Method for improving survival rate of corn haploid doubled buds

Abstract

The invention discloses a method for increasing the survival rate of corn haploid doubled buds, and belongs to the technical field of agricultural biology. According to the method for increasing the survival rate of the corn haploid doubled buds, the survival rate of the haploid buds is increased by adjusting the germination temperature, the illumination time, the agent treatment concentration, the flushing water temperature and the flushing frequency, simplifying the procedure, reducing the transplanting frequency and removing the seedling hardening process.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and more particularly to a method for improving the survival rate of corn haploid doubling sprouts. Background Art

[0002] Haploid breeding technology is of great significance in the genetic improvement of maize. The most critical factor in the successful restoration of the growth vitality of the D0 generation seedlings during the DH line breeding process. However, the survival rate of haploids after doubling by chemical methods is usually low, which poses a great challenge to haploid breeding work. Since the D0 generation seedlings are weak from the beginning, any type of stress will lead to a decrease in the survival rate of the D0 generation seedlings. At present, the commonly used haploid doubling methods mainly use chemical doubling methods such as colchicine treatment, but these methods still have certain limitations in improving the survival rate of seedlings. One or more of the following problems are common: (1) Haploid seeds become moldy or rotten due to bacterial infection during the germination process, resulting in the death of young shoots; (2) Poor control of germination time leads to excessive elongation of the coleoptile and reduced bud vitality. During the bud cutting process, the bud breakage rate is greatly increased, resulting in the death of the seedlings; (3) After bud cutting, the buds are directly immersed in colchicine and DMSO solutions, and the bud wounds are not buffered and adapted, causing serious damage to the cells at the incision and greatly reducing the survival rate; (4) The chemical agents colchicine and DMSO are mostly prepared in tap water, and the low temperature affects the survival rate of seedlings; (5) Excessive concentrations of colchicine and DMSO lead to decreased cell viability and also affect other normal physiological metabolism; (6) After doubling, the agent is not rinsed properly, the agent remains or the rinsing water temperature is too low, which affects the growth and development of the young shoots; (7) After transplanting to the seedling pot for hardening and then transplanting to the field, the two transplants increase the damage rate of the young shoots and seedlings, which is not conducive to root growth and reduces the survival rate of the seedlings. Based on this, there is an urgent need for an innovative and effective method to improve the survival rate of seedlings after haploid doubling.

[0003] Therefore, providing a method for improving the survival rate of doubled corn haploid sprouts is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] In view of this, the present invention provides a method for improving the survival rate of haploid doubling sprouts of corn, which significantly improves the survival rate of haploid sprouts, thereby improving the efficiency and quality of haploid breeding of corn. The survival rate of haploid sprouts is improved by optimizing the original haploid doubling process and each process processing method, changing the germination temperature, light exposure time, agent treatment concentration, flushing water temperature, flushing times, simplifying the procedure, reducing the number of transplanting times, and removing the seedling hardening process.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for improving the survival rate of doubled corn haploid sprouts, comprising the following specific steps:

[0007] (1) Soak the haploid seeds selected from the quasi-haploid seeds in a 0.3% potassium permanganate solution for 15 minutes (which can effectively kill bacteria and insect eggs on the surface of the seeds), then rinse them with clean water and germinate them; the germination tray, germination paper and germination covering towel all need to be disinfected with a 3% sodium hypochlorite solution in advance;

[0008] (2) The temperature in the germination room is controlled at 25-28°C, the humidity is controlled at 70%, the light duration is 8-10 hours / day, the light intensity is 3000LX, and the temperature is controlled at 28°C 2 days before germination; after 2 days, the light duration is set to 6-8 hours / day, the light intensity is set to 3000LX, and the temperature is set to 25°C; to prevent the buds from growing too fast and too long and missing the best vitality period of the young buds;

[0009] (3) Seedlings with coleoptile length of about 2 cm are most suitable for colchicine and DMSO treatment, and the vigor of the young buds is the strongest at this time; before colchicine and DMSO treatment, a sterile blade fixed on a scalpel blade holder is used to cut the bud tissue about 1 cm from the tip. At this time, the coleoptile length is optimal and it is not easy to break after cutting;

[0010] (4) placing the cut buds in a mesh bag and labeling them, soaking them in room temperature ddH2O for 2 hours to allow the wounds of the cut buds to be buffered and adapted to a certain extent, thereby reducing the excessive stimulation and damage that may be caused by direct contact with colchicine and DMSO solutions; then transferring them to an aqueous solution of 0.06% colchicine and 0.6% DMSO for chromosome doubling for 7 hours;

[0011] (5) Rinse the soaked sprouts three times in room temperature ddH2O prepared in advance to remove residual colchicine and DMSO solution, with each rinse lasting 10-15 minutes; after rinsing, rinse and disinfect with 2% sodium hypochlorite solution;

[0012] (6) Soak the rinsed young shoots in a rooting powder solution diluted to 500-1000 times for 2 hours, rinse them once in room temperature ddH2O to remove the rooting powder solution, and rinse for 5 minutes;

[0013] (7) After flushing, the quasi-diploid seedlings are directly transplanted to the field without going through a complicated and tedious seedling hardening process. At the same time, the number of transplanting times is reduced, which reduces the damage rate and mortality rate of the seedlings.

[0014] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for improving the survival rate of doubled haploid sprouts of corn, which has the following beneficial effects:

[0015] (1) Significantly improve the survival rate of haploid seedlings; by disinfecting before germination and optimizing the treatment concentration of chemical agents, the survival rate of young shoots can be effectively improved.

[0016] (2) The excessive doubling damage to seedlings caused by residual colchicine and DMSO solution was significantly reduced, thereby further improving the survival rate of young shoots. By optimizing the haploid doubling program, setting the post-doubling rinse to 3 times, and using room temperature ddH2O water for rinsing, the damage to young shoots caused by low water temperature and unclean water quality can be effectively reduced, thereby improving the survival rate of young shoots.

[0017] (3) Improve the efficiency of haploid breeding of corn; young shoots with high survival rates provide more options and possibilities for subsequent breeding work, which can significantly shorten the breeding cycle and improve breeding efficiency.

[0018] (4) Directly transplanting seedlings into the field can reduce damage during the secondary hardening and transplanting process and further improve the survival rate of seedlings.

[0019] (5) The operation is simple, the procedure is simple, and the cost is greatly reduced; the chemical doubling method of the present invention is relatively simple to operate, does not require complex equipment and technology, optimizes the procedure and reduces the production cost, and is suitable for large-scale application in corn haploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The accompanying drawing is a flow chart of the method for improving the survival rate of maize haploid doubled sprouts of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0023] Example 1

[0024] The flowchart of the method for improving the survival rate of doubled haploid shoots of maize is shown in Figure 1 .

[0025] 1. Preliminary preparation for haploid doubling

[0026] 1. Quasi-haploid grains: A number of quasi-haploid grains obtained through a specific screening and identification process are selected as the experimental material basis for this example.

[0027] 2. Chemical reagents: Prepare enough potassium permanganate to prepare a 0.3% potassium permanganate solution. Sodium hypochlorite is used to prepare a 3% sodium hypochlorite solution to disinfect germination-related supplies, and a 2% sodium hypochlorite solution for subsequent rinsing and disinfection of young shoots. Colchicine and dimethyl sulfoxide (DMSO) are used to prepare an aqueous solution with a concentration of 0.06% colchicine and 0.6% DMSO for chromosome doubling treatment of young shoots. Rooting powder is diluted to 500 times according to the product instructions to promote rooting of young shoots.

[0028] 3. Other materials: Several germination trays, germination papers and germination covering towels to provide a seedling room for seed germination. Several mesh bags to place the cut young shoots. Scalpels and matching blade holders equipped with sterile blades for cutting young shoots. Sufficient deionized water (ddH2O) for soaking, rinsing and other operations.

[0029] 2. Haploid doubling time and results

[0030] (I) Seed pretreatment

[0031] 1. According to the predetermined formula, accurately weigh an appropriate amount of potassium permanganate and dissolve it in deionized water to prepare a potassium permanganate solution with a concentration of 0.3%.

[0032] 2. Soak the haploid seeds selected from the quasi-haploid seeds in the prepared potassium permanganate solution for 15 minutes. During this period, the potassium permanganate solution can effectively kill the bacteria and insect eggs attached to the surface of the seeds, ensuring a healthy environment for the subsequent germination process.

[0033] 3. After 15 minutes, rinse the soaked haploid seeds carefully with clean water to ensure that there is no potassium permanganate residue on the surface of the seeds to avoid affecting subsequent germination. The results are shown in Table 1.

[0034] Table 1 Effects of different concentrations of potassium permanganate solution immersion disinfection on seed germination

[0035] Solution concentration 0.1％ 0.2％ 0.3％ 0.4％ 0.5％ 0.6％ Mold rate 7％ 5％ 1％ 1％ 0 0 Ungerminated rate 9％ 6％ 3％ 9％ 11％ 15％ Germination rate 91％ 94％ 97％ 91％ 89％ 85％

[0036] (II) Germination environment preparation and seed germination

[0037] 1. Use 3% sodium hypochlorite solution to fully disinfect the germination tray, germination paper and germination covering towel in advance. Cover the disinfected germination tray with germination paper and then cover it with disinfected towel to create a relatively sterile environment for seed germination. The results are shown in Table 2.

[0038] Table 2 Effects of different germination environments on germination results

[0039]

[0040] 2. Place the pre-treated haploid grains evenly on the germination paper of the germination tray, and then place the germination tray into the germination room.

[0041] 3. In the germination room, strictly control the environmental conditions. For the first 2 days, set the temperature to 28℃, the humidity to 70%, and set the light time to 8 hours / day and the light intensity to 3000LX. In such an environment, the seeds begin to germinate gradually and the young shoots grow slowly.

[0042] 4. After 2 days, adjust the environmental parameters of the germination room. Set the light duration to 6 hours / day, keep the light intensity at 3000LX, and adjust the temperature to 25°C. Through this staged environmental regulation, the buds can be prevented from growing too fast and too long and missing the best vitality period of the young buds, so that the young buds can grow healthily at a suitable rhythm. The results are shown in Table 3.

[0043] Table 3 Effects of different germination environments on germination results

[0044]

[0045] Variable time and temperature treatment can provide seeds with a suitable temperature environment during the germination stage to promote rapid germination and shorten the working time. Variable time and temperature treatment after 2 days can better control the growth rate of young shoots, making it easier to cut and double the buds during the optimal length period.

[0046] (III) Preparation for young shoots

[0047] 1. Continue to observe the growth of the young shoots. When the coleoptile of the young shoots is found to be about 2 cm long, it is determined that the seedlings at this time are most suitable for colchicine and DMSO treatment, because the young shoots are most active at this stage.

[0048] 2. Before the treatment with colchicine and DMSO, a sterile blade fixed on a scalpel blade holder was used to cut the bud tissue about 1 cm from the tip. Since the coleoptile is of moderate length at this time, it is not easy to break after cutting, and it can provide a suitable incision position for subsequent treatment. The results are shown in Table 4.

[0049] Table 4 Effects of different coleoptile lengths after bud cutting and doubling

[0050] Bud length (cm) 1 2 3 Survival rate 87％ 92％ 85％

[0051] (IV) Chromosome doubling treatment

[0052] 1. Place the cut sprouts carefully in a mesh bag and attach corresponding labels for later identification.

[0053] 2. First, soak the mesh bag containing the young shoots in ddH2O at room temperature for 2 hours. This step can buffer and adapt the wound of the cut shoots to a certain extent, reducing the excessive stimulation and damage that may be caused by direct contact with colchicine and DMSO solution.

[0054] After 3.2 hours, the mesh bag was transferred to a pre-prepared treatment tank with a concentration of 0.06% colchicine and 0.6% DMSO for chromosome doubling treatment, and the treatment time was set to 7 hours. During this treatment process, colchicine and DMSO acted synergistically to promote the doubling of chromosomes in the young shoots, laying the foundation for obtaining quasi-diploid young shoots. The results are shown in Table 5.

[0055] Table 5 Effects of different germination environments on germination results

[0056]

[0057] (V) Subsequent processing

[0058] After the 1.7-hour chromosome doubling treatment, the soaked sprouts were taken out of the treatment tank and rinsed in ddH2O prepared in advance at room temperature (20-25°C). The rinses were performed 3 times, each time for 15 minutes, in order to completely remove the residual colchicine and DMSO solution on the surface of the sprouts and avoid the adverse effects of these chemicals on the subsequent growth of the sprouts. The results are shown in Tables 6 and 7.

[0059] Table 6 Effects of doubling the soaking time

[0060] Soaking time (h) 6 7 8 9 10 Doubling rate (diploid) 8％ 25％ 24％ 20％ 13％

[0061]

[0062] The results in Table 6 show that the survival rate of seedlings reached an ideal effect of 98% after being soaked in the agent for 6 hours and 7 hours, but the doubling rate after soaking for 7 hours was significantly higher than that after soaking for 6 hours.

[0063] Table 7 Effect of different flushing time and flushing water temperature after doubling

[0064] Rinse time 5 15 25 Deformed seedlings 5％ 1％ 1％ Survival rate 90％ 97％ 89％ Flushing temperature Less than 10℃ 15-20℃ 20-25℃ Survival rate 85％ 93％ 98％

[0065] 2. After the deionized water rinse, the young shoots were rinsed and disinfected with 2% sodium hypochlorite solution to further ensure the sanitary condition of the young shoots and reduce the risk of bacterial infection. The results are shown in Table 8.

[0066] Table 8 Effects of different treatments on seedling survival rate

[0067]

[0068] (VI) Rooting treatment

[0069] 1. Soak the sprouts treated above in a rooting powder solution diluted to 500 times for 2 hours. Under the action of the rooting powder solution, the sprouts can take root better, providing a good root foundation for subsequent growth after transplanting to the field. The results are shown in Table 9.

[0070] Table 9 Effects of different treatments on seedling survival rate

[0071]

[0072] 2. After 2 hours of soaking, take the sprouts out of the rooting powder solution and rinse them once in room temperature ddH2O for 5 minutes to ensure that the rooting powder solution is removed to prevent it from interfering with the subsequent growth of the sprouts.

[0073] (VII) Transplanting to the field

[0074] 1. The washed quasi-diploid seedlings were directly transplanted to the field. During the transplanting process, the injury rate and mortality rate of the seedlings were effectively reduced because there was no need to go through a complicated and tedious seedling hardening process and the number of transplanting times was reduced. The results are shown in Table 10.

[0075] Table 10 Comparison of different transplanting and planting methods after doubling

[0076]

[0077] 2. After transplanting, the growth of young shoots in the field was continuously observed, and it was found that the quasi-diploid young shoots treated with the technical solution of the present invention showed a good growth trend in the field, with normal root development and strong plant growth. This preliminarily verified the effectiveness of the technical solution of the present invention in improving the survival rate of young shoots, promoting the healthy growth of young shoots, and simplifying the operating procedures.

[0078] Conclusion

[0079] Through the detailed operation and observation of the results of this embodiment, it can be seen that the technical solution provided by the present invention effectively achieves the goals of killing pathogens and eggs, promoting the healthy growth of young shoots, achieving good chromosome doubling effects, rooting treatment, and reducing the damage rate and mortality rate of young shoots through sufficient preliminary preparation, chemical reagent treatment with appropriate concentrations, environmental regulation, and simplified operating procedures. This provides a feasible and advantageous technical method for corn haploid doubling and planting.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the survival rate of doubled haploid sprouts of corn, characterized in that: The specific steps are as follows: (1) Soak the haploid seeds in a 0.3% potassium permanganate solution for 15 minutes, then rinse with clean water before germination; the germination tray, germination paper, and germination covering towel all need to be disinfected with a 3% sodium hypochlorite solution in advance; (2) The temperature in the germination room is controlled at 25-28°C, the humidity is controlled at 70%, the light duration is 8-10 hours / day, the light intensity is 3000LX, and the temperature is controlled at 28°C 2 days before germination; after 2 days, the light duration is set to 6-8 hours / day, the light intensity is set to 3000LX, and the temperature is set to 25°C; (3) Seedlings with 2-cm coleoptile length were treated with colchicine and DMSO; before colchicine and DMSO treatment, the bud tissue was cut 1 cm from the tip with a sterile blade fixed in a scalpel blade holder; (4) Place the cut shoots in a mesh bag and label them, soak them in room temperature ddH2O for 2 hours, and then transfer them to an aqueous solution of 0.06% colchicine and 0.6% DMSO for chromosome doubling for 7 hours; (5) Rinse the soaked sprouts three times in room temperature ddH2O prepared in advance to remove residual colchicine and DMSO solution, with each rinse lasting 10-15 minutes; after rinsing, rinse and disinfect with 2% sodium hypochlorite solution; (6) Soak the rinsed young shoots in a rooting powder solution diluted to 500-1000 times for 2 hours, rinse them once in room temperature ddH2O to remove the rooting powder solution, and rinse for 5 minutes; (7) The washed quasi-diploid shoots are directly transplanted to the field.

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