Novel corn haploid immature embryo tissue culture doubling method

Abstract

The invention discloses a novel corn haploid immature embryo tissue culture doubling method, and belongs to the field of plant breeding. The technical problem to be solved by the invention is how to obtain a simpler corn haploid breeding method. The corn haploid immature embryo chromosome doubling and seedling forming method provided by the invention comprises the following steps: taking a corn inbred line as a female parent, taking a corn haploid induction line as a male parent, inducing to obtain a corn immature embryo, inoculating the corn immature embryo into a double-layer culture medium, and culturing to obtain the corn haploid immature embryo chromosome doubling and seedling forming method. Obtaining seedlings formed by chromosome doubling of the corn haploid immature embryos; the double-layer culture medium is composed of a seedling culture medium and a doubling culture medium located on the seedling culture medium. According to the method provided by the invention, chromosome doubling and seedling formation of the maize haploid immature embryos can be completed in one step, and the maize haploid immature embryos do not need to be transferred to a seedling formation culture medium after chromosome doubling, so that a large amount of manpower is saved, and the method is expected to be used in production practice.

Description

Technical Field

[0001] The present application belongs to the field of breeding and is a novel method for doubling haploid immature embryos of corn by tissue culture. Background Art

[0002] Corn is one of the most important food crops and cash crops in the world. Corn breeding is of great significance for ensuring national food security and promoting sustainable and healthy agricultural development. Corn hybrid breeding plays a key role in increasing yield, improving traits, and promoting germplasm innovation. The basis for utilizing corn hybrid vigor is to select and breed excellent corn inbred line materials. Traditional corn breeding generally requires 6 to 7 generations of continuous self-pollination to obtain nearly homozygous inbred lines. This process is time-consuming and labor-intensive, and the breeding cycle is long. However, through the method of corn haploid breeding, corn materials are induced to produce haploids and their chromosomes are doubled. In theory, only 2 generations are needed to obtain homozygous materials, which greatly improves the efficiency of corn material selection. DH breeding plays an important role in corn breeding and is increasingly valued and favored by breeding units at home and abroad.

[0003] DH breeding technology mainly includes three links: induction, identification and doubling of haploids. Inducing haploids means using pollen from the haploid induced line of corn to pollinate other corn materials, that is, the induced line is used as the male parent and other corn materials are used as the female parent to induce the production of maternal haploids. Identification means distinguishing diploids from haploids in induced corn kernels mainly by using methods such as color marking, morphological differences and oil content differences. The R1-nj color marker is currently the main color marker used to identify haploids. Corn kernels carrying R1-nj produce and accumulate anthocyanins during development. The induced line carrying R1-nj pollinates corn materials without color markings, and the resulting heterozygous diploid embryos carry the R1-nj marker, while the resulting haploid embryos do not contain the R1-nj marker. Therefore, haploid kernels without anthocyanin color can be identified by observing the scutellum of the embryo. Common methods for doubling maize haploid chromosomes include tissue culture doubling, seed soaking, root soaking, bud soaking, and growth point injection. Among them, the doubling efficiency of the tissue culture doubling method is high, and more fertile maize haploids can be obtained. At present, the tissue culture doubling method generally requires two steps, which require the use of a doubling medium and a seedling medium, respectively. First, the maize haploid embryos are placed on the doubling medium for doubling, and then the embryos are transferred to the seedling medium after doubling for a period of time. This process is labor-intensive, cumbersome to operate, time-consuming, and inefficient. Therefore, optimizing and improving the tissue culture doubling method is of great significance to improving the doubling efficiency of haploids and large-scale breeding. Summary of the invention

[0004] The technical problem to be solved by the present application is: how to obtain a simpler method for haploid breeding of corn.

[0005] In order to solve the above technical problems, the present application provides a method for chromosome doubling and seedling formation of haploid immature embryos of corn in haploid corn breeding, the method comprising the following steps: S1) Haploid induction: using a maize inbred line as the female parent and a maize haploid induced line as the male parent to induce maize immature embryos, wherein the maize immature embryos include maize haploid immature embryos and maize heterozygous diploid immature embryos; S2) inoculating the maize immature embryos into a double-layer culture medium for culturing to obtain maize seedlings formed by chromosome doubling of the maize haploid immature embryos; The double-layer culture medium consists of a seedling culture medium and a doubling culture medium located on the seedling culture medium; the doubling culture medium includes a 1 / 2MS culture medium and a doubling agent, and the seedling culture medium includes a 1 / 2MS culture medium; the inoculation includes the step of contacting the corn immature embryo with the doubling culture medium in the double-layer culture medium, but not with the seedling culture medium.

[0006] The corn inbred lines include but are not limited to: Zheng 58, Chang 7-2, Huang Zao 4, Jing 92, Qi 319 and the like.

[0007] The maize haploid induction lines include but are not limited to: haploid induction line CAU5, haploid induction line CAU6, Agricultural University High Induction No. 1, Jilin High Induction Line No. 3, Stock6, etc.

[0008] In some embodiments of the present application, the corn inbred line is Chang 7-2.

[0009] In some embodiments of the present application, the haploid induction line is the haploid induction line CAU5 and the haploid induction line CAU6.

[0010] Furthermore, the doubling medium may consist of 1 / 2MS medium and a doubling agent.

[0011] Furthermore, the seedling culture medium is 1 / 2MS culture medium.

[0012] Furthermore, the inoculation may be performed by inserting 1 / 3 to 1 / 2 of the corn immature embryo into the double-layer culture medium with the radicle facing downward and the plumule facing upward, so that the corn immature embryo contacts the added culture medium in the double-layer culture medium but does not contact the seedling culture medium.

[0013] The doubling agent includes a chemical doubling agent and an auxiliary agent. The chemical doubling agent includes but is not limited to colchicine, methyl amine glufosinate (APM), pronamide, dinitroaniline (trifluralin) and / or oryzalin. The auxiliary agent includes but is not limited to dimethyl sulfoxide (DMSO), Tween-80 and / or cytokinin, etc.

[0014] The use methods of the doubling agent include, but are not limited to: seed soaking method, bud soaking method, root soaking method, heart dripping method, injection method and / or tissue culture method.

[0015] Furthermore, in the method described, the solutes of the 1 / 2MS culture medium include MS salt, sucrose and a coagulant, the content of the MS salt is 2.37 g / L, the content of the sucrose is 30 g / L, and the solvent is water; the doubling agent includes colchicine and dimethyl sulfoxide.

[0016] Furthermore, in the method described above, the coagulant may specifically be plant gel or agar.

[0017] In some embodiments of the present application, the coagulant is agar, and the content of the agar may be 6.5 g / L.

[0018] In some embodiments of the present application, the doubling agent is composed of colchicine and dimethyl sulfoxide. In the doubling agent, the ratio of colchicine to dimethyl sulfoxide can be 0.05 g colchicine: 0.25 mL dimethyl sulfoxide.

[0019] Furthermore, in the method described above, in the doubling agent, the ratio of colchicine to dimethyl sulfoxide can be 0.05 g colchicine: 0.25 mL dimethyl sulfoxide.

[0020] Furthermore, in the method, in the doubling culture medium, the content of colchicine may be 0.05 g / L, and the content of dimethyl sulfoxide may be 0.25 mL / L.

[0021] Furthermore, a dye may be added to the doubling medium or the seedling medium, so as to distinguish the doubling medium and the seedling layer medium according to the color, and facilitate the young embryo inoculation operation.

[0022] Furthermore, dyes of different colors can be added to the doubling medium and the seedling medium, respectively, so as to distinguish the doubling medium from the seedling medium and facilitate the young embryo inoculation operation.

[0023] Furthermore, no dye may be added to the doubling medium and the seedling medium. Both the doubling medium and the seedling medium are transparent, and stratification can be observed by naked eyes after solidification.

[0024] Furthermore, in the method, the height of the doubled culture medium in the culture container may be 4 to 6 mm.

[0025] Furthermore, in the method, the volume ratio of the seedling medium to the doubling medium may be 5:1 to 5:2.

[0026] Furthermore, in the method, the height of the doubled culture medium in the culture container may be 5 mm.

[0027] Furthermore, in the method described above, the volume ratio of the seedling culture medium to the doubling culture medium may be 10:3.

[0028] Furthermore, in the method described above, the culture conditions in step S2) may be: a light cycle of 16 hours of light and 8 hours of darkness, a temperature of 26° C. to 28° C., and a humidity of 30%.

[0029] Furthermore, the culturing time in step S2) may be 7 to 10 days from the time of inoculation into the double-layer culture medium.

[0030] Furthermore, in the method described above, S2) also includes the step of screening seedlings formed after chromosome doubling of haploid corn immature embryos.

[0031] In some embodiments of the present application, the haploid induction line has an R1-nj marker. The method for distinguishing haploids from diploids is: inoculating maize immature embryos into a double-layer culture medium, and after the seedlings are formed, distinguishing the immature embryos according to the color of the immature embryos, and the seedlings with colorless embryo scutellums are haploids, and the seedlings with color in the embryo scutellums are diploids.

[0032] In some embodiments of the present application, because diploid seedling result data is required, the maize haploid immature embryos are not identified in advance. Instead, the maize immature embryos are inoculated, and after seedlings are formed, the haploid and heterozygous diploid are distinguished by the color of the embryo shield.

[0033] The present application also provides the double-layer culture medium.

[0034] The present application also provides the use of the double-layer culture medium in any of the following: A1), application of the culture medium in doubling maize haploid immature embryos; A2), application of the culture medium in haploid breeding of corn; A3) Application of the culture medium in plant breeding.

[0035] In the present application, the MS salt is also referred to as MS medium (without sucrose and agar). In some embodiments of the present application, the composition and proportion of the MS salt in the 1 / 2 MS medium are shown in Table 1.

[0036] Table 1. Composition of MS salts in 1 / 2 MS medium and their content in 1 / 2 MS medium (in mg / L)

[0037] After haploid induction, the easy and rapid screening of haploid grains is one of the important links in haploid breeding engineering. Currently, there are many methods for haploid identification, and the commonly used methods include: plant morphology, cytogenetics, genetic marker identification and other methods.

[0038] (1) Identification of haploids using plant morphology The most basic method for identifying haploids is morphological identification. Since haploids contain only one set of chromosomes, they are characterized by smaller cell volumes, smaller leaf areas, smaller flower stems, smaller seeds and other nutritional organs than diploids, and shorter plants. However, this method takes a long time to identify haploids, and the growth of plants is easily affected by the environment, resulting in identification errors. In addition, during the identification of haploid crops, they need to be planted in the field, which can easily lead to a waste of arable land.

[0039] In addition, there is a method for identifying immature embryos by morphology. After pollination of the induced line, young ears are taken for embryo peeling to obtain corn immature embryos; the corn immature embryos are sorted in order of length, width or area from small to large, and the top 20 to 30% of the immature embryos are selected to obtain haploid corn immature embryos. A large part of the heterozygous diploid immature embryos are eliminated before the color of the immature embryos is expressed, which advances the time for haploid selection, improves the efficiency of selection, and advances the time for haploid selection. However, this method has a certain probability of misselection and omission, and requires certain identification and selection experience to be applicable to large-scale haploid screening.

[0040] (2) Combining cytogenetics to identify haploids: Observing the root tip cells or meiotic cells of plants by chromosome pressing is the most intuitive method to identify haploids. By observing whether the number of chromosomes is equal to the number of gametes, it can be determined whether the plant is haploid. However, the experimental steps involved in this method are cumbersome and inefficient, and it is not suitable for engineering screening and identification.

[0041] (3) Using genetic markers to identify haploids (3-1) R1-nj marking At present, most of the haploid genetic markers used by major breeding companies and research institutes are based on the genetic marker method of the R1-nj gene. This gene can synthesize anthocyanins in the aleurone layer of the corn endosperm and the scutellum of the embryo, and a distinct purple color can be observed with the naked eye. Using the haploid induction line containing this genetic marker, the haploid kernels obtained show that the embryo is not colored, and the endosperm is partially colored, so it is easy to screen out from the corn kernels.

[0042] (3-2) MAGIC system The MAGIC system is a new haploid identification system recently developed by the Chen Shaojiang team and the Chen Rumei team using optimized anthocyanin markers. Compared with the R1-nj marker, MAGIC1 has an accuracy rate of 99.1% in identifying haploids, and can identify haploids 12 days after pollination, making it more suitable for large-scale haploid screening.

[0043] (3-3) High oil content marker The oil content of heterozygous diploid and haploid grains obtained by hybridizing high-oil induction lines with ordinary corn is very different. Therefore, using nuclear magnetic resonance instruments or near-infrared instruments to detect haploid grains can greatly shorten the detection time compared to manual identification (Li et al., 2009). Further improving the induction rate and oil content of oil-type induction lines can not only improve the haploid induction efficiency, but also improve the efficiency and accuracy of identification, thereby realizing engineered haploid breeding.

[0044] (3-4) Fluorescent protein labeling After the transgenic plants containing fluorescent markers (YFP, GFP, etc.) are hybridized with the created haploid induction lines, the haploid induction lines containing stable fluorescent markers are obtained. The hybrids obtained from this induction line can be easily identified and screened for haploid grains with the help of fluorescence observation tools such as fluorescent stereo microscopes and laser confocal microscopes.

[0045] (3-5) Betalain synthesis system RUBY color labeling The betalain synthesis system RUBY was introduced into the maize haploid induction line to obtain the RUBY induction line. The induced immature embryos can show color 10 days after pollination. The ones that show color are heterozygous diploids, and the ones that do not show color are haploids. During the in vitro culture of immature embryos, haploid immature embryos can be identified by naked eye observation. Compared with the R1-nj color marker, the RUBY color marker shows color earlier and is easier to identify haploids, which is conducive to its application in tissue culture doubling.

[0046] The haploid induction lines CAU5 and CAU6 described in this application carry R1-nj mark.

[0047] The beneficial technical effects of this application are as follows: The method provided in the present application can complete the chromosome doubling and seedling formation of corn haploid immature embryos in one step, without the need to transfer to a seedling formation medium after chromosome doubling, thus saving a lot of manpower and being expected to be used in production practice.

[0048] In addition, the colchicine concentration used in the one-step doubling method is lower, which is beneficial to the health of operators and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the color development of maize embryos after being cultured on doubling medium for 24 hours. The R1-nj color marker is used to identify maize embryos. The embryos with purple-red scutellum are diploid, and those without purple-red are haploid.

[0050] Figure 2 To insert the maize haploid immature embryos upright into the seedling culture medium.

[0051] Figure 3 To prepare the completed double-layer culture medium, the upper layer is colored for easy observation.

[0052] Figure 4 This is a double-layer culture medium without added dye. It can be seen in the figure that there is a clear stratification between the upper culture medium (doubling culture medium) and the lower culture medium (seedling culture medium).

[0053] Figure 5 This is a schematic diagram of the situation in which the maize embryo is inoculated into the upper culture medium (doubling medium). The root of the maize embryo is facing downward, and 1 / 3 to 1 / 2 of the embryo is inserted upright into the upper culture medium without touching the lower culture medium (seedling medium).

[0054] Figure 6 This is the rooting condition of corn immature embryos after being cultured in a double-layer culture medium for a period of time. The radicle of the immature embryo grows and extends into the lower culture medium (seedling medium).

[0055] Figure 7 This is a schematic diagram of the growth of corn embryos after being cultured in a double-layer culture medium for a period of time. DETAILED DESCRIPTION

[0056] In corn DH breeding, tissue culture doubling is a commonly used method for doubling corn haploids. The current basic doubling method is a two-step doubling method, that is, first peeling the corn immature embryo from the corn, placing it in a doubling medium for doubling treatment, then identifying the haploid by color, and then transferring the corn haploid immature embryo to a seedling medium and culturing it to seedlings. This method is implemented as a control group of the present application. There is an obvious problem with the two-step doubling method: the transfer medium requires a lot of labor and time, so the present application proposes a one-step doubling method, specifically using a double-layer culture medium, without the need to transfer the culture medium, to complete the doubling and seedling of the corn immature embryo.

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] The corn in the following implementation case ( Zea mays L.) haploid doubling material Chang 7-2, which is a public material and has been disclosed in the document "Hua Fuping, Shen Weimin, Zhang Yi, et al. Characteristics and utilization of excellent corn inbred line Chang 7-2 [J]. Henan Agricultural Science, 2004, (09): 11-13." The public can obtain it from the applicant. This material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0060] The corn ( Zea mays L.) Haploid induction lines CAU5 and CAU6, both of which have the R1-nj marker, were released from the project of Professor Chen Shaojiang of China Agricultural University and were disclosed in the document "Jiao Yanyan. Evaluation of maize haploid induction lines and contemporary genetic effect research. [D]. China Agricultural University, 2017." The public can obtain this material from the applicant. This material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0061] In the following examples, the MS salt is a Solebol product with the product number M8521, also known as MS medium (without agar and sucrose).

[0062] In the following examples, the sucrose is Solebao product, item number S8271; In the following examples, the dimethyl sulfoxide is a product of Solebao, product number D8370; In the following examples, the agar is a Coolabo product, item number CA1331.

[0063] In the following embodiments, the double-layer culture medium (solid form) includes a doubling culture medium (located in the upper layer) and a seedling culture medium (located in the lower layer), the doubling culture medium includes a 1 / 2MS culture medium and a doubling agent, and the seedling culture medium includes a 1 / 2MS culture medium. The formula of the 1 / 2MS culture medium is: MS salt 2.37g / L, sucrose 30g / L, agar (as a coagulant) 6.5g / L, and the rest is water, and the pH is 5.8. The components of the maize haploid doubling agent include colchicine and dimethyl sulfoxide, wherein in the two-step method, the content of the doubling agent in the doubling culture medium is: colchicine 0.1g / L, dimethyl sulfoxide 20mL / L; in the one-step method, the content of the doubling agent in the doubling culture medium is: colchicine 0.05g / L, dimethyl sulfoxide 0.25mL / L.

[0064] In this embodiment, the culture medium components of each treatment group are as follows: Control group: The control group was a two-step doubling method. In the first step, the maize immature embryos were inoculated on the doubling medium for doubling treatment. In the second step, the maize haploid immature embryos were transferred to the seedling medium.

[0065] Treatment group: The treatment group was a one-step doubling method, using a double-layer culture medium, and the maize immature embryos were upright inoculated in the doubling medium of the double-layer culture medium, and the maize immature embryos were not in contact with the seedling culture medium during inoculation.

[0066] In the following embodiments, the specification of the culture bottle (the bottle body is made of glass) is 240 mL, with a diameter of 62 mm and a height of 90 mm.

[0067] In the present application, the composition and proportion of MS salts in 1 / 2 MS medium are shown in Table 1.

[0068] Table 1. Composition of MS salts in 1 / 2 MS medium and their content in 1 / 2 MS medium (in mg / L)

[0069] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test, and P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) indicated a very significant difference.

[0070] Example 1: Two-step method for doubling maize embryo tissue culture 1.1. Inducing the production of maize haploid embryos Chang 7-2 was used as the female material, and the induced lines CAU5 and CAU6 were used as the male material. The materials were sown in Beijing in May 2024. The male material was sown at different times to facilitate the flowering period of the parent materials. In July, the female material was bagged and the filaments were cut. The male induced line material was over-pollinated, and the pollinated male parent and pollination date were recorded at the same time. The female ear was harvested 16 days after pollination. At this time, the size of the young embryo was about 5mm. The young embryo was peeled from the ear by aseptic operation for in vitro culture of the young embryo.

[0071] 1.2. Preparation of doubled medium for two-step tissue culture The medium for doubling tissue culture in two-step method is divided into doubling medium and seedling medium. The composition of doubling medium is as follows: the content of MS salt is 2.37g / L, the content of sucrose is 30g / L, the content of agar is 6.5g / L, the content of dimethyl sulfoxide is 20mL / L, the content of colchicine is 0.1g / L, and the rest is water, and the pH value is 5.8. The composition of seedling medium is as follows: the content of MS salt is 2.37g / L, the content of sucrose is 30g / L, the content of agar is 6.5g / L, and the rest is water, and the pH value is 5.8.

[0072] 1.3. Inoculation doubling and transfer culture medium The maize embryos were peeled out and laid flat on the doubling medium, and the date and time were recorded. The maize embryos were doubling treated for 24 hours. The paternal induction line materials all had R1-nj markers. After 24 hours of light culture, the scutellum of the maize embryos of the heterozygous diploids would show obvious purple-red color, while the scutellum of the maize embryos of the haploids would not show purple-red color. Figure 1 As shown in the figure, the maize embryo scutellum without purple-red mark is haploid. Therefore, the maize haploid embryos are identified and selected according to the color of the maize embryo scutellum, and the haploid embryos are transferred to the seedling medium with the radicle part facing downward. The 1 / 3 to 1 / 2 part of the embryo is inserted upright into the seedling medium of the culture bottle, and 6 to 8 embryos are inoculated in each culture bottle. Figure 2 The culture conditions were as follows: a photoperiod of 16 h light (14,000 lux) and 8 h dark, a temperature of 26°C to 28°C, and a humidity of 30%.

[0073] 1.4. Seedling cultivation and field transplanting The day when the young embryos were inoculated into the doubling medium was recorded as day 0 (i.e., culture day 0). After 7 to 10 days of culture, the number of seedlings was counted, transferred to plug trays, and hardened outdoors for about 10 to 14 days. When the seedlings grew to 3 to 5 leaves, they were transplanted to the field.

[0074] The criteria for judging the statistical range of seedlings after 7 to 10 days of cultivation are as follows: Seedlings: Seedlings grow to the state of two leaves and one heart or three leaves; No seedlings: The growth of the immature embryo stops, the immature embryo becomes vitrified or grows callus tissue, and the immature embryo only takes root but does not sprout.

[0075] Example 2: One-step method for doubling maize embryo tissue culture 2.1. Inducing the production of maize haploid embryos Chang 7-2 was used as the female material, and the induced lines CAU5 and CAU6 were used as the male material. The materials were sown in Beijing in May 2024. The male material was sown at different times to facilitate the flowering period of the parent materials. In July, the female material was bagged and the filaments were cut. The male induced line material was over-pollinated, and the pollinated male parent and pollination date were recorded at the same time. The female ear was harvested 16 days after pollination. At this time, the size of the young embryo was about 5mm. The young embryo was peeled from the ear by aseptic operation for in vitro culture of the young embryo.

[0076] 2.2. One-step preparation of double-layer culture medium The double-layer culture medium is composed of the upper doubling culture medium and the lower seedling culture medium. The ratio of the lower culture medium to the upper culture medium is 10:3, and the height of the upper culture medium is 5mm. In a 240mL culture bottle, the lower culture medium is 40mL and the upper culture medium is 12mL. Use a liquid dispensing pump to accurately add 40mL of the seedling culture medium to the culture bottle. After the culture medium cools naturally, use a liquid dispensing pump to accurately add 12mL of the doubling culture medium to the same bottle. After cooling, the upper and lower layers of the culture medium can be observed.

[0077] The upper culture medium is a doubling culture medium, which includes 1 / 2 MS culture medium and a doubling agent. The lower culture medium is a seedling culture medium, which includes 1 / 2 MS culture medium. Among them, the composition of 1 / 2 MS culture medium is: the content of MS salt is 2.37g / L, the content of sucrose is 30g / L, the content of agar is 6.5g / L, and the rest is water, and the pH value is adjusted to 5.8. The final concentration of the doubling agent in the doubling culture medium is 0.25mL / L of dimethyl sulfoxide and 0.05g / L of colchicine.

[0078] Depending on whether a stain is added, bilayer cultures can be divided into the following three types: (1) Adding dye to the doubling medium or seedling medium Add dye to the doubling medium or seedling medium, such as Figure 3 shown. Figure 3 The doubling medium in the double-layer culture medium shown contains Alexander staining solution, and the added amount is 0.1mL / L. Its function is to facilitate the distinction between the doubling medium and the seedling medium. In addition, the doubling medium is dyed to observe whether the components of the doubling medium will penetrate into the seedling medium. Experiments have shown that the seedling medium can only be observed to be stained by a small amount of dye after the medium is prepared and left to stand for about 2 months. Therefore, it is believed that during the use of the double-layer culture medium, the doubling agent in the doubling medium will not penetrate into the lower layer.

[0079] (2) Add different colored dyes to the doubling medium and seedling medium respectively Different colored dyes are added to the doubling medium and the seedling medium respectively, so that the doubling medium and the seedling medium can be distinguished conveniently by observing the colors.

[0080] (3) No dye is added to the doubling medium and seedling medium At the same time, no dye is added to the doubling medium and the seedling medium. Both the doubling medium and the seedling medium are transparent, and stratification can be observed by the naked eye after solidification ( Figure 4 ).

[0081] 2.3. Double inoculation treatment and seedling formation Remove the young embryo of corn with the radicle facing downward. Figure 5 As shown, 1 / 3 to 1 / 2 of the young corn embryo is inserted upright into the double-layer culture medium with the radicle facing downward and the plumule facing upward. The young corn embryo only contacts the doubling culture medium but not the seedling culture medium. 6 to 8 young corn embryos are inoculated in each culture bottle. The inoculation date is recorded and placed in a biochemical culture box (placed on a tissue culture rack). The culture conditions are: a light cycle of 16 hours of light (14,000 lux) and 8 hours of darkness, a temperature of 26°C to 28°C, and a humidity of 30%. After inoculation, the young embryo grows a radicle after 2 to 3 days of culture in the doubling culture medium. The radicle then grows downward and extends into the seedling culture medium, as shown in FIG. Figure 6 and Figure 7 As shown. Before the young embryo radicle extends into the seedling medium, it only absorbs nutrients from the doubling medium, and the doubling process is completed during this period. When the radicle enters the seedling medium, it mainly absorbs nutrients from the lower layer of the seedling medium. When the young embryo enters the seedling stage, the doubling medium has little effect on the seedling, so the doubling and seedling can be completed in one step.

[0082] The method for distinguishing haploids and diploids is to inoculate all the immature embryos of corn into a double-layer culture medium. After they have grown into seedlings, the immature embryos are identified based on their color. The seedlings with colorless embryo scutellum are haploids, and the seedlings with color in the embryo scutellum are diploids.

[0083] Since this experiment required diploid seedling result data, the maize haploid embryos were not identified in advance. Instead, the maize embryos were inoculated, and the color of the embryo shield was used to distinguish haploids and heterozygous diploids after the seedlings were formed. Generally speaking, in actual production, it is necessary to distinguish haploids from diploids.

[0084] 2.4. Seedling cultivation and field transplanting The day when the young embryo was inoculated into the double-layer culture medium was recorded as day 0. After 7 to 10 days of culture, the color mark of the embryo scutellum was also used to identify and select the maize haploid seedlings. The seedlings with purple-red embryo scutellum were heterozygous diploids, while those without purple-red were haploids. The number of haploid seedlings was counted, and the haploid seedlings were transferred to plug trays and hardened outdoors for 10 to 14 days. After the seedlings grew to 3 to 5 leaves, they were transplanted to the field.

[0085] During the process of the immature embryo growing into a seedling, the heterozygous diploid immature embryo will also accumulate anthocyanins and appear purple-red. It can be identified by observing the color of the embryo shield, and no additional identification or marking is required.

[0086] For seedling evaluation, the number of maize seedlings that emerged from two-step doubling and one-step doubling methods were counted. The statistical results are shown in Table 2. The calculation formula is: seedling rate = number of seedlings / number of inoculations.

[0087] The results showed that the seedling rate of maize haploids doubled by the two-step method was higher than that of the one-step method. This was because, under the same culture conditions, the maize haploid embryos grew slowly during the one-step doubling process and needed 3 days or more to grow radicles. The doubling treatment time was long, which had a greater impact on seedlings. However, the maize diploid embryos grew faster and only needed 2 days to grow radicles. They could extend into the lower layer earlier and subsequently enter the seedling growth stage. The doubling treatment time was short, and the stress of the doubling agent on the embryos was reduced, so the seedling rate was higher.

[0088] The criteria for judging the statistical range of seedlings after 7 to 10 days of cultivation are as follows: Seedlings: Seedlings grow to the state of two leaves and one heart or three leaves; Seedling failure: The growth of the immature embryo stagnates, the immature embryo becomes vitrified or grows callus tissue, the immature embryo only takes root but does not sprout, and the embryo swells into a ball and stops developing.

[0089] Table 2. Number and seedling rate of maize immature embryos treated with two doubling methods

[0090] Note: The sample size in the table refers to the number of culture bottles.

[0091] 2.5. Results and analysis of double-layer culture medium doubling treatment By observing the growth of young embryos during the one-step doubling process, it was found that it only took about 2 days for the diploid to grow from inoculation to radicle growth to seedling medium, and only about 7 to 10 days for subsequent seedlings. It takes 3 days for the haploid radicle to grow to the lower layer, and the subsequent rooting and plumule growth are slower, and it takes 12 to 14 days to seedling. The experimental results showed that the survival rate of haploids doubled by the two-step method was 95%, while the survival rate of haploids doubled by the one-step method was 63%, but the survival rate of diploids doubled in the one-step method was 96%. The reason for the analysis is that there are ploidy differences between haploids and diploids. At the same doubling agent concentration, haploids have weaker tolerance to doubling agents and are more susceptible to the stress of doubling agents, resulting in slow growth. After 3 days of doubling treatment, it also has a greater impact on the subsequent seedling growth. More young embryos cannot develop into seedlings, resulting in a lower seedling rate. For the two-step method, the doubling treatment time is about 24 hours. Although the concentration of the doubling agent is higher, it can still maintain a good seedling rate. Therefore, in the subsequent improvement of the one-step doubling method, controlling the doubling time and the concentration of the doubling agent will be the main content.

[0092] Example 3: Quantification of the ratio and dosage of the double-layer culture medium 3.1. Inducing haploid immature embryos in maize Chang 7-2 was used as the female material, and the induced lines CAU5 and CAU6 were used as the male material. The materials were sown in Beijing in August 2024. The male material was sown at different times to facilitate the flowering period of the parent materials. In October, the female material was bagged and the filaments were cut. The male induced line material was over-pollinated, and the pollinated male parent and pollination date were recorded at the same time. The female ear was harvested 20 days after pollination. At this time, the size of the young embryo was about 5mm. The young embryo was peeled from the ear by aseptic operation for in vitro culture of the young embryo.

[0093] 3.2. Preparation of double-layer culture medium with different heights of upper culture medium The double-layer culture medium consists of the doubling culture medium on the upper layer and the seedling culture medium on the lower layer. A series of double-layer culture media with different amounts of upper culture medium were set up. The following is the experimental treatment plan: Group 1: The amount of upper culture medium was 8 mL. In a 240 mL culture bottle, the height of the upper culture medium was measured to be approximately 4.0 mm.

[0094] Group 2: The amount of upper culture medium was 10 mL. In a 240 mL culture bottle, the height of the upper culture medium was measured to be approximately 4.5 mm.

[0095] Group 3: The amount of upper culture medium was 12 mL. In a 240 mL culture bottle, the height of the upper culture medium was measured to be approximately 5.0 mm.

[0096] Group 4: The amount of upper culture medium used was 14 mL. In a 240 mL culture bottle, the height of the upper culture medium was measured to be approximately 5.5 mm.

[0097] Group 5: The amount of upper culture medium was 16 mL. In a 240 mL culture bottle, the height of the upper culture medium was measured to be approximately 6.0 mm.

[0098] The amount of the lower culture medium is 40 mL. Use a liquid dispensing pump to accurately add 40 mL of seedling culture medium into the culture bottle. After the culture medium cools naturally, use a liquid dispensing pump to accurately add 8 mL, 10 mL, 12 mL, 14 mL and 16 mL of doubled culture medium respectively. After cooling, the upper and lower layers of the culture medium can be observed.

[0099] The upper culture medium is a doubling culture medium, which includes 1 / 2 MS culture medium and a doubling agent. The lower culture medium is a seedling culture medium, which includes 1 / 2 MS culture medium. Among them, the composition of 1 / 2 MS culture medium is: the content of MS salt is 2.37g / L, the content of sucrose is 30g / L, the content of agar is 6.5g / L, and the rest is water, and the pH value is adjusted to 5.8. The final concentration of the doubling agent in the doubling culture medium is 0.25mL / L of dimethyl sulfoxide and 0.05g / L of colchicine.

[0100] 3.3. Double inoculation treatment and seedling formation The young corn embryos were peeled out, with the radicle part facing downwards. 1 / 3 to 1 / 2 of the young corn embryos were inserted upright into the double-layer culture medium in the direction of the radicle downwards and the plumule upwards. The young corn embryos only contacted the upper culture medium but not the lower culture medium. 6 to 8 young corn embryos were inoculated in each culture bottle. The inoculation date was recorded and placed in a biochemical culture box (placed on a tissue culture rack). The culture conditions were: a light cycle of 16 hours of light (14,000 lux) and 8 hours of darkness, a temperature of 26°C to 28°C, and a humidity of 30%. After inoculation, the growth of the radicle of the young embryo in the upper culture medium was observed, and the elongation of the radicle of the young embryo was recorded every 12 hours, and the time when the radicle began to enter the lower culture medium was recorded.

[0101] Method for distinguishing haploid and diploid: Inoculate all maize immature embryos into a double-layer culture medium, and after culturing for about 24 hours, identify the immature embryos based on their coloration. Seedlings with colorless embryo scutellum are haploid, and seedlings with color in the embryo scutellum are diploid.

[0102] 3.4. Observation results of rooting after young embryo inoculation Table 3. Relationship between the time when the upper culture medium (doubling culture medium) at different heights and the radicle of the young embryos elongated into the lower culture medium (seedling culture medium)

[0103] According to the experimental results, the height of the upper culture medium (doubling culture medium) is in the range of 4 to 6 mm, the volume ratio of the lower culture medium (seedling culture medium) to the upper culture medium (doubling culture medium) is 5:1 to 5:2, and from the start of inoculation, it takes about 48 hours for the radicle of the diploid young embryo to enter the lower culture medium, and it takes about 72 hours for the radicle of the haploid young embryo to enter the lower culture medium. Whether it is a diploid young embryo or a haploid young embryo, there is no significant difference between the treatments with different upper culture medium heights. Therefore, in the double-layer culture medium, the height of the upper culture medium is in the range of 4 to 6 mm, which has little effect on the doubling treatment time of the young embryo. If the doubling treatment time needs to be adjusted, the height of the upper culture medium should be set lower or higher.

[0104] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for chromosome doubling and seedling formation of haploid immature embryos of corn in haploid corn breeding, characterized in that: The method comprises: S1) Haploid induction: using a maize inbred line as the female parent and a maize haploid induced line as the male parent to induce maize immature embryos, wherein the maize immature embryos include maize haploid immature embryos and maize heterozygous diploid immature embryos; S2) inoculating the maize immature embryos into a double-layer culture medium for culturing to obtain maize seedlings formed by chromosome doubling of the maize haploid immature embryos; The double-layer culture medium consists of a seedling culture medium and a doubling culture medium located on the seedling culture medium; the doubling culture medium includes a 1 / 2MS culture medium and a doubling agent, and the seedling culture medium includes a 1 / 2MS culture medium; the inoculation includes the step of contacting the corn immature embryo with the doubling culture medium in the double-layer culture medium, but not with the seedling culture medium.

2. The method according to claim 1, characterized in that The solutes of the 1 / 2MS culture medium include MS salt, sucrose and a coagulant, the content of the MS salt is 2.37 g / L, the content of the sucrose is 30 g / L, and the solvent is water; the doubling agent includes colchicine and dimethyl sulfoxide.

3. The method according to claim 2, characterized in that In the doubling agent, the ratio of colchicine to dimethyl sulfoxide can be 0.05 g colchicine: 0.25 mL dimethyl sulfoxide.

4. The method according to any one of claims 1 to 3, characterized in that In the doubling culture medium, the content of colchicine is 0.05 g / L, and the content of dimethyl sulfoxide is 0.25 mL / L.

5. The method according to any one of claims 1 to 4, characterized in that The height of the doubling culture medium in the culture container is 4-6 mm.

6. The method according to any one of claims 1 to 5, characterized in that The volume ratio of the seedling culture medium to the doubling culture medium is 5:1 to 5:

2.

7. The method according to any one of claims 1 to 6, characterized in that: S2) The culture conditions in the step are: a light cycle of 16 hours of light and 8 hours of darkness, a temperature of 26° C. to 28° C., and a humidity of 30%.

8. The method according to any one of claims 1 to 7, characterized in that: S2) also includes the step of screening seedlings formed after chromosome doubling of haploid maize immature embryos.

9. The double-layer culture medium according to any one of claims 1 to 8.

10. The use of the double-layer culture medium according to claim 9, wherein the use is selected from any one of the following: A1), application of the culture medium in doubling maize haploid immature embryos; A2), application of the culture medium in haploid breeding of corn; A3) Application of the culture medium in plant breeding.

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