A method for doubling maize haploid immature embryo tissue culture

Abstract

The invention discloses a method for doubling the tissue culture of a maize haploid immature embryo, comprising the following steps: S1 configures a liquid doubling medium, takes MS salt, sucrose, NAA, calcium chloride, and dimethyl sulfoxide respectively, and mixes and stirs in DDH2O until dissolved, sequentially performs pH acid adjustment, sterilization treatment, and cooling treatment, adds colchicine and mixes, and the mixed solution is dispensed into a culture dish to form a liquid doubling medium, and the culture dish is kept in a light-shielding environment; S2 inoculates and doubles, and each portion of liquid doubling medium strips out a corresponding number of maize haploid immature embryos and places them on the same filter paper, and the filter paper is soaked in a liquid doubling medium for doubling treatment. The method of the present invention can shorten the time for inoculating immature embryos, and only needs to soak the immature embryos in a liquid culture medium, and 1min can complete the inoculation of 5000 immature embryos, reducing labor and instrument costs. Secondly, by using the method of immersion treatment, the immature embryos can be more fully exposed to colchicine and improve the doubling rate.

Description

Technical Field

[0001] The invention relates to the technical field of corn breeding, in particular to a method for doubling corn haploid immature embryos through tissue culture. Background Art

[0002] The haploid breeding cycle of corn is short, and it takes two generations to go from hybrid to pure line (i.e. DH line), while conventional breeding takes at least seven generations. The haploid breeding process is divided into induction and doubling. The commonly used doubling methods include tissue culture doubling, root immersion method, growth point injection method, bud immersion method, seed soaking method, and heart dripping method. Among them, tissue culture doubling can further shorten the pure line acquisition cycle to 180 days, and the efficient doubling method is widely used in commercial breeding. The current tissue culture doubling method is to use tweezers to inoculate the haploid embryos into a solid culture medium containing colchicine (see Figure 1 ), embryo side down, gently handle and place, and spread them flat on the surface of the culture medium one by one.

[0003] Since the doubling culture medium currently used is mostly solid culture medium, which is manually inoculated to the surface, each person inoculates an average of 700-800 embryos per hour, which is inefficient. If 5,000 embryos are doubled every day, it will take more than 6 hours to complete, which is difficult to meet the needs of large-scale breeding. Summary of the Invention

[0004] In order to solve the technical problems raised in the above-mentioned background technology, the present invention provides a method for doubling haploid immature embryos in tissue culture of maize. The method can optimize and formulate a liquid culture medium based on the commonly used solid doubling culture medium, and adopt the method of soaking the immature embryos to achieve chromosome doubling of the haploid immature embryos, shorten the inoculation time, reduce labor and instrument costs, and improve the processing throughput of the immature embryos to meet the needs of commercial breeding.

[0005] The present invention is implemented by the following technical solution: a method for doubling maize haploid immature embryos through tissue culture, comprising the following steps:

[0006] S1. Prepare liquid doubling medium by mixing MS salts, sucrose, NAA, calcium chloride, and dimethyl sulfoxide in ddH2O until dissolved.

[0007] The solution is sequentially subjected to pH adjustment, sterilization, and cooling treatments, and filtered colchicine is added and mixed;

[0008] The mixed solution was dispensed into corresponding culture dishes to form liquid doubling culture medium, and the culture dishes were kept in a light-shielding environment;

[0009] S2. Inoculation doubling: For each portion of liquid doubling medium, first peel out a corresponding number of maize haploid immature embryos and place them together on the same filter paper. Then, soak the filter paper in the liquid doubling medium prepared in S1 to double the maize haploid immature embryos. Record the time and amount of medium required for inoculation of the immature embryos.

[0010] Based on the above content, in order to further improve the above scheme, in this scheme, in step S1: the content of MS salt is 3.46 g / L-3.66 g / L, the content of sucrose is 28 g / L-33 g / L, the content of NAA is 0.07 mg / L-0.13 mg / L, the content of calcium chloride is 0.45 g / L-0.53 g / L, the content of colchicine is 0.18 g / L-0.24 g / L, and the content of dimethyl sulfoxide is 18 ml-22 ml.

[0011] As a further improvement of the above scheme, in step S1, the content of MS salt is 3.56 g / L, the content of sucrose is 30 g / L, the content of NAA is 0.1 mg / L, the content of calcium chloride is 0.5 g / L, the content of colchicine is 0.2 g / L, and the content of dimethyl sulfoxide is 20 ml.

[0012] As a further improvement of the above scheme, in step S1, potassium hydroxide solution is added to the dissolved solution to adjust the Ph to 5.6-5.8, sterilized at 117°C-123°C for 14min-16min, and cooled to 38°C-43°C.

[0013] As a further improvement of the above scheme, in step S1, potassium hydroxide solution is added to the dissolved solution to adjust the pH to 5.7, sterilized at 121°C for 15 minutes, cooled to 40°C, and 20-40 ml of filtered colchicine is added and mixed.

[0014] As a further improvement of the above scheme, in step S1, a peristaltic pump is used to dispense the mixed liquid into corresponding culture dishes to form liquid doubling culture medium, ensuring that the mixed liquid content in each dish is 15 mL, and the culture dishes are kept in a light-shielding environment.

[0015] As a further improvement of the above solution, in step S2, the number of immature embryos placed on a single piece of filter paper is 190-220.

[0016] As a further improvement of the above solution, in step S2, the filter paper is immersed in the liquid doubling culture medium for 12-26 hours.

[0017] As a further improvement of the above solution, in step S2, the filter paper is immersed in the liquid doubling culture medium for 24 hours.

[0018] As a further improvement of the above scheme, it also includes: S3, seedling culture, inoculating the doubly treated embryos into a seedling culture medium to culture seedlings, transplanting the seedlings into hole trays to slow down the growth, and planting them in the field when they grow to five leaves and one heart.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention first adds doubling reagents colchicine and dimethyl sulfoxide into a liquid growth medium to prepare a liquid doubling medium, then peels the immature embryos onto filter paper, clamps the filter paper with the immature embryos with tweezers, and places the filter paper into the liquid doubling medium for full immersion. This method can shorten the time for inoculating the immature embryos, and the inoculation of 5000 immature embryos can be completed in 1 minute, thereby reducing labor and instrument costs. In addition, the immature embryos can be more fully exposed to colchicine by adopting the immersion treatment method, thereby improving the doubling rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the structure of a commonly used solid doubling culture medium;

[0022] Figure 2 A schematic flow chart of the method for tissue culture doubling of maize haploid immature embryos provided by the present invention;

[0023] Figure 3 This is a schematic diagram of the state of peeling the haploid immature embryos onto filter paper during inoculation in the doubling method of the present invention;

[0024] Figure 4 This is a schematic diagram of the state of immersing the immature embryos and filter paper into the liquid doubling medium during inoculation in the doubling method of the present invention;

[0025] Figure 5 for Figure 2 A schematic cross-sectional view of a box and other structures capable of providing a light-shielding environment for the culture dish, provided after the liquid doubling culture medium is prepared in step S1;

[0026] Figure 6 for Figure 5 A schematic cross-sectional view of the middle box cover in a flipped-open state on the box body;

[0027] Figure 7 for Figure 6 A schematic diagram of the structure enlarged in the middle;

[0028] Figure 8 for Figure 6 Schematic diagram of the structure enlarged at point B.

[0029] Description of main symbols:

[0030] 1. Box body; 2. Box cover; 3. Carrier plate; 4. Culture dish slot; 5. Worm; 6. Worm wheel; 7. Drive shaft; 8. First sprocket; 9. Second sprocket; 10. Cylinder body; 11. Screw; 12. Limit rod; 13. Limit slot; 14. First gear; 15. First rack; 16. Support arm; 17. Support plate; 18. Second gear; 19. Rocker arm; 20. Clamp; 100. Culture dish. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0032] Example 1

[0033] Please combine Figures 2 to 4 The method for doubling maize haploid embryos through tissue culture comprises the following steps:

[0034] S1. Prepare liquid doubling medium

[0035] S11. Take 3.46 g / L of MS salt, 28 g / L of sucrose, 0.07 mg / L of NAA, 0.45 g / L of calcium chloride, and 18 ml of dimethyl sulfoxide and mix them in DDH2O until dissolved.

[0036] In S11:

[0037] NAA refers to naphthaleneacetic acid, a plant growth regulator. NAA can be used to promote plant growth, root development, and fruit ripening. It is widely used as a plant growth regulator in horticulture and agriculture. The preparation method of NAA in this example is as follows: 0.07g of NAA is pre-dissolved in 0.1M potassium hydroxide (take 0.56g potassium hydroxide and dissolve it in 100mL of DD H2O), the volume is adjusted to 100mL with DDH2O, and stored at 4°C in the dark.

[0038] S12. Add 0.1 M potassium hydroxide solution to the dissolved solution to adjust the pH to 5.6, then sterilize at 117°C for 14 minutes, cool to 38°C, add 0.18 g / L filtered colchicine, and mix thoroughly.

[0039] In S12: The colchicine preparation method is as follows: take 1 g of colchicine, add 100 mL of DD H2O to dissolve, filter and sterilize with a 0.22 μm filter, and store at 4°C in the dark.

[0040] S13. Place each culture dish on a sterile operating table and use a peristaltic pump to dispense the mixed solution into the corresponding culture dishes to form liquid doubling culture medium, ensuring that the mixed solution content in each dish is 15 mL. Keep the culture dishes in a light-shielded environment until ready for use.

[0041] The specification of the culture dish used in this embodiment is 90*25 mm, and the diameter of the filter paper is also 90 mm.

[0042] S2. Inoculation doubling: For each portion of liquid doubling medium, first peel off about 200 maize haploid immature embryos and place them together on the same filter paper. Then soak the filter paper in the liquid doubling medium prepared in S1 for 12 hours to double the maize haploid immature embryos. Record the time and amount of medium required for inoculation of the immature embryos.

[0043] S3. Seedling culture: inoculate the doubly treated embryos into the seedling culture medium to culture seedlings. After 7 days of seedling formation, transplant them into a plug tray filled with peat to slow down the growth of the seedlings. When they grow to five leaves and one heart, they are transplanted into the field.

[0044] Example 2

[0045] Please combine Figures 2 to 4 The method for doubling maize haploid immature embryos through tissue culture in Example 2 comprises the following steps:

[0046] S1. Prepare liquid doubling medium

[0047] S11. Take 3.66 g / L of MS salt, 33 g / L of sucrose, 0.13 mg / L of NAA, 0.53 g / L of calcium chloride, and 22 ml of dimethyl sulfoxide and mix them in DDH2O until dissolved.

[0048] In S11:

[0049] The NAA was prepared by pre-dissolving 0.13 g of NAA in 0.1 M potassium hydroxide (0.56 g of potassium hydroxide was dissolved in 100 mL of DD H2O), adjusting the volume to 100 mL with DD H2O, and storing at 4°C in the dark.

[0050] S12. Add 0.1 M potassium hydroxide solution to the dissolved solution to adjust the pH to 5.8, then sterilize at 123°C for 16 minutes, cool to 43°C, add 0.24 g / L filtered colchicine, and mix thoroughly.

[0051] In S12: The colchicine preparation method is as follows: take 1 g of colchicine, add 100 mL of DD H2O to dissolve, filter and sterilize with a 0.22 μm filter, and store at 4°C in the dark.

[0052] S13. Place each culture dish on a sterile operating table and use a peristaltic pump to dispense the mixed solution into the corresponding culture dishes to form liquid doubling culture medium, ensuring that the mixed solution content in each dish is 15 mL. Keep the culture dishes in a light-shielded environment until ready for use.

[0053] The specification of the culture dish used in this embodiment is 90*25 mm, and the diameter of the filter paper is also 90 mm.

[0054] S2. Inoculation doubling: For each portion of liquid doubling medium, first peel out about 220 maize haploid immature embryos and place them together on the same filter paper. Then soak the filter paper in the liquid doubling medium prepared in S1 for 26 hours to double the maize haploid immature embryos. Record the time and amount of medium required for inoculation of the immature embryos.

[0055] S3. Seedling culture: inoculate the doubly treated embryos into the seedling culture medium to culture seedlings. After 7 days of seedling formation, transplant them into a plug tray filled with peat to slow down the growth of the seedlings. When they grow to five leaves and one heart, they are transplanted into the field.

[0056] Example 3

[0057] Please combine Figures 2 to 4 The method for doubling the maize haploid immature embryos through tissue culture in Example 3 comprises the following steps:

[0058] S1. Prepare liquid doubling medium

[0059] S11. Take 3.56 g / L of MS salt, 30 g / L of sucrose, 0.1 mg / L of NAA, 0.5 g / L of calcium chloride, and 20 ml of dimethyl sulfoxide and mix them in DDH2O until dissolved.

[0060] In S11:

[0061] The NAA was prepared by pre-dissolving 0.1 g of NAA in 0.1 M potassium hydroxide (0.56 g of potassium hydroxide was dissolved in 100 mL of DD H2O), adjusting the volume to 100 mL with DD H2O, and storing at 4°C in the dark.

[0062] S12. Add 0.1 M potassium hydroxide solution to the dissolved solution to adjust the pH to 5.7, then sterilize at 121°C for 15 minutes, cool to 40°C, add 0.2 g / L filtered colchicine, and mix thoroughly.

[0063] In S12: The colchicine preparation method is as follows: take 1 g of colchicine, add 100 mL of DD H2O to dissolve, filter and sterilize with a 0.22 μm filter, and store at 4°C in the dark.

[0064] S13. Place each culture dish on a sterile operating table and use a peristaltic pump to dispense the mixed solution into the corresponding culture dishes to form liquid doubling culture medium, ensuring that the mixed solution content in each dish is 15 mL. Keep the culture dishes in a light-shielded environment until ready for use.

[0065] The specification of the culture dish used in this embodiment is 90*25 mm, and the diameter of the filter paper is also 90 mm.

[0066] S2. Inoculation doubling: For each portion of liquid doubling medium, first peel off about 200 maize haploid immature embryos and place them together on the same filter paper. Then soak the filter paper in the liquid doubling medium prepared in S1 for 24 hours to double the maize haploid immature embryos. Record the time and amount of medium required for inoculation of the immature embryos.

[0067] S3. Seedling culture: inoculate the doubly treated embryos into the seedling culture medium to culture seedlings. After 7 days of seedling formation, transplant them into a plug tray filled with peat to slow down the growth of the seedlings. When they grow to five leaves and one heart, they are transplanted into the field.

[0068] Example 4

[0069] This example is a comparative example of Example 3, specifically to explore the effects of the solid doubling medium and the liquid doubling medium of Example 3 on the doubling flux and doubling effect of maize haploid immature embryos.

[0070] 1. Obtaining maize haploid embryos

[0071] In 2020, at the Hainan breeding base, the induced line CAU5 was used as the male parent and Zhengdan 958 as the female parent. The female parent was sown on September 30, and the male parent was planted in two phases, on October 5 and October 10, respectively, to allow the two parental lines to flower at the same time. The female parent was emasculated, and the pollination time was recorded. Sixteen days after pollination, the ears were removed and the embryos were peeled.

[0072] 2. Screening and doubling of maize haploid embryos using solid and liquid doubling media

[0073] The solid doubling medium contained 3.56 g / L of MS salts, 30 g / L of sucrose, 0.1 mg / L of NAA, 0.5 g / L of calcium chloride, 0.2 g / L of colchicine, 2% of dimethyl sulfoxide, and 7 g of agar.

[0074] The liquid doubling medium contained 3.56 g / L MS salts, 30 g / L sucrose, 0.1 mg / L NAA, 0.5 g / L calcium chloride, 0.2 g / L colchicine, and 2% dimethyl sulfoxide.

[0075] The exfoliated embryos were inoculated into solid and liquid doubling medium, respectively. The time and amount of medium required to inoculate 5,000 embryos were recorded (see Table 1 below, which is a comparison table of the time and amount of medium required to inoculate 5,000 embryos into solid and liquid doubling medium, respectively). The cells were treated for 24 hours under full illumination, a temperature of 24° C., and a humidity of 65%.

[0076] Treatment Vaccination time Culture medium dosage Solid doubling medium 6-7h 1000mL Liquid doubling medium 1-2 minutes 160mL

[0077] Table 1

[0078] Haploid embryos were selected based on the color of their scutellum. Those with purple-red scutellum were heterozygous diploids, and those with colorless scutellum were haploids. They were inoculated into seedling culture medium for cultivation. After 7 days, they were transplanted into hole trays filled with peat to slow down the growth of the seedlings. When they grew to five leaves and one heart, they were planted in the field.

[0079] 3. Effects of solid and liquid doubling media on the doubling effect of maize haploid immature embryos

[0080] After about 45-50 days of field growth, the pollen shedding rate and pollination rate of the haploid plants were counted, as shown in Table 2.

[0081]

[0082] Table 2 is a comparison table of the effects of solid and liquid doubling medium on haploid embryo doubling

[0083] As shown in Tables 1-2 above, there was little difference in pollen shedding and pollination rates between solid and liquid doubling medium for haploid embryos after doubling. Liquid doubling medium achieved slightly higher haploid plant doubling efficiency than solid doubling medium. However, liquid doubling medium offered significant advantages in inoculation time and medium dosage. Inoculation of 5,000 embryos took only 1-2 minutes, shortening inoculation time and increasing the throughput of doubling embryos to meet the needs of commercial breeding.

[0084] Example 5

[0085] Please combine Figures 5 to 8 This embodiment is a preferred embodiment of Embodiment 3 and specifically provides a light-shielding environment for the prepared liquid doubling culture medium prior to inoculation to maintain its activity. Specifically, a box 1 is provided on a sterile operating table to temporarily shield the culture dish 100 from light. The box 1 has an opening at the top, and a reversible lid 2 is provided at the opening to seal it. A carrier plate 3 for holding the culture dish 100 is provided within the box 1. The carrier plate 3 is provided with a dish placement slot 4 adapted to accommodate the culture dish 100.

[0086] The box body 1 is provided with an action mechanism. When the box cover 2 is flipped open, the action mechanism can drive the carrier plate 3 to move toward the top opening of the box body 1 so that the culture dish 100 can be taken and placed.

[0087] The action mechanism includes a worm 5, which is fixed on one side of the box cover 2, and its two ends protrude from the side of the box cover 2 respectively, and is rotatably inserted into the two ends of the inner wall of the box body 1. The box cover 2 can be flipped relative to the top side of the box body 1 with the worm 5 as the axis to realize the flipping opening and closing of the box cover 2 at the opening of the box body 1.

[0088] A transmission shaft 7 is rotatably inserted on the wall of the box body 1. The top of the transmission shaft 7 cooperates with the worm 5, and a first sprocket 8 is fixed to the bottom. A cylinder 10 is rotatably inserted on the inner bottom wall of the box body 1. The bottom of the cylinder 10 extends into the box wall of the box body 1 and is fixed with a second sprocket 9. The first sprocket 8 and the second sprocket 9 are connected by a chain drive. A screw 11 is threadedly inserted on the top of the cylinder 10. The top of the screw 11 is fixed to the bottom of the carrier plate 3. A limiting groove 13 is provided at the bottom of the carrier plate 3. A limiting rod 12 parallel to the screw 11 is slidingly provided in the limiting groove 13. The bottom of the limiting rod 12 is fixed to the inner bottom wall of the box body 1. The number of limiting rods 12 corresponds to the position and number of the dish grooves 4.

[0089] That is, when the box cover 2 is flipped open, the worm 5 drives the worm wheel 6, the transmission shaft 7, and the first sprocket 8 to rotate. The first sprocket 8 drives the second sprocket 9 and the barrel 10 to rotate through the chain, so that the barrel 10 and the screw 11 interact with each other in a threaded manner. Under the limiting action of the limiting rod 12 and the limiting groove 13, the screw 11 is axially extended from the barrel 10 to drive the carrier plate 3 to move toward the top opening of the box 1, so as to facilitate the placement and removal of the culture dish 100. Conversely, when the box cover 2 is flipped closed, the screw 11 can be indirectly retracted axially from the barrel 10 to drive the carrier plate 3 to move away from the top opening of the box 1, so as to better store and accommodate the culture dish 100.

[0090] Furthermore, in order to improve the stability of the carrier plate 3 in storing the culture dish 100, this embodiment provides a self-locking mechanism on the carrier plate 3. The self-locking mechanism is driven by the carrier plate 3 to move in a direction away from the top opening of the box body 1, and can lock and fix the culture dish 100 in the dish storage groove 4.

[0091] Specifically, the self-locking mechanism includes a second rack (not shown) provided on the limiting rod 12, a first gear 14 matched with the second rack is installed at the bottom of the carrier plate 3 through a hanging plate, and a first rack 15 matched with the first gear 14 is slidably inserted on the carrier plate 3. The first rack 15 and the second rack are parallel to each other and are respectively located on both sides of the first gear 14. The top of the first rack 15 extends into the carrier plate 3 and is fixed with a support arm 16. The top of the support arm 16 is fixed with a support plate 17 located in the dish placement groove 4, and the culture dish 100 is placed Placed on the support plate 17, third racks (not marked) are provided on both sides of the support arm 16, and two second gears 18 are provided in the carrier plate 3 relative to the support arm 16. The second gears 18 are respectively matched with the two third gears. The second gears 18 are coaxially fixed with rocker arms 19 that extend obliquely into the dish placement slot 4, and the two rocker arms 19 are located on both sides of the support plate 17. The two rocker arms 19 are connected to clamps 20 through springs, and a clamping space for clamping and fixing the two sides of the culture dish 100 is formed between the two clamps 20.

[0092] That is to say, when the box cover 2 is flipped and closed, the screw 11 is indirectly retracted axially from the barrel 10 to drive the carrier plate 3 to move in the direction away from the top opening of the box body 1. The limiting rod 12 can also drive the first gear 14 to rotate through the second rack thereon while limiting the position, so that the first gear 14 drives the first rack 15, the support arm 16, and the support plate 17 to move downward as a whole, which not only makes the culture dish 100 move further downward and accommodated in the dish receiving groove 4, but the downward movement of the support arm 16 can also drive the second gears 18 on both sides to rotate through the third rack, so that the two rocker rods 19 drive the two clamping plates 20 to approach each other, thereby achieving clamping and fixing of both sides of the culture dish 100, improving the stability of the carrier plate 3 in storing the culture dish 100, and avoiding accidental falling and damage. On the contrary, when the box cover 2 is flipped open, the screw 11 can be indirectly extended axially from the barrel 10 to drive the carrier plate 3 to move toward the direction close to the top opening of the box body 1, thereby not only allowing the culture dish 100 to extend from the dish placement slot 4, but also releasing the clamping state of the clamping plate 20 on the culture dish 100, so as to facilitate the removal and placement of the culture dish 100, which is convenient and reliable.

[0093] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for doubling maize haploid immature embryo tissue culture, characterized in that: The steps include: S1. Prepare liquid doubling medium, take MS salt, sucrose, NAA, calcium chloride, and dimethyl sulfoxide respectively, mix and stir in DDH2O until dissolved; The dissolved solution is sequentially subjected to pH acidity adjustment, sterilization treatment and cooling treatment, and the filtered colchicine is added and mixed; The mixed solution is dispensed into corresponding culture dishes to form liquid doubling medium, and the culture dishes are kept in a light-shielding environment; S2, inoculation doubling, for each portion of liquid doubling medium, first peel off a corresponding number of maize haploid immature embryos and place them together on the same filter paper, then soak the filter paper in the liquid doubling medium prepared in S1 to perform doubling treatment on the maize haploid immature embryos, and record the time required for inoculating the immature embryos and the amount of medium; In step S1, the culture medium is placed on a sterile operating table and then the mixed liquid is packaged. The sterile operating table is provided with a box body that can temporarily shield the culture dish from light. The top of the box body has an opening, and a box cover that can be turned over and closed at the opening of the box body is provided. A carrier plate for carrying the culture dish is provided in the box body, and a dish placement groove suitable for inserting the culture dish is provided on the carrier plate. An action mechanism is provided on the box body, and when the box cover is flipped open, the action mechanism can drive the carrier plate to move toward the top opening of the box body; The action mechanism includes a worm, which is fixed on one side of the box cover, and its two ends protrude from the side of the box cover and are rotatably plugged into the two ends of the inner wall of the box body. A transmission shaft is rotatably inserted on the box wall of the box body, the top of the transmission shaft cooperates with the worm, and a first sprocket is fixed on the bottom. A cylinder is rotatably inserted on the bottom wall of the box body, the bottom of the cylinder extends into the box wall of the box body and is fixed with a second sprocket, the first sprocket and the second sprocket are connected by a chain transmission, a screw is threadedly inserted on the top of the cylinder, the top of the screw is fixed to the bottom of the carrier plate, a limiting groove is provided at the bottom of the carrier plate, a limiting rod parallel to the screw is slidably provided in the limiting groove, the bottom of the limiting rod is fixed on the bottom wall of the box body, and the number of the limiting rods corresponds to the position and number of the setting grooves; When the box cover is flipped open, the worm gear drives the worm wheel, the transmission shaft and the first sprocket to rotate, and the first sprocket drives the second sprocket and the barrel to rotate through the chain, so that the barrel and the screw rod interact with each other, and under the limiting action of the limiting rod and the limiting groove, the screw rod extends axially from the barrel to drive the carrier plate to move toward the top opening of the box body; A self-locking mechanism is provided on the carrier plate, and the self-locking mechanism is driven by the carrier plate to move in a direction away from the top opening of the box body, so as to lock and fix the culture dish in the dish placement groove; The self-locking mechanism includes a second rack arranged on the limit rod, a first gear matched with the second rack is installed at the bottom of the carrier plate through a hanging plate, a first rack matched with the first gear is slidably inserted on the carrier plate, the first rack and the second rack are parallel to each other and are respectively located on both sides of the first gear, the top of the first rack extends into the carrier plate and is fixed with a support arm, a support plate located in the dish placement groove is fixed on the top of the support arm, the culture dish is placed on the support plate, third racks are arranged on both sides of the support arm, two second gears are arranged relatively with respect to the support arm in the carrier plate, the two second gears are respectively matched with the two third gears, swing rods obliquely extending into the dish placement groove are coaxially fixed on the two second gears, and the two swing rods are located on both sides of the support plate, and clamping plates are connected to the two swing rods through springs, and a clamping space for clamping and fixing the two sides of the culture dish is formed between the two clamping plates; When the box cover is flipped and closed, the screw rod is indirectly retracted axially from the barrel to drive the carrier plate to move in the direction away from the top opening of the box body, and the limiting rod drives the first gear to rotate through the second rack, so that the first gear drives the first rack, the support arm, and the support plate to move downward as a whole, so that the culture dish moves downward and is accommodated in the dish placement groove. At the same time, the downward movement of the support arm can also drive the second gears on both sides to rotate through the third rack, so that the two swing rods drive the two clamping plates to approach each other, so as to achieve clamping and fixing of the two sides of the culture dish; In step S1, the content of MS salt is 3.46g / L-3.66g / L, the content of sucrose is 28g / L-33g / L, the content of NAA is 0.07mg / L-0.13mg / L, the content of calcium chloride is 0.45g / L-0.53g / L, the content of colchicine is 0.18g / L-0.24g / L, and the content of dimethyl sulfoxide is 18ml-22ml.

2. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S1, the content of MS salt was 3.56 g / L, the content of sucrose was 30 g / L, the content of NAA was 0.1 mg / L, the content of calcium chloride was 0.5 g / L, the content of colchicine was 0.2 g / L, and the content of dimethyl sulfoxide was 20 ml.

3. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S1, potassium hydroxide solution is added to the dissolved solution to adjust the Ph to 5.6-5.8, sterilized at 117°C-123°C for 14min-16min, and cooled to 38°C-43°C.

4. The device for doubling the maize haploid immature embryo tissue culture as claimed in claim 3, characterized in that: In step S1, potassium hydroxide solution is added to the dissolved solution to adjust the Ph to 5.7, sterilized at 121° C. for 15 min, cooled to 40° C., and 20-40 ml of filtered colchicine is added and mixed.

5. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S1, a peristaltic pump is used to dispense the mixed solution into corresponding culture dishes to form a liquid doubling culture medium, ensuring that the content of the mixed solution in each dish is 15 mL, and the culture dishes are kept in a light-shielding environment.

6. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S2, the number of immature embryos placed on a single filter paper is 190-220.

7. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S2, the filter paper is immersed in the liquid doubling medium for 12-26 hours.

8. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: In step S2, the filter paper is immersed in the liquid doubling medium for 24 hours.

9. The device for doubling the maize haploid immature embryo tissue culture according to claim 1, characterized in that: Also includes: S3. Seedling culture: inoculate the doubly treated embryos into seedling culture medium to culture seedlings. After the seedlings are formed, transplant them into plug trays for acclimatization. When they grow to five leaves and one heart, they are planted in the field.

Images