A method for breeding alfalfa

Abstract

The present application belongs to the technical field of plant breeding, and particularly relates to a method for breeding alfalfa. The method comprises the following steps: 1) land preparation and seeding; 2) parent alfalfa growth management; 3) promoting the present budding and flowering of the parent alfalfa (accelerating the conversion of vegetative growth to reproductive growth) and pollination; 4) promoting seed plumping and maturation; 5) seed harvesting and seed treatment; 6) land preparation and summer seeding of the first generation seeds; 7) summer growth and development management of the first generation alfalfa; 8) present budding, pollination and seed harvesting of the first generation alfalfa; 9) autumn seeding and winter protection of the second generation alfalfa seeds. The method can significantly shorten the alfalfa breeding cycle, effectively solve the problem of low alfalfa breeding efficiency in China, and perfect the grass breeding technology system in China, which has far-reaching significance for the development of grass and animal husbandry, food security and ecological environment protection in Henan and even in China.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to a method for increasing the number of generations in alfalfa breeding. Background Technology

[0002] Alfalfa, a typical autotetraploid cross-pollinated crop, is an important representative of high-quality forage. In conventional alfalfa breeding, under field conditions, only one seed propagation generation can usually be completed per year. The selection of superior varieties often requires more than 6 years of cycle selection, and it generally takes more than 10 years for a variety to obtain plant variety rights. The long breeding cycle seriously restricts the process of new variety development.

[0003] Generation-skipping technology is a key technology for shortening the crop breeding process. Currently, generation-skipping technologies in crop breeding are mainly divided into three categories: First, off-site generation-skipping, which utilizes the climatic differences at different latitudes to achieve multiple generations of breeding in a single year. For example, Sanya in Hainan, China, known as the "Southern Breeding Silicon Valley," allows for the cultivation of crops such as rice and corn in winter, shortening the breeding cycle by one-third to one-half. Soybeans, using a "winter-spring-summer-autumn" model, can complete five generations of breeding in a year in Hainan, doubling the efficiency compared to traditional methods. Second, facility-based environmental control generation-skipping, which artificially creates suitable growing environments to achieve generation-skipping by optimizing light conditions, precisely controlling temperature, and implementing precise water and fertilizer management. Third, in-situ generation-skipping in special environments. The dry-hot valley climate and short-day characteristics of Yuanmou in Yunnan Province significantly enhance the generation-skipping effect for short-day crops; for example, corn can achieve 2-3 generations of breeding per year in the dry-hot valley of Yuanmou, Yunnan.

[0004] The economics of the three types of generation techniques differ significantly. Off-site generation techniques suffer from high breeding costs, large geographical spans, and management difficulties; controlled-environment generation techniques are prohibitively expensive (e.g., generation in a fully artificial climate chamber costs approximately $500 per generation); while in-situ generation techniques relying on the local natural environment are the most economically viable option. If regional natural conditions can be fully utilized, and alfalfa can be planted two generations per year through technological means, the breeding process can be effectively accelerated, breeding efficiency improved, and breeding costs reduced, addressing a core pain point in the research field.

[0005] Currently, first and second generation propagation techniques are widely used in the global alfalfa breeding field. For example, the United States uses a "north-breeding, south-propagation" model to achieve two generations of alfalfa breeding per year, but it basically cannot achieve seed production in the second generation; by combining year-round photoperiod-controlled greenhouse generation propagation, 4-5 generations of propagation can be completed. For example, the salt-tolerant alfalfa variety Oaxaca 2000, bred by the US Department of Agriculture (USDA), shortened the breeding cycle from 8 years to 3 years by combining 5 generations of greenhouse generation propagation technology with molecular marker-assisted selection (MAS) screening technology. In my country, research on alfalfa generation propagation technology is mostly concentrated in the arid northern regions and the hot southern regions. For example, Mengcao Ecology, in conjunction with the Yunnan Yuanmou Research and Development Center, is using the local dry and hot valley climate advantages to carry out generation propagation of alfalfa and other forage resources, and integrating the light and heat resources of the north and south, which is expected to increase the efficiency of alfalfa hybrid breeding by 2 times; the alfalfa breeding team of China Agricultural University can enable alfalfa to complete a generation in 3-4 months indoors by artificially controlling light and temperature. However, no relevant reports or literature were found on in-situ generation propagation technology using special environments.

[0006] Henan Province, located in the transitional zone between northern and southern my country, currently lacks research on alfalfa generation under natural conditions. However, breakthroughs have been achieved in field generation technology for crops such as wheat, corn, and peanuts in the Huang-Huai transitional climate zone. Peanuts (short-day crop): In Kaifeng, a "first-generation greenhouse mulching planting, second-generation field summer sowing mulching planting" model has been implemented, achieving two generations per year. In Zhumadian, two peanut harvests per year in Henan have been achieved solely through mulching. Corn (short-day crop): In the Nanyang Basin, simple techniques such as mulching and ripening promotion have enabled in-situ generation of corn breeding. In Zhengzhou, a combination of seedling transplanting in raised beds with nutrient pots and field mulching has also achieved the goal of generational increase in corn. Wheat (long-day crop): Heading and flowering require more than 12 hours of sunlight per day. In northern Henan, spring-type, weak-spring-type, and semi-winter-type varieties are selected and sown before June 15. Combined with cold storage vernalization treatment, supplemental lighting control, and greenhouse management, summer sowing and generation increase of wheat can be achieved. In the southern Huang-Huai region, seeds are harvested before June 4, dried, and then sown. Combined with cold storage vernalization, preheating treatment, and conventional transplanting management, natural generation increase of wheat can be achieved.

[0007] Therefore, it is evident that in Henan, a transitional zone between northern and southern my country, both short-day and long-day crops can potentially achieve in-situ generation increases through technological measures. Consequently, alfalfa, as a long-day crop, has a high probability of achieving in-situ generation increases under natural conditions in Henan. The feasibility is based on the following: Firstly, perennial alfalfa requires at least 12 hours of sunlight to bolt and flower normally, with a complete generation cycle (from greening to seed maturity) totaling 3-4.5 months. Spring-sown alfalfa flowers about one month later than greening alfalfa. If the second generation requires about 1.5 months for seed sowing and early seedling growth, then the complete generation cycle for the second generation totals 4.5-6 months. Therefore, alfalfa has two generations per year totaling 7.5-10.5 months. Secondly, Henan Province has two "effective long-day cycles" throughout the year (February-June and July-November), with an average annual sunshine duration of 1848.0-2488.7 hours and a frost-free period of 189-240 days (about 6-8 months). In fact, the alfalfa growing season is longer than the frost-free period; in Henan, alfalfa has approximately 9 months of growing time per year. Therefore, the natural conditions in Henan, such as sunshine, temperature, and frost-free period, basically meet the requirements for alfalfa generation.

[0008] Although the possibility of increasing the number of generations of alfalfa in Henan Province is promising, several challenges remain in practice: Summers are prone to high temperatures, droughts, or torrential rains, while winters may encounter strong cold air masses, all of which can negatively impact alfalfa growth, development, flowering, and fruiting. Furthermore, current summer-sown alfalfa cultivation and management techniques, high-temperature pollination efficiency enhancement techniques, and alfalfa seed dormancy breaking techniques are still immature. The technology for regulating the transition from vegetative to reproductive growth in alfalfa also has shortcomings, and there is a lack of clear standards for sowing time and crop rotation. All these factors increase the difficulty of implementing generational breeding. Therefore, this invention aims to achieve the goal of increasing the number of generations of alfalfa in Henan Province (two generations per year) under natural conditions by developing methods for protecting alfalfa seedling growth, shortening the alfalfa growth period, and improving pollination efficiency. Summary of the Invention

[0009] Currently, there are two approaches to alfalfa breeding: one is to achieve two generations of alfalfa cultivation in one year through the "north-south propagation" model, and the other is to increase the number of generations by controlling photoperiod and temperature indoors. The "north-south propagation" model can only achieve the propagation of the second generation and it is difficult to achieve seed production in the second generation. Moreover, the round-trip transportation costs are high, losses are significant, and it is troublesome. Indoor control of photoperiod and temperature for increasing the number of generations is very costly and the seed production is very limited.

[0010] To overcome the technical problems of the two aforementioned schemes and accelerate the breeding of alfalfa varieties, this invention provides a method for advancing alfalfa breeding generations, including the following measures: promoting early budding and flowering of alfalfa through field water control and the application of growth regulators; improving pollination efficiency and pod setting rate through artificial assisted self-pollination, hybridization, and insect-assisted hybridization; directly sowing seeds harvested in summer after breaking dormancy; implementing effective summer pest and disease control and preventing damage to alfalfa from rainstorms and hail during the rainy season; applying stress-resistant agents to improve the summer resistance of alfalfa; improving summer pollination efficiency and pod setting rate through artificial assisted self-pollination, hybridization, and insect-assisted hybridization under suitable conditions; ensuring summer seed quality through fertilization and irrigation; harvesting mature seeds and sowing in autumn (September-October); and comprehensively utilizing efficient alfalfa planting techniques to achieve generational breeding of alfalfa with two planting and harvesting seasons per year.

[0011] The method described in this invention fully utilizes the advantages of Henan Province, which has two long day cycles and a long frost-free period, to develop and apply technologies such as shortening the growth period of alfalfa, summer alfalfa germination and seedling cultivation management, improving the stress resistance of alfalfa seedlings, assisted pollination, and seedling overwintering, and to establish a breeding and generation technology system for alfalfa in Henan Province under natural conditions.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for increasing the number of generations of alfalfa breeding, comprising three main parts: parent alfalfa breeding and cultivation, first-generation breeding and cultivation, and second-generation breeding and cultivation.

[0013] Specifically, the method includes the following steps: 1) Land preparation and sowing (autumn sowing, or spring sowing, or winter sowing). 2) Growth management of parent alfalfa; 3) Promotes budding and flowering of parent alfalfa (accelerating the transition from vegetative growth to reproductive growth) and pollination; 4) Promotes seed filling and maturation; 5) Seed harvesting and treatment; 6) Land preparation and summer sowing of first-generation seeds; 7) Summer growth and development management of first-generation alfalfa; 8) First generation alfalfa budding, pollination and seed harvesting; 9) Sowing second-generation alfalfa seeds in late autumn and winter protection.

[0014] Specifically, in step 1), when preparing the land, first remove weeds and pests, then apply sufficient base fertilizer (preferably 50 kg of nitrogen, phosphorus and potassium compound fertilizer per acre), then plow the land to a depth of 25-30 cm until the soil is loose and level, and compact the soil; then, dig drainage ditches around the land to ensure drainage and prevent water from accumulating after heavy rain.

[0015] Specifically, in step 1), the sowing method is to use hole sowing, with a row spacing of 60cm and a plant spacing of 40cm; plump yellow seeds are preferred, and one or two seeds are sown alternately in consecutive holes, with a sowing depth of 1-2cm, and the seeds are compacted after sowing.

[0016] Specifically, in step 2), the principle of alfalfa regrowth and growth management is that as long as it can grow normally, no irrigation or fertilization is needed; in addition to normal weed control and pest and disease control, the key point is water control. If there is too much rain, timely drainage should be done; if the alfalfa in the seedbed does not experience severe drought in the spring, no watering should be done. The cultivation and management of alfalfa should be carried out in accordance with DB41 / T 1807-2019 "Technical Regulations for Alfalfa Production". The difference from DB41 / T 1807-2019 is that the soil relative field water holding capacity should be maintained at 50% to 60%; the soil should be kept relatively dry during the critical period of alfalfa transition from vegetative growth to reproductive growth (i.e., the early budding stage).

[0017] Specifically, in step 2), for pest and disease control, select the corresponding highly effective and low-toxicity insecticides (such as imidacloprid and abamectin) and fungicides (such as chlorothalonil and thiophanate-methyl) according to the actual types of pests and diseases, and carry out precise control at the early stage of disease or pest occurrence.

[0018] Specifically, in step 3), when promoting budding and flowering, the soil moisture content in the 0-40cm root layer should be maintained at 50%-60% of field capacity during the branching stage to the early budding stage of alfalfa. When the soil moisture content continues to decrease to 50% of field capacity, supplemental irrigation should be started. Avoid soil moisture content being lower than 45% of field capacity for a long period of time to prevent excessive drought from hindering bud development and causing premature leaf senescence and shedding.

[0019] Specifically, in step 3), when promoting budding and flowering, spray with fluopyram (CAG, dosage 1.0-1.5g / L) or chlormequat chloride (DPC, dosage 0.25-0.45g / L) or 1.8% sodium nitrophenolate (CSN, 4000-5000 times dilution) when the plant height is 55cm-60cm or the number of stem nodes is 8-10. At the same time, supplement with spraying potassium dihydrogen phosphate (0.25%-0.3%), borax (0.08%-0.12%), and ammonium molybdate (0.025%-0.04%).

[0020] Preferably, in step 3), when promoting budding and flowering, hormones ABA, ethephon, or 6-BA can also be applied; wherein the dosage of 6-BA is 10-20 mg / L.

[0021] Specifically, in step 3), flowering and pollination occur from mid-April to early May (early May specifically); to improve pollination efficiency, the following pollination method is adopted: Self-pollination: Self-pollination is achieved by using bagging artificial pollination technology or by isolating individual plants in a net house with leafcutter bees or bumblebees. Hybrid pollination: Hybrid pollination is achieved by using leafcutter bees, bumblebees, or other assisted cross-pollination techniques, or by artificial assisted hybridization techniques or open pollination under natural conditions.

[0022] The specific steps are as follows: For self-pollination, use a 60-mesh net bag to expose 1 / 4 of the floret in the late budding stage. Perform artificial pollination multiple times as needed, until the flag petal opens but the keel petal remains closed. For single-plant self-pollination, a 60-mesh isolation net can be used to isolate bumblebees for assisted pollination. For assisted hybridization with leafcutter bees or bumblebees, use a 60-mesh isolation net to isolate the parent alfalfa in the late budding stage, then introduce leafcutter bees or bumblebees for assisted pollination. Artificial assisted hybridization involves manually removing the emasculation from the female parent, manually collecting pollen from the male parent, and manually transferring the pollen to the female pistil by gently touching the stigma with a pollen-covered cotton swab. Open pollination under natural conditions refers to the natural pollination of alfalfa plants by bees, insects, or other animals within a radius of at least 1 kilometer where no alfalfa is planted or isolated.

[0023] Specifically, in step 4), after pollination is completed, water once (drip irrigation depth 10cm-20cm), spray with potassium dihydrogen phosphate (0.25%~0.3%), and at the same time prevent lodging. The seeds mature in late May to early July, and then they are harvested.

[0024] Specifically, in step 5), once the pods have grown to full size, they can be harvested when more than 70% of the pods have turned brown. The pods are then threshed, and the seeds are immediately screened and processed after threshing.

[0025] Specifically, in step 5), the seed treatment involves selecting plump alfalfa seeds free from pests and diseases, and treating them using the friction-rubbing method and the freezing method to improve the germination rate. During the harvesting and threshing process, the land should be prepared and drainage ditches should be made in accordance with the conventional sowing requirements (same as the land preparation method in step 1).

[0026] Preferably, in step 5), the seeds obtained after threshing are subjected to friction and temperature-changing treatment; Friction treatment: lightly rub the seed coat with sandpaper 50 times to break the hard kernel; variable temperature treatment: treat the seeds in liquid nitrogen for 10 seconds and then remove them to allow them to return to room temperature.

[0027] Specifically, in step 6), land preparation should be completed before harvesting; to prevent waterlogging in summer, drainage should be done well to ensure that no water remains after heavy rain; sufficient base fertilizer should be applied during land preparation (50 kg of alfalfa-specific compound fertilizer per acre). Sowing should be done as early as possible, at the latest one week after the harvest; hill sowing should be used. When sowing, treat the seeds with liquid nitrogen for 10 seconds, then remove them and let them cool to room temperature before sowing. The sowing method is the same as in step 1.

[0028] Specifically, in step 6), sowing is generally carried out in the field from the end of May to the beginning of July.

[0029] Preferably, when sowing and raising seedlings in the field, protective sowing techniques are adopted, specifically sowing annual protective crops such as Sudan grass, sorghum, and forage corn.

[0030] Preferably, during the seedling stage in the field, heat damage can be alleviated by adding substances such as silicon and salicylic acid.

[0031] Specifically, in step 7), shading and irrigation are used to cool the seedlings; Post-seedling management includes: a) shading; b) water control, requiring minimal watering as long as the seedlings can grow normally; c) timely drainage; d) spraying agents to enhance stress resistance (including potassium dihydrogen phosphate and brassinolide); e) weed control; f) disease and pest control.

[0032] Specifically, step 8) includes steps such as preventing lodging, promoting budding, promoting flowering, assisting pollination, natural pollination, promoting seed filling, and harvesting seeds; among these, alfalfa is prone to lodging in summer, so anti-lodging devices are set up to prevent lodging; in order to accelerate budding and flowering, agents that promote budding and flowering are sprayed.

[0033] Preferably, in step 8), flowering and pollination generally take place from late July to early September. Depending on the purpose and environmental conditions, self-pollination or hybridization methods such as natural pollination, bee-assisted pollination, or artificial pollination are used to pollinate as early as possible.

[0034] Preferably, in step 8), the soil relative field capacity is maintained at 50%–60%; when the plant height is 30cm–40cm or the number of stem nodes is 6–8, a bud-promoting agent (specifically CAG 1.0–1.5g / L or DPC 0.25–0.45g / L or 1.8% sodium nitrophenolate (CSN, diluted 4000–5000 times)) is sprayed. Preferably, in step 8), when promoting bud formation, hormones ABA, ethephon, or 6-BA may also be applied; wherein the dosage of 6-BA is 10–20 mg / L. At the same time, apply supplementary foliar sprays of potassium dihydrogen phosphate (0.25%–0.3%), borax (0.08%–0.12%), and ammonium dimolybdate (0.025%–0.04%) during the late branching stage to 7–10 days before budding. Finally, spraying with 1-2 mg / L brassinolide can improve summer stress resistance.

[0035] Preferably, in step 8), the following pollination method is used: Self-pollination: Self-pollination is achieved by using bagging artificial pollination technology or by isolating individual plants in a net house with leafcutter bees or bumblebees. Hybrid pollination: Hybrid pollination is achieved by using leafcutter bees, bumblebees, or other assisted cross-pollination techniques, or by artificial assisted hybridization techniques or open pollination under natural conditions.

[0036] The specific steps are as follows: For self-pollination, use a 60-mesh net bag to expose 1 / 4 of the floret in the late budding stage. Perform artificial pollination multiple times as needed, until the flag petal opens but the keel petal remains closed. For single-plant self-pollination, a 60-mesh isolation net can be used to isolate bumblebees for assisted pollination. For assisted hybridization with leafcutter bees or bumblebees, use a 60-mesh isolation net to isolate the parent alfalfa in the late budding stage, then introduce leafcutter bees or bumblebees for assisted pollination. Artificial assisted hybridization involves manually removing the emasculation from the female parent, manually collecting pollen from the male parent, and manually transferring the pollen to the female pistil by gently touching the stigma with a pollen-covered cotton swab. Open pollination under natural conditions refers to the natural pollination of alfalfa plants by bees, insects, or other animals within a radius of at least 1 kilometer where no alfalfa is planted or isolated.

[0037] Specifically, in step 8), to improve pollination efficiency, artificial or bee-assisted pollination is used; to ensure high-quality seed filling and maturation, timely irrigation is provided during droughts, and potassium dihydrogen phosphate (0.25%–0.3% by mass) is sprayed on individual plants; when the temperature is above 37℃ and the sun is strong, shading and irrigation are provided to cool the plants and prevent lodging. The seeds are typically sown and threshed between late August and mid-October.

[0038] Specifically, in step 9), the land preparation for second-generation seeds to be sown in late autumn should be completed before harvesting; the treatment method for second-generation seeds is the same as that in step 5), and the land preparation and sowing method for second-generation seeds to be sown in autumn is the same as that for first-generation seeds; no shading or irrigation for cooling is required during seedling emergence and seedling management, and irrigation should be carried out in time if there is drought.

[0039] Specifically, in step 9), after treating the second-generation seeds, sow them as early as possible, select different types of film and design different covering methods, and cover them with mulch film to keep them warm after sowing; before winter, when the soil freezes at night and thaws during the day, water them thoroughly to seal them from freezing.

[0040] Preferably, in step 9), the second-generation seeds should be harvested as early and quickly as possible, with some second-generation seeds being harvested in mid-to-late October or even early November; the harvesting method for the second-generation seeds is the same as that for the first-generation seeds, and the seed treatment method is the same as that in step 5).

[0041] Compared with the prior art, the advantages of the present invention are: Henan's climate and soil characteristics are representative of the Central Plains region of my country. The achievements of its field research, such as germplasm screening standards, seed dormancy breaking technology, suitable sowing period schemes, technology for regulating the transition from vegetative growth to reproductive growth, field management technology for high temperature and drought (or rainstorm), integrated pest management schemes, and assisted pollination technology, can directly benefit surrounding provinces and provide replicable and scalable technical paradigms for alfalfa breeding and industrial development in the entire Central Plains and Huanghuai regions.

[0042] The alfalfa breeding generation method proposed in this invention was studied under natural field conditions in Henan Province. It not only addresses the practical needs of low alfalfa breeding efficiency and forage shortage in the region, but also improves the theoretical system of grass breeding in the Huang-Huai region. It has both important scientific theoretical value and agricultural production value, and has profound significance for the development of grass animal husbandry, food security and ecological environment protection in Henan and even the Central Plains region of my country. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods in the following examples are generally carried out under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the raw materials and reagents used are all commercially available products.

[0045] The alfalfa breeding generation method described in this invention is applicable to alfalfa breeding or cultivation in multiple regions of my country, for example: a. The entire provinces of Hubei and Henan in Central China, northern Hunan (north of Changde and Yueyang), and northern Jiangxi (around Jiujiang). b. The entire provinces of Anhui, Jiangsu, and Shandong in East China, northern Zhejiang (north of Hangzhou), and Shanghai; c. All of Hebei, Shanxi, Beijing, and Tianjin in North China.

[0046] Especially applicable to the broad Central Plains region: the entire Henan province (core), southern Shanxi: Yuncheng and Linfen, southern Hebei: Handan and Xingtai, southwestern Shandong: Heze, Jining, Zaozhuang, and western Linyi, northern Anhui: Fuyang, Bozhou, Huaibei, Suzhou, and northern Bengbu, and northern Jiangsu: Xuzhou and Suqian.

[0047] Example 1

[0048] Example 1 provides a method for increasing the number of generations of alfalfa breeding. This example mainly focuses on the cultivation of alfalfa. The specific steps are as follows: 1. Land preparation and sowing 1.1 Land preparation: Before deep plowing, remove as many weeds as possible, apply phorate-treated soil to control underground pests such as mole crickets, grubs, and cutworms, and apply sufficient base fertilizer (generally 50 kg of NPK compound fertilizer per acre). Deep plow to a depth of 25-30 cm, harrow until the soil is loose and level, and finally compact the soil until an adult's shoe sinks 1-2 cm into the soil. Note that high-viscosity soils do not require compaction.

[0049] Meanwhile, drainage ditches were dug around the land to ensure proper drainage and prevent waterlogging after heavy rains. Other procedures were carried out using conventional seed sowing methods.

[0050] 1.2 Sowing: Before sowing, investigate the soil moisture. If the moisture is poor, irrigate in advance. Use hill sowing in early October in autumn, with a row spacing of 60cm and a plant spacing of 40cm. Select plump yellow seeds and sow 1 or 2 seeds per hill alternately, at a depth of 1-2cm. After sowing, compact the soil.

[0051] 2. Growth Management of Parent Alfalfa The principle of alfalfa regrowth and growth management is that as long as it can grow normally, irrigation and fertilization are not necessary. In addition to normal weed control and pest and disease control, the key point is water control. If there is excessive rainfall, timely drainage should be ensured. If the alfalfa in the seedbed does not experience severe drought in the spring, it should not be watered.

[0052] In accordance with DB41 / T 1807-2019 "Technical Regulations for Alfalfa Production", the field cultivation and management of parent alfalfa should be carried out. The difference from DB41 / T 1807-2019 "Technical Regulations for Alfalfa Production" is that the soil relative field water holding capacity should be maintained at 50% to 60%.

[0053] During the critical period when parent alfalfa transitions from vegetative growth to reproductive growth (i.e., the early budding stage), the soil must be kept relatively dry.

[0054] 3. Promote budding and flowering (accelerate the transition from vegetative growth to reproductive growth) and pollination. Spray a certain concentration of CAG, DPC, and CSN during the later stages of alfalfa vegetative growth; at the same time, apply a certain concentration of potassium dihydrogen phosphate, borax, and ammonium molybdate as an auxiliary spray 7-10 days before budding at the end of branching stage; and spray an appropriate concentration of brassinolide to improve summer stress resistance.

[0055] The specific steps are as follows: 3.1 Maintaining soil relative field capacity With the goal of promoting early budding and flowering of the parent alfalfa, the soil moisture content in the 0-40cm root layer of the parent alfalfa should be maintained at 50%-60% of field capacity during the branching stage to the pre-budding stage. Supplementary irrigation should be started when the soil moisture content continues to drop to 50% of field capacity. Avoid soil moisture content that is below 45% of field capacity for a long period of time to prevent excessive drought from hindering bud development and causing premature leaf senescence and shedding.

[0056] 3.2 Spraying agents to promote budding and flowering When the plant height reaches 55-60cm or the number of stem nodes reaches 8-10, spray with flubendiamide (CAG, 1.5g / L), dichlorvos (DPC, 0.30g / L), or a 4000-fold dilution of 1.8% sodium nitrophenolate, applying 5ml per plant. Simultaneously, spray with a solution of 0.3% potassium dihydrogen phosphate, 0.08% borax, and 0.025% ammonium molybdate, applying 5ml per plant. Hormones ABA, ethephon, or 6-BA can also be applied; the dosage of 6-BA is 10-20 mg / L.

[0057] 3.3 Pollination Flowering and pollination occur from mid-April to early May. To achieve different seed production goals and improve pollination efficiency, for self-pollination, a 60-mesh small net bag is used, with 1 / 4 of the floret exposed in the bag during the late budding stage. Artificial pollination is performed multiple times while the flag petal is open but the keel petal is not, along with some natural pollination. For hybrid combinations, a 60-mesh isolation net is used to isolate the parent alfalfa during the late budding stage, and bumblebees are introduced to assist in pollination.

[0058] 4. Promotes seed filling and maturation After pollination, water once with drip irrigation to a depth of 10cm. Spray each alfalfa plant with 5ml of 0.3% potassium dihydrogen phosphate. At the same time, prevent lodging. The pods formed by pollination will mature in late May to early July.

[0059] 5. Seed harvesting and treatment Harvesting takes place from late May to early July. Seeds are harvested when the pods turn brown and then threshed. Immediately after threshing, seeds are screened and treated. Seed treatment involves selecting plump, disease-free alfalfa seeds and treating them using a friction-rubbing method followed by freezing.

[0060] Specifically, in this embodiment, the seeds obtained after threshing are subjected to friction and temperature-changing treatment.

[0061] Friction treatment involves gently rubbing the seed coat with sandpaper 50 times to break up the hard kernel. Temperature change treatment involves immersing the seeds in liquid nitrogen for 10 seconds, then immediately removing them and allowing them to return to room temperature before direct sowing.

[0062] During the harvesting and threshing process, the land should be prepared according to the conventional sowing requirements. In addition, the land should be 10cm higher than the surrounding area, and drainage ditches should be constructed.

[0063] 6. Land preparation and summer sowing of first-generation seeds Complete land preparation before harvesting, following the same steps as in 1.1. Prepare drainage ditches to prevent waterlogging in summer.

[0064] Sowing should be done as early as possible, at the latest one week after the harvest. Use hill sowing, with a row spacing of 60cm and a plant spacing of 20cm.

[0065] When sowing, treat the seeds with liquid nitrogen for 10 seconds, then remove them and let them cool to room temperature before sowing. The sowing method is the same as in step 1.2.

[0066] The specific steps are as follows: Sowing is generally done from late May to early July: use hill sowing, with a sowing depth of 1cm, row spacing of 60cm, and plant spacing of 20cm. Provide shade and watering.

[0067] 7. Summer growth and development management of first-generation alfalfa After sowing, the main methods to cool the seedlings are shading and irrigation.

[0068] Post-seedling management includes: (1) shading; (2) water control; the requirement is to avoid watering as much as possible as long as the seedlings can grow normally; (3) timely drainage; (4) spraying stress-resistant agents such as brassinolide and potassium dihydrogen phosphate; (5) weed control; (6) disease and pest control.

[0069] The specific steps are as follows: Two shading schemes were adopted: one was to use 100% shading netting during the seedling emergence period, and then switch to 50% shading netting after seedling emergence; the other was to use 50% shading netting continuously until the first generation alfalfa reached the 5-layer true leaf stage.

[0070] After sowing, drip irrigation is used twice a day, once in the morning and once in the evening, for 20 minutes each time, to effectively reduce soil temperature.

[0071] During the 2-3 true leaf stage of the first-generation alfalfa seedlings, foliar spray with stress-resistance agents such as brassinolide and 0.2% potassium dihydrogen phosphate. Specifically, spray each plant with 2 mg / L brassinolide and 5 ml of 0.2% potassium dihydrogen phosphate to improve summer stress resistance.

[0072] Summer is the rainy season, so if there is water accumulation, drain it promptly. Follow DB41 / T 1807-2019 "Technical Regulations for Alfalfa Production" to control weeds and prevent summer pests and diseases, and take precautions against rain, hail, and lodging.

[0073] 8. First-generation alfalfa budding, pollination, and seed harvesting Step 8. This mainly includes steps such as preventing lodging, promoting budding, promoting flowering, assisting pollination, natural pollination, promoting seed filling, and harvesting seeds, as detailed below: Alfalfa is prone to lodging in summer, so anti-lodging devices should be installed to prevent it. The method for treating first-generation seeds is the same as in step 5.

[0074] 8.1 Promote bud formation and flowering Flowering and pollination typically occur from late July to early September. Depending on the purpose and environmental conditions, self-pollination or hybridization methods such as natural pollination, bee-assisted pollination, or artificial pollination should be used, and pollination should be carried out as early and as quickly as possible. Specific procedures are as follows: 8.1.1 Maintain soil relative field capacity at 50%–60%.

[0075] 8.1.2 When the plant height is 30cm-40cm or the number of stem nodes is 6-8, spray with an agent to promote budding and flowering, as in 3.2.

[0076] 8.1.3 Spraying with 2 mg / L brassinolide can improve summer stress resistance.

[0077] 8.2 Improve summer pollination efficiency To achieve seed production and improve pollination efficiency, self-pollination and cross-pollination are employed, as detailed below: Self-pollination: Self-pollination is achieved using bagging and artificial pollination techniques.

[0078] Cross-pollination: Cross-pollination is achieved by using leafcutter bees or bumblebees in net rooms to assist in cross-pollination, or by artificial hybridization techniques.

[0079] Since natural pollination efficiency is low in summer, this application optimizes the pollination period and pollination conditions (temperature below 35°C) to improve summer pollination efficiency (summer pollination efficiency is key to pod formation).

[0080] 8.3 Ensure high-quality seed filling and maturation Spray 5 ml of 0.3% potassium dihydrogen phosphate per plant; irrigate promptly during drought; provide shade and irrigation to cool the plant when the temperature is above 37℃ or the sun is too strong, and prevent lodging.

[0081] 8.4 Harvesting and Threshing The seeds are generally harvested and threshed from late August to mid-October. The harvesting steps are the same as in step 5.

[0082] 9. Second-generation seeds: late autumn sowing and winter protection For second-generation seeds, land preparation for late autumn sowing should be completed before harvest. The land preparation and sowing methods for second-generation seeds are the same as for first-generation seeds, only the sowing time is different. No shading or irrigation for cooling is required during emergence and seedling management; sprinkler or drip irrigation can be used if drought occurs.

[0083] After treating the second-generation seeds, sow them as early as possible, selecting appropriate film types and designing different covering methods. After sowing, cover with mulch to retain heat. Before winter, when the soil freezes at night and thaws during the day, water thoroughly to seal the ground.

[0084] Specifically, sowing takes place from mid-September to October 20th in autumn, and the seeds turn green again in the spring of the following year to enter the next generation cycle (including steps such as greening and growth management, budding and flowering, pollination, seed harvesting and seed treatment).

[0085] Seed treatment is the same as in step 5. Sowing is the same as in step 6, except that shading is not required.

[0086] Specifically, second-generation seeds should be harvested as early and quickly as possible. Some second-generation seeds may be harvested in mid-to-late October, or even early November. The harvesting method for second-generation seeds is the same as that for first-generation seeds, and the seed treatment method is the same as in step 5.

[0087] At the same time, take precautions against frost damage during winter.

[0088] Application Example 1 At the alfalfa variety resource nursery of the Henan Provincial Modern Agricultural Research and Development Base, following the steps 2. Spring alfalfa growth management and 3. Methods for promoting budding and flowering in Example 1, the stubble alfalfa was managed to promote regrowth and budding and flowering.

[0089] Observations revealed that some alfalfa plants began flowering on April 13, 2026. Flowering plants were marked starting April 13, with a marking period of one week. The first batch of seeds was harvested on May 27, and the harvesting, treatment, and sowing methods were performed according to the method described in Example 1. Similar to May 27, seeds were harvested and sown on June 15, 2026, and June 30, 2026, respectively.

[0090] Seedlings emerge 5-7 days after sowing. Field management is carried out according to DB41 / T 1807-2019 "Technical Regulations for Alfalfa Production". Various reagents are sprayed according to the method for promoting budding and flowering in summer in Example 1 to accelerate growth, budding and flowering.

[0091] The plant population established on May 27, 2026, saw some plants budding on July 15, 2026, and reaching the initial flowering stage on July 19, when pollination began. Some plants underwent artificial self-pollination for seven consecutive days with bagging, while others were naturally pollinated. Pods emerged on July 25, and some mature pods were observed on August 21. The mature pods yielded plump seeds after threshing, and the plants were still in full bloom.

[0092] The plant population established on June 15, 2026, saw some plants bud and bloom on July 19, and by July 22, they had reached the initial flowering stage and started pollination. Similarly, some plants were bagged for seven consecutive days for artificial self-pollination, while others were naturally pollinated. Pods appeared on July 26, and by August 21, there were already many pods. Some pods matured on August 26. The mature pods yielded plump seeds after threshing, and the plant was still in its peak flowering period.

[0093] The plant population established on June 30, 2026, saw a large number of plants budding on August 21. Natural pollination was used, and the plant population reached the initial flowering stage on August 28, with some plants producing pods. The pods matured and were harvested around the end of September or the beginning of October.

[0094] The harvested seeds were treated with friction and liquid nitrogen and sown in late September to early October 2026. They were managed according to conventional alfalfa emergence, seedling stage and overwintering management methods.

[0095] Since alfalfa has indeterminate inflorescences, once it reaches the reproductive stage, it will continue to grow, bud, flower, and bear fruit as long as the growth and development conditions are suitable, until the pods mature before winter when alfalfa is no longer suitable for growth. The embodiments of this invention only describe the growth or cultivation process during a specific growth period (i.e., from the appearance of buds to the discovery of mature pods). Those skilled in the art will know that the generation method described in this embodiment is only used to illustrate the invention and should not be regarded as limiting the scope of the invention.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for increasing the number of generations in alfalfa breeding, characterized in that, Includes the following steps: 1) Land preparation and sowing; 2) Growth management of parent alfalfa; 3) Promotes budding, flowering, and pollination of the parent alfalfa; 4) Promotes seed filling and maturation; 5) Seed harvesting and treatment; 6) Land preparation and summer sowing of first-generation seeds; 7) Summer growth and development management of first-generation alfalfa; 8) First generation alfalfa budding, pollination and seed harvesting; 9) Sowing second-generation alfalfa seeds in late autumn and winter protection.

2. The method according to claim 1, characterized in that, In step 1), when preparing the land, first remove weeds and pests, then apply sufficient base fertilizer, then turn the soil to a depth of 25-30 cm until the soil is loose and level, and compact the soil; then, dig drainage ditches around the land to ensure drainage and prevent water from accumulating after heavy rain. In step 1), the sowing method is hole sowing, with a row spacing of 60cm and a plant spacing of 40cm; plump yellow seeds are preferred, and one or two seeds are sown alternately in consecutive holes, with a sowing depth of 1-2cm, and the soil is compacted after sowing.

3. The method according to claim 1, characterized in that, In step 3), when promoting budding and flowering, the soil moisture content in the 0-40cm root layer should be maintained at 50%-60% of field capacity during the branching stage to the pre-budding stage of alfalfa. When the soil moisture content continues to drop to 50% of field capacity, supplemental irrigation should be started. Avoid soil moisture content being lower than 45% of field capacity for a long period of time to prevent excessive drought from hindering bud development and causing premature leaf senescence and shedding. In step 3), when promoting budding and flowering, spray with flubendiamide (CAG, dosage 1.0-1.5 g / L) or chlormequat chloride (DPC, dosage 0.25-0.45 g / L) or 1.8% sodium nitrophenolate (CSN, 4000-5000 times dilution) when the plant height is 55cm-60cm or the number of stem nodes is 8-10. At the same time, supplement with spraying potassium dihydrogen phosphate (0.25%-0.3%), borax (0.08%-0.12%), and ammonium molybdate (0.025%-0.04%). In step 3), when promoting bud formation and flowering, hormones ABA, ethephon, or 6-BA can also be applied; the dosage of 6-BA is 10-20 mg / L.

4. The method according to claim 1, characterized in that, In step 3), flowering and pollination occur from mid-April to early May (specifically, early May). To improve pollination efficiency, the following pollination method is used: Self-pollination: Self-pollination is achieved by using bagging artificial pollination technology or by isolating individual plants in a net house with leafcutter bees or bumblebees. Hybrid pollination: Hybrid pollination is achieved by using leafcutter bees or bumblebees to assist cross-pollination, or by artificial hybridization techniques or open pollination under natural conditions.

5. The method according to claim 1, characterized in that, In step 5), the seed treatment involves selecting plump alfalfa seeds free from pests and diseases, and treating them using a friction-rubbing method and a freezing method to improve the germination rate.

6. The method according to claim 1, characterized in that, In step 6), land preparation should be completed before harvesting; to prevent waterlogging in summer, drainage should be ensured to prevent water accumulation after heavy rain; sufficient base fertilizer should be applied during land preparation. Sowing should be done as early as possible, at the latest one week after the harvest; hill sowing should be used. When sowing, treat the seeds with liquid nitrogen for 10 seconds, then remove them and let them cool to room temperature before sowing, using the same method as step 1). In step 6), sowing is generally carried out in the field from the end of May to the beginning of July.

7. The method according to claim 1, characterized in that, In step 7), shading and irrigation are used to cool the seedlings; Post-seedling management includes: a) shading; b) water control; c) timely drainage; d) spraying agents to improve stress resistance; e) weed control; f) disease and pest control.

8. The method according to claim 1, characterized in that, Step 8) includes steps such as preventing lodging, promoting budding, promoting flowering, assisting pollination, natural pollination, promoting seed filling, and harvesting seeds; among them, alfalfa is prone to lodging in summer, so anti-lodging devices are set up to prevent lodging; in order to accelerate budding and flowering, agents that promote budding and flowering are sprayed (same as step 3).

9. The method according to claim 8, characterized in that, In step 8), to improve pollination efficiency, artificial or bee-assisted pollination is used (same as step 4); to ensure high-quality seed filling and maturation, timely irrigation is provided during droughts, and potassium dihydrogen phosphate (mass fraction 0.25% to 0.3%) is sprayed on individual plants. When the temperature is above 37℃ and the sun is strong, shading and irrigation are provided to cool the plants and prevent lodging. Harvesting and threshing typically take place from late August to mid-October.

10. The method according to claim 1, characterized in that, In step 9), the land preparation for second-generation seeds to be sown in late autumn should be completed before harvesting; the treatment method for second-generation seeds is the same as that in step 5), and the land preparation and sowing method for second-generation seeds to be sown in autumn is the same as that for first-generation seeds; no shading or irrigation for cooling is required during seedling emergence and seedling management, and irrigation should be carried out in time if there is drought.