A method for creating maize germplasm with low mycotoxin and high starch content by gene editing technology and application thereof

Abstract

The present application belongs to the technical field of genetic breeding, and particularly relates to a method for efficiently creating new germplasm of maize with low mycotoxin and high starch content by using gene editing technology and application thereof. The method comprises using CRISPR / Cas9 multi-gene editing technology and genetic transformation method, and in the genetic transformation process, one or more of betaine and calcium chloride are contained in the infection medium. On the basis of the research on the existing starch synthesis and ear rot related functional genes, the high-throughput gene editing technology is used to efficiently and randomly aggregate a large number of excellent sites, and in the genetic transformation process, appropriate concentrations of betaine and calcium chloride are added, so that more numbers are edited in a single transgenic event, the efficiency is better, the diversity degree is effectively improved, more excellent site combination modes are generated, a large number of new germplasm with high starch content and resistance to diseases such as ear rot are efficiently and large-scale produced, and the method has good application value in gene editing breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic breeding, and particularly relates to a method for efficiently creating new maize germplasm with low mycotoxin and high starch content by using gene editing technology and application thereof. BACKGROUND

[0002] Gene editing can cause point mutations in the coding region of a gene to weaken and delete the function of the gene, and can also cause fragment deletion in the promoter element and UTR region through a double-target system to regulate gene expression and further regulate gene function. In recent years, the CRISPR / Cas9 system has been widely used for gene editing in various species due to its high targeting specificity, simple and fast operation, and other advantages, and has achieved large-scale mutant resource creation in rice and soybeans. However, these large-scale gene editing studies mainly use CRISPR / Cas9 as a substitute for traditional mutation methods, and there is a lack of in-depth research on how to improve the high-throughput system from target design to mutation sequence detection, reduce costs, and explore the rules of the impact of this emerging technology on the genome. Professor Jianbing Yan of Huazhong Agricultural University used high-throughput single-target gene editing technology to obtain mutant plants with multiple phenotypes, including plant height, ear position, stress, and grain agronomic traits, by high-throughput gene editing of several hundred gene coding regions, which can verify the function of multiple genes. However, the gene editing efficiency needs to be further improved. At the same time, it is mainly used for gene function verification and cannot achieve high-throughput material creation. Only by achieving high-throughput excellent material creation can the optimal breeding material be selected from it, and the genetic improvement of maize varieties can be truly realized.

[0003] High-throughput gene editing technology is currently mainly used for gene function verification and cannot achieve high-throughput breeding material creation. Only by achieving high-throughput excellent material creation can the optimal breeding material be selected from it, and the genetic improvement of maize varieties can be truly realized. With the continuous optimization and improvement of maize genetic transformation efficiency, the number of verified functional genes is increasing, and it is possible to achieve shape improvement through targeted gene editing technology. However, current gene editing is mainly used for single or several gene function modification and cannot achieve large-scale efficient aggregation of excellent functions, resulting in low innovation rate and low selectivity of new germplasm, which limits the development of germplasm innovation.

[0004] Therefore, seeking a method for efficiently creating new maize germplasm by using gene editing technology is the key to realizing efficient aggregation of excellent functional sites in maize, improving the innovation rate and selectivity of new germplasm. SUMMARY

[0005] In order to solve the above problems, the application provides a method for efficiently creating new germplasm of maize with low mycotoxin and high starch content by using gene editing technology and application thereof. In the process of genetic transformation, by adding appropriate amounts of betaine and calcium chloride in the infection medium, the probability of site editing in a single transgenic event is greatly improved, and at the same time, the diversification and efficient aggregation of excellent sites with high starch content and low mycotoxin content (anti-eared grain rot, etc.) are realized, which greatly improves the speed and efficiency of creating excellent new germplasm, and is a fast and efficient method for creating new breeding materials at present. Based on the above research, the patent is completed.

[0006] In order to achieve the above object, the application adopts the following technical scheme:

[0007] In the first aspect, the application provides a method for improving the efficiency of gene editing in maize, which comprises using CRISPR / Cas9 multi-gene editing technology and genetic transformation method, and in the process of genetic transformation, the infection medium contains one or more of betaine and calcium chloride.

[0008] Further, the method specifically comprises the following steps:

[0009] S1, selecting a target gene and designing gRNA;

[0010] S2, using the CRISPR / Cas9 system, obtaining a double-target knockout vector by means of enzyme cutting and connection, gene recombination, and transforming Agrobacterium in an equal amount to prepare an Agrobacterium mixed pool;

[0011] S3, genetic transformation: the Agrobacterium mixed pool obtained in S2 is transformed into a maize transformant by genetic transformation, and positive seedlings are obtained by herbicide resistance screening; wherein, in the process of genetic transformation, the infection medium contains 80-120 mM betaine and / or 30-60 mM calcium chloride;

[0012] S4, planting and screening homozygous edited plants, then counting the editing efficiency, and observing the phenotype.

[0013] Further, in S1, according to the sequence information of the maize genome, specific sgRNA sequences are designed for the genes related to eared grain rot, low mycotoxin and auxin content (predicted to be related to low mycotoxin);

[0014] Or,

[0015] In the step S2, the sgRNA prepared in the step S1 is integrated into the CRISPR / Cas9 vector, and after sequencing verification and construction of the correct one, a plurality of double-target knockout vectors are formed;

[0016] The plurality of double-target knockout vectors are transformed into Agrobacterium EHA105, and the Agrobacterium is mixed in an equal amount to obtain a mixed pool which is used for subsequent genetic transformation.

[0017] Further, the Agrobacterium EHA105 has a volume of 80-100 uL.

[0018] Further, in the step S2, the CRISPR / Cas9 vector is a binary expression vector, preferably, the binary expression vector is pCXB053.

[0019] Further, in the step S3, the genetic transformation specifically comprises the following steps:

[0020] S3-1, placing the corn embryo into the suspension liquid containing 1.5-2 mL, and treating for 25-35 min;

[0021] S3-2, sucking off the treatment liquid, adding the infection medium, and standing for infection for 3-8 min;

[0022] S3-3, transferring the infected corn embryo to the co-culture medium, and culturing in the dark at 22-24 °C for 3 days, then transferring the corn embryo to the resting medium, and culturing in the dark at 27-29 °C for 5-7 d;

[0023] S3-4, taking the corn embryo after the culture in S3-3 ends, cutting off the radicle, and transferring to the screening medium containing 5 mg / L Bialaphos for culture for 14 d, then transferring to the screening medium containing 8 mg / L Bialaphos for culture for 14 d, to obtain the resistant callus;

[0024] S3-5, transferring the resistant callus to the differentiation medium, and culturing under light at 24-26 °C, 5000 lx, for three weeks, to obtain the regenerated seedling.

[0025] Further, the infection medium comprises 1 / 2MS, sucrose 68.5 g / L, glucose 36 g / L, L-proline 0.115 g / L, acetosyringone 200 mM, cysteine 200 mg / L, Agrobacterium mixed liquid with OD600 of 0.5-1, betaine 100 mM, and / or calcium chloride 50 mM.

[0026] Further, the infection medium comprises 1 / 2MS, sucrose 68.5 g / L, glucose 36 g / L, L-proline 0.115 g / L, acetosyringone 200 mM, cysteine 200 mg / L, Agrobacterium mixed liquid with OD600 of 0.5-1, betaine 100 mM, and calcium chloride 50 mM.

[0027] In a second aspect, the application provides the use of the above method in creating new germplasm in plant breeding.

[0028] In a third aspect, the application provides the use of the above method in creating new germplasm of corn with low mycotoxin content and high starch content.

[0029] In a fourth aspect, the present application provides a method for creating new germplasm of maize with low mycotoxin content and high starch content, which uses the above method to edit the ZmGBSSI, Shrunken 2, Brittle 2, ZmDOF36, ZmTPS9, ZmAuxRP1, TAR4-1, TAR4-3, Yucca2, Yucca4, ZmSIZ1a, ZmSIZ1b, ZmFER1, ZmTSB2, ZmTSB1, ZmIGS, AUX2, TAR2, Yucca6, AGPLS3 genes of maize, and obtains after screening.

[0030] The beneficial technical effects of the one or more technical solutions described above are:

[0031] (1) The present application selects multiple starch synthesis and ear rot related functional genes from the perspective of improving the efficiency of the technology itself, including the genes related to starch content and ear rot that have been studied: ZmGBSSI, Shrunken 2, AGPLS3, Brittle2, ZmDOF36, ZmTPS9, ZmAuxRP1, ZmSIZ1a, ZmSIZ1b, ZmFER1, and the genes related to prediction: ZmTAR4-1, ZmTAR4-3, Yucca2, Yucca4, ZmTSB2, ZmTSB1, ZmIGS, ZmAUX2, ZmTAR2, ZmYUC6. Using high-throughput gene editing technology, a large number of excellent sites are efficiently and randomly aggregated, and an appropriate concentration of betaine and calcium chloride is added during genetic transformation, so that the number of editing sites in a single transgenic event is more, the efficiency is better, the diversification degree is higher, more excellent site combination modes are generated, and a large number of new germplasm with high starch content and low mycotoxin content (resistance to ear rot and other diseases) are efficiently and scaled. It is an efficient application method for creating new germplasm required for breeding by using gene editing. At the same time, by performing field multi-point and multiple repetition of phenotype identification, large-scale, systematic and precise screening of excellent planting resources required for breeding can be realized, and the resources can be quickly and efficiently applied to breeding. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and their description serve to explain the present application, and do not constitute improper limitations on the present application.

[0033] Figure 1 The average number of edits in a single event in the transformation event obtained by treating betaine and calcium chloride in the embodiments of the present application, wherein the concentration of betaine is 100 mM and the concentration of calcium chloride is 50 mM;

[0034] Figure 2For the high starch content mutants and starch content identified and screened in the gene editing materials in the embodiments of the present application, zml-cr11 and zml-cr22 are selected from the transformation events obtained by betaine treatment; zml-cr42 and zml-cr56 are selected from the transformation events obtained by calcium chloride treatment; zml-cr74 and zml-cr92 are selected from the transformation events obtained by betaine + calcium chloride treatment. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to limit the scope of the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0037] The embodiments of the present application will be described in detail below with reference to the embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and the guidelines given in the present application are preferred. The experimental methods can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or according to the known experimental methods in the art.

[0038] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range, unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0039] Glossary:

[0040] In the present application, the multi-gene editing technology refers to a technology for simultaneously editing multiple sites in a single transgenic event using CRISPR / Ca9 gene editing technology.

[0041] In the application, the gene editing efficiency is the polymeric editing efficiency, which is the number of vectors corresponding to the editing of multiple target sites in one transgenic event using CRISPR / Cas9 mediated multi-gene editing technology.

[0042] In the application, the target site is the target point.

[0043] Example 1

[0044] A method for efficiently creating new germplasm with low mycotoxin and high starch content by using gene editing technology

[0045] I. Experimental materials

[0046] 1. Zea mays inbred line KN5585 immature embryos;

[0047] The backbone vector used for gene editing target is pCXB053, and the Agrobacterium tumefaciens strain is EHA105 (purchased from Shanghai Weidi);

[0048] Plant grain starch content and mycotoxin content detection kit

[0049] Culture medium: YP medium, infection medium, co-culture medium, rest medium, screening medium 1, screening medium 2, differentiation medium, and rooting medium. The specific components are shown in Table 1.

[0050] Table 1 Specific components of different types of MS medium

[0051]

[0052]

[0053] The above trace components are prepared as stock solutions. A large amount of solid components are weighed on a balance, dissolved in appropriate deionized water, adjusted to pH, and then sterilized at high temperature and pressure for 20 min. When the temperature decreases to about 50℃, the liquid components that are not resistant to high temperature need to be filtered and sterilized using a bacteria filter, and then added to the culture medium using a disposable needle tube. Pour into a culture dish. Then store in a 4℃ refrigerator for standby.

[0054] II. Experimental methods

[0055] 1. Select target genes and design gRNA

[0056] 1.1 Gene selection: 20 genes related to high starch content and low mycotoxin were selected, including 6 starch-related genes and 14 ear rot-related genes. Studies have shown that the reduction of auxin content in the ear is beneficial to improve the resistance of corn to ear rot disease, so the genes related to auxin content were also selected as the pre-editing genes of the present application, including Yucca2, Yucca4, Yucca6, TAR4-1, TAR4-3, AUX2, TAR2, ZmIGS, ZmTSB1, ZmTSB2. The names and gene numbers of the genes are shown in Table 2.

[0057] Table 2 Names and gene numbers of genes

[0058]

[0059]

[0060] 1.2 Batch target synthesis

[0061] Batch target synthesis was carried out by double target of each vector. The genes were divided into negative regulation and positive regulation. For the 13 negative regulation genes, 2 target points were designed in the coding region to cause frame shift mutation, 13 double target vectors were constructed for each gene to obtain partial or complete deletion of gene function. 3'UTR (3' Untranslated Region) is the untranslated region at the tail end of the gene after transcription, which plays an important role in the regulation process after transcription. It has important influence on gene expression, mRNA stability, positioning and post-transcriptional modification. In the present application, for the 7 positive regulation genes, the method of double target in 3'UTR region was adopted to knock out the fragment, 2 pairs (4 targets) were designed for each gene, 2 double vectors were constructed for each gene, and a total of 14 vectors were constructed to obtain the variant germplasm with up-regulated expression of gene protein. A total of 27 double target vectors are needed, and new germplasm materials with high starch and low mycotoxin content are expected to be created. The specific primers and sequences are shown in Table 3.

[0062] Table 3 Specific primers and sequences

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] 1.3 High-throughput vector construction

[0069] The target gene is designed and specific sgRNA sequence is screened by CRISPR-P 2.0 software, and BsaI enzyme site is added respectively, which is used for synthesizing primers for preparing double-target vector. The vector construction method is referred to [Zhang M, Wang J, Yan J, et al. Creation of maize diversity allelic variation by CRISPR / Cas9 gene editing fragment knockout technology [J]. Shandong Agricultural Science, 2024, 56(09): 1-5. DOI:10.14083 / j.issn.1001-4942.2024.09.001.]. The target backbone vector is pCXB053 (specific reference doi:10.1105 / tpc.19.00934.), and the double-target vector is reserved after sequencing.

[0070] 1.4 Agrobacterium culture

[0071] The constructed plasmid was transformed into 100 uL Agrobacterium EHA105, and the successfully transformed Agrobacterium was activated in YP liquid medium. When the OD600 value was between 0.5 and 1 (the OD600 value used in this experiment was 0.6), 100 uL of bacterial solution was mixed to prepare Agrobacterium pool for the next genetic transformation.

[0072] 1.5 High-throughput genetic transformation

[0073] The plasmid-transformed Agrobacterium pool was transformed into the receptor inbred line KN5585. During the genetic transformation process, the infection medium was treated with betaine and / or calcium chloride. Treatment 1: 100 mM betaine, treatment 2: 50 mM calcium chloride, treatment 3: 100 mM betaine + 50 mM calcium chloride, and normal AS suspension without betaine and calcium chloride was used as a control. As shown in Table 4:

[0074] Table 4 Infection medium treatment method

[0075]

[0076] The suspension was 1 / 2MS + sucrose 68.5 g / L + glucose 36 g / L + L-proline 0.115 g / L.

[0077] The suspension containing AS was 1 / 2MS + sucrose 68.5 g / L + glucose 36 g / L + L-proline 0.115 g / L + acetyl-syringone (AS) 200 mM + cysteine 200 mg / L.

[0078] Specifically, immature embryos of about 1 mm of fresh peeled corn inbred line KN5585 (inbred line bred by Jiangsu Unicrop Biotech Co., Ltd.) were used as materials, and the peeled corn embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension or treatment solution (see table below) to maintain the osmotic pressure of the immature embryos, and about 150 immature embryos were treated within 30 min; the suspension was aspirated, and the remaining corn embryos in the tube were then added with 1.0 mL of Agrobacterium-containing infection medium and placed for 5 min. The concentration of the bacterial solution in the infection medium was diluted to OD600 between 0.5 and 1, and the concentration used in this experiment was 0.6.

[0079] After the centrifuge tube was suspended, the immature embryos were poured into the co-culture medium, and the excess Agrobacterium solution on the surface was aspirated with a pipette, and co-cultured at 23°C in the dark for 3 days. After co-culturing, the immature embryos were transferred to the resting medium and cultured at 28°C in the dark for 6 days, and then placed in screening medium 1 containing 5 mg / L Bialaphos (a kind of herbicide), and the screening was started for 2 weeks, and then transferred to screening medium 2 containing 8 mg / L Bialaphos for 2 weeks of screening to obtain resistant callus.

[0080] The resistant callus was transferred to the differentiation medium and cultured at 25°C under 5000 lx light for 3 weeks; the small seedlings differentiated from the differentiation medium were transferred to the rooting medium and cultured at 25°C under 5000 lx light until rooting; the seedlings were transferred to pots for growth, and after a certain growth stage, they were transplanted to a greenhouse, and the offspring seeds were harvested after 3-4 months.

[0081] 1.6 The positive seedlings obtained were taken to the modern greenhouse in Xishuangbanna, and were carefully cared for and managed to collect seeds. Then, the transgenic system was removed by separation to obtain homozygous transformation events, and not less than 80 homozygous transformation events were obtained for each treatment.

[0082] 1.7 The leaves of the experimental materials in the field were taken, and DNA was extracted. At the same time, 23 pairs of primers were designed according to the target editing position for identification of gene editing types. The primers used are as shown in Table 5.

[0083] Table 5 Identification primer sequences

[0084]

[0085]

[0086] Each processing obtained gene editing material randomly picked 30 (total 120) to identify the type of editing, each single plant with 23 pairs of primers for PCR amplification after sanger sequencing, to obtain accurate editing type. Statistics site editing number, statistical rules: each vector has two targets, one of the two target sites occurs editing (base deletion, insertion, substitution, etc.), two occur editing or two targets between the corresponding fragment editing (fragment deletion, replacement, etc.), any of the above cases are considered to be edited by the vector to play a role and counted as 1, that is, the number of editing refers to the number of single transformation event vector editing Figure 1 ).

[0087] The results are shown in Figure 1 , whether it is the addition of betaine or calcium chloride alone, or both, the number of single transformation event editing is significantly higher than the control, both add the number of editing sites. Compared with the control, after the treatment of betaine alone, the editing efficiency is improved by 22.6%, after the treatment of calcium chloride alone, the editing efficiency is improved by 15.1%, betaine and calcium chloride synergistically promote the efficiency of gene editing, compared with the control group, it is improved by 81%. Therefore, betaine and calcium chloride can effectively promote the efficiency of target site polymerization editing, improve the efficiency of gene editing, which provides a new way for the innovation of maize germplasm.

[0088] 1.8 The homozygous gene editing material seeds are planted in the field (Shandong) for multi-point and multi-repetition of field phenotype identification screening, and pollen is collected for seed detection of starch content and mycotoxin content. The content of aflatoxin B1, which is well known and relatively important, represents the content of mycotoxin. New germplasm materials with good comprehensive performance, high starch and low mycotoxin, etc. are screened Figure 2, Table 6). Six gene editing materials with relatively high starch content were identified in the screening, two of which were selected from the transformation events obtained by betaine treatment, two from calcium chloride treatment, and two from betaine + calcium chloride treatment. Shandong belongs to the Huanghuaihai region with variable climate conditions, so the experimental points with more serious spike grain rot were selected from the repeated environment to determine the mycotoxin content of the grains, and seven germplasm with very low mycotoxin content were selected (less than 3 ug / kg is not easy to measure, represented as <3 ug / kg) (Table 6), two of which were from the transformation events obtained by betaine treatment, two from calcium chloride treatment, and three from betaine + calcium chloride treatment. Due to the limitation of sample size, we have not screened enough gene editing materials with high starch and low mycotoxin content, so we cannot statistically compare which treatment obtains more germplasm with high starch and low mycotoxin content. However, it can be determined that betaine, calcium chloride and betaine + calcium chloride treatment can effectively increase the number of edits in the average individual transformation event, providing a new idea for efficient directional and precise breeding of new germplasm materials.

[0089] Table 6 Six gene editing materials with relatively high starch content identified in the screening

[0090] Name Mycotoxin content (ug / KG) Treatment KN5585 6.2 Control zml-cr14 <3 Betaine 100 mM zml-cr16 <3 Betaine 100 mM zml-cr19 <3 Calcium chloride 50 mM zml-cr44 <3 Calcium chloride 50 mM zml-cr48 <3 Betaine 100 mM + Calcium chloride 50 mM zml-cr76 <3 Betaine 100 mM + Calcium chloride 50 mM zml-cr96 <3 Betaine 100 mM + Calcium chloride 50 mM

[0091] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method of increasing gene editing efficiency in maize, comprising, The method comprises using CRISPR / Cas9 multi-gene editing technology and genetic transformation method, and 80-120 mM betaine and 30-60 mM calcium chloride are contained in the infection medium in the genetic transformation process.

2. The method for improving gene editing efficiency in maize as described in claim 1, characterized in that, Specifically, the method comprises the following steps: S1, selecting a target gene and designing gRNA; S2, using the CRISPR / Cas9 system, obtaining a double-target knockout vector through enzyme cutting and connection and gene recombination, and then mixing equal amounts of agrobacterium to obtain a mixed agrobacterium after single transformation of the agrobacterium; S3, genetic transformation: the mixed agrobacterium obtained in S2 is transformed into a corn transformation body through genetic transformation, and positive seedlings are obtained through herbicide resistance screening; S4, planting and screening homozygous edited plants to count the editing efficiency and observe the phenotype.

3. The method of increasing gene editing efficiency in maize of claim 2, wherein, In the step S1, according to the corn genome sequence information, specific sgRNA sequences are designed for ear rot, low mycotoxin and auxin content related genes; Or, In the step S2, the sgRNA prepared in the step S1 is integrated into a CRISPR / Cas9 vector, and after sequencing verification and confirmation of correct construction, a plurality of double-target knockout vectors are formed; The plurality of double-target knockout vectors are transformed into agrobacterium EHA105, and the agrobacterium is mixed in equal amounts to obtain a mixed pool of bacteria, which is used for subsequent genetic transformation.

4. The method of improving gene editing efficiency in maize of claim 3, wherein, In the step S2, the CRISPR / Cas9 vector is a binary expression vector.

5. The method of improving gene editing efficiency in maize of claim 4, wherein, The binary expression vector is pCXB053.

6. The method for improving gene editing efficiency in maize of claim 2, wherein, In the step S3, the genetic transformation specifically comprises the following steps: S3-1, placing the corn embryo into a suspension containing 1.5-2 mL, and treating for 25-35 min; S3-2, removing the treatment liquid, adding an infection medium, and standing for 3-8 min for infection; S3-3, transferring the infected corn embryo to a co-culture medium, and culturing in the dark at 22-24 DEG C for 3 days, and then transferring the corn embryo to a resting medium, and culturing in the dark at 27-29 DEG C for 5-7 d; S3-4, removing the radicle of the corn embryo after the culture in S3-3, and transferring to a selection medium containing 5 mg / L Bialaphos for culture for 14 d, and then transferring to a selection medium containing 8 mg / L Bialaphos for culture for 14 d, to obtain resistant callus; S3-5, transferring the resistant callus to a differentiation medium, and culturing under light at 24-26 DEG C and 5000 lx for 3 weeks to obtain regenerated seedlings.

7. The method for improving gene editing efficiency in maize of claim 6, wherein, The infection medium comprises 1 / 2MS, sucrose 68.5 g / L, glucose 36 g / L, L-proline 0.115 g / L, acetyl-syringone 200 mM, cysteine 200 mg / L, agrobacterium mixed liquid with OD600 of 0.5-1, betaine 100 mM and calcium chloride 50 mM.

8. Application of the method of claim 1 in creating plant breeding germplasm.

9. Application of the method of claim 1 in creating corn germplasm with low mycotoxin and high starch content.

10. A method of creating a low mycotoxin and high starch content maize germplasm, characterized by, The method of claim 1 is used to edit the genes of ZmGBSSI, Shrunken 2, Brittle 2, ZmDOF36, ZmTPS9, ZmAuxRP1, TAR4-1, TAR4-3, Yucca2, Yucca4, ZmSIZ1a, ZmSIZ1b, ZmFER1, ZmTSB2, ZmTSB1, ZmIGS, AUX2, TAR2, Yucca6, AGPLS3 of corn, and after screening, the following are obtained.

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