A method for efficiently doubling corn haploid

Abstract

The present invention discloses a method for efficiently doubling haploid maize, and belongs to the field of breeding. The technical problem to be solved by the present invention is how to solve the problem of low DH production efficiency when colchicine is used to double haploid immature embryos. To solve the above technical problem, the present invention provides a composition, which includes glycine, vitamin B1, niacin and vitamin B6. In the composition, the ratio of glycine: vitamin B1: niacin: vitamin B6 is 20:1:5:5. The present invention also provides an application of the composition in doubling haploid immature embryos of maize. The present invention provides a feasible solution for improving the tissue culture identification and doubling efficiency of haploid immature embryos acted on by colchicine and realizing efficient doubling of haploids, which can greatly improve the efficiency of haploid breeding technology.

Description

Technical Field

[0001] The invention belongs to the field of breeding, and in particular relates to a method for efficiently doubling corn haploids. Background Art

[0002] The continuous growth of the world's population has put increasing pressure on human development. How to solve the growing demand for food has become a difficult problem that countries around the world have to face. Under the constraints of limited arable land and water resources, increasing crop yields has become a basic way to solve the food problem. The use of hybrid vigor has made outstanding contributions to increasing crop yields, ensuring food security and protecting natural resources. The combination of corn hybrids originated in the United States in the 1920s. Compared with open-pollinated corn varieties, the yield of hybrids is 15% higher. After nearly 30 years of promotion and popularization, by the 1950s, most corn production areas in the United States began to plant hybrid corn. According to incomplete statistics, the annual planting area of ​​corn hybrids in my country in recent years is about 600 million mu, and corn has become my country's largest crop. In corn breeding, the traits of the parent inbred lines themselves and their compatibility often determine the potential of the offspring hybrids. Therefore, the selection of inbred lines with excellent traits is the basis for the use of corn hybrid vigor. In traditional breeding, it takes at least 8 generations to obtain an almost homozygous inbred line through self-pollination or sister-pollination, which is time-consuming and labor-intensive. However, through the DH (doubled haploid) breeding method, it only takes 2 generations to obtain a theoretically completely homozygous line, which greatly improves the efficiency of line selection and is increasingly valued. DH breeding plays an increasingly important role in the commercial breeding process of corn, and has become the core technology of modern corn breeding together with molecular breeding technology and transgenic technology. DH breeding, that is, (double) haploid breeding, refers to the process of using plant haploids to quickly produce pure lines. Therefore, achieving efficient doubling of corn haploids is one of the most direct ways to accelerate pure line production, which can effectively improve the efficiency of commercial breeding. Summary of the invention

[0003] The technical problem to be solved by the present invention is: how to solve the problem of low DH production efficiency when colchicine is used to double haploid immature embryos.

[0004] To solve the above technical problems, in a first aspect, the present invention provides a composition for improving the haploid doubling efficiency of corn, wherein the active ingredients of the composition are glycine, vitamin B1, niacin and vitamin B6.

[0005] The improving the maize haploid doubling efficiency may be improving the maize haploid doubling efficiency of colchicine.

[0006] Furthermore, in the above composition, the ratio of glycine: vitamin B1: niacin: vitamin B6 is 20:1:5:5.

[0007] In order to solve the above technical problems, in a second aspect, the present invention provides a maize haploid doubling agent, wherein the active ingredients of the maize haploid doubling agent include colchicine and the above composition.

[0008] The maize haploid doubling agent is a composition for doubling the maize haploid chromosome.

[0009] Furthermore, in the above-mentioned maize haploid doubling agent, the ratio of colchicine: glycine: vitamin B1: niacin: vitamin B6 is 50-400:20:1:5:5.

[0010] Furthermore, the corn haploid doubling agent also includes dimethyl sulfoxide.

[0011] In order to solve the above technical problems, in a third aspect, the present invention provides an application of the above composition, wherein the application is selected from any one of the following:

[0012] A1), application of the composition in increasing the doubling of haploid immature embryos of maize;

[0013] A2), use of the composition in preparing a product for increasing the doubling of haploid immature embryos of maize;

[0014] A3), application of the composition in haploid breeding of corn;

[0015] A4), use of the composition in preparing a product of haploid corn breeding;

[0016] A5) Use of the composition in plant breeding.

[0017] In order to solve the above technical problems, in a fourth aspect, the present invention provides an application of the above-mentioned maize haploid doubling agent, wherein the application is selected from any one of the following:

[0018] P1), application of the maize haploid doubling agent in doubling maize haploid immature embryos;

[0019] P2), application of the maize haploid doubling agent in maize haploid breeding;

[0020] P3) Application of the maize haploid doubling agent in plant breeding.

[0021] To solve the above technical problems, in a fifth aspect, the present invention provides a method for efficiently doubling corn haploids, the method comprising using the above-mentioned corn haploid doubling agent to treat corn haploid immature embryos to obtain corn with doubled chromosomes.

[0022] Furthermore, in the above method, the doubling of the maize haploid immature embryos using the maize haploid doubling agent comprises the following steps: inoculating the maize haploid immature embryos into a culture medium containing the maize haploid doubling agent for culturing.

[0023] Furthermore, in the above method, the content of colchicine in the culture medium containing the corn haploid doubling agent is 0.1 g / L, the volume content of DMSO is 20 mL / L, the content of glycine is 2.0 mg / L, the content of vitamin B1 is 0.1 mg / L, the content of niacin is 0.5 mg / L, and the content of vitamin B6 is 0.5 mg / L.

[0024] In one embodiment of the present invention, the composition of the culture medium containing the corn haploid doubling agent is: 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, 0.1 g / L of colchicine, 20 mL / L of DMSO, 2.0 mg / L of glycine, 0.1 mg / L of vitamin B1, 0.5 mg / L of niacin, 0.5 mg / L of vitamin B6, and the rest is water, and the pH value is 5.8.

[0025] Furthermore, in the above method, the maize haploid immature embryos are inoculated into the culture medium containing the maize haploid doubling agent for culturing for 24 hours, and the culture conditions are full light, temperature 26° C., and humidity 60%.

[0026] Furthermore, in the above method, the maize haploid immature embryo is a maize haploid immature embryo peeled off 12-20 days after pollination after hybridization with a haploid induction line as the male parent and the induced material, and specifically can be a maize haploid immature embryo peeled off 15 days after pollination.

[0027] The method for efficiently doubling corn haploids provided by the present invention specifically comprises the following steps:

[0028] First, obtain germplasm with high-frequency chemical doubling, that is, inbred lines that are easy to double after treatment with chemical agents, such as colchicine. Use this material as the induced material, and the induction line carrying the R1-nj color mark as the male parent. Take the pollen of the induction line and pollinate it with the induced material, and record the pollination time. On the same day after pollination, take the induced hybrid ears, peel the embryos, and place them on the control medium and the doubling medium for treatment.

[0029] After doubling treatment, haploid immature embryos with colorless scutellum were selected from the control medium and the doubling medium, respectively, and transcriptome sequencing was performed. The differentially expressed genes in the haploid immature embryos of the control group and the doubling group were compared and analyzed to explore the key substances that affect haploid doubling.

[0030] Combined with the transcriptome sequencing results, the key substances that may affect the haploid doubling efficiency were combined with conventional doubling agents, and the haploid embryos of other genetic background materials were treated. At the same time, blank controls and different content gradient controls were set up, and the haploids treated with different doubling agents were grown into seedlings. Combined with important indicators such as pollen shedding rate, fruit setting rate and DH productivity, we finally obtained a doubling agent that can be used for efficient haploid doubling.

[0031] In the above method, the germplasm with high-frequency chemical doubling can be an inbred line that is easy to double after colchicine treatment, such as any one of the inbred lines such as Zheng 58, B73, Qi 319, etc.

[0032] In the above method, the male parent of the haploid induction line can be selected from any one of the haploid induction lines having the R1-nj marker, such as CAU5.

[0033] In the above method, doubling the maize haploid immature embryos with a maize haploid doubling agent comprises the following steps:

[0034] 1) Using the induced maize material as the female parent and the haploid induced line with the R1-nj marker as the male parent for artificial pollination hybridization;

[0035] 2) 12-20 days after pollination, the hybrid ears are taken and young embryos are peeled off, and the embryos are inoculated into the culture medium containing the maize haploid doubling agent and the corresponding control culture medium not containing the doubling agent for doubling treatment;

[0036] 3) Select haploid embryos with colorless scutellum from the embryos treated for 12 to 72 hours and culture them into seedlings.

[0037] 4) After the seedlings obtained from the seedling cultivation are planted, they are self-pollinated to obtain haploid chromosome-doubled corn DH seeds.

[0038] In the above method, in the artificial pollination hybridization, to ensure that the flowering periods of the parents meet, the female parent can be planted for one period, and the male parent can be sown for multiple periods (e.g., 2-3 periods) around the sowing period of the female parent. In the artificial pollination hybridization, the female parent is emasculated (the filaments are cut off) during the flowering period, the female ear is strictly bagged, and the pollen of the male parent is taken for artificial over-pollination, and the pollination time is recorded.

[0039] In the above method, the culture conditions for the doubling treatment are full light, temperature 26° C., and humidity 60%.

[0040] In the above method, the seedling culture adopts a common basic culture medium for plant tissue culture, such as any one of MS solid culture medium and 1 / 2MS solid culture medium.

[0041] In the above method, the time for the seedling culture can be 2 days to 7 days, specifically 4 days.

[0042] In the above method, the culture conditions for the seedling culture are alternating 16-hour light period / 8-hour dark period, a temperature of about 26° C., and a humidity of about 60%.

[0043] In the above method, the seedlings obtained from the seedling cultivation are planted in the field. Before planting, they can be hardened and cultivated. Specifically, normal embryo seedlings are transplanted into nutrient pots filled with peat soil for hardening and cultivation after growing to 2-3 leaves and one heart, and are planted in the field after growing to 5 leaves and one heart.

[0044] In the above method, the control culture medium uses a common basal culture medium for plant tissue culture as a basal culture medium, such as one of MS solid culture medium and N6 solid culture medium.

[0045] In the above method, the culture medium of the maize haploid doubling agent is, on the basis of the basic culture medium, added with 0.05-0.4 g / L of colchicine and 2% of dimethyl sulfoxide by volume.

[0046] In the above method, the maize haploid embryo is a maize haploid embryo peeled off 12-20 days after pollination after hybridization with a haploid induction line as the male parent and the induced material, and specifically can be a maize haploid embryo peeled off 15 days after pollination.

[0047] In the above method, the doubling treatment time may be 12 hours to 72 hours, specifically 24 hours.

[0048] In the above method, the key substances affecting haploid doubling mined in combination with transcriptome sequencing results may be glycine, vitamin B1, niacin, vitamin B6, and the like.

[0049] The method for doubling the haploid of corn provided by the present invention can effectively improve the doubling rate of colchicine treatment by applying a combination of glycine, vitamin B1, niacin and vitamin B6 doubling agent in the doubling process of immature embryos, greatly improve the haploid doubling rate and seed setting rate, thereby improving the DH production efficiency. A feasible scheme is provided for improving the identification and doubling efficiency of haploid immature embryo tissue culture under the action of colchicine and realizing efficient doubling of haploids, which can greatly improve the efficiency of haploid breeding technology. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] The maize (Zea mays L.) haploid high-frequency chemically doubled material Zheng 58 in the following implementation case has been disclosed in the document "Wei Wenxing. Analysis of genomic differences between Zheng 58 and Ye 478 maize inbred lines [J]. Maize Science, 2010, 18(03)57-60." The public can obtain it from the Beijing Academy of Agriculture and Forestry Sciences. This material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0053] The maize (Zea mays L.) haploid induction line CAU5 in the following embodiment has the R1-nj marker, which was issued by Professor Chen Shaojiang of China Agricultural University and was 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 for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0054] In the following examples, the maize inbred lines Jinghuangou 1 and Jing J2418 were preserved for this experiment. The maize (Zea mays L.) inbred lines Jinghuangou 1 (Plant New Variety Rights Application Announcement No. 20201000753) and Jing J2418 (Plant New Variety Rights Application Announcement No. 20201000757) were both bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences. The public can obtain them from Beijing Academy of Agricultural and Forestry Sciences to repeat the experiments in this application and cannot be used for other purposes.

[0055] The sucrose and agarose in the following examples are both products of Beijing Jipteng Biotechnology Co., Ltd.

[0056] The glycine (CAS No. 56-40-6), vitamin B1 (CAS No. 59-43-8), niacin CAS No. 59-67-6, vitamin B6 (CAS: 65-23-6), and colchicine in the following implementation cases are all products of Beijing Boyuhang Biotechnology Co., Ltd.

[0057] In the following examples, dimethyl sulfoxide was purchased from Beijing Jiputeng Biotechnology Co., Ltd., CAS: 67-68-5.

[0058] In the following implementation case, the doubling identification medium is composed of MS solid medium + corn haploid doubling agent, wherein the MS solid medium specifically contains 3.0 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, and a pH value of 5.8 (i.e., the formula is MS salt 3.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.8); MS salt is a product of Shanghai Yuhan Biotechnology Co., Ltd., with a product catalog number of 140225.

[0059] The active ingredients of the corn haploid doubling agent include colchicine, dimethyl sulfoxide, glycine, vitamin B1, niacin and vitamin B6, wherein the ratio of colchicine: glycine: vitamin B1: niacin: vitamin B6 is 50-400: 20: 1: 5: 5.

[0060] In this embodiment, the culture medium composition of each treatment group is as follows:

[0061] CK: The content of MS salt is 3.0 g / L, the content of sucrose is 30 g / L, the content of agar is 7.5 g / L, and the rest is water. The pH value is 5.8.

[0062] Q1: The content of MS salt is 3.0g / L, the content of sucrose is 30g / L, the content of agar is 7.5g / L, the content of colchicine is 0.1g / L, the content of DMSO is 20mL / L, the rest is water, and the pH value is 5.8.

[0063] Q1+T: The content of MS salt is 3.0g / L, the content of sucrose is 30g / L, the content of agar is 7.5g / L, the content of colchicine is 0.1g / L, the content of DMSO is 20mL / L, the content of glycine is 2.0mg / L, the content of vitamin B1 is 0.1mg / L, the content of niacin is 0.5mg / L, the content of vitamin B6 is 0.5mg / L, and the rest is water. The pH value is 5.8.

[0064] In the following implementation cases, the seedling culture medium is a 1 / 2MS solid culture medium, specifically a culture medium containing 1.5 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, and the rest water, with a pH value of 5.8 (i.e., the formula is MS salt 1.5 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.8).

[0065] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0066] Example 1: Exploration of the mechanism of haploid doubling and mining of key influencing factors

[0067] 1.1. Induction of maize haploid embryos

[0068] In 2021, in Beijing, Zheng 58 was used as the female parent (induced material) and the induction line CAU5 was used as the male parent to induce haploids. The male parent CAU5 was planted in two phases, the first phase was sown on April 25, and the second phase was sown on April 30; the sowing period of the female parent was the same as that of the first phase of the male parent so that the flowering period would coincide. The female parent uniformly cut the filaments, strictly emasculated, and over-pollinated with the male parent induction line, and the uniform pollination time was recorded. The young embryos 15 days after pollination (recorded as DAP15 days) were used as materials for in vitro culture of young embryos.

[0069] 1.2 Identification and doubling of maize haploid embryos

[0070] The immature embryos obtained in 1.1, which were recorded as DAP15, were cultured in a culture medium and divided into the following two treatment groups according to different culture medium formulas:

[0071] CK: The CK group served as the control group, and its culture medium specifically contained 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, and the rest was water, with a pH value of 5.8.

[0072] Q1: Q1 group was used as the treatment group, and its culture medium specifically contained 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, 0.1 g / L of colchicine, 2% (v / v) of DMSO, and the rest was water, with a pH value of 5.8.

[0073] The culture time of each treatment group was 24 hours. The culture conditions were full light, temperature 26°C, and humidity 60%. CAU5 all had R1-nj markers. After 24 hours of embryo culture, the scutellum of the heterozygous diploid embryos appeared purple-red due to the R1-nj marker. The haploid embryos only contained one set of chromosomes of the maternal material and did not contain the R1-nj marker, so the scutellum of the haploid embryos was colorless. Haploid embryos were selected according to the color of the scutellum and inoculated on the seedling medium for seedling culture. The culture conditions were 16 hours of light period / 8 hours of dark period alternation, temperature 26°C, and humidity 60%. Placed in the culture room, seedling culture was carried out for 4 days. In addition, 3 haploid embryos from CK and Q1 were selected, each with more than 500 mg, placed in liquid nitrogen, and stored in a -80°C refrigerator for transcriptome sequencing.

[0074] 1.3. Sequencing analysis of doubled transcriptome of haploid young embryos

[0075] Transcriptome sequencing was performed on three replicates of the above CK and Q1 treatments after 24 hours, and then differentially expressed genes were screened and GO enrichment was performed. The results showed (Table 1) that compared with the non-doubling control group, the differentially expressed genes after doubling treatment were enriched in glutathione metabolic and transport processes (glutathione metabolic process, glutathione transferase activity), nicotinamide nucleotide biosynthetic process, vitamin binding (vitamin binding, vitamin B6 binding) and hormone synthesis pathways in addition to chromosome replication and cell division-related pathways. Glycine is an important component of glutathione, and nicotinic acid is an important precursor substance for nicotinamide nucleotide biosynthesis, also known as nicotinic acid, which is highly related to the subsequent vitamin binding pathway. Therefore, it is speculated that changes in the content of glycine, nicotinic acid and other vitamins may affect the haploid doubling efficiency.

[0076] Table 1 GO enrichment analysis of CK VS Q1

[0077]

[0078] Example 2: Effect of externally applied doubling agent on doubling efficiency of haploid immature embryos

[0079] To further verify the effect of glycine, vitamin B1, niacin and vitamin B6 combined doubling agent on haploid doubling, we used glycine, vitamin B1, niacin and vitamin B6 combined doubling agent to verify the haploid chemical doubling effect with Jinghuangou No. 1 and Jing J2418 as the mother parent. The specific experimental schedule is as follows:

[0080] In 2022, in Hainan, Jinghuangou No. 1 and JingJ2418 were used as female parents (induced materials), and the induction line CAU5 was used as the male parent to induce haploids. The female parents were sown on October 15. The male parents were sown at two different times, on October 15 and 20, respectively. The female parents were uniformly cut for filaments, strictly emasculated, and over-pollinated with the male parent induction line, and the pollination was recorded at the same time. The young embryos were peeled 15 days after pollination. They were cultured in the following culture media:

[0081] CK: The CK group served as a control group, and its culture medium specifically contained 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, and the rest was water, with a pH value of 5.8.

[0082] Q1: Q1 group was used as the treatment group, and its culture medium contained 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, 0.1 g / L of colchicine, 20 mL / L of DMSO, and the rest was water, with a pH value of 5.8.

[0083] Q1+T: The Q1+T group was used as the treatment group. Its culture medium contained 3.0 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, 0.1 g / L of colchicine, 20 mL / L of DMSO, 2.0 mg / L of glycine, 0.1 mg / L of vitamin B1, 0.5 mg / L of niacin, 0.5 mg / L of vitamin B6, and the rest was water. The pH value was 5.8.

[0084] The culture time of each treatment group is 24 hours. The culture conditions are full light, temperature of about 26°C, and humidity of about 60%. CAU5 has an R1-nj marker. After 24 hours of embryo culture, the scutellum of the heterozygous diploid embryo appears purple-red due to the R1-nj marker. The haploid embryo and double haploid (DH) embryo only contain one set of chromosomes of the maternal material, without the R1-nj marker, so the scutellum of the haploid embryo is colorless. The haploid embryos (the scutellum of the embryo is colorless) are selected according to the color of the scutellum and inoculated into the seedling medium for seedling culture. The seedling medium is composed of 1 / 2MS solid culture medium, specifically a culture medium containing 1.5g / L MS salt, 30g / L sucrose, 7.5g / L agar, and pH 5.8. The culture conditions are 16 hours of light period / 8 hours of dark period alternation, temperature of about 26°C, and humidity of about 60%. Place in the culture room and culture for 7 days. Then carry out outdoor hardening, and transplant to the field when the seedlings are 3-5 leaves. When the haploid plants are masculine and silking, the male spike dew and pollen registration of each treatment are investigated. For specific investigation indicators, refer to Penghao Wu et al, New Insights into the Genetics of Haploid Male Fertility in Maize 2017. The female spike is strictly bagged, and the haploid is strictly self-pollinated after the pollen is shed. After mature harvest, the key indicators such as haploid dew score, pollen score, pollen shed rate, fruit set rate, average fruit set number, DH productivity, etc. are counted to evaluate the doubling efficiency of each treatment haploid. The grains obtained by self-pollination of haploids with loose pollen are double haploids (DH). The specific results are shown in Table 2.

[0085] Pollen shedding rate = number of haploid plants shedding pollen / total number of haploid plants × 100%

[0086] Fruiting rate = number of fruiting haploid plants / number of haploid plants with loose pollen × 100%

[0087] DH productivity = number of fruiting haploid plants / total number of haploid plants × 100%.

[0088] Table 2. Effects of different treatment groups on doubling of haploid embryos

[0089]

[0090] Comparison of the doubling of haploid young embryos in the non-doubling treatment group CK, the conventional doubling treatment group Q1 and the new doubling treatment group Q1+T showed that when colchicine was not added, the drug dew scores of Jinghuangou No. 1 and Jing J2418 were 0.36 and 0.31 respectively, and the loose powder scores were both lower than 0.5, and the final DH productivity was also below 10%. After adding the doubling agent, the doubling efficiency was significantly improved. Compared with the conventional doubling medium Q1, the new doubling treatment group Q1+T was slightly lower than Q1 in drug dew score and loose powder score, but significantly improved in loose powder rate, among which Jinghuangou No. 1 increased by 16.79% and Jing J2418 increased by 7.93%. In addition, in terms of average fruiting rate, the Q1+T treatment group was also higher than Q1. In Jinghuangou No. 1, the average fruiting rate of the Q1+T treatment group was as high as 85.04%, and the average number of fruits in the Q1+T treatment group was more than 30 in the two material backgrounds. Finally, the DH productivity of the Q1+T treatment group in the two material backgrounds was as high as 69.68% and 56.49%, respectively, and the DH productivity of the Q1 treatment group was less than 50%. In summary, the exogenous application of glycine, vitamin B1, niacin and vitamin B6 combined with conventional doubling agents can effectively improve the haploid doubling rate, average fruit number and final DH productivity.

[0091] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, contents and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of a composition for improving the doubling efficiency of maize haploids, the application being selected from any one of the following: A1) Use of the composition in increasing the doubling of haploid immature embryos of maize; A2) Use of the composition in preparing a product for increasing the doubling of haploid immature embryos of maize; The composition consists of glycine, vitamin B1, niacin and vitamin B6; In the composition, the working concentration of glycine is 2.0 mg / L, the working concentration of vitamin B1 is 0.1 mg / L, the working concentration of niacin is 0.5 mg / L, and the working concentration of vitamin B6 is 0.5 mg / L.

2. Application of a composition for improving the doubling efficiency of maize haploids, the application being selected from any one of the following: A3) Use of the composition in haploid breeding of corn; A4) Use of the composition in preparing a product of haploid corn breeding; The composition consists of glycine, vitamin B1, niacin and vitamin B6; In the composition, the working concentration of glycine is 2.0 mg / L, the working concentration of vitamin B1 is 0.1 mg / L, the working concentration of niacin is 0.5 mg / L, and the working concentration of vitamin B6 is 0.5 mg / L.

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