL of primer F solution (10 μM), 0.5 μL of primer R solution (10 μM), and the balance is ddH2O. 2×GC buffer (2.5 mM): Takala company;

[0072] Reaction conditions: 95 °C for 5 min; 95 °C for 30 s, 53 °C for 30 s, 72 °C for 40 s, 34 cycles; 72 °C for 5 min; store at 4 °C;

[0073] (3) Take the PCR amplification product obtained in step (2) and perform 1.2% agarose gel electrophoresis.

[0074] The results are shown in Figure 3, it can be seen from the DNA polymorphism amplification results that the amplified bands of the mutant plants after radiation treatment are quite different. The polymorphism of the 8 selected primers ranges from 31.58% to 72.73%. In particular, the polymorphism amplified by primer P-11 is the highest, reaching 72.73%.

[0075] The results amplified by 8 primers were standardized to 0-1 for the similarity coefficient between lines, and cluster analysis was performed using SPSS. The results are shown in Figure 4 , it can be found that among the 31 mutant strains, except for M20-6-2, M0-7, M0-3 and M10-3-2 clustering with CK in one branch, other mutant strains have significant genetic differences from CK. This indicates that 2-HG can be used as a DNA repair inhibitor to induce random mutations in DNA fragments, resulting in a relatively high mutation rate, laying a foundation for breeding new germplasm resources.

[0076] The above has described the present invention in detail. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any variations, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A composition for Co-60 γ-ray irradiation mutagenesis breeding of Dendrocalamus brandisii, characterized in that The composition consists of an irradiation pretreatment solution and an irradiation post-treatment solution: The irradiation pretreatment solution consists of a solute and a solvent. The solvent is 10 mM MES buffer with a pH value of 6.5, and the solute and its content are 5 mM magnesium chloride and 5 mM L-hydroxyproline; The irradiation post-treatment solution consists of a solute and a solvent. The solvent is 10 mM MES buffer with a pH value of 6.5, and the solute and its content are 10 - 20 μm 2-hydroxyglutaric acid, 100 mM glutathione, and 200 mM vitamin C.

2. The composition according to claim 1, wherein The concentration of 2-hydroxyglutaric acid in the irradiation post-treatment solution is 10 μm.

3. A composition for Co-60 γ-ray irradiation mutagenesis breeding of Dendrocalamus brandisii, characterized in that, The composition is the irradiation post-treatment solution described in Claim 1 or 2.

4. A breeding method of Dendrocalamus brandisii by Co-60 γ-ray irradiation mutagenesis, characterized in that, It includes the steps of soaking the seeds of Dendrocalamus brandisii in the irradiation pretreatment solution described in Claim 1 or 2 until they are fully swollen, irradiating them with Co-60 γ-rays at a semi-lethal dose, and then treating them with the irradiation post-treatment solution described in Claim 1 or 2.

5. The method according to claim 4, wherein The treatment with the irradiation post-treatment solution is soaking in the dark at 25°C for 3 - 6 hours.

6. The method according to claim 5, wherein The treatment with the irradiation post-treatment solution is soaking in the dark at 25°C for 6 hours.

7. The method according to any one of claims 4-6, characterized in that, The semi-lethal dose is 96 Gy.

8. The method according to claim 7, wherein The sweet dragon bamboo is Dendrocalamus brandisii ( Dendrocalamus brandisii (Munro) Kurz).

9. Use of the composition according to any one of Claims 1 - 3 in the Co-60 γ-ray irradiation mutagenesis breeding of Dendrocalamus brandisii.

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