Celosia cristata proton radiation mutation breeding method and application

Abstract

The invention discloses a cockscomb proton radiation mutation breeding method and application, belongs to the field of plant mutation breeding, and aims to better meet the requirements of cockscomb mutation breeding. The method comprises the following steps: (a) placing cockscomb seeds to be treated in an independent space of an irradiation tray, and shielding an aluminum film on the surface of the independent space; then, the tray cover plate is placed on the aluminum film, and the cover plate dividing piece and the base plate dividing piece are arranged correspondingly; finally, the tray cover plate and the tray base plate are connected into a whole through screws, and assembling of the irradiation tray is completed. And (b) placing the irradiation tray filled with the cockscomb seeds to be treated in a vacuum target chamber, and irradiating with protons. According to the method, protons are adopted to carry out cockscomb proton radiation mutation breeding for the first time, the radiation mutation biological effect of the cockscomb is deeply explored, and the mutation breeding method not only can effectively expand the heritable variation spectrum of the cockscomb, but also is beneficial to breeding excellent varieties with novel characters.

Description

Technical Field

[0001] The present application relates to the field of plant mutation breeding, and specifically to a method and application of proton radiation mutation breeding of Celosia cristata. Background Art

[0002] Celosia cristata, belonging to the Amaranthaceae family, is a popular annual herb. Known for its unique flower morphology and bright colors, Celosia cristata is widely planted and appreciated around the world. Its flowers are diverse in shape and rich in color, including red, yellow, pink, etc., bringing rich colors and vivid visual effects to parks, gardens and urban greening. In addition to its high ornamental value, Celosia cristata also shows significant potential in edible and medicinal uses.

[0003] In garden applications, cockscomb shows its unique advantages. Short-stemmed varieties are often used as the base of patterned flower beds, especially in the decoration of large flower beds during various festivals or major events. With its rich colors and unique shapes, it adds a gorgeous and vivid visual effect to the flower beds. Medium and tall-stemmed cockscomb varieties are suitable for arranging courtyards, as the main plants of flower mirrors or small flower beds, or decorated beside courtyard roads, or even planted in the corners of courtyards. When the breeze blows, the corolla sways lightly, adding endless fun and beauty to the environment.

[0004] In order to meet people's demand for novel flower varieties, researchers are constantly exploring innovative breeding techniques. For example, Chinese patent application CN201110078292.7 discloses a method for breeding new varieties of cockscomb using distant hybridization and backcrossing technology, and its operating steps are: a. Select distant parents and perform hybridization and hormone treatment; b. Select hybrids and backcross with another cultivated variety; c. Select hybrid offspring; d. Stabilize the variety (line). This method has the following advantages: 1. The excellent traits of wild species of Celosia argentea are hybridized and combined into new varieties to form new varieties with multiple spikes, such as Qiansuihong; 2. New traits generated from the combination of Celosia argentea and Celosia argentea, such as Hongtalin, Huangtalin, Honghuoju, and Dwarf Multi-Crown; 3. The new variety (line) not only has new ornamental characteristics, but also has strong disease resistance.

[0005] Chinese patent application CN201510196554.8 discloses a method for breeding new varieties of Celosia cristata by using epigenetic principles and distant hybridization, and the steps are as follows: a. distant hybridization of crown cultivars and Celosia chinensis and hormone treatment to obtain progeny F1-1; b. hybridization of crown cultivars and Vietnam red to obtain progeny F1-2; c. composite cross between F1-1 and F1-2 to obtain progeny CCF1; d. backcrossing CCF1 with crown cultivar Celosia cristata; e. artificially controlling the different growth of three types of inflorescences, spike, crown, and phoenix tail, by different temperatures and light, and selecting hybrid offspring for directional breeding; f. after 5 generations of breeding, a new variety of Celosia cristata with three types of inflorescences, spike, crown, and phoenix tail, is stabilized. By adopting this method, a new variety of ornamental Celosia cristata with multiple inflorescences can be bred.

[0006] As mentioned above, although the traditional hybrid breeding method has laid the foundation for the diversification of cockscomb varieties, its progress is relatively slow and it is difficult to quickly meet people's growing demand for novelty and diversity of cockscomb. With the development of science and technology, modern breeding technologies such as radiation mutagenesis, ion implantation, and genetic modification have gradually become an important means to promote the innovation of cockscomb varieties, accelerate the breeding process of new varieties, and expand the possibility of cockscomb breeding.

[0007] Radiation-induced mutagenesis breeding technology has significant advantages in rapidly creating new genotypes and improving specific traits due to its high mutation rate, short breeding cycle, and large random heritability. This technology can significantly increase the mutation rate in a relatively short period of time and create materials with extremely rich genetic variability, thus providing an economical and efficient way for plant breeding. However, the randomness of radiation-induced mutagenesis and the possible deterioration are also its limitations that cannot be ignored. This requires an assessment of the radiation sensitivity of the plant to determine the appropriate radiation dose in order to increase the acquisition rate and survival rate of excellent variant individuals.

[0008] For this reason, a new method and / or device is urgently needed to better meet the needs of Celosia cristata mutation breeding. Summary of the invention

[0009] The invention object of the present application is to provide a method and application of proton radiation induced mutation breeding of Celosia cristata, so as to better meet the needs of Celosia cristata mutation breeding.

[0010] In order to achieve the above purpose, this application adopts the following technical solutions:

[0011] A method for breeding cockscomb by proton radiation mutagenesis, comprising the following steps:

[0012] (a) Irradiation tray assembly

[0013] The Celosia cristata seeds to be processed are placed in an independent space of the irradiation tray, and an aluminum film is placed on the surface of the independent space; then, the tray cover is placed on the aluminum film, and the cover partitions and the chassis partitions are arranged correspondingly; finally, the tray cover and the tray chassis are connected as a whole by screws to complete the assembly of the irradiation tray;

[0014] (b) Proton irradiation

[0015] The irradiation tray containing the Celosia cristata seeds to be treated was placed in a vacuum target chamber and irradiated with protons at a irradiation dose of 1.00×10 15 ions / cm 2 ~1.00×10 17 ions / cm 2 .

[0016] In the step b, during the proton radiation treatment, the maximum temperature on the sample is less than or equal to 33°C.

[0017] In the step a, after the tray cover and the tray bottom plate are connected as one body by screws, the cockscomb seeds to be processed in each independent space are in the corresponding independent space and cannot flow between the independent spaces.

[0018] The irradiation tray comprises a tray chassis, an aluminum film, and a tray cover;

[0019] The tray chassis is circular, and a plurality of chassis dividers are arranged in the opening of the tray chassis, and the chassis dividers divide the tray chassis into a plurality of independent spaces;

[0020] The tray cover includes a cover ring and a cover partition matching the chassis partition, and the cover partition is in multiple numbers;

[0021] The cover plate ring is in a circular shape, and the cover plate partition is arranged inside the cover plate ring. The cover plate partition is connected to the cover plate ring as a whole. The use of this structure can avoid the cover plate partition from blocking the independent space in the tray chassis and meet the irradiation requirements;

[0022] The aluminum film is located between the pallet bottom plate and the pallet cover plate and the aluminum film can shield the independent space;

[0023] The tray bottom plate and the tray cover plate are detachably connected.

[0024] The pallet bottom plate and the pallet cover plate are connected with each other through screws or bolts.

[0025] In the step b, the irradiation tray containing the Celosia cristata seeds to be treated is placed in a vacuum chamber with a vacuum degree of 10 -5Pa vacuum target chamber, irradiated with protons with energy of 1 MeV. The energy of protons after penetrating the 7 μm thick aluminum film is about 620 keV, and the irradiation dose is 3.32×10 15 ions / cm 2 ~1.66×10 16 ions / cm 2 .

[0026] In step b, the irradiation dose is 1.66×10 16 ions / cm 2 .

[0027] In the step b, the irradiation tray is placed in a vertical direction, and the protons pass through the aluminum film of the irradiation tray in a horizontal direction to irradiate the crown flower seeds in the irradiation tray.

[0028] The cockscomb seeds irradiated with protons in step b are taken out, soaked in warm water at 20° C. for 1 hour, and then soaked in a polyethylene glycol solution for 1 hour to complete the soaking treatment.

[0029] The following steps are also included:

[0030] (c) Cockscomb cultivation

[0031] Taking out the cockscomb seeds irradiated by protons in step b, soaking them in warm water, and then soaking them in a polyethylene glycol solution;

[0032] The soaked seeds are sown, and then the cultivated cockscombs are selected and bred.

[0033] Application of the aforementioned Celosia proton radiation mutagenesis breeding method.

[0034] This method was used for the mutation breeding of Celosia cristata.

[0035] In response to the aforementioned problems, the present application provides a method for breeding cockscomb by proton radiation induced mutagenesis.

[0036] On the one hand, proton rays are positively charged particle rays. They are produced by accelerators, and their energy can be precisely controlled. When irradiated by protons, the energy loss of proton rays in the material is relatively even, and the depth distribution is relatively uniform. Compared with other rays, proton rays have higher linear energy transfer (LET) and can produce highly concentrated ions and free radicals in a relatively small area, which causes them to produce localized damage on DNA. Due to its precise control ability, proton rays show potential in targeted mutagenesis.

[0037] At present, the research on radiation-induced mutagenesis breeding of Celosia is relatively limited, especially the systematic research on the sensitivity, dose effect and biological effects of proton irradiation as a radiation source.

[0038] On the other hand, the selection of plant radiation mutagenesis dose is affected by many factors such as plant species, variety, growth stage, organ and radiation source characteristics. There are significant differences in the sensitivity of different test materials to radiation.

[0039] In the field of radiation-induced mutation breeding, dose rate and irradiation method have a significant impact on the efficiency and effect of inducing plant mutations. The dose rate is defined as the rate of transfer of radiation energy per unit time. According to its value, it can be divided into high dose rate (acute irradiation) and low dose rate (chronic irradiation). High-dose rate irradiation can cause rapid and strong biological effects in plants, but the duration and scope of these effects are relatively limited. In contrast, low-dose rate irradiation induces biological effects that last longer and have a wider range of effects.

[0040] The researchers evaluated the effects of dose rate on irradiated plants by conducting in-depth analysis of various characteristics of irradiated plants (such as mortality, growth rate, and fertility). The results revealed that dose rate has a significant effect on the degree of radiation damage to plants. Experiments have shown that although high-dose rate irradiation can more effectively induce plant mutations, it will also cause plants to suffer more serious radiation damage, thereby affecting their survival rate and other growth indicators. On the contrary, low-dose rate gamma-ray irradiation can induce more effective mutations while reducing radiation damage. For example, Nagatomi et al. reported in their study that flower color mutants can be effectively obtained by chronic gamma-ray irradiation of chrysanthemums, demonstrating the application potential of low-dose rate irradiation in plant mutation breeding.

[0041] Repeated irradiation and cumulative irradiation have been proven to be effective methods to increase the mutation rate and expand the mutation spectrum. Annegret et al. combined multiple irradiation and repeated irradiation with in vitro culture technology to successfully screen out commercially valuable mutants from tetraploid sterile peony flowers. Yang Aizhu's research found that by 60 Repeated irradiation of dahlias with Co-γ rays can achieve a higher mutation rate than single irradiation. Shen Shoujiang et al. pointed out that the slow irradiation effect is closely related to the daily irradiation dose. Wen et al. found through research that the mutation of chrysanthemums after progressive irradiation is more obvious.

[0042] These research results not only deepen the understanding of the mechanism of radiation-induced mutagenesis, but also provide effective methods and strategies for using radiation-induced mutagenesis technology to improve crop varieties. By combining high and low dose rate irradiation and repeated and cumulative irradiation strategies, researchers can effectively expand the genetic variation spectrum while controlling the possible negative effects of radiation on plants, providing important technical support for agricultural production and plant breeding research.

[0043] Different flower varieties have different sensitivities to radiation doses. Low doses of radiation can stimulate plant growth, while high doses can inhibit plant growth and even cause plant death. Radiation mutagenesis can cause many changes in flowers, including changes in plant height, leaf number, chlorophyll content, flower color, petal shape and other morphological characteristics. At the same time, radiation can also affect seed germination rate, seedling vitality and survival rate.

[0044] Cockscomb is a widely planted plant with high economic and ornamental value in China. Its variety innovation is of great significance to improving the competitiveness of China's ornamental plant market. This application first proposed the use of protons for proton radiation mutagenesis breeding of Cockscomb, and deeply explored the biological effects of radiation mutagenesis of Cockscomb, determined its sensitivity to different radiation sources and the optimal radiation dose, which can not only effectively expand the genetic variation spectrum of Cockscomb, but also help to cultivate excellent varieties with novel traits.

[0045] The experimental results show that the effect of proton irradiation on the growth and development of Celosia cristata also has a dose effect. 15 ions / cm 2 and 1.11×10 16 ions / cm 2 The germination rates of the treatments with the doses were significantly lower than those of the control group, decreasing by 19.74% and 25%, respectively, while the 1.66×10 16 ions / cm 2 There was no significant difference between the dose treatment and the control group. With the increase of irradiation dose, the number of roots per plant, root length, plant height and stem diameter decreased significantly. The number of leaves was 3.32×10 15 ions / cm 2 The dose increased significantly by 36.36% and then decreased with the increase of dose. 16 ions / cm 2 There was no significant difference between the control group and the control group at the dose of 1.11×10 16 ions / cm 2 The dose effect was the most significant.

[0046] The length, width and height of the corolla decreased with the increase of irradiation dose, and the degree of variation increased. The high dose had a more significant effect. The petal chromaticity analysis showed that irradiation increased the L* value, the distribution of a* and b* values ​​became more discrete, and the flower color changed from bright orange-red to pink and purple tones. The color diversity increased, and the higher the dose, the more obvious the change. The effect of proton irradiation on the number of flowers per plant and the number of flower clusters at the top showed a "double saddle" nonlinear trend, 1.11×10 16 ions / cm 2When the dose dropped to the minimum, irradiation significantly increased the variation of flowering number. In terms of physiology and biochemistry, with the increase of irradiation dose, the chlorophyll a content did not change much, the chlorophyll b content increased slightly, and the chlorophyll a / b ratio first decreased and then increased. The soluble protein and sugar content was 1.11×10 16 ions / cm 2 The malondialdehyde (MDA) content showed a trend of first increasing and then decreasing, 1.11×10 16 ions / cm 2 The peak value was reached at the dose of 3.32×10 15 , 1.11×10 16 and 1.66×10 16 ions / cm 2 The mutation rates of different dosages were 39.76%, 34.67% and 53.57%, which were much higher than the 8.57% of the control group. The mutation types involved the morphology and color variation of stems, leaves and inflorescences, and different dosages showed different characteristics.

[0047] The inventors found through comparative experiments that: 60 Co-γ-ray irradiation, proton irradiation and N + There are obvious differences in the effects of proton irradiation on the mutagenic effect of Celosia cristata. Among them, in terms of mutagenic efficiency, proton irradiation at 1.66×10 16 ions / cm 2 The best performance was achieved at the dose of N, with the mutation rate induced reaching 53.57% and the number of surviving strains being 56; + Injection at 6×1016ions / cm 2 The mutation rate induced by the dose was 40.54%, and the number of surviving strains was 37, which was second only to proton irradiation. + The distribution of mutations induced by injection was relatively balanced, covering stems, leaves, inflorescences and other aspects, while the mutations induced by γ-ray irradiation were relatively concentrated in inflorescences; in terms of the number of excellent mutants created, 26 strains were obtained by proton irradiation, and N + 14 strains were obtained by injection. 60 Eleven strains were obtained by Co-γ-ray irradiation, indicating that proton irradiation is the most efficient in creating new germplasm of Celosia cristata.

[0048] Furthermore, the inventors believe that the selection of an appropriate radiation mutagenesis dose requires a balance between the mutagenesis efficiency and the plant survival rate. The dose needs to be high enough to obtain effective genetic variation, but not too high to cause large-scale death of plant cells. Generally, a higher survival rate (e.g., 50%-60%) is conducive to the screening and identification of mutants. Under this premise, a radiation dose that can maintain a higher mutagenesis rate should be selected as much as possible to improve the mutagenesis efficiency. Therefore, the proton irradiation of the present application is the preferred method for radiation mutagenesis breeding of Celosia cristata. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0050] Figure 1 The irradiation trays filled with plant seeds and the temperature monitoring diagram of the trays during irradiation; Figure 1 a is a picture of the irradiation tray filled with plant seeds. Figure 1 b is Figure 1 a Image of the irradiated tray after covering with aluminum film, Figure 1 c is the temperature monitoring diagram of the tray during irradiation.

[0051] Figure 2 This is the effect of different doses of proton irradiation on the germination rate of Celosia cristata in weeks 1-4.

[0052] Figure 3 The effect of different doses of proton irradiation on the germination rate of Celosia cristata.

[0053] Figure 4 This is an electron microscope scan of Celosia cristata leaves after proton irradiation.

[0054] Figure 5 This is the three-dimensional distribution diagram of the chromaticity values ​​of cockscomb petals after proton irradiation at different doses.

[0055] Figure 6 Effects of different doses of proton irradiation on the content of photosynthetic pigments in Celosia cristata.

[0056] Figure 7 Effects of different doses of proton irradiation on the soluble protein and soluble sugar contents of Celosia cristata.

[0057] Figure 8 This is the effect of different doses of proton irradiation on the MDA content of Celosia cristata. DETAILED DESCRIPTION

[0058] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0059] Any feature disclosed in this specification, unless otherwise stated, can be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0060] Example 1

[0061] 1. Test Materials

[0062] 1.3MV tandem electrostatic accelerator (for producing protons).

[0063] 2. Celosia cristata L.cv.'Shengshi'. Capitulate inflorescence, red corolla, 1,000-seed weight 17.5465g.

[0064] 2. Technical Process

[0065] 1. Place the cockscomb seeds in a tray (such as Figure 1 The surface is covered with an aluminum film about 7 μm thick.

[0066] Figure 1 a shows a diagram of an irradiation tray filled with plant seeds; Figure 1 As shown, it includes a tray chassis, an aluminum film, and a tray cover. The tray chassis is circular, and a plurality of chassis dividers are arranged in the opening of the tray chassis, and the chassis dividers divide the tray chassis into a plurality of independent spaces.

[0067] The pallet cover includes a cover ring and a cover divider matched with a chassis divider, and there are a plurality of cover dividers. The cover ring is in a circular ring shape, and the cover divider is arranged inside the cover ring. The cover divider is connected to the cover ring as a whole. With this structure, it is possible to avoid the cover divider from blocking the independent space in the pallet chassis.

[0068] When in use, place cockscomb seeds in the tray, and then cover the surface of the independent space with aluminum film. Then, place the tray cover on the aluminum film, and arrange the cover partitions and the chassis partitions in correspondence. Finally, connect the tray cover and the tray chassis with screws.

[0069] 2. Place the tray with seeds vertically in a vacuum chamber with a vacuum degree of 10 -5 Pa vacuum target chamber, irradiated with 1 MeV protons, the energy of protons after penetrating 7 μm thick aluminum film is about 620 keV. It has been measured that the highest temperature on the sample during radiation treatment does not exceed 33 ° C.

[0070] 3. Set three radiation doses, the radiation doses are 3.32×10 15 , 1.11×10 16, 1.66×10 16 ions / cm 2 Seeds not treated with proton radiation were used as controls.

[0071] 4. Each proton radiation dose was used to treat 50 seeds each time, repeated 3 times, for a total of 450 seeds.

[0072] 5. Before planting, first soak the seeds in 20℃ warm water for 1 hour, and then soak them in 100mg / L polyethylene glycol solution for 1 hour.

[0073] 6. Plant the soaked seeds in the greenhouse of Southwest University of Science and Technology. The size of the flower pots used for planting is: 13 cm high and 15 cm in diameter. The experimental soil is a mixture of garden soil, peat soil and perlite in a ratio of 5:4:1. The flower pots are placed according to a randomized block design and set up three replicates, with 50 seeds per replicate, and a total of 600 seeds are sown (including seeds in the control group).

[0074] 7. At a specific time before the flowering of cockscomb, the seed germination rate, plant survival rate, plant height, stem thickness, number of leaves per plant, leaf length, leaf width, and number of leaf variants were counted. After flowering, the number of flowers, corolla diameter, petal width, petal length, corolla height, corolla width, and petal chromaticity were counted. The leaf surface morphology was observed using a cryo-electron microscope. Chlorophyll content, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase (CAT) activity were determined (see Appendix IV for detailed experimental methods).

[0075] 8. The results of each index in this test are expressed as mean ± standard deviation and significance. Three replicates are set for each index, and each replicate is at least 3 parallels. Data statistics are performed using Microsoft Excel 2010 software, and the degree of difference between treatments is tested using SPSS software. The difference analysis between groups is performed using one-way analysis of variance and T test. Microsoft Excel 2010 software is used for drawing.

[0076] 3. Experimental Results

[0077] 1. Effect of proton irradiation on germination of Celosia cristata

[0078] In the 1st, 2nd and 3rd week, the control group (0.00ions / cm 2 ) had no significant difference in germination rate compared with the irradiation treatment group. Figure 2 As shown, at week 4, 3.32×10 15 , 1.11×10 16 ions / cm 2 The germination rate of the treatment group was significantly lower than that of the control group, 1.66×1016 ions / cm 2 There was no significant difference between the treatment group and the control group.

[0079] Figure 3 The effects of different doses of proton irradiation on the germination rate of Celosia cristata are given. Figure 3 As shown in the figure, the germination rate of Celosia cristata showed a more complex trend with the change of irradiation dose. This nonlinear pattern shows that with the increase of dose, the germination rate first decreased and then increased.

[0080] 2. Effects of proton irradiation on the roots of Celosia cristata

[0081] 3.32×10 15 , 1.11×10 16 , 1.66×10 16 ions / cm 2 The number of roots per plant and the root length of the dose treatment group were significantly lower than those of the control group.

[0082] Table 1 Effects of different doses of proton irradiation on the root number and root length of Celosia cristata

[0083] <![CDATA[Dose ions / cm 2 > 0 <![CDATA[3.32×10 15 ]]> <![CDATA[1.11×10 16 ]]> <![CDATA[1.66×10 16 ]]> Number of roots per plant 40.5±5.78a 32.5±11.15b 33.7±7.24b 28.2±5.49b Variation of root number per plant 35～53 16～49 24～45 20～36 Root length / cm 5.11±1.21a 3.42±0.66b 3.52±0.6b 4.18±1.51b Root length variation 3.03～7.05 2.23～4.35 2.36～4.34 1.32～6.61

[0084] 3. Effect of proton irradiation on stem growth of Celosia cristata

[0085] Proton irradiation 3.32×10 15 , 1.11×10 16 , 1.66×10 16 ions / cm 2 The plant height and stem diameter of the dose treatment group were significantly lower than those of the control group.

[0086] Table 2 Effects of different doses of proton irradiation on stem diameter and plant height of Celosia cristata

[0087]

[0088] 4. Effects of proton irradiation on the growth of Celosia cristata leaves

[0089] 3.32×10 15 ions / cm 2 Under the dosage, the number of leaves per plant increased significantly, and the leaf length and width decreased significantly compared with the control group. 16 ions / cm 2 Under the dosage, the number of leaves per plant did not change significantly, but the leaf length and leaf width decreased significantly. 16 ions / cm 2 Under different dosages, the number of leaves and leaf width per plant did not differ significantly, but the leaf length decreased significantly. 15 , 1.11×1016 ions / cm 2 Under the dosage, the leaf area decreased significantly, and the ratio of leaf width to leaf length did not change significantly. 16 ions / cm 2 Under the different dosages, the ratio of leaf width to leaf length increased significantly, and the leaf area decreased significantly.

[0090]

[0091] 5. Effect of proton irradiation on the surface morphology of Celosia cristata leaves

[0092] Figure 4 The electron microscope scanning image of Celosia cristata leaves after proton irradiation is given. Figure 4 a1- Figure 4 a4 represents the scanning electron micrograph of leaves in the control group: Figure 4 a1. Figure 4 a2 shows the leaf surface of the control group (×700); Figure 4 a3. Figure 4 a4 shows the stomata of leaves in the control group (×2000);

[0093] Figure 4 b1- Figure 4 b4 represents 3.32×10 15 ions / cm 2 Proton irradiated leaf scanning electron microscope image: Figure 4 b1. Figure 4 b2 is 3.32×10 15 ions / cm 2 Proton irradiation of leaf surface (×700); Figure 4 b3. Figure 4 b4 is 3.32×10 15 ions / cm 2 Proton irradiated leaf stomata (×2000);

[0094] Figure 4 c1- Figure 4 c4 represents 1.11×10 16 ions / cm 2 Proton irradiated leaf scanning electron microscope image: Figure 4 c1. Figure 4 c2 is 1.11×10 16 ions / cm 2 Proton irradiation of leaf surface (×700); Figure 4 c3, Figure 4 c4 is 1.11×10 16 ions / cm 2 Proton irradiated leaf stomata (×2000);

[0095] Figure 4 d1- Figure 4 d4 represents 1.66×10 16 ions / cm 2 Proton irradiated leaf scanning electron microscope image: Figure 4 d1, Figure 4 d2 is 1.66×10 16 ions / cm 2 Proton irradiation of leaf surface (×700); Figure 4 d3, Figure 4 d4 is 1.66×10 16 ions / cm 2 Proton irradiated leaf stomata (×2000).

[0096] Under electron microscope observation, the leaves of Celosia scomb in the control group were slightly wrinkled, with fewer stomata, most of which were open (a1-a4). 15 ions / cm 2 After proton irradiation, the leaf surface wrinkles increased and deepened, the number of stomata increased (b1, b2), the size of some stomata slightly increased, and the degree of stomatal closure increased (b3, b4). 16 ions / cm 2 After proton irradiation, the wrinkles on the leaves of Celosia cristata became deeper (c1, c2), the stomata morphology became more three-dimensional and the stomata size became significantly larger, which was significantly different from the control group (c3, c4). 16 ions / cm 2 After proton irradiation, the wrinkle degree of Celosia cristata leaves ranged from 1.11×10 16 ions / cm 2 There was a difference between the treatment group and the control group (d1, d2), and the grooves between the folds widened, the number of stomata increased, and the degree of stomatal closure deepened (d3, d4).

[0097] Under electron microscope observation, the stomata edges of the cockscombs in the control group were relatively smooth and mostly open (a3, a4). 15 ions / cm 2 After proton irradiation, the pores became flatter, the size of some pores increased slightly, and the pore edges became irregular. 16 ions / cm 2 After proton irradiation, the pore morphology becomes more three-dimensional, the pores are visually thickened, and the pore openings are more obvious, and the internal morphology of the pores can be clearly seen (d3, d4). 16 ions / cm 2After proton irradiation, the degree of stomatal closure increased and the stomatal shape became clearer.

[0098] 6. Effect of proton irradiation on the corolla size of Celosia cristata

[0099] As shown in Table 4, with the increase of proton irradiation dose, the length, width and height of cockscomb flower corolla showed a downward trend. 15 ions / cm 2 After irradiation with 1.11×10 16 ions / cm 2 When the highest dose was 1.66×10 16 ions / cm 2 After irradiation, the length, width and height of the corolla of Celosia were significantly lower than those in the control group.

[0100]

[0101] 7. Effect of proton irradiation on the flowering of Celosia cristata

[0102] When the irradiation dose is 3.32×10 15 ions / cm 2 When the number of flowers per plant and the number of flowers in the top flower cluster were not significantly different from those in the control group. 16 ions / cm 2 The number of flowers per plant and the number of flowers in the top cluster were significantly lower than those in the control group. 16 ions / cm 2 When the number of flowers per plant was 1.57 × 10 cm, it was not significantly different from that in the control group, while the number of flowers in the top cluster remained at a low level.

[0103]

[0104] 8. Effect of proton irradiation on the chromaticity of Celosia cristata petals

[0105] Figure 5 The three-dimensional distribution diagram of chromaticity value of Celosia cristata petals after proton irradiation with different doses is given.

[0106] Control group (0ions / cm 2 ) had relatively consistent petal colors and higher saturation. Compared with the control group, 3.32×10 15 ions / cm 2The red-green and yellow-blue values ​​of the irradiated group were more dispersed, and the saturation range increased compared with the control group. 16 ions / cm 2 The distribution of chromaticity values ​​changes more obviously. Although the saturation of the petals increases in amplitude, the average value does not change much. 16 ions / cm 2 When the color is adjusted, it can be observed that the color changes tend to be consistent and the saturation decreases.

[0107] Table 6 Effect of different doses of proton irradiation on the color difference of Celosia cristata

[0108]

[0109] Table 7 Effect of different doses of proton irradiation on the chromaticity value of Celosia cristata

[0110]

[0111] 9. Effect of proton irradiation on the content of photosynthetic pigments in Celosia cristata

[0112] Figure 6 The effects of different doses of proton irradiation on the contents of photosynthetic pigments in Celosia cristata are given.

[0113] After irradiation, 3.32×10 15 ions / cm 2 The dosage slightly decreased the chlorophyll a content, increased the chlorophyll b content, and decreased the ratio of chlorophyll a to b. Increasing the dosage to 1.01×1016ions / cm 2 When the chlorophyll a content was the same as the control group, the chlorophyll b content increased, and the ratio of chlorophyll a to b decreased. The highest dose was 1.66×10 16 ions / cm 2 The chlorophyll a content increased, the chlorophyll b content remained the same, and the ratio of chlorophyll a to b increased. 16 ions / cm 2 The carotene content in the dose treatment groups did not change much.

[0114] 10. Effects of proton irradiation on the content of primary metabolites in Celosia cristata

[0115] Figure 7 The effects of different doses of proton irradiation on the contents of soluble protein and soluble sugar in Celosia cristata were given.

[0116] The soluble protein content of the control group was 5.57 mg / g, and the soluble sugar content was 0.38 mg / g. As the proton irradiation dose increased to 3.32×10 15ions / cm 2 , the soluble protein content increased to 6.90 mg / g, and the soluble sugar content increased to 0.60 mg / g. 16 ions / cm 2 , the soluble protein content slightly increased to 6.94 mg / g, and the soluble sugar content increased to 0.75 mg / g. 16 ions / cm 2 The soluble protein content decreased to 6.49 mg / g, and the soluble sugar content also decreased to 0.56 mg / g. The soluble protein and soluble sugar contents of the irradiation treatment groups at each dose were significantly higher than those of the control group.

[0117] 11. Effect of proton irradiation on the content of malondialdehyde in Celosia cristata

[0118] Figure 8 The effects of different doses of proton irradiation on the MDA content in Celosia cristata are given.

[0119] The MDA content showed a trend of first increasing and then decreasing under different proton irradiation doses. 15 ions / cm 2 , the MDA content increased slightly. When the dose increased to 1.11×10 16 ions / cm 2 The MDA content increased significantly when 16 ions / cm 2 Under the dose, the MDA content decreased but did not reach a significant level.

[0120] 12. Effect of proton irradiation on antioxidant enzyme activities in Celosia cristata

[0121] For SOD activity, at 1.11×10 16 ions / cm 2 At the radiation dose of , the activity was significantly increased compared with the control group. The activity of POD and CAT was 1.66×10 16 ions / cm 2 The most significant effect was observed at radiation doses of .

[0122] Table 8 Effects of different doses of proton irradiation on antioxidant enzyme activities of Celosia cristata

[0123]

[0124] 13. Phenotypic variation of Celosia cristata after proton irradiation

[0125] In the proton irradiation experiment on Celosia cristata, as the irradiation dose increased, the phenotypic variation rate increased significantly compared with the non-irradiated control group.

[0126] Table 9 Phenotypic variation and frequency after different doses of proton irradiation

[0127]

[0128] 14. Construction of a mutant library of Celosia cristata plants after proton irradiation

[0129] This experiment obtained 25 excellent mutants with obvious variation and great ornamental value. The variation types included leaf morphology, inflorescence type variation, inflorescence structure variation, inflorescence color variation, etc., see Table 10.

[0130] Table 10 Characteristics of excellent mutants of Celosia cristata M1 after proton irradiation

[0131]

[0132]

[0133]

[0134]

[0135] IV. Appendix

[0136] 1. The determination method of biological traits of Celosia cristata is shown in Table 11.

[0137] Table 11 Statistics and determination methods of biological traits of Celosia cristata

[0138]

[0139] Germination rate (%) = number of seeds germinated / number of seeds sown × 100%

[0140] Survival rate (%) = final number of surviving plants / number of seeds sown × 100%

[0141] Flowering plant rate (%) = number of flowering plants / total number of plants × 100%

[0142] Red-green color difference Δa * =a * Treatment group-a * Control group

[0143] Yellow-blue difference Δb * =b * Treatment group-b * Control group

[0144] Lightness color difference ΔL * =L* Treatment group-L * Control group

[0145] Total color difference ΔE * =[(ΔL * ) 2 +(Δa * ) 2 +(Δb * ) 2 ] 1 / 2

[0146] Correlation between germination rate and irradiation dose: Taking different irradiation doses x as the independent variable and germination rate y under different doses as the dependent variable, the linear regression equation y=a+bx (where b is the regression coefficient and a is a constant) is used to calculate the germination dose of Celosia cristata.

[0147] The correlation coefficient formula was used to calculate the correlation coefficient between germination rate and irradiation dose, and a correlation graph between germination rate and irradiation dose was drawn. The correlation coefficient formula is as follows:

[0148]

[0149] 2. Leaf surface morphology observation

[0150] Leaves treated with different radiation doses were sampled and treated in a freeze dryer model FD-1A-50 for 7-10 hours to dry the leaves to almost no moisture, and then sent to the analysis and testing center for electron microscopy scanning. All samples were sprayed with gold using SC7620, and then observed for morphological changes under a tungsten filament scanning electron microscope model EVO18. The surface morphological characteristics of leaves at each dose were observed under an appropriate field of view.

[0151] 3. Determination of chlorophyll and active ingredients in Celosia cristata

[0152] The chlorophyll content was determined by alcohol extraction method; the malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) method; the superoxide dismutase (SOD) activity was determined by nitroblue tetrazolium (NBT) redox method; the peroxidase (POD) activity was determined by guaiacol method; and the catalase (CAT) activity was determined by UV spectrophotometry.

[0153] The present invention is not limited to the above-mentioned specific embodiments, but extends to any new features or any new combination disclosed in this specification, as well as any new method or process steps or any new combination disclosed.

[0154] Those skilled in the art should understand that the above embodiments are only for illustrating the present invention and do not limit the present invention. Any modification, equivalent substitution and change made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0155] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

Claims

1. A method for breeding cockscomb by proton radiation mutagenesis, characterized in that: The steps include: (a) Irradiation tray assembly The Celosia cristata seeds to be processed are placed in an independent space of the irradiation tray, and an aluminum film is placed on the surface of the independent space; then, the tray cover is placed on the aluminum film, and the cover partitions and the chassis partitions are arranged correspondingly; finally, the tray cover and the tray chassis are connected as a whole by screws to complete the assembly of the irradiation tray; (b) Proton irradiation The irradiation tray containing the Celosia cristata seeds to be treated was placed in a vacuum target chamber and irradiated with protons at a irradiation dose of 1.00×10 15 ions / cm 2 ~ 1.00×10 17 ions / cm 2 .

2. The breeding method according to claim 1, characterized in that: The irradiation tray comprises a tray chassis, an aluminum film, and a tray cover; The tray chassis is circular, and a plurality of chassis dividers are arranged in the opening of the tray chassis, and the chassis dividers divide the tray chassis into a plurality of independent spaces; The tray cover includes a cover ring and a cover partition matching the chassis partition, and the cover partition is in multiple numbers; The cover plate ring is in a circular shape, and the cover plate partition is arranged inside the cover plate ring. The cover plate partition is connected to the cover plate ring as a whole. The use of this structure can avoid the cover plate partition from blocking the independent space in the tray chassis and meet the irradiation requirements; The aluminum film is located between the pallet bottom plate and the pallet cover plate and the aluminum film can shield the independent space; The tray bottom plate and the tray cover plate are detachably connected.

3. The breeding method according to claim 1 or 2, characterized in that: In the step b, the irradiation tray containing the Celosia cristata seeds to be treated is placed in a vacuum chamber with a vacuum degree of 10 -5 Pa vacuum target chamber, irradiated with protons with energy of 1 MeV. The energy of protons after penetrating 7 μm thick aluminum film is about 620 keV, and the irradiation dose is 3.32×10 15 ions / cm 2 ~ 1.66×10 16 ions / cm 2 .

4. The breeding method according to claim 3, characterized in that: In step b, the irradiation dose is 1.66×10 16 ions / cm 2 .

5. The breeding method according to claim 1, characterized in that: The cockscomb seeds irradiated with protons in step b are taken out, soaked in warm water at 20° C. for 1 hour, and then soaked in a polyethylene glycol solution for 1 hour to complete the soaking treatment.

6. The breeding method according to any one of claims 1 to 5, characterized in that: The following steps are also included: (c) Cockscomb cultivation Taking out the cockscomb seeds irradiated by protons in step b, soaking them in warm water, and then soaking them in a polyethylene glycol solution; The soaked seeds are sown, and then the cultivated cockscombs are selected and bred.

7. Use of the method for proton radiation-induced mutagenesis breeding of Celosia cristata according to any one of claims 1 to 6.

8. The use according to claim 7, characterized in that: This method was used for the mutation breeding of Celosia cristata.

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