Ionizing radiation breeding method for improving mutation rate of cucumbers

Abstract

The invention belongs to the technical field of mutation breeding, and provides an ionizing radiation breeding method for improving the cucumber mutation rate, which comprises the following steps: A, enabling the radicle end of a cucumber seed to face upwards, and performing radiation treatment by adopting a heavy ion beam 12C6 + to obtain a primary mutation seed; taking out the primary mutagenesis seeds, and performing nitrogen ion implantation treatment to obtain secondary mutagenesis seeds; b, accelerating germination of the secondary mutagenesis seeds to obtain mutagenesis cucumber seedlings; planting and culturing mutagenized cucumber seedlings to obtain M1-generation groups, counting variation types and quantity by taking cucumber seeds which are not subjected to mutagenizing treatment as a contrast, and selfing variation single plants to reserve seeds; c, continuously planting to obtain an M2-generation group, selecting single plants showing the last-generation characters and new variant single plants from the M2-generation group, selfing and reserving seeds, continuously planting to obtain an M3-generation group, selecting single plants showing the last-generation characters from the M3-generation group, selfing and reserving seeds. According to the method, the mutation rate of cucumbers is increased, the genetic stability is high, and the breeding time can be shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of mutation breeding and relates to a mutation breeding method for cucumber. Background Art

[0002] Cucumber is one of the important vegetable crops. Due to natural erosion and long-term directional improvement, the genetic background of cucumber has become increasingly narrow, and its genetic diversity has decreased, which has caused certain obstacles to cucumber breeding work, resulting in cucumber production being extremely susceptible to pests and diseases, and the yield and quality being reduced. Therefore, the innovation of new germplasm is needed.

[0003] Cucumber is a crop with a relatively narrow genetic basis, with a mutation rate of only 3% to 8%. The means of creating new breeding materials are backward and the discovery of excellent new genes is slow. There are currently several solutions to the above problems: 1. Distant hybridization of cucumber with wild cucumber or other crops in the same genus, expanding the genetic basis from their distant hybrid offspring, and constructing new breeding materials; 2. Chemical mutagenesis, using chemical mutagens such as EMS (ethyl methanesulfonate) to treat cucumber seedlings or seeds to cause mutations, and screening positive mutations from their offspring; 3. Molecular design breeding, through gene editing technology or transgenic technology, knock out or insert related gene fragments, and then obtain regenerated plants with specific traits through genetic transformation; 4. 60Co-γ-ray radiation mutagenesis, when the nucleic acid molecules of cucumber are hit by radiation, it causes changes in its DNA structure or breaks. When the broken DNA fragments are repaired, they may be joined in a new way, causing the chromosomes where the DNA is located to be disconnected, and may also cause chromosome duplication, inversion, and deletion, causing the irradiated cucumbers to mutate; in addition, there are methods such as space transportation. However, all of the above schemes have some problems: the distant hybridization in the existing technology will cause reproductive barriers, and the hybrid offspring will not produce seeds, so it needs to be combined with technologies such as embryo rescue, which is a lot of work; the mutagenic agents used in chemical mutagenesis are highly toxic, and the DNA of the test materials is only point mutations, and there is no large-scale breakage and recombination of chromosomes; molecular breeding technology has high requirements and high costs, and the subsequent safety verification is cumbersome and time-consuming; the utilization efficiency of the mutagenic source in 60Co-γ ray mutagenesis is low, radioactive waste is generated, and it is not environmentally friendly; and the traits of the offspring induced by 60Co-γ ray mutagenesis are unstable, and the variant traits are often not inherited; the cost of space transportation is high.

[0004] Heavy ion beams can induce a variety of diverse mutant materials due to their high energy transfer line density and unique ionization peaks. They are centralized and directional, have high energy utilization efficiency, do not produce radioactive waste, and are environmentally friendly new mutagenic sources. In addition, the ionizing radiation dose is easy to accurately control and has strong repeatability. However, different plant species have significant differences in radiation sensitivity to the same mutagenic source, and their sensitivities to different mutagenic sources are also different. However, cucumbers currently have a low mutagenic efficiency problem. Summary of the invention

[0005] The invention provides an ionizing radiation breeding method for improving the mutation rate of cucumbers. The method improves the mutation rate of cucumbers, shortens the breeding time, and successfully breeds new varieties of positive mutations that can be stably inherited.

[0006] The technical solution of the present invention is achieved in this way: An ionizing radiation breeding method for improving the mutation rate of cucumbers comprises the following steps: A. Place the cucumber seeds with the radicle facing upwards and use heavy ion beam 12 C 6+ Radiation treatment was performed to obtain the primary mutagenesis seeds; among them, heavy ion beam 12 C 6+ The implantation dose is 10-80Gy and the ion energy is 80MeV·um -1 , linear energy transfer density LET = 30keV·um -1 , range 17mm, dose rate 60Gy·min -1 ; Take out the primary mutagenesis seeds, place them in an ion implanter, and perform nitrogen ion implantation to obtain secondary mutagenesis seeds; wherein the acceleration current of the ion implanter is 2-3 mA, the ion implantation time is 30 s, and the acceleration voltage of the ion implanter is 30 KeV; wherein the nitrogen ion implantation requires an atmospheric pressure of 8×10 - 4 pa; B. Germinating the secondary mutagenized seeds to obtain mutagenized cucumber seedlings; planting and cultivating the mutagenized cucumber seedlings to obtain the M1 generation population, and taking the unmutated cucumber seeds as the control, recording the agronomic traits, counting the types and quantities of mutations, and self-pollinating the mutant plants for seed preservation; C. Continue to plant the mutant seeds obtained from the mutant plants obtained in step B to obtain an M2 generation population, select plants that exhibit the characteristics of the previous generation and new mutant plants from the M2 generation population, self-pollinate and save seeds, continue to plant, obtain an M3 generation population, select plants that exhibit the characteristics of the previous generation from the M3 generation population, and self-pollinate and save seeds.

[0007] Preferably, the heavy ion beam 12 C 6+ The implantation dose was 60 Gy.

[0008] Preferably, the specific steps of germinating the secondary mutagenized seeds in step B to obtain the induced cucumber seedlings include: soaking the seeds in a warm water of 55°C-60°C for 8-12 minutes, stirring them at a uniform speed during the period, cooling them naturally and soaking them for 5-12 hours, taking them out and washing off the mucus on the surface of the secondary mutagenized seeds with ddH2O, fishing them out and draining them, wrapping them with moist gauze, germinating them at a constant temperature of 30°C, and sowing them in plug trays after 24 hours to obtain the induced cucumber seedlings.

[0009] Preferably, the specific steps of germinating the secondary mutagenized seeds in step B to obtain the induced cucumber seedlings include: soaking the seeds in a warm water of 55°C-60°C for 10 minutes, stirring them at a constant speed during the period, cooling them naturally and soaking them for 8 hours, taking them out and washing off the mucus on the surface of the secondary mutagenized seeds with ddH2O, fishing them out and draining them, wrapping them with moist gauze, germinating them at a constant temperature of 30°C, and sowing them in plug trays after 24 hours.

[0010] Preferably, the cucumber seeds are dry and plump seeds harvested in the current year.

[0011] Preferably, conventional cultivation management is performed during the planting period.

[0012] Preferably, the plug tray V 草炭 :V 珍珠岩 =9:1.

[0013] The working principle and beneficial effects of the present invention are: 1. The present invention treats cucumber seeds with two ion sources at specific doses to cause mutations in their offspring. Through field screening and identification, the genetic basis of cucumbers can be rapidly and safely expanded, genetic resources can be enriched, and new breeding materials can be created.

[0014] 2. The present invention uses HIRFL accelerator and ion implanter to realize the utilization of 12 C 6+ and N + The mutagenesis rate of cucumber seeds was increased. Among them, heavy ions 12 C 6+ With high energy transfer line density and unique ionization peak, N + The present invention has the characteristics of a wide mutation spectrum and easy stabilization of mutant traits. The present invention combines the two methods, and only 2-4 generations of mutant traits are required to achieve genetic stability and a high forward mutation rate, which can induce a variety of mutations in cucumber test materials. The technology has the characteristics of centralized orientation, high energy utilization efficiency, no radioactive waste, and an environmentally friendly new mutagenic source; and the radiation dose of the mutagenic source is easy to accurately control and has strong repeatability. However, the mutagenic source in 60Co-γ ray mutagenesis has low utilization efficiency, produces radioactive waste, and is unfriendly to the environment; and the traits of the offspring induced by 60Co-γ ray are unstable, and the variant traits are often not inherited, etc. The present invention avoids this shortcoming.

[0015] 3. The present invention first adopts 12 C 6+ Mutagenesis, followed by N + Mutagenesis significantly increases the mutagenesis rate and makes it easier to screen out stable and inherited positive mutations. This is because 12 C 6+ Mutagenesis first introduces a large number of mutations, increasing the diversity of the genome, and N +Mutagenesis is based on this, and mild mutations are carried out, which are gradually optimized and finally the ideal mutants are screened out. + Mutagenesis eliminates some undesirable mutations through precise repair mechanisms, thus improving the stability of mutations. + Mutagenesis, again 12 C 6+ Mutagenesis, this method has a higher mutagenesis rate, this is because N + The mutagenesis efficiency of induced mutation is low and may not produce sufficient genetic diversity. + Mutagenesis mainly produces point mutations and deletion or insertion of small fragments, and the mutation types are relatively simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 This is a schematic diagram of placing cucumber seeds of the present invention into a nitrogen ion injection tray.

[0018] Figure 2 Shown are the germination conditions at different nitrogen ion injection times.

[0019] Figure 3 This is a schematic diagram of dried cucumber seeds placed in a single layer in a plastic tray with the radicle end facing upward.

[0020] Figure 4 This is a picture of the cotyledon damage of M1 generation cucumber.

[0021] Figure 5 This is a comparison chart of the growth of M1 generation seedlings.

[0022] Figure 6 This is a partial variation diagram of the M1 generation at the seedling stage.

[0023] Figure 7 The graph shows the dynamic changes in the growth potential of cucumber plants in each treatment group of the M1 generation.

[0024] Figure 8 This is a diagram of the variation types of M1 generation plants.

[0025] Fig. 9 This is a diagram of the fruit variation types of the M1 generation.

[0026] Fig.10 This is a comparison chart of the M2 generation's variable traits.

[0027] Fig.11 This is a picture of the male flower with its petals closed.

[0028] in, Figure 2CK, 10s, 20s, 30s, 40s, 50s, 60s, and 70s respectively represent cucumber seeds without nitrogen ion implantation and cucumber seeds with different ion implantation times (10s, 20s, 30s, 40s, 50s, 60s, and 70s); Figure 5 The upper one in the middle is single radiation, and the lower one is double radiation; Figure 6 In the figure, A, B, C, and D represent leaf wrinkles, leaf mosaic, leaf notch, and two leaves on one petiole, respectively; Figure 7 CK indicates cucumber seeds that have not been subjected to any mutagenic treatment, and 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, and 80Gy indicate different 12 C 6+ Radiation injection doses (10 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy, 70 Gy, and 80 Gy) were given to cucumber seeds. 10 Gy+30s, 20 Gy+30s, 30 Gy+30s, 40 Gy+30s, 50 Gy+30s, 60 Gy+30s, 70 Gy+30s, and 80 Gy+30s represent different 12 C 6+ Cucumber seeds were injected with nitrogen ions for 30s after radiation injection doses (10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, 80Gy); Figure 8 1 means the cucumber stem becomes flat and leaves are opposite; 2 means the leaves are abnormal in color and uneven; 3 means the branching becomes stronger; 4 means double growth points; 5 means the stalk burrs disappear; Fig. 9 ① indicates that the melon thorns are sparse; ② indicates that there are multiple melons in one section; ③ indicates that the melon strips become longer; ④ indicates that the melon handles become shorter; the left side of ③ indicates the control group, and the right side indicates the variant type of melon strip lengthening; Fig.10 In the middle, a represents the comparison of leaf fold variation traits of the M2 generation, the right side represents the control group, and the left side represents the leaf fold variation traits of the M2 generation; b represents the variation trait of melon strip lengthening in the M2 generation, with the left side representing the control group and the right side representing the variation trait of melon strip lengthening in the M2 generation; c represents the variation trait of the M2 generation with increased serration of leaf margins and shortened internodes, the right side represents the control group, and the left side represents the variation trait of the M2 generation with increased serration of leaf margins and shortened internodes; d represents the variation trait diagram of M2 generation melon stripe shortening. The left side represents the control group, and the right side represents the variation trait of M2 generation melon stripe shortening. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 1. Test materials: Dry and plump cucumber seeds harvested in the current year: inbred line HGD-1 (the public can obtain this biological material from the Institute of Biophysics of Hebei University of Technology).

[0031] 2. Single nitrogen ion (N + ) Injection test The nitrogen ion implantation was completed in the ion implantation machine of the Institute of Biophysics, Hebei University of Technology. Dry and plump cucumber seeds were selected and placed in the injection tray (such as Figure 1 ), placed in a vacuum target chamber, nitrogen ion implantation requires an atmospheric pressure of 8×10 -4 pa.

[0032] The number of ions is derived according to the following formula I = Q / t (charge per unit time) I=V×e×N / t N=I×t / V×e I is the acceleration current of the ion implanter, set to 2-3 mA, t is the duration of the acceleration voltage (ion implantation time), V is the acceleration voltage of the ion implanter, set to 30 KeV, and e is the electron constant 1.6×10-9. The number of ions per unit area is N / S, S is the area of ​​the irradiated seed, and the radius is 4 cm according to the size of the injection disk of the ion implanter.

[0033] According to the formula, the dosage can be expressed by the ion injection time. The ion injection time was 10s, 20s, 30s, 40s, 50s, 60s, and 70s, with cucumber seeds without nitrogen ion injection as the control, and a total of 8 experimental treatments were set up.

[0034] After the ion implantation experiment, 50 cucumber seeds from each of the eight experimental treatments were taken, soaked for 6 hours, and then placed in a 30°C incubator for germination. This was repeated three times, and the germination rates were counted after 24 hours.

[0035] In biological radiation, the low-energy ion injection dose (half-lethal dose) that allows half of the plants to survive after radiation treatment is used as an indicator of mutagenic sensitivity. Figure 2 , for the germination conditions at different nitrogen ion injection times, according to Figure 2The statistical results in Table 1 show that as the nitrogen ion injection dose increases, the damage to cucumber seeds will become more and more serious; when the ion injection time is 30s, about half of the seeds die, so the median lethal dose of cucumber is 30s. For cucumbers, when nitrogen ion injection is 30s, the radiation damage to its DNA and its own repair reach a balance, and the probability of mutation in the offspring is the highest, so the optimal nitrogen ion injection time for cucumbers is 30s.

[0036] 3. Secondary mutagenesis seeds Place the dried cucumber seeds in a single layer in a culture dish (such as Figure 3 ), multiple culture dishes were placed in a single layer on the TR4 biological radiation terminal, facing the vacuum sealing film, and the shallow heavy ion beam biological radiation terminal (TR4) of the HIRFL accelerator of the Lanzhou Heavy Ion Accelerator National Laboratory was used to generate 12 C 6+ The beam passes through the nickel window, ionization chamber, and air to irradiate the seed embryo. During the radiation process, the beam intensity is used to count the number of particles. When the predetermined number of particles is reached, the beam switch is turned off to achieve quantitative radiation. Positioning radiation is achieved by moving the sample and beam position. TR4 radiation parameters are: ion energy 80MeV·um -1 , energy transfer line density LET = 30keV·um-1, range 17mm, dose rate 60Gy·min -1 , the radiation injection doses were 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, and 80Gy, with the unirradiated seeds from the same batch as the control; Then the heavy ions 12 C 6+ The irradiated seeds were divided into two parts, one part was not treated, and the other part was injected with nitrogen ions by an ion implanter for mutagenesis (heavy ion 12 C 6+ The irradiated seeds were stored under normal temperature and pressure, and subjected to nitrogen ion mutagenesis treatment again the next day), forming a group containing 16 mutagenesis treatment combinations, namely 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, 80Gy, 10Gy+30s, 20Gy+30s, 30Gy+30s, 40Gy+30s, 50Gy+30s, 60Gy+30s, 70Gy+30s, 80Gy+30s; 120-150 seeds were treated for 3 times, and 30s indicated the nitrogen ion injection time; the seeds subjected to the second mutagenesis treatment were obtained as the second mutagenesis seeds; The nitrogen ion implantation was completed in the ion implantation machine of the Institute of Biophysics of Hebei University of Technology. The specific steps were as follows: the cucumber seeds were placed in an injection tray and placed in a vacuum target chamber. The nitrogen ion implantation required an atmospheric pressure of 8×10-4 pa, the number of ions is derived according to the following formula: I = Q / t (charge per unit time) I=V×e×N / t N=I×t / V×e I is the acceleration current of the ion implanter, which is 2-3 mA (the acceleration current of the ion implanter is unstable and fluctuates within this range), t is the duration of the acceleration voltage (ion implantation time), V is the acceleration voltage of the ion implanter, which is set to 30 KeV, and e is the electronic constant 1.6×10 -9 The number of ions per unit area is N / S, where S is the area of ​​the irradiated seed and the radius is 4 cm according to the size of the injection disk of the ion implanter; According to the formula, the dose can be expressed by the ion implantation time.

[0037] Among them, the shallow heavy ion beam biological radiation terminal (TR4) of the HIRFL accelerator of the Lanzhou Heavy Ion Accelerator National Laboratory is mainly composed of an ion source (ECR), a sector focusing cyclotron (SFC), a separated sector cyclotron (SSC), radioactive beam lines (RIBLL1, RIBLL2), and heavy ion cooling storage rings (CSRm, CSRe). 12 C 6+ The radiation device is debugged and tested as a mutagenic source. When the beam current is stable and uniform, the dried cucumber seeds for test are placed there.

[0038] 4. Obtain the M1 generation population Germination of secondary mutagenesis seeds to obtain mutagenesis cucumber seedlings: The above-mentioned control group and 16 radiation groups were germinated, namely CK, 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, 80Gy, 10Gy+30s, 20Gy+30s, 30Gy+30s, 40Gy+30s, 50Gy+30s, 60Gy+30s, 70Gy+30s, and 80Gy+30s, with 120-150 seeds in each group and repeated 3 times.

[0039] The specific steps include: soaking the seeds of each group in 55℃ warm water for 10 minutes, stirring at a constant speed during the period, cooling naturally and soaking for 8 hours, taking out and washing the mucus on the surface of the seeds with ddH2O, taking out and draining, wrapping with wet gauze and placing in a culture dish, placing in a constant temperature box (30℃) for germination, and sowing in a plug tray after 24 hours to obtain the induced cucumber seedlings; wherein, V in the plug tray 草炭 :V 珍珠岩 =9:1; The mutated cucumber seedlings were planted and cultivated, and conventional cultivation management was performed during the planting period to obtain the M1 generation population. At the same time, the cucumber seeds that were not mutated were used as the control, and the agronomic traits were recorded, and the types and quantities of mutations were counted. The mutant single plants were strictly self-pollinated and kept as seeds; The specific details of routine cultivation management are as follows: (1) Soil requirements for the planting area: sandy loam with sufficient base fertilizer, double-row film-covered cultivation on high ridges, ridge spacing of 80 cm, ridge width of 50-60 cm, and plant spacing of 30-35 cm; (2) After planting, pay attention to temperature and humidity control, maintain 28-32℃ during the day, 15-17℃ in the first half of the night, and 12-14℃ in the second half of the night; (3) Pay attention to timely topdressing and foliar fertilizer application on the basis of ensuring sufficient base fertilizer, especially calcium fertilizer supplementation; (4) Hang vines in time, remove side branches and tendrils to reduce ineffective nutrient consumption, and remove old and diseased leaves in time to facilitate ventilation and light transmission and reduce the occurrence of diseases and insect pests; (5) Harvest in time according to the growth of the plant to maintain a balance between vegetative growth and reproductive growth.

[0040] 5. Effects of radiation on phenotypic traits of M1 5.1 Effects of radiation on the seedling rate and deformed seedling rate of M1 After 7 days of sowing in the plug tray, the number of seedlings and the number of seedlings with deformed cotyledons were counted (some seedlings that failed to germinate were not sown and were not counted in the sowing amount). The results are shown in Table 2 below.

[0041] As can be seen from Table 2 above, after radiation treatment, the seedling rate decreased. When the radiation treatment was 70Gy+30s, the seedling rate was the lowest. The deformed seedling rate increased. The cotyledon deformity rate was the highest when the radiation treatment was 70Gy. The main manifestations were small cotyledons, yellow leaf margins, serrated notches, and slenderness. This shows that a certain dose of radiation can cause certain damage to seeds, which is most directly reflected in the cotyledons (such as Figure 4 , the left and right pictures are both pictures of M1 generation cucumber cotyledon damage).

[0042] 5.2 Effects of radiation on the M1 seedling stage The field traits of plants are another important indicator of the biological effects of radiation-induced mutagenesis, which show certain physiological damage and genetic variation in the radiation period.

[0043] After investigating the morphology of cucumber seedlings, it was found that the growth of 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy, 80Gy, 10Gy+30s, and 20Gy+30s radiation treatments was basically the same; the growth of 30Gy+30s, 40Gy+30s, 50Gy+30s, 60Gy+30s, 70Gy+30s, and 80Gy+30s radiation treatments was basically the same, but the latter was 2-3 days slower than the former. The double treatment of high-dose heavy ion carbon radiation and nitrogen ion injection had a greater effect on the cucumber seedling stage than the single heavy ion carbon radiation, as shown in Figure 2. Figure 5 , is a comparison of the seedling growth of the two (the upper one is single radiation, the lower one is double radiation).

[0044] As cucumbers grow, some of the true leaves of cucumber plants begin to show deformities, such as leaf wrinkles, leaf mosaics, leaf notches, and two leaves on one stalk. Figure 6 , which is a partial variation of the M1 generation at the seedling stage.

[0045] 5.3 Effects of radiation treatment on the growth potential of cucumber plants After the test materials were planted, the height of the cucumber plants was measured on May 11 and May 20, 2022, and the average daily growth height was calculated to measure the difference in plant growth potential. The results are shown below Figure 7 As can be seen from the figure, the growth potential of cucumber plants in each treatment group presents a dynamic change curve. Compared with the control, the 80Gy+30s radiation treatment resulted in the highest growth potential, and the 50Gy+30s radiation treatment resulted in the lowest growth potential of cucumber plants. A certain dose of radiation treatment can change the growth potential of cucumber plants.

[0046] 5.4 Effects of radiation treatment on the vegetative growth of cucumber As the plants grow, some changes in the seedling stage will gradually disappear, and new changes will appear. Compared with the control group, the radiation group has the following changes: 1. The cucumber stem becomes flat and the leaves are opposite; 2. The leaf color is abnormal and the leaves are uneven; 3. The branching becomes stronger; 4. Double growth points; 5. The stalk burrs disappear. Figure 8 It is the variation type of M1 generation plants.

[0047] 5.5 Effects of radiation treatment on commercial cucumber characteristics During the peak fruiting period, the plants in the treatment group were investigated and various types of variation were found in individual plants, such as sparse thorns on the cucumber fruits, longer stems, lighter colors, shorter handles, and multiple fruits on one node. Fig. 9 , which is the fruit variation type of the M1 generation.

[0048] According to statistics (see Table 3), there are more negative mutations in the M1 generation, including wrinkled and uneven leaves, double growth points, and enhanced branching. There are also variations such as opposite leaves and sparse thorns on the stems and melons. There are also positive mutations, including longer melon strips, shorter melon handles, and multiple melons in one section. In the 80Gy+30s dose treatment group, a total of 138 plants with mutations were found, but most of them were negative mutations. In the 60Gy+30s and 70Gy+30s dose treatment groups, the mutation rate was also high, 16%-18.1%, and there were quite a few positive mutations such as shorter melon handles. In the 10Gy+30s dose treatment group, two melon strips had mutations of longer melon strips. According to the statistics of the trait variation of the M1 generation, the mutation rate of the offspring of double radiation is higher than that of single radiation, and there are more types of mutations. The 60Gy+30s double radiation is the appropriate radiation dose for cucumbers. It is more possible to screen out meaningful mutation types from its offspring.

[0049] 6. Effects of radiation on field traits of the M2 generation Select the mutant seeds obtained from all the variant types of individual plants in M1 and continue to plant them to obtain the M2 generation population, specifically: From all the variant plants of M1, 4 plants with opposite leaves, 2 plants with light stripes on the melon, 1 plant with elongated melon stripes, 2 plants with deformed leaves and flowers, 3 plants with sparse melon thorns, 6 plants with wrinkled leaves, 7 plants with shortened melon handles, 4 plants with double growth points, and 2 plants with strong branches were selected, totaling 9 variant types and 31 plants for planting observation in the M2 generation. All variant plants were cultivated into 32 plants respectively, and the variant traits in the M1 generation were observed by comparison to see whether they can be inherited.

[0050] After a growing season of investigation and identification (see Fig.10 , which is the variant trait of the M2 generation). Most variant types in the M1 generation were not inherited in the M2 generation, and all returned to normal traits. This shows that ion radiation can cause certain damage to cucumber DNA, showing various types of mutations in the M1 generation, but they will be repaired during the growth and development process, resulting in the inability to stably inherit the mutations. However, there are two traits in the M2 generation that are consistent with the M1 generation: 1. Among the 6 leaf wrinkle strains planted in the M2 generation, 4 strains maintained this variant trait in the M2 generation ( Fig.10 a), among which the 60Gy+30s radiation dose strain had the highest. However, this variation is a negative variation and cannot be directly applied to breeding, but can be used as a material for cucumber gene research. 2. A strain with lengthened cucumber strips planted in the M2 generation maintained this variation trait in the M2 generation ( Fig.10 b), also the offspring of the 60Gy+30s radiation dose. In addition, a new variant type appeared in the M2 generation of the 60Gy+30s radiation dose, namely, the leaf margins became serrated, the internodes became shorter, and the melon strips became shorter ( Fig.10c, d). This indicates that when DNA is exposed to ionizing radiation, there will be a certain percentage of error repair during the repair process, which also provides a basis for creating new germplasm.

[0051] 7. Effects of radiation on field traits of the M3 generation From the M2 generation population, select individual plants that exhibit the traits of the previous generation and new variant individual plants, self-pollinate and keep seeds, continue to plant, obtain the M3 generation population, select individual plants that exhibit the traits of the previous generation from the M3 generation population, self-pollinate and keep seeds, the specific steps are as follows: The M3 generation planted a single plant of the M2 generation variant type: Ⅰ, wrinkled leaves; Ⅱ, longer melon strips; Ⅲ, deep serrations on the leaf edges and shorter melon strips; Ⅳ, the melon thorns of the M1 generation became sparse, and the M2 generation returned to normal. 50 plants of each variant type were cultivated. After a growing season of observation, the M3 generation of types Ⅰ, Ⅱ, and Ⅲ all showed the characteristics of the previous generation, indicating that the above mutations caused by radiation can be inherited; a single plant with closed male flower petals appeared in the Ⅳ type plant ( Fig.11 , both the left and right pictures show the male flowers with closed petals).

[0052] Subsequently, the M4 generation population was obtained, and the M4 generations of types I, II and III all showed the characteristics of the previous generation.

[0053] 8. Conclusion Heavy ions 12 C 6+ Irradiation and ion implantation of N + Injecting double mutagenesis can induce mutations in cucumber genes. Through two years and three generations of observation, screening and statistics, 60Gy+30s double radiation was determined to be the optimal radiation dose for cucumbers. From its offspring, the following stable heritable variations were screened out: Ⅰ, wrinkled leaves; Ⅱ, longer cucumber strips; Ⅲ, deep serrations on the edges of leaves, shorter cucumber strips, etc., among which Ⅱ and Ⅲ type variations can be directly used as parents for breeding; Ⅰ type variation can be used as material for research on radiation mutagenesis related mechanisms.

[0054] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An ionizing radiation breeding method for improving the mutation rate of cucumbers, characterized in that: The steps include: A. Place the cucumber seeds with the radicle facing upwards and use heavy ion beam 12 C 6+ Radiation treatment was performed to obtain the primary mutagenesis seeds; among them, heavy ion beam 12 C 6+ The implantation dose is 10-80Gy and the ion energy is 80MeV·um -1 , linear energy transfer density LET = 30keV·um -1 , range 17mm, dose rate 60Gy·min -1 ; Take out the primary mutagenesis seeds, place them in an ion implanter, and perform nitrogen ion implantation to obtain secondary mutagenesis seeds; wherein the acceleration current of the ion implanter is 2-3 mA, the ion implantation time is 30 s, and the acceleration voltage of the ion implanter is 30 KeV; B. Germinating the secondary mutagenized seeds to obtain mutagenized cucumber seedlings; planting and cultivating the mutagenized cucumber seedlings to obtain the M1 generation population, and taking the unmutated cucumber seeds as the control, recording the agronomic traits, counting the types and quantities of mutations, and self-pollinating the mutant plants for seed preservation; C. Continue to plant the mutant seeds obtained from the mutant plants obtained in step B to obtain an M2 generation population, select plants that exhibit the characteristics of the previous generation and new mutant plants from the M2 generation population, self-pollinate and save seeds, continue to plant, obtain an M3 generation population, select plants that exhibit the characteristics of the previous generation from the M3 generation population, and self-pollinate and save seeds.

2. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 1, characterized in that: The heavy ion beam 12 C 6+ The implantation dose was 60 Gy.

3. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 1, characterized in that: The specific steps of germinating the secondary mutagenized seeds in step B to obtain the induced cucumber seedlings include: soaking the seeds in a warm water of 55°C-60°C for 8-12 minutes, stirring at a constant speed during the period, cooling naturally and soaking for 5-12 hours, taking out and washing off the mucus on the surface of the secondary mutagenized seeds with ddH2O, taking out and draining, wrapping with moist gauze, germinating at a constant temperature of 30°C, and sowing in a plug tray after 24 hours to obtain the induced cucumber seedlings.

4. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 1, characterized in that: The specific steps of germinating the secondary mutagenized seeds in step B to obtain the induced cucumber seedlings include: soaking the seeds in a 55°C-60°C warm water for 10 minutes, stirring at a constant speed during the period, cooling naturally and soaking for 8 hours, taking out and washing off the mucus on the surface of the secondary mutagenized seeds with ddH2O, taking out and draining, wrapping with moist gauze, germinating at a constant temperature of 30°C, and sowing in a plug tray after 24 hours.

5. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 1, characterized in that: The cucumber seeds are dry and plump seeds harvested in the current year.

6. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 1, characterized in that: Conventional cultivation management was performed during the planting period.

7. The ionizing radiation breeding method for improving the mutation rate of cucumber according to claim 3, characterized in that: The plug tray V 草炭 :V 珍珠岩 =9:1.

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