CN105494082A - Method for identifying haploids based on forms of maize immature embryos - Google Patents

Abstract

The invention discloses a method for identifying haploids based on the morphology of maize immature embryos. The method comprises the following steps: using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, the young ears are taken and the embryos are peeled off to obtain corn immature embryos; Sorting in large order, selecting and sorting the top 20-30% corn immature embryos to obtain maize haploid immature embryos. It is proved by experiments that the method of the present invention can realize the selection of haploids in the stage of immature corn embryos, and a large part of heterozygous diploid immature embryos will be eliminated before the color expression of immature embryos, which advances the selection of haploids. The time is improved, the efficiency of selection is improved, the breeding time is further shortened, the follow-up cost is also saved, and the economic benefit is improved. Moreover, the method provided by the invention has the prospect of automatically selecting haploids, and accelerates the process of engineering breeding of maize haploids.

Description

A method for haploid identification based on the morphology of maize immature embryos

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for identifying haploids based on the morphology of immature maize embryos.

Background technique

Corn is the largest crop planted in my country. In 2012, the sown area of corn exceeded 34 million hm2. More than 97% of the corn acreage in my country uses hybrid corn (LiJ, 2009). Breeding of superior maize inbred lines is the basis and key to utilizing heterosis and breeding superior hybrids in maize. However, the traditional breeding method needs 7 to 8 generations to obtain a relatively stable inbred line, while the haploid breeding technology only needs 2 generations (WeberDF, 2014). Therefore, haploid breeding technology, as a method to quickly obtain pure lines, has been applied on a large scale by many seed companies at home and abroad, and has become one of the three core technologies of modern corn breeding comparable to transgenic technology and molecular marker-assisted breeding technology. One (Chen Shaojiang et al., 2009).

With the large-scale application of haploid breeding technology, it is extremely important to further improve its efficiency and engineering. The haploid technical process can be divided into the following four links: ① generation of haploid; ② identification of haploid; ③ doubling of haploid; ④ utilization of DH line. Among them, the induction of haploid is crucial to the generation of DH line (doubledhaploid, DH), which determines the number of haploid obtained. Haploid identification is an important link in the engineering of haploid breeding technology, and the accuracy and speed of its identification directly affect the large-scale application of haploid breeding technology.

The factory production of DH lines through tissue culture is another important way to realize haploid engineering breeding. Existing methods for identifying haploids include chromosome counting on root tip tablets, flow cytometry analysis of nucleic acid content, detection of SSR and SNP molecular markers, R1-nj color markers, grain oil markers, grain weight, etc. (Antoine-MichardSandBeckertM, 1997; Chen Shaojiang et al., 2003; BarretPetal, 2008; MelchingerAEetal, 2013; XuXetal, 2013; BartonJEetal, 2014; SmelserAetal, 2015). These methods are mainly effective on grains and plants, but under tissue culture conditions, there is no efficient method for how to realize rapid and accurate selection of haploids.

Contents of the invention

One object of the present invention is to provide a method for identifying or assisting in identifying immature maize haploid embryos.

The method for identifying or assisting in identifying immature corn haploid embryos provided by the present invention is as follows 1) or 2) or 3):

1) Using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of width from small to large, and select and sort For the top 20-30% corn immature embryos, obtain or candidate to obtain maize haploid immature embryos;

2) using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of area from small to large, and select and sort For the top 20-30% corn immature embryos, obtain or candidate to obtain maize haploid immature embryos;

3) using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of length from small to large, and select and sort For the first 20-30% of corn immature embryos, maize haploid immature embryos are obtained or selected.

In the above method, the selection and ranking are the top 30% young corn embryos.

In the above method, the length of the young corn embryo is 2.0-7.0 mm.

In the above method, the corn haploid induction line is Nongda Gaoyu No. 5 or Nongda Gaoyou Gaoyu No. 3; the female parent is a corn inbred line or a corn hybrid.

In the above method, the corn inbred line is B73 or Mo17; the corn hybrid is Zhengdan 958 or B73/Mo17;

The B73/Mo17 is a hybrid offspring of B73 as the female parent and Mo17 as the male parent.

In the above method, the number of young corn embryos is 86-1734; specifically, the number of young corn embryos is 86, 189, 428, 335, 411, 474, 1642 or 1734.

Another object of the present invention is to provide a new application of the above method.

The invention provides the application of the above method in cultivating maize haploid plants.

A final object of the present invention is to provide a method for breeding maize haploid plants.

The method for cultivating corn haploid plants provided by the invention comprises the following steps,

1) Using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; carry out the corn immature embryos according to the order of length or width or area from small to large Sorting, selecting and sorting the top 20-30% corn immature embryos, obtaining or candidate maize haploid immature embryos;

2) The maize haploid immature embryos are doubled on the doubling medium, cultured, and maize haploid plants are selected according to the plant type and/or stalk color.

It is proved by experiments that the method of the present invention can realize the selection of haploids at the immature embryo stage of corn, and a large part of heterozygous diploid immature embryos will be eliminated before the color expression of immature embryos, and the haploid selection will be advanced in advance. The time is improved, the efficiency of selection is improved, the breeding time is further shortened, the follow-up cost is also saved, and the economic benefit is improved. Moreover, the method provided by the invention has the prospect of automatically selecting haploids, and accelerates the process of engineering breeding of maize haploids.

Description of drawings

Figure 1 is a photograph of a haploid immature embryo. Among them, the red arrow points to the haploid immature embryo.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The maize haploid induction line Nongda Gaoyu No. 5 (CAU5) is the male parent in the following examples, in the literature "Dong, X., et al. (2014)."Marker-assisted selection and evaluation of highoilinvivohaploidinducersinmaize."MolecularBreeding34(3):1147 -1158.", publicly available from China Agricultural University.

Maize (Zeamays L.) hybrid Zhengdan 958 is a product of Beijing Si Nong Seed Industry Co., Ltd., and the implementation standard is GB4404.1-2008.

The maize haploid inducible line Nongda Gaoyou Gaoyu No. 3 (CHOI3) is the male parent in the following examples. , Doctoral Dissertation", which is available to the public from China Agricultural University.

Maize inbred lines B73 and Mo17 are the female parents in the following examples, in the literature "LeeM, SharopovaN, BeavisWD, et al. Expanding the genetic map of maize with the intermated B73 × Mo17 (IBM) population [J]. Plantmolecularbiology, 2002, 48 (5-6): 453-461.", publicly available from China Agricultural University.

MS salt is a product of Shanghai Yuhan Biotechnology Co., Ltd., the product catalog number is 140225.

B73/Mo17 is the F1 individual obtained by crossing B73 as the female parent and Mo17 as the male parent. In the literature "LeeM, SharopovaN, BeavisWD, et al. -6):453-461.", publicly available from China Agricultural University.

Embodiment 1, a kind of method for identifying haploid based on maize immature embryo morphology

1. Identification method of haploid immature embryos

1. Hybridization

(1) Hybrid parent

B73 was used as the female parent, and the induced line CAU5 was used as the male parent, and the hybrid offspring A was obtained;

Mo17 was used as the female parent, and the induced line CAU5 was used as the male parent, and the hybrid offspring B was obtained;

B73 was used as the female parent, and the induced line CHOI3 was used as the male parent, and the hybrid offspring C was obtained;

Mo17 was used as the female parent, and the induced line CHOI3 was used as the male parent, and the hybrid offspring D was obtained.

(2) The specific method of hybridization

In the spring of 2015, the female parent and male parent were planted at Beijing Shangzhuang Experimental Station, and field pollination was carried out during the pollination period: after most of the filaments of the female parent materials B73 and Mo17 came out, the induced lines CAU5 and CHOI3 were used in excess on the same day for the female parent. Materials B73 and Mo17 were pollinated; young ears were taken 12-23 days after pollination to obtain hybrid progenies with young embryos of 2-7 mm in length.

2. Selection of pseudo-haploid immature embryos

(1) Embryo peeling and taking pictures

Get the young ears of the hybrid progeny A, hybrid progeny B, hybrid progeny C, and hybrid progeny D whose young embryos are 2.0-7.0 mm long, and peel off the embryos the next day and take pictures (you can temporarily take pictures after the young ears are retrieved). Put it at 4°C, but not for too long). The specific steps are as follows: peel off the bract leaves of the young ears, pick off the filaments, insert them from the top with tweezers, disinfect with 75% alcohol mist and enter the ultra-clean bench, fill 1500ml of 75% alcohol with a clean large beaker, put the corn in, Stir well and sterilize for a while, during which the corn is stirred several times. After the disinfection is completed, take it out, and start to peel the embryos after the alcohol evaporates to obtain corn immature embryos, and place them on the doubling medium, and take pictures of each petri dish with a stereo microscope.

(2) Select pseudo-haplotype

According to the photographing situation, calculate the length of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the length from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

According to the photographing situation, calculate respectively the width of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the width from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

According to the photographing situation, calculate the area of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the area from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

2. Identification of haploid plants

1. The doubling of haploid immature embryos: the pseudo-haploid immature embryos (Fig. 1) selected in the above step 1 and the remaining corn immature embryos are doubled in the medium (MS salt is 3.0g/L, sucrose is 40g/L , agar is 7.5g/L, Tween 80 is 1‰, dimethyl sulfoxide is 2%), doubling in light culture (cultivation time is 1-4 days, temperature is 26-30 ℃), after doubling of immature embryos. Due to the pollen intuition effect, because the heterozygous diploid contains the A1A2C1C2R1-nj gene from the male parent, the expression of purple pigment can be clearly seen on the scutellum of the embryo, while the immature haploid embryo does not contain the dominant gene from the male parent. Therefore, the scutellum of the embryo is colorless. Before being transferred to the regeneration medium for regeneration, most of the heterozygous diploids can be eliminated through the colorless scutellum of the embryo, leaving a very small number of heterozygotes.

2. Growing seedlings: Transfer the doubling immature embryos to regeneration medium (containing MS salt 3.0 g/L, sucrose 30 g/L, agar 7.5 g/L) to grow seedlings. Note that the embryo is facing up at this time. Culture in the dark at 28°C for 3-5 days, and then culture in normal light to obtain seedlings of hybrid progeny; about 10-14 days later, when the seedlings have two leaves and one heart, move them to culture bottles in time, and the culture bottles are MS medium (containing MS Salt 3.0g/L, sucrose 30g/L, agar 7.5g/L). After 7-10 days, the root system of the seedlings is developed, and when the four leaves are in one heart, the seedlings need to be moved to a suitable environment such as a greenhouse in time for hardening.

3. Seedling hardening: move the seedlings of the above-mentioned hybrid progeny to the nutrient bowl, and harden the seedlings in a suitable place such as a greenhouse. When transplanting seedlings, pay attention to rinse the root medium before putting it into the nutrient soil (nutrient soil configuration: nutrient soil: vermiculite = 3:1). During seedling hardening, pay attention to initial shading, water and fertilizer supply, and disease prevention and control. Carbendazim can be used to water once a little.

4. Transplanting: After hardening for about a week, wait for the seedlings to resume growth, at the 5-6 leaf stage, choose rainy weather or evening, and transplant them into field or greenhouse flowerpots. Pay attention to field management to ensure timely and sufficient supply of water and fertilizer. When the powder is loosened, bagging and self-pollination are carried out to obtain haploid doubled plants (double haploid).

5. Identification of haploid plants

Since the plants of the CAU5 and CHOI3 inducible lines are colorless, they cannot be used to distinguish haploid plants from heterozygous diploid plants. At this time, maize haploid plants can be selected according to the plant type: tall plants or scattered leaves It is a heterozygous diploid plant, while the plant is short or the leaves are raised to a haploid plant.

3. The efficiency of identifying haploids based on the morphology of maize immature embryos

According to the identification result of haploid plant, use A to represent to select 20% immature embryo group; Use B to represent to select 30% immature embryo group; Wrong selection rate=heterozygous diploid immature embryo number/ Total number of pseudo-haploid immature embryos; missed selection rate = number of missed haploid immature embryos/total number of embryos. The misselection rate and misselection rate of haploid selection based on the three morphological indicators were counted respectively.

1. The results of identifying haploid immature embryos based on area are shown in Table 1: It can be seen from the table that, on the whole, the misselection rate of identifying haploid immature embryos based on area of 30% of the immature embryo group is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on area to identify haploid immature embryos is significantly lower than that of selecting 20% immature embryo group based on area identification Missing rate of haploid immature embryos.

Table 1. Statistical results of area-based misselection rate and omission rate

2. The results of identifying haploid immature embryos based on length are shown in Table 2: It can be seen from the table that, on the whole, the misselection rate of identifying haploid immature embryos based on length in 30% of the immature embryo group is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on length to identify haploid immature embryos was significantly lower than that of selecting 20% immature embryo group based on length identification Missing rate of haploid immature embryos.

Table 2. Statistical results of length-based misselection rate and missed selection rate

3. The results of identifying haploid immature embryos based on width are shown in Table 3: It can be seen from the table that, on the whole, the misselection rate of selecting 30% of immature embryos based on width to identify haploid immature embryos is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on width to identify haploid immature embryos was significantly lower than that of selecting 20% immature embryo group based on width identification Missing rate of haploid immature embryos.

Table 3. Statistical results of width-based wrong selection rate and missed selection rate

In summary: In this example, the inbred lines B73 and Mo17 were used as the female parent to carry out hybridization to obtain immature embryos of the hybrid offspring. It has been proved by experiments that the method for identifying haploid immature embryos of the present invention can be used for self-fertilization Screening or auxiliary screening of haploid immature embryos obtained from hybrid offspring obtained from the female parent.

Example 2, application of the method for identifying haploids based on the morphology of immature maize embryos

1. Identification method of haploid immature embryos

1. Hybridization

(1) Hybrid parent

Zhengdan 958 was used as the female parent, and the induced line CAU5 was used as the male parent, and crossed to obtain the hybrid offspring E;

Zhengdan 958 was used as the female parent, and the induced line CHOI3 was used as the male parent, and crossed to obtain the hybrid offspring F;

B73/Mo17 was used as the female parent, and the induced line CAU5 was used as the male parent, and crossed to obtain the hybrid offspring G;

B73/Mo17 was used as the female parent, and the induced line CHOI3 was used as the male parent, and the hybrid progeny H was obtained.

(2) The specific method of hybridization

In the spring of 2015, the female parent and male parent were planted at the Shangzhuang Experimental Station in Beijing, and field pollination was carried out during the pollination period: after most of the female parent materials Zhengdan 958 and B73/Mo17 had all the filaments, the inducible lines CAU5 and CHOI3 pollinated the female parent materials Zhengdan 958 and B73/Mo17; harvested young ears 12-23 days after pollination to obtain hybrid offspring with young embryos of 2-7 mm in length.

2. Selection of pseudo-haploid immature embryos

(1) Embryo peeling and taking pictures

Get the young ears of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H whose young embryos are 2.0-7.0 mm long, and peel off the embryos the next day and take pictures (you can temporarily take pictures after the young ears are retrieved). Put it at 4°C, but not for too long). The specific steps are as follows: peel off the bract leaves of the young ears, pick off the filaments, insert them from the top with tweezers, disinfect with 75% alcohol mist and enter the ultra-clean bench, fill 1500ml of 75% alcohol with a clean large beaker, put the corn in, Stir well and sterilize for a while, during which the corn is stirred several times. After the disinfection is completed, take it out, and start to peel the embryos after the alcohol evaporates to obtain corn immature embryos, and place them on the doubling medium, and take pictures of each petri dish with a stereo microscope.

(2) Select pseudo-haplotype

According to the photographing situation, calculate the length of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the length from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

According to the photographing situation, calculate respectively the width of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the width from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

According to the photographing situation, calculate the area of the corn immature embryos of the hybrid progeny E, hybrid progeny F, hybrid progeny G, and hybrid progeny H obtained in the above step 1, and sort the corn immature embryos according to the area from small to large, Mark the immature embryos ranked as the top 20% (select the 20% immature embryo group) and the top 30% (select the 30% immature embryo group), which are pseudo-haploid immature embryos.

2. Identification of haploid plants

1, the doubling of haploid immature embryos: the pseudo-haploid immature embryos selected in the above step 1 and the remaining corn immature embryos were placed in the doubling medium (MS salt is 3.0g/L, sucrose is 40g/L, agar is 7.5 g/L, Tween 80 at 1‰, and dimethyl sulfoxide at 2%), cultured under light (1-4 days of culture, temperature at 26-30° C.) to obtain doubling immature embryos. Due to the pollen intuition effect, because the heterozygous diploid contains the A1A2C1C2R1-nj gene from the male parent, the expression of purple pigment can be clearly seen on the scutellum of the embryo, while the immature haploid embryo does not contain the dominant gene from the male parent. Therefore, the scutellum of the embryo is colorless. Before being transferred to the regeneration medium for regeneration, most of the heterozygous diploids can be eliminated through the colorless scutellum of the embryo, leaving a very small number of heterozygotes.

2. Growing seedlings: Transfer the doubling immature embryos to regeneration medium (containing MS salt 3.0 g/L, sucrose 30 g/L, agar 7.5 g/L) to grow seedlings. Note that the embryo is facing up at this time. Culture in the dark at 28°C for 3-5 days, and then culture in normal light to obtain seedlings of hybrid progeny; about 10-14 days later, when the seedlings have two leaves and one heart, move them to culture bottles in time, and the culture bottles are MS medium (containing MS Salt 3.0g/L, sucrose 30g/L, agar 7.5g/L). After 7-10 days, the root system of the seedlings is developed, and when the four leaves are in one heart, the seedlings need to be moved to a suitable environment such as a greenhouse in time for hardening.

3. Seedling hardening: move the seedlings of the above-mentioned hybrid progeny to the nutrient bowl, and harden the seedlings in a suitable place such as a greenhouse. When transplanting seedlings, pay attention to rinse the root medium before putting it into the nutrient soil (nutrient soil configuration: nutrient soil: vermiculite = 3:1). During seedling hardening, pay attention to initial shading, water and fertilizer supply, and disease prevention and control. Carbendazim can be used to water once a little.

4. Transplanting: After hardening for about a week, wait for the seedlings to resume growth, at the 5-6 leaf stage, choose rainy weather or evening, and transplant them into field or greenhouse flowerpots. Pay attention to field management to ensure timely and sufficient supply of water and fertilizer. When the powder is loosened, bagging and self-pollination are carried out to obtain haploid doubled plants (double haploid).

5. Identification of haploid plants

Since the plants of the CAU5 and CHOI3 inducible lines are colorless, they cannot be used to distinguish haploid plants from heterozygous diploid plants. At this time, maize haploid plants can be selected according to the plant type: tall plants or scattered leaves It is a heterozygous diploid plant, while the plant is short or the leaves are raised to a haploid plant.

3. The efficiency of identifying haploids based on the morphology of maize immature embryos

According to the identification results of haploid plants, use A to represent the selection of 20% of the immature embryo group; use B to represent the selection of 30% of the immature embryo group; misselection rate=the number of heterozygous diploids in pseudo-haploids)/pseudo-haploids Total number of somatic immature embryos; missed selection rate = number of missed haploid immature embryos/total number of embryos. The misselection rate and misselection rate of haploid selection based on the three morphological indicators were counted respectively.

1. The results of identifying haploid immature embryos based on area are shown in Table 4: It can be seen from the table that, on the whole, the misselection rate of selecting 30% of immature embryos based on area to identify haploid immature embryos is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on area to identify haploid immature embryos is significantly lower than that of selecting 20% immature embryo group based on area identification Missing rate of haploid immature embryos.

Table 4. Statistical results of misselection rate and omission rate based on area

2. The results of identifying haploid immature embryos based on length are shown in Table 5: It can be seen from the table that, on the whole, the misselection rate of identifying haploid immature embryos based on length in 30% of the immature embryo group is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on length to identify haploid immature embryos was significantly lower than that of selecting 20% immature embryo group based on length identification Missing rate of haploid immature embryos.

Table 5. Statistical results of wrong selection rate and missed selection rate based on length

3. The results of identifying haploid immature embryos based on width are shown in Table 6: It can be seen from the table that, on the whole, the misselection rate of selecting 30% of immature embryos based on width to identify haploid immature embryos is slightly higher than that of The misselection rate of selecting 20% immature embryo group based on width to identify haploid immature embryos is significantly lower than that of selecting 20% immature embryo group based on area identification Missing rate of haploid immature embryos.

Table 6. Statistical results of wrong selection rate and missed selection rate based on width

In summary: In this example, the hybrid Zhengdan 958 and B73/Mo17 were used as the female parent to perform hybridization to obtain immature embryos of the hybrid offspring. It has been proved by experiments that the method of the present invention can be used to obtain hybrids using the hybrid as the female parent. Screening of haploid immature embryos from hybrid progenies.

Claims (8)

1. A method for distinguishing or assisting in distinguishing maize haploid immature embryos, which is as follows 1) or 2) or 3): 1) Using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of width from small to large, and select and sort For the top 20-30% corn immature embryos, obtain or candidate to obtain maize haploid immature embryos; 2) using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of area from small to large, and select and sort For the top 20-30% corn immature embryos, obtain or candidate to obtain maize haploid immature embryos; 3) using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; sort the corn immature embryos according to the order of length from small to large, and select and sort For the first 20-30% of corn immature embryos, maize haploid immature embryos are obtained or selected. 2. The method according to claim 1, characterized in that: the selection and ranking are the top 30% corn immature embryos. 3. The method according to claim 1 or 2, characterized in that: the length of the young corn embryo is 2.0-7.0 mm. 4. The method according to any one of claims 1-3, characterized in that: the maize haploid induction line is Nongda Gaoyu No. 5 or Nongda Gaoyou Gaoyu No. 3; Cross-line or corn hybrid. 5. The method according to any one of claims 1-4, characterized in that: said corn inbred line is B73 or Mo17; said corn hybrid is Zhengdan 958 or B73/Mo17; The B73/Mo17 is a hybrid offspring of B73 as the female parent and Mo17 as the male parent. 6. The method according to any one of claims 1-5, characterized in that: the number of young corn embryos is 86-1734. 7. Application of the method according to any one of claims 1-6 in cultivating maize haploid plants. 8. A method for cultivating corn haploid plants, comprising the steps of: 1) Using the maize haploid induction line as the male parent to pollinate the female parent, and after pollination, take the young ears and peel off the embryos to obtain corn immature embryos; carry out the corn immature embryos according to the order of length or width or area from small to large Sorting, selecting and sorting the top 20-30% corn immature embryos, obtaining or candidate maize haploid immature embryos; 2) The maize haploid immature embryos are doubled on the doubling medium, cultured, and maize haploid plants are selected according to the plant type and/or stalk color.