A new method for doubling of maize haploid young embryo by tissue culture

Abstract

The application discloses a new type of maize haploid young embryo tissue culture doubling method and belongs to the field of plant breeding. The technical problem to be solved by the application is how to obtain a more simple maize haploid breeding method. To this end, the application provides a maize haploid young embryo chromosome doubling and seedling forming method, the method comprises the following steps: taking a maize inbred line as a female parent, taking a maize haploid induction line as a male parent to induce a maize young embryo, inoculating the maize young embryo into a double-layer culture medium for culture, and obtaining seedlings formed by chromosome doubling of the maize haploid young embryo; the double-layer culture medium is composed of a seedling forming culture medium and a doubling culture medium located on the seedling forming culture medium. The method provided by the application can complete chromosome doubling and seedling forming of the maize haploid young embryo in one step, and the maize young embryo does not need to be transferred to a seedling forming 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 maize haploid immature embryos through tissue culture. Background Art

[0002] Maize is one of the world's most important food and cash crops. Maize breeding is crucial for ensuring national food security and promoting sustainable and healthy agricultural development. Maize hybrid breeding plays a key role in increasing yield, improving traits, and promoting germplasm innovation. The fundamental basis for harnessing heterosis in maize is the selection and breeding of superior inbred lines. Traditional maize breeding typically requires six to seven generations of continuous selfing to obtain nearly homozygous inbred lines, a time-consuming and labor-intensive process with a long breeding cycle. However, haploid doubling (DH) breeding, which induces haploid production in maize material and doubles its chromosomes, theoretically requires only two generations to obtain homozygous material, greatly improving the efficiency of maize breeding. DH breeding plays a crucial role in maize breeding and is gaining increasing attention and favor among breeders both domestically and internationally.

[0003] DH breeding technology mainly involves three steps: haploid induction, identification, and doubling. Inducing haploids refers to using pollen from a maize haploid induction line to pollinate other maize materials, that is, using the induction line as the male parent and other maize materials as the female parent to induce the production of maternal haploids. Identification refers to distinguishing diploids from haploids in induced maize kernels 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. Maize kernels carrying R1-nj produce and accumulate anthocyanins during development. When an induction line carrying R1-nj is pollinated with a maize material without a color marker, 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. The commonly used methods for doubling maize haploid chromosomes include tissue culture doubling, seed soaking, root soaking, bud soaking, and growing point injection. Among them, the doubling efficiency of the tissue culture doubling method is higher, and more fertile maize haploids can be obtained. At present, the tissue culture doubling method generally requires two steps, namely, doubling medium and seedling medium. 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, complicated to operate, time-consuming, and inefficient. Therefore, optimizing and improving the tissue culture doubling method is of great significance to improving haploid doubling efficiency and large-scale breeding. Summary of the Invention

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

[0005] 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 breeding of corn, the method comprising the following steps:

[0006] S1) Haploid induction: using a maize inbred line as the female parent and a maize haploid induction 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;

[0007] 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;

[0008] 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 embryo with the doubling culture medium in the double-layer culture medium but not with the seedling culture medium.

[0009] The corn inbred lines include but are not limited to: Zheng 58, Chang 7-2, Huang Zao Si, Jing 92, Qi 319 and the like.

[0010] 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.

[0011] In some embodiments of the present application, the corn inbred line is Chang 7-2.

[0012] In some embodiments of the present application, the haploid induction line is the haploid induction line CAU5 and the haploid induction line CAU6.

[0013] Furthermore, the doubling medium may consist of 1 / 2 MS medium and a doubling agent.

[0014] Furthermore, the seedling culture medium is 1 / 2MS culture medium.

[0015] Furthermore, the inoculation can be performed by inserting 1 / 3 to 1 / 2 of the corn embryo into the double-layer culture medium with the radicle facing downward and the plumule facing upward, so that the corn embryo contacts the added culture medium in the double-layer culture medium but does not contact the seedling culture medium.

[0016] The doubling agents include chemical doubling agents and adjuvants. Chemical doubling agents include, but are not limited to, colchicine, methylaminophosphine (APM), pronamide, trifluralin, and / or oryzalin. Adjuvants include, but are not limited to, dimethyl sulfoxide (DMSO), Tween-80, and / or cytokinins.

[0017] 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.

[0018] Furthermore, in the method, 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.

[0019] Furthermore, in the method described above, the coagulant may specifically be plant gel or agar.

[0020] In some embodiments of the present application, the coagulant is agar, and the content of the agar may be 6.5 g / L.

[0021] 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 0.05g colchicine:0.25mL dimethyl sulfoxide.

[0022] Furthermore, in the method, the ratio of colchicine to dimethyl sulfoxide in the doubling agent can be 0.05g colchicine:0.25mL dimethyl sulfoxide.

[0023] Furthermore, in the method, the content of colchicine in the doubling culture medium may be 0.05 g / L, and the content of dimethyl sulfoxide may be 0.25 mL / L.

[0024] Furthermore, a dye can be added to the doubling medium or the seedling medium to facilitate the differentiation of the doubling medium and the seedling layer medium according to color, thereby facilitating the young embryo inoculation operation.

[0025] 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.

[0026] 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 with the naked eye after solidification.

[0027] Further, in the method, the height of the doubling medium in the culture vessel can be 4 to 6 mm.

[0028] Further, in the method, the volume ratio of the seedling culture medium to the doubling medium can be 5:1 to 5:2.

[0029] Further, in the method, the height of the doubling medium in the culture vessel can be 5 mm.

[0030] Further, in the method, the volume ratio of the seedling culture medium to the doubling medium can be 10:3.

[0031] Further, in the method, the conditions of the culture in S2) can be: a light cycle of 16 h light and 8 h darkness, a temperature of 26 to 28℃, and a humidity of 30%.

[0032] Further, the time of the culture in S2) can be 7 to 10 days from inoculation to the double-layer medium.

[0033] Further, in the method, S2) further comprises a step of screening the seedlings formed by doubling the chromosomes of the haploid maize embryo.

[0034] In some embodiments of the present application, the haploid induction line has R1-nj markers. The method for identifying the haploid and the diploid is: inoculating the maize embryo into a double-layer medium, and identifying the embryo according to the color of the embryo after the seedling is formed. The seedling without color of the embryo is a haploid, and the seedling with color of the embryo is a diploid.

[0035] In some embodiments of the present application, because the data of the seedling formation of the diploid is needed, the maize haploid embryo is not identified in advance, but inoculated, and then the haploid and the heterozygous diploid are distinguished by the color of the embryo after the seedling is formed.

[0036] The present application also provides the double-layer medium.

[0037] The present application also provides the use of the double-layer medium in any one of the following:

[0038] A1), the use of the medium in doubling of maize haploid embryo;

[0039] A2), the use of the medium in maize haploid breeding;

[0040] A3), the use of the medium in plant breeding.

[0041] 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 MS salt in the 1 / 2 MS medium are as shown in Table 1.

[0042] Table 1, Composition and content of MS salt in 1 / 2 MS medium (unit: mg / L)

[0043]

[0044] After haploid induction, it is one of the important links of haploid breeding engineering to screen out haploid seeds simply and quickly. There are many methods for identifying haploids, including plant morphology, cytogenetics, genetic marker identification, etc.

[0045] (1) Using plant morphology to identify haploids

[0046] The most basic method for identifying haploids is morphological identification. Since haploids contain only one set of chromosomes, they exhibit smaller cell size, smaller leaf area, flower stem, and seed of the plant, and shorter plant height compared to diploids. However, this method requires a long time to identify haploids, and the growth of plants is easily affected by the environment, causing identification errors. In addition, during the identification of crop haploids, it is necessary to plant in the field, which can easily cause waste of cultivated land.

[0047] In addition, there is a method of identifying young embryos. After pollination of the induction line, young ears are stripped to obtain corn embryos; the corn embryos are sorted in order of length, width, or area from small to large, and the corn embryos ranked in the top 20 to 30% are selected to obtain corn haploid embryos. A large number of heterozygous diploid embryos are eliminated before the color of the young embryos is expressed, which advances the time of haploid selection and improves the efficiency of 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.

[0048] (2) Identifying haploids by combining cytogenetics: observing plant root tip cells or meiotic cells by chromosome pressing is the most intuitive method for identifying haploids, which can determine whether the plant is a haploid by observing whether the number of chromosomes is equal to the number of gametes. However, this method involves complicated experimental steps and has low efficiency, and is not suitable for engineering screening and identification.

[0049] (3) Using genetic markers to identify haploids

[0050] (3-1) R1-nj marker

[0051] Currently, most major breeding companies and research institutes use haploid genetic markers based on the R1-nj gene. This gene synthesizes anthocyanins in the aleurone layer of the corn endosperm and the scutellum of the embryo, resulting in a distinct purple color visible to the naked eye. Using a haploid induction line containing this genetic marker, haploid kernels are obtained that exhibit a non-pigmented embryo and partially pigmented endosperm, making them easily distinguishable from other corn kernels.

[0052] (3-2) MAGIC system

[0053] The MAGIC system is a novel haploid identification system recently developed by the teams of Chen Shaojiang and Chen Rumei using optimized anthocyanin markers. Compared to the R1-nj marker, MAGIC1 achieves 99.1% accuracy in haploid identification and can identify haploids as early as 12 days after pollination, making it more suitable for large-scale haploid screening.

[0054] (3-3) High oil trait marker

[0055] Heterozygous diploid and haploid kernels obtained by hybridizing high-oil-producing induced lines with conventional corn exhibit significant differences in oil content. Therefore, using nuclear magnetic resonance (NMR) or near-infrared (NIR) instruments to analyze haploid kernels significantly reduces detection time compared to manual identification (Li et al., 2009). Further improving the induction rate and oil content of oil-producing induced lines would not only increase haploid induction efficiency but also enhance the efficiency and accuracy of identification, thereby enabling engineered haploid breeding.

[0056] (3-4) Fluorescent protein labeling

[0057] By crossing transgenic plants containing fluorescent markers (YFP, GFP, etc.) with the created haploid induction lines, a haploid induction line containing a stable fluorescent marker is obtained. The hybrids obtained from this induction line can be easily identified and screened for haploid grains using fluorescence observation tools such as fluorescence stereo microscopes and laser confocal microscopes.

[0058] (3-5) Betalain synthesis system RUBY color marking

[0059] The RUBY betalain biosynthesis system was introduced into a maize haploid induction line to generate the RUBY induction line. The induced embryos began to color as early as 10 days after pollination. Those that did color were heterozygous diploids, while those that did not were haploids. During in vitro embryo culture, haploid embryos could be visually identified. Compared to the R1-nj color marker, the RUBY color marker develops color earlier, making it easier to identify haploids, facilitating its application in tissue culture doubling.

[0060] The haploid induction lines CAU5 and CAU6 described in this application carry R1-nj mark.

[0061] The beneficial technical effects of this application are as follows:

[0062] The method provided in this application can complete the chromosome doubling and seedling formation of corn haploid embryos in one step, without the need to transfer to seedling culture medium after chromosome doubling, saving a lot of manpower and is expected to be used in production practice.

[0063] 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

[0064] Figure 1 This is the color development of maize embryos after 24 hours of culture on doubling medium. Maize embryos were identified using R1-nj color markers. Embryos with purple-red scutellum were diploid, while those without purple-red were haploid.

[0065] Figure 2 The method is to insert the maize haploid embryos upright into the seedling culture medium.

[0066] Figure 3 The double-layer culture medium is prepared and the upper layer is colored for easy observation.

[0067] Figure 4 This is a double-layer culture medium without added dye. The figure shows a clear stratification between the upper culture medium (doubling culture medium) and the lower culture medium (seedling culture medium).

[0068] Figure 5 This is a schematic diagram of the situation where maize embryos are inoculated into the upper culture medium (doubling culture medium). The root of the maize embryo faces 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 culture medium).

[0069] Figure 6 This is the rooting situation 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 culture medium).

[0070] Figure 7 This is a schematic diagram of the growth of maize embryos after being cultured in a double-layer culture medium for a period of time. DETAILED DESCRIPTION

[0071] 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, in which the corn embryos are first peeled from the corn, placed in a doubling medium for doubling treatment, and then the haploids are identified by color. The corn haploid embryos are then transferred to a seedling culture medium and cultured until they are seedlings. This method is implemented as a control group in this application. The two-step doubling method has an obvious problem: the transfer culture medium requires a lot of labor and time, so this application proposes a one-step doubling method, which specifically uses a double-layer culture medium. It does not require a transfer culture medium to complete the doubling and seedling formation of the corn embryos.

[0072] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0073] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0074] The corn in the following implementation case ( Zea mays L.) haploid doubling material Chang 7-2. This maize inbred line material is a public material and has been disclosed in the document "Hua Fuping, Shen Weimin, Zhang Yi, et al. Characteristics and utilization of the excellent maize 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.

[0075] The corn in the following examples ( Zea mays L.) Haploid induction lines CAU5 and CAU6, both of which have the R1-nj marker, were developed as a research project by Professor Chen Shaojiang of China Agricultural University and were previously published 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 for use in repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0076] 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).

[0077] In the following examples, the sucrose is Solebao product, product number S8271;

[0078] In the following examples, the dimethyl sulfoxide is a product of Solebol, product number D8370;

[0079] In the following examples, the agar is a Coolabo product, product number CA1331.

[0080] In the following examples, the double-layer culture medium (solid form) includes a doubling medium (on the upper layer) and a seedling medium (on the lower layer). The doubling medium includes 1 / 2 MS medium and a doubling agent, and the seedling medium includes 1 / 2 MS medium. The 1 / 2 MS medium has a formulation of 2.37 g / L MS salts, 30 g / L sucrose, 6.5 g / L agar (as a coagulant), and the remainder is water, with a pH of 5.8. The maize haploid doubling agent includes colchicine and dimethyl sulfoxide. In the two-step method, the doubling agent content in the doubling medium is: 0.1 g / L colchicine and 20 mL / L dimethyl sulfoxide; in the one-step method, the doubling agent content in the doubling medium is: 0.05 g / L colchicine and 0.25 mL / L dimethyl sulfoxide.

[0081] In this example, the culture medium components of each treatment group are as follows:

[0082] 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.

[0083] 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.

[0084] In the following examples, 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.

[0085] In this application, the composition and proportion of MS salts in 1 / 2 MS medium are shown in Table 1.

[0086] Table 1. Composition of MS salts in 1 / 2 MS medium and their content in 1 / 2 MS medium (in mg / L)

[0087]

[0088] The following examples used GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation and tested using t-test. P < 0.05 (*) indicates a significant difference, and P < 0.001 (***) indicates an extremely significant difference.

[0089] Example 1: Two-step method for doubling maize embryo tissue culture

[0090] 1.1, Inducing production of maize haploid young embryos

[0091] Take Chang 7-2 as the female material, and take the induction lines CAU5 and CAU6 as the male material, sow the material in Beijing in May 2024, sow the male material at different times to facilitate the meeting of the flowering periods of the female and male materials. In July, bag the female material, cut the silk, and pollinate with the male induction line material, and record the pollinated male and the pollination date. Harvest the female ear 16 days after pollination, when the size of the young embryo is about 5 mm, and peel the young embryo from the ear through aseptic operation for in vitro culture of the young embryo.

[0092] 1.2, Medium preparation for two-step tissue culture doubling

[0093] The medium for two-step tissue culture doubling is divided into doubling medium and seedling medium. The composition of the doubling medium is as follows: the content of MS salt is 2.37 g / L, the content of sucrose is 30 g / L, the content of agar is 6.5 g / L, the content of dimethyl sulfoxide is 20 mL / L, the content of colchicine is 0.1 g / L, and the rest is water, with a pH value of 5.8. The composition of the seedling medium is as follows: the content of MS salt is 2.37 g / L, the content of sucrose is 30 g / L, the content of agar is 6.5 g / L, and the rest is water, with a pH value of 5.8.

[0094] 1.3, Inoculation doubling treatment and medium transfer

[0095] Place the peeled maize young embryo on the doubling medium, record the date and time, and perform doubling treatment on the maize young embryo for 24 h. The male induction line material has an R1-nj marker. After 24 h of illumination culture, the scutes of hybrid diploid maize young embryos will present obvious purple red color, while the scutes of haploid maize young embryos will not present purple red color, as shown in FIG. 1B. The maize young embryo scutes without purple red marker are haploid. Therefore, according to the color of the maize young embryo scutes, distinguish and select the maize haploid young embryo, and transfer the haploid young embryo to the seedling medium, with the radicle part downward, and 1 / 3 to 1 / 2 of the embryo upright inserted into the seedling medium of the culture bottle, as shown in FIG. 1C. Inoculate 6 to 8 young embryos in each culture bottle. Figure 1 Figure 2 The culture conditions are as follows: a light cycle of 16 h light (14000 lux) and 8 h darkness, a temperature of 26-28°C, and a humidity of 30%.

[0096] 1.4, Seedling culture and field transplanting

[0097] The day when the young embryo is inoculated into the doubling medium is recorded as day 0 (i.e., 0 days of culture). After 7-10 days of culture, count the number of seedlings, transfer to a plug tray, and acclimate in the outdoor for about 10-14 days, and then transplant to the field when the seedlings grow to 3-5 leaves.

[0098] ​The criteria for judging the statistical range of seedlings after 7 to 10 days of cultivation are as follows:

[0099] Seedlings: Seedlings grow to the stage of two leaves and one heart or three leaves;

[0100] Unfertilized seedlings: The growth of the young embryo stagnates, the young embryo becomes vitrified or grows callus tissue, and the young embryo only takes root but does not sprout.

[0101] Example 2: One-step method for doubling maize embryo tissue culture

[0102] 2.1. Induction of maize haploid embryos

[0103] Chang 7-2 was used as the female parent, and the induction lines CAU5 and CAU6 were used as the male parent. The material was sown in Beijing in May 2024. The male parent was sown at a different time to allow for the two parent lines to flower at the same time. In July, the female parent was bagged, the filaments cut, and then over-pollinated with the induction line of the male parent. The pollinated male parent and pollination date were recorded. The female fruit clusters were harvested 16 days after pollination. At this time, the embryos were approximately 5 mm in size. The embryos were aseptically removed from the clusters and cultured in vitro.

[0104] 2.2. One-step preparation of double-layer culture medium

[0105] The double-layer culture medium consists of an upper layer of doubling medium and a lower layer of seedling medium. The ratio of the lower layer to the upper layer is 10:3, and the upper layer is 5 mm high. In a 240 mL culture bottle, use 40 mL of the lower layer and 12 mL of the upper layer. Use a liquid dispensing pump to accurately add 40 mL of the seedling medium to the bottle. After the medium cools naturally, use a liquid dispensing pump to accurately add 12 mL of the doubling medium to the same bottle. After cooling, the upper and lower layers of the culture medium can be observed.

[0106] The upper layer of the culture medium is the doubling medium, which includes 1 / 2 MS medium and a doubling agent. The lower layer of the culture medium is the seedling medium, which includes 1 / 2 MS medium. The 1 / 2 MS medium is composed of 2.37 g / L MS salts, 30 g / L sucrose, 6.5 g / L agar, and the remainder is water. The pH is adjusted to 5.8. The final concentrations of the doubling agent in the doubling medium are 0.25 mL / L dimethyl sulfoxide and 0.05 g / L colchicine.

[0107] Depending on whether a stain is added, bilayer cultures can be divided into the following three types:

[0108] (1) Add dye to doubling medium or seedling medium

[0109] 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 stain at a rate of 0.1 mL / L. This serves to facilitate differentiation between the doubling medium and the seedling medium. Furthermore, staining the doubling medium is used to observe whether components of the doubling medium permeate into the seedling medium. Experimental results indicate that only after approximately two months of preparation and stagnant culture medium can a small amount of dye stain the seedling medium. Therefore, it is believed that during use of the double-layer culture medium, the doubling agent in the doubling medium will not permeate into the lower layer.

[0110] (2) Add different colored dyes to the doubling medium and seedling medium respectively

[0111] 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 by observing the colors.

[0112] (3) No dye is added to the doubling medium and seedling medium

[0113] 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 with the naked eye after solidification ( Figure 4 ).

[0114] 2.3. Inoculation doubling treatment and seedling formation

[0115] Peel out the young embryo of corn with the radicle facing downwards. Figure 5 As shown, 1 / 3 to 1 / 2 of the maize embryo is placed upright in a double-layer culture medium with the radicle facing downward and the plumule facing upward. The maize embryo only contacts the doubling medium and not the seedling medium. Six to eight maize embryos are inoculated into each culture bottle. The inoculation date is recorded and the flask is placed in a biochemical incubator (placed on a tissue culture rack). Culture conditions are: a photoperiod 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 embryo grows a radicle after 2 to 3 days of culture in the doubling medium. The radicle then grows downward and extends into the seedling medium, as shown in the figure. Figure 6 and Figure 7 As shown in the figure, before the young embryo's radicle extends into the seedling medium, it only absorbs nutrients from the doubling medium, completing the doubling process during this period. Once the radicle enters the seedling medium, it primarily absorbs nutrients from the underlying seedling medium. As the young embryo enters the seedling stage, the doubling medium has little effect on seedling formation, allowing doubling and seedling formation to be completed in one step.

[0116] Method for distinguishing haploids from diploids: Inoculate all maize embryos into a double-layer culture medium. After they have grown into seedlings, identify the embryos based on their coloration. Seedlings with colorless embryo scutellum are haploids, and seedlings with colored embryo scutellum are diploids.

[0117] Because this experiment required diploid seedling data, haploid maize embryos were not identified in advance. Instead, maize embryos were inoculated and, after seedling formation, haploid and heterozygous diploids were distinguished by the color of the embryo scutellum. Generally speaking, in actual production, it is necessary to distinguish between haploid and diploid plants.

[0118] 2.4. Seedling cultivation and field transplanting

[0119] The day the embryos were inoculated into the double-layer culture medium was designated as day 0. After 7 to 10 days of culture, haploid maize seedlings were identified and selected using the color markings of the embryonic scutellum. Seedlings with purple-red embryonic scutellum were heterozygous diploids, while those without purple-red embryonic scutellum 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. Once the seedlings had 3 to 5 leaves, they were transplanted into the field.

[0120] 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 simply observing the color of the embryo shield, without the need for additional identification or labeling.

[0121] 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 inoculated seeds.

[0122] Results showed that the seedling establishment rate of maize haploids obtained with the two-step doubling method was higher than that obtained with the one-step method. This is because, under the same culture conditions, the maize haploid embryos grow more slowly during the one-step doubling process, requiring three days or longer to develop radicles. This prolonged doubling treatment significantly impacts seedling establishment. In contrast, the maize diploid embryos grow faster, developing radicles in just two days. These embryos can penetrate the underlying stratum earlier and subsequently enter the seedling establishment stage. The doubling treatment takes less time, reducing the stress of the doubling agent on the embryos, resulting in a higher seedling establishment rate.

[0123] The criteria for judging the statistical range of seedlings after 7 to 10 days of cultivation are as follows:

[0124] Seedlings: Seedlings grow to the stage of two leaves and one heart or three leaves;

[0125] Seedling failure: The growth of the young embryo stagnates, the young embryo becomes vitrified or grows callus tissue, the young embryo only takes root but does not sprout, and the embryo swells into a ball and stops developing.

[0126] Table 2. Number and seedling rate of maize immature embryos treated with two doubling methods

[0127]

[0128] Note: The sample size in the table refers to the number of culture bottles.

[0129] 2.5. Results and analysis of double-layer culture medium doubling treatment

[0130] 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 culture medium, and subsequent seedling formation only took about 7 to 10 days. In contrast, it took 3 days for the haploid radicle to grow to the lower layer, and subsequent rooting and plumule growth were slower, requiring 12 to 14 days to reach seedling formation. 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 this was analyzed to be due to the ploidy difference between haploids and diploids. At the same doubling agent concentration, haploids have weaker tolerance to the doubling agent and are more susceptible to the stress of the doubling agent, resulting in slow growth. Furthermore, after 3 days of doubling treatment, the subsequent seedling growth was also greatly affected, with more young embryos unable to 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.

[0131] Example 3. Quantification of the ratio and dosage of the double-layer culture medium

[0132] 3.1. Induction of Maize Haploid Embryos

[0133] Chang 7-2 was used as the female parent, and the induction lines CAU5 and CAU6 were used as the male parent. The material was sown in Beijing in August 2024. The male parent was sown at a different time to allow for the two parent lines to flower at the same time. In October, the female parent was bagged, the filaments cut, and then over-pollinated with the male induction line. The pollinated male parent and pollination date were recorded. The female fruit clusters were harvested 20 days after pollination. At this point, the embryos were approximately 5 mm in size. The embryos were aseptically removed from the clusters and cultured in vitro.

[0134] 3.2. Preparation of double-layer culture medium with different heights of upper culture medium

[0135] The double-layer culture medium consists of an upper doubling medium and a lower seedling medium. A series of double-layer culture media with different upper medium dosages were set up. The following is the experimental treatment plan:

[0136] Group 1: The amount of upper layer culture medium was 8 mL. In a 240 mL culture bottle, the height of the upper layer culture medium was measured to be approximately 4.0 mm.

[0137] Group 2: The amount of upper layer culture medium was 10 mL. In a 240 mL culture bottle, the height of the upper layer culture medium was measured to be approximately 4.5 mm.

[0138] Group 3: The amount of upper layer culture medium was 12 mL. In a 240 mL culture bottle, the height of the upper layer culture medium was measured to be approximately 5.0 mm.

[0139] Group 4: The amount of upper layer culture medium was 14 mL. In a 240 mL culture bottle, the height of the upper layer culture medium was measured to be approximately 5.5 mm.

[0140] Group 5: The amount of upper layer culture medium was 16 mL. In a 240 mL culture bottle, the height of the upper layer culture medium was measured to be approximately 6.0 mm.

[0141] The volume of the lower layer culture medium is 40 mL. Use a liquid dispensing pump to accurately add 40 mL of seedling culture medium to 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 the doubling culture medium, respectively. After cooling, the upper and lower layers of the culture medium can be observed.

[0142] The upper layer of the culture medium is the doubling medium, which includes 1 / 2 MS medium and a doubling agent. The lower layer of the culture medium is the seedling medium, which includes 1 / 2 MS medium. The 1 / 2 MS medium is composed of 2.37 g / L MS salts, 30 g / L sucrose, 6.5 g / L agar, and the remainder is water. The pH is adjusted to 5.8. The final concentrations of the doubling agent in the doubling medium are 0.25 mL / L dimethyl sulfoxide and 0.05 g / L colchicine.

[0143] 3.3. Inoculation doubling treatment and seedling formation

[0144] Remove the maize embryos, with the radicle facing downward. Place one-third to one-half of the embryo upright in a double-layered culture medium, with the radicle facing downward and the plumule facing upward. The embryos should contact only the upper layer of culture medium, not the lower layer. Inoculate 6 to 8 maize embryos per flask. Record the date of inoculation and place the embryos in a biochemical incubator (placed on a tissue culture rack). Culture conditions include a photoperiod 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, observe the growth of the radicles in the upper layer of culture medium. Record radicle elongation every 12 hours, and note the time when the radicles begin to enter the lower layer of culture medium.

[0145] Method for distinguishing haploids from diploids: Inoculate all maize embryos into a double-layer culture medium. After culturing for about 24 hours, identify the embryos based on their coloration. Seedlings with colorless embryo scutellum are haploids, and seedlings with colored embryo scutellum are diploids.

[0146] 3.4 Observation results of rooting after young embryo inoculation

[0147] Table 3. Relationship between the time of the upper culture medium (doubling culture medium) at different heights and the elongation of the young embryo radicle into the lower culture medium (seedling culture medium)

[0148]

[0149] Experimental results show that when the height of the upper culture medium (doubling medium) ranges from 4 to 6 mm and the volume ratio of the lower culture medium (seedling medium) to the upper culture medium (doubling medium) is 5:1 to 5:2, it takes approximately 48 hours for the radicle of a diploid embryo to enter the lower culture medium from the time of inoculation, and approximately 72 hours for the radicle of a haploid embryo to enter the lower culture medium. There are no significant differences between treatments with different upper culture medium heights for either diploid or haploid embryos. Therefore, in a double-layer culture, an upper culture medium height between 4 and 6 mm has little effect on the doubling time of the embryo. If the doubling time needs to be adjusted, the upper culture medium height should be set lower or higher.

[0150] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for chromosome doubling and seedling formation of haploid immature embryos of corn in haploid breeding, characterized in that: The method comprises: S1) Haploid induction: using a maize inbred line as the female parent and a maize haploid induction 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 / 2 MS culture medium and a doubling agent, and the seedling culture medium includes a 1 / 2 MS culture medium; the inoculation includes the step of contacting the maize embryo with the doubling culture medium in the double-layer culture medium but not with the seedling culture medium; The solutes of the 1 / 2 MS culture medium include MS salts, sucrose and a coagulant.

2. The method according to claim 1, characterized in that 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 claim 2 or 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 3, 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 3, 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 3, characterized in that: The culture conditions in step S2) are as follows: a photoperiod of 16 h light and 8 h dark, a temperature of 26° C. to 28° C., and a humidity of 30%.

8. The method according to claim 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. An application, characterized in that: The application is the application of the double-layer culture medium according to claim 9 in doubling maize haploid immature embryos.

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