Use of zmhscf1 protein and encoding gene thereof in regulating callus proliferation and plant regeneration

Abstract

Provided is a use of ZmHSCF1 protein and an encoding gene thereof in regulating callus proliferation and plant regeneration. Overexpression of ZmHSCF1 gene can promote callus formation and proliferation in maize, and can significantly increase the callus induction rate, the proportion of resistant calluses, the regeneration efficiency, and the transformation efficiency.

Description

Application of ZmHSCF1 protein and its encoding gene in regulating callus proliferation and plant regeneration

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application No. 202510282895.0, filed on March 11, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the fields of genetic engineering and transgenic technology, specifically relating to the application of ZmHSCF1 protein and its encoding gene in regulating callus proliferation and plant regeneration. Background Technology

[0004] With the advent of the Molecular Breeding 4.0 era, the use of biotechnologies, including transgenics, gene editing, and diffraction techniques, for targeted, precise, and rapid design, improvement, and creation of new crop lines has become a major approach to addressing the balance between population, resources, environment, and food security. From PEG-mediated transformation, gene gun methods, Agrobacterium-mediated transformation, pollen tube introduction, to nanomagnetic bead methods, plant transformation methods have continuously evolved. However, Agrobacterium-mediated transformation remains the most widely used transformation method in transgenics and gene editing due to its ease of operation, low cost, good genetic stability, and mature and relatively stable system.

[0005] However, the genotype dependence of Agrobacterium-mediated transformation recipients remains a bottleneck for the further application of this method in modern agricultural breeding based on molecular design breeding. Even with relatively stable and efficient genetic transformation systems, recipient genotypes still require multiple generations of backcrossing with superior parents due to issues such as poor agronomic traits. However, backcrossing efficiency is limited by the genetic distance between the target gene and the desirable traits, and the process is time-consuming and labor-intensive. More importantly, it is difficult to achieve the transfer of a single target gene, which may alter one or more desirable traits in the breeding parents, affecting breeding outcomes. Therefore, breaking down the genotype dependence barrier, avoiding backcrossing, and establishing a crop genetic transformation system using commercially available inbred lines as transformation recipients will greatly facilitate the better application of modern biotechnologies such as transgenics, gene editing and its derivatives, and synthetic biology in molecular design breeding, providing technical support for the efficient creation of new breeding materials.

[0006] Invention Overview

[0007] The technical problem to be solved by this invention is how to promote the formation or proliferation of plant callus tissue and improve the efficiency of plant genetic transformation. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] To address the aforementioned technical problems, this invention first provides a novel use for the ZmHSCF1 protein.

[0009] This invention provides the use of ZmHSCF1 protein in any of the following (A1)-A14):

[0010] A1) Promotes the formation of callus tissue in plant explants;

[0011] A2) Prepare products that promote the formation of callus tissue in plant explants;

[0012] A3) Promotes the proliferation of callus tissue in plant explants;

[0013] A4) Prepare products that promote the proliferation of callus tissue in plant explants;

[0014] A5) Increase the proportion of positive callus for the target gene in plant explants;

[0015] A6) Prepare products that increase the proportion of positive callus for the target gene in plant explants;

[0016] A7) Improve plant regeneration ability;

[0017] A8) Prepare products that enhance plant regeneration capabilities;

[0018] A9) Improve the efficiency of plant genetic transformation;

[0019] A10) Prepare products that improve the efficiency of plant genetic transformation;

[0020] A11) Plant genetic transformation;

[0021] A12) Preparation of plant genetic transformation products;

[0022] A13) Plant breeding;

[0023] A14) Preparation of plant breeding products;

[0024] The ZmHSCF1 protein is a1), a2), a3), or a4):

[0025] a1) The amino acid sequence is that of the protein shown in sequence 4;

[0026] a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in sequence 4;

[0027] a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4, which are related to the efficiency of plant callus formation or regeneration or genetic transformation.

[0028] a4) Proteins that share 75% or more of the amino acid sequence shown in Sequence 4, are derived from maize, and are associated with plant callus formation or regeneration or genetic transformation efficiency.

[0029] Sequence 4 consists of 485 amino acid residues.

[0030] In the protein described in a2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0031] In some embodiments, the fusion protein is formed by fusing DsRed fluorescent protein and ZmHSCF1 protein.

[0032] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0033] In the protein described in a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences that have 75% or more, or 76% or more, or 77% or more, or 78% or more, or 79% or more, or 80% or more, or 81% or more, or 82% or more, or 83% or more, or 84% or more, or 85% or more, or 86% or more, or 87% or more, or 88% or more, or 89% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more identity with the amino acid sequence shown in Sequence 4 of the present invention.

[0034] The proteins described in a1), a2), a3), or a4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0035] To address the aforementioned technical problems, this invention provides new applications for biomaterials related to the ZmHSCF1 protein.

[0036] This invention provides the use of biomaterials related to the ZmHSCF1 protein in any of the following A1)-A14):

[0037] A1) Promotes the formation of callus tissue in plant explants;

[0038] A2) Prepare products that promote the formation of callus tissue in plant explants;

[0039] A3) Promotes the proliferation of callus tissue in plant explants;

[0040] A4) Prepare products that promote the proliferation of callus tissue in plant explants;

[0041] A5) Increase the proportion of positive callus for the target gene in plant explants;

[0042] A6) Prepare products that increase the proportion of positive callus for the target gene in plant explants;

[0043] A7) Improve plant regeneration ability;

[0044] A8) Prepare products that enhance plant regeneration capabilities;

[0045] A9) Improve the efficiency of plant genetic transformation;

[0046] A10) Prepare products that improve the efficiency of plant genetic transformation;

[0047] A11) Plant genetic transformation;

[0048] A12) Preparation of plant genetic transformation products;

[0049] A13) Plant breeding;

[0050] A14) Preparation of plant breeding products;

[0051] The biological material is a nucleic acid molecule encoding the ZmHSCF1 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ.

[0052] The nucleic acid molecule is a gene as shown in B1) or B2) below:

[0053] B1) The DNA molecule shown in sequence 2 or sequence 3;

[0054] The nucleotide sequence defined by B2) has 75% or more identity with that defined by B1) and is a DNA molecule encoding the ZmHSCF1 protein described above.

[0055] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0056] Those skilled in the art can readily mutate the nucleotide sequence encoding the ZmHSCF1 protein of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence encoding the ZmHSCF1 protein, as long as they encode the ZmHSCF1 protein and have the same function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0057] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in Sequence 4 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0058] The aforementioned 75% or more identity can be defined as having an identity of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0059] In the above applications, the expression cassette refers to DNA capable of expressing the ZmHSCF1 protein in host cells. This DNA may include not only a promoter to initiate ZmHSCF1 transcription but also a terminator to terminate ZmHSCF1 transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters; and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0060] In the above applications, the vector can be a plasmid, granule, bacteriophage, or viral vector. The recombinant vector can be a vector containing the ZmHSCF1 gene expression cassette constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3′ untranslated region of the foreign gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3′ end of the mRNA precursor. Similar functions exist in the untranslated regions transcribed at the 3′ end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nosine synthase gene) and plant genes (such as the soybean storage protein gene). When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signal and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed. This can involve adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic marker genes (such as the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methotrexate, and the EPSPS gene for resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0061] In some embodiments, the recombinant vector may be a pCAMBAIA3300-ZmHSCF1 overexpression vector. The pCAMBAIA3300-ZmHSCF1 overexpression vector is obtained by replacing the DNA fragment between the two XcmI restriction sites in the overexpression vector pCAMBAIA3300 with the DNA molecule shown in sequence 3 (ZmHSCF1 gene), while keeping the other sequences of the overexpression vector pCAMBAIA3300 unchanged.

[0062] In some embodiments, the recombinant vector may be a pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector. The pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector is obtained by replacing the DNA fragment between the AvrII and SpeI restriction sites in the overexpression vector pCAMBAIA3300 with the DNA molecule shown in Sequence 5, while keeping the other sequences of the overexpression vector pCAMBAIA3300 unchanged.

[0063] In the above applications, the microorganisms can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Specifically, the bacteria can be from genera such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, but are not limited to these. For example, the bacteria can be *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. The fungus may be a yeast, and the yeast may come from genera such as *Saccharomyces cerevisiae*, *Kluyveromyces* (e.g., *Kluyveromyces lactis*), *Pichia pastoris* (e.g., *Pichia pastoris*), *Schizosaccharomyces pombe* (e.g., *Schizosaccharomyces pombe*), and *Hansenula* (e.g., *Hansenula polymorpha*), but is not limited thereto. The fungus may also come from genera such as *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp., but is not limited thereto. The actinomycetes may originate from genera such as *Streptomyces* sp., *Nocardia* sp., *Micromonospora* sp., *Streptosporangium* sp., *Actinoplanes* sp., and *Thermoactinomyces* sp., but are not limited to these. The algae may originate from genera such as *Fucus* sp., *Achnanthes* sp., *Amphiprora* sp., *Amphora* sp., *Ankistrodesmus* sp., *Asteromonas* sp., and *Boekelovia* sp., but are not limited to these. The viruses may be rotavirus, herpesvirus, influenza virus, adenovirus, etc., but are not limited to these.

[0064] The recombinant microorganisms refer to those obtained by manipulating and modifying the genes of a target microorganism, resulting in a functional change. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0065] In some embodiments, the recombinant microorganism is Agrobacterium EHA105 containing the above-mentioned pCAMBAIA3300-ZmHSCF1 overexpression vector.

[0066] In some embodiments, the recombinant microorganism is Agrobacterium EHA105 containing the above-mentioned pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector.

[0067] In the above applications, the transgenic plant tissues may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0068] In the above applications, the transgenic plant organs can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0069] In the above applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0070] The genetic transformation described above is Agrobacterium-mediated genetic transformation.

[0071] To address the aforementioned technical problems, the present invention also provides a method for Agrobacterium-mediated plant genetic transformation.

[0072] The method for Agrobacterium-mediated plant genetic transformation provided by the present invention includes the following steps: infecting plant explants with Agrobacterium containing a target vector to obtain infected explants; the target vector expresses the above-mentioned ZmHSCF1 protein.

[0073] In the above method, the target vector also expresses the target protein. The number of target proteins can be one, two, or more. The target protein can be an endogenous plant protein or an exogenous protein (such as DsRed protein).

[0074] The number of target vectors can be one, two, or more. The target protein and the ZmHSCF1 protein can be expressed using one target vector, or they can be expressed using two or more different target vectors respectively.

[0075] In some embodiments, the target protein is a protein that can increase plant yield, stress tolerance, or disease resistance.

[0076] In some implementations, the target protein is DsRed fluorescent protein.

[0077] In some embodiments, the target vector is either the pCAMBAIA3300-ZmHSCF1 overexpression vector or the pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector described above.

[0078] In some embodiments, the Agrobacterium containing the target vector is Agrobacterium EHA105 containing the above-mentioned pCAMBAIA3300-ZmHSCF1 overexpression vector or the above-mentioned pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector.

[0079] The above method also includes the step of culturing the infected explants to obtain regenerated plants. The culturing method can refer to the method in the literature "Liu S, Qiao J, Zhang S, Lu M, Yang Y, Lai J, Guo Y, Shi Y. Application of uniconazole in improving the high-throughput genetic transformation efficiency in maize. Plant Sci. 2024 Dec; 349:112270. doi:10.1016 / j.plantsci.2024.112270.Epub 2024Sep 28.PMID:39349145."

[0080] In some implementations, the cultivation method may include the following steps:

[0081] 1) The infected explants were cultured in a co-culture medium to obtain co-cultured explants;

[0082] 2) The co-cultured explants were cultured in a resistance selection medium to obtain resistant callus;

[0083] 3) The resistant callus was cultured in a predifferentiation medium to obtain callus blocks with regenerated buds;

[0084] 4) The callus tissue blocks with regenerated buds are cultured in a differentiation medium to obtain differentiated seedlings or tissue blocks with leaves.

[0085] 5) The differentiated seedlings or tissue blocks with leaves are cultured in a differentiation subculture medium to obtain resistant regenerated plants;

[0086] 6) The resistant regenerated plants are cultured in a rooting medium to obtain regenerated plants.

[0087] In some implementations, the explant is a maize embryo.

[0088] To address the aforementioned technical problems, the present invention also provides new applications for the above-mentioned methods.

[0089] This invention provides the application of the above method in any of the following B1)-B6):

[0090] B1) Promotes the formation of callus tissue in plant explants;

[0091] B2) Promotes the proliferation of callus tissue in plant explants;

[0092] B3) Increase the proportion of positive callus for the target gene in plant explants;

[0093] B4) Improve plant regeneration ability;

[0094] B5) Improve the efficiency of plant genetic transformation;

[0095] B6) Plant breeding.

[0096] The promotion of callus formation in plant explants as described above is manifested in increasing the callus emergence rate of plant explants.

[0097] The above-mentioned promotion of plant explant callus proliferation is manifested in increasing the area of ​​callus formed by plant explants.

[0098] The aforementioned increase in the proportion of positive callus for the target gene in plant explants is reflected in the increase in the proportion of resistant callus in plant explants.

[0099] The improvement of plant regeneration capacity described above is reflected in the improvement of plant regeneration efficiency.

[0100] The calculation methods for the above-mentioned callus formation rate, the above-mentioned resistant callus ratio, the above-mentioned regeneration efficiency, and the above-mentioned transformation efficiency can all refer to the methods in the literature "Liu S, Shi Y, Liu F, Guo Y, Lu M. LaCl3 treatment improves Agrobacterium-mediated immature embryo genetic transformation frequency of maize. Plant Cell Rep. 2022 Jun; 41(6):1439-1448. doi:10.1007 / s00299-022-02867-w. Epub 2022 Apr 4. PMID:35376997."

[0101] The above-described plant genetic transformations are genotype-independent plant genetic transformations.

[0102] The plants mentioned above can be dicotyledonous or monocotyledonous.

[0103] Furthermore, the plant in question is a monocotyledonous plant.

[0104] Furthermore, the monocotyledonous plant is maize.

[0105] Furthermore, the maize includes maize breeding parent backbone inbred lines that are difficult to convert.

[0106] In some implementations, the maize is a maize inbred line LH244, PH4CV, or Zheng58.

[0107] The regenerated plants produced by the above method are also within the scope of protection of this invention.

[0108] This invention, through the inoculation of maize immature embryos with Agrobacterium containing a vector expressing the ZmHSCF1 gene, revealed that compared to a control vector not expressing the ZmHSCF1 gene, explants overexpressing the ZmHSCF1 gene exhibited significantly higher callus formation, larger callus area, a significantly increased proportion of resistant callus, and significantly improved regeneration and transformation efficiency. In particular, it enabled the acquisition of transgenic positive genetic material from maize breeding parents, a backbone inbred line that is difficult to transform. This invention is the first to discover that overexpression of the ZmHSCF1 gene can promote callus formation in explants, especially positive callus formation, thereby enabling the regeneration of recipient genotypes that are difficult to transform, overcoming the Agrobacterium-mediated genotype-dependent bottleneck. This invention not only facilitates the genetic transformation of commercially available parental inbred lines currently promoted in agricultural production, but also effectively avoids the long-term backcrossing and transformation barriers after transformation with non-breeding parental recipients. It is of great significance for improving the transformation efficiency and the number of high-quality transformants of plants, especially important maize breeding parents that are difficult to transform, as well as for creating new breeding materials more accurately, quickly, and at low cost through molecular design breeding mediated by Agrobacterium-mediated transgenic or gene editing. Attached Figure Description

[0109] Figure 1 shows a schematic diagram of the vector structure. A is a schematic diagram of the control vector structure overexpressing the DsRed gene. B is a schematic diagram of the vector structure overexpressing the ZmHSCF1 gene. C is a schematic diagram of the vector structure simultaneously overexpressing the DsRed and ZmHSCF1 genes. D is a schematic diagram of the gene editing vector structure for ZmHSCF1 gene knockout.

[0110] Figure 2 shows the phenotypes and statistical analysis results of ZmHSCF1-regulated callus formation and proliferation. A shows the callus phenotypes after 1 day of co-culture and 20 days of screening culture. B shows the statistical results of callus emergence rate. C shows the statistical results of callus area.

[0111] Figure 3 shows the phenotypic and statistical analysis results of ZmHSCF1-regulated resistant callus proportion. A shows the observation results of RFP-positive callus after 14 days of screening culture under a microscope. B shows the statistical results of the RFP-positive callus proportion.

[0112] Figure 4 shows the phenotypic and statistical analysis results of ZmHSCF1 regulating maize regeneration capacity. A shows the callus phenotype after 20 days of differentiation culture. B shows the statistical results of regeneration efficiency.

[0113] Figure 5 shows the genotype-independent genetic transformation achieved by ZmHSCF1 through regulating the formation of resistant callus in maize backbone inbred lines. A shows the phenotype and microscopic observation results of callus tissue after 20 days of screening culture (PH4CV). B shows the phenotype and microscopic observation results of callus tissue after 20 days of screening culture (Zheng 58). C shows the statistical results of callus emergence rate. D shows the statistical results of the proportion of resistant callus. E shows PH4CV regenerated plants and T0 generation bar-positive seedlings. F shows Zheng 58 regenerated plants and T0 generation bar-positive seedlings. G shows the statistical results of transformation efficiency. Embodiments of the present invention

[0114] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0115] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, all quantitative experiments in the following examples are repeated three or more times.

[0116] The maize inbred line LH244 in the following examples is described in the literature “Yue Yang, Jianxin Shi, Limei Chen, Wenhan Xiao, Jingjuan Yu, ZmEREB46, a maize ortholog of Arabidopsis WAX INDUCER1 / SHINE1, is involved in the biosynthesis of leaf epicuticular very-long-chain waxes and drought tolerance, Plant Science, Volume 321, 2022, 111256, ISSN 0168-9452.”

[0117] The maize inbred line PH4CV in the following examples is described in the literature “Xiaoping Gong, Xiaoyang Liu, Qingchun Pan, Guohua Mi, Fanjun Chen and Lixing Yuan, Combined physiological, transcriptome, and genetic analysis reveals a molecular network of nitrogen remobilization in maize, Journal of Experimental Botany, Volume 71, May 2020, Pages 5061-5073”.

[0118] The maize inbred line Zheng58 in the following examples is described in the literature “Yang Z,Li X,Zhang N,Zhang YN,Jiang HW,Gao J,Kuai BK,Ding YL,Huang XQ.Detection of quantitative trait loci for kernel oil and protein concentration in a B73 and Zheng58 maize cross.Genet Mol Res.2016Sep 30;15(3).doi:10.4238 / gmr.15038951.PMID:27706793.”.

[0119] The Escherichia coli strain DH5α(DE3) competent cells in the following examples are products of TransGen Biotech Ltd.

[0120] The Agrobacterium tumefaciens strain EHA105 in the following examples is described in the literature “Chen, S.; Songkumarn, P.; Liu, J.; Wang, G.-LA Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics. Plant Physiol. 2009, 150, 1111-1121.”

[0121] The overexpression vector pCAMBAIA3300 used in the following examples is described in the literature “Boxin Liu and others, Manipulating ZmEXPA4 expression ameliorates the drought-induced prolonged anthesis and silking interval in maize, The Plant Cell, Volume 33, Issue 6, June 2021, Pages 2058-2071, https: / / doi.org / 10.1093 / plcell / koab083.”

[0122] The carrier pBUE411 and intermediate carrier pCBC-MT1T2 in the following embodiments are both described in the literature "Xing, HL". # ,Dong,L. # ,Wang,ZP,Zhang,HY,Han,CY,Liu,B.,Wang,XC,and Chen,QJ*(2014).A CRISPR / Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol 14,327."

[0123] The culture medium, its components, and culture conditions involved in the Agrobacterium-mediated genetic transformation method for maize immature embryos in the following examples are all described in the literature "Liu S, Qiao J, Zhang S, Lu M, Yang Y, Lai J, Guo Y, Shi Y. Application of uniconazole in improving the high-throughput genetic transformation efficiency in maize. Plant Sci. 2024 Dec;349:112270. doi:10.1016 / j.plantsci.2024.112270.Epub 2024Sep 28.PMID:39349145."

[0124] The calculation methods and formulas for callus rate, RFP-positive callus ratio, regeneration efficiency and transformation efficiency involved in the Agrobacterium-mediated genetic transformation method for maize immature embryos in the following examples are all recorded in the literature "Liu S, Shi Y, Liu F, Guo Y, Lu M. LaCl3 treatment improves Agrobacterium-mediated immature embryo genetic transformation frequency of maize. Plant Cell Rep. 2022 Jun;41(6):1439-1448. doi:10.1007 / s00299-022-02867-w.Epub 2022Apr 4.PMID:35376997."

[0125] Example 1: Preparation of control vector, ZmHSCF1 overexpression vector, and CRISPR / Cas9 vector with ZmHSCF1 as the target gene.

[0126] I. ZmHSCF1 gene-related sequences

[0127] The ZmHSCF1 gene involved in this invention is derived from maize (Zea may L.), and its sequence in the maize genome is shown in Sequence 2, which consists of 3086 nucleotides; the CDS sequence of the ZmHSCF1 gene is shown in Sequence 3, which consists of 1458 nucleotides; both Sequence 2 and Sequence 3 encode the ZmHSCF1 protein shown in Sequence 4, which consists of 485 amino acid residues.

[0128] II. Preparation of control vector, ZmHSCF1 overexpression vector, and CRISPR / Cas9 vector with ZmHSCF1 as the target gene

[0129] 1. Preparation of pCAMBAIA3300-DsRed control vector

[0130] The DNA fragment between the two XcmI restriction sites in the overexpression vector pCAMBAIA3300 was replaced with the DNA molecule shown in Sequence 1 (the red fluorescent gene DsRed, which serves as a reporter gene), while keeping the other sequences of the overexpression vector pCAMBAIA3300 unchanged, to obtain the recombinant vector pCAMBAIA3300-DsRed, which serves as the control vector in the genetic transformation experiment.

[0131] 2. Preparation of pCAMBAIA3300-ZmHSCF1 overexpression vector

[0132] The DNA fragment between the two XcmI restriction sites in the overexpression vector pCAMBAIA3300 was replaced with the DNA molecule (ZmHSCF1 gene) shown in sequence 3, while keeping the other sequences of the overexpression vector pCAMBAIA3300 unchanged, to obtain the overexpression vector pCAMBAIA3300-ZmHSCF1.

[0133] 3. Preparation of pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector

[0134] The DNA fragment between the AvrII and SpeI restriction sites in the overexpression vector pCAMBAIA3300 was replaced with the DNA molecule shown in Sequence 5, while keeping the other sequences of the overexpression vector pCAMBAIA3300 unchanged, resulting in the pCAMBAIA3300-DsRed-ZmHSCF1 overexpression vector. The DNA molecule shown in Sequence 5 is composed of the red fluorescent gene DsRed, the Ubi promoter, and the ZmHSCF1 gene, in sequence.

[0135] 4. Preparation of the CRISPR / Cas9 gene editing vector pBUE411-pCBC-MTaTb with ZmHSCF1 as the target gene

[0136] The preparation method of the CRISPR / Cas9 gene editing vector pBUE411-pCBC-MTaTb with ZmHSCF1 as the target gene was described in the literature "Xing, HL". # ,Dong,L. # Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, and Chen, QJ* (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327. The specific steps are as follows:

[0137] 1) Based on the ZmHSCF1 gene sequence, sgRNA target sequences were designed, and two sgRNA targets were finally designed, denoted as Ta target and Tb target, respectively.

[0138] The target sequence for Ta is as follows: 5'-AGAAGGCTTCGAGTAGGCCA-3' (sequence 6).

[0139] The Tb target sequence is as follows: 5'-AGGCCCCCAGTACTTGAGCG-3' (sequence 7).

[0140] 2) The pBUE411 vector was digested with the restriction endonuclease BsaI to obtain the digested vector. Four-primer amplification was performed using two pairs of primers with pCBC-MT1T2 as a template. The F0 and R0 primer concentrations were diluted tenfold from the working concentrations and amplified by PCR. The ligation fragment was recovered from the gel. Then, the ligation fragment and the digested vector were ligated using T4 ligase to obtain the recombinant plasmid. The recombinant plasmid was sent for sequencing, and the correctly sequenced recombinant plasmid was named pBUE411-pCBC-MTaTb. The recombinant plasmid pBUE411-pCBC-MTaTb can express Cas9 protein and two sgRNAs targeting the ZmHSCF1 gene. The target sites of the two sgRNAs targeting the ZmHSCF1 gene are Ta and Tb, respectively.

[0141] The two primer pairs are as follows:

[0142] BsF: (Sequence 8)

[0143] F0: 5'-TAGAAGGCTTCGAGTAGGCCAGTTTTAGAGCTAGAAATAGC-3' (sequence 9).

[0144] In this sequence, the bolded bases are the recognition sites of BsaI, and the underlined bases are the Ta sequence (the reverse complementary sequence of the sequence shown at positions 72-91 of sequence 3).

[0145] R0: 5'-AACAGGCCCCAGTACTTGAGCGGCTTCTTGGTGCC-3' (sequence 10).

[0146] BsR: (Sequence 11).

[0147] In this sequence, the bolded bases are the recognition sites of BsaI, and the underlined bases are the Tb sequence (the reverse complementary sequence of the sequence shown at positions 569-588 in sequence 3).

[0148] Example 2: Application of ZmHSCF1 protein in regulating callus formation and proliferation

[0149] I. Effects and Analysis of ZmHSCF1 Overexpression on Callus Formation and Proliferation

[0150] The Agrobacterium-mediated genetic transformation of maize immature embryos was employed (referring to the method in the literature "Liu S, Qiao J, Zhang S, Lu M, Yang Y, Lai J, Guo Y, Shi Y. Application of uniconazole in improving the high-throughput genetic transformation efficiency in maize. Plant Sci. 2024 Dec;349:112270. doi:10.1016 / j.plantsci.2024.112270.Epub 2024Sep 28.PMID:39349145."). The recombinant vectors pCAMBAIA3300-DsRed, pCAMBAIA3300-ZmHSCF1, and pBUE411-pCBC-MTaTb prepared in Example 1 were transformed into the maize inbred line LH244. The specific steps include the following: The recombinant vectors pCAMBAIA3300-DsRed, pCAMBAIA3300-ZmHSCF1, and pBUE411-pCBC-MTaTb prepared in Example 1 were transformed into Agrobacterium tumefaciens strain EHA105, respectively. After identification, recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105, pCAMBAIA3300-ZmHSCF1 / EHA105, and pBUE411-pCBC-MTaTb / EHA105 were obtained, respectively. The recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105, pCAMBAIA3300-ZmHSCF1 / EHA105, and pBUE411-pCBC-MTaTb / EHA105 were then used to infect maize embryos (approximately 1.5 mm in size) 12 days after pollination. After infection with Agrobacterium, embryos were placed on co-culture medium with the scutellum facing upwards, 30 embryos per dish. After one day of culture, they were transferred to selection medium containing Bar resistance as shown in Figure 2. After 14 days of culture, callus tissues transformed with recombinant vectors pCAMBAIA3300-DsRed, pCAMBAIA3300-ZmHSCF1, and pBUE411-pCBC-MTaTb were observed, and the callus rate and callus area were calculated. The callus tissue transformed with the pCAMBAIA3300-DsRed vector was designated as DsRed. OE Callus transformed with pCAMBAIA3300-ZmHSCF1 vector is designated as ZmHSCF1. OE The callus transformed with the pBUE411-pCBC-MTaTb vector is denoted as zmhscf1. CRSIPR .

[0151] The results are shown in Figures 2A and 2B. The results show that, compared to the pCAMBAIA3300-DsRed vector, the callus rate after transformation with the pCAMBAIA3300-ZmHSCF1 vector significantly increased from 91.7% to 97.8%, while the callus rate after transformation with the pBUE411-pCBC-MTaTb vector significantly decreased to 91.9%. Furthermore, the callus area statistics are shown in Figure 2C. The results show that, compared to the pCAMBAIA3300-DsRed vector, the callus area after transformation with the pCAMBAIA3300-ZmHSCF1 vector increased from 1.20 cm² to 97.8%. 2 Significantly increased by 1.60cm 2 The callus area after transformation with pBUE411-pCBC-MTaTb vector was significantly reduced to 1.10 cm². 2 This indicates that overexpression of ZmHSCF1 can promote callus formation.

[0152] II. Effects and Analysis of ZmHSCF1 Overexpression on Positive Callus Formation

[0153] The Agrobacterium-mediated genetic transformation of maize immature embryos was employed (referring to the method in the literature "Liu S, Qiao J, Zhang S, Lu M, Yang Y, Lai J, Guo Y, Shi Y. Application of uniconazole in improving the high-throughput genetic transformation efficiency in maize. Plant Sci. 2024 Dec;349:112270. doi:10.1016 / j.plantsci.2024.112270.Epub 2024Sep 28.PMID:39349145."). The recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 prepared in Example 1 were transformed into maize inbred line LH244. The specific steps include the following: The recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 prepared in Example 1 were transformed into Agrobacterium tumefaciens strain EHA105, respectively. After identification, recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105 and pCAMBAIA3300-DsRed-ZmHSCF1 / EHA105 were obtained, respectively. The recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105 and pCAMBAIA3300-DsRed-ZmHSCF1 / EHA105 were then used to infect maize embryos (approximately 1.5 mm in size) 12 days after pollination. After infection with Agrobacterium, embryos were placed on co-culture medium with the scutellum facing upwards, 30 embryos per dish. After one day of culture, they were transferred to selection medium containing bar resistance as shown in Figure 2. After 14 days of culture, the transient expression of RFP in callus transformed with recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 was observed using fluorescence microscopy, and the proportion of RFP-positive callus was statistically analyzed. The callus transformed with pCAMBAIA3300-DsRed vector was designated as DsRed. OE Callus transformed with pCAMBAIA3300-DsRed-ZmHSCF vector is designated as ZmHSCF1. OE .

[0154] The results are shown in Figure 3. The results indicate that, compared to the pCAMBAIA3300-DsRed vector, the transient expression of RFP in callus was significantly enhanced after transformation with the pCAMBAIA3300-DsRed-ZmHSCF1 vector, and the proportion of RFP-positive callus increased significantly from 30.3% to 51.5%. This demonstrates that overexpression of ZmHSCF1 can promote the formation of positive callus tissue.

[0155] III. Effects and Analysis of ZmHSCF1 Overexpression on Callus Differentiation Capacity

[0156] The callus tissue formed in step one after culturing in a selection medium containing bar resistance for 14 days was transferred to a predifferentiation medium containing bar resistance. After culturing for 12 days, the callus tissue was transferred to a differentiation medium containing bar resistance. After culturing for 20 days, the differentiation of the callus tissue transformed with recombinant vectors pCAMBAIA3300-DsRed, pCAMBAIA3300-ZmHSCF1, and pBUE411-pCBC-MTaTb was observed, and the regeneration efficiency was calculated.

[0157] The results are shown in Figure 4. The results indicate that, compared to the pCAMBAIA3300-DsRed vector, the regeneration efficiency of callus tissue significantly increased after transformation with the pCAMBAIA3300-ZmHSCF1 vector, rising from 10.9% to 14.8%. Conversely, transformation with the pBUE411-pCBC-MTaTb vector decreased callus differentiation capacity, and the regeneration efficiency significantly decreased to 7.1%. This demonstrates that overexpression of ZmHSCF1 can promote the formation and proliferation of positive callus tissue, thereby improving callus differentiation capacity and regeneration efficiency.

[0158] IV. Effects and Analysis of ZmHSCF1 Overexpression on Transformation Efficiency of Difficult-to-Transform Maize Breeding Parent Backbone Inbred Lines

[0159] The Agrobacterium-mediated genetic transformation of maize immature embryos was employed (referring to the method in the literature "Liu S, Qiao J, Zhang S, Lu M, Yang Y, Lai J, Guo Y, Shi Y. Application of uniconazole in improving the high-throughput genetic transformation efficiency in maize. Plant Sci. 2024 Dec;349:112270. doi:10.1016 / j.plantsci.2024.112270.Epub 2024Sep 28.PMID:39349145."). The recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 prepared in Example 1 were transformed into the maize inbred line PH4CV (paternal parent of Xianyu 335) and the maize inbred line Zheng58 (maternal parent of Zhengdan 958), respectively. The specific steps include the following: The recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 prepared in Example 1 were transformed into Agrobacterium tumefaciens strain EHA105, respectively. After identification, recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105 and pCAMBAIA3300-DsRed-ZmHSCF1 / EHA105 were obtained, respectively. The recombinant Agrobacterium tumefaciens pCAMBAIA3300-DsRed / EHA105 and pCAMBAIA3300-DsRed-ZmHSCF1 / EHA105 were then used to infect maize embryos (approximately 1.5 mm in size) 12 days after pollination. After infection with Agrobacterium, embryos were placed on co-culture medium with the scutellum facing upwards, 30 embryos per dish. After one day of culture, they were transferred to selection medium containing bar resistance as shown in Figure 2. After 20 days of culture, the transient expression of RFP in callus transformed with recombinant vectors pCAMBAIA3300-DsRed and pCAMBAIA3300-DsRed-ZmHSCF1 was observed using fluorescence microscopy, and the callus rate and the proportion of RFP-positive callus were calculated. Callus transformed with bar resistance for 20 days was then subjected to pre-differentiation culture, differentiation culture, subculture, and rooting culture to obtain T0 generation regenerated plants. Bar positivity was identified using bar test strips (Shanghai Youlong Biotechnology Co., Ltd., catalog number AA1032-LS), and the transformation efficiency was calculated. Callus transformed with pCAMBAIA3300-DsRed vector was designated as DsRed. OE Callus transformed with pCAMBAIA3300-DsRed-ZmHSCF vector is designated as ZmHSCF1.OE .

[0160] The results are shown in Figure 5. The results show that, with the pCAMBAIA3300-DsRed vector as the control, the transient expression of RFP in the callus tissues formed by PH4CV and Zheng58 transformed with pCAMBAIA3300-DsRed-ZmHSCF1 vector was significantly enhanced. The callus rates also increased significantly from 14.5% and 21.5% to 45.5% and 48.2%, respectively. Furthermore, the proportion of RFP-positive callus increased significantly from 12.9% and 5.4% to 38.5% and 32.5%, respectively (Figures 5A, B, C, and D). Regarding transformation efficiency, using the pCAMBAIA3300-DsRed vector as a control, the transformation efficiency of the PH4CV maize inbred line transformed with the pCAMBAIA3300-DsRed-ZmHSCF1 vector significantly increased from 0.9% to 9.6%, and the transformation efficiency of the Zheng58 maize inbred line transformed with the pCAMBAIA3300-DsRed-ZmHSCF1 vector significantly increased from 0 to 7.2% (Figures 5E, F, and G). This indicates that overexpression of ZmHSCF1 can significantly improve transformation efficiency by promoting the formation and proliferation of positive callus in the backbone inbred lines of maize breeding parents that are difficult to transform.

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. Industrial applicability

[0162] This invention is the first to discover that overexpression of the ZmHSCF1 gene can promote callus formation and proliferation, and significantly improve callus emergence rate, the proportion of resistant callus, regeneration efficiency, and transformation efficiency. In particular, it enables the acquisition of transgenic positive genetic material from maize breeding parents, a difficult-to-transform backbone inbred line. This invention is of great significance for improving the transformation efficiency and the number of high-quality transformants in plants, especially difficult-to-transform important maize breeding parents and other recalcitrant maize inbred lines, as well as for achieving more precise, rapid, and low-cost creation of new breeding materials through Agrobacterium-mediated transgenic or gene-editing-led molecular design breeding.

Claims

1. Application of ZmHSCF1 protein in any of the following (A1)-A14): A1) Promotes the formation of callus tissue in plant explants; A2) Prepare products that promote the formation of callus tissue in plant explants; A3) Promotes the proliferation of callus tissue in plant explants; A4) Prepare products that promote the proliferation of callus tissue in plant explants; A5) Increase the proportion of positive callus for the target gene in plant explants; A6) Prepare products that increase the proportion of positive callus for the target gene in plant explants; A7) Improve plant regeneration ability; A8) Prepare products that enhance plant regeneration capabilities; A9) Improve the efficiency of plant genetic transformation; A10) Prepare products that improve the efficiency of plant genetic transformation; A11) Plant genetic transformation; A12) Preparation of plant genetic transformation products; A13) Plant breeding; A14) Preparation of plant breeding products; The ZmHSCF1 protein is a1), a2), a3), or a4): a1) The amino acid sequence is that of the protein shown in sequence 4; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in sequence 4; a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4, which are related to the efficiency of plant callus formation or regeneration or genetic transformation. a4) Proteins that share 75% or more of the amino acid sequence shown in Sequence 4, are derived from maize, and are associated with plant callus formation or regeneration or genetic transformation efficiency.

2. Application of biomaterials related to ZmHSCF1 protein in any of the following A1)-A14): A1) Promotes the formation of callus tissue in plant explants; A2) Prepare products that promote the formation of callus tissue in plant explants; A3) Promotes the proliferation of callus tissue in plant explants; A4) Prepare products that promote the proliferation of callus tissue in plant explants; A5) Increase the proportion of positive callus for the target gene in plant explants; A6) Prepare products that increase the proportion of positive callus for the target gene in plant explants; A7) Improve plant regeneration ability; A8) Prepare products that enhance plant regeneration capabilities; A9) Improve the efficiency of plant genetic transformation; A10) Prepare products that improve the efficiency of plant genetic transformation; A11) Plant genetic transformation; A12) Preparation of plant genetic transformation products; A13) Plant breeding; A14) Preparation of plant breeding products; The biological material is a nucleic acid molecule encoding the ZmHSCF1 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ. The ZmHSCF1 protein is a1), a2), a3), or a4): a1) The amino acid sequence is that of the protein shown in sequence 4; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in sequence 4; a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4, which are related to the efficiency of plant callus formation or regeneration or genetic transformation. a4) Proteins that share 75% or more of the amino acid sequence shown in Sequence 4, are derived from maize, and are associated with plant callus formation or regeneration or genetic transformation efficiency.

3. The application according to claim 2, characterized in that: The nucleic acid molecule is a gene as shown in B1) or B2) below: B1) The DNA molecule shown in sequence 2 or sequence 3; B2) has 75% or more identity with the nucleotide sequence defined by B1) and is a DNA molecule encoding the ZmHSCF1 protein.

4. The application according to any one of claims 1-3, characterized in that: The genetic transformation is Agrobacterium-mediated genetic transformation.

5. The application according to any one of claims 1-4, characterized in that: The plant in question is a monocotyledonous plant.

6. The application according to claim 5, characterized in that: The monocotyledonous plant mentioned is maize.

7. The application according to claim 6, characterized in that: The maize includes maize inbred lines LH244, PH4CV, and Zheng58.

8. A method for Agrobacterium-mediated plant genetic transformation, comprising the following steps: infecting plant explants with Agrobacterium containing a target vector to obtain infected explants; wherein the target vector expresses ZmHSCF1 protein; The ZmHSCF1 protein is a1), a2), a3), or a4): a1) The amino acid sequence is that of the protein shown in sequence 4; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in sequence 4; a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4, which are related to the efficiency of plant callus formation or regeneration or genetic transformation. a4) Proteins that share 75% or more of the amino acid sequence shown in Sequence 4, are derived from maize, and are associated with plant callus formation or regeneration or genetic transformation efficiency.

9. The method according to claim 8, characterized in that: The method further includes the step of culturing the infected explants to obtain regenerated plants.

10. The method according to claim 8 or 9, characterized in that: The plant in question is a monocotyledonous plant.

11. The method according to claim 10, characterized in that: The monocotyledonous plant mentioned is maize.

12. The application or method according to claim 11, characterized in that: The maize includes maize inbred lines LH244, PH4CV, and Zheng58.

13. The application of the method according to any one of claims 8-12 in any one of the following B1)-B6): B1) Promotes the formation of callus tissue in plant explants; B2) Promotes the proliferation of callus tissue in plant explants; B3) Increase the proportion of positive callus for the target gene in plant explants; B4) Improve plant regeneration ability; B5) Improve the efficiency of plant genetic transformation; B6) Plant breeding.

14. The application according to claim 13, characterized in that: The genetic transformation is Agrobacterium-mediated genetic transformation.

15. The application according to claim 13 or 14, characterized in that: The plant in question is a monocotyledonous plant.

16. The application according to claim 15, characterized in that: The monocotyledonous plant mentioned is maize.

17. The application according to claim 16, characterized in that: The maize includes maize inbred lines LH244, PH4CV, and Zheng58.

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