Sweet osmanthus radiation mutation breeding method

Abstract

The invention relates to an osmanthus fragrans radiation mutation breeding method, and aims to solve the technical problem that a high-quality and high-efficiency osmanthus fragrans mutation breeding system and method are lacked at present. On the basis of long-term experimental research, an appropriate radiation mutagenic agent amount of osmanthus fragrans under < 60 > Co-gamma rays is screened out, a to-be-germinated branch of a perennial osmanthus fragrans arbor which grows robustly is cut in spring to serve as a scion, and grafting cultivation is performed after top end wax sealing so as to construct a mutant library; by combining phenotypic character characteristics (including quantitative characters and quality characters) which are selected after survival of sweet-scented osmanthus and are easy to identify effectively, a rapid radiation mutation breeding system and method taking sweet-scented osmanthus branches as materials are constructed, and a technical guarantee is provided for creating new sweet-scented osmanthus germplasm by utilizing 60Co-gamma ray radiation mutation. Powerful support is provided for improvement and transformation and upgrading of the osmanthus fragrans industry. The method is rapid, safe and efficient, high in survival rate and success rate, low in cost and easy to popularize and implement when applied to new sweet-scented osmanthus strain breeding.

Description

Technical Field

[0001] The invention relates to the technical field of flower breeding, and in particular to a radiation-induced mutation breeding method for sweet osmanthus. Background Art

[0002] Osmanthus fragrans( Osmanthus fragrans Lour.) belongs to the genus Oleaceae ( Osmanthus ), one of the ten famous traditional Chinese flowers, is famous for its beautiful shape, color and fragrance. It is an excellent garden tree species with both ornamental and practical value. However, the fruiting rate of osmanthus is low, the seeds are difficult to germinate, and there is a lack of a mature genetic transformation system. In addition, the breeding of osmanthus varieties mainly relies on bud mutation and artificial selection of sowing seedlings and cutting seedlings. These methods have limited genetic variation and low breeding efficiency, which greatly restricts the breeding process of new osmanthus varieties.

[0003] Radiation mutagenesis breeding refers to the use of physical factors such as X-rays, gamma rays, beta rays and neutrons to induce mutations in the genetic material of organisms, screen excellent mutants from them, and then breed new varieties. It has the characteristics of high mutation frequency, wide mutation range and shortened breeding years. At present, the difficulties of radiation mutagenesis breeding of osmanthus mainly include low mutagenesis efficiency, difficult to control the direction of mutation, imperfect mutant identification methods and long breeding cycle. In order to solve these difficulties, it is necessary to continuously explore and optimize radiation mutagenesis technology, and at the same time combine modern biotechnology to jointly promote the selection and breeding of new osmanthus varieties.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] Although radiation-induced mutagenesis breeding has shown great potential in plant variety improvement, the inventors have found based on long-term practical research that there are still some challenges in the selection of radiation sources and precise control of dosage, mutation detection and screening, offspring breeding, and radiation safety. In view of this, the present disclosure provides a method for radiation-induced mutagenesis breeding of osmanthus. Taking into account the penetration ability, sensitivity and biological effects of different types of radiation (such as X-rays, gamma rays, neutrons, etc.) on different tissue parts of osmanthus, as well as the mutagenic effect, etc., a suitable radiation source and an optimal radiation dose are screened, and an efficient radiation-induced mutagenesis breeding system for osmanthus is constructed in combination with the phenotypic characteristics of osmanthus, providing methods and ideas for osmanthus germplasm innovation and new variety breeding. Specifically, it mainly includes the following steps: (1)In spring, cut disease- and pest-free branches with the same size and nutritional status from healthy and robust osmanthus plants as scions. Remove all leaves, moisturize them, and store them in a cool place to keep their moisture. The branches to germinate have 4 or more germinating buds; (2)Use 60 Co-γ rays to irradiate the young osmanthus branches. The dose of the irradiation treatment is 20 - 35 Gy, and the dose rate is 2 - 3 Gy / min; (3)Mix rosin and beeswax in a ratio of 1:1 - 2, add water, heat it until it is completely melted, and after it cools, quickly immerse the top of the branches to germinate for wax sealing; (4)Use the method of cut grafting to graft the branches to germinate onto 2-year-old rootstocks with a trunk diameter of 0.5 - 1 cm; (5)Observe the phenotypic traits of the surviving strains after grafting, select materials with significant differences in phenotypic traits from the control plants as target mutants, and compare the richness of variation.

[0006] In some embodiments of the present disclosure, in the step (1), the length of the young osmanthus branches is 8 - 10 cm, and the diameter is 0.8 - 1.2 cm.

[0007] In some embodiments of the present disclosure, in the step (4), the rootstock includes Chionanthus retusus.

[0008] In some embodiments of the present disclosure, the osmanthus varieties include any one of 'Zanghuage', 'Shishan Gui', 'Zhuangyuan Hong', and 'Rixiang Gui'.

[0009] In some embodiments of the present disclosure, the radiation dose of 'Zanghuage' is 20 - 21 Gy; the radiation dose of 'Shishan Gui' is 24 - 25 Gy; the radiation dose of 'Zhuangyuan Hong' is 23 - 24 Gy; the radiation dose of 'Rixiang Gui' is 30 - 31 Gy.

[0010] In some embodiments of the present disclosure, in the step (5), the phenotypic traits include qualitative traits and quantitative traits.

[0011] In some embodiments of the present disclosure, the quantitative traits include one or more of leaf length, leaf width, leaf shape index, leaf thickness, petiole length, number of lateral veins, and leaf color; the qualitative traits include one or more of young shoot color, leaf shape, leaf texture, glossiness, type of leaf margin serrations, number of leaf margin serrations, morphology of leaf margin serrations, leaf margin undulation, leaf blade rolling degree, leaf blade flatness, leaf tip, leaf base, petiole color, and obviousness of reticulate veins.

[0012] In some embodiments of the present disclosure, the optimal radiation dose for osmanthus flowers is 20 Gy to 30 Gy. At this optimal radiation dose, it can retain a certain graft survival rate and at the same time induce more abundant variant traits.

[0013] One or more technical solutions provided in the embodiments of the present disclosure have at least any one of the following technical effects or advantages: 1. Based on long-term experimental research, the 60 Co-γ rays sensitive to young osmanthus branches and the radiation mutagenesis dose with good mutagenic effects are screened. After apical wax sealing, grafting and cultivation are carried out. Combining the phenotypic trait characteristics (including quantitative traits and qualitative traits) that are easy to effectively identify and the richness of phenotypic variation selected after the osmanthus survives, a rapid radiation mutagenesis breeding system and method using osmanthus branches as materials are constructed, providing a technical approach for 60 creating new osmanthus germplasms by Co-γ ray radiation mutagenesis in the future, and providing strong support for the improvement and transformation and upgrading of the osmanthus industry.

[0014] 2. The method of the present disclosure can quickly and simply obtain variant materials, which are applied to the breeding of new osmanthus strains quickly, safely and efficiently, with simple operation, high survival rate and success rate, low cost, and easy to promote and implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a diagram showing the graft survival situation of scions of 'Zanghuage', 'Shishan Gui', 'Zhuangyuan Hong' and 'Rixiang Gui' after being irradiated with different doses of 60 Co-γ rays in an embodiment of the present disclosure.

[0016] Figure 2 It is the morphological screening and spectral characteristics of unique variations of osmanthus surviving plant lines in an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to better understand the technical solutions of the present application, the above technical solutions will be described in detail below in combination with the accompanying drawings of the specification and specific embodiments.

[0018] In the following embodiments, the instrument equipment involved is all conventional instrument equipment unless otherwise specified; the reagents involved are all commercially available conventional reagents unless otherwise specified; the detection methods involved are all conventional methods unless otherwise specified.

[0019] Example 1. Verification test on radiation mutagenesis breeding of osmanthus In this verification test, all the young osmanthus branches used were from Huangchuan Jinguiyuan in Huangchuan County, Xinyang City, Henan Province, including osmanthus of 4 different varieties, namely 'Zanghuage','Shishan Gui', 'Zhuangyuan Hong' and 'Rixiang Gui'. The rootstocks used were 2-year-old seedling of Chionanthus retusus with a trunk diameter of 0.5 - 1 cm. The specific steps are as follows: 1. Pre-irradiation treatment of young branches of Osmanthus fragrans In mid-March in spring, select the pest-free, similar-sized and nutritionally-similar germinating branches of perennial arbors of 4 Osmanthus fragrans varieties with strong growth. According to the standard from the top to the bottom of the branches, trim these branches into scions with an average length of 10 cm and a diameter of about 0.8 cm, remove all the leaves, and ensure that there are at least 4 germinating buds on each scion. Immediately after harvesting, wrap the branches with a wet towel and cover them with plastic wrap to maintain the humidity and freshness of the branches. Place these wrapped branches in a shady place for subsequent irradiation treatment.

[0020] 2. 60 Co-γ radiation mutagenesis treatment of young branches of Osmanthus fragrans Use 60 Co-γ rays to irradiate the collected young branches of Osmanthus fragrans at the Zhengzhou Isotope Irradiation Center. The radiation dose rate is about between 2 and 3 Gy / min, and 6 gradient radiation doses are set, which are 0 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy and 60 Gy respectively. Among them, 0 Gy is the blank control (without irradiation), each dose is a treatment, and there are a total of 6 treatments. There are 10 - 20 branches for each treatment, and each dose is repeated 3 times.

[0021] 3. Grafting of scions of young branches of Osmanthus fragrans and observation of survival rate Add a little rosin and beeswax to the container in a ratio of 1:2, add an appropriate amount of water, and heat it in the microwave oven until it melts completely. After it cools down, quickly immerse the top of the irradiated Osmanthus fragrans branches for wax sealing, and immediately take them out to ensure that a thin and transparent wax layer evenly covers the top of the scions, preventing water loss in the branches and the invasion of pests and diseases.

[0022] Use Chionanthus retusus as the rootstock and adopt the cut grafting method for grafting: Cut the base of the scion into 2 wedge-shaped cut surfaces with equal lengths, about 2 - 3 cm long. Use an electric shear to cut the rootstock at a suitable position, trim the cut surface flat, and use a grafting knife to vertically split the middle of the rootstock cut surface, with a depth slightly longer than the scion cut surface. Then insert the cut scion, align one side of the cambium, and expose about 0.5 - 1.0 mm of the upper end of the scion cut surface. Wrap and tie it tightly from bottom to top with a grafting film strip. The grafted seedlings are divided into different zones according to different varieties, and signs and numbers are hung on the scions respectively. After grafting, the test trees are all managed according to the conventional method. Observe and count the relative survival rate of the scions every month until it stabilizes, observe the phenotypic traits of the surviving plants, and record the plants with variation after survival.

[0023] Example 2. Experiment verification on the influence of irradiation on the survival of Osmanthus fragrans scions and its semi-lethal dose Based on the relative survival rates of Osmanthus fragrans scions at different doses in Example 1, the correlation coefficient between the relative survival rate and the dose was calculated, and the linear regression equation Y = a + bX was fitted to derive the radiation dose corresponding to a 50% survival rate (LD50) of Osmanthus fragrans branches. Based on this, the semi-lethal doses of 'Zanghuage', 'Shishan Gui', 'Zhuangyuan Hong', and 'Rixiang Gui' Osmanthus fragrans scions were determined respectively, so as to screen out 60 the appropriate dose range of Co-γ ray irradiation for Osmanthus fragrans. The results are as Figure 1 shown.

[0024] As can be seen from Figure 1 -A, with the increase of the radiation dose, the relative survival rate of the 'Zanghuage' scions generally showed a downward trend. The relative survival rate dropped sharply to 24% under the 20 Gy treatment, which was significantly lower than that of the control. Under the 30 Gy treatment, the relative survival rate increased to 33%. Under the 40 and 50 Gy treatments, the relative survival rate decreased continuously to 20% and 8% respectively. Under the 60 Gy treatment, the relative survival rate increased slightly compared with that under the 50 Gy treatment, reaching 12%. The relative survival rates at different radiation doses were fitted with the radiation dose to obtain the linear fitting equation y = 78.2 - 1.36x, from which the semi-lethal dose of 'Zanghuage' was derived as 20.7 Gy.

[0025] As can be seen from Figure 1 -B, with the increase of the dose, the relative survival rate of the 'Shishan Gui' scions gradually decreased. The relative survival rates under the 20, 30, 40, 50, and 60 Gy treatments were 42%, 37%, 30%, 11%, and 4% respectively. The relative survival rates at different radiation doses were fitted with the radiation dose to obtain the linear fitting equation y = 87.1 - 1.51x, from which the semi-lethal dose of 'Shishan Gui' was derived as 24.6 Gy.

[0026] As can be seen from Figure 1 -C, with the increase of the dose, the relative survival rate of the 'Zhuangyuan Hong' scions gradually decreased. The relative survival rates under the 20, 30, 40, 50, and 60 Gy treatments were 44%, 37%, 9%, 6%, and 6% respectively. The relative survival rates at different radiation doses were fitted with the radiation dose to obtain the linear fitting equation y = 86.9 - 1.60x, from which the semi-lethal dose of 'Zhuangyuan Hong' was derived as 23.1 Gy.

[0027] As can be seen from Figure 1-D shows that with the increase of dose, the relative survival rate of the 'Rixianggui' scion generally shows a downward trend. The relative survival rate drops to 48% under the 20 Gy treatment, while it rises to 79% under the 30 Gy treatment, and continuously drops to 27%, 9%, and 8% under the 40, 50, and 60 Gy treatments. Fitting the relative survival rates at different radiation doses with the radiation dose, the linear fitting equation y = 97.9 - 1.58x is obtained, and the semi-lethal dose of 'Rixianggui' is derived as 30.3 Gy.

[0028] Example 3. Sensitivity verification of different osmanthus varieties to 60 Co-γ ray irradiation Calculate the semi-lethal dose of different osmanthus varieties through the constructed linear regression equation. Generally speaking, the semi-lethal doses of the three varieties 'Zanghuage', 'Shishan Gui', and 'Zhuangyuan Hong' are all between 20 Gy and 25 Gy, while that of 'Rixianggui' is 30.3 Gy, indicating that its radiation resistance ability is stronger than that of the other three varieties. The semi-lethal doses of 'Zanghuage', 'Shishan Gui', and 'Zhuangyuan Hong' under 60 Co-γ ray irradiation are 20.7 Gy, 24.6 Gy, and 23.1 Gy respectively, showing similar sensitivities to the irradiation semi-lethal dose, but relatively speaking, 'Zanghuage' is more sensitive.

[0029] Example 4. 60 Experimental study on the morphology of osmanthus scions irradiated by Co-γ rays Detecting genetic variation from morphological or phenotypic traits is the most direct and simple method. After the grafted plant lines grow stably, by measuring the quality traits such as the color of the tender shoots of the newly grown mature leaves, leaf shape, leaf texture, glossiness, type of leaf margin serrations, number of leaf margin serrations, morphology of leaf margin serrations, leaf margin undulation, leaf blade curling degree, leaf blade flatness, leaf tip, leaf base, petiole color, and obviousness of veinlets, as well as the quantitative traits such as leaf length, leaf width, leaf shape index, leaf thickness, petiole length, number of lateral veins, and leaf color (L*, a*, b*), initially screen out the materials with significant morphological differences from the control plants as target mutants, and count the phenotypic variation of plants under different varieties and different dose treatments to screen out the varieties and dose ranges with high variation richness.

[0030] The results are as Figure 2 shown. 60The phenotypic variations induced by Co-γ rays in osmanthus mainly focus on two traits: leaf shape and leaf color, among which the proportion of leaf shape variation is higher than that of leaf color variation. In the leaf shape variation, the leaf cleavage phenotype is dominant; in the leaf color variation, the frequency of leaf yellowing is the highest. Specifically, the leaves of 'Zanghuage' without radiation mutagenesis are dark green, wide, dense, and oval-shaped. After radiation mutagenesis, the leaves show shrinkage, slightly concave leaf tips, and yellowing of leaf color. The leaves of 'Shishan Gui' without radiation mutagenesis are dark green, thick leathery, shiny, oval to obovate, with serrations. After radiation mutagenesis, the leaf margins become round and leaf cleavage occurs. The leaves of 'Zhuangyuan Hong' without radiation mutagenesis are dark green, thick leathery, shiny, lanceolate-oblong, with serrations. After radiation mutagenesis, the leaves become irregular and leaf cleavage occurs. The leaves of 'Rixiang Gui' without radiation mutagenesis are green, hard leathery, slightly shiny, obovate or oblanceolate, entire. After radiation mutagenesis, leaf cleavage, notches, and yellowing and purpling of leaf color occur.

[0031] Among the four osmanthus varieties, the mutation rate of 'Rixiang Gui' is the highest, followed by 'Shishan Gui'. The mutation rates of 'Zanghuage' and 'Zhuangyuan Hong' are the same and the lowest. When the radiation dose is 20 Gy and 30 Gy, the mutation rate of osmanthus is the highest.

[0032] Therefore, by comprehensively considering the semi-lethal dose and the dose range of rich variation of each osmanthus variety, the appropriate radiation dose for radiation mutagenesis of osmanthus is determined to be 20 - 30 Gy.

[0033] Although some preferred embodiments of the present invention application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention application.

[0034] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of its inventive concept. Thus, if these modifications and variations to the present invention fall within the scope of the claims of this application and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A method for radiation-induced mutagenesis breeding of sweet osmanthus fragrans, characterized in that: The following steps are involved: (1) In spring, a branch to be sprouted that is free of pests and diseases and has the same size and nutritional status is cut from a healthy Osmanthus fragrans plant as a scion, all leaves are removed, the branch is treated to retain moisture, and is placed in a cool place to retain moisture, and the branch to be sprouted has more than 4 sprouting buds; (2) Utilization 60 The osmanthus fragrans young branches are irradiated with Co-γ rays, wherein the radiation treatment dose is 20-35 Gy and the dose rate is 2-3 Gy / min; (3) Mix rosin and beeswax in a ratio of 1:1-2, add water, heat until completely melted, and after cooling, quickly immerse the top of the branch to be sprouted in the mixture for wax sealing; (4) grafting the shoots to be germinated onto a 2-year-old rootstock with a trunk diameter of 0.5 to 1 cm using a cut grafting method; (5) Observe the phenotypic traits of the surviving grafted plants, select the materials with significant differences in phenotypic traits from the control plants as target mutants, and compare the variation richness.

2. The method for radiation-induced mutagenesis breeding of sweet osmanthus according to claim 1, characterized in that: In step (1), the length of the osmanthus fragrans young branch is 8-10 cm and the diameter is 0.8-1.2 cm.

3. The method for radiation-induced mutation breeding of sweet osmanthus according to claim 1, characterized in that: In the step (4), the rootstock includes a tassel tree.

4. The method for radiation-induced mutagenesis breeding of sweet osmanthus according to claim 1, characterized in that: The osmanthus varieties include any one of 'Canghuage', 'Shishangui', 'Zhuangyuanhong' and 'Rixianggui'.

5. The method for radiation-induced mutation breeding of sweet osmanthus according to claim 4, characterized in that: The radiation dose of the 'Canghuage' is 20~21 Gy; the radiation dose of the 'Shishangui' is 24~25 Gy; the radiation dose of the 'Zhuangyuanhong' is 23~24 Gy; and the radiation dose of the 'Rixianggui' is 30~31 Gy.

6. The method for radiation-induced mutagenesis breeding of sweet osmanthus according to claim 1, characterized in that: In step (5), the phenotypic traits include qualitative traits and quantitative traits.

7. The method for radiation-induced mutagenesis breeding of sweet osmanthus according to claim 6, characterized in that: The quantitative traits include one or more of leaf length, leaf width, leaf shape index, leaf thickness, petiole length, lateral vein logarithm, and leaf color; the quality traits include one or more of young shoot color, leaf shape, leaf quality, glossiness, leaf edge serration type, leaf edge serration number, leaf edge serration morphology, leaf edge curvature, leaf curling degree, leaf flatness, leaf tip, leaf base, petiole color, and reticulate vein visibility.

8. The method for radiation-induced mutagenesis breeding of sweet osmanthus according to claim 1, characterized in that: The optimal radiation dose of the sweet osmanthus is 20 Gy to 30 Gy.

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