Agrobacterium tumefaciens-mediated maize immature embryo high-throughput genetic transformation method and application thereof

Abstract

The invention discloses a high-throughput genetic transformation method of agrobacterium tumefaciens-mediated maize immature embryos and application of the high-throughput genetic transformation method. According to the method, uniconazole is added into a differentiation / regeneration subculture medium, an optimization system suitable for corn immature embryo genetic transformation is established, and efficient and stable genetic transformation of corn can be effectively achieved in an agrobacterium tumefaciens-mediated mode. Analysis on transformation data of transformation vectors in a year before and after optimization shows that the regeneration plant obtaining proportion is obviously improved to 20.04% after optimization from 4.5% before optimization; the annual average conversion efficiency is obviously improved from 2.19% before optimization to 7.48% after optimization; and the average conversion period is shortened from 112 days before optimization to 93 days after optimization.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a high-throughput genetic transformation method for Agrobacterium-mediated maize immature embryos and its application. In particular, by adding uniconazole components to the differentiation / regeneration subculture medium, the chlorophyll content of the leaves of the regenerated plants, the stem diameter and the stem strength of the regenerated plants are significantly increased, thereby improving the acquisition rate of the regenerated plants, ultimately improving the transformation efficiency of Agrobacterium-mediated genetic transformation of maize immature embryos, shortening the transformation cycle, and saving production costs. Background Art

[0002] As one of the three major staple food crops, maize is currently the crop with the widest planting area and the highest yield; in addition, maize is also an important feed crop and industrial raw material. Therefore, increasing maize yield plays a crucial role in ensuring food security and meeting market demand. With the continuous growth of the population and the increasing living standards of people, it is necessary to meet both the basic problem of people having enough to eat and the quality requirement of eating well. In recent years, with the continuous maturity of modern molecular biology and bioengineering technologies (molecular design), especially the birth and development of gene editing technology and synthetic biology, in-depth analysis of maize gene functions and excavation of key candidate genes and excellent allelic genes for yield increase, stress resistance, high quality and excellent flavor are the keys to solving the problem; molecular design breeding has become an important way to solve the food supply demand.

[0003] However, whether it is the analysis of maize gene function and the excavation of key candidate genes, or the use of molecular design for maize molecular breeding, an efficient, rapid and stable maize genetic transformation system is an insurmountable technical foundation at present. However, due to the relatively complex maize genome, the presence of a large number of repetitive sequences, gene function redundancy, and a large number of genes whose biological functions remain to be analyzed, there is still a lack of an efficient and fast high-throughput maize genetic transformation system. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to achieve maize genetic transformation efficiently and stably.

[0005] To solve the above technical problems, the present invention first provides a genetic transformation method for Agrobacterium-mediated maize immature embryos.

[0006] The genetic transformation method for Agrobacterium-mediated maize immature embryos provided by the present invention comprises the following steps:

[0007] 1) Infecting maize immature embryos with Agrobacterium containing a target vector to obtain infected immature embryos;

[0008] 2) Culturing the infected immature embryos in a co-culture medium to obtain co-cultured immature embryos;

[0009] 3) Cultivate the co-cultured young embryos in a resistant callus induction medium to obtain resistant callus;

[0010] 4) Cultivate the resistant callus in a pre-differentiation medium to obtain a callus mass with regenerated buds growing;

[0011] 5) Cultivate the callus mass with regenerated buds growing in a differentiation / regeneration medium to obtain differentiated small seedlings or a tissue mass with leaves growing;

[0012] 6) Cultivate the differentiated small seedlings or the tissue mass with leaves growing in a differentiation / regeneration subculture medium to obtain resistant regenerated plants; the differentiation / regeneration subculture medium contains uniconazole;

[0013] 7) Cultivate the resistant regenerated plants in a rooting medium to obtain T 0 -generation plants.

[0014] In the above method, the concentration of uniconazole in the differentiation / regeneration subculture medium can be 0.02 - 0.06 mg / L or 0.02 - 0.045 mg / L or 0.045 - 0.06 mg / L, preferably 0.045 mg / L.

[0015] Furthermore, in the above 1), the infection method includes the following steps:

[0016] 1-1) Activate and culture the Agrobacterium containing the target vector on an Agrobacterium activation medium to obtain activated Agrobacterium;

[0017] 1-2) Shake and mix the activated Agrobacterium in an infection liquid medium to obtain an Agrobacterium resuspension;

[0018] The infection liquid medium includes 2.16 - 3.44 g / L MS basal salts, 8 - 12 mL / L 100X MS organics, 2 - 4 mg / L 2,4-D, 10 - 30 g / L sucrose, 10 - 30 g / L glucose, 0.1 - 0.15 g / L proline, 0.2 - 0.4 mM acetosyringone, 1.7 - 3.4 mg / L silver nitrate, 7 - 10 g / L agar powder;

[0019] 1-3) Immerse the maize young embryos in the Agrobacterium resuspension to obtain infected young embryos.

[0020] In 2), the co-culture medium comprises 2.16 - 3.44 g / L of MS basal salts, 8 - 12 mL / L of 100X MS organics, 2 - 4 mg / L of 2,4-D, 10 - 30 g / L of sucrose, 10 - 30 g / L of glucose, 0.1 - 0.15 g / L of proline, 0.2 - 0.4 mM of acetosyringone, 1.7 - 3.4 mg / L of silver nitrate, and 7 - 10 g / L of agar powder.

[0021] In 3), the resistant callus induction medium comprises 2.16 - 4.33 g / L of MS basal salts, 8 - 12 mL / L of 100X MS organics, 10 - 30 g / L of sucrose, 1 - 2 g / L of proline, 0.5 - 1 mg / L of 2,4-D, 0.5 - 1 g / L of casein hydrolysate, 2 - 3 mg / L of picloram, 1.7 - 3.4 mg / L of silver nitrate, 3 - 7 mg / L of bilanafos, 100 - 200 mg / L of ticarcillin, and 3 - 4 g / L of phytagel.

[0022] In 4), the pre-differentiation medium comprises 2.16 - 4.33 g / L of MS basal salts, 0.8 - 1.2 mL / L of 1000X MS organics, 10 - 30 g / L of glucose, 10 - 30 g / L of maltose, 0.1 - 0.2 g / L of aspartic acid, 0.1 - 0.15 g / L of inositol, 3 - 7 mg / L of bilanafos, 100 - 200 mg / L of ticarcillin, and 3.0 - 4.0 g / L of phytagel.

[0023] In 5), the differentiation / regeneration medium comprises 2.16 - 4.33 g / L of MS basal salts, 0.8 - 1.2 mL / L of 1000X MS organics, 10 - 30 g / L of glucose, 10 - 30 g / L of maltose, 0.1 - 0.2 g / L of aspartic acid, 0.1 - 0.15 g / L of inositol, 3 - 7 mg / L of bilanafos, 100 - 200 mg / L of ticarcillin, and 3.0 - 4.0 g / L of phytagel.

[0024] In 6), the differentiation / regeneration subculture medium comprises 2.16 - 4.33 g / L of MS basal salts, 0.8 - 1.2 mL / L of 1000X MS organics, 10 - 30 g / L of glucose, 10 - 30 g / L of maltose, 0.1 - 0.2 g / L of aspartic acid, 0.1 - 0.15 g / L of inositol, 0.02 - 0.06 mg / L of uniconazole, 3 - 7 mg / L of bilanafos, 100 - 200 mg / L of ticarcillin, and 3 - 4 g / L of phytagel.

[0025] In the above (7), the rooting medium comprises 2.16 - 4.33 g / L of MS basal salts, 1 - 2 mL / L of 100X MS organic, 10 - 30 g / L of sucrose, 0.1 - 0.15 mg / L of naphthylacetic acid, 0 - 5.0 mg / L of bilanafos, 100 - 200 mg / L of ticarcillin and 3 - 4 g / L of phytagel.

[0026] Furthermore, in the above (1 - 1), the Agrobacterium activation medium consists of a solvent and solutes. The solvent is double-distilled water, and the solutes and their concentrations are as follows: 5 g / L of yeast extract, 10 g / L of peptone, 5 g / L of sodium chloride, 15 g / L of agar. It is sterilized at 121 °C for 15 minutes. When the medium cools to 50 °C, rifampicin and kanamycin are added, and their concentrations are 25 mg / L and 50 mg / L respectively. The pH of the Agrobacterium activation medium is 7.0.

[0027] In the above (1 - 2), the infection medium consists of a solvent and solutes. The solvent is double-distilled water, and the solutes and their concentrations are: 2.16 g / L of MS basal salts, 10 mL / L of 100X MS organic, 3 mg / L of 2,4-D, 20 g / L of sucrose, 10 g / L of glucose, 0.115 g / L of proline, 0.2 mM of acetosyringone, 3.4 mg / L of silver nitrate, 7 g / L of agar powder. The pH of the infection medium is 5.2.

[0028] In the above (2), the co-culture medium consists of a solvent and solutes. The solvent is double-distilled water, and the solutes and their concentrations are: 2.16 g / L of MS basal salts, 10 mL / L of 100X MS organic, 3 mg / L of 2,4-D, 20 g / L of sucrose, 10 g / L of glucose, 0.115 g / L of proline, 0.2 mM of acetosyringone, 3.4 mg / L of silver nitrate, 7 g / L of agar powder. The pH of the co-culture medium is 5.8.

[0029] In the above (3), the resistant callus induction medium consists of a solvent and solutes. The solvent is double-distilled water, and the solutes and their concentrations are: 4.33 g / L of MS basal salts, 10 mL / L of 100X MS organic, 30 g / L of sucrose, 1.38 g / L of proline, 0.5 mg / L of 2,4-D, 0.5 g / L of casein hydrolysate, 2.2 mg / L of picloram, 3.4 mg / L of silver nitrate, 5.0 mg / L of bilanafos, 100 mg / L of ticarcillin and 3.0 g / L of phytagel. The pH of the resistant callus induction medium is 5.8.

[0030] In the above item 4), the pre-differentiation medium consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 ml / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 5.0 mg / L bilanafos, 100 mg / L ticarcillin, and 3.0 g / L phytagel. The pH of the pre-differentiation medium is 5.8.

[0031] In the above item 5), the differentiation / regeneration medium consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 ml / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 5.0 mg / L bilanafos, 100 mg / L ticarcillin, and 3.0 g / L phytagel. The pH of the differentiation / regeneration medium is 5.8.

[0032] In the above item 6), the subculture medium for differentiation / regeneration consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 ml / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 0.045 mg / L uniconazole, 5.0 mg / L bilanafos, 100 mg / L ticarcillin, and 3.0 g / L phytagel. The pH of the subculture medium for differentiation / regeneration is 5.8.

[0033] In the above item 7), the rooting medium consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 2.16 g / L MS basal salts, 2 ml / L 100X MS organic, 15 g / L sucrose, 0.1 mg / L naphthaleneacetic acid, 5.0 mg / L bilanafos, 100 mg / L ticarcillin, and 3.0 g / L phytagel.

[0034] The 100X MS organic consists of a solvent and a solute. The solvent is double-distilled water, and the solute and its concentration are as follows: glycine 2.0 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 1 mg / L.

[0035] The 1000X MS organic consists of a solvent and a solute. The solvent is double-distilled water, and the solute and its concentration are as follows: glycine 2.0 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.1 mg / L.

[0036] In the above method, in the step 1), the immature embryo can be an immature embryo obtained by peeling from an immature ear; the immature ear can be an immature ear picked when the size of the maize immature embryo is 1.5 - 2 mm.

[0037] In the step 1 - 2), the OD of the Agrobacterium resuspension 660 can be 0.6 - 0.8.

[0038] In the step 1 - 3), the soaking time can be 30 - 60 min, specifically 30 min.

[0039] In the step 2), the culture conditions are as follows: dark culture at 20 - 25 °C for 1 - 2 days, specifically dark culture at 22 °C for 1 day.

[0040] In the step 3), the culture conditions are as follows: dark culture at 25 - 28 °C for 10 - 14 days, specifically dark culture at 28 °C for 14 days.

[0041] In the step 4), the culture conditions are as follows: weak light culture at 25 - 28 °C for 10 - 14 days, specifically culture at 25 °C under a light cycle of white light for 16 h / dark for 8 h for 12 days.

[0042] In the steps 5) and 6), the culture conditions are as follows: weak light culture at 25 - 28 °C for 20 - 30 days, specifically culture at 25 °C under a light cycle of white light for 16 h / dark for 8 h for 20 - 30 days.

[0043] In the step 7), the culture conditions are as follows: weak light culture at 25 - 28 °C for 7 - 14 days, specifically culture at 25 °C under a light cycle of white light for 16 h / dark for 8 h for 7 - 14 days.

[0044] To solve the above technical problems, the present invention also provides a kit for maize genetic transformation.

[0045] The kit for maize genetic transformation provided by the present invention includes the above - mentioned co - culture medium and / or the above - mentioned resistant callus induction medium and / or the above - mentioned pre - differentiation medium and / or the above - mentioned differentiation / regeneration medium and / or the above - mentioned differentiation / regeneration sub - culture medium and / or the above - mentioned rooting medium and / or the above - mentioned infection liquid medium.

[0046] To solve the above technical problems, the present invention finally provides a new use of the above method or the above kit.

[0047] The present invention provides the application of the above method or the above kit in any one of the following a1) - a22):

[0048] a1) Maize genetic transformation;

[0049] a2) Preparing a product for maize genetic transformation;

[0050] a3) High-throughput genetic transformation of maize;

[0051] a4) Preparation of products for high-throughput genetic transformation of maize;

[0052] a5) Maize tissue culture;

[0053] a6) Preparation of products for maize tissue culture;

[0054] a7) Improving the genetic transformation efficiency of maize;

[0055] a8) Preparation of products for improving the genetic transformation efficiency of maize;

[0056] a9) Improving the high-throughput genetic transformation efficiency of maize;

[0057] a10) Preparation of products for improving the high-throughput genetic transformation efficiency of maize;

[0058] a11) Improving the tissue culture efficiency of maize;

[0059] a12) Preparation of products for improving the tissue culture efficiency of maize;

[0060] a13) Increasing the chlorophyll content in the leaves of maize plants regenerated from genetic transformation / tissue culture;

[0061] a14) Preparation of products for increasing the chlorophyll content in the leaves of maize plants regenerated from genetic transformation / tissue culture;

[0062] a15) Increasing the stem diameter of maize plants regenerated from genetic transformation / tissue culture;

[0063] a16) Preparation of products for increasing the stem diameter of maize plants regenerated from genetic transformation / tissue culture;

[0064] a17) Increasing the stem strength of maize plants regenerated from genetic transformation / tissue culture;

[0065] a18) Preparation of products for increasing the stem strength of maize plants regenerated from genetic transformation / tissue culture;

[0066] a19) Increasing the regeneration rate (proportion of regenerated plants) of maize plants regenerated from genetic transformation / tissue culture;

[0067] a20) Preparation of products for increasing the regeneration rate (proportion of regenerated plants) of maize plants regenerated from genetic transformation / tissue culture;

[0068] a21) Shortening the maize culture cycle;

[0069] a22) Preparation of products for shortening the maize culture cycle.

[0070] In any of the above-mentioned methods, kits or applications, the maize variety can be the maize elite inbred line ND101.

[0071] In the present invention, an optimized system suitable for genetic transformation of maize immature embryos was established by adding uniconazole to the differentiation / regeneration subculture medium, and efficient and stable genetic transformation of maize can be effectively achieved through the Agrobacterium-mediated method. Analysis of the transformation data of the transformation vectors in each of the year before and after optimization found that the proportion of regenerated plants obtained increased significantly from 4.5% before optimization to 20.04% after optimization; the annual average transformation efficiency increased significantly from 2.19% before optimization to 7.48% after optimization; and the average transformation cycle was shortened from 112 days before optimization to 93 days after optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is a schematic structural diagram of the recombinant expression vector pCAMBIA-DsRed.

[0073] Figure 2 A shows the phenotypes of the regenerated plants obtained by culturing in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group) for 3 weeks, respectively. Figure 2 B shows the statistical results of the stem diameters of the regenerated plants obtained by culturing in the control group and the experimental group for 3 weeks in different transformation batches. The measurement positions of the stem diameters are as shown by the red arrows in Figure 2 A. The data were analyzed by the analysis of variance method of t-test, ***P<0.001. Figure 2 C shows the statistical results of the relative chlorophyll content (SPAD) in the leaves of the regenerated plants obtained by culturing in the control group and the experimental group for 3 weeks in different transformation batches. The data were analyzed by the analysis of variance method of t-test, **0.05<P<0.01.

[0074] Figure 3 A is a paraffin section diagram of the cross-section of the stem nodes of the regenerated plants obtained by culturing in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group) for 3 weeks, respectively. Figure 3 B shows the statistical results of the number of vascular bundles in the cross-section of the stem nodes of the regenerated plants obtained by culturing in the control group and the experimental group for 3 weeks, respectively. The data were analyzed by the analysis of variance method of t-test, *0.01<P<0.05. Figure 3 C and Figure 3 D shows the statistical results of the puncture force measurement at the stem nodes of the regenerated plants obtained by culturing in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group) for 3 weeks, respectively, using a plant stem strength tester. The data were analyzed by the analysis of variance method of t-test, ***P<0.001.

[0075] Figure 4 A is the phenotype of the regenerated plants obtained by culturing for 30 days in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group). Figure 4 B is the statistical result of the proportion of regenerated plants obtained from the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group). The data was analyzed by the variance analysis method of t-test, **0.001 < P < 0.01. Figure 4 C is the identification result of positive regenerated plants from the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group). Figure 4 D is the statistical result of the transformation efficiency of the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group).

[0076] Figure 5 A is the statistical result of the proportion of regenerated plants obtained from the high-throughput genetic transformation system before and after optimization. Figure 5 B is the statistical result of the transformation efficiency of the high-throughput genetic transformation system before and after optimization. Figure 5 C is the statistical result of the transformation cycle of the high-throughput genetic transformation system before and after optimization. The data was analyzed by the variance analysis method of t-test, ***P < 0.001. Specific implementation manners

[0077] The present invention will be further described in detail below in combination with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0078] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0079] The maize elite inbred line ND101 in the following examples is described in the literature "Liu F, Cheng J, Liu X, Wang XQ. High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis. Int J Mol Sci. 2021 Nov 19; 22(22):12487. doi:10.3390 / ijms222212487. PMID:34830369; PMCID:PMC8619409. and 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." and can be obtained from the Research Center of Crop Functional Genomics and Molecular Breeding, China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0080] The Agrobacterium tumefaciens strain EHA105 in the following examples is described in the literature "Chen, S.; Songkumarn, P.; Liu, J.; Wang, G.-L. A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics. Plant Physiol. 2009, 150, 11111121." and can be obtained from the Research Center of Crop Functional Genomics and Molecular Breeding, China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0081] The overexpression vector pCAMBAIA3300 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." The public can obtain it from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0082] The gene editing vector pBUE411 in the following examples is described in the literature "Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14:327.doi:10.1186 / s12870-014-0327-y. PMID:25432517; PMCID:PMC4262988." The public can obtain it from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0083] The reagents and their sources involved in the following examples are as follows: MS basal salts (MS medium, Sigma - M524), pyridoxine hydrochloride (Sigma - 47862), thiamine hydrochloride (Sigma - 47858), inositol (Sigma - 15125), potassium nitrate (Sigma - P8291), ammonium sulfate (Simga - A3920), potassium dihydrogen phosphate (Amresco - 0781), magnesium sulfate heptahydrate (Sigma - M7506), calcium chloride dihydrate (Sigma - C7902), manganese sulfate (Simga - M7899), zinc sulfate heptahydrate (Simga - Z0251), potassium iodide (Simga - P8166), copper sulfate pentahydrate (domestic), sodium molybdate dihydrate (domestic), cobalt chloride hexahydrate (domestic), ferrous sulfate heptahydrate (Simga - F8633), disodium ethylenediaminetetraacetate (Na 2EDTA, Sigma - E6635), sucrose (domestic product), glucose (domestic product), maltose (domestic product), sodium chloride (domestic product), sorbitol (Sigma - S3889), hydrolyzed casein (Sigma - C7290), glutamine (Sigma, G8540), proline (Sigma - P5607), 2,4 - D (Phytotech - D309), picloram (Sigma - D5575), Phytagel (Phytotech - G3251), hygromycin (Phytotech - H385), 6 - benzylaminopurine (Sigma - B3408), naphthaleneacetic acid (Sigma - N0640), uniconazole (Wako - 212 - 00981), bilanafos (Wako - 022 - 15413), acetosyringone (Sigma - D134406), ticarcillin (domestic product), silver nitrate (Sigma - S7276), peptone (Sigma - P5905), yeast extract (Sigma - Y1625), kanamycin (Sigma - E004000), agar (Sigma - A1296).

[0084] The 100X MS organic in the following examples consists of a solvent and solutes. The solvent is double - distilled water, and the solutes and their concentrations are as follows: glycine 2.0 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 1 mg / L.

[0085] The 1000X MS organic in the following examples consists of a solvent and solutes. The solvent is double - distilled water, and the solutes and their concentrations are as follows: glycine 2.0 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.1 mg / L.

[0086] The Agrobacterium activation medium in the following examples is the YET medium, which consists of a solvent and solutes. The solvent is double - distilled water, and the solutes and their concentrations are as follows: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, agar 15 g / L. Sterilize at 121 °C for 15 minutes. When the medium cools to 50 °C, add rifampicin and kanamycin, and their concentrations are 25 mg / L and 50 mg / L respectively. The pH value of the YET medium is 7.

[0087] Example 1. Preparation of media required for Agrobacterium - mediated genetic transformation of immature embryos of maize inbred line ND101 I. Infection liquid medium IM

[0088] The infection liquid medium IM designed in the present invention is for the genetic transformation of immature embryos of maize inbred line ND101.

[0089] The infection liquid medium IM is composed of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 2.16 g / L MS basal salts, 10 mL / L 100X MS organic, 3 mg / L 2,4-D, 20 g / L sucrose, 10 g / L glucose, 0.115 g / L proline, 0.2 mM acetosyringone, 3.4 mg / L silver nitrate, 7 g / L agar powder, and the pH is 5.2.

[0090] II. Co-culture medium

[0091] The co-culture medium CCM designed for the genetic transformation of immature embryos of maize inbred line ND101 is the co-culture medium CCM.

[0092] The co-culture medium CCM is composed of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 2.16 g / L MS basal salts, 10 mL / L 100X MS organic, 3 mg / L 2,4-D, 20 g / L sucrose, 10 g / L glucose, 0.115 g / L proline, 0.2 mM acetosyringone, 3.4 mg / L silver nitrate, 7 g / L agar powder, and the pH is 5.8.

[0093] III. Resistant callus induction medium

[0094] The resistant callus induction medium SM designed for the genetic transformation of immature embryos of maize inbred line ND101 is the resistant callus induction medium SM.

[0095] The resistant callus induction medium SM is composed of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 10 mL / L 100X MS organic, 30 g / L sucrose, 1.38 g / L proline, 0.5 mg / L 2,4-D, 0.5 g / L casein hydrolysate, 2.2 mg / L picloram, 3.4 mg / L silver nitrate, 5.0 mg / L bilanafos, 100 mg / L ticarcillin and 3.0 g / L phytagel, and the pH is 5.8.

[0096] IV. Pre-differentiation medium

[0097] The pre-differentiation medium PRM designed for the genetic transformation of immature embryos of maize inbred line ND101 is the pre-differentiation medium PRM.

[0098] The pre-differentiation medium PRM consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 mL / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 5.0 mg / L bilanafos, 100 mg / L ticarcillin and 3.0 g / L phytagel, with a pH of 5.8.

[0099] V. Differentiation / Regeneration Medium

[0100] The differentiation / regeneration medium DM designed in the present invention for the genetic transformation of immature embryos of maize inbred line ND101 is the differentiation / regeneration medium DM.

[0101] The differentiation / regeneration medium DM consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 mL / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 5.0 mg / L bilanafos, 100 mg / L ticarcillin and 3.0 g / L phytagel, with a pH of 5.8.

[0102] VI. Subculture Medium for Differentiation / Regeneration

[0103] The subculture medium DSM for the genetic transformation of immature embryos of maize inbred line ND101 designed in the present invention is the subculture medium DSM for differentiation / regeneration.

[0104] The subculture medium DSM for differentiation / regeneration consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 4.33 g / L MS basal salts, 1 mL / L 1000X MS organic, 10 g / L glucose, 20 g / L maltose, 0.15 g / L aspartic acid, 0.1 g / L inositol, 0.045 mg / L uniconazole, 5.0 mg / L bilanafos, 100 mg / L ticarcillin and 3.0 g / L phytagel, with a pH of 5.8.

[0105] VII. Rooting Medium

[0106] The rooting medium RM designed in the present invention for the genetic transformation of immature embryos of maize inbred line ND101 is the rooting medium RM.

[0107] The rooting medium RM consists of a solute and a solvent. The solvent is double-distilled water, and the solute and its concentration are as follows: 2.16 g / L MS basal salts, 2 mL / L 100X MS organic, 15 g sucrose, 0.1 mg / L naphthylacetic acid, 5.0 mg / L bilanafos, 100 mg / L ticarcillin and 3.0 g / L phytagel, with a pH of 5.8.

[0108] Example 2: Establishment of an Agrobacterium-mediated genetic transformation system for immature embryos of maize inbred line ND101

[0109] I. Construction of recombinant expression vector

[0110] The DsRed gene shown in Sequence 1 was inserted between the single cleavage sites of Xcm I of the pCAMBAIA3300 vector to obtain the recombinant expression vector pCAMBIA3300-DsRed. The structural schematic diagram of the recombinant expression vector pCAMBIA3300-DsRed is as shown in Figure 1 As shown. It can be seen from the figure that the recombinant expression vector pCAMBIA3300-DsRed contains an expression cassette in which the DsRed gene is driven by the Ubi1-Promotor promoter and an expression cassette in which the Bar gene (herbicide resistance selection marker gene) is driven by the 35S Promotor promoter.

[0111] II. Agrobacterium-mediated genetic transformation method for immature embryos of maize inbred line ND101 (experimental group)

[0112] 1. Preparation of Agrobacterium

[0113] The recombinant expression vector pCAMBIA-DsRed constructed in Step 1 was introduced into Agrobacterium EHA105. After identification, Agrobacterium EHA105 containing pCAMBIA-DsRed was obtained. The bacterial solution of Agrobacterium EHA105 containing pCAMBIA-DsRed was streaked on the Agrobacterium activation medium and cultured for 48 hours under dark conditions at 28°C.

[0114] 2. Preparation of Agrobacterium resuspension

[0115] The Agrobacterium on the Agrobacterium plate in Step 1 was collected and inoculated into the infection medium IM in Example 1, and shaken and mixed evenly to obtain an Agrobacterium resuspension with an OD 660nm of 0.6 - 0.8.

[0116] 3. Infection

[0117] The immature embryos of maize NDA101 were collected and transferred to a sterile centrifuge tube containing the Agrobacterium resuspension, and incubated for 30 minutes under dark conditions at 22°C.

[0118] The method for obtaining the immature embryos of maize ND101 is as follows:

[0119] 1) Acquisition and picking of young ears

[0120] According to the literature "Liu S, Shi Y, Liu F, Guo Y, Lu M. LaCl 3The method reported in "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." was used to sow maize inbred line ND101 in the field respectively. At 9-12 days after artificial pollination, when the size of maize immature embryos was 1.5-2.0 mm, the young ears were harvested.

[0121] 2) Dissect immature embryos

[0122] Dissect immature embryos from the young ears obtained in step 1 according to the method reported in the literature "Sidorov V, Duncan D. Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol Biol. 2009; 526: 47-58. doi: 10.1007 / 978-1-59745-494-0_4. PMID: 19378003."

[0123] 4. Co-culture

[0124] Collect the immature embryos immersed in the Agrobacterium suspension after removing the Agrobacterium liquid as cleanly as possible after the incubation in step 3 onto the co-culture medium CCM in Example 1, and suck the residual Agrobacterium liquid on the surface of the immature embryos with sterilized filter paper. Then, disperse the immature embryos with a sterilized scalpel and place them with the scutellum facing up, and culture them under dark conditions at 22 °C for 1 day.

[0125] 5. Induction of resistant callus

[0126] Transfer the maize immature embryos after co-culture in step 4 to the resistant callus induction medium SM in Example 1 using a sterile scalpel, and still place them flat on the surface of the resistant callus induction medium SM with the scutellum side facing up, and culture them under dark conditions at 28 °C for 14 days.

[0127] 6. Pre-differentiation

[0128] Transfer the resistant callus formed in step 5 to the pre-differentiation medium PRM in Example 1 using sterilized forceps. Keep a distance of 1 cm between the calli and place them evenly on the medium. After covering the surface of the petri dish with a layer of medical gauze, culture them under weak light conditions of 25°C, 16 hours of light / 8 hours of darkness for 12 days.

[0129] 7. Differentiation / Regeneration

[0130] Transfer the callus pieces after the pre-differentiation culture in step 6 to the differentiation / regeneration medium DM in Example 1 using sterilized forceps. Keep a distance of 1.5 cm between each callus piece and place them evenly on the surface of the medium. Culture them under weak light conditions of 25°C, 16 hours of light / 8 hours of darkness for 20 - 30 days.

[0131] 8. Subculture of Differentiation

[0132] Transfer the tissue pieces with regenerated buds after the differentiation culture in step 7 to the subculture medium DSM for differentiation / regeneration in Example 1 using sterilized forceps. Keep a distance of 1.5 cm between each tissue piece and place them evenly on the surface of the medium. Culture them under weak light conditions of 25°C, 16 hours of light / 8 hours of darkness for 20 - 30 days.

[0133] 9. Rooting

[0134] Separate the regenerated plants formed by subculture of differentiation in step 8 from the tissue pieces using sterilized forceps, insert them into the rooting medium RM in Example 1, and culture them under weak light conditions of 25°C, 16 hours of light / 8 hours of darkness for 7 - 14 days to obtain transgenic positive regenerated plants.

[0135] III. Agrobacterium-mediated Genetic Transformation Method for Immature Embryos of Maize Inbred Line ND101 (Control Group)

[0136] Remove uniconazole from the subculture medium DSM for differentiation / regeneration in step 8 of step two, and the remaining steps are the same as those in step two.

[0137] IV. Analysis of Genetic Transformation Effect

[0138] 1. Observation of Phenotypes of Regenerated Plants

[0139] Observe the phenotypes of the regenerated plants after 3 weeks of subculture in the subculture medium for differentiation / regeneration (control group) and the subculture medium for differentiation / regeneration supplemented with uniconazole (experimental group) respectively.

[0140] The results are as Figure 2 shown in A. The results show that the regenerated plants obtained in the subculture medium for differentiation / regeneration supplemented with uniconazole (experimental group) are significantly sturdier than those obtained in the subculture medium for differentiation / regeneration without uniconazole (control group).

[0141] 2. Detection of the Stem Diameter of Regenerated Plants

[0142] Use a vernier caliper to measure the stem node diameters of the regenerated plants after 3 weeks of subculture in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group). A total of 11 regenerated plants were counted in the control group and 16 regenerated plants were counted in the experimental group.

[0143] The results are as Figure 2 shown in Figure B. The results show that the average stem node diameter of the regenerated plants after 3 weeks of subculture in the control group is 2.61 mm, and the average stem node diameter of the regenerated plants after 3 weeks of subculture in the experimental group is 4.23 mm.

[0144] 3. Detection of the Relative Chlorophyll Content in the Leaves of Regenerated Plants

[0145] Use a SPAD-502PLUS chlorophyll analyzer to measure the relative chlorophyll content in the leaves of the regenerated plants after 3 weeks of subculture in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group). A total of 11 regenerated plants were counted in the control group and 16 regenerated plants were counted in the experimental group.

[0146] The results are as Figure 2 shown in Figure C. The results show that the average relative chlorophyll content in the leaves of the regenerated plants after 3 weeks of subculture in the control group is 16.78 (SPAD), and the average relative chlorophyll content in the leaves of the regenerated plants after 3 weeks of subculture in the experimental group is 22.84 (SPAD).

[0147] 4. Detection of the Number of Vascular Bundles in the Cross-Section of the Stem Nodes of Regenerated Plants

[0148] The number of vascular bundles in the cross-section of the stem nodes of the regenerated plants after 3 weeks of subculture in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group) was counted separately. A total of 4 regenerated plants were counted in the control group, and a total of 6 regenerated plants were counted in the experimental group. The specific steps are as follows: Use a sharp surgical blade to cut 2-mm transverse tissues from the stem nodes of the regenerated plants obtained after 3 weeks of subculture in the control group and the experimental group, and then place them in 4% paraformaldehyde and fix them overnight at 4°C; then dehydrate the samples successively in 50%, 60%, 70%, 80%, 90%, and 100% ethanol; subsequently, soak the samples in xylene for clarification. Melt the paraffin at 65°C, add the samples, and after impregnating the wax for 48 h, embed the samples in the paraffin; take out the wax block for trimming, fix it on the microtome, start sectioning with a thickness of 8 μm, spread the sections in a 42°C constant temperature water bath, and pick up the sections with polylysine-coated slides; then bake the sections at 42°C for 2 h; put the sections into xylene for dewaxing, and successively put them into 100%, 90%, 80%, 70%, 50% ethanol, and PBS for rehydration; put the sections into safranin staining solution for 1 h, wash off the surface staining solution with PBS, then put them into fast green staining solution for 10 min, and wash off the surface staining solution with PBS. Then drop glycerol on the slide and cover it with a coverslip. This method for preparing paraffin sections is described in the literature "Zhang Y, Wang Y, Feng X, Zhang S, Xu X, Li L, Niu S, Bo Y, Wang C, Li Z, Xia G and Zhang H (2021) Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice. Nat. Commun. 12: 2523." Observe with an optical microscope Olympus BX53 and take pictures with an Olympus DP72 camera. Use the graphic image annotation tool LabelImg (this labeling software is described in the literature "Tzutalin D (2015) LabelImg. GitHub repository. 6.") to label and count the number of vascular bundles in the cross-section of the stem nodes of the regenerated plants.

[0149] The results are as Figure 3 shown in Figure 3 Figures A and B, and the results show that: the average number of vascular bundles in the cross-section of the stem nodes of the regenerated plants after 3 weeks of subculture in the control group was 62.25, and the average number of vascular bundles in the cross-section of the stem nodes of the regenerated plants after 3 weeks of subculture in the experimental group was 86.17.

[0150] 5. Detection of puncture force at the stem nodes of regenerated plants

[0151] The puncture force of the stem nodes of the regenerated plants after 3 weeks of subculture in the differentiation / regeneration subculture medium (control group) and the differentiation / regeneration subculture medium supplemented with uniconazole (experimental group) was measured using a plant stem strength meter SY-S03 (Shiya Technology Co., Ltd., Shijiazhuang, China). A total of 14 regenerated plants were counted in the control group and 19 regenerated plants were counted in the experimental group.

[0152] The results are as Figure 3 shown in Figure 3 Figures C and 2 D. The results show that the average puncture force of the stem nodes of the regenerated plants after 3 weeks of subculture in the control group was 1.53 N / mm 2 , and the average puncture force of the stem nodes of the regenerated plants after 3 weeks of subculture in the experimental group was 2.93 N / mm.

[0153] 6. Proportion of regenerated plants obtained and transformation efficiency

[0154] The proportion of regenerated plants obtained and the transformation efficiency were counted for the control group and the experimental group, respectively.

[0155] Proportion of regenerated plants obtained = number of regenerated plants transplanted / number of co-cultured immature embryos × 100%.

[0156] Transformation efficiency = number of bar-positive regenerated plants / number of co-cultured immature embryos × 100%.

[0157] Among them, the bar-positive regenerated plants were obtained by PCR amplification using primers bar-F: ATGAGCCCAGAACGACGC and bar-R: TCAAATCTCGGTGACGGG to obtain regenerated plants with a size of 552 bp.

[0158] The results are as Figure 4 shown. The results show that the average proportion of regenerated plants obtained in the control group was 11.6%, and the average proportion of regenerated plants obtained in the experimental group was 18.2%; the average transformation efficiency in the control group was 9.4%, and the average transformation efficiency in the experimental group was 15.6%.

[0159] Example 3. Verification of a high-throughput genetic transformation system for Agrobacterium-mediated immature embryos of maize inbred line ND101 I. Construction of a recombinant expression vector

[0160] 1. Construction of a gene editing vector

[0161] Construct a gene editing vector for editing the target genes shown in Table 1 according to the gene editing vector construction method in the literature "Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14:327. doi:10.1186 / s12870-014-0327-y. PMID:25432517; PMCID:PMC4262988." The specific steps are as follows: Replace the ccdb in the pBUE411 vector with the target sequence GGTGCCCTTCCGATCAACA corresponding to the target gene shown by the gene number Zm00001d040567, and insert this target sequence between the single digestion sites of BasI in the pBUE411 vector to obtain the Zm00001d040567 gene editing vector for maize transformation. According to the same method, ligate the target sequences designed according to other target genes into the pBUE411 vector to obtain other gene editing vectors for maize transformation.

[0162] Table 1

[0163]

[0164]

[0165] 2. Construction of overexpression vector

[0166] Construct the overexpression vectors with the target genes being GRMZM2G355846, Zm00001d040567, Zm00001d028949, Zm00001d033316, Zm00001d008901, Zm00001d038690, Zm00001d008899, Zm00001d023353, Zm00001d052340, Zm00001d005748, Zm00001d024762, Zm00001d043389, Zm00001d006944, Zm00001d038048, Zm00001d036879, Zm00001d022451, Zm00001d012697, Zm00001d000080, Zm00001d048460, Zm00001d007167, Zm00001d012696, Zm00001d023365, Zm00001d023360, Zm00001d052544, Zm00001d023631, Zm00001d053293, Zm00001d030955 or GRMZM2G125656 (the sequences of each target gene are from the Gramene database (https: / / ensembl.gramene.org / Zea_mays / Info / Index)) according to the construction method of the recombinant expression vector 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." The specific steps are as follows: Insert the sequence of the target gene with the gene number GRMZM2G35584 between the double digestion sites of XhoI and KpnI in the pCAMBAIA3300 vector to obtain the GRMZM2G35584 overexpression vector for maize transformation. According to the same method, insert the sequences of other target genes in Table 2 into the pCAMBAIA3300 vector to obtain other overexpression vectors for maize transformation.

[0167] II. High-throughput genetic transformation optimization method (after optimization) of immature embryos of maize inbred line ND101 mediated by Agrobacterium

[0168] The 37 recombinant expression vectors constructed in Step 1 (2 overexpression vectors respectively targeting Zm00001d008901 and Zm00001d040567, and 35 gene editing vectors respectively targeting Zm00001d040567, Zm00001d028949, Zm00001d033316, Zm00001d008901, Zm00001d032155, Zm00001d041422, Zm00001d044553, Zm00001d029075, Zm00001d038690, Zm00001d008899, Zm00001d023353, Zm00001d052340, Zm00001d005748, Zm00001d043389, Zm00001d010743, Zm00001d020496, Zm00001d036879, Zm00001d046175, Zm00001d015325, Zm00001d050422, Zm00001d020497, Zm00001d022451, Zm00001d012697, Zm00001d000080, Zm00001d022272, Zm00001d012696, Zm00001d012696, Zm00001d023365, Zm00001d008833, Zm00001d023360, Zm00001d052544, Zm00001d023631, Zm00001d030955, Zm00001d033295 and Zm00001d023504) were used for Agrobacterium-mediated genetic transformation of immature embryos of maize inbred line ND101 according to the method in Step 2 of Example 2.

[0169] III. High-throughput genetic transformation method of immature embryos of maize inbred line ND101 mediated by Agrobacterium (before optimization)

[0170] The 32 recombinant expression vectors constructed in Step 1 (26 overexpression vectors respectively targeting Zm00001d033316, Zm00001d008901, Zm00001d038690, Zm00001d008899, Zm00001d023353, Zm00001d023353, Zm00001d005748, Zm00001d024762, Zm00001d043389, Zm00001d006944, Zm00001d038048, Zm00001d018799, Zm00001d036879, Zm00001d022451, Zm00001d012697, Zm00001d000080, Zm00001d048460, Zm00001d007167, Zm00001d010459, Zm00001d023365, Zm00001d023360, Zm00001d052544, Zm00001d023631, Zm00001d053293, Zm00001d030955, Zm00001d002641 and 6 gene editing vectors respectively targeting Zm00001d002641, Zm00001d027455, Zm00001d018799 (T01), Zm00001d018799 (T03), Zm00001d052416 and Zm00001d044285) were used for Agrobacterium-mediated genetic transformation of immature embryos of maize inbred line ND101 according to the method in Step 3 of Example 2.

[0171] IV. Analysis of genetic transformation effects before and after optimization

[0172] According to the method described in item 6 of Step 4 of Example 2, the obtained proportion of regenerated plants, transformation efficiency, and transformation period (the time from Agrobacterium infection of maize immature embryos to obtaining Bar-positive T 0 regenerated plants) obtained by the genetic transformation methods before and after optimization were compared and analyzed.

[0173] The results are as Figure 5 shown. The results show that the average obtained proportion of regenerated plants, average transformation efficiency, and average transformation period of the genetic transformation method before optimization were 4.50%, 2.19%, and 112 days respectively, while those of the genetic transformation method after optimization were 20.04%, 7.48%, and 93 days respectively. Compared with the genetic transformation method before optimization, the obtained proportion of regenerated plants and transformation efficiency of the genetic transformation method after optimization were significantly improved, and the transformation period was significantly shortened.

[0174] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. A method for genetic transformation of maize immature embryos mediated by Agrobacterium, comprising the following steps: 1) Infecting maize immature embryos with Agrobacterium containing the target vector to obtain infected immature embryos; 2) culturing the infected immature embryos in a co-culture medium to obtain co-cultured immature embryos; 3) culturing the immature embryos after co-cultivation in a resistance callus induction medium to obtain resistant callus tissue; 4) culturing the resistant callus in a pre-differentiation medium to obtain a callus mass with regenerated buds growing thereon; 5) culturing the callus mass with regenerated buds in a differentiation / regeneration medium to obtain differentiated seedlings or tissue mass with leaves; 6) culturing the differentiated seedlings or tissue blocks with leaves in a differentiation / regeneration subculture medium to obtain resistant regenerated plants; the differentiation / regeneration subculture medium contains uniconazole; 7) Cultivating the resistant regenerated plants in a rooting medium to obtain T0 generation plants.

2. The method according to claim 1, characterized in that: In 1), the infection method comprises the following steps: 1-1) activating the Agrobacterium containing the target vector on an Agrobacterium activation medium to obtain activated Agrobacterium; 1-2) shaking and mixing the activated Agrobacterium in the infection liquid culture medium to obtain an Agrobacterium resuspension; The infection liquid culture medium includes 2.16-3.44 g / L MS basic salts, 8-12 mL / L 100X MS organic, 2-4 mg / L 2,4-D, 10-30 g / L sucrose, 10-30 g / L glucose, 0.1-0.15 g / L proline, 0.2-0.4 mM acetosyringone, 1.7-3.4 mg / L silver nitrate, and 7-10 g / L agar powder; 1-3) Immersing maize inbred line immature embryos in the Agrobacterium resuspension solution to obtain infected immature embryos.

3. The method according to claim 1 or 2, characterized in that: In the above 2), the co-cultivation medium includes 2.16-3.44 g / L MS basal salts, 8-12 mL / L 100X MS organic, 2-4 mg / L 2,4-D, 10-30 g / L sucrose, 10-30 g / L glucose, 0.1-0.15 g / L proline, 0.2-0.4 mM acetosyringone, 1.7-3.4 mg / L silver nitrate, and 7-10 g / L agar powder.

4. The method according to any one of claims 1 to 3, characterized in that: In the above 3), the resistant callus induction medium includes 2.16-4.33 g / L MS basal salts, 8-12 mL / L 100X MS organic, 10-30 g / L sucrose, 1-2 g / L proline, 0.5-1 mg / L 2,4-D, 0.5-1 g / L hydrolyzed casein, 2-3 mg / L piclopidine, 1.7-3.4 mg / L silver nitrate, 3-7 mg / L bialaphos, 100-200 mg / L timentin and 3-4 g / L plant gel.

5. The method according to any one of claims 1 to 4, characterized in that: In the above 4), the pre-differentiation medium includes 2.16-4.33 g / L MS basal salts, 0.8-1.2 mL / L 1000X MS organic, 10-30 g / L glucose, 10-30 g / L maltose, 0.1-0.2 g / L aspartic acid, 0.1-0.15 g / L inositol, 3-7 mg / L bialaphos, 100-200 mg / L timentin and 3.0-4.0 g / L plant gel.

6. The method according to any one of claims 1 to 5, characterized in that: In the above 5), the differentiation / regeneration medium includes 2.16-4.33 g / L MS basal salts, 0.8-1.2 mL / L 1000X MS organic, 10-30 g / L glucose, 10-30 g / L maltose, 0.1-0.2 g / L aspartic acid, 0.1-0.15 g / L inositol, 3-7 mg / L bialaphos, 100-200 mg / L timentin and 3.0-4.0 g / L phytagel; Or, in 6), the differentiation / regeneration subculture medium includes 2.16-4.33 g / L MS basal salts, 0.8-1.2 mL / L 1000X MS organic, 10-30 g / L glucose, 10-30 g / L maltose, 0.1-0.2 g / L aspartic acid, 0.1-0.15 g / L inositol, 0.02-0.06 mg / L uniconazole, 3-7 mg / L bisabolamide, 100-200 mg / L timentin and 3-4 g / L phytagel.

7. The method according to any one of claims 1 to 6, characterized in that: In the above 7), the rooting medium comprises 2.16-4.33 g / L MS basal salt, 1-2 mL / L 100X MS organic, 10-30 g / L sucrose, 0.1-0.15 mg / L naphthylacetic acid, 0-5.0 mg / L bipropylamine, 100-200 mg / L timentin and 3-4 g / L plant gel.

8. The method according to any one of claims 1 to 7, characterized in that: In 1), the immature embryo is an immature embryo obtained by peeling off a young ear; the young ear is an young ear picked when the size of the corn immature embryo is 1.5-2 mm; Or, in 1-2), the OD of the Agrobacterium resuspension is 660 0.6-0.8; Or, in 1-3), the soaking time is 30-60 min; Or, in 2), the culture conditions are as follows: dark culture at 20-25° C. for 1-2 days; Or, in 3), the culture conditions are as follows: dark culture at 25-28° C. for 10-14 days; Or, in 4), the culture conditions are as follows: 25-28°C in weak light for 10-14 days; Or, in 5), the culture conditions are as follows: 25-28°C in weak light for 25-30 days; Or, in 6), the culture conditions are as follows: 25-28°C in weak light for 25-30 days; Or, in 7), the culture conditions are as follows: 25-28° C. under weak light for 7-14 days.

9. A kit for genetic transformation of corn, comprising the co-cultivation medium described in any one of claims 1 to 8 and / or the resistant callus inducing medium described in any one of claims 1 to 8 and / or the pre-differentiation medium described in any one of claims 1 to 8 and / or the differentiation / regeneration medium described in any one of claims 1 to 8 and / or the differentiation / regeneration subculture medium described in any one of claims 1 to 8 and / or the rooting medium described in any one of claims 1 to 8 and / or the infection solution described in any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the kit according to claim 9 in any one of the following a1) to a22): a1) Genetic transformation of maize; a2) preparing products of genetic transformation of maize; a3) High-throughput genetic transformation of maize; a4) preparing products for high-throughput genetic transformation of corn; a5) Maize tissue culture; a6) preparing products of corn tissue culture; a7) Improving the efficiency of genetic transformation of maize; a8) preparing products that improve the efficiency of corn genetic transformation; a9) Improving the efficiency of high-throughput genetic transformation of maize; a10) preparing products for improving the efficiency of high-throughput genetic transformation of corn; a11) improving the efficiency of corn tissue culture; a12) preparing products that improve corn tissue culture efficiency; a13) increasing the chlorophyll content in leaves of corn genetically transformed / tissue culture regenerated plants; a14) preparing a product for increasing the chlorophyll content in leaves of genetically transformed / tissue culture regenerated maize plants; a15) increasing the stem diameter of genetically transformed / tissue culture regenerated maize plants; a16) preparing a product for increasing the stem diameter of corn genetically transformed / tissue culture regenerated plants; a17) Improving the stalk strength of corn genetically transformed / tissue culture regenerated plants; a18) preparing a product for improving the stalk strength of corn genetically transformed / tissue culture regenerated plants; a19) Improving the efficiency of corn genetic transformation / tissue culture regeneration plants; a20) preparing products for improving the yield of corn genetic transformation / tissue culture regeneration plants; a21) shortening the corn cultivation cycle; a22) preparing a product that shortens the corn cultivation cycle.

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