Construction method of efficient development and culture system for wheat haploid immature embryos

Abstract

The invention discloses a construction method of a wheat haploid immature embryo efficient development culture system. The construction method comprises the following steps: obtaining wheat * corn hybrid caryopses; preparing a melatonin mother solution, and sealing and storing in an environment of-20 DEG C for later use; preparing a 1 / 2MS solid culture medium, cooling and sealing for later use; inoculating the sterilized immature embryos into a 1 / 2 MS solid culture medium with the hypocotyls facing downwards under an anatomical lens on an ultra-clean workbench; carrying out dark culture for 14-20 days under the condition that the temperature is 23-25 DEG C; transferring well germinated wheat haploid seedlings into a 1 / 2MS culture medium without melatonin, and culturing until the haploid seedlings have 3-5 leaves. The melatonin is introduced into a wheat haploid immature embryo culture system, the suitable concentration range and action mechanism of the melatonin are determined, an efficient, stable and standardized culture system is constructed, and technical support is provided for large-scale production of wheat double haploids (DH).

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to a method for constructing a high-efficiency germination and seedling culture system for wheat haploid embryos. Background Technology

[0002] Double haploid (DH) technology can make hybrid materials homozygous and stable in the first generation. It is a core common technology for upgrading conventional breeding and molecular breeding. The key to its large-scale application lies in the efficient production of DH lines. Wheat × maize hybridization induced haploid (chromosome elimination method) has become the most widely used haploid induction technology in wheat breeding at home and abroad because it has no strict genotype restrictions and high induction rate. Its technical efficiency is mainly determined by three major indicators: embryo yield, emergence rate and doubling rate. Among them, the emergence rate (the in vitro germination rate of haploid embryos) is the core bottleneck restricting the large-scale production of DH lines.

[0003] Traditional wheat haploid embryo culture relies on hormone-free 1 / 2MS solid medium, which has three major problems: First, the germination rate is low, averaging only 34.00%-39.50%; second, seedling emergence is not concentrated, with significant differences in the size of haploid seedlings within the same culture system, and the germination period is as long as 7-9 days, requiring multiple seedling transfers, which is cumbersome; third, the subsequent workload is large, and uneven seedling growth increases the difficulty of cultivating strong seedlings and raises the risk of contamination.

[0004] Existing techniques attempt to improve seedling emergence rate by adding exogenous hormones (such as 6-BA and gibberellin), but the effects are limited (only slight increases) and the problem of uniform emergence remains unresolved. The core reason for this is the lack of specific regulatory hormones targeting the developmental characteristics of wheat haploid embryos. Melatonin, a naturally occurring substance widely found in plants, has been shown to promote seed germination, enhance stress resistance, and regulate metabolic balance. It can significantly improve seedling emergence rate under drought stress, but no studies have yet applied it to in vitro culture systems of wheat haploid embryos, and its optimal concentration and mechanism of action in this system remain unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an efficient wheat haploid embryo development culture system. This method introduces melatonin into the wheat haploid embryo culture system, clarifies its suitable concentration range and mechanism of action, and constructs an efficient, stable, and standardized culture system to provide technical support for the large-scale production of wheat haploid embryos.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A method for constructing a high-efficiency culture system for the development of wheat haploid embryos includes the following steps: Step 1: Obtaining wheat × maize hybrid caryopsis: Harvest wheat × maize hybrid ears that have been cultured for about 2 weeks, remove the hybrid caryopsis, pre-screen caryopsis containing embryos, and isolate wheat haploid embryos after sterilization. Step 2: Prepare melatonin stock solution: Weigh 500 mg of melatonin powder, dissolve it in 10 mL of anhydrous ethanol, and then filter it through a 0.22 μm filter membrane to remove bacteria, obtaining a melatonin stock solution with a concentration of 50 mg / mL. Store it in a sealed container at -20℃ for later use. Step 3: Prepare 1 / 2 MS solid culture medium: Adjust the pH of the culture medium to 5.7-5.9 with 0.1M NaOH, then sterilize at 121℃ for 20 minutes and cool to 48-52℃; Next, add 200 μL of melatonin stock solution per liter of culture medium in a clean bench, mix well, dispense into culture dishes, cool, and seal for later use. Step 4: Wheat haploid embryo inoculation: Under a dissecting microscope on a clean bench, inoculate the sterilized immature embryos with the hypocotyl facing down into 1 / 2 MS solid culture medium; Step 5, Primary Culture: Cultivate in the dark for 14 days at a temperature of 23-25℃ to obtain wheat haploid seedlings (1 leaf). Step 6, Seedling Culture: Transfer well-germinated wheat haploid seedlings to 1 / 2 MS medium without melatonin and culture them until the haploid seedlings grow to 3-5 leaves.

[0007] Further: In step one, the sterilization process involves soaking the caryopsis fruit in a 15% sodium hypochlorite solution for 30 minutes to achieve sterilization, stirring once every 5 minutes during the process. After sterilization, the fruit is rinsed 3-5 times with sterile water. The 15% sodium hypochlorite solution is prepared by mixing 150 mL of sodium hypochlorite stock solution with 850 mL of sterile water.

[0008] Furthermore, in step two, the melatonin stock solution is aliquoted into 1 mL tubes for storage to avoid repeated freeze-thaw cycles.

[0009] Furthermore: In step three, the final concentration of melatonin in the 1 / 2MS solid culture medium is 10 mg / L.

[0010] Furthermore: In step three, the height of the 1 / 2 MS solid culture medium in the petri dish is approximately 7 mm.

[0011] Furthermore: In step four, the inoculation density of the wheat haploid embryos is 15-20 embryos / 9 cm culture dish, and the interembryonic spacing is greater than 1 cm.

[0012] In summary, the present invention has the following beneficial effects: Firstly, this invention introduces melatonin into the wheat haploid embryo culture system, clarifies its suitable concentration range and mechanism of action, and constructs an efficient, stable, and standardized culture system to provide technical support for the large-scale production of wheat DH. Secondly, this invention improves the embryo emergence rate, reduces the workload of transferring the embryos to the differentiation medium for further culture, and reduces the workload of rescuing the embryos. Thirdly, it is easy to operate and the method is easy to standardize, and it has broad application prospects in the rescue of immature embryos in wheat haploid breeding; Fourth, the core configuration method of this invention is precise and controllable: Step 2 clarifies the raw material ratio, sterilization method and storage conditions of the melatonin mother liquor to avoid contamination or activity decay of the mother liquor; Step 3 clarifies the pH of the culture medium, sterilization parameters, melatonin addition amount and final concentration to ensure that the performance of each batch of culture medium is consistent, thus solving the problem of insufficient standardization in traditional culture systems. Attached Figure Description

[0013] Figure 1 This is a flowchart of the operation of the present invention. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the accompanying drawings.

[0015] In the description of this invention, it should be understood that the terms "upper", "lower", "left", "right", "front", "rear", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0016] Example 1, referring to Figure 1 Effects of different hormones on the germination rate of wheat haploid embryos Experimental materials: Wheat × maize hybrid caryopsis (containing haploid embryos) of Yunmai 112 DH line. Experimental design: Nine hormone treatments (gibberellin, auxin, cytokinin, melatonin, 6-BA, dicamba, 2,4-D) and a blank control were set up. Two concentration gradients were set up for each hormone. At least 150 immature embryos were inoculated for each treatment, and the experiment was repeated 3 times.

[0017] A method for constructing a high-efficiency culture system for the development of wheat haploid embryos includes the following steps: Step 1: Obtaining wheat × maize hybrid caryopsis: Take out wheat hybrid ears that have been cultured for about two weeks in an artificial climate incubator with working parameters of 23℃ temperature, 80% humidity, 10000 Lx light intensity, 14 h light duration / 10 h dark duration, peel off the hybrid caryopsis, and pre-screen caryopsis containing embryos. Next, the hazelnuts are immersed in a 15% sodium hypochlorite solution prepared by 150 mL of sodium hypochlorite stock solution and 850 mL of sterile water for 30 minutes for sterilization. During the process, the hazelnuts are stirred once every 5 minutes. After sterilization, they are rinsed with sterile water 3-5 times. Step 2: Prepare melatonin stock solution: Weigh 500 mg of melatonin powder, dissolve it in 10 mL of anhydrous ethanol, and then filter it through a 0.22 μm filter membrane to sterilize it, obtaining a melatonin stock solution with a concentration of 50 mg / mL. Dispense the melatonin stock solution into 1 mL tubes, seal and store them in an environment of -20℃ for later use, avoiding repeated freeze-thaw cycles. Step 3: Prepare 1 / 2MS solid culture medium: Adjust the pH of the culture medium to 5.8 with 0.1M NaOH, then sterilize at 121℃ for 20 minutes and cool to 48℃; Next, in a clean bench, add 100 μL / 200 μL of melatonin stock solution per liter of culture medium to make the final concentration of melatonin in the 1 / 2MS solid culture medium 5 mg / L / 10 mg / L. Mix well and dispense into 9 cm culture dishes, while ensuring that the height of the 1 / 2MS solid culture medium in the culture dish is about 7 mm. After cooling, seal for later use. Step 4: Wheat haploid embryo inoculation: On a clean bench, under a dissecting microscope, sterilized immature embryos with their hypocotyls facing down are inoculated into 1 / 2 MS solid culture medium containing different concentrations of different hormones. At the same time, the inoculation density of wheat haploid embryos is 15 embryos / 9 cm culture dish, and the interembryonic spacing is greater than 1 cm. Step 5, Primary Culture: Cultivate in the dark at 23℃ for 20 days to obtain wheat haploid seedlings (1 leaf). Step 6, Seedling Culture: Transfer the haploid seedlings that have developed to 1 leaf to 1 / 2 MS medium without melatonin and culture them until they have 3-5 leaves.

[0018] The experimental results are summarized in Table 1. Table 1: Effects of different hormone concentrations and 1 / 2 MS solid medium on germination rate of wheat haploid embryos (Yunmai 112) in May-June 2024 The results show that the 1 / 2 MS solid medium with added melatonin significantly improved the germination rate, increasing by 21.26% and 28.81% compared to the control (1 / 2 MS solid medium without any hormones). The 1 / 2 MS solid medium containing melatonin showed a faster germination rate, and the 10 mg / L melatonin treatment had the highest germination rate of 62.81%, which was significantly better than other hormone treatments.

[0019] Example 2: Effects of different concentrations of melatonin on the germination rate of wheat haploid embryos Experimental materials: Same as in Example 1 (Yunmai 112DH series); Experimental design: Eight melatonin concentration gradients were set up: 1, 2, 5, 10, 20, 40, 80, and 100 mg / L. The number of embryos inoculated for each treatment was no less than 160.

[0020] A method for constructing a high-efficiency culture system for the development of wheat haploid embryos includes the following steps: Step 1: Obtaining wheat × maize hybrid caryopsis: Take out wheat hybrid ears that have been cultured for about two weeks in an artificial climate incubator with working parameters of 23℃ temperature, 80% humidity, 10000Lx light intensity, 14 h light duration / 10 h dark duration, peel off the hybrid caryopsis, and pre-screen caryopsis containing embryos. Next, the hazelnuts are immersed in a 15% sodium hypochlorite solution prepared by 150 mL of sodium hypochlorite stock solution and 850 mL of sterile water for 30 minutes for sterilization. During the process, the hazelnuts are stirred once every 5 minutes. After sterilization, they are rinsed with sterile water 3-5 times. Step 2: Prepare melatonin stock solution: Weigh 500 mg of melatonin powder, dissolve it in 10 mL of anhydrous ethanol, and then filter it through a 0.22 μm filter membrane to sterilize it, obtaining a melatonin stock solution with a concentration of 50 mg / mL. Dispense the melatonin stock solution into 1 mL tubes, seal and store them at -20℃ for later use, avoiding repeated freeze-thaw cycles. Step 3: Prepare 1 / 2MS solid culture medium: Adjust the pH of the culture medium to 5.8 with 0.1M NaOH, then sterilize at 121℃ for 20 minutes and cool to 50℃; Next, in a clean bench, add 20 μL / 40 μL / 100 μL / 200 μL / 400 μL / 800 μL / 1600 μL / 2000 μL of melatonin stock solution per liter of culture medium, so that the final concentration of melatonin in the 1 / 2MS solid culture medium is 1 mg / L / 2 mg / L / 5 mg / L / 10 mg / L / 20 mg / L / 40 mg / L / 80 mg / L / 100 mg / L. Mix well and dispense into 9 cm culture dishes, while ensuring that the height of the 1 / 2MS solid culture medium in the culture dish is about 7 mm. After cooling, seal for later use. Step 4: Wheat haploid embryo inoculation: On a clean bench, under a dissecting microscope, sterilized immature embryos with their hypocotyls facing down are inoculated into 1 / 2 MS solid medium containing different concentrations of melatonin. At the same time, the inoculation density of wheat haploid embryos is 18 embryos / 9 cm culture dish, and the interembryonic spacing is greater than 1 cm. Step 5, Primary Culture: Cultivate in the dark at 24℃ for 14 days to obtain wheat haploid seedlings (1 leaf). Step 6, Seedling Culture: When the wheat haploid seedlings have grown to 3 leaves, transfer them to 1 / 2 MS medium without melatonin and culture for 7-10 days until they are ready for transplanting.

[0021] The experimental results are summarized in Table 2. Table 2: Effects of different concentrations of melatonin on the germination rate of haploid wheat embryos (Yunmai 112) in June-July 2024 on 1 / 2 MS solid medium. The results show that 1 / 2 MS solid medium supplemented with 10 mg / L melatonin significantly improved the germination rate. Within the concentration range of 10-20 mg / L, the germination rate remained above 60%. However, the germination rate gradually decreased after the concentration exceeded 40 mg / L. At 100 mg / L, the germination rate (31.09%) was lower than the control, exhibiting an inhibitory effect. Furthermore, 10 mg / L was the optimal concentration, resulting in the highest germination rate. Concentrations that were too high or too low were detrimental to embryo germination. Compared to the control group (1 / 2 MS solid medium without any hormones), the germination rate was increased by 31.15% and 25.03%, respectively.

[0022] Example 3: Effect of 10 mg / L melatonin on the germination rate of haploid embryos of different wheat varieties (lines) Experimental materials: wheat × maize hybrid caryopsis (containing haploid embryos) from 25 different wheat varieties (lines) Experimental design: Each variety (line) was set up with a treatment group (10 mg / L melatonin + 1 / 2 MS medium) and a control group (hormone-free 1 / 2 MS medium). Each treatment was inoculated with 400 immature embryos, replicated 4 times, and cultured in the dark for 14 days. Germination rate and average germination period were recorded.

[0023] A method for constructing a high-efficiency culture system for the development of wheat haploid embryos includes the following steps: Step 1: Obtaining wheat × maize hybrid caryopsis: Take out wheat hybrid ears that have been cultured for about two weeks in an artificial climate incubator with working parameters of 23℃ temperature, 80% humidity, 10000 Lx light intensity, 14 h light duration / 10 h dark duration, peel off the hybrid caryopsis, and pre-screen caryopsis containing embryos. Next, the hazelnuts are immersed in a 15% sodium hypochlorite solution prepared by 150 mL of sodium hypochlorite stock solution and 850 mL of sterile water for 30 minutes for sterilization. During the process, the hazelnuts are stirred once every 5 minutes. After sterilization, they are rinsed with sterile water 3-5 times. Step 2: Prepare melatonin stock solution: Weigh 500 mg of melatonin powder, dissolve it in 10 mL of anhydrous ethanol, and then filter it through a 0.22 μm filter membrane to sterilize it, obtaining a melatonin stock solution with a concentration of 50 mg / mL. Dispense the melatonin stock solution into 1 mL tubes, seal and store them at -20℃ for later use, avoiding repeated freeze-thaw cycles. Step 3: Prepare 1 / 2MS solid culture medium: Adjust the pH of the culture medium to 5.8 with 0.1M NaOH, then sterilize at 121℃ for 20 minutes and cool to 52℃; Next, in a clean bench, add 200 μL of melatonin stock solution per liter of culture medium to make the final concentration of melatonin in the 1 / 2MS solid culture medium 10 mg / L. Mix well and dispense into 9 cm culture dishes, while ensuring that the height of the 1 / 2MS solid culture medium in the culture dish is about 7 mm. After cooling, seal for later use. Step 4: Wheat haploid embryo inoculation: On a clean bench, under a dissecting microscope, inoculate the sterilized immature embryos with the hypocotyl facing down into 1 / 2 MS solid medium containing 10 mg / L melatonin, while ensuring that the inoculation density of wheat haploid embryos is 20 embryos / 9cm culture dish, with an interembryonic spacing of more than 1cm. Step 5, Primary Culture: Incubate in the dark at 24℃ for 14 days to obtain wheat haploid seedlings (1 leaf). Step 6, Seedling Culture: When the wheat haploid seedlings have grown to 3 leaves, transfer them to 1 / 2 MS medium without melatonin and culture for 7-10 days until they are ready for transplanting.

[0024] The experimental results are summarized in Table 3. Table 3: Effects of 1 / 2 MS solid medium containing 10 mg / L melatonin on germination rate of wheat haploid embryos (different varieties / lines) in July-August 2024 Note: Average duration (days) = the average time from the germination of the first embryo to the germination of the last embryo in each of the 20 dishes.

[0025] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make inventive modifications to this embodiment as needed, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for constructing a high-efficiency development culture system for wheat haploid embryos, characterized in that, Includes the following steps: Step 1: Obtaining wheat × maize hybrid caryopsis: Harvest wheat × maize hybrid ears that have been cultured for about 2 weeks, remove the hybrid caryopsis, pre-screen caryopsis containing embryos, and isolate wheat haploid embryos after sterilization. Step 2: Prepare melatonin stock solution: Weigh 500 mg of melatonin powder, dissolve it in 10 mL of anhydrous ethanol, and then filter it through a 0.22 μm filter membrane to remove bacteria, obtaining a melatonin stock solution with a concentration of 50 mg / mL. Store it in a sealed container at -20℃ for later use. Step 3: Prepare 1 / 2 MS solid culture medium: Adjust the pH of the culture medium to 5.7-5.9 with 0.1M NaOH, then sterilize at 121℃ for 20 minutes and cool to 48-52℃; Next, add 200 μL of melatonin stock solution per liter of culture medium in a clean bench, mix well, dispense into culture dishes, cool, and seal for later use. Step 4: Wheat haploid embryo inoculation: Under a dissecting microscope on a clean bench, inoculate the sterilized immature embryos with the hypocotyl facing down into 1 / 2 MS solid medium containing melatonin; Step 5, Primary Culture: Under dark culture at a temperature of 23-25℃ for 14-20 days to obtain wheat haploid seedlings; Step 6, Seedling Culture: Transfer well-germinated wheat haploid seedlings to 1 / 2 MS medium without melatonin and culture them under light conditions until the haploid seedlings grow 3-5 leaves.

2. The method for constructing a high-efficiency development culture system for wheat haploid embryos according to claim 1, characterized in that: In step one, the sterilization process involves soaking the caryopsis fruit in a 15% sodium hypochlorite solution for 30 minutes, stirring once every 5 minutes during the process. After sterilization, the fruit is rinsed 3-5 times with sterile water. The 15% sodium hypochlorite solution is prepared by mixing 150 mL of sodium hypochlorite stock solution with 850 mL of sterile water.

3. The method for constructing a high-efficiency development culture system for wheat haploid embryos according to claim 1, characterized in that: In step two, the melatonin stock solution is aliquoted into 1mL tubes for storage to avoid repeated freeze-thaw cycles.

4. The method for constructing a high-efficiency development culture system for wheat haploid embryos according to claim 1, characterized in that: In step three, the final concentration of melatonin in the 1 / 2MS solid culture medium is 10 mg / L.

5. The method for constructing a high-efficiency development culture system for wheat haploid embryos according to claim 1, characterized in that: In step three, the height of the 1 / 2MS solid culture medium in the petri dish is approximately 7 mm.

6. The method for constructing a high-efficiency development culture system for wheat haploid embryos according to claim 1, characterized in that: In step four, the inoculation density of the wheat haploid embryos is 15-20 embryos / 9cm culture dish, and the interembryonic spacing is greater than 1cm.

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