A method for efficient genetic transformation of rice that overcomes genotype dependence and a dedicated culture medium

Abstract

This invention discloses a method for efficient genetic transformation of rice that overcomes genotype dependence and a dedicated culture medium, relating to the field of biotechnology. The dedicated culture medium comprises KCl, KH₂PO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, disodium ethylenediaminetetraacetate, FeSO₄·7H₂O, MnSO₄·H₂O, ZnSO₄·7H₂O, H₃BO₃, KI, CoCl₂·6H₂O, CuSO₄·5H₂O, Na₂MoO₄·2H₂O, glutamine, aspartic acid, arginine, glycine, cyclohexanehexyl alcohol, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, and a carbon source. Using this dedicated culture medium can increase the number of rice cells containing the NiR gene. 375 S 453 The genetic transformation efficiency of haplotype rice varieties is of great significance for overcoming the bottleneck of rice genotype dependence, promoting functional genomics research and molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for efficient genetic transformation of rice that overcomes genotype dependence and a special culture medium. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops and a key model plant for plant gene function research and genetic improvement. Genetic transformation technology is a crucial prerequisite for rice gene function analysis, the development and application of gene editing tools, and bio-breeding. Currently, Agrobacterium-mediated transformation is the mainstream method for rice genetic transformation, and it can achieve efficient genetic transformation in some model japonica rice varieties (such as Zhonghua 11, Nipponbare, and Kitaake). However, currently used transformation methods still cannot meet the needs of efficient transformation for most varieties, and genotype dependence remains a problem that urgently needs to be addressed. This bottleneck is prominently manifested in the significant differences in regeneration capacity among different rice varieties: First, it is known that a considerable number of rice varieties, especially those with large commercial scale, such as the famous high-quality japonica rice variety Koshihikari and the female parent of the super rice variety Liangyoupei 9, Pei'ai 64S, exhibit very low plant regeneration capacity in their callus tissue under existing conventional transformation systems, and some varieties even have no plant regeneration capacity at all; Second, despite years of efforts by researchers, such as optimizing transformation strategies, trying different transformation processes (e.g., shortening subculture time), and optimizing culture medium formulations, the plant regeneration problem of some transformation-resistant varieties has not yet been effectively solved. This widespread regeneration barrier severely restricts the development of rice functional genomics research and modern biological breeding, specifically in the following ways: (1) It is impossible to directly verify gene function and study regulatory mechanisms in excellent germplasm with excellent and important agronomic traits (such as stress resistance, high yield, and high quality) but difficult to transform, so it is impossible to deeply understand the value of important genes under 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, which greatly limits the application scope and efficiency of molecular design breeding; (3) It is difficult to efficiently mine excellent genes for rich genetic diversity resources.

[0003] For a long time, researchers have conducted extensive research to address the genotype-dependent problem, including optimizing culture medium composition (such as adjusting hormone ratios and adding specific compounds), improving Agrobacterium co-culture conditions, screening for highly embryogenic callus, targeting specific easily transformable genotypes, or developing alternative methods (such as gene gun methods). However, most of these efforts have failed to fundamentally solve the genotype-dependent problem. The core reason is the insufficient understanding of the universal core molecular mechanisms and key factors regulating rice regeneration ability, leading to 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 a cross between Koshihikari (japonica rice, no regeneration ability) and Kasalath (indica rice, high regeneration ability) to map-locate the major QTL PSR1 (Promoter of Shoot Regeneration 1) gene (i.e., the NiR gene) located at 45.4 cM on the short arm of chromosome 1 of rice, and confirmed the variation in the promoter region of Koshihikari and the substitution of two amino acids in the gene coding region (E 375 D and S 453 The presence of the A site significantly reduces NiR enzyme activity (to only 62.5% of that in Kasalath), affecting nitrogen assimilation efficiency, impairing cell viability, and leading to regeneration defects. NiR is responsible for catalyzing nitrite (NO2)... - ) is reduced to ammonium (NH4) +The reduced 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 callus, while the compound was undetectable in the culture medium of subcultured Kasalath callus. Based on this discovery, introducing the NiR gene from Kasalath into Koshihikari significantly improved its regeneration capacity (Nishimura A, Ashikari M, Lin S, Takashi T, 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 at present. The main reasons are: (1) low operability, that is, first use NiR to transform varieties with low regeneration capacity, and then transform the target gene, or transform two vectors together, which is time-consuming and laborious, and it is difficult to achieve large-scale genetic transformation; (2) the genetic variation of NiR gene in different varieties lacks comprehensive analysis, and the correlation between NiR enzyme activity and transformation efficiency still needs more extensive verification; (3) based on nitrogen metabolism process, whether a universal culture medium can be designed to avoid the problem of low NiR activity has not been explored in depth.

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

[0005] The purpose of this invention is to provide a highly efficient genetic transformation method for rice that overcomes genotype dependence and a dedicated culture medium, thereby solving the problems existing in the prior art. Using this dedicated culture medium can fundamentally solve the regeneration barrier caused by low NiR activity and increase the number of rice cells containing the NiR gene. 375 S 453 Genetic transformation efficiency of haplotype rice varieties.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a high-efficiency genetic transformation medium for rice that overcomes genotype dependence. By weight, the high-efficiency genetic transformation medium for rice comprises the following components: KCl 2500-3500 parts, KH₂PO₄ 150-250 parts, MgSO₄·7H₂O 350-420 parts, CaCl₂·2H₂O 400-500 parts, disodium ethylenediaminetetraacetate 35-40 parts, FeSO₄·7H₂O 25-30 parts, MnSO₄·H₂O 15-20 parts, ZnSO₄·7H₂O 7-10 parts, H₃BO₃ 5-7 parts, KI 0.5-1 part, CoCl₂·6H₂O 0.025 parts, CuSO₄·5H₂O 0.025 parts, and 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 cyclohexanehexol, 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 rice high-efficiency genetic transformation culture medium, by weight, comprises the following components: 3000 parts KCl, 200 parts KH2PO4, 400 parts MgSO4·7H2O, 450 parts CaCl2·2H2O, 38 parts disodium ethylenediaminetetraacetate, 28 parts FeSO4·7H2O, 18 parts MnSO4·H2O, 8 parts ZnSO4·7H2O, 6 parts H3BO3, 0.75 parts KI, 0.025 parts CoCl2·6H2O, 0.025 parts CuSO4·5H2O, and 0.025 parts Na2MoO4·2H2O. 0.25 parts, 900 parts glutamine, 250 parts aspartic acid, 350 parts arginine, 75 parts glycine, 150 parts cyclohexanehexol, 2 parts thiamine hydrochloride, 1 part pyridoxine hydrochloride, 1 part nicotinic acid, and 25,000 parts carbon source.

[0009] Furthermore, the carbon source is sucrose.

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

[0011] Furthermore, the rice high-efficiency genetic transformation medium improves the efficiency of rice genetic transformation by mitigating regeneration barriers 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 genetic transformation of rice using the above-mentioned special culture medium for efficient genetic transformation of rice.

[0013] Furthermore, the rice high-efficiency genetic transformation medium improves the efficiency of rice genetic transformation by mitigating regeneration barriers caused by low NiR enzyme activity.

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

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

[0016] After disinfection, rice seeds were transferred to the rice high-efficiency genetic transformation medium for induction culture to obtain callus tissue;

[0017] The callus tissue containing the target gene was infected by Agrobacterium and then screened to obtain transformed callus tissue; the screening was carried out on the rice high-efficiency genetic transformation medium.

[0018] The transformed callus tissue was transferred to the rice high-efficiency genetic transformation medium for plant regeneration culture, and then subjected to hardening treatment to obtain transgenic rice plants.

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

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

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

[0022] Both the plant regeneration culture and the hardening treatment were carried out under cold fluorescent lamp conditions at 28℃.

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

[0024] This invention develops a highly efficient genetically modified rice culture medium (Bypass-N) that overcomes genotype dependence. By altering the medium's composition (completely replacing inorganic nitrogen with amino acids), it bypasses the functional impairment of NiR enzymes at the metabolic pathway level without involving any gene manipulation. This fundamentally solves the problem caused by low NiR activity (E... 375 S 453 Regeneration impairment caused by type ( ). Transformation experiments confirmed that Bypass-N medium, in E 375 S 453 The representative variety, Yueguang, significantly improved transformation and regeneration efficiency. The amino acid nitrogen source metabolic pathway provided by the Bypass-N medium is independent of NiR function; therefore, its effect is unaffected by the rice's endogenous NiR genotype (E). 375 S 453 Or D 375 A453 (restrictions)

[0025] The Bypass-N medium provided by this invention can directly replace standard regeneration media (such as MS) for genetic transformation. This method is particularly suitable for E cells, which account for a large proportion of genetic transformation. 375 S 453 The availability of rice germplasm (especially temperate japonica rice) makes it possible to directly verify gene function, introduce superior genes, and conduct molecular design breeding in these important germplasm resources. This is of great significance for overcoming the bottleneck of rice genotype dependence and promoting functional genomics research and molecular breeding. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The figures show the results of genetic transformation of the *Vibrant Light* material using Bypass-N and MS media, respectively. A shows the results of coleoptiles induced under white light in different media; B shows the GFP signal detection results of GFP-positive cells in callus tissue cultured on different media during antibiotic selection, under white light and ultraviolet light; C shows the results of assessing regeneration capacity at 26 days on different media; and D shows the GFP signal detection results of buds regenerated on Bypass-N medium and callus tissue that did not regenerate on MS medium under white light and ultraviolet light, respectively.

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

[0029] Figure 3 A pie chart showing the genotype frequencies of the 375 SNPs in the NiR gene from 3825 Chinese rice germplasms; among them, 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 This is a schematic diagram of the amino acid sequence and key functional domains of ferroredoxin-nitrite reductase protein. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0036] In plant tissue culture, nitrogen metabolism mainly involves two key processes: inorganic nitrogen metabolism and amino acid metabolism. Inorganic nitrogen is primarily metabolized as nitrate (NO3). - ) and ammonium salts (NH4) + After being absorbed by cells in its original form, nitrate reductase (NR) and nitrite reductase (NiR) gradually reduce it to NH4. + NH4 +Subsequently, glutamine is synthesized by binding with glutamate via the glutamine synthetase (GS) pathway. Glutamine, in turn, reacts with α-ketoglutaric acid via glutamate synthetase (GOGAT) to form two molecules of glutamate. This GS-GOGAT cycle is the main pathway for ammonium assimilation in plant cells. Simultaneously, NH4+... + Glutamate can also be directly converted to glutamate via the glutamate dehydrogenase (GDH) pathway, reacting with α-ketoglutarate. However, this pathway contributes little under normal physiological conditions. As a core molecule in nitrogen metabolism, glutamate plays a crucial role. On one hand, it transfers its amino group to other carboxylic acids (such as pyruvate and oxaloacetate) via transaminases to synthesize various amino acids (such as alanine and aspartic acid). On the other hand, it provides substrates for the GS-GOGAT cycle, maintaining efficient intracellular nitrogen recycling. In amino acid metabolism, intermediates of the TCA cycle (such as α-ketoglutarate, oxaloacetate, and pyruvate) provide the carbon skeleton for amino acid synthesis, while carbohydrate and carboxylic acid metabolism (such as malic acid and citric acid) provide necessary precursors for this process. Furthermore, exogenously added organic nitrogen (such as amino acids) or endogenous protein degradation products can directly participate in metabolism through transamination or deamination reactions, achieving efficient nitrogen reuse. This series of metabolic processes not only provides essential nitrogen and amino acids for plant cell growth, but is also closely coupled with energy metabolism (such as the TCA cycle and oxidative phosphorylation), ensuring normal cell proliferation and differentiation in tissue culture. By optimizing the ratio and form of inorganic and organic nitrogen in the culture medium and regulating the efficiency of the GS-GOGAT cycle, the nitrogen utilization efficiency and the synthesis of target products in the tissue culture system can be significantly improved.

[0037] Nitrogen metabolism disorder is one of the factors causing rice regeneration impairment. Based on this characteristic, this invention designs a transformation medium formulation that avoids inorganic nitrogen metabolism. The core design principle is to completely eliminate nitrate nitrogen (NO3). - ) and ammonium nitrogen (NH4) + This approach mitigates the risk of nitrogen metabolism disorders at the source by introducing a quaternary amino acid nitrogen system: glutamine (the main amino acid donor, directly involved in the GS / GOGAT cycle) + aspartic acid (providing the carbon skeleton and amino group, 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 components, supplemented by other nutrients, the regeneration process eliminates the need for nitrogen metabolism, directly utilizing the NH2 provided by amino acids. -The culture medium formulation is shown in Table 1 and named Nitrogen metabolism-bypassing, abbreviated as Bypass-N.

[0038] Table 1 Bypass-N medium formulation

[0039]

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

[0041] Example 1

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

[0043] Table 2 Bypass-N medium formulation for Example 1

[0044]

[0045] Example 2

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

[0047] Table 3. Bypass-N medium formulation for Example 2

[0048]

[0049] Example 3

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

[0051] Table 4. Bypass-N medium formulation for Example 3

[0052]

[0053] The Bypass-N culture medium from Example 1 will be used as an example for functional verification.

[0054] Example 4

[0055] The Koshihikari rice material was used for experimental verification: genetic transformation of Koshihikari was performed using Bypass-N medium (Example 1) and conventional MS medium, respectively. The transformation method using Bypass-N medium is as follows:

[0056] 1. Seed disinfection treatment

[0057] Remove the husks from mature seeds and discard any seeds with spots. Transfer the dehulled seeds to a 50 mL centrifuge tube, rinse the seeds with 70% (v / v) ethanol for 1 minute, discard the ethanol, rinse the seeds with sterile ultrapure water, then add 20 wt% sodium hypochlorite solution, shake on a shaker for 30 minutes, discard the sodium hypochlorite solution, rinse the seeds with sterile ultrapure water, and blot dry the seed surface.

[0058] 2. Callus induction

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

[0060] 3. Co-culture of Agrobacterium infection and callus tissue

[0061] (1) Agrobacterium strains containing the target gene (i.e., GFP gene) construct were stored in YEB medium containing 15% (v / v) glycerol. 50 μL of the Agrobacterium stock solution was inoculated into a sterile 15 mL centrifuge tube containing 5 mL of YEB medium (with 5 μL of rifampicin solution and 5 μL of kanamycin solution added), and incubated overnight on a shaker at 140 rpm until the bacterial culture reached OD. 600 Once the value reaches 1.0, the Agrobacterium tumefaciens bacterial solution is obtained and ready for use.

[0062] (2) Transfer the Agrobacterium tumefaciens bacterial suspension to a centrifuge tube, centrifuge at 4000 × g for 5 minutes, discard the supernatant, resuspend the Agrobacterium tumefaciens precipitate in liquid N6-AS medium, and adjust the bacterial suspension concentration to achieve the desired OD value. 600 When the value reaches 0.02, an Agrobacterium suspension is obtained.

[0063] (3) Transfer 15 g of embryogenic callus to a sterile 50 mL centrifuge tube, add 25 mL of Agrobacterium suspension obtained in step (2), gently shake the centrifuge tube by hand for 3 minutes, discard the bacterial solution, and use a scraper to transfer the callus to sterile wiping paper to absorb excess liquid.

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

[0065] 4. Screening of transformed callus tissue

[0066] (1) Transfer the co-cultured callus to a sterile centrifuge tube containing sterile ultrapure water, shake gently for a few seconds and discard the water, repeat the washing 4 times; during the last washing, add carbenicillin solution to the ultrapure water to make the final concentration 250 mg / L.

[0067] (2) After cleaning, the callus tissue was transferred to sterile wiping paper to absorb excess moisture, and then transferred to Bypass-N medium, with 20 callus tissue pieces placed in each culture dish. The culture dishes were sealed with medical tape and cultured in the dark at 28°C for 20 days. Actively growing transformed callus tissue was screened.

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

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

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

[0071] (2) Transfer the seedlings that have differentiated into 125 mL Erlenmeyer flasks containing 40 mL MS-RG medium and place them under a cold fluorescent lamp at 28℃ to promote root growth. After 10-14 days, transplant them into flower pots.

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

[0073] The preparation methods for the culture medium and reagents used in this embodiment 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 bring the volume to 100 mL, and store at 4℃.

[0075] Vitamin-glycine solution (1000×): Weigh 500 mg thiamine hydrochloride, 100 mg pyridoxine hydrochloride, 100 mg nicotinic acid and 200 mg glycine, dissolve in 100 mL 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℃.

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

[0078] Kanamycin solution (50 mg / mL): Weigh 500 mg of kanamycin, dissolve it in 10 mL of ultrapure water, filter and sterilize, then dispense into 10 portions and store at -20℃.

[0079] Rifampicin solution (50 mg / mL): Weigh 500 mg of rifampicin, dissolve it in 10 mL of ultrapure water, filter and sterilize, then dispense into 10 portions and store at -20℃.

[0080] Carbenicillin solution (250 mg / mL): Weigh 25 g of carbenicillin, dissolve it in 100 mL of ultrapure water, filter and sterilize, then dispense into 100 portions and store at -20℃.

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

[0082] N6-AS medium: Add 4 g N6 salt (Phytotechnology), 1 mL copper / molybdenum / cobalt solution, 1 mL vitamin-glycine solution, 1 mL potassium iodide solution, 1 mL 2,4-dichlorophenoxyacetic acid solution, 100 mg inositol, 1 g casein amino acids, and 30 g sucrose to 800 mL ultrapure water. Adjust the volume to 1 L with ultrapure water and adjust the pH to 5.6. Add 4.0 g / L gellan gel when preparing solid N6-AS medium. After autoclaving and cooling to approximately 65°C, add acetylsuccinone solution to achieve a final concentration of 200 μM.

[0083] MS-RG medium: Add 2.165 g MS salt, 1 mL vitamin-glycine solution, 100 mg inositol and 15 g sucrose to 800 mL ultrapure water, bring the volume to 1 L, adjust the pH to 5.6, and add 2.5 g / L gellan gel. Autoclave at 121℃ for 15 minutes.

[0084] A one-to-one comparison of the transformation process of Koshihikari cultivar using Bypass-N and conventional MS medium was conducted, and it was found that Bypass-N medium significantly improved the Agrobacterium-mediated transformation efficiency and overcame the regeneration barrier. Figure 1 (See Table 5). Compared to traditional MS medium, this novel medium, which bypasses nitrogen metabolism limitations, showed no significant difference between Bypass-N and MS during the entire transformation process, from coleoptile induction to screening (GFP tracer signal). Figure 1 In the regeneration and differentiation process, Bypass-N demonstrated the advantage that: while all regeneration failed in MS medium, Bypass-N successfully induced shoot differentiation within a 26-day regeneration period. Figure 1 (C) GFP expression in regenerated tissue confirms 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%), and the average regeneration efficiency of Bypass-N (30.52%) was also higher than that of MS (10.61%). By adding amino acids to replace the nitrogen source, Bypass-N avoids the inhibition of inorganic nitrogen metabolism and provides a universal genetic transformation scheme for nitrogen metabolism-deficient germplasm.

[0085] Table 5. Quantitative analysis results of conversion and regeneration efficiency in three independent experiments.

[0086]

[0087] Example 5: Genotyping analysis of the NiR gene 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. A schematic diagram of the rice NiR gene structure and the alternative splicing transcript structure is shown below. Figure 2 .

[0089] This ferroredoxin-nitrite reductase protein contains three key functional domains: 1. Chloroplast transport peptide region ( Figure 4 (Marked with a single wavy line), the N-terminal signal sequence mediates the directional transport of newly synthesized proteins to the chloroplast stroma, after which the protease cleaves them. 2. Ferroredoxin binding region ( Figure 4 (Highlighted by double wavy lines), specifically binds to ferredoxin, accepting electrons to catalyze nitrite reduction. 3. 4Fe-4S cluster ( Figure 4 (with double underscores), its function is as an iron-sulfur auxiliary group, through Fe 3+ / Fe2+ Valence state changes involve electron transfer, commonly occurring at the active site of oxidoreductases. Previous studies have confirmed that the amino acid difference between positions 375 (E / D) and 453 (S / A) in the NiR gene is key to determining NiR activity. Koshihikari-type: E 375 S 453 It exhibits low activity (enzyme activity is only 62.5% of that of the Kasalath type); Kasalath type (Kasalath-type is D) 375 A 453 It exhibits high activity (recombinase activity is 1.6 times higher than that of Koshihikari). Analysis of whole-genome SNP data from 3024 global rice germplasm accessions provided by the International Rice Research Institute (IRRI) database 3K RG (3000 Rice Genome Project, Wang et al., 2018 Nature) revealed that the NiR gene D... 375 A 453 The haplotype frequency is 68.02%, while E 375 S 453 The haplotype frequency is 29.23%, as shown in Table 6.

[0090] Table 6. Statistical table of NiR genotyping of 3024 rice germplasms (IRRI database)

[0091]

[0092] Note: "-" indicates that the database does not contain the information.

[0093] Furthermore, this invention analyzed two key variation sites of the NiR gene in 4726 rice germplasm accessions from different databases (RiceVarMap v2.0). The genotype frequencies of these two sites in different Indica and Japonica rice types (Table 7) indicate that Kasalath-type (D 375 A 453 The majority of haplotypes in rice are of the Indica type, accounting for D% respectively. 375 (80.6%) and A 453 (98.6%); Koshihikari-type (E 375 S 453 The vast majority are of the Japonica type, accounting for E... 375 (97.3%) and S 453 (97.3%).

[0094] Genotype frequency results from two databases demonstrate strong differentiation between indica and japonica rice at this haplotype locus.

[0095] Table 7. Genotype frequencies at loci 375 and 453 in 4726 rice germplasms.

[0096]

[0097] Furthermore, based on the genomic data of 4419 newly published modern rice varieties (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: This invention (40083159.) performed genotypic analysis on the 375 and 453 loci of the NiR gene (Tables 8-1 and 8-2). The results showed that no variation was detected at the 453 locus in any of the samples. By removing 48 materials with missing genotypes at the 375 locus through SNP calling, 4064 valid germplasm accessions were obtained (complete data are shown in Tables 8-1 and 8-2). Analysis of these 4064 rice germplasm accessions covering 32 geographical regions showed (Tables 9-1 and 9-2): 1. Temperate gene fixation. In temperate regions with latitude >35°N (Heilongjiang, Jilin, and Liaoning provinces in China; Japan; South Korea), E 375 The alleles exhibited a nearly fixed distribution, with an average frequency of 98.6 ± 1.1% (χ² test, all p < 0.001). A core fixed region was formed in the three northeastern provinces, with the Koshihikari-type (E) allele being dominant. 375 The frequency was >99.3%, significantly higher than the average level (57.8%, p<0.001). 2. In tropical / subtropical regions at latitudes <26°N (Guangdong Province; Fujian Province; IRRI), D 375 It became the absolutely dominant genotype (frequency >92.8%, p<0.001). The Yangtze River basin (31-34°N) exhibited a transitional pattern: Jiangsu (87.5% E... 375 → Shanghai (43.5% E375) → Hubei (3.0% E) 375), showing E due to the decrease in latitude. 375 The frequency drops sharply (r=0.94, p<0.001). 3.E 375 The frequency showed a highly significant positive correlation 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%. Allelic diversity in temperate regions (He=0.08) was significantly lower than in non-temperate regions (He=0.32, p<0.01), suggesting that strong selection pressure led to a decrease in genetic diversity. This invention processed 3825 Chinese rice germplasms in E / D... 375 The loci were classified according to Haplotype and a haplotype distribution map of China was created. Figure 3 ), of which Koshihikari-type (E 375 Haplotype (G / G) is concentrated in the region north of the Yangtze River in China, accounting for 58.22%. Kasalath (D) 375 The proportions of the three types were C / C (29.52%), C / T (0.24%), and T / T (11.9%), respectively. The intermediate type (E / D)... 375 The percentages were G / C (0.1%) and G / T (0.03%), respectively. Overall, 58.22% of modern Chinese rice varieties belong to the Koshihikari-type (E) 375 (Type 435). Although there is no definitive data at position 435 indicating whether the NiR gene has low enzyme activity, there is a potential issue of low NiR activity in 58.22% of the varieties.

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

[0099]

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

[0101]

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

[0103]

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

[0105]

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

[0107] To further verify the presence of the NiR gene E 375 S 453 Genetic transformation characteristics of haplotype (Kasalath-type, G / T) rice: This invention screened 50 germplasm accessions carrying this haplotype from the 3k-RG rice core germplasm bank (see Table 10 for details), and performed genetic transformation using the universal vector pCAMBIA1305-Ubi-GFP (publicly available), following the same method as in Example 4. Observation of each stage of the overall transformation revealed a low overall transformation efficiency. 40 accessions (80%) showed transformation efficiencies <10% in both MS and N6 media, with 19 accessions (38%) failing to produce regenerated seedlings at all. Only 10 accessions (20%) showed transformation efficiencies >10%. Detailed analysis of the reasons for the low transformation efficiency revealed that the low-efficiency accessions exhibited continuous callus proliferation but no bud differentiation during the regeneration stage (MS basal medium), indicating inhibited organogenesis. This result is consistent with E... 375 S 453 This is consistent with the theory that haplotypes lead to reduced NiR enzyme activity; low-activity NiR results in the accumulation of toxic nitrites and prevents the formation of NH4. + Ultimately, this inhibits regeneration capacity, and 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 *Viburnum bisporum* var. *yueguang* based on the 3K core germplasm bank.

[0109]

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

[0111] This invention selected nine recalcitrant (Kashihikari type), six Kasalath type, and one heterozygous (Kasalath / Koshihikari) rice varieties to test the effectiveness and versatility of bypass-N medium in the transformation process. The genetic transformation method described in Example 4 was used, with the universal vector pCAMBIA1305-Ubi-GFP (publicly available) for genetic transformation. All rice varieties were successfully transformed, and the transformation efficiency is shown in Table 11.

[0112] Table 11 Assessment of the effectiveness and versatility of Bypass-N medium

[0113]

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

Claims

1. A special culture medium for rice genetic transformation that overcomes genotype dependence, characterized in that, The components of the rice genetic transformation culture medium are as follows: KCl 2500-3500 mg / L, KH2PO4 150-250 mg / L, MgSO4·7H2O 350-420 mg / L, CaCl2·2H2O 400-500 mg / L, disodium ethylenediaminetetraacetate 35-40 mg / L, FeSO4·7H2O 25-30 mg / L, MnSO4·H2O 15-20 mg / L, ZnSO4·7H2O 7-10 mg / L, H3BO3 5-7 mg / L, KI 0.5-1 mg / L, CoCl2·6H2O 0.025 mg / L, CuSO4·5H2O 0.025 mg / L, Na2MoO4·2H2O 0.25 mg / L, glutamine 800-1000 mg / L. mg / L, aspartic acid 200-300 mg / L, arginine 300-400 mg / L, glycine 50-100 mg / L, cyclohexanehexol 100-200 mg / L, thiamine hydrochloride 2 mg / L, pyridoxine hydrochloride 1 mg / L, nicotinic acid 1 mg / L, and carbon source 20,000-30,000 mg / L.

2. The rice genetic transformation culture medium according to claim 1, characterized in that, The components of the rice genetic transformation culture medium are as follows: KCl 3000 mg / L, KH2PO4 200 mg / L, MgSO4·7H2O 400 mg / L, CaCl2·2H2O 450 mg / L, disodium ethylenediaminetetraacetate 38 mg / L, FeSO4·7H2O 28 mg / L, MnSO4·H2O 18 mg / L, ZnSO4·7H2O 8 mg / L, H3BO3 6 mg / L, KI 0.75 mg / L, CoCl2·6H2O 0.025 mg / L, CuSO4·5H2O 0.025 mg / L, Na2MoO4·2H2O 0.25 mg / L, glutamine 900 mg / L, aspartic acid 250 mg / L, arginine 350 mg / L, glycine 75 mg / L, cyclohexanehexol 150 mg / L. mg / L, thiamine hydrochloride 2 mg / L, pyridoxine hydrochloride 1 mg / L, nicotinic acid 1 mg / L and carbon source 25,000 mg / L.

3. The rice genetic transformation culture medium according to claim 1 or 2, characterized in that, The carbon source is sucrose.

4. The application of a rice genetic transformation-specific culture medium as described in any one of claims 1-3 in improving the efficiency of rice genetic transformation.

5. The application according to claim 4, characterized in that, The rice genetic transformation-specific culture medium improves the efficiency of rice genetic transformation by mitigating regeneration barriers caused by low NiR enzyme activity.

6. A method for overcoming genotype-dependent rice genetic transformation, characterized in that, The method includes the step of genetically transforming rice using the rice genetic transformation culture medium as described in any one of claims 1-3.

7. The rice genetic transformation method according to claim 6, characterized in that, The rice genetic transformation-specific culture medium improves the efficiency of rice genetic transformation by mitigating regeneration barriers caused by low NiR enzyme activity.

8. The rice genetic transformation method according to claim 6, characterized in that, The rice variety contains the NiR gene E. 375 S 453 Haploid varieties.

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