Efficient rice genetic transformation method capable of overcoming genotype dependence and special culture medium

Abstract

The invention discloses an efficient rice genetic transformation method capable of overcoming genotype dependence and a special culture medium, and relates to the technical field of biology. The special culture medium is prepared from KCl, KH2PO4, MgSO4. 7H2O, CaCl2. 2H2O, disodium ethylene diamine tetraacetate, FeSO4. 7H2O, MnSO4. H2O, ZnSO4. 7H2O, H3BO3, KI, CoCl2. 6H2O, CuSO4. 5H2O, Na2MoO4. 2H2O, glutamine, aspartic acid, arginine, glycine, inositol, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and a carbon source. By adopting the special culture medium, the genetic transformation efficiency of a rice variety containing the NiR gene E375S453 haplotype can be improved, and the special culture medium has important significance in overcoming the bottleneck of rice genotype dependence and promoting functional genome research and molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a high-efficiency rice genetic transformation method and a special culture medium for overcoming genotype dependence. Background Art

[0002] Rice (Oryza sativa L.) is a major food crop and a key model plant for studying plant gene function and genetic improvement. Genetic transformation technology is a crucial prerequisite for elucidating rice gene function, developing and applying gene editing tools, and promoting biotechnology breeding. Currently, Agrobacterium-mediated genetic transformation is the mainstream method for rice transformation, achieving high efficiency in some model japonica rice varieties (such as Zhonghua 11, Nipponbare, and Kitaake). However, currently used transformation methods still cannot meet the requirements for efficient transformation in most varieties, and genotype dependence remains an urgent issue that needs to be addressed. This bottleneck is most evident in the significant differences in regeneration capacity among different rice varieties: First, it is known that a considerable number of rice varieties, especially those produced on a large commercial scale, such as the famous high-quality japonica rice variety Koshihikari and the female parent Pei'ai 64S of the super rice variety Liangyoupeijiu, have very low plant regeneration capacity from their callus tissue under the existing conventional transformation system, and some varieties even have no plant regeneration capacity; Second, despite years of efforts by researchers, such as optimizing transformation strategies, trying different transformation processes (such as shortening the subculture time), and optimizing culture medium formulations, the plant regeneration problem of some transformation-resistant varieties has not been effectively solved. This widespread regeneration disorder has severely restricted the development of rice functional genomics research and modern biobreeding, specifically: (1) it is impossible to directly verify gene function and study regulatory mechanisms in excellent germplasms that have excellent and important agronomic traits (such as stress resistance, high yield, and high quality) but are difficult to transform, so it is impossible to deeply understand the value of important genes in different genetic backgrounds; (2) excellent genes (such as disease resistance, insect resistance, herbicide resistance, and nutrient efficient utilization genes) or gene editing tools are difficult to efficiently and quickly introduce into various excellent varieties or local specialty varieties widely used in production, greatly limiting the application scope and efficiency of molecular design breeding; (3) it is difficult to achieve efficient mining of excellent genes for rich genetic diversity resources.

[0003] For a long time, researchers have conducted extensive explorations to solve the problem of genotype dependence, including: optimizing culture medium components (such as adjusting hormone ratios, adding specific compounds), improving Agrobacterium co-cultivation conditions, screening for highly embryogenic callus, relying on specific easily transformed genotypes, or developing alternative methods (such as gene guns). However, most of these efforts have failed to fundamentally solve the problem of genotype dependence. The core reason is that there is insufficient understanding of the universal core molecular mechanisms and key factors that regulate rice regeneration ability, resulting in optimization strategies that are often empirical and genotype-specific, making it difficult to achieve universal promotion and application across varieties. Previous researchers used a population derived from the hybridization of Koshihikari (japonica rice Koshihikari, no regeneration ability) and Kasalath (indica rice, high regeneration ability) to map-clone the major effect QTL PSR1 (Promoter of Shoot Regeneration 1) gene (i.e., NiR gene) at 45.4 cM on the short arm of rice chromosome 1, and confirmed the promoter region variation of Koshihikari and the substitution of two amino acids in the gene coding region (E 375 D and S 453 A site) leads to a significant decrease in NiR enzyme activity (only 62.5% of Kasalath), affecting nitrogen assimilation efficiency and impairing cell activity, thus leading to regeneration defects. NiR is responsible for catalyzing nitrite (NO2 - ) is reduced to ammonium (NH4 +Decreased NiR activity directly affects this reduction reaction, leading to the accumulation of toxic nitrite. Nitrite levels as high as 1.2 μmol / g FW were detected in the culture medium of subcultured Koshihikari calli, whereas this compound was undetectable in the culture medium of subcultured Kasalath calli. Based on this discovery, introducing the NiR gene from Kasalath into Koshihikari significantly improved its regeneration ability (Nishimura A, Ashikari M, Lin S, TakashiT, Angeles ER, Yamamoto T, Matsuoka M. Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems. Proc Natl Acad Sci US A. 2005 Aug 16;102(33):11940-4. doi: 10.1073 / pnas.0504220102. Epub 2005 Aug 9. PMID: 16091467; PMCID: PMC1187985). However, the method of using this mechanism to improve rice transformation efficiency has not been widely used. The main reasons are: (1) low operability, that is, first using NiR to transform varieties with low regeneration ability, and then transforming the target gene, or co-transforming two vectors, is time-consuming and labor-intensive, making it difficult to achieve large-scale genetic transformation; (2) there is a lack of comprehensive analysis of the genetic variation of NiR genes in different varieties, and the relationship between NiR enzyme activity and transformation efficiency still needs to be more widely verified; (3) based on the nitrogen metabolism process, whether a universal culture medium can be designed to circumvent the problem of low NiR activity has not been deeply explored.

[0004] Therefore, developing a truly efficient, stable and broadly adaptable genetic transformation method that is not restricted by rice genotype is of great significance for overcoming the bottleneck of rice genotype dependence and promoting functional genomic research and molecular breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for efficient genetic transformation of rice that overcomes genotype dependence and a special culture medium to solve the problems of the above-mentioned prior art. The use of this special culture medium can fundamentally solve the regeneration disorder caused by low NiR activity and improve the efficiency of rice containing NiR gene E 375 S 453 Genetic transformation efficiency of haplotype-dependent rice varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a special medium for efficient genetic transformation of rice for overcoming genotype dependence. The special medium comprises the following components in parts by weight: 2500-3500 parts of KCl, 150-250 parts of KH2PO4, 350-420 parts of MgSO4·7H2O, 400-500 parts of CaCl2·2H2O, 35-40 parts of disodium edetate, 25-30 parts of FeSO4·7H2O, 15-20 parts of MnSO4·H2O, 7-10 parts of ZnSO4·7H2O, 35-7 parts of H3BO, 0.5-1 part of KI, 0.025 parts of CoCl2·6H2O, 0.025 parts of CuSO4·5H2O, 0.025 parts of Na2MoO4·2H2O 0.25 parts, 800-1000 parts of glutamine, 200-300 parts of aspartic acid, 300-400 parts of arginine, 50-100 parts of glycine, 100-200 parts of cyclohexane hexol, 2 parts of thiamine hydrochloride, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid and 20,000-30,000 parts of carbon source.

[0008] Preferably, the special medium for efficient genetic transformation of rice comprises the following components in parts by weight: 3000 parts of KCl, 200 parts of KH2PO4, 400 parts of MgSO4·7H2O, 450 parts of CaCl2·2H2O, 38 parts of disodium edetate, 28 parts of FeSO4·7H2O, 18 parts of MnSO4·H2O, 8 parts of ZnSO4·7H2O, 36 parts of H3BO, 0.75 parts of KI, 0.025 parts of CoCl2·6H2O, 0.025 parts of CuSO4·5H2O, 0.025 parts of Na2MoO4·2H2O 0.25 parts, 900 parts of glutamine, 250 parts of aspartic acid, 350 parts of arginine, 75 parts of glycine, 150 parts of cyclohexane hexol, 2 parts of thiamine hydrochloride, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid and 25,000 parts of carbon source.

[0009] Furthermore, the carbon source is sucrose.

[0010] The present invention also provides application of the above-mentioned special medium for efficient genetic transformation of rice in improving the efficiency of genetic transformation of rice.

[0011] Furthermore, the special medium for efficient genetic transformation of rice improves the genetic transformation efficiency of rice by improving the regeneration disorder caused by low NiR enzyme activity.

[0012] The present invention also provides a method for efficient genetic transformation of rice that overcomes genotype dependence, comprising the step of genetically transforming rice using the above-mentioned special culture medium for efficient genetic transformation of rice.

[0013] Furthermore, the special medium for efficient genetic transformation of rice improves the genetic transformation efficiency of rice by improving the regeneration disorder caused by low NiR enzyme activity.

[0014] Furthermore, the rice contains the NiR gene E 375 S 453 Haplotype variety.

[0015] Furthermore, the genetic transformation specifically comprises the following steps:

[0016] sterilizing the rice seeds and then transferring them to the special medium for efficient genetic transformation of rice for induction culture to obtain callus tissue;

[0017] The callus tissue is infected with Agrobacterium containing the target gene, and after co-cultivation, transformed callus tissue is screened; the screening is performed on the special medium for efficient genetic transformation of rice;

[0018] The transformed callus tissue is transferred to the special medium for efficient genetic transformation of rice for plant regeneration culture, and then subjected to seedling hardening treatment to obtain transgenic rice plants.

[0019] Furthermore, the induction culture is carried out in a dark environment at 28°C; and / or

[0020] The co-cultivation is carried out in a dark environment at 22°C; and / or

[0021] The screening was performed in a dark environment at 28°C; and / or

[0022] The plant regeneration culture and the seedling hardening treatment were both carried out under a cold fluorescent light condition at 28°C.

[0023] The present invention discloses the following technical effects:

[0024] This paper develops a rice high-efficiency genetic special culture medium (Bypass-N) that overcomes genotype dependence. By changing the culture medium components (completely replacing inorganic nitrogen with amino acids), the functional disorder of NiR enzyme is bypassed from the metabolic pathway without involving any genetic manipulation. This fundamentally solves the problem of low NiR activity (E 375 S 453 Transformation experiments confirmed that Bypass-N medium can effectively inhibit the growth of E 375 S 453 The amino acid nitrogen source metabolism pathway provided by Bypass-N medium does not depend on NiR function, so its effect is not affected by the rice endogenous NiR genotype (E 375 S 453 or D 375 A453 ) restrictions.

[0025] The Bypass-N medium provided by the present invention can directly replace the standard regeneration medium (such as MS) for genetic transformation. This method is particularly suitable for E 375 S 453 The study of rice germplasm (especially temperate japonica rice) makes it possible to directly verify gene function, introduce excellent genes and conduct molecular design breeding in these important germplasm resources, which is of great significance for overcoming the bottleneck of rice genotype dependence and promoting functional genomics research and molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Figures 1 and 2 show the experimental results of genetic transformation of Koshihikari materials using Bypass-N medium and MS medium, respectively. Figure A shows the results of coleoptiles induced on different culture media under white light conditions. Figure B shows the results of GFP signal detection of GFP-positive cells by imaging under white light and ultraviolet light during antibiotic selection of callus cultured on different culture media. Figure C shows the results of evaluating regeneration capacity at 26 days on different culture media. Figure D shows the results of GFP signal detection of buds regenerated on Bypass-N medium and non-regenerated callus on MS medium under white light and ultraviolet light, respectively.

[0028] Figure 2 Schematic diagram of the rice NiR gene structure and alternative splicing transcript structure, where * represents two core amino acid variation sites 375 and 453;

[0029] Figure 3 The pie chart shows the genotype frequency of SNPs at 375 sites of NiR gene in 3825 Chinese rice germplasms; Kashihari-type: G / G genotype (E 375 ), Kasalath-type: C / C+C / T+T / T genotype (D 375 ), Intermediate-type: G / T+G / C genotype;

[0030] Figure 4 Schematic diagram of the amino acid sequence of ferredoxin-nitrite reductase protein and its key functional domains. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] In the process of plant tissue culture, the utilization of nitrogen metabolism mainly includes two key processes: inorganic nitrogen metabolism and amino acid metabolism. Inorganic nitrogen is nitrate (NO3 - ) and ammonium salts (NH4 + ) is absorbed by cells and then gradually reduced to NH4 by the catalysis of nitrate reductase (NR) and nitrite reductase (NiR). + NH4 +Subsequently, glutamine is combined with glutamate through the glutamine synthetase (GS) pathway to produce glutamine. Glutamine then reacts with α-ketoglutaric acid through glutamate synthase (GOGAT) to produce two molecules of glutamate. This GS-GOGAT cycle is the main pathway for ammonium assimilation in plant cells. At the same time, NH4 + Glutamate can also be produced directly by reacting with α-ketoglutarate via the glutamate dehydrogenase (GDH) pathway, but this pathway contributes minimally under normal physiological conditions. As a core molecule in nitrogen metabolism, glutamate, through transaminases, transfers its amino group to other carboxylic acids (such as pyruvate and oxaloacetate) to synthesize various amino acids (such as alanine and aspartate). It also provides substrate for the GS-GOGAT cycle, maintaining efficient nitrogen recycling within the cell. During amino acid metabolism, TCA cycle intermediates (such as α-ketoglutarate, oxaloacetate, and pyruvate) provide the carbon skeleton for amino acid synthesis, while sugar metabolism and carboxylic acid metabolism (such as malate and citric acid) provide essential precursors for this process. Furthermore, exogenously added organic nitrogen (such as amino acids) or endogenous protein degradation products can be directly metabolized through transamination or deamination reactions, achieving efficient nitrogen recycling. This series of metabolic processes not only provides essential nitrogen and amino acids for plant cell growth but is also tightly coupled with energy metabolism (such as the TCA cycle and oxidative phosphorylation) to ensure normal cell proliferation and differentiation in tissue culture. By optimizing the ratio and addition form of inorganic and organic nitrogen in the culture medium and regulating the efficiency of the GS-GOGAT cycle, nitrogen utilization efficiency and target product synthesis in tissue culture systems can be significantly improved.

[0037] Nitrogen metabolism disorder is one of the factors that cause rice regeneration problems. Based on this characteristic, the present invention designed a conversion medium formula that avoids inorganic nitrogen metabolism. The core design principle is to completely eliminate nitrate nitrogen (NO3 - ) and ammonium nitrogen (NH4 + ), avoiding the risk of nitrogen metabolism disorders from the source; introducing a four-membered amino acid nitrogen system: glutamine (the main amino group donor, directly involved in the GS / GOGAT cycle) + aspartic acid (provides a carbon skeleton and amino groups, promoting purine synthesis) + arginine (a polyamine precursor, stimulating cell differentiation) + glycine (a one-carbon unit donor, supporting chlorophyll synthesis). With amino acids as the main component, supplemented with other nutrients, the regeneration process does not require nitrogen metabolism, and the NH2 provided by amino acids is directly utilized. -The designed culture medium formula is shown in Table 1 and is named Nitrogen metabolism-bypassing, abbreviated as Bypass-N.

[0038] Table 1 Bypass-N medium formula

[0039]

[0040] Note: This culture medium does not contain any nitrate nitrogen (NO3 - ) or ammonium nitrogen (NH4 + ) source of inorganic salts, the nitrogen source is provided entirely by a specified amino acid combination; pH range: 5.2-5.8.

[0041] Example 1

[0042] A Bypass-N medium for efficient genetic transformation of rice that overcomes genotype dependence, the specific formula of which is shown in Table 2.

[0043] Table 2 Bypass-N medium formula of Example 1

[0044]

[0045] Example 2

[0046] A Bypass-N medium for efficient genetic transformation of rice that overcomes genotype dependence, the specific formula of which is shown in Table 3.

[0047] Table 3 Bypass-N medium formula of Example 2

[0048]

[0049] Example 3

[0050] A Bypass-N medium for efficient genetic transformation of rice that overcomes genotype dependence, the specific formula of which is shown in Table 4.

[0051] Table 4 Bypass-N medium formula of Example 3

[0052]

[0053] The function verification is performed below using the Bypass-N culture medium of Example 1 as an example.

[0054] Example 4

[0055] Experimental verification was conducted using Koshihikari as the rice material: Genetic transformation of Koshihikari material was performed using the Bypass-N medium of Example 1 and conventional MS medium. The transformation method using Bypass-N medium was as follows:

[0056] 1. Seed disinfection

[0057] Remove the hulls of mature seeds and discard any spotted seeds. Transfer the hulled seeds to a 50 mL centrifuge tube and rinse with 70% (v / v) ethanol for 1 minute. Discard the ethanol and rinse with sterile ultrapure water. Add 20 wt% sodium hypochlorite solution and shake on a shaker for 30 minutes. Discard the sodium hypochlorite solution and rinse with sterile ultrapure water. Absorb the surface moisture from the seeds.

[0058] 2. Callus Induction

[0059] The sterilized seeds were transferred to a solid callus induction medium (Bypass-N medium) and cultured in a dark environment at 28°C to induce callus tissue.

[0060] 3. Agrobacterium Infection and Callus Co-cultivation

[0061] (1) Store the Agrobacterium strain containing the target gene (i.e., GFP gene) construct in YEB medium containing 15% (v / v) glycerol. Take 50 μL of the Agrobacterium stock solution and inoculate it into a sterile 15 mL centrifuge tube containing 5 mL of YEB medium (add 5 μL of rifampicin solution and 5 μL of kanamycin solution), place it on a shaker at 140 rpm and culture overnight until the bacterial solution OD reaches 0. 600 When the value reaches 1.0, the Agrobacterium culture solution is obtained and set aside.

[0062] (2) Transfer the Agrobacterium culture solution to a centrifuge tube, centrifuge at 4000 × g for 5 minutes, discard the supernatant, resuspend the Agrobacterium pellet in liquid N6-AS medium, and adjust the concentration of the culture solution to OD 600 The value reached 0.02, and Agrobacterium suspension was obtained.

[0063] (3) Transfer 15 g of embryogenic callus to a sterile 50 mL centrifuge tube, add 25 mL of the Agrobacterium suspension obtained in step (2), and gently shake the centrifuge tube for 3 minutes. Discard the suspension and use a scraper to transfer the callus to a sterilized wipe to remove excess liquid.

[0064] (4) Transfer the callus tissue from the wipe paper to solid N6-AS medium covered with sterile filter paper and culture it in a dark environment at 22°C for 3-4 days.

[0065] 4. Transformed Callus Screening

[0066] (1) Transfer the co-cultured callus tissue to a sterile centrifuge tube containing sterile ultrapure water, shake gently for a few seconds, discard the water, and repeat the washing process four times; during the final washing, add carbenicillin solution to the ultrapure water to a final concentration of 250 mg / L.

[0067] (2) Transfer the washed callus onto sterile wipes to remove excess water. Place 20 calli per dish in Bypass-N medium. Seal the dish with medical tape and incubate at 28°C in the dark for 20 days. Actively growing transformed callus is screened.

[0068] The transformed callus blocks obtained by screening were transferred to fresh Bypass-N medium and cultured under the same conditions for 5 days for the second round of screening.

[0069] 5. Differentiation of resistant callus buds and hardening of transgenic plants

[0070] (1) The resistant callus after the second round of screening was transferred to the plant regeneration medium (Bypass-N medium), placed in an environment of 28°C, and cultured under cold fluorescent light for 20 days.

[0071] (2) Transfer the differentiated seedlings to a 125 mL Erlenmeyer flask containing 40 mL of MS-RG medium and culture them under a cold fluorescent light at 28°C to promote root growth. After 10-14 days, transplant them into a flower pot.

[0072] The method for genetic transformation of Koshihikari materials using conventional MS medium is the same as above, with the only difference being that the Bypass-N medium is replaced by MS medium.

[0073] The preparation methods of the culture medium and reagents used in this example are as follows:

[0074] 2,4-Dichlorophenoxyacetic acid (2,4-D) solution (2 mg / mL): Weigh 200 mg of 2,4-dichlorophenoxyacetic acid, add 20 mL of 0.1 N sodium hydroxide solution, stir magnetically until completely dissolved, then add 80 mL of ultrapure water to make up to 100 mL. Store at 4°C.

[0075] Vitamin-glycine solution (1000×): Weigh 500 mg of thiamine hydrochloride, 100 mg of pyridoxine hydrochloride, 100 mg of niacin, and 200 mg of glycine, dissolve in 100 mL of ultrapure water, and store at 4°C.

[0076] Copper / molybdenum / cobalt solution (1000×): Weigh 25 mg of copper sulfate pentahydrate (CuSO4·5H2O), 250 mg of sodium molybdate dihydrate (NaMoO4·2H2O), and 25 mg of cobalt chloride hexahydrate (CoCl2·6H2O), dissolve in 1000 mL of ultrapure water, and store at 4°C.

[0077] Potassium iodide solution (0.8 mg / mL): Weigh 80 mg of potassium iodide and dissolve it in 100 mL of ultrapure water. Store at 4°C.

[0078] Kanamycin solution (50 mg / mL): Weigh 500 mg of kanamycin and dissolve it in 10 mL of ultrapure water. After filtration sterilization, aliquot into 10 portions and store at -20°C.

[0079] Rifampicin solution (50 mg / mL): Weigh 500 mg of rifampicin and dissolve it in 10 mL of ultrapure water. After filtration sterilization, divide it into 10 portions and store at -20°C.

[0080] Carbenicillin solution (250 mg / mL): Weigh 25 g of carbenicillin and dissolve it in 100 mL of ultrapure water. After filtration and sterilization, aliquot into 100 portions and store at -20°C.

[0081] YEB medium: Each liter of medium contains 5 g of peptone, 1 g of yeast extract, 5 g of beef extract, 0.443 g of magnesium sulfate heptahydrate (MgSO4·7H2O), and 5 g of sucrose. Adjust the pH to 7.0 and store at room temperature after autoclaving.

[0082] N6-AS medium: To 800 mL of ultrapure water, add 4 g of N6 salts (Phytotechnology), 1 mL of copper / molybdenum / cobalt solution, 1 mL of vitamin-glycine solution, 1 mL of potassium iodide solution, 1 mL of 2,4-dichlorophenoxyacetic acid solution, 100 mg of inositol, 1 g of casamino acids, and 30 g of sucrose. Add ultrapure water to 1 L and adjust the pH to 5.6. To prepare solid N6-AS medium, add 4.0 g / L of gellan gum. After autoclaving, cool the medium to approximately 65°C and add acetosyringone solution to a final concentration of 200 μM.

[0083] MS-RG medium: Add 2.165 g MS salts, 1 mL vitamin-glycine solution, 100 mg inositol, and 15 g sucrose to 800 mL of ultrapure water. Bring the volume to 1 L with ultrapure water. Adjust the pH to 5.6 and add 2.5 g / L gellan gum. Autoclave at 121°C for 15 minutes.

[0084] A one-to-one comparison of the transformation process of Koshihikari cultivar using Bypass-N and conventional MS medium revealed that Bypass-N medium significantly improved Agrobacterium-mediated transformation efficiency and overcame regeneration barriers ( Figure 1 Compared with the traditional MS medium, this new medium that bypasses the limitation of nitrogen metabolism showed no significant difference between Bypass-N and MS in the whole transformation process from coleoptile induction to screening (GFP tracing signal, Figure 1 In the process of regeneration and differentiation, Bypass-N showed the following advantages: all regeneration failed in MS medium, while Bypass-N successfully induced bud differentiation within the 26-day regeneration period ( Figure 1 Middle C). GFP expression in regenerated tissues confirmed the completion of the transformation event ( Figure 1 (D). Three independent transformation experiments further verified that the average transformation efficiency of Bypass-N (27.89%) was higher than that of MS (6.98%). The average efficiency of regenerated shoots from Bypass-N (30.52%) was also higher than that from MS (10.61%). By adding amino acids as a nitrogen source, Bypass-N circumvents the blockage of inorganic nitrogen metabolism, providing a universal genetic transformation solution for germplasm with nitrogen metabolism defects.

[0085] Table 5 Quantitative analysis of transformation and regeneration efficiency in three independent experiments

[0086]

[0087] Example 5 Analysis of genotyping of NiR genes in existing rice germplasm

[0088] The rice NiR gene (LOC_Os01g25484) encodes ferredoxin-nitrite reductase (NiR), and its amino acid sequence is shown in SEQ ID NO. 1. The rice NiR gene structure and the schematic diagram of the alternative splicing transcript structure are shown in Figure 2 .

[0089] The ferredoxin-nitrite reductase protein contains three key functional domains: 1. Chloroplast transit peptide region ( Figure 4 The N-terminal signal sequence mediates the directional transport of newly synthesized proteins to the chloroplast matrix, and the protease removes them after transport is completed. 2. Ferredoxin binding region ( Figure 4 3. 4Fe-4S cluster ( Figure 4 double underlined in the middle), which functions as an iron-sulfur auxiliary group, through Fe 3+ / Fe2+ The change in valence state transfers electrons, which is common in the active center of oxidoreductases. Previous studies have confirmed that the difference in amino acids at positions 375 (E / D) and 453 (S / A) of the NiR gene is the key to determining NiR activity. 375 S 453 The activity is low (the enzyme activity is only 62.5% of that of Kasalath type); Kasalath type (Kasalath-type is D 375 A 453 , which is highly active (the recombinase activity is 1.6 times higher than that of Koshihikari). According to the whole genome SNP data analysis of 3024 global rice germplasms provided by the International Rice Research Institute (IRRI) database 3K RG (3000 Rice Genome Project, Wang et al., 2018 Nature), it was found that NiR gene D 375 A 453 The haplotype frequency was 68.02%, while E 375 S 453 The haplotype frequency was 29.23%, as shown in Table 6.

[0090] Table 6 Statistics of NiR genotyping of 3024 rice accessions (IRRI database)

[0091]

[0092] Note: “-” means the value does not exist in the database.

[0093] Furthermore, the present invention analyzed the two key variable sites of NiR gene in 4726 rice germplasms from different databases (RiceVarMap v2.0). The genotype frequencies of these two sites in different Indica and Japonica types of rice (Table 7) showed that Kasalath-type (D 375 A 453 ) The haplotypes are mostly Indica type rice with the proportions of D 375 (80.6%) and A 453 (98.6%); Koshihikari-type (E 375 S 453 ) Most of them are Japonica type, accounting for E 375 (97.3%) and S 453 (97.3%).

[0094] The genotype frequency results from the two databases demonstrated that there was strong indica-japonica differentiation at this haplotype locus in indica and japonica rice.

[0095] Table 7 Statistics of genotype frequencies at loci 375 and 453 in 4726 rice accessions

[0096]

[0097] Again, based on the newly published genomic data of 4419 modern rice cultivars (3873 domestic and 246 foreign) (Ma X, Wang H, Yan S, Zhou C, Zhou K, Zhang Q, Li M, Yang Y, Li D, Song P, Tang C, Geng L, Sun J, Ji Z, Sun X, Zhou Y, Zhou P, Cui D, Han B, Jing X, He Q, Fang W, Han L. Large-scale genomic and phenomic analyses of modern cultivars empower future rice breeding design. Mol Plant. 2025 Apr 7;18(4):651-668. doi: 10.1016 / j.molp.2025.03.007. Epub 2025 Mar 13. PMID: 40083159.), the present invention conducted genotyping analysis on sites 375 and 453 of the NiR gene (Table 8-1 and Table 8-2). The results showed that no variation at site 453 was detected in all samples. 48 materials with missing genotypes at site 375 were eliminated by SNP calling, and 4064 effective germplasms were obtained (complete data are shown in Table 8-1 and Table 8-2). Analysis of these 4064 rice germplasms covering 32 geographical regions showed (Table 9-1 and Table 9-2): 1. Temperate gene fixation phenomenon. In temperate regions with latitudes > 35°N (Heilongjiang Province, Jilin Province and Liaoning Province in China; Japan; South Korea), E 375 The alleles showed a nearly fixed distribution, with an average frequency of 98.6±1.1% (χ² test p<0.001). The three northeastern provinces formed a core fixed area, Koshihikari-type (E 375 ) frequency >99.3%, significantly higher than the average level (57.8%, p<0.001). 2. In tropical / subtropical areas with latitude <26°N (Guangdong Province; Fujian Province; IRRI), D 375 became the absolute dominant genotype (frequency > 92.8%, p < 0.001). The Yangtze River Basin (31-34°N) showed a transition zone: Jiangsu (87.5% E 375 )→Shanghai(43.5%E375)→Hubei(3.0% E 375), showing the E caused by the decrease in latitude 375 The frequency dropped drastically (r=0.94, p<0.001). 375 Frequency was significantly positively correlated with latitude (r = 0.94*, slope = 2.1%·latitude -1 , R 2 =0.88), indicating that for every 1°N increase in latitude, E 375 The frequency increased by 2.1%. The allele diversity in the temperate zone (He=0.08) was significantly lower than that in the non-temperate zone (He=0.32, p<0.01), suggesting that strong selection pressure led to a decrease in genetic diversity. 375 The loci were classified according to Haplotype and a haplotype distribution map of China was made ( Figure 3 ), of which Koshihikari-type (E 375 ) that is, Haplotype (G / G) is concentrated in the area north of the Yangtze River in China, accounting for 58.22%. Kasalath (D 375 ) types accounted for C / C (29.52%), C / T (0.24%) and T / T (11.9%). Intermediate type (E / D 375 ) accounted for G / C (0.1%) and G / T (0.03%) respectively. Overall, 58.22% of modern Chinese rice varieties belong to Koshihikari-type (E 375 Although there is no definitive data at locus 435 indicating whether the NiR gene is characterized by low enzyme activity, it is possible that 58.22% of the varieties may have low NiR activity.

[0098] Table 8-1 Rice germplasm NiR gene E / D 375 Geographical distribution characteristics of allelic variation abroad

[0099]

[0100] Table 8-2 Rice germplasm in NiR gene E / D 375 Geographical distribution characteristics of domestic allelic variation at the locus

[0101]

[0102] Table 9-1 Rice NiR gene E / D 375 Locus allelic variation in the geographical distribution of rice germplasm and the characteristics of selection for temperate adaptation (abroad)

[0103]

[0104] Table 9-2 Rice NiR gene E / D 375Locus allelic variation in the geographical distribution of rice germplasm and the characteristics of selection for temperate adaptation (domestic)

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[0106] Example 6 Evaluation of genetic transformation efficiency and regeneration characteristics of 50 Koshihikari rice accessions based on the 3K core germplasm library

[0107] To further verify the presence of NiR gene E 375 S 453 The genetic transformation characteristics of haplotype (Kasalath-type, G / T) rice, the present invention screened 50 germplasms carrying this haplotype from the 3k-RG rice core germplasm library (see Table 10 for details), and genetically transformed with the universal vector pCAMBIA1305-Ubi-GFP (publicly available), using the same method as Example 4. Through observation of each stage of the overall transformation, it was found that the overall transformation efficiency was low. The transformation efficiency of 40 germplasms (80%) in MS and N6 culture media was <10%, of which 19 (38%) failed to obtain regenerated seedlings at all. Only 10 germplasms (20%) had a transformation efficiency >10%. A detailed analysis of the reasons for the low transformation efficiency showed that the germplasms with low transformation efficiency showed continued callus proliferation but no differentiation of buds in the regeneration stage (MS basal medium), indicating that the organogenesis process was blocked. This result is consistent with E 375 S 453 The haplotype is consistent with the theory that the low activity of NiR leads to the accumulation of toxic nitrite and the inability to form NH4 + , ultimately inhibiting the regeneration ability. This result confirms the regulatory mechanism by which nitrogen metabolism disorders inhibit regeneration.

[0108] Table 10 Evaluation of genetic transformation efficiency and regeneration characteristics of 50 Koshihikari rice accessions based on the 3K core germplasm bank

[0109]

[0110] Example 7 Evaluation of the effectiveness and adaptability of Bypass-N medium for transformation of recalcitrant rice varieties

[0111] The present invention tested the effectiveness and adaptability of bypass-N medium in the transformation process for nine recalcitrant (Kashihikari), six Kasalath, and one hybrid (Kasalath / Koshihikari) rice varieties. Transformation was performed using the genetic transformation method described in Example 4 and the universal vector pCAMBIA1305-Ubi-GFP (publicly available). All tested rice varieties were effectively transformed, with transformation efficiencies shown in Table 11.

[0112] Table 11 Evaluation of the effectiveness and adaptability of Bypass-N culture medium

[0113]

[0114] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A special medium for efficient genetic transformation of rice that overcomes genotype dependence, characterized in that: The special medium for efficient genetic transformation of rice comprises the following components in parts by weight: 2500-3500 parts of KCl, 150-250 parts of KH2PO4, 350-420 parts of MgSO4·7H2O, 400-500 parts of CaCl2·2H2O, 35-40 parts of disodium edetate, 25-30 parts of FeSO4·7H2O, 15-20 parts of MnSO4·H2O, 7-10 parts of ZnSO4·7H2O, 35-7 parts of H3BO, 0.5-1 part of KI, 0.025 parts of CoCl2·6H2O, 0.025 parts of CuSO4·5H2O, 0.025 parts of Na2MoO4·2H2O 0.25 parts, 800-1000 parts of glutamine, 200-300 parts of aspartic acid, 300-400 parts of arginine, 50-100 parts of glycine, 100-200 parts of cyclohexane hexol, 2 parts of thiamine hydrochloride, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid and 20,000-30,000 parts of carbon source.

2. The special medium for efficient genetic transformation of rice according to claim 1, characterized in that The special medium for efficient genetic transformation of rice includes the following components, in parts by weight: 3000 parts of KCl, 200 parts of KH2PO4, 400 parts of MgSO4·7H2O, 450 parts of CaCl2·2H2O, 38 parts of disodium ethylenediaminetetraacetic acid, 28 parts of FeSO4·7H2O, 18 parts of MnSO4·H2O, 8 parts of ZnSO4·7H2O, 36 parts of H3BO, 0.75 parts of KI, 0.025 parts of CoCl2·6H2O, 0.025 parts of CuSO4·5H2O, 0.25 parts of Na2MoO4·2H2O, 900 parts of glutamine, 250 parts of aspartic acid, 350 parts of arginine, 75 parts of glycine, 150 parts of cyclohexanehexol, 2 parts of thiamine hydrochloride, 1 part of pyridoxine hydrochloride, 1 part of nicotinic acid, and 25,000 parts of a carbon source.

3. The special medium for efficient genetic transformation of rice according to claim 1 or 2, characterized in that: The carbon source is sucrose.

4. Use of the special medium for efficient genetic transformation of rice according to any one of claims 1 to 3 in improving the efficiency of genetic transformation of rice.

5. The use according to claim 4, characterized in that The special medium for efficient genetic transformation of rice improves the genetic transformation efficiency of rice by improving the regeneration obstacle caused by low NiR enzyme activity.

6. A method for efficient genetic transformation of rice that overcomes genotype dependence, characterized in that: The method comprises the step of genetically transforming rice using the special medium for efficient genetic transformation of rice according to any one of claims 1 to 3.

7. The method for efficient genetic transformation of rice according to claim 6, characterized in that: The special medium for efficient genetic transformation of rice improves the genetic transformation efficiency of rice by improving the regeneration obstacle caused by low NiR enzyme activity.

8. The method for efficient genetic transformation of rice according to claim 6, characterized in that: The rice contains the NiR gene E 375 S 453 Haplotype variety.

9. The method for efficient genetic transformation of rice according to claim 6, characterized in that: Described genetic transformation specifically comprises the following steps: sterilizing the rice seeds and then transferring them to the special medium for efficient genetic transformation of rice for induction culture to obtain callus tissue; The callus tissue is infected with Agrobacterium containing the target gene, and after co-cultivation, transformed callus tissue is screened; the screening is performed on the special medium for efficient genetic transformation of rice; The transformed callus tissue is transferred to the special medium for efficient genetic transformation of rice for plant regeneration culture, and then subjected to seedling hardening treatment to obtain transgenic rice plants.

10. The method for efficient genetic transformation of rice according to claim 9, characterized in that: The induction culture is carried out in a dark environment at 28°C; and / or The co-cultivation is carried out in a dark environment at 22°C; and / or The screening was performed in a dark environment at 28°C; and / or The plant regeneration culture and the seedling hardening treatment were both carried out under a cold fluorescent light condition at 28°C.

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