High-yield and high-efficiency seed production method suitable for breeding artemisia apiacea variety by derivative pedigree method
By adopting derivative genealogy method and group selection method in Artemisia annua breeding, combined with tissue culture and field management, the problems of gene homozygity and consistency in Artemisia annua breeding were solved, efficient and stable seed production methods were achieved, and Artemisia annua was improved Market acceptance and continuous seed production capacity of varieties.
Patent Information
- Application Number
- CN202411489696.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-01-07
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Among the existing breeding methods of Artemisia annua breeding, the genealogy and its derivative methods have problems such as low gene homozygation, poor progeny consistency, low selection efficiency and insufficient seed production volume during the seed production process, resulting in a relatively high market acceptance of varieties. Low.
The derivative genealogy method is used to select excellent single plants for tissue culture expansion and expansion, and the parent is selected in combination with the group selection method. By performing specific field management and flowering adjustment in the field, pollination efficiency and seed harvesting volume are improved, and a high consistency production is formed. Use species.
A high yield and efficient seed production method was achieved, with a continuity of progeny reaching 98.3%, and the seed production volume was much higher than the traditional mixed seed harvest method, and it had stability and convenience for many years.
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Figure CN119256962A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic breeding, and in particular to a high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties using a derived pedigree method. Background Art
[0002] Artemisia annua is a strictly outcrossing crop, with a self-pollination rate of less than 5%, and some breeding materials even less than 3‰. At present, the main breeding methods for Artemisia annua varieties promoted in the market are mainly based on traditional pedigree method, hybrid method and its derivative methods. In recent years, modern breeding technologies such as radiation breeding, ploidy breeding, and molecular marker-assisted breeding have been gradually applied to Artemisia annua variety breeding, greatly improving the breeding process of high-yield and high-resistant Artemisia annua varieties. However, no matter what breeding method is adopted, the selection of excellent individual plants or groups is an indispensable means, and it is also an important way to breed good varieties.
[0003] Due to the strict outcrossing nature of Artemisia annua, the pedigree method and its derivative methods for breeding Artemisia annua seeds must be based on systematic selection to achieve a certain purity (phenotypic identification purity ≥85%), and then retain excellent individual plants for mixed pollination to obtain seeds after removing impurities, inferior plants, and diseases. This method of seed breeding can not only avoid the decline in vitality of Artemisia annua due to self-pollination for many generations, but also produce a certain amount of seeds for field production during the breeding process.
[0004] By studying the breeding methods and seed production methods of approved or registered Artemisia annua varieties, the researchers found that most of the predecessors used the mixed method for seed production (see Appendix Figure 1 ), which also reflects from the side that Artemisia annua, as a strictly outcrossing crop, cannot be self-pollinated to produce high-purity seeds. However, the mixed method of seed production still has the following problems: (1) Due to the genetic polymorphism of Artemisia annua, even if the phenotype of the population is consistent, its genetic homozygosity is still not high, and the consistency and stability of its offspring after mixed pollination are still not strong, which is why we often observe a large number of hybrid plants in field cultivation. (2) Since the seed breeding is based on the selection of excellent single plants for mixed pollination, and the production of strains is based on the isolation pollination (self-pollination) of selected single plants, the amount of seeds produced by self-pollination of single plants is limited, and the number of superior single plants after removing impurities, inferior plants and diseases is even smaller, which determines that the seed breeding volume of a single strain will not be too large and is not suitable for large-scale seed supply. (3) Although the mixed selection method and its derivative methods can obtain a large number of seeds for production, their selection efficiency is lower than that of the pedigree method and the derived pedigree method, and the purity of the selected offspring is not high, resulting in the market acceptance of the variety being far less than the former. Based on the problems faced by the pedigree method and the derived pedigree method in the process of Artemisia annua seed production, it is very meaningful to develop a high-yield and efficient seed production method suitable for the derived pedigree method to select Artemisia annua varieties. Summary of the invention
[0005] In view of the above problems, the present invention provides a high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by the derivative pedigree method.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by a derived pedigree method, which specifically comprises the following steps: S1: Acquisition of maternal material: In the field cultivation of Artemisia annua, the method of selecting excellent individual plants by the derivative pedigree method is adopted, the best individual plants selected after the last individual plant selection are selected, and young shoots are taken as explants in the budding stage to be propagated in the tissue culture room; in early April of the second year, the cover is opened to slow down the seedlings, and after 3-5 days, the seedlings are transplanted to the outdoor floating nutrient tray for seedling hardening, and then transplanted to the field in early May as the maternal parent; the derivative pedigree method is a one-time mixed selection method plus a continuous 3-year individual plant selection method; S2: Obtaining male parent materials: spray the inflorescence of the best single plant selected after the last single plant selection in step S1 with NaCl solution before flowering, bag the inflorescence or isolate the whole plant after the surface moisture is slightly dry, and collect the seeds individually; sow all in the second year, transplant to potted pots 5 days before the mother plant is transplanted, and place in the open space around the isolated seed production nursery, slightly higher than the conventional fertilizer and water management; pass phenotypic identification 15 days before budding, remove impurities, inferior plants and diseases, and select plants with the same or similar phenotypes as the parents as male parents; S3: Seed production: S31: Field preparation and plant-row configuration: After the hardening of the female parent seedlings is completed, select a field with medium to high fertility and convenient drainage and irrigation as the seed production field, and plant in ridges after rotary tillage and land preparation; in order to increase the seed production per unit area and prevent weeds, plant the female parent in the whole field in the early stage, and after the pre-bud phenotype identification is completed, thin the female parent with an interval of 3 plants according to the row ratio of 2:1 to reserve the male parent area; move the male parent selected in step S2 to the reserved male parent area and simply fix it to prevent it from tipping over; at this time, the male parent is planted at a higher position than the female parent, which is conducive to wind pollination to improve the pollination effect; S32: Field management: Fertilizer and water management should be carried out at a level slightly higher than the production management level during the entire growth period; avoid excessive nitrogen fertilizer application, and add sulfur-based potassium fertilizer in appropriate amounts during the clustering period; pay attention to field drainage in spring and summer, and prevent stem rot, stem base rot and root rot in hot and humid weather. For the initial outbreak, spray 450 times of 5% oxadipate aqueous solution once every 7 days for 3 times in a row; after July, pay attention to the prevention and control of chrysanthemum gall midges and green leafhoppers. Spray the whole plant with 1500 times of 40% dimethoate emulsifiable concentrate for chrysanthemum gall midges, and use 10,000 times of 25% thiamethoxam for green leafhoppers; S33: Flowering period regulation: mainly regulate the flowering period of the male parent. When the male parent’s flowering period is late, spray 90% gibberellin 100,000 times solution and 0.1% potassium dihydrogen phosphate after budding, which can advance the flowering period by 1-4 days. S34: Seed harvesting: Harvest the mother plant 7 days before the seeds are fully mature, dry in the sun until the seeds are fully mature, thresh, bag and store at 4°C for use as production seeds.
[0007] Furthermore, in step S2, the height of the potted plant pot is 0.6 m.
[0008] Furthermore, in step S31, the ridge width is 0.8 m, and the ridge height is 0.3-0.4 m.
[0009] Furthermore, in step S31, the plant spacing is 0.8-1.2 m.
[0010] Furthermore, in step S31, the cultivation position of the male parent is 0.6 m higher than that of the female parent.
[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses tissue culture technology to expand the preferred female parent in large quantities based on the selection of the derivative pedigree method, and combines the group selection method to select the male parent, and finally produces a large number of production seeds. The seed production method of the present invention has a much higher seed production volume than the traditional mixed seed harvesting method, and because the source of the female parent is single and the genetic background is consistent, the consistency of its offspring is high. In addition, the seed production method of the present invention can achieve large-scale seed production for many consecutive years, which has greater advantages than the traditional mixed seed harvesting method in terms of the convenience of continuous seed production for many years and the stability of yield. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 It is a diagram of several approved Artemisia annua varieties breeding and seed production and preservation methods found by the present invention. DETAILED DESCRIPTION
[0013] In order to make the purpose 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.
[0014] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.
[0015] Example 1 This embodiment provides a high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by derived pedigree method, which specifically comprises the following steps: S1: Acquisition of maternal material: In the field cultivation of Artemisia annua, the method of selecting excellent individual plants by the derivative pedigree method is adopted, the best individual plants selected after the last individual plant selection are selected, and young shoots are taken as explants in the budding stage to be propagated in the tissue culture room; in early April of the second year, the cover is opened to slow down the seedlings, and after 3-5 days, the seedlings are transplanted to the outdoor floating nutrient tray for seedling hardening, and then transplanted to the field in early May as the maternal parent; the derivative pedigree method is a one-time mixed selection method plus a continuous 3-year individual plant selection method; S2: Obtaining male parent materials: spray the inflorescence of the best single plant selected after the last single plant selection in step S1 with NaCl solution before flowering, and bag the inflorescence or isolate the whole plant after the surface moisture is slightly dry, and collect the seeds individually; sow all in the second year, transplant to potted pots (0.6m high) 5 days before the transplanting of the mother parent (seedling height is about 15cm), and place in the open space around the isolated seed production nursery, slightly higher than the conventional fertilizer and water management; 15 days before budding, pass phenotypic identification, remove impurities, inferior plants and diseases, and select plants with the same or similar phenotypes as the parents as male parents; S3: Seed production: S31: Field preparation and plant-row configuration: After the hardening of the female parent seedlings is completed, select a field with medium to high fertility and convenient drainage and irrigation as the seed production field, and plant in ridges after rotary tillage and land preparation. The ridge width is 0.8m and the ridge height is 0.3-0.4m; the plant spacing is adjusted according to the size of the female parent plant, generally 0.8-1.2m; in order to increase the seed production per unit area and prevent weeds, the female parent is planted in the whole field in the early stage. After the pre-bud phenotype identification is completed, the female parent is thinned at an interval of 3 plants according to the row ratio of 2:1 to reserve the male parent area; the male parent selected in step S2 is moved to the reserved male parent area and simply fixed to prevent tipping; at this time, the male parent cultivation position is 0.6m higher than the female parent, which is conducive to wind pollination to improve the pollination effect; S32: Field management: Fertilizer and water management should be carried out at a level slightly higher than the production management level during the entire growth period; avoid excessive nitrogen fertilizer application, and add sulfur-based potassium fertilizer in appropriate amounts during the clustering period; pay attention to field drainage in spring and summer, and prevent stem rot, stem base rot and root rot in hot and humid weather. For the initial outbreak, spray 450 times of 5% oxadipate aqueous solution once every 7 days for 3 times in a row; after July, pay attention to the prevention and control of chrysanthemum gall midges and green leafhoppers. Spray the whole plant with 1500 times of 40% dimethoate emulsifiable concentrate for chrysanthemum gall midges, and use 10,000 times of 25% thiamethoxam (Actamethasone) for green leafhoppers; S33: Regulation of flowering period: Previous studies have shown that the flowering period of a single Artemisia annua plant is about 15 days. The inflorescence of the whole plant opens from the outside to the inside, and the single inflorescence opens from top to bottom. The female flowers on the outer layer of the capitula bloom 1-3 days earlier than the bisexual flowers in the middle. The pollination period is 3-6 days after flowering and lasts for 3-6 days. The flowers bloom around 10:00 am on a sunny day and are delayed until after 10:30 on cloudy days.
[0016] Taking advantage of this characteristic, the male parent can be transplanted 5 days in advance, so that the male parent's pollen peak period can coincide with the opening period of the outer / upper high-quality inflorescence of the female parent as much as possible, thereby improving seed quality. Because Artemisia annua has a long flowering period and a large amount of pollen, the same line of parents basically do not need to adjust the flowering period. If you want to maximize the seed production, the main focus is on adjusting the male parent's flowering period. When the male parent's flowering period is late, spraying 90% gibberellin 100,000 times solution and 0.1% potassium dihydrogen phosphate after budding can advance the flowering period by 1-4 days, and the inflorescence will increase significantly, which is more conducive to the male parent's pollen release; S34: Seed harvesting: Harvest the mother plant 7 days before the seeds are fully mature, dry in the sun until the seeds are fully mature, thresh, bag and store at 4°C for use as production seeds.
[0017] Experimental Example 1: Optimization of key links 1. Optimization of tissue culture propagation methods and propagation efficiency Because the parental materials often have a large number of chromosome disorders and other mutations during tissue culture propagation, the hybridization of seed-producing parents and the reduction of offspring consistency and stability. In order to reduce the mutation rate, this study used stem segments with buds as explants, and compared two tissue culture propagation methods: direct induction of rooting and seedlings from explants - axillary bud / stem segment propagation, and direct induction of budding from organs - adventitious bud subculture propagation - induction of rooting and seedlings. The ratio of exogenous induction hormones was optimized by examining the propagation efficiency, mutation rate, and mutation type.
[0018] The results are shown in Table 1. The principle of direct induction of rooting and seedling formation from explants - propagation of axillary buds / stem segments is similar to that of cuttings, and no mutants were found in the propagation offspring. By optimizing the hormone formula, it was found that adding 0.05 mg / L 6-BA on the basis of 0.5 mg / L NAA can effectively promote the occurrence of axillary buds, thereby improving the propagation efficiency. However, the method of inducing budding through direct organ generation - propagation of adventitious buds - inducing rooting and seedling formation has more than 3% flat stem variability, and the propagation efficiency is lower without delaying the seed production phenological period (210 days from inoculation to seedling emergence), which is not suitable as a means of maternal propagation.
[0019] Table 1 Comparison of tissue culture propagation methods and propagation efficiency
[0020] 2. Optimization of isolation method and NaCl solution concentration In order to reduce the specific recognition ability of the stigma and improve the self-pollination rate, it is necessary to spray the inflorescence that is about to bloom with NaCl solution to harvest as many self-pollination seeds as possible from the selected superior individual plants. This study optimized the concentration of NaCl solution by setting different concentration gradients of NaCl solution. In addition, the effects of two different isolation methods, single plant isolation pollination or inflorescence bag isolation pollination, on the fruiting rate of Artemisia annua were compared.
[0021] The results are shown in Table 2. Spraying 0.8-1.0% NaCl solution can significantly increase the self-pollination and fruiting rate of Artemisia annua. Too high NaCl concentration or no spraying is not conducive to its self-pollination and fruiting. Compared with the inflorescence bag isolation method, the single plant overall isolation method can greatly increase the seed yield. This may be related to the high temperature and poor air permeability in the bag during the flowering period caused by bagging, or it may be related to the fact that the inflorescence is easily affected by rainfall from pollination to maturity, resulting in mildew of the inflorescence.
[0022] Table 2 Effects of isolation method and NaCl solution concentration on selfed seed yield
[0023] Experimental Example 2 Evaluation of seed production effect This embodiment uses tissue culture technology to expand the female parent in large quantities on the basis of the selection of the derivative pedigree method, combines the group selection method to select the male parent, and finally produces a large number of production seeds. The method is temporarily named "maternal control method". In order to compare the advantages and disadvantages of the "maternal control method" and the traditional mixed seed collection method, the two methods are used for seed production, and the number of parents, seed production and offspring consistency of the two methods are compared (as shown in Table 3). Among them, the "total number of single plants in the selected strains" is the number of offspring transplanted and survived after the single plants selected by the pedigree method are isolated and pollinated. In the "maternal control method", it only refers to the male parent without weeds. The corresponding parents are obtained after the selected strains are weeded, inferior and diseased. The number of female parents available for transplantation in the "maternal control method" is much higher than the actual number of cultivation, and its cultivation number is limited by the number of male parents (female parent: male parent = 14:1). The consistency of offspring is determined by examining the plant type, leaf type and stem color indicators among the 1,000 cultivated seed-producing offspring. If there is no obvious difference in the three when observed with the naked eye, they are judged to be consistent.
[0024] As shown in Table 3, the traditional mixed seed harvesting method is affected by the number of self-pollinated offspring per plant, and the seed production is very limited, and the offspring consistency is only 83.4%. However, due to the large number of maternal parents, the "maternal control method" has a much larger number of maternal parents, so even if the converted single plant seed production (seed production / number of parent or maternal plants) is far less than the traditional mixed seed harvesting method, the seed production is still much higher than the latter, and because the maternal parent source is single and the genetic background is consistent, the offspring consistency is as high as 98.3%.
[0025] Table 3 Comparison of two seed production methods
[0026] Experimental Example 3 Continuous Seed Production The pedigree method and the derived pedigree method use the traditional mixed seed harvesting method, which inevitably retains a large amount of genetic heterogeneity during seed production, resulting in a further reduction in the gene homozygosity of the offspring, making it impossible to continuously select the superior plant group in the offspring through mixed selection or group selection for mixed pollination and seed production. The desirable method is to continue to select excellent individual plants for isolated pollination in the offspring of system selection mixed pollination, form a new system group, and then select the superior system group for mixed pollination. The advantage of this is that a certain amount of genetic heterogeneity can be retained to avoid the decline of vitality caused by continuous self-pollination for multiple generations; the disadvantage is that system selection needs to be carried out every year, and the seed production volume is still limited by the number of parents.
[0027] The "maternal control method" is used for seed production. The source of the maternal parent is the best single plant in the system, and the source of the paternal parent is the excellent single plant group among the self-pollinated offspring of the maternal parent. In a similar way to "backcrossing", the excellent traits of the best single plant (maternal parent) are maintained. When continuous seed production is required, there are two optional ways: Approach 1: Continue to use the mother plant of the first year of seed production as the mother plant, and the father plant is the offspring of the excellent single plant group among the self-pollinated offspring of the first year of seed production, which is further selected by the group (isolated by cuttings in different places, mixed pollination and seed preservation). The obtained offspring can be stored at 4℃ for many years as the father plant. In a similar way to "backcrossing", the excellent traits of the best single plant (the mother plant of the first year of seed production) are maintained. The advantage of this is that the consistency of the offspring can be further improved by improving the purity of the father plant group. While ensuring the high consistency of the offspring, a certain amount of genetic heterogeneity is retained to ensure the strong vitality and stable yield of the offspring.
[0028] Approach 2: The self-pollination offspring of the first-year seed production mother are selected as the plant selection object, and the best single plant is selected, and the new mother parent (the self-pollination offspring of the first-year seed production mother parent) is formed by tissue culture and propagation; the father parent is the offspring of the excellent single plant group among the self-pollination offspring of the first-year seed production mother parent, which is further selected by the group (isolated mixed pollination by cuttings in different places), and the obtained offspring can be stored at 4℃ for many years as the father parent. In a similar way to "sister cross", the various excellent traits of the best single plant (the self-pollination offspring of the first-year seed production mother parent) are maintained. The advantage of this is that the homozygosity of the mother parent gene and the purity of the father parent group are further improved, while ensuring the high consistency of the offspring, a certain degree of genetic heterogeneity is retained, ensuring the strong vitality and stable yield of the offspring.
[0029] The specific method to be adopted is determined by the genetic purity of the original single plant (the first year seed production mother). If the purity is high, the method 1 is adopted to avoid serious self-pollination depression; if the purity is slightly lower, the method 2 is adopted to further improve the consistency of the offspring. However, no matter which method is adopted, the "maternal control method" can achieve large-scale seed production for many consecutive years, which has greater advantages than the traditional mixed seed harvesting method in terms of convenience and stability of yield and quality for many consecutive years.
[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by derivative pedigree method, characterized by: The specific steps include: S1: Acquisition of maternal material: In the field cultivation of Artemisia annua, the method of selecting excellent individual plants by the derivative pedigree method is adopted, the best individual plants selected after the last individual plant selection are selected, and young shoots are taken as explants in the budding stage to be propagated in the tissue culture room; in early April of the second year, the cover is opened to slow down the seedlings, and after 3-5 days, the seedlings are transplanted to the outdoor floating nutrient tray for seedling hardening, and then transplanted to the field in early May as the maternal parent; the derivative pedigree method is a one-time mixed selection method plus a continuous 3-year individual plant selection method; S2: Obtaining male parent materials: spray the inflorescence of the best single plant selected after the last single plant selection in step S1 with NaCl solution before flowering, bag the inflorescence or isolate the whole plant after the surface moisture is slightly dry, and collect the seeds individually; sow all in the second year, transplant to potted pots 5 days before the mother plant is transplanted, and place in the open space around the isolated seed production nursery, slightly higher than the conventional fertilizer and water management; pass phenotypic identification 15 days before budding, remove impurities, inferior plants and diseases, and select plants with the same or similar phenotypes as the parents as male parents; S3: Seed production: S31: Field preparation and plant-row configuration: After the hardening of the female parent seedlings is completed, select a field with medium to high fertility and convenient drainage and irrigation as the seed production field, and plant in ridges after rotary tillage and land preparation; in order to increase the seed production per unit area and prevent weeds, plant the female parent in the whole field in the early stage, and after the pre-bud phenotype identification is completed, thin the female parent with an interval of 3 plants according to the row ratio of 2:1 to reserve the male parent area; move the male parent selected in step S2 to the reserved male parent area and simply fix it to prevent it from tipping over; at this time, the male parent is planted at a higher position than the female parent, which is conducive to wind pollination to improve the pollination effect; S32: Field management: Fertilizer and water management should be carried out at a level slightly higher than the production management level during the entire growth period; avoid excessive nitrogen fertilizer application, and add sulfur-based potassium fertilizer in appropriate amounts during the clustering period; pay attention to field drainage in spring and summer, and prevent stem rot, stem base rot and root rot in hot and humid weather. For the initial outbreak, spray 450 times of 5% oxadipate aqueous solution once every 7 days for 3 times in a row; after July, pay attention to the prevention and control of chrysanthemum gall midges and green leafhoppers. Spray the whole plant with 1500 times of 40% dimethoate emulsifiable concentrate for chrysanthemum gall midges, and use 10,000 times of 25% thiamethoxam for green leafhoppers; S33: Flowering period regulation: mainly regulate the flowering period of the male parent. When the male parent’s flowering period is late, spray 90% gibberellin 100,000 times solution and 0.1% potassium dihydrogen phosphate after budding, which can advance the flowering period by 1-4 days. S34: Seed harvesting: Harvest the mother plant 7 days before the seeds are fully mature, dry in the sun until the seeds are fully mature, thresh, bag and store at 4°C for use as production seeds.
2. A high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by derivative pedigree method according to claim 1, characterized in that: In step S2, the height of the potted plant pot is 0.6 m.
3. The high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by the derivative pedigree method according to claim 1, characterized in that: In step S31, the ridge width is 0.8 m and the ridge height is 0.3-0.4 m.
4. The high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by the derivative pedigree method according to claim 1, characterized in that: In step S31, the plant spacing is 0.8-1.2 m.
5. The high-yield and high-efficiency seed production method suitable for breeding Artemisia annua varieties by the derivative pedigree method according to claim 1, characterized in that: In step S31, the cultivation position of the male parent is 0.6 m higher than that of the female parent.