Cultivation method for rapid vernalization of winter wheat to accelerate annual iteration

Abstract

The invention discloses a cultivation method for rapid vernalization of winter wheat to accelerate annual iteration, and belongs to the technical field of plant growth regulation and control. The cultivation method comprises the following steps of disinfection and germination, low-temperature vernalization, transplanting and cultivation, and drying and harvesting. The method has the beneficial effects that compared with other current wheat growth acceleration methods, the technical process and the required environment of each stage are simple, and the (semi) winter wheat breeding efficiency is greatly improved. The temperature or photoperiod does not need to be repeatedly adjusted, and vernalization can be performed by adopting short photoperiod / low illumination intensity, so that energy is saved, and the method is environment-friendly and suitable for large-scale popularization. The cultivation method is green, efficient, low in power consumption, low in requirement for environment equipment and simple and convenient. The method is suitable for being used in large-scale plant factories, and meanwhile, a method for rapid vernalization of winter wheat to accelerate annual accelerated iteration of wheat is provided for scientific research institutions, enterprises and public institutions with limited conditions.

Description

Technical Field

[0001] The invention relates to the technical field of plant growth regulation and control, and in particular to a cultivation method for rapid vernalization of winter wheat to accelerate annual iteration. Background Art

[0002] Winter wheat accounts for more than 93% of the wheat planting area in my country. Compared with other crops, its biggest feature is that it needs to undergo vernalization. Vernalization is a phenomenon that plants must experience a period of continuous low temperature to transition from the vegetative growth stage to the reproductive growth stage. Otherwise, the plants will delay or even stop flowering and remain in the vegetative stage for a long time. Wheat can be divided into spring wheat, facultative wheat and winter wheat due to their different needs for the vernalization process. Due to climate and geographical factors, winter wheat is the main planting type in my country's wheat planting areas, and the global winter wheat planting area also accounts for about 75% of the total wheat planting area. The growth period of winter and semi-winter wheat is about 220-270 days. Under normal circumstances, only one generation can be harvested in the field in a year, which is also one of the important factors that lead to the slow progress of wheat breeding. Because vernalization is an inevitable and long process in the process of completing the life cycle of winter wheat, current research on accelerated wheat breeding often focuses on the accelerated iteration research of spring wheat, while research on rapid, efficient and green vernalization of winter wheat to promote acceleration is rarely reported. Therefore, a technical solution to shorten the vernalization time to accelerate flowering and achieve the purpose of winter wheat generation needs to be solved urgently.

[0003] Winter wheat flowering is mainly regulated by the interaction of four signal pathways: photoperiod pathway, vernalization pathway, gibberellin pathway and autonomous pathway. The first two pathways are mainly regulated by external factors, and the latter two are affected by the plant's own developmental characteristics. Therefore, we consider that we can affect the transcription and expression of wheat genes by adjusting the photoperiod and vernalization conditions, causing physiological changes, increasing the growth rate and accelerating wheat flowering. At the same time, the environmental facilities and conditions of domestic scientific research institutions and other units are different. High-precision plant incubators are expensive, plant incubators that can provide low temperature under light conditions often consume a lot of electricity, and there is a lack of effective industry standards for low-temperature vernalization light and temperature. There is also a lack of effective methods to promote the annual acceleration of wheat iterations through rapid and efficient vernalization. Therefore, a simple, convenient, efficient and low-cost method for winter wheat generation is imminent.

[0004] There are currently no research reports on the vernalization of wheat seedlings to further reduce the growth period. For example, the patent application document of the method of accelerating the hybrid breeding process of strong winter wheat in 1 year and 4 generations by using embryo culture technology is disclosed in the publication number CN111631130A. This method needs to be cultured to the tillering stage like wheat grown naturally in the field and then vernalized. It is necessary to repeatedly culture and transplant and change the culture environment. Therefore, the operation steps are cumbersome and the efficiency is low. Another example is the patent application document of the publication number CN112655492A, which discloses a semi-winter wheat three-site two-generation plus generation breeding method. The semi-winter wheat three-site two-generation plus generation breeding method needs to be cultivated in three places, and the operation process is relatively complicated, and the labor cost and other costs are high. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to solve the problem that the existing winter wheat vernalization breeding operation steps are complicated and the cost is high.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention provides a cultivation method for winter wheat rapid vernalization to accelerate annual iteration, comprising the following steps:

[0008] (1) Disinfection and germination: After disinfection, winter wheat seeds are immersed in pure water for germination and culture to obtain wheat seedlings;

[0009] (2) Low-temperature vernalization: The wheat seedlings were moved to a low-temperature light incubator for vernalization. The temperature was set at 2-13°C. The lighting used was LED cold / warm white light. The photoperiod was 22-8 h light / 2-16 h dark. The light intensity was set at 1000-10000 lux. 1 / 4-1 / 2 Hogland nutrient solution was applied twice a week. The vernalization time was 14-35 days.

[0010] (3) Transplanting and cultivation: Transplant the vernalized wheat to the breeding acceleration platform for cultivation. Set the temperature to 20-25°C, use a photoperiod of 20-23 h light / 4-1 h dark, and set the light intensity to 10,000-30,000 lux. Apply 1 / 4-1 / 2 Hogland nutrient solution 2-3 times a week. Keep cutting off excess tillers before wheat heading, and only keep 1-4 strong tillers per plant.

[0011] (4) Drying and harvesting: Wheat grains are harvested 15 to 25 days after flowering, dried at 30 to 37°C for 3 to 5 days, and then dormancy-breaking treatment is performed on the wheat grains. Subsequently, the wheat grains are germinated for vernalization and breeding of the next generation.

[0012] Preferably, the winter wheat in step (1) is any one of winter wheat, semi-winter wheat and weak winter wheat.

[0013] Preferably, the disinfection treatment in step (1) specifically refers to disinfection with a 2% to 10% sodium hypochlorite aqueous solution for 2 to 15 minutes, followed by washing with pure water for 4 to 6 times.

[0014] Preferably, the culture in step (2) is carried out by hydroponics or soil culture, the hydroponics nutrient solution uses 1 / 4 to 1 / 2 Hogland, and the soil ratio of soil culture is nutrient soil: vermiculite: sand = 1 to 3: 0.5 to 2: X, 0.25≤X≤1.

[0015] Preferably, the light in step (3) adopts any one of LED light source cold white light, blue spectrum monochromatic light, red spectrum monochromatic light, and red:blue=1:1 / 2:1 / 3:1 / 1:2 / 1:3 composite light.

[0016] Preferably, in step (3), the photoperiod is 22 h light / 2 h dark.

[0017] Preferably, the light intensity in step (3) is set to 10000 lux.

[0018] Preferably, in step (3), 1 / 2 Hogland nutrient solution is applied twice a week.

[0019] Preferably, the dormancy-breaking treatment in step (4) is specifically soaking in 0.1-0.3% hydrogen peroxide solution, 1% potassium hydrogen sulfate or 1% gibberellin for 20-24 hours, 4-6 hours or 20-24 hours.

[0020] Preferably, the dormancy-breaking treatment in step (4) is specifically performed by soaking in pure water at room temperature for 16 to 30 hours and then placing at 2 to 6° C. for 20 to 30 hours to break the dormancy.

[0021] The beneficial effects of the present invention are:

[0022] (1) Compared with the natural cultivation environment in the field, the growing period of winter wheat is significantly shortened.

[0023] (2) Compared with other current methods for accelerating wheat growth, the technical process and required environment at each stage are simple, which greatly improves the breeding efficiency of (semi-)winter wheat.

[0024] (3) During the vernalization stage, seedlings are vernalized. When a certain number of days have passed, there is no need to observe whether the plant morphology has completed vernalization. At the same time, vernalization of green seedlings avoids the situation where seeds start vernalization with only one unfolded leaf and grow weakly, thereby improving the survival rate after transplanting.

[0025] (4) At the same time, there is no need to repeatedly adjust the temperature or photoperiod and a short photoperiod / low light intensity can be used for vernalization, which saves energy, is green and environmentally friendly, and is suitable for large-scale promotion. For breeding bases with ordinary conditions, the vernalization method used in the present invention does not require the purchase of an artificial climate box or a low-temperature plant incubator, and the vernalization process can be completed using an ordinary light-equipped freezer. The present invention is simple to operate and low in cost, and is very suitable for use in plant factories.

[0026] (5) This cultivation method is green and efficient, has low energy consumption, low requirements on environmental equipment, and is simple. This method is suitable for use in large-scale plant factories, and also provides a method for rapid vernalization of winter wheat to accelerate the annual iteration of wheat for scientific research institutions and enterprises with limited conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 are wheat growth and development indicators after vernalization in Examples 1-5 of the present invention, A is Example 1, B is Example 2, C is Example 3, D is Example 4, and E is Example 5; different lowercase letters (a, b, c, d) indicate significant differences (P<0.05) between different treatment groups at the same sampling time, and the same letters indicate no significant differences;

[0028] Figure 2 The growth and development of wheat seedlings of Bainong 207 at 28 days of vernalization in Examples 1-4 of the present invention and Comparative Example 1;

[0029] Figure 3 After the vernalization is completed in Example 1 of the present invention, the wheat of Bainong 207 is transplanted to the breeding acceleration platform to cultivate each growth period;

[0030] Figure 4 The wheat ear pictures of Bainong 207 after Example 1 (A), Example 2 (B), Example 3 (C), and Example 4 (D) of the present invention are shown. Comparative Example 1 was still in the vegetative stage and had no ear structure until the experimental contact.

[0031] Figure 5 Example 1 of the present invention harvests wheat grains in the grain filling stage in advance and then dries them at 35° C. for 3 days.

[0032] Figure 6 The germination of wheat seeds after breaking dormancy after drying in Example 1 of the present invention is as follows: a. germination of wheat seeds after breaking dormancy by hydrogen peroxide method; b. germination of wheat seeds after breaking dormancy by gibberellin; c. germination of wheat seeds treated at 4°C for 24 hours after being soaked in pure water at room temperature for 24 hours.

[0033] Figure 7 This is a schematic diagram of the rapid vernalization and periodic iterative cultivation process of winter wheat in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0036] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0037] Embodiment 1:

[0038] A method for cultivating winter wheat by rapid vernalization to accelerate annual iteration, comprising the following steps:

[0039] (1) Wheat seed germination and seedling culture

[0040] The fresh winter wheat variety "Bainong 207" kept in the field of this laboratory was used as the experimental object of this example. Bainong 207 is a variety included in the National Catalogue of Excellent Crop Varieties Promotion (2023). The specific experimental treatment is as follows: Use 5% NaClO aqueous solution to disinfect for 120s, then rinse repeatedly with double distilled water 6 times, and add a small amount of pure water to culture for 12h for germination. Lay 6 layers of filter paper in a clean culture dish, add pure water to soak the filter paper, select seeds with consistent germination in the refrigerator, and evenly distribute the wheat seeds with their abdomens facing down in a clean culture dish (150mm in diameter, with 6 layers of filter paper of appropriate size on the bottom), and place the culture dish in a light culture room (100μmol m -2 s -1 , 25℃, 16h light / 8h dark), and 20mL pure water was injected every day with a pipette to keep the filter paper wet. After 3 days, seedlings with the same height were selected and re-transplanted into a culture box with 15-hole plug trays containing 1 / 2 Hoagland nutrient solution, with 2 wheat seedlings planted in each hole. The wheat seedlings were cultured in light and water for 4 days.

[0041] (2) Vernalization of wheat seedlings

[0042] Wheat seedlings with uniform growth were selected and moved to a low-temperature light incubator for formal experiments. The incubator temperature was set to 4°C, the photoperiod was set to 22h light / 2h dark, the light intensity was set to 10,000 lux, and they were transplanted to a nutrient solution containing 1 / 2 Hoagland (250mL) supplemented twice a week. The wheat seedlings were cultured for 28 days to complete the vernalization process.

[0043] (3) Accelerated platform cultivation of wheat seedling breeding

[0044] The vernalized wheat was transplanted to the breeding acceleration platform for soil culture (soil ratio: nutrient soil: vermiculite: sand = 2:1:1), the temperature was set to 22°C (±1.5°C), the lighting used was LED cold white light, the photoperiod was 22h light / 2h dark, the light intensity was set to 10000 lux, and 1 / 2 Hogland nutrient solution was applied twice a week until the wheat head emerged, and the excess tillers were cut off, leaving only 1-2 strong tillers per plant.

[0045] (4) Early harvest of wheat grains

[0046] The wheat grains are harvested in advance about 10 days after flowering, dried at 30°C for 3 days, and then soaked in 0.3% hydrogen peroxide solution for 24 hours to break dormancy. Then, the grains can be germinated for the next generation of cultivation.

[0047] Embodiment 2:

[0048] The difference between this embodiment and embodiment 1 is that during the vernalization of wheat seedlings in step (2), the photoperiod is set to 8 h light / 16 h dark.

[0049] Embodiment 3:

[0050] The difference between this embodiment and embodiment 2 is that during the vernalization of wheat seedlings in step (2), the light intensity is set to 1000 lux.

[0051] Embodiment 4:

[0052] The difference between this embodiment and embodiment 1 is that during the vernalization of wheat seedlings in step (2), the light intensity is set to 1000 lux.

[0053] Embodiment 5:

[0054] The difference between this embodiment and embodiment 1 is that a germination experiment is performed on the grains harvested in embodiment 1.

[0055] Comparative Example 1:

[0056] The difference between this comparative example and Example 1 is that during the vernalization of the wheat seedlings in step (2), the temperature is set to 22°C.

[0057] Embodiment 6:

[0058] The growth and development indicators of wheat seedlings at the end of the vernalization stage in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 were measured; the heading, heading and flowering times of wheat in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 were counted; and the germination rate measurement experiment was performed on the grains harvested in Example 5.

[0059] Embodiment 7:

[0060] The difference between this embodiment and embodiment 4 is that the wheat variety is Yannong 999 (a semi-winter wheat variety).

[0061] Embodiment 8:

[0062] The difference between this embodiment and embodiment 4 is that the wheat variety is Jimai 22 (a semi-winter wheat variety).

[0063] Embodiment 9:

[0064] The difference between this embodiment and embodiment 4 is that the wheat variety is Zhongmai 895 (a semi-winter wheat variety).

[0065] Embodiment 10:

[0066] The difference between this embodiment and embodiment 7 is that the vernalization temperature is 7°C.

[0067] Experimental results: Table 1 shows the comparison of vernalization environmental conditions of various embodiments and comparative examples. It can be seen from Table 1 that high / low temperature, long / short photoperiod and high / low light intensity are included. Figure 1 The wheat growth and development indicators after vernalization in each embodiment and comparative example are shown. Figure 1 As shown in A, the fresh weight of the aboveground part, the fresh weight of the underground part and the plant height of Comparative Example 5 after the vernalization were significantly higher than those of Examples 1, 2, 3 and 4, which indicates that the growth of wheat seedlings slowed down during the vernalization stage, which is consistent with Figure 2 The observed results are consistent with Figure 1 As shown in A, there is no significant difference in plant height among the treatment groups of Examples 1, 2, 3 and 4. Figure 1 As shown in Figure C, the fresh weight of the underground part of Examples 3 and 4 is significantly higher than that of Examples 1 and 2. This may be due to the lower / less light received by Groups 3 and 4, which further promotes the development of wheat roots. This is consistent with the result that the root length of Example 4 is significantly higher than that of other Examples and higher than that of the treatment groups of Examples 1, 2, and 3. Figure 1As shown in B, the root length of wheat seedlings in the treatment group of Comparative Example 5 is significantly lower than that in the treatment groups of Examples 1, 2, 3 and 4, but the fresh weight of the underground part is significantly higher than that in the treatment groups of Examples 1, 2, 3 and 4, which shows that the root development of the treatment group of Comparative Example 5 is more developed. The root length of the treatment group of Example 4 is the longest, and the fresh weight of the underground part of wheat in the treatment groups of Examples 3 and 4 is significantly higher than that in Examples 1 and 2, which may indicate that the insufficient light intensity further promotes the root development of wheat. In general, in the treatments of each embodiment, there is little difference in the growth and development of wheat after vernalization, and the growth difference is mainly concentrated in the root, which is caused by different light intensity or photoperiod. The growth and development stage of Comparative Example 5 shows the growth state of wheat that has not been vernalized, that is, it stays in the nutritional stage, and there is no phenotype such as jointing or flagging that marks the transition of wheat from the growth stage to the reproductive stage.

[0068] like Figure 3 The results show that after vernalization is completed, the wheat is transplanted to the breeding acceleration platform to cultivate various growth periods, including the booting period (flag picking), heading period and flowering period. The results in Table 2 show the statistics of wheat heading, heading and flowering time in each embodiment and comparative example, and the flowering of wheat with 50% biological replicates is usually used as the standard for reaching the flowering period. Example 1 and Example 4 both reached the flowering period at 88d, and Example 2 and Example 3 both reached the flowering period within 94d. This result significantly shortens the growth period of wheat compared with the growth of wheat under natural conditions in the field (an average of 175 to 195d is required to reach the flowering period, and the data comes from the 5-year field observation results of Science Island, Hefei City, Anhui Province from 2019 to 2024). The results in Table 2 prove that Example 1, Example 2, Example 3, and Example 4 can all complete the accelerated growth of wheat under this method to meet the needs of generation. At the same time, we observed that the wheat in the comparative example 1 treatment group still remained in the growth stage and did not move towards the reproductive stage. This is because the wheat in the comparative example 1 treatment group did not undergo the vernalization process necessary for its life cycle. Figure 4 The results show the wheat ear pictures of Bainong 207 after Example 1, Example 2, Example 3, and Example 4. Comparative Example 1 was still in the vegetative period and had no ear structure at the end of the experiment. Figure 5 The results are for wheat seeds after harvesting and drying at 35℃ for 3 days. Figure 6The results show the germination of wheat seeds after breaking dormancy. Wheat seeds that broke dormancy by hydrogen peroxide method, wheat seeds that broke dormancy by gibberellin method and wheat seeds that broke dormancy by temperature change method can all make wheat germinate and turn white. This shows that the wheat seeds obtained by this method can continue to be cultivated according to this method to achieve the purpose of increasing generations. Among other semi-winter / winter varieties, the cultivation speed of a generation of wheat using this method is faster. For example, in Examples 7, 8, and 9, the flowering period using this method is within 80 days. As shown in Example 10, the use of this method in Yannong 999 wheat can further shorten the flowering time under higher vernalization temperature conditions. The above results show that the use of this method can complete the cultivation of 3 to 5 generations of winter wheat a year under relatively simple technical processes and environmental equipment conditions.

[0069] Table 1 Comparison of vernalization conditions in various embodiments and comparative examples.

[0070]

[0071] Table 2 Statistics of heading, booting and flowering time of wheat in various embodiments and comparative examples.

[0072]

[0073] V in the table represents that the wheat is in the vegetative stage.

[0074] Embodiment 11:

[0075] The difference between this embodiment and embodiment 1 is that:

[0076] In step (2), the vernalization temperature was set to 2°C, the photoperiod was 16 h light / 8 h dark, the light intensity was 5000 lux, and the vernalization time was 35 days;

[0077] In step (3), the temperature was set to 20°C, the photoperiod was 23 h light / 1 h dark, and the light intensity was set to 20,000 lux.

[0078] The rest is the same as in Example 1.

[0079] Embodiment 12:

[0080] The difference between this embodiment and embodiment 1 is that:

[0081] In step (2), the vernalization temperature was set to 13°C, the photoperiod was 20 h light / 4 h dark, the light intensity was 6000 lux, and the vernalization time was 14 days;

[0082] In step (3), the temperature was set to 25°C, the photoperiod was 20 h light / 4 h dark, and the light intensity was set to 30,000 lux.

[0083] The rest is the same as in Example 1.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cultivating winter wheat by rapid vernalization to accelerate annual iteration, characterized in that: The following steps are involved: (1) Disinfection and germination: After disinfection, winter wheat seeds are immersed in pure water for germination and culture to obtain wheat seedlings; (2) Low-temperature vernalization: The wheat seedlings were moved to a low-temperature light incubator for vernalization. The temperature was set at 2-13°C. The lighting used was LED cold / warm white light. The photoperiod was 22-8 h light / 2-16 h dark. The light intensity was set at 1000-10000 lux. 1 / 4-1 / 2 Hogland nutrient solution was applied twice a week. The vernalization time was 14-35 days. (3) Transplanting and cultivation: Transplant the vernalized wheat to the breeding acceleration platform for cultivation. Set the temperature to 20-25°C, use a photoperiod of 20-23 h light / 4-1 h dark, and set the light intensity to 10,000-30,000 lux. Apply 1 / 4-1 / 2 Hogland nutrient solution 2-3 times a week. Keep cutting off excess tillers before wheat heading, and keep 1-4 strong tillers per plant. (4) Drying and harvesting: Wheat grains are harvested 15 to 25 days after flowering, dried at 30 to 37°C for 3 to 5 days, and then dormancy-breaking treatment is performed on the wheat grains. Subsequently, the wheat grains are germinated for vernalization and breeding of the next generation.

2. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: The winter wheat in step (1) is any one of winter wheat, semi-winter wheat and weak winter wheat.

3. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: The disinfection treatment in step (1) specifically refers to disinfection with a 2% to 10% sodium hypochlorite aqueous solution for 2 to 15 minutes, followed by washing with pure water for 4 to 6 times.

4. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: In step (2), the culture is carried out by hydroponics or soil culture, the hydroponics nutrient solution uses 1 / 4 to 1 / 2 Hogland, and the soil ratio of soil culture is nutrient soil: vermiculite: sand = 1 to 3: 0.5 to 2: X, 0.25≤X≤1.

5. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: In step (3), the light source adopts any one of LED light source cold white light, blue spectrum monochromatic light, red spectrum monochromatic light, and red:blue=1:1 / 2:1 / 3:1 / 1:2 / 1:3 composite light.

6. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: In step (3), the photoperiod is 22 h light / 2 h dark.

7. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: In step (3), the light intensity is set to 10000 lux.

8. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: In step (3), 1 / 2 Hogland nutrient solution was applied twice a week.

9. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: The dormancy-breaking treatment in step (4) is specifically performed by soaking the plants in 0.1-0.3% hydrogen peroxide solution, 1% potassium hydrogen sulfate or 1% gibberellin for 20-24 hours, 4-6 hours or 20-24 hours.

10. The method for cultivating winter wheat by rapid vernalization to accelerate annual iteration according to claim 1, characterized in that: The dormancy-breaking treatment in step (4) is specifically to soak the cells in pure water at room temperature for 16 to 30 hours and then place them at 2 to 6° C. for 20 to 30 hours to break the dormancy.

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