A method for accelerating alfalfa breeding using photoperiod induction

Abstract

This application provides a method for accelerating alfalfa breeding using photoperiod induction. The method uses the following conditions to induce flowering of alfalfa seedlings: daylight duration: 20-22 hours; light quality ratio: white light, blue light, red light, and far-red light; light intensity: 10,000-40,000 lx; temperature: 23-26 degrees Celsius. This method can overcome the temporal and spatial limitations of alfalfa breeding, allowing the harvest of four to six generations of seeds within a year, and has promising application prospects in alfalfa breeding research and production.

Description

Technical Field

[0001] The present application belongs to the field of plant breeding. Specifically, the present application provides a method for accelerating alfalfa breeding by utilizing photoperiod induction. Background Art

[0002] Alfalfa (Medicago sativa L.) is an autotetraploid, insect-pollinated, cross-pollinated perennial legume forage grass. Rich in nutrients, high yield, and palatable, it earns the nickname "King of Forage Grasses." In the field, flowering and harvesting times vary depending on factors such as location, climate, planting management, and variety. Generally, spring-sown alfalfa takes four to six months to mature, while autumn-sown alfalfa takes even longer. The progress of alfalfa breeding in my country and research on flowering, fertilization, and fruiting are limited by time and space.

[0003] Photoperiod refers to the alternating lengths of light and dark periods during the diurnal cycle. Photoperiodism in plants refers to the response of plants to the length of day and night (Liu Li, 2004). Alfalfa is a long-day plant, and long-day conditions promote early flowering (Chen Xiang, 2016). Light quality has a positive impact on plant flowering and fruiting. Studies have found that red, blue, and far-red light can promote early flowering (Sun Miao, 2023; Wu Xiaoxiao, 2023).

[0004] Alfalfa seed breeding has always relied on natural field conditions, which has limited the breeding process and research on flowering, fertilization and fruiting of alfalfa in my country. Currently, there is little research on induced flowering of alfalfa. Summary of the Invention

[0005] This application utilizes long days and different light qualities to promote early flowering of alfalfa, in order to obtain better conditions, establish an alfalfa induction flowering and early fruiting system, accelerate the alfalfa breeding process, and promote the generation and reproduction of alfalfa seeds.

[0006] In one aspect, the present application provides a method for accelerating alfalfa breeding using photoperiod induction, wherein the following conditions are used to induce flowering of alfalfa seedlings:

[0007] Daylight hours: 20-22 hours;

[0008] Light quality ratio: white light, blue light, red light, far-infrared light;

[0009] Light intensity: 10000-40000lx;

[0010] Temperature: 23-26 degrees Celsius.

[0011] Furthermore, the sunshine duration is 22 hours.

[0012] Further, the temperature is 26 degrees Celsius.

[0013] Furthermore, the light intensity is 11000-32000lx.

[0014] Furthermore, the light quality ratio is selected from one of the following (1)-(3):

[0015] (1) 450-465nm wavelength light: 34.9%, 450-480nm wavelength light: 51.53%, 615-650nm wavelength light: 13.2%, 730nm wavelength light: 0.27%;

[0016] (2) 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%;

[0017] (3) 380-399nm wavelength light: 0.06%, 400-499nm wavelength light: 29.28%, 500-599nm wavelength light: 15.38%, 600-699nm wavelength light: 44.81%, 700-780nm wavelength light: 10.47%.

[0018] Furthermore, the method uses the following conditions to induce flowering of alfalfa seedlings:

[0019] Sunshine duration: 22 hours;

[0020] Light quality ratio: 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%;

[0021] Light intensity: 11780lx;

[0022] Temperature: 26 degrees Celsius.

[0023] Furthermore, the induction of flowering of alfalfa seedlings lasts for 18-24 days.

[0024] Furthermore, the induction of flowering of alfalfa seedlings lasted for 22 days.

[0025] Furthermore, the alfalfa variety is selected from Zhongnong No. 1, Zhongmu No. 1, Avia or Knight No. 2, and the autumn dormancy level range is 2.4-8.

[0026] In another aspect, the present application provides application of the above method in alfalfa breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown are the plant heights of different plant materials at day 0 of induction (left) and the flowering induction rates under various culture conditions.

[0028] Figure 2 It was shown that the seedlings of Zhongnong No. 1 (ZN-A) germinated and grew for 45 days under 14hWL conditions were induced to flower at 22 days under 22hFCL1 and 22hFCL2 conditions, while no flower was induced under 14hWL as the control condition.

[0029] Figure 3 It showed that no flower bud differentiation was found when the plant materials were cultured for 5 days under the control conditions of 14hWL in the same period.

[0030] Figure 4 The results showed that under the four induction conditions, ZM-2 had flower buds at 16 days and flowered at 20-23 days.

[0031] Figure 5 The results showed that it took 29 days for Avia seedlings (AWY-A) to flower, 29 days for Knight 2 seedlings (QS2-A) and Zhongmu No. 1 seedlings (ZM-A) to form flower buds and 33 days for flower to bloom.

[0032] Figure 6 It showed that ZM-2 induced flower buds to appear on 16 days and flowering on 22 days; ZM-1 induced flower buds to appear on 21 days and flowering on 29 days; ZM-A induced flower buds to appear on 29 days and flowering on 33 days.

[0033] Figure 7 The seedlings of about 20 cm long (ZM-1) cut from the single plant A of Zhongmu No. 1 and the seedlings of about 20 cm long (ZM-3) cut from the single plant B of Zhongmu No. 1 were induced to flower for 30 days under 14hWL conditions. They were then directly induced under 22hFCL2 conditions. ZM-3 flowered after 16 days of induction, and ZM-1 flowered after 22 days of induction.

[0034] Figure 8 Shown are the changes in single plant A cutting seedlings (ZM-1) of Zhongmu No. 1 after pollination under 22hFCL2 induction conditions. DETAILED DESCRIPTION

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and in no way limit the scope of protection of the present invention.

[0036] Example 1 Different induction conditions promote early flowering of alfalfa

[0037] In this experiment, six culture conditions were set up (Table 1), and seven different alfalfa plant materials were selected for induction (Table 2). The number of seedlings induced to flower under different culture conditions is shown in Table 3. The flowering induction rate was between 75% and 100%. Seedlings of about 20 cm in length were selected for induction for different varieties ( Figure 1 Among them, the seedlings of Zhongnong No. 1 (ZN-A) germinated and grew for 45 days under 14hWL conditions were induced to flower on 22 days under 22hFCL1 and 22hFCL2 conditions, while no flower was produced under 14hWL as the control condition ( Figure 2 To exclude the possibility that the late flowering occurred under the 14hWL condition rather than the absence of flowering, the plant materials were cultured for 5 days under the 14hWL control condition during the same period, but no flower bud differentiation was found ( Figure 3 The results showed that 14h white light conditions could not induce alfalfa to flower, while 22h white light, blue light, red light, far-red light and other four-color light conditions with different ratios could promote early flowering of alfalfa.

[0038] On this basis, we selected a single plant A of Zhongmu No. 1 and cut the seedlings of about 20 cm (ZM-2) to compare the flowering induction of alfalfa under different 22h light quality ratio conditions. We found that under 22hFCL2, 22hFCL3, 22hFCL4, and 22hFCL5 conditions, ZM-2 could bloom in the non-flowering season. Although flower buds appeared in ZM-2 under the four induction conditions at 16 days and flowering took 20-23 days ( Figure 4 ), but it bloomed relatively early under 22hFCL2 and 22hFCL4 conditions, and the consistency of flowering time of the same batch of seedlings was relatively high, and the plants grew relatively strong, while it bloomed relatively late under 22hFCL3 and 22hFCL5 conditions, and the consistency of flowering time of the same batch of seedlings was relatively poor, with the maximum difference of flowering time of the same batch of seedlings being 7 days.

[0039] In summary, 22hFCL1, 22hFCL2, and 22hFCL4 were superior in inducing alfalfa flowering among the six induction conditions, inducing flowering within 20-22 days, with relatively high consistency in flowering time and robust plant growth. In addition to white light, all three conditions included blue, red, and far-red light, with either red or blue light accounting for a significant proportion, suggesting that red and blue light may be important light sources for inducing alfalfa flowering.

[0040] Table 1. 6 culture conditions

[0041]

[0042] Note: The measurement was performed using a DELIXI DLY-1802 illuminometer at a distance of 5 cm under the light without any obstruction by plants.

[0043] Table 2. Plant material

[0044]

[0045] Table 3. Statistics of the number of induced flowering seedlings

[0046]

[0047] Example 2: Flowering induction of seedlings of different varieties

[0048] Under the induction condition of 22hFCL2, seedlings of about 20cm in low, medium and high autumn dormancy were selected, namely QS2-A (Knight 2, 2.4), ZM-A (Zhongmu No. 1, 3) and AWY-A (Aweiya, 8) for induction of flowering. It was found that Aweiya seedlings (AWY-A) flowered on the 29th day of induction, Knight 2 seedlings (QS2-A) and Zhongmu No. 1 seedlings (ZM-A) flower buds appeared on the 29th day of induction, and flowered on the 33rd day of induction ( Figure 5 ). This shows that seedlings of different varieties with a length of about 20 cm can all bloom under the induction conditions of FCL2 for 22 h.

[0049] Example 3: Flowering induction of seedlings of different growth ages

[0050] Under the induction condition of 22hFCL2, Zhongmu No. 1 seedlings (ZM-A), Zhongmu No. 1 single plant A annual cutting seedlings (ZM-1) and Zhongmu No. 1 single plant A cutting seedlings (ZM-2) were selected for flowering induction. It was found that ZM-2 induced flower buds on 16 days and flowered on 22 days; ZM-1 induced flower buds on 21 days and flowered on 29 days; ZM-A induced flower buds on 29 days and flowered on 33 days ( Figure 6 The induced flowering time of the cutting seedlings of Zhongmu No. 1 A single plant was earlier than that of the clipping seedlings, and the induced flowering time of the clipping seedlings of Zhongmu No. 1 A single plant was earlier than that of the seed seedlings.

[0051] Seedlings treated in Example 4 were induced to flower

[0052] The seedlings of Zhongmu No. 1 plant A cut to about 20 cm (ZM-1) and the seedlings of Zhongmu No. 1 plant B cut to about 20 cm (ZM-3) were induced under 14hWL conditions for 30 days without flowering. Then they were directly induced under 22hFCL2 conditions. ZM-3 flowered after 16 days of induction, and ZM-1 flowered after 22 days of induction ( Figure 7 This indicates that large seedlings that grow normally in the non-flowering season can still flower if induced directly, indicating that the biomass conditions for inducing flowering are within a certain range. Whether the plants are induced directly when they grow to 20 cm or continue to grow after 20 cm and then induced, the biomass conditions for inducing flowering are achieved.

[0053] Example 5 Pollination and Fruiting Time under 22hFCL2 Conditions

[0054] Under the induction condition of 22hFCL2, the single-plant A cutting seedlings of Zhongmu No. 1 (ZM-1) were induced to bloom for 29 days. After flowering, they were bagged for pollination and fruiting. It was found that pods began to form 5 days after pollination. The pods showed a spiral upward growth from bottom to top. At this time, the stigma and style at the top of the pods could be clearly observed to be gradually wilting and losing water; the pods completed the upward spiral growth (elongation growth) 7 days after pollination, and the color of the pods was light green. At this time, the style and stigma at the top of the pods did not disappear, but showed a more wilted and water-losing state; the pods were full and plump 15 days after pollination; the pods began to turn yellow and brown 24 days after pollination, and the stigma and style at the top of the pods also began to turn brown and shriveled; the pods became shriveled and lost water 27 days after pollination; the pods matured 33 days after pollination, and the brown-yellow seeds could be harvested. At this time, the style and stigma at the top of the pods could still be observed to be wrinkled, wilted and shriveled ( Figure 8 ).

[0055] In summary, the 22hFCL1, 22hFCL2, and 22hFCL4 conditions performed better in inducing flowering time in alfalfa, with durations ranging from 20 to 22 days, with no significant differences. All three induction conditions shared the common characteristic of including four colors of light (white, red, blue, and far-red). Compared to 22hFCL1 and 22hFCL4, seedlings induced to flower under 22hFCL2 developed more branches and new shoots. Therefore, 22hFCL2 was selected for experimental verification of inducing flowering in seedlings of different varieties and inducing flowering and pollination and fruiting in seedlings of different ages. The results showed that under 22hFCL2, seedlings approximately 20 cm in length could be induced to flower in 22 to 33 days, and mature seeds could be harvested around 33 days, for a total of 55 to 66 days to complete a single seed generation. Seeds germinated under 14hWL conditions grow for about 45 days to produce seedlings approximately 20 cm in length. Induced under 22hFCL2 conditions flower in 22-33 days, for a total of 67-78 days from seed to induced flowering. Therefore, using 22hFCL2 conditions can overcome the temporal and spatial limitations of alfalfa breeding, enabling the multi-generational propagation of alfalfa seeds, yielding 4-6 generations of seeds per year.

Claims

1. A method for accelerating alfalfa breeding by photoperiod induction, characterized in that: The method uses the following conditions to induce flowering of alfalfa seedlings, which lasts for 18-24 days: Sunshine duration: 22 hours; Light quality ratio: 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%; Light intensity: 11780lx; Temperature: 26 degrees Celsius.

2. The method according to claim 1, wherein the inducing flowering of the alfalfa seedlings lasts for 22 days.

3. The method according to claim 1 or 2, wherein the alfalfa variety is selected from the alfalfa varieties with a fall dormancy level ranging from 2.4 to 8, namely, Zhongnong No. 1, Zhongmu No. 1, Avia or Knight No.

2.

4. Application of the method according to any one of claims 1 to 3 in alfalfa breeding.

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