A method for accelerated generation breeding of alfalfa

Abstract

The application provides an alfalfa accelerated generation breeding method, which comprises (1) seed germination: alfalfa seeds are cultured for 1 week to germinate to obtain seedlings; (2) seedling culture: the seedlings are hydroponically cultured for 1-2 weeks, and then transplanted into vermiculite for culture for 2 weeks; (3) culture in the seedling stage: culture in the vermiculite for 4 weeks; (4) flowering induction: induction for 1-3 weeks to the full flowering stage; (5) full flowering stage culture: culture for 3-4 weeks to the end flowering stage; (6) seed maturation: culture for 4 weeks to seed maturation. The method can effectively shorten the time required for alfalfa breeding by adjusting the culture conditions, light conditions and pollination parameters, so that alfalfa can complete a generation in 3-4 months in the room.

Description

Technical Field

[0001] This application belongs to the field of plant breeding, specifically, this application provides a method for accelerated generation breeding of alfalfa. Background Technology

[0002] Alfalfa (Medicago sativa L.) is an autotetraploid plant that is cross-pollinated by insects. As a perennial leguminous forage, it has strong vitality and adaptability, can grow in various soil conditions, and has a certain tolerance to climate change. It is known for its rich nutritional value, high yield and good palatability, and is hailed as the "King of Forage".

[0003] Under natural field conditions, the flowering and harvesting time of alfalfa is affected by factors such as geographical location, climate, planting management, and variety. Spring-sown alfalfa typically requires 4-6 months to harvest mature seeds. Autumn-sown alfalfa, due to lower temperatures and reduced daylight hours, requires harvesting the next generation of seeds in June or July of the following year. Therefore, in alfalfa breeding, only one seed propagation generation is usually completed in the field each year. The selection of superior varieties often requires more than 6 years of cycle selection; therefore, accelerating the seed propagation generation within a single year is a key factor determining the speed of alfalfa breeding.

[0004] Multigenerational propagation is a reproductive process that exceeds the reproductive cycle and number of offspring of the species itself. With artificial intervention, the alfalfa breeding cycle can be shortened by changing the photoperiod of the alfalfa growth environment and introducing bees for pollination. Summary of the Invention

[0005] To explore accelerated breeding techniques for alfalfa to propagate multiple generations in a year, this study: 1) investigated and obtained the conditions for rapid growth of alfalfa from seed germination to mature plants, enabling flowering induction 8 weeks after sowing; 2) studied bee release techniques during alfalfa flowering, considering its cross-pollination and insect-pollinated characteristics, and established an indoor insect pollination technology for alfalfa; 3) previous findings showed that alfalfa showed good growth performance at all stages of a generation under FCL2 light quality, but the high cost of the light panels and limited space in the incubator made it unsuitable for large-scale alfalfa generation propagation. Given that FCL4 can also induce flowering, but is not suitable for seedling stage and post-fruiting plant growth, we explored suitable light quality and photoperiod conditions for different growth stages of alfalfa for generation propagation. This system has advantages such as being economical, applicable, and suitable for large-scale propagation; 4) through testing on 20 alfalfa varieties from home and abroad, the results showed that this system can enable alfalfa to complete a generation indoors in 3-4 months. This system has significant application value for shortening the alfalfa breeding cycle and accelerating the alfalfa breeding process.

[0006] On the one hand, this application provides a method for accelerated generation breeding of alfalfa, the method comprising:

[0007] (1) Seed germination: Alfalfa seeds were cultured in a germination box for 1 week to obtain seedlings;

[0008] (2) Seedling stage cultivation: Cultivate seedlings in hydroponics for 1-2 weeks, then transplant them into vermiculite for 2 weeks;

[0009] (3) Seedling stage cultivation: Cultivate in vermiculite with nutrient solution for 4 weeks;

[0010] (4) Inducing flowering: Inducing flowering for 1-3 weeks until the peak flowering period;

[0011] (5) Cultivation during peak flowering period: Cultivate for 3-4 weeks until the end of flowering period;

[0012] (6) Seed maturation: Cultivate for 4 weeks until the seeds mature.

[0013] Further, in step (2), the seedlings were transplanted into 0.1× Hoagland nutrient solution at 25℃, with 14h light / 10h darkness, and hydroponically cultured for 1-2 weeks; or transplanted into vermiculite at 25℃, with 14h light / 10h darkness, and cultured for 2 weeks. The light quality conditions were: 450nm-465nm: 96.06%, 450nm-480nm: 1.44%, 615nm-650nm: 2.5%, and the total light intensity was 19000lx-32000lx. Nutrient solution was applied every other day.

[0014] Further, in step (3), the culture was carried out at 25°C for 14 hours under light and 10 hours under darkness for 4 weeks. The light quality conditions were as follows: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, 700nm-780nm: 10.47%; the total light intensity was 19000lx-32000lx, and the nutrient solution was applied every other day.

[0015] Further, in step (4), the culture was carried out at 26°C for 22 hours of light and 2 hours of darkness for 1-3 weeks, with the following light quality conditions: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, 700nm-780nm: 10.47%; and the total light intensity was 19000lx-32000lx.

[0016] Furthermore, in step (4), when most of the materials reach the initial flowering stage, the beehive is placed in the cultivation room or net room, and the number of bees is observed. The size of the beehive outlet is adjusted according to the number of materials for pollination.

[0017] Furthermore, regular cleaning is necessary to prevent the occurrence of pests and diseases.

[0018] Furthermore, in step (5), the light quality conditions for 18 hours of illumination / 6 hours of darkness are as follows: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, 700nm-780nm: 10.47%; and the total illumination intensity is 19000lx-32000lx.

[0019] Furthermore, in step (6), the seed is cultured for 4 weeks under 14 hours of light and 10 hours of darkness until it matures. The light quality conditions are: 450nm-465nm: 96.06%, 450nm-480nm: 1.44%, 615nm-650nm: 2.5%, and the total light intensity is 19000lx-32000lx.

[0020] Furthermore, in step (1), the germination chamber containing moistened filter paper was incubated at 25°C for 14 hours under light and 8 hours under darkness, with light quality conditions of 450nm-465nm: 100%, for one week.

[0021] On the other hand, this application provides the application of the above-mentioned method in the breeding of alfalfa varieties. Attached Figure Description

[0022] Figure 1 Displays a roadmap for generational breeding technology;

[0023] Figure 2A Part a shows the phenotype of plants that were transplanted into 0.1× Hoagland nutrient solution for 1 week after germination on moist filter paper; Part b shows the phenotype of plants that were sown in soil and watered with nutrient solution every week for 3 weeks.

[0024] Figure 2B The images show that alfalfa plants cultured under FCL4 conditions have yellowing leaf edges (parts a and b); plants under FCL3 conditions grow normally (parts c and d); plants cultured under FCL4 conditions for 2 weeks were transferred to FCL3 light conditions and recovered to a healthy growth state (parts e and f).

[0025] Figure 2C The plants were grown in soil and watered with nutrient solution every week. The plants that had grown for 8 weeks did not flower after being induced for 2 weeks under FCL4 light conditions (parts a and b). The plants were planted in vermiculite and watered with nutrient solution every other day. The plants that had grown for 8 weeks flowered after being cultivated for 2 weeks under FCL4 light conditions (part ce).

[0026] Figure 2DDisplays bee activity at different times of the day (Part a, 8:00 AM; Part b, 11:00 AM; Part c, 3:00 PM);

[0027] Figure 2E The results show that after flowering, under FCL4 conditions with 22h light / 2h darkness, alfalfa plants died (ac section), while under FCL4 conditions with 18h light / 6h darkness, the plants grew normally (d and e sections).

[0028] Figure 2F The results showed that after the plants produced fruit, the alfalfa plants placed under FCL4 conditions with 18 hours of light and 6 hours of darkness died (part a), while the plants placed under FCL3 conditions with 14 hours of light and 10 hours of darkness survived and grew healthily (part b).

[0029] Figure 3A This shows the germination of different seed varieties on moist filter paper;

[0030] Figure 3B This shows the results of transplanting seedlings of different varieties into 0.1× Hoagland nutrient solution and culturing them for 1 week;

[0031] Figure 3C This shows the transplanting and cultivation of different commercial varieties in vermiculite;

[0032] Figure 3D Showing the percentage of each alfalfa variety tested;

[0033] Figure 3E The data shows that most plants entered the initial flowering stage after one week of flowering induction (first row: different alfalfa varieties underwent flowering induction for 0 days; second row: plants were induced for 1 week under FCL4 conditions, and most plants developed flower buds and entered the initial flowering stage; third row: plants entered the full flowering stage after 2 weeks of induction, and some plants formed pods).

[0034] Figure 3F Displays the pollination and fruit set of bees (First row: After the initial flowering period, seal the cultivation rack with mesh and place the purchased pollination-specific bees inside; Second row: Bee pollination status; Third row: Pollination completed, adjust the photoperiod of the plant and continue cultivation).

[0035] Figure 3G Displays the fruit set status of some varieties (First row: Xinmu No. 3, Longmu 801; Second row: Da Yinhang, Bara416; Third row: Huanghou, Gongnong No. 1; Fourth row: Baimu 202, WL-168); Detailed Implementation

[0036] Example 1: Generation Cultivation Technology Route

[0037] The next generation breeding technology route is as follows: Figure 1 As shown:

[0038] 1) Seed germination (1 week of cultivation): After sterilization, seeds are placed in a germination box containing moistened filter paper and cultured at 25℃ under WL conditions (14h light / 8h darkness, 450nm-465nm 100%) for 1 week. Figure 1 (part a).

[0039] 2) Seedling stage cultivation conditions (hydroponics for 1-2 weeks, transplanting to vermiculite for 2 weeks): Transplant 1-week-old healthy seedlings into 0.1× Hoagland nutrient solution ( Figure 1 Part b), 25℃, 14h light / 10h darkness, hydroponic growth for 1-2 weeks; transplant seedlings into vermiculite ( Figure 1 Part C), water with nutrient solution (Miracle Grove macro-element water-soluble fertilizer general type) every other day, 25℃, 14h light / 10h dark, culture for 2 weeks; light quality ratio is FCL3: white light (450nm-465nm) 96.06%, blue light (450nm-480nm) 1.44%, red light (615nm-650nm) 2.5%; total light intensity is 32000lx, LED tubes (Foshan Ms Max Technology Co., Ltd. LED tubes).

[0040] 3) Seedling stage cultivation (4 weeks): Plant 4-week-old seedlings in vermiculite, water with nutrient solution (Miracle Growing Multi-element Water-soluble Fertilizer General Type) every other day, at 25℃, 14h light / 10h dark, light quality ratio: FCL4: (380nm-399nm) 0.06%, (400nm-499nm) 29.28%, (500nm-599nm) 15.38%, (600-699nm) 44.81%, (700nm-780nm) 10.47%; total light intensity 32000lx, LED tubes (Foshan Ms Max Technology Co., Ltd. LED tubes); cultivation temperature 26℃), growth for 4 weeks ( Figure 1 (part d).

[0041] 4) Flowering induction in mature plants (1-3 weeks of cultivation): Adjust the photoperiod to 22h light / 2h darkness, with the light quality ratio as follows: FCL4: (380nm-399nm) 0.06%, (400nm-499nm) 29.28%, (500nm-599nm) 15.38%, (600-699nm) 44.81%, (700nm-780nm) 10.47%; total light intensity is 32000 lx; Figure 1 (part e); the cultivation temperature is 26℃, and flowering is induced for 1-3 weeks until the peak flowering period ( Figure 1 (part f).

[0042] 5) Initial flowering stage (1 week after induction of flowering): When most materials reach the initial flowering stage, place the pollination-specific bee (Italian bee) hive in the rearing room or net room, observe the number of bees, and adjust the hive outlet size according to the number of materials. Figure 1 (the g and h parts).

[0043] 6) Clean once a week to prevent pests and diseases.

[0044] 7) After peak flowering (3-4 weeks of cultivation): After peak flowering, adjust the photoperiod to 18h light / 6h darkness, with the light quality ratio as follows: FCL4: (380nm-399nm) 0.06%, (400nm-499nm) 29.28%, (500nm-599nm) 15.38%, (600-699nm) 44.81%, (700nm-780nm) 10.47%; total light intensity is 32000 lx; ​​ensure normal plant growth and flowering. Figure 1 (the i part).

[0045] 8) Seed maturation stage (4 weeks of cultivation): After the end of the flowering period, adjust the photoperiod to 14h light / 10h dark until seed maturity (4 weeks). The light quality ratio is FCL3: white light (450nm-465nm) 96.06%, blue light (450nm-480nm) 1.44%, red light (615nm-650nm) 2.5%; the total light intensity is 32000lx.

[0046] Example 2: Process and Results of Generation Cultivation

[0047] (1) Alfalfa seeds germinate faster and have a higher germination rate on moist filter paper than in soil; they grow healthily under light quality FCL3 conditions within 4 weeks of age, while under FCL4 conditions, the leaf edges of the plants wither and growth is inhibited.

[0048] Germination on moist filter paper: Disinfect seeds with sodium hypochlorite for 30 minutes (for varieties with high seed hardness, such as 'yellow alfalfa', disinfect with sulfuric acid for 5 minutes beforehand, discard the solution, and rinse repeatedly with sterile water to remove the sulfuric acid). Rinse repeatedly with sterile water to ensure no sodium hypochlorite residue remains, then air dry on filter paper. Line a germination box with two layers of sterilized filter paper, add 5 ml of sterile water, and place the dried seeds in the germination box. Incubate at 25℃ with 14 hours of light / 10 hours of darkness for one week. Once the seeds have grown to their first trifoliate leaf (approximately one week), transfer them to 0.1× Hoagland's solution for hydroponic growth, incubating at 25℃ with 14 hours of light / 10 hours of darkness, changing the nutrient solution every other day for one week.

[0049] Germination in soil: Sow the seeds directly into the soil (nutrient soil: vermiculite: black soil = 1:1:1), water thoroughly, and maintain a moisture content of over 60%. Water with nutrient solution once a week, and cultivate under 14 hours of light / 10 hours of darkness, or full white light conditions for 3 weeks.

[0050] The culture conditions were set at 25℃, with 14 hours of light and 10 hours of darkness. The effects of light quality ratios FCL5 and FCL2 on plant growth were investigated.

[0051] Hogland nutrient solution formula: Use 100× modified Hogland nutrient solution (product number: NS1010-5L, containing three mother solutions A, B, and C) to prepare 0.1× nutrient solution. If you need to prepare 1L of working solution, take 1ml of each of solution A, B, and C and add them to 997ml of sterile water and mix well to obtain 1L of 0.1× Hogland nutrient solution with a pH value of 5.8±0.1.

[0052] The results show that:

[0053] 1) After disinfection and hardening treatment, the germination rate of seeds on filter paper was significantly higher than that in soil culture. After germination on moist filter paper for one week, followed by hydroponics for one week, there was no significant difference in plant size and growth compared to soil culture after three weeks, indicating that hydroponics was significantly more effective than soil culture. Figure 2A ).

[0054] 2) Plants under 4 weeks old grown healthily under FCL3 conditions, while plants cultured under FCL4 conditions showed withered leaf edges and inhibited growth. Figure 2B ).

[0055] (2) Eight-week-old plants were placed in an FCL3 light quality ratio environment with 22 hours of light and 2 hours of darkness for photoperiodic induction in both vermiculite and soil. It was found that vermiculite-cultured plants developed flower buds after 1-2 weeks of induction, while soil-cultured plants required more than 4 weeks, possibly due to slower growth under soil conditions. Figure 2C ).

[0056] (3) After the initial flowering stage, seal the cultivation rack with mesh with a 0.5cm aperture; place the purchased pollination-specific bee (Italian bee) hive inside the mesh. Observe the number of bees at different times each day and adjust the hive outlet size accordingly. In addition, the number of active bees is greatly affected by the ambient temperature; as the ambient temperature rises throughout the day, the number of bees leaving the hive increases significantly. Figure 2D ).

[0057] (4) After the peak flowering period, a light quality ratio of FCL4 and a photoperiod of 22h light / 2h darkness will cause plant dehydration and death; a photoperiod of 18h light / 6h darkness will allow the plant to grow normally. Figure 2E ).

[0058] (5) At the end of the flowering period, place the plants under FCL3 conditions for 14 hours of light / 10 hours of darkness to ensure plant survival after seed maturation. Figure 2F ).

[0059] Example 3: Photoperiod-induced generation of 20 alfalfa varieties

[0060] This experiment selected yellow alfalfa and 20 different purple alfalfa varieties (Table 3-1) for testing. After sterilization, the seeds were placed in germination boxes containing filter paper moistened with sterile water for cultivation. Figure 3A After one week of cultivation, the seedlings were transplanted into 0.1× Hoagland nutrient solution for hydroponic culture. Figure 3B ).

[0061] Table 3-1 Information on 20 commercial products of experimental materials

[0062]

[0063] Seedling stage cultivation (1-2 weeks of hydroponics, 2 weeks of cultivation in vermiculite): After about 1 week of hydroponic cultivation, seedlings are transplanted to vermiculite (10×11cm pots) and placed in a greenhouse for cultivation. Cultivation conditions: light quality of FCL3, photoperiod of 14h light / 10h darkness, for 2 weeks; water with nutrient solution prepared with Flower More No. 1 (balanced general-purpose) fertilizer every other day.

[0064] Seedling stage cultivation (4 weeks): Place 4-week-old plants under FCL4 conditions, with 14 hours of light and 10 hours of darkness, for 4 weeks; water with a nutrient solution prepared with Flower More No. 1 (balanced general-purpose) fertilizer every other day. Figure 3C ).

[0065] Flowering induction: 130 eight-week-old plants from 20 varieties were subjected to flowering induction using an induction system previously screened in the laboratory. The induction conditions used were 22h FCL4 (lighting time 6:00-4:00, light duration 22h; light quality ratio: 380nm-399nm 0.06%, 400nm-499nm 29.28%, 500nm-599nm 15.38%, 600nm-699nm 44.81%, 700nm-780nm 10.47%; total light intensity 32000lx; culture temperature 23℃). During the flowering induction period, the plants were irrigated with a nutrient solution prepared with Flower More No. 1 fertilizer once a day.

[0066] Early flowering stage pollination with bees: Nine days after flowering induction (when most materials enter the early flowering stage), release bees through a net for random pollination. Figure 3F And 6 days after releasing the bees (when most plants are in full bloom), the light duration was adjusted to 18 hours to ensure normal plant growth.

[0067] Statistical analysis revealed that most of the 20 varieties tested had already flowered and borne fruit. Figure 3G In summary, after germination, most commercial varieties are hydroponically cultured and then transferred to vermiculite culture. By altering the duration of light exposure to induce flowering and introducing random pollination by bees, the time required for one growth cycle (from seed germination to seed harvest) of alfalfa under natural conditions can be significantly shortened, thus completing the multigenerational culture.

Claims

1. A method for accelerated generation breeding of alfalfa, characterized by, The method comprises: (1) Seed germination: cultivate alfalfa seeds in a germination box containing wet filter paper at 25°C under 14h light / 8h dark conditions for 1 week to obtain seedlings; the light quality conditions are: 450nm-465nm, and the light intensity is 16000lx-20000lx; (2) Seedling culture: transplant the seedlings into 0.1x Hoagland nutrient solution at 25°C under 14h light / 10h dark, and hydroponically culture for 1-2 weeks; transplant into vermiculite at 25°C under 14h light / 10h dark, and culture for 2 weeks; the light quality conditions are: white light 450nm-465nm: 96.06%, blue light 450nm-480nm: 1.44%, and red light 615nm-650nm: 2.5%; the total light intensity is 19000lx-32000lx, and the nutrient solution is watered every other day; (3) Seedling culture: culture in vermiculite at 25°C under 14h light / 10h dark for 4 weeks; the light quality conditions are: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, and 700nm-780nm: 10.47%; the total light intensity is 19000lx-32000lx, and the nutrient solution is watered every other day; (4) Flowering induction: induce flowering under 26°C, 22h light / 2h dark for 1-3 weeks to the full flowering stage; the light quality conditions are: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, and 700nm-780nm: 10.47%; the total light intensity is 19000lx-32000lx; when most of the materials reach the initial flowering stage, place the beehive in the culture room, observe the number of bees, and adjust the size of the beehive outlet according to the number of materials to pollinate; (5) Full flowering stage culture: culture under 18h light / 6h dark for 3-4 weeks to the end of flowering; the light quality conditions are: 380nm-399nm: 0.06%, 400nm-499nm: 29.28%, 500nm-599nm: 15.38%, 600-699nm: 44.81%, and 700nm-780nm: 10.47%; the total light intensity is 19000lx-32000lx; (6) Seed maturation: culture under 14h light / 10h dark for 4 weeks to seed maturation; the light quality conditions are: white light 450nm-465nm: 96.06%, blue light 450nm-480nm: 1.44%, and red light 615nm-650nm: 2.5%; the total light intensity is 19000lx-32000lx.

2. The method of claim 1, further comprising regular cleaning to prevent disease and pest occurrence.

3. Application of the method of claim 1 or 2 in alfalfa variety breeding.

Images