Chrysanthemum new variety breeding and efficient cultivation method and application thereof

Abstract

The invention belongs to but not limited to the technical field of mutation breeding, and particularly relates to a new chrysanthemum variety breeding and efficient cultivation method and application thereof. Firstly, domestic chrysanthemum varieties are collected and introduced, varieties with important prospects are selected for propagation culture, and a tissue culture vegetative propagation line is established. And then performing functional verification on the gene with the flower type or flower color regulation function to determine whether the gene has the effect of improving flowers or not. On the basis, a tissue culture regeneration system of the selected chrysanthemum variety is established, mutagenesis treatment is carried out on the established asexual reproduction line by utilizing a radiation mutagenesis technology to obtain a mutant strain, the flower type or flower color and agronomic traits of the mutant strain are comprehensively evaluated, and a new material conforming to a breeding target is selected. Meanwhile, a genetic engineering means is tried to improve the flower color of the chrysanthemum.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of induced breeding, and in particular relates to a new chrysanthemum variety selection and efficient cultivation method and application thereof. Background Art

[0002] In recent years, with the development of society and the improvement of people's living standards, my country's flower industry has developed rapidly, mainly reflected in the increase in flower varieties and quantity. However, looking at domestic flower enterprises, there is still a problem of neglecting the original innovation of new varieties, and most new varieties are still introduced from abroad. Therefore, increasing the original breeding of flowers in my country and forming a flower breeding technology platform with Chinese characteristics will have a huge driving effect on my country's flower industry.

[0003] Chrysanthemum is one of the four major cut flowers and has many varieties. However, due to the gradual decline in its ornamental characteristics, adaptability and stress resistance during the long-term cultivation process (especially asexual reproduction), it is not conducive to the development of the chrysanthemum industry and cannot meet the needs of the chrysanthemum industrialization development. Therefore, how to improve chrysanthemum varieties has become a bottleneck issue affecting the progress of chrysanthemum industrialization.

[0004] my country's flower breeding mainly adopts conventional methods such as artificial hybridization, natural hybridization, and bud mutation selection. Moreover, many flower cultivars in my country are highly heterozygous allopolyploids with very complex genotypes, ever-changing hybrid offspring, and very extensive separation. There is often a phenomenon of super-parenting, and the direction of variation cannot be controlled, making it difficult to obtain satisfactory results. Moreover, the traits required for conventional breeding are limited by the genetic background. It takes more than 10 years of breeding time to obtain a new variety, which cannot meet the needs of production. Since the current cultivated chrysanthemum varieties are all heterozygous polyploids, sexual reproduction cannot be used for research, and radiation mutagenesis technology can specifically change the flower shape and color without changing the original agronomic traits. Therefore, the use of radiation mutagenesis and tissue culture combined with genetic engineering technology has great advantages in obtaining the desired useful traits such as flower color and morphology. Compared with conventional breeding, plant genetic engineering methods not only break the limitations of interspecific differences, make up for the defects of conventional genetic breeding, and accelerate the integration of excellent trait genes between plants of the same species and useful genes from completely different sources into the same plant, but also obtain satisfactory agricultural traits in a short time (4-6 years).

[0005] Flower color is another important economic trait of flower crops. The substances that determine the flower color of plants are mainly substances such as anthocyanins. Anthocyanins are water-soluble flavonoids with rich colors (from yellow to blue) widely distributed in plants (Tanaka, 2006). Its biosynthesis pathway is relatively conserved in plants.

[0006] With the rapid development of genomics and molecular biology techniques of model plants such as Arabidopsis thaliana and antirrhinum, people have not only explored the synthesis pathways of anthocyanins, the regulatory pathways and mechanisms of floral organ development, but also cloned related genes, providing a theoretical basis and many useful genetic resources for the molecular breeding of flower crops.

[0007] Therefore, a new chrysanthemum variety breeding and efficient cultivation method are needed. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for breeding a new chrysanthemum variety and a high-efficiency cultivation method and application thereof.

[0009] The present invention is achieved by a method for breeding and efficiently cultivating new chrysanthemum varieties, comprising the following steps:

[0010] Step 1: Establish tissue culture asexual propagation lines, verify the function of genes that regulate flower shape or color, and clarify whether they have the function of improving flowers;

[0011] Step 2: establishing a tissue culture regeneration system for the selected chrysanthemum variety, and using radiation mutagenesis technology to perform mutagenesis treatment on the established asexual reproduction system to obtain mutant strains;

[0012] Step three: conduct a comprehensive evaluation of the flower shape or color and agronomic traits of the mutant strains to select new varieties that meet the breeding goals.

[0013] Furthermore, in the step 2, when establishing the chrysanthemum regeneration system, a culture medium formula with different combinations of auxin and cytokinin is selected, and plant materials such as roots, leaves, and petioles are used as explants for cultivation. After several months of cultivation, the growth status of the explants is observed and counted.

[0014] Furthermore, leaves were selected as explants, and a combination of BA and NAA hormones was used as culture regulators.

[0015] Furthermore, 2 mg / L BA and 0.25 mg / L NAA were selected as the culture conditions.

[0016] Furthermore, on the basis of establishing an efficient and stable regeneration system, the obtained asexual reproduction lines were screened for tissue culture traits; through screening and cultivation, chrysanthemum varieties numbered H7, H10 and H15 were selected to enter the molecular breeding stage.

[0017] Furthermore, in molecular breeding, genetic engineering and 60 Improvement of chrysanthemum by using Coγ-ray radiation induced mutation breeding technology.

[0018] Further, in 60When Coγ-ray radiation mutagenesis was used, the semi-lethal dose of radiation was first screened; 50 and 100 units of radiation were used to irradiate the leaves of chrysanthemum H7 and H15, respectively, for a total of more than 20,000 explants; after cultivation, callus tissue with differentiation ability was removed from the browned tissue, and after continuous subculture, callus tissue with good growth vitality was obtained, and finally regenerated plants were obtained.

[0019] Another object of the present invention is to provide a method for breeding new chrysanthemum varieties and efficient cultivation, and its application in breeding new chrysanthemum varieties and efficient cultivation.

[0020] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0021] First, the present invention first collects and introduces domestic chrysanthemum varieties, selects varieties with important prospects for expansion and cultivation, and establishes tissue culture asexual propagation systems. Then, the genes with flower type or color regulation function are functionally verified to clarify whether they have the effect of improving flowers. On this basis, a tissue culture regeneration system of the selected chrysanthemum variety is established, and the established asexual propagation system is mutagenized using radiation mutagenesis technology to obtain mutants, and the flower type or color and agronomic traits of the mutants are comprehensively evaluated to select new materials that meet the breeding goals. At the same time, attempts are made to improve the chrysanthemum color using genetic engineering means.

[0022] Second, the expected benefits and commercial value of the technical solution of the present invention after conversion are as follows: after patent conversion, the research and development cycle of new chrysanthemum varieties can be effectively shortened, and higher economic benefits and commercial value can be generated, which are specifically manifested in the following aspects:

[0023] 1. Improve variety quality: Through breeding with new technologies, new varieties that are resistant to diseases and pests, resistant to transportation, and have strong adaptability can be cultivated, thereby increasing the survival rate and ornamental value of flowers and enhancing market competitiveness.

[0024] 2. Reduce production costs: The application of new technologies helps to reduce the use of fertilizers and pesticides, reduce labor intensity, improve production efficiency, and thus reduce overall production costs.

[0025] 3. Expand market share: New flower varieties with independent intellectual property rights can fill market gaps, meet the diverse needs of consumers, further expand market share and increase product added value.

[0026] 4. Promote industrial upgrading: The promotion of new technologies will drive the upgrading of the entire flower industry chain, promote the development of the traditional flower industry towards modernization, scale and standardization, and form a new economic growth point.

[0027] 5. Enhance international competitiveness: Through technological innovation, break the monopoly of foreign varieties, realize the transformation from import to export, and enhance the competitiveness and influence of my country's flowers in the international market.

[0028] Third, the technical solution of the present invention solves a technical problem that people have been eager to solve but have never succeeded in solving: traditional chrysanthemum breeding techniques are mainly based on hybridization, and collected germplasm resources are used to select trait advantages. However, since the traits of germplasm materials are relatively similar and the probability of natural mutation is very low, it is difficult to find specific trait genes. This patented technology uses radiation mutagenesis and genetic engineering methods to select the required trait genes in a targeted manner, thereby achieving relatively directional breeding, effectively improving the success rate, accuracy and efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of a method for breeding new chrysanthemum varieties and efficient cultivation provided by an embodiment of the present invention;

[0030] Figure 2 Schematic diagram of 50 fresh cut chrysanthemum flowers provided by an embodiment of the present invention;

[0031] Figure 3 This is a schematic diagram of the zoning expansion and cultivation of fresh cut chrysanthemum flowers provided by an embodiment of the present invention;

[0032] Figure 4 It is a schematic diagram of a tissue culture asexual propagation system provided by an embodiment of the present invention;

[0033] Figure 5 is a schematic diagram of growth conditions of different explants provided by an embodiment of the present invention;

[0034] Figure 6 Schematic diagram of the regeneration of some chrysanthemum varieties provided in the embodiments of the present invention; A: callus; B: regenerated buds; C: rooted regenerated plants;

[0035] Figure 7 is a schematic diagram of wound healing induced by radiation provided by an embodiment of the present invention;

[0036] Figure 8 is a schematic diagram of a regenerated plant after differentiation provided by an embodiment of the present invention;

[0037] Fig. 9 It is a schematic diagram of the transplanted radiated chrysanthemum seedlings provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] like Figure 1 As shown, the embodiment of the present invention provides a method for breeding and efficiently cultivating new chrysanthemum varieties, which is characterized by comprising the following steps:

[0040] Step 1: Establish tissue culture asexual propagation lines, verify the function of genes that regulate flower shape or color, and clarify whether they have the function of improving flowers;

[0041] Step 2: establishing a tissue culture regeneration system for the selected chrysanthemum variety, and using radiation mutagenesis technology to perform mutagenesis treatment on the established asexual reproduction system to obtain mutant strains;

[0042] Step three: conduct a comprehensive evaluation of the flower shape or color and agronomic traits of the mutant strains to select new varieties that meet the breeding goals.

[0043] Chrysanthemum is a typical ornamental plant, and flower shape and color are the key features that determine its commercial value. Through **tissue culture technology**, explants such as stem tips, leaves or petals of chrysanthemums are inoculated on the culture medium under sterile conditions to induce callus tissue and sprouts, and finally form asexual reproduction lines. This process can achieve rapid reproduction and maintain genetic consistency, effectively solving the problem of long traditional chrysanthemum breeding cycles. In addition, through gene function verification, the target genes that affect the regulation of flower shape and color are analyzed to determine whether they have application value in improving flower characteristics, providing a theoretical basis for subsequent genetic improvement.

[0044] The regeneration system is the core link of plant tissue culture, which ensures efficient regeneration under controlled conditions. By optimizing factors such as culture medium formula, hormone concentration, light and temperature, a **tissue culture regeneration system** suitable for the selected chrysanthemum variety is established. This system not only improves the regeneration rate of chrysanthemum, but also lays the foundation for genetic improvement and mutagenesis. The successful establishment of the regeneration system means efficient conversion from explants to complete plants, further realizing the rapid production of large quantities of high-quality seedlings.

[0045] Radiation mutagenesis is a commonly used method of plant breeding. It mainly causes random mutations in plant cell DNA through physical radiation (such as gamma rays and X-rays), thereby changing the characteristics of plants. Based on the asexual reproduction system of chrysanthemum, the chrysanthemum regenerated plants are treated with radiation mutagenesis technology to induce genetic mutations. Since radiation mutagenesis has a broad spectrum, it can produce diverse mutations in flower shape, flower color, flowering period and stress resistance, providing rich materials and possibilities for the selection and breeding of new varieties.

[0046] ###**4. Screening and comprehensive evaluation of mutant strains**

[0047] The mutants produced by radiation mutagenesis have a large diversity of traits. At this stage, **phenotypic screening** is used to comprehensively evaluate the flower shape, flower color and agronomic traits of chrysanthemum mutants. This includes observing the petal shape, color depth, flower diameter size and other characteristics of the mutants, as well as evaluating their growth potential, branching, flowering cycle and stress resistance and other agronomic indicators. Through quantitative evaluation and statistical analysis, chrysanthemum mutants with excellent performance in flower shape, flower color and comprehensive traits are selected to meet the breeding goals.

[0048] In order to further confirm the regulatory effect of the target gene on flower shape or color, molecular biological analysis is performed on the selected excellent mutant strains to verify the correlation between their gene expression and mutant traits. This process can be completed through technical means such as **gene expression profile analysis** or **qPCR**. By clarifying the functions of key genes, the molecular mechanism of flower trait improvement can be revealed, providing theoretical support for the cultivation of new chrysanthemum varieties and providing genetic resources for subsequent variety improvement.

[0049] The selected new varieties of chrysanthemum need to achieve their best performance through scientific cultivation and management. In the efficient cultivation method, the optimized cultivation medium, light control, temperature and humidity management, water and fertilizer regulation and other measures are combined to promote the healthy growth and high-quality flowering of chrysanthemums. For example, by adjusting the photoperiod, the directional flowering period of chrysanthemums can be controlled to improve the ornamental value; through reasonable fertilization and pest and disease control, the yield and stress resistance of new varieties can be improved. In addition, the promotion of large-scale production of tissue culture seedlings of asexual reproduction systems is conducive to the rapid promotion and application of new chrysanthemum varieties.

[0050] (1) Collection and introduction of chrysanthemum varieties

[0051] like Figure 2 As shown, the present invention introduces and collects 50 excellent varieties of fresh cut chrysanthemums from home and abroad for zoning expansion and cultivation. At the same time, 10 new varieties that meet market demand are selected for expansion and cultivation experiments.

[0052] (2) Introduction, collection and propagation of excellent chrysanthemum germplasm resources at home and abroad

[0053] On the basis of obtaining functional color improvement genes, the germplasm resources of excellent chrysanthemum varieties at home and abroad were introduced and collected. Chrysanthemum flower type and color genetic variation are rich, and different groups have been formed through continuous artificial breeding. Different individuals in the same group may have similar genetic backgrounds, and the gene homology controlling flower type and color is relatively high. During breeding, the corresponding group can be selected as the basic material for breeding according to different breeding goals. Therefore, the introduction and collection of existing chrysanthemum resources is an important prerequisite for chrysanthemum breeding. The present invention has collected more than 50 chrysanthemum varieties with important market prospects for flower type and color as basic materials. In order to establish an efficient regeneration system for chrysanthemum, the sterile seedlings of the above chrysanthemum varieties were first obtained, and the tissue culture asexual propagation system of these varieties was successfully established ( Figure 4 ).

[0054] (3) Establishment of an efficient and stable chrysanthemum regeneration system

[0055] Establishing a stable and efficient regeneration system is an important prerequisite for chrysanthemum breeding by genetic engineering or radiation. When establishing the chrysanthemum regeneration system, we selected culture medium formulas with different combinations of auxin and cytokinin (Table 1), and used plant materials such as roots, leaves, and petioles as explants for culture. After several months of culture and observation of the growth of explants, we found that different types of explants had the highest regeneration frequency in culture medium containing BA and NAA ( Figure 5 ), among which leaves have the highest regeneration frequency. Therefore, leaves were selected as explants, and BA and NAA were used as culture regulators.

[0056] Table 1 Different hormone combinations

[0057]

[0058] Subsequently, in order to obtain the highest regeneration frequency and optimal culture conditions, the ratio of BA and NAA was studied in more detail. The combinations of different ratios are shown in Table 2. The culture results showed that the regeneration frequency of explants was the highest when 2 mg / L BA and 0.25 mg / L NAA were added, and this concentration was finally selected as the culture condition.

[0059] Table 2 The medium formula and growth statistics used in the establishment of the chrysanthemum regeneration system

[0060]

[0061] (4) Screening out five chrysanthemum varieties with good tissue culture traits

[0062] On the basis of establishing an efficient and stable regeneration system, we further screened the tissue culture traits of the obtained asexual propagation lines. Through screening and cultivation, the chrysanthemum varieties numbered H7, H10 and H15 have good tissue culture traits such as high callus induction rate, high regeneration frequency, large number of buds induced on explants, and vigorous growth. After the buds are cut from the explants, they are transferred to the rooting medium for rooting culture. The seedlings are easy to root and have a high rooting rate. Finally, the above chrysanthemum varieties are selected to enter the molecular breeding stage.

[0063] (5) Adoption 60 Obtaining chrysanthemum mutants by Coγ-ray radiation-induced breeding

[0064] In molecular breeding, genetic engineering and 60 Coγ-ray radiation induced mutation breeding technology is used to improve chrysanthemums to increase the probability of successful directional selection. In the process of chrysanthemum transgenic, not enough transgenic regenerated plants were obtained, so it could not meet the breeding needs. 60 When Coγ-ray radiation mutagenesis was performed, a semi-lethal dose of radiation was first screened. The screening results showed that when the radiation dose was 50, the explants were less browned and the frequency of regeneration and budding was higher, but the probability of mutations might be reduced; when the radiation dose was 100, the browning degree of the explants deepened, indicating that the degree of damage to the plant cells deepened, but it would also increase the frequency of mutant individuals. In order to increase the probability of successful breeding, 50 and 100 units of radiation were used to irradiate the leaves of chrysanthemum H7 and H15, respectively, for a total of more than 20,000 explants. After cultivation, callus tissue with differentiation ability was removed from the browned tissue, and after continuous subculture, callus tissue with good growth vitality was obtained ( Figure 7 ), and finally obtained regenerated plants.

[0065] According to statistics, a total of more than 2,000 regenerated plants were obtained, and these plants were preserved as resources ( Figure 8 ) to preserve germplasm resources for commercial propagation.

[0066] (6) Screening new chrysanthemum varieties whose flower colors and flower shapes meet the breeding goals

[0067] The sterile seedlings obtained by radiation mutagenesis were transferred into pots after seedling hardening and planted in Kunming Hongzhihua Horticulture Co., Ltd. Fig. 9 The regenerated seedlings grew well and vigorously, and the color, shape and other characteristics of the flowers were identified after they bloomed.

[0068] The invention is mainly applied to the technical field of flower breeding, and has obvious advantages particularly for chrysanthemums.

[0069] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for breeding new chrysanthemum varieties and efficient cultivation, characterized in that: The following steps are involved: Step 1: Establish tissue culture asexual propagation lines, verify the function of genes that regulate flower shape or color, and clarify whether they have the function of improving flowers; Step 2: establishing a tissue culture regeneration system for the selected chrysanthemum variety, and using radiation mutagenesis technology to perform mutagenesis treatment on the established asexual reproduction system to obtain mutant strains; Step three: conduct a comprehensive evaluation of the flower shape or color and agronomic traits of the mutant strains to select new varieties that meet the breeding goals.

2. The method for breeding new chrysanthemum varieties and efficiently cultivating the same as claimed in claim 1, characterized in that: In the step 2, when establishing the chrysanthemum regeneration system, a culture medium formula with different combinations of auxin and cytokinin is selected, and plant materials such as roots, leaves, petioles, etc. are used as explants for cultivation. After several months of cultivation, the growth status of the explants is observed and counted.

3. The method for breeding new chrysanthemum varieties and efficiently cultivating the same as claimed in claim 2, characterized in that: Leaves were selected as explants, and BA and NAA were used as culture regulators.

4. The method for breeding new chrysanthemum varieties and efficiently cultivating the same as claimed in claim 3, characterized in that: 2 mg / L BA and 0.25 mg / L NAA were selected as the culture conditions.

5. The method for breeding new chrysanthemum varieties and efficiently cultivating the same as claimed in claim 1, characterized in that: On the basis of establishing an efficient and stable regeneration system, the obtained asexual reproduction lines were screened for tissue culture traits; through screening and culture, chrysanthemum varieties numbered H7, H10 and H15 were selected to enter the molecular breeding stage.

6. The method for breeding new chrysanthemum varieties and efficiently cultivating them according to claim 5, characterized in that: In molecular breeding, genetic engineering and 60 Improvement of chrysanthemum by using Coγ-ray radiation induced mutation breeding technology.

7. The method for breeding new chrysanthemum varieties and efficiently cultivating them according to claim 6, characterized in that: exist 60 When Coγ-rays were used for mutagenesis, the semi-lethal dose of radiation was first screened; 50 and 100 units of radiation were used to irradiate the leaves of chrysanthemum H7 and H15, respectively, for a total of more than 20,000 explants; After cultivation, callus tissue with differentiation ability was removed from the browned tissue. After continuous subculture, callus tissue with good growth vitality was obtained, and finally regenerated plants were obtained.

8. Use of the method for breeding new chrysanthemum varieties and efficient cultivation as claimed in any one of claims 1 to 6 in breeding new chrysanthemum varieties and efficient cultivation.

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