Corn haploid doubling one-step planting method

Abstract

The invention discloses a corn haploid doubling one-step planting method, and belongs to the technical field of agricultural biology. The invention discloses a corn haploid doubling one-step planting method. D0-generation seedlings after doubling are directly transplanted to a field. According to the method, the operation process is simplified, the working efficiency is improved, the production cost is reduced, the method is suitable for large-scale development, the damage rate of the seedlings is reduced, and the survival rate of the seedlings is improved.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and more particularly to a one-step planting method for doubling haploid corn. Background Art

[0002] Haploid breeding technology induces haploid plants and doubles chromosomes to quickly obtain homozygous inbred lines. This process greatly shortens the breeding cycle compared to conventional breeding methods. It only takes one generation to reach the homozygous state, thereby quickly locking in the target trait. The commonly used haploid doubling methods are mainly colchicine treatment and tissue culture doubling methods, but both methods require a series of procedures from haploid seed screening, indoor germination and cutting, colchicine treatment doubling, residual agent washing, indoor transplanting and seedling hardening, and then transplanting to the field. The existing haploid doubling technology process generally has the following multiple problems: (1) complex procedures; (2) large workload; (3) the operation process is not easy to master and is not easy to understand; (4) low efficiency and high production cost; (5) two transplants cause great damage to seedlings and increase seedling mortality. This brings great challenges to haploid breeding work. Based on this, there is an urgent need for an innovative method to optimize and simplify the haploid doubling process to improve work efficiency and reduce production costs.

[0003] Therefore, providing a one-step colonization method for doubling the haploid of corn 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 one-step planting method for doubling haploids of corn, which is a simple and efficient haploid doubling procedure, thereby improving the efficiency and quality of haploid breeding of corn.

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

[0006] The method of the present invention is a "one-step planting method"; the maize haploid doubling process can be divided into three stages: 1) germination stage; 2) doubling stage; 3) planting stage. This method mainly innovates and simplifies the third stage, the planting stage.

[0007] Simplified haploid doubling process:

[0008] The first stage is the germination stage: (1) screening of haploid seeds; (2) disinfection of haploid seeds by soaking them in a 3% hydrogen peroxide solution for 10 minutes; (3) spreading them on a germination tray, covering them with germination paper and a wet towel, and placing them in a germination room at a temperature of 25-30°C, a humidity of 70%, and a light intensity of 3000LX.

[0009] The second stage, the doubling stage: (1) When the coleoptile grows to 2 cm, cut it with a sterilized surgical blade at 1 cm from the tip of the bud. Buds that are too short are easily damaged, and buds that are too long are easily broken during the doubling process; (2) Soak in a 0.1% colchicine solution for chromosome doubling for 7 hours; (3) Rinse the residual colchicine with ddH2O at room temperature (20-25°C) for 3 times, with each rinse lasting 15 minutes for the best;

[0010] The third stage is the planting stage: the doubled D0 seedlings are directly transplanted to the field, eliminating the many intermediate steps of transplanting to the seedling pots for hardening. Watering should be carried out within 2 hours after transplanting to ensure sufficient water. Watering should be carried out once a day for 3 consecutive days. After that, the field management is the same as that of ordinary fields.

[0011] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a one-step corn haploid doubling colonization method, which has the following beneficial effects:

[0012] (1) Simplify the operation process: The simplified process reduces the cumbersome intermediate links, and operators can master and execute more quickly, thereby improving the overall fluency of the work.

[0013] (2) Improve work efficiency: Efficient operation can complete more tasks in a unit of time, speed up production progress, and meet breeding needs.

[0014] (3) Reduce production costs: You can get more output with the same input, or reduce input while keeping the output unchanged, thereby improving efficiency.

[0015] (4) Suitable for large-scale development: give full play to the scale effect, improve resource utilization, reduce production costs and enhance competitiveness.

[0016] (5) Reduce seedling damage rate: Reducing the number of transplanting times of D0 generation seedlings can ensure the healthy growth of seedlings and reduce the losses caused by seedling damage.

[0017] (6) Improve seedling survival rate: Seedlings with high survival rate provide more options and possibilities for subsequent breeding work, significantly shorten the breeding cycle and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The accompanying drawings show the improved haploid doubling process of the present invention and the original haploid doubling process;

[0020] Figure 2 The accompanying drawing shows that the present invention adopts the improved "one-step planting method" to directly transplant the doubled young shoots into the field;

[0021] Figure 3 The attached figure shows the seedlings being transplanted twice after the original haploid was doubled;

[0022] A is the first transplanting to the seedling pot after doubling and hardening the seedlings in the seedling shed; B is the second transplanting to the field after the seedlings have been hardened for 5-7 days and have grown to 3-4 leaves. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] A one-step planting method for doubling the haploid of maize and the original haploid doubling process Figure 1 .

[0026] A one-step corn haploid doubling colonization method comprises the following steps:

[0027] 1) Germination stage: (1) Selection of haploid seeds; (2) Disinfection of haploid seeds: Soak in 3% hydrogen peroxide solution for 10 minutes; disinfect for 5, 15, and 20 minutes as a control; (3) Spread on a germination tray, cover with germination paper and a wet towel, and place in a germination room at a temperature of 25-30°C, a humidity of 70%, and a light intensity of 3000LX. The germination rate results at different disinfection times are shown in Table 1.

[0028] Table 1 Effect of immersion disinfection with 3% hydrogen peroxide solution on seed germination

[0029] Disinfection time (min) 5 10 15 20 Mold rate 7％ 3％ 2％ 2％ Ungerminated rate 1％ 1％ 5％ 7％ Germination rate 91％ 96％ 93％ 91％

[0030] 2) Doubling stage: (1) When the coleoptile grows to 2 cm, cut it with a sterilized surgical blade at 1 cm from the tip of the bud. If the bud is too short, it is easy to be damaged, and if the bud is too long, it is easy to break during the doubling process; (2) Soak in a 0.1% colchicine solution for chromosome doubling treatment for 7 hours; (3) Rinse the residual colchicine with ddH2O at room temperature (20-25°C) for 3 times, each time for 15 minutes. In Table 2-5, except for the limited parameters that have changed, the remaining parameters are the optimal parameters of the present invention mentioned above.

[0031] Table 2 Effects of different coleoptile lengths after bud cutting and doubling

[0032] Bud length (cm) 1 2 3 Survival rate 87％ 92％ 85％

[0033] Table 3 Effects of doubling the concentration of colchicine

[0034] Colchicine concentration 0.05％ 0.1％ 0.15％ 0.2％ Doubling rate (diploid) 10％ 25％ 18％ 15％ Double the effect Doubling is not complete Double Complete Over-doubling Over-doubling Deformed seedlings 0 0 6％ 11％

[0035] Table 4 Effects of doubling the soaking time

[0036]

[0037] Table 5 Effect of different flushing time and flushing water temperature after doubling

[0038] Rinse time (minutes) 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％

[0039] Note: ddH2O water temperature measurement, the laboratory tap water supply source is underground deep well water. After ddH2O is prepared by a water maker, the direct use temperature is less than 10°C, 3 hours later the use temperature is 15-20°C, and 5 hours later the use temperature is 20-25°C (close to room temperature).

[0040] 3) Planting stage: The doubled D0 seedlings are directly transplanted to the field, eliminating the need for many intermediate transplanting to the seedling pots for seedling hardening. Watering is carried out promptly within 2 hours after transplanting to ensure sufficient water. Watering is carried out once a day for 3 consecutive days. After that, the field management is the same as that of ordinary fields. The comparison results of different transplanting and planting methods after doubling are shown in Table 6.

[0041] Table 6 Comparison of different transplanting and planting methods after doubling

[0042]

[0043] (1) After the three F1 populations were hybridized and induced, 1,000 haploid grains with uniform and full grains were selected from each population, totaling 3,000 samples. (2) According to the comparison of the experiment and the summary of years of work experience, one person can plant 0.5 mu of land in one day. According to the calculation of 20 cm plant spacing, one person can plant 3,031 seedlings in one day; one person can plant one seedling in the seedling pot in an average of 2 minutes, and can only plant 240 seedlings in one day. It takes 12-13 people to complete the same number of samples on the same day. (3) Using the one-step fixed value method, one person can complete the transplanting of all samples in one day (8 hours of working time); using the seedling hardening and transplanting method, it takes 1 day to transplant to the seedling pot, 7-10 days to harden the seedlings (including 2-3 days for the first transplant to slow down the seedlings), 1 day to transplant the seedling pot to the field, and 3-5 days to slow down the seedlings in the field. Therefore, it takes a total of 10-15 days to transplant to the field after hardening the seedlings. (4) The seedling hardening and transplanting method also requires additional greenhouses, supporting environmental temperature monitoring equipment, seedling pots, seedling soil, and many other investments.

[0044] The "one-step planting method" transplants the doubled young shoots directly into the field ( Figure 2 ), which not only saves the complicated intermediate links, but also improves efficiency and reduces production costs. One-time transplanting reduces the probability of damage to young shoots.

[0045] Comparative Example 1

[0046] The original haploid doubling method comprises the following steps:

[0047] 1) Germination stage: (1) Screening of haploid seeds; (2) Spreading the soaked germination paper flat on the table and placing the seeds in a row with an interval of 5 cm. After placing, roll up the germination paper along one side, tie it with a rubber band, and soak it in a 3% hydrogen peroxide solution for 20 minutes; (3) Put the sterilized seeds in a sink filled with ddH2O water, set the sink to a temperature of 30°C, a humidity of 60%, and germinate under natural light.

[0048] 2) Doubling stage: (1) When the coleoptile grows to 2-3 cm, cut it 1 cm from the bud tip with a sterilized surgical blade; (2) Soak it in a 0.2% colchicine solution for 10 hours to perform chromosome doubling treatment; (3) Rinse the residual colchicine with tap water (water temperature about 10°C) for 3 times, each rinse lasting 5 minutes.

[0049] 3) Planting stage: The D0 seedlings are manually transplanted into disposable seedling pots using peat soil. After transplanting, the seedling pots are transferred to the seedling shed. After manual fine management, such as spraying water and applying rooting powder, the D0 seedlings are transplanted into the field after 7-10 days of hardening.

[0050] After doubling the original haploid, the seedlings were transplanted twice ( Figure 3 ), the seedlings are hardened in the nursery shed for 7-10 days, which is not only time-consuming and labor-intensive, but also increases production and management costs and is inefficient; at the same time, the two transplanting processes increase the probability of seedling damage and death.

[0051] 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 one-step corn haploid doubling colonization method, characterized in that: The following steps are involved: 1) Germination stage: (1) Selection of haploid seeds; (2) Disinfection of haploid seeds: Soak in 3% hydrogen peroxide solution for 10 minutes; (3) Spread on a germination tray, cover with germination paper and a wet towel, and place in a germination room at a temperature of 25-30°C, a humidity of 70%, and a light intensity of 3000LX; 2) Doubling stage: (1) When the coleoptile grows to 2 cm, cut it at 1 cm from the bud tip with a sterilized surgical blade; (2) Soak in a 0.1% colchicine solution for 7 hours to double the chromosomes; (3) Rinse with residual colchicine, rinse three times with 20-25°C ddH2O, each rinse for 15 minutes; 3) Planting stage: The doubled D0 seedlings are directly transplanted into the field.

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