Genetic transformation method of recalcitrant rice variety based on agrobacterium tumefaciens mediation

Abstract

The invention relates to the field of plant genetic engineering, and discloses a recalcitrant rice variety genetic transformation method based on agrobacterium tumefaciens mediation, which comprises the following steps: 1) carrying out illumination culture on rice seeds in an induction culture medium to obtain calluses; 2) culturing the activated agrobacterium in a suspension culture medium, and adding the callus to obtain an infected callus; (3) carrying out dark culture on the infected callus in a screening culture medium; 4) selecting resistant infection calluses without browning death, and carrying out light-dark alternate culture in a differential medium; and 5) after the resistant infection callus forms a regeneration seedling, transferring the regeneration seedling into a rooting culture medium for alternate light and dark culture to obtain a plant. The efficient, stable and repeatable agrobacterium tumefaciens-mediated genetic transformation method suitable for recalcitrant rice varieties is established by systematically optimizing culture conditions, culture media and the like at the stages of induction, screening, differentiation, rooting and the like, and the transformation efficiency can be remarkably improved.

Description

A Genetic Transformation Method for Resilient Rice Varieties Based on Agrobacterium-mediated Transformation Technical Field

[0001] This invention relates to the field of plant genetic engineering, and more particularly to a method for genetic transformation of recalcitrant rice varieties based on Agrobacterium-mediated transformation. Background Technology

[0002] Genetic transformation in rice is a core technology for functional genomics research and precision molecular breeding. Agrobacterium-mediated transformation has become the most widely used method in plant genetic transformation due to its mature operation and stable transformation (Ozawa, 2009, 2012; Wuand Sui, 2019), providing strong support for gene function verification, genetic improvement, and new variety breeding.

[0003] Currently, the efficiency of rice genetic transformation is highly dependent on genotype, with significant differences between different varieties. Generally, japonica rice varieties (such as Nipponbare and Zhonghua 11) exhibit high transformation efficiency due to their excellent tissue culture characteristics. Under optimized culture systems (such as NB medium) and transformation conditions, they can achieve callus differentiation rates exceeding 80% and stable resistant callus formation rates. In contrast, indica rice varieties, especially the backbone parents such as 93-11, Zhongzao 39, and Zhongzu 141, as well as most hybrid rice varieties, are generally recalcitrant rice, with extremely low genetic transformation efficiency, sometimes less than 5%, becoming a key bottleneck restricting their application in functional studies and molecular design breeding.

[0004] Taking 93-11 (Oryza sativa L. ssp. indica) as an example, this variety is a widely used excellent backbone parent in indica rice hybrid breeding in my country, possessing outstanding agronomic traits and important genetic value. However, during tissue culture, it exhibits problems such as low callus induction rate, difficulty in embryogenic callus formation, easy browning or excessive lignification, and a lack of dense granular callus with high regeneration potential, seriously affecting subsequent Agrobacterium infection and plant regeneration. Simultaneously, 93-11 has low sensitivity to Agrobacterium infection, low T-DNA transfer and integration efficiency, and is easily inhibited by strong plant defense responses (such as reactive oxygen species bursts and phenolic accumulation) to promote Agrobacterium attachment and stable T-DNA integration. Furthermore, this variety is highly sensitive to commonly used screening agents (such as hygromycin and glufosinate), often being "mis-screened" and lost during the screening stage due to slow cell growth; during the differentiation stage, it generally suffers from low green seedling rate and a high proportion of albino or deformed seedlings, further reducing the rate of obtaining fertile transgenic plants. Similarly, other stubborn indica rice varieties such as Guanglu Dwarf No. 4, Zhongjia No. 3, TN1, and Shuhui 527 have extremely low conversion efficiency and may even fail to emerge.

[0005] Given that heterosis in indica-japonica hybrids is a crucial pathway to increasing rice yield, and since most high-yielding hybrid rice varieties and their parents currently contain indica rice lineage, overcoming the technical bottleneck of low genetic transformation efficiency in recalcitrant indica rice is of significant strategic importance for advancing rice functional genomics research, accelerating molecular design breeding, and cultivating groundbreaking high-yielding and high-quality new varieties. Therefore, there is an urgent need to develop a highly efficient Agrobacterium-mediated genetic transformation system that can effectively overcome genotype limitations and is specifically applicable to recalcitrant indica rice varieties such as 93-11, providing key technical support for precision rice breeding. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an Agrobacterium-mediated genetic transformation method for recalcitrant rice varieties. By systematically optimizing the culture conditions and culture media during induction, screening, differentiation, and rooting stages, this invention establishes a highly efficient, stable, and reproducible Agrobacterium-mediated genetic transformation method suitable for recalcitrant rice varieties, significantly improving transformation efficiency.

[0007] The specific technical solution of the present invention includes: a genetic transformation method for recalcitrant rice varieties based on Agrobacterium-mediated transformation, which includes the following steps: 1) Inducing callus: sterilized rice seeds are cultured under light in an induction medium for 7-10 days to obtain callus tissue; the induction medium contains: maltose 25-35 g / L, MS macro-element mixture 4-5 g / L, and L-proline 2.5-3 g / L.

[0008] Compared to traditional indica rice genetic transformation methods, this invention provides a scheme to optimize the callus induction rate of mature rice embryos. Specifically, the following optimizations are made during the callus induction stage: (a) The callus induction time is shortened from the usual 15 days to 7-10 days of full-light culture. During this stage, callus cells undergo vigorous division, have thinner cell walls, and are metabolically active, making them more susceptible to infection by exogenous vectors such as Agrobacterium, ensuring that the callus tissue remains undifferentiated and has high infectivity; (b) This invention has found that the type of carbon source in the culture medium has a significant impact on callus induction, somatic embryogenesis, and plant regeneration. Studies have shown that the callus induction and differentiation rates of the medium using maltose as the carbon source are significantly better than those using sucrose. Therefore, this invention selects maltose as the carbon source in the induction medium, screening medium, and differentiation medium; (c) The N6D macronutrient mixture in the conventional culture medium is replaced with the MS macronutrient mixture. The nitrogen source of the N6D macronutrient mixture is mainly nitrate nitrogen, with very low or no ammonium nitrogen content; the potassium and phosphorus contents have also been optimized and adjusted. In contrast, the MS macronutrient mixture has higher nitrogen and potassium contents, with ammonium nitrogen and nitrate nitrogen coexisting, and a higher concentration of inorganic salts. The MS macronutrient mixture provides a higher concentration of nutrients during the callus induction stage, which is beneficial for initiating dedifferentiation and has a better induction effect. (d) In conventional rice callus induction medium, the proline concentration is usually 0.1-1.0 g / L. This invention increases the proline content to 2.5-3 g / L. During tissue culture, proline, as an important osmotic regulator, can accumulate in the cytoplasm, maintain the osmotic balance of cells, protect cell structure and function, thereby improving the explant's adaptability to culture conditions and promoting callus formation. The proline accumulation level is positively correlated with the embryogenicity of callus; callus with high proline content is more likely to regenerate plants. Proline plays a positive role in rice callus induction through mechanisms such as osmotic regulation, antioxidation, nutrient supply, and potential signal regulation.

[0009] 2) Suspension, infection and co-culture of Agrobacterium: Activated Agrobacterium was cultured in suspension medium, and callus tissue was added for infection to obtain infected callus tissue, which was then cultured in the dark.

[0010] 3) Screening: The infected callus tissue after dark culture was cultured in the dark in the screening medium. The dried infected callus tissue without Agrobacterium contamination on the screening medium was transferred to another screening medium for continued dark culture. The screening medium contained: PEG6000 25-30g / L and maltose 25-35g / L.

[0011] This invention discovered that PEG6000 can increase the osmotic pressure of the culture medium, simulating a mild stress environment, allowing cells to gradually adapt to subsequent selection pressures. However, further research revealed that excessively high PEG6000 concentrations can easily lead to severe cell dehydration, thereby inhibiting callus growth. Therefore, this invention controls the PEG6000 content in the screening medium at a low level (25-30 g / L).

[0012] 4) Differentiation: Select resistant callus tissues that have not browned and died in 3) and culture them in light and dark alternately in differentiation medium containing 25-35 g / L maltose.

[0013] 5) Rooting: The resistant callus tissue that is about to turn green forms a regenerated seedling. It is then transferred to a rooting medium containing 0.05-0.15 mg / L NAA and cultured under alternating light and dark conditions until a complete plant is formed.

[0014] During the rooting stage, this invention adjusts the concentration of NAA in the rooting medium, whereas conventional rooting media do not contain NAA. NAA's effect on plant organogenesis follows a pattern of promotion at low concentrations and inhibition at high concentrations. For adventitious root induction, there exists an optimal concentration window; excessively high or low concentrations will lead to decreased effectiveness. Low concentrations of NAA (0.05-0.15 mg / L) effectively induce adventitious root formation and the expression of cell differentiation-related genes (such as AUX / IAA and ARF family genes), promoting their dedifferentiation and the formation of root primordia. High NAA concentrations (>0.5-1.0 mg / L) overload the intracellular auxin signaling pathway, leading to excessive cell division, disordered swelling, and the formation of callus tissue rather than structured roots. Therefore, this invention adds NAA to the rooting medium, limiting its concentration to 0.05-0.15 mg / L, to promote adventitious root formation and improve transplant survival rate.

[0015] Furthermore, this invention employs different light conditions at different stages. In the rice genetic transformation process, using specific light conditions (full light, full darkness, and alternating light and dark) at different stages is a key optimized environmental control method aimed at maximizing transformation efficiency, screening effect, and regeneration power. Specifically: Callus induction stage: full light culture. Light helps inhibit excessive hydration or browning of callus tissue, improving its regeneration potential and ensuring high infectivity of the callus tissue.

[0016] Co-culture phase: Complete darkness culture enhances the efficiency of Agrobacterium-mediated T-DNA transfer. Light induces plants to produce reactive oxygen species (ROS) and phenolic compounds, which may inhibit Agrobacterium activity or interfere with T-DNA integration. A dark environment can alleviate this stress and promote interaction between Agrobacterium and plant cells.

[0017] Selection stage: Complete darkness culture is used primarily to increase selection pressure and effectively eliminate untransformed cells. This avoids interference with photosynthesis; under dark conditions, cells rely entirely on the carbon source in the culture medium for survival and cannot produce energy through photosynthesis, thus being fully exposed to selection pressure. Untransformed cells, lacking resistance genes, have suppressed metabolic activity and die more quickly.

[0018] Differentiation and rooting stage: alternating light and dark cultivation to simulate the natural environment, activate organogenesis genes, and promote bud differentiation and root formation.

[0019] Preferably, in step 1), the rice seeds are recalcitrant rice varieties; further examples include 93-11, Guanglu Dwarf No. 4, Zhongjia No. 3, TN1, and Shuhui 527.

[0020] The temperature for the light culture is 25-35℃.

[0021] Preferably, in step 1), the induction medium comprises: 4-5 g / L MS macro-element mixture, MS... min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 2.5-3 g / L, hydrolyzed casein 0.4-0.8 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, water.

[0022] Preferably, the activation of Agrobacterium includes: streaking Agrobacterium (EHA105) onto LB medium containing 45-55 mg / L kanamycin and 45-55 mg / L rifampin, and culturing at 25-30°C.

[0023] Preferably, in step 2), the concentration of Agrobacterium in the suspension culture medium before infection is OD600 = 0.1-0.5; the infection time is 5-10 min; and the dark culture is carried out in a closed environment at 24-28℃ for 2-4 days.

[0024] Preferably, in step 2), the suspension culture medium comprises: 25-35 g / L sucrose, 0.3-0.7 g / L hydrolyzed casein, 8-12 mL / L Fe-EDTA, 45-55 mL / L AAM AminoAcid, 45-55 mL / L AAM macro, 4-6 mL / L AAM micro, and 4-6 mL / L AAM organic.

[0025] Preferably, in step 3), the two dark incubations are performed at 24-28°C for 8-12 days.

[0026] Preferably, in step 3), the screening medium comprises: 4-5 g / L MS macro-element mixture, MS... min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 0.4-0.6 g / L, hydrolyzed casein 0.2-0.4 g / L, PEG6000 25-30 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, water.

[0027] Preferably, in step 4), the temperature for alternating light and dark culture is 24-28℃, the light culture cycle is 10-14h, the dark culture cycle is 10-14h, and the total culture time is 25-35 days.

[0028] Preferably, in step 4), the differentiation medium comprises: MS macro-element mixture 4-5 g / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, hydrolyzed casein 1.5-2.5 g / L, maltose 25-35 g / L, sorbitol 25-35 g / L, NAA 0.2-0.3 mg / L, KT stock solution 1-3 mL / L, gum powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, and water.

[0029] Preferably, in step 5), the height of the regenerated seedling is 3-4 cm; the temperature for alternating light and dark culture is 24-28℃, the light culture cycle is 10-14 h, the dark culture cycle is 10-14 h, and the total culture time is 13-17 days.

[0030] Preferably, in step 5), the rooting medium comprises: 4-5 g / L MS macro-element mixture, MS... min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, sucrose 15-25 g / L, gum powder 4-6 g / L, water.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: Through a series of optimizations of the culture medium and process conditions in stages such as callus induction, screening, differentiation and rooting, the process of the present invention is simple and can significantly improve the induction rate and emergence rate of embryogenic callus tissue in cultivated varieties with a high proportion of indica rice lineage, such as 93-11, as well as recalcitrant rice varieties such as hybrid rice. It can greatly improve the efficiency of obtaining transgenic plants of recalcitrant rice varieties and shorten the cycle of obtaining transgenic rice plants. Attached Figure Description

[0032] Figure 1 shows a photograph of the genetic transformation process of rice variety 93-11 mediated by Agrobacterium; Figure 1A shows aseptic sowing; Figure 1B shows callus induction; Figure 1C shows infection; Figure 1D shows screening; Figure 1E shows differentiation; and Figure 1F shows rooting.

[0033] Figure 2 shows the identification results of 93-11 transgenic positive seedlings; Figure 2A shows the PCR amplification results of hygromycin-specific primers; Figure 2B shows the PCR amplification results of G418-specific primers. M represents 5000 bp marker DNA; Lanes 1-12 are transgenic plants; Lane 13 is a non-transgenic plant.

[0034] Figure 3 shows the genetic transformation and emergence statistics of rice variety 93-11; Figure 3A shows the callus emergence rate and positive emergence rate; Figure 3B shows the callus emergence rate and positive emergence rate of G418 and hygromycin-resistant varieties.

[0035] Figure 4 shows the state of 93-11 callus tissue in different screening media; Figure 4A is the control group, with basic screening medium; Figure 4B is experimental group 1, with basic screening medium + 10 g / L PEG6000; Figure 4C is experimental group 2, with basic screening medium + 30 g / L PEG6000; Figure 4D is experimental group 3, with basic screening medium + 60 g / L PEG6000. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments.

[0037] The general embodiment is a genetic transformation method for recalcitrant rice varieties based on Agrobacterium-mediated transformation, which includes the following steps: 1) Inducing callus: sterilized rice seeds are cultured under light in an induction medium for 7-10 days to obtain callus tissue; the induction medium contains: maltose 25-35 g / L, MS macro-element mixture 4-5 g / L, and L-proline 2.5-3 g / L.

[0038] In some preferred embodiments, in step 1), the rice seeds are recalcitrant rice varieties; further, they are 93-11, Guanglu Dwarf No. 4, Zhongjia No. 3, TN1, Shuhui 527, etc.

[0039] In some preferred embodiments, in step 1), the temperature of the light culture is 25-35°C.

[0040] In some preferred embodiments, in step 1), the induction culture medium comprises: 4-5 g / L MS macro-element mixture, MS minStock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 2.5-3 g / L, hydrolyzed casein 0.4-0.8 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, water.

[0041] 2) Suspension, infection and co-culture of Agrobacterium: Activated Agrobacterium was cultured in suspension medium, and callus tissue was added for infection to obtain infected callus tissue, which was then cultured in the dark.

[0042] In some preferred embodiments, step 2) involves activating Agrobacterium by streaking Agrobacterium (EHA105) onto LB medium containing 45-55 mg / L kanamycin and 45-55 mg / L rifampin, and culturing at 25-30°C.

[0043] In some preferred embodiments, in step 2), the concentration of Agrobacterium in the suspension culture medium before infection is OD600 = 0.1-0.5; the infection time is 5-10 min; and the dark culture is carried out in a closed environment at 24-28℃ for 2-4 days.

[0044] In some preferred embodiments, in step 2), the suspension culture medium comprises: 25-35 g / L sucrose, 0.3-0.7 g / L hydrolyzed casein, 8-12 mL / L Fe-EDTA, 45-55 mL / L AAM AminoAcid, 45-55 mL / L AAM macro, 4-6 mL / L AAM micro, and 4-6 mL / L AAM organic.

[0045] 3) Screening: The infected callus tissue after dark culture was cultured in the dark in the screening medium. The dried infected callus tissue without Agrobacterium contamination on the screening medium was transferred to another screening medium for continued dark culture. The screening medium contained: PEG6000 25-30g / L and maltose 25-35g / L.

[0046] In some preferred embodiments, in step 3), the two dark incubations are performed at 24-28°C for 8-12 days.

[0047] In some preferred embodiments, in step 3), the screening medium comprises: 4-5 g / L MS macro-element mixture, MS minStock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 0.4-0.6 g / L, hydrolyzed casein 0.2-0.4 g / L, PEG6000 25-30 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, water.

[0048] 4) Differentiation: Select resistant callus tissues that have not browned and died in 3) and culture them in light and dark alternately in differentiation medium containing 25-35 g / L maltose.

[0049] In some preferred embodiments, in step 4), the temperature for alternating light and dark culture is 24-28°C, the light culture cycle is 10-14h, the dark culture cycle is 10-14h, and the total culture time is 25-35 days.

[0050] In some preferred embodiments, in step 4), the differentiation medium comprises: MS macro-element mixture 4-5 g / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, hydrolyzed casein 1.5-2.5 g / L, maltose 25-35 g / L, sorbitol 25-35 g / L, NAA 0.2-0.3 mg / L, KT stock solution 1-3 mL / L, gum powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, and water.

[0051] 5) Rooting: The resistant callus tissue that is about to turn green forms a regenerated seedling. It is then transferred to a rooting medium containing 0.05-0.15 mg / L NAA and cultured under alternating light and dark conditions until a complete plant is formed.

[0052] In some preferred embodiments, in step 5), the height of the regenerated seedling is 3-4 cm; the temperature for alternating light and dark culture is 24-28℃, the light culture cycle is 10-14 h, the dark culture cycle is 10-14 h, and the total culture time is 13-17 days.

[0053] In some preferred embodiments, in step 5), the rooting medium comprises: 4-5 g / L MS macro-element mixture, MS... min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, sucrose 15-25 g / L, gum powder 4-6 g / L, water.

[0054] Example 1 (1) Induction of callus: Select mature and plump rice seeds (rice variety 93-11), remove the husk, soak in 70% alcohol for 2 minutes, pour off the alcohol, and rinse 5 times with sterile water. Add 50% Blue Moon disinfectant (with Tween-20 added) and soak for 30 minutes, shaking in a constant temperature shaker at 120 rpm, and rinse 5 times with sterile water. Inoculate the disinfected seeds onto a culture dish lined with filter paper to filter out excess water, then transfer to a new culture dish lined with filter paper and place it on a clean bench to dry. Select a thickened sterile culture dish with a diameter of 90 mm and a height of 40 mm to prepare the induction medium. Select seeds with normal endosperm and no sterile spots and spread them evenly on the induction medium. Inoculate about 100 seeds per dish and culture at 30℃ for 8 days (Figure 1A).

[0055] Table 1: Induction Culture Medium Preparation method of induction medium: First, add the formula in Table 1 to 900mL of distilled water, adjust the pH to 5.8 with 1N KOH, add distilled water to make up to 1L, and autoclave.

[0056] (2) Agrobacterium streaking activation: 3 days before infection, Agrobacterium (EHA105) was streaked on LB medium containing 50 mg / L kanamycin and 50 mg / L Rifampicin and cultured at 28°C.

[0057] (3) Suspension, infection, and co-culture of Agrobacterium: Before infection, the activated Agrobacterium was scraped into the suspension culture medium and cultured at 28°C with shaking at 180 mpm, and the bacterial concentration was adjusted to OD600=0.3. The callus tissue induced for 8 days (Figure 1B) was placed into the Agrobacterium suspension and infected for 10 min (Figure 1C). The bacterial solution was discarded, and the particles were spread on a sterile culture dish containing 10 layers of filter paper. The top layer of filter paper was discarded, ensuring that the remaining filter paper was slightly moistened. The culture dish containing the infected callus tissue and filter paper was sealed and transferred to a 26°C incubator for continued dark culture for 3 days.

[0058] Table 2: Suspension Culture Medium Preparation method of suspension culture medium: First, add the formula in Table 2 to 900 mL of distilled water, adjust the pH to 5.2 with 1N KOH, add distilled water to a final volume of 1 L, and autoclave. When using, add 1 mL AS to each 1 L of suspension culture medium and store temporarily at 4℃.

[0059] (4) After screening and co-culture, the infected callus tissue was placed on the screening medium and cultured in the dark at 26°C for 10 days. The dried callus particles without Agrobacterium contamination on the screening medium were transferred to a new screening medium and placed in an incubator at 26°C for another 10 days of dark culture (Figure 1D).

[0060] Table 3: Screening Culture Media Preparation method of screening medium: First, add the formula in Table 3 to 900 mL of distilled water, adjust the pH to 5.8 with 1N KOH, and add distilled water to a final volume of 1 L. Autoclave. When using, add 1.5 mL of carboxybenzyl and 1 mL of hygromycin (final concentration 450 mg / L carboxybenzyl and 50 mg / L hygromycin) to each 1 L of screening medium, pour into petri dishes and store temporarily at 4℃, about 20-30 mL per dish.

[0061] (5) Select resistant callus that has not browned and died and transfer it into differentiation medium. Culture at 26℃ (12h light / 12h dark) for 30 days (Figure 1E).

[0062] Table 4: Differentiation Culture Medium Preparation method of differentiation medium: First, add the formula in Table 4 to 900 mL of distilled water, adjust the pH to 5.8 with 1N KOH, and add distilled water to a final volume of 1 L. Autoclave. When using, add 1.5 mL of carboxybenzyl and 1 mL of hygromycin (final concentration 450 mg / L carboxybenzyl and 50 mg / L hygromycin) to each 1 L of screening medium, pour into petri dishes and store temporarily at 4℃, about 20-30 mL per dish.

[0063] (6) When the rooted and greening resistant callus forms a 3-4 cm tall regenerated seedling on the differentiation medium, it is transferred to the rooting medium and cultured at 26℃ (12h light / 12h dark cycle) until a complete plant is formed, about 15 days (Fig. 1F).

[0064] Table 5: Rooting Culture Medium Preparation method of rooting medium: First, add the formula in Table 5 to 900mL of distilled water, adjust the pH to 5.8 with 1N KOH, add distilled water to make up to 1L, autoclave, and dispense into rooting tubes.

[0065] The formulations of some components in the culture media in Tables 1-5 above are as follows: (1) MS min The formula for the stock solution is shown in Table 6: Table 6 Preparation method: Prepare separate stock solutions (1000X) for Cu, Co, and Mo: 2.5g CuSO4·5H2O, 2.5g CoCl2·6H2O, and 25g Na2Mo4·2H2O, and bring the volume to 100mL; add 200uL of separate stock solutions (1000X) for Cu, Co, and Mo to 200mL of AAM micro-volume (200X).

[0066] (2) The formulation of MS organic is shown in Table 7: Table 7 (3) The formulation of Fe-EDTA is shown in Table 8: Table 8 Preparation method: Na2-EDTA and FeSO4·7H2O were first prepared into solutions separately, then mixed and stored at 70℃ for 2 hours (Fe 2+ (Chelate), boil until the color turns dark yellow, and store in a brown bottle.

[0067] (4) AAM's numerous formulations are shown in Table 9: Table 9 (5) The formulation of trace amounts of AAM is shown in Table 10: Table 10 Preparation method: Prepare separate stock solutions (1000X) for Cu, Co, and Mo: 2.5g CuSO4·5H2O, 2.5g CoCl2·6H2O, and 25g Na2Mo4·2H2O, and bring the volume to 100 mL; add 200 μL of separate stock solutions (1000X) for Cu, Co, and Mo to 200 mL of AAM micro-volume (200X).

[0068] (6) The formula for AAM organic is shown in Table 11: Table 11 (7) The formulation of AAM AminoAcid is shown in Table 12: Table 12 (8) Kanamycin (50mg / mL): Dissolve 5g of powder directly in 100mL of ddH2O; store at -20℃.

[0069] (9) Rifampicin (50mg / mL): Dissolve 5g of powder directly in 100mL of DMSO; store at -20℃.

[0070] (10) Carbenicillin (300 mg / mL): Dissolve 30 g of powder directly in 100 mL of ddH2O; store at -20 °C.

[0071] (11) Hygromycin B (50 mg / mL), catalog number: 10843555001.

[0072] (12) As stock solution (15mg / mL, about 80μmol / L): Dissolve 0.3g of powder in DMSO first, then make up to 20mL with ddH2O; store at -20℃.

[0073] (13) 2,4-D stock solution (1 mg / mL): Add 100 mg 2,4-D to 1 mL 1N KOH and stir for 5 min. Add distilled water while stirring until 2,4-D is completely dissolved. Make up to 100 mL and store in a brown bottle at 4 °C.

[0074] (14) NAA (0.5 mg / mL): Dissolve 100 mg NAA in NaOH, add ddH2O to make up to 200 mL, and store at 4 °C.

[0075] (15) Kinetin (KT) stock solution (1 mg / mL): Dissolve 1 mg of powder directly in 1 mL of DMSO (zero the solution directly using a 2 mL EP tube and weigh it). The working concentration is 2 mg / L, and it should be prepared and used immediately.

[0076] (16) 1N KOH: Dissolve 5.6g KOH powder in water, add ddH2O to make up to 100mL, and store at room temperature.

[0077] Example 2: Genetic Transformation of Rice Using 93-11 as Material 1.1 Obtaining the Transgenic T0 Generation Using mature seeds of 93-11 as material, the rice was cultured under 24-hour full light for 8 days, and the callus induction rate exceeded 90% (Figure 1B). Approximately 500 callus tissues were used for Agrobacterium infection, co-culture, screening, differentiation, and rooting culture. Transgenic seedlings were obtained in about 3 months (using the method of Example 1).

[0078] 1.2 Pre-transplanting acclimatization culture of transgenic T0 generation seedlings The above-mentioned transgenic T0 generation seedlings were thoroughly cleaned of residual culture medium from the roots with sterile water, and then the plants were placed in clean water to completely submerge the roots. After acclimatization in outdoor environment for 3 days, they can be transplanted into the soil.

[0079] 1.3 Statistics on the emergence rate of transgenic T0 generation According to the above genetic transformation process, the emergence rate (%) is calculated as follows: total number of seedlings / total number of infected callus tissues × 100.

[0080] 1.4 Resistance Identification of Transgenic Seedlings Young leaves of transgenic T0 generation rice seedlings were minced and placed in 2 mL centrifuge tubes. DNA was extracted from the leaves using the CTAB method. Based on the hygromycin and G418 genome sequences provided on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), the following primers for hygromycin detection were designed: Hyg-jc-F: 5'-CTGCCCGCTGTTCTACAACCGG-3'; Hyg-jc-R: 5'-GGAGCATATACGCCCGGAGTC-3'; G418-jc-F: 5'-CAAGATGGATTGCACGCAGG-3'; G418-jc-R: 5'-TTCAGTGACAACGTCGAGCA-3'. The DNA extracted from the rice leaves was amplified by PCR using the above primers (using Vazyme, 2×Rapid Taq Master enzymes). The amplified fragments of hygromycin (481 bp) and G418 (249 bp) were analyzed by gel electrophoresis, and positive plants were screened based on the amplified bands (Figure 2).

[0081] Between 2022 and 2025, a total of 39 transformation projects were conducted using variety 93-11 as the material. The callus emergence rate ranged from 0.6% to 28.6%, and the positive rate of transgenic seedlings ranged from 14.29% to 100%. Figure 2A shows that with the maturity of the technology, the average callus emergence rate and average positive rate of transgenic seedlings increased year by year, reaching 9.18% and 71.51% in 2025, respectively. In addition, this invention also compared the callus emergence rate and positive rate of 93-11 genetic transformation against G418 and hygromycin eukaryotic resistance. The results showed that the average callus emergence rate of G418 was higher than that of hygromycin resistance, but the positive rate of transgenic seedlings was lower than that of hygromycin resistance.

[0082] Furthermore, between 2022 and 2025, this invention utilized its transgenic process to study over 20 recalcitrant indica rice varieties other than 93-11, and statistically analyzed the emergence rates of over 30 transgenic TN1 and Huazhan rice varieties. All transgenic experiments used approximately 350 callus tissues for Agrobacterium infection, with hygromycin as the eukaryotic resistance (Table 1). Addressing the technical challenge of genetic transformation of "recalcitrant" rice, the transgenic system of this invention demonstrated exceptional efficiency. In the indica rice varieties TN1 and Huazhan, the average emergence rates jumped to 24.14% and 20.61%, respectively (Table 13), significantly exceeding the previous level of less than 5%, thus overcoming the transformation efficiency bottleneck that has long constrained gene function research.

[0083] Table 13: Summary of TN1 and Huazhan genetic transformation efficiency Example 3: Optimized Screening Medium with Added PEG6000 and Determination of its Optimal Concentration In plant genetic transformation, the efficiency of the screening medium directly determines the rate of positive transformants. Traditional screening media often face the problem of "escape" due to insufficient screening pressure, or the problem of inhibited growth of transformed cells due to excessive screening pressure. This invention aims to improve the rate of resistant callus acquisition, reduce the false positive rate, and promote its subsequent regeneration capacity by adding a novel synergist, PEG6000, to conventional screening media and optimizing its concentration. To verify the effect of reagent PEG6000 and determine its optimal working concentration, this invention induced callus using 93-11 rice seeds and set up the following experimental groups: Control group: Basic screening medium (the difference from the screening medium in Example 1 is that it does not contain reagent PEG6000).

[0084] Experimental group 1: Basic screening medium + reagent PEG6000, concentration 10g / L.

[0085] Experimental group 2: Basic screening medium + reagent PEG6000, concentration 30g / L.

[0086] Experimental group 3: Basic screening culture medium + reagent PEG6000, concentration 60g / L.

[0087] All culture media were prepared with the same basic formulation and screened for 2 weeks using the same batch of 93-11 rice embryogenic callus under the same culture conditions (complete darkness, 26°C). Each group had at least 3 replicates, with each replicate inoculated with approximately 100 callus blocks.

[0088] After the culture was completed, the following key indicators of each group were statistically analyzed and compared as shown in Table 14.

[0089] Table 14: Effects of different PEG6000 concentrations on 93-11 resistant callus The data comparison in the table above shows that: (1) Compared with the control, the resistant callus acquisition rate of all experimental groups with added PEG6000 was significantly improved, proving that PEG6000 can effectively enhance the screening pressure and improve the screening efficiency of the transformants (Figure 4, Table 14).

[0090] (2) PEG6000 showed obvious concentration dependence. The amount of resistant callus in experimental group 1 (10g / L PEG6000) and experimental group 2 (30g / L PEG6000) was significantly higher than that in the control group by 26%, the callus state was the best, and the proportion of regenerable healthy resistant callus was as high as 83% (Figure 4, Table 14), which laid a solid foundation for subsequent high-efficiency seedling growth.

[0091] In experimental group 3 (60g / L PEG6000), the quality of callus tissue decreased, and the proportion of regenerable healthy resistant callus decreased to 39% (Figure 4, Table 14), which was not conducive to subsequent regeneration.

[0092] In summary, this invention establishes a highly efficient and precise rice genetic transformation screening system by adding PEG6000 to the screening medium and determining its optimal concentration gradient of 1-3%. This scheme can maximize the enrichment of true transformed cells while maintaining their vigorous regenerative potential, which is one of the key steps for the success of this transformation method.

[0093] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for genetic transformation of recalcitrant rice varieties based on Agrobacterium-mediated transformation, characterized in that... include: 1) Rice seeds were cultured under light for 7-10 days in an induction medium containing 25-35 g / L maltose, 4-5 g / L MS macro-element mixture, and 2.5-3 g / L L-proline to obtain callus tissue; 2) Activated Agrobacterium was cultured in a suspension medium, and the callus tissue was added to obtain infected callus tissue, which was then cultured in the dark; 3) The infected callus tissue was cultured in the dark in a selection medium containing 25-30 g / L PEG6000 and 25-35 g / L maltose; 4) Resistant infected callus tissue without browning and death was selected and cultured in alternating light and dark in a differentiation medium containing 25-35 g / L maltose; 5) Once the resistant infected callus tissue formed regenerated seedlings, they were transferred to a rooting medium containing 0.05-0.15 mg / L NAA and cultured in alternating light and dark to develop into plants.

2. The method according to claim 1, characterized in that: In step 1), the rice seeds are recalcitrant rice varieties; the temperature for light cultivation is 25-35℃.

3. The method according to claim 1 or 2, characterized in that: In step 1), the induction medium comprises: 4-5 g / L MS macro-element mixture, MS min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 2.5-3 g / L, hydrolyzed casein 0.4-0.8 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, water.

4. The method according to claim 1, characterized in that: In step 2), the concentration of Agrobacterium in the suspension culture medium before infection is OD600 = 0.1-0.5; the infection time is 5-10 min; and the dark culture is carried out in the dark for 2-4 days under closed conditions at 24-28℃.

5. The method according to claim 1 or 4, characterized in that: In step 2), the suspension culture medium comprises: 25-35 g / L sucrose, 0.3-0.7 g / L hydrolyzed casein, 8-12 mL / L Fe-EDTA, 45-55 mL / L AAM AminoAcid, 45-55 mL / L AAM macro, 4-6 mL / L AAM micro, and 4-6 mL / L AAM organic.

6. The method according to claim 1, characterized in that: In step 3), the two dark incubations are performed at 24-28℃ for 8-12 days.

7. The method according to claim 1 or 6, characterized in that: In step 3), the screening medium comprises: 4-5 g / L MS macro-element mixture, MS min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, 2,4-D stock solution 1.5-2.5 mL / L, L-proline 0.4-0.6 g / L, hydrolyzed casein 0.2-0.4 g / L, PEG6000 25-30 g / L, maltose 25-35 g / L, gum powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, water.

8. The method according to claim 1, characterized in that: In step 4), the temperature for the alternating light and dark culture is 24-28℃, the light culture cycle is 10-14h, the dark culture cycle is 10-14h, and the total culture time is 25-35 days.

9. The method according to claim 1 or 8, characterized in that: In step 4), the differentiation medium comprises: MS macro-element mixture 4-5 g / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, hydrolyzed casein 1.5-2.5 g / L, maltose 25-35 g / L, sorbitol 25-35 g / L, NAA 0.2-0.3 mg / L, KT stock solution 1-3 mL / L, gelatin powder 4-6 g / L, carboxybenzyl 400-500 mg / L, hygromycin 45-55 mg / L, and water.

10. The method according to claim 1, characterized in that: In step 5), the height of the regenerated seedlings is 3-4 cm; the temperature for the light-dark alternating culture is 24-28℃, the light culture period is 10-14 h, the dark culture period is 10-14 h, and the total culture time is 13-17 days; the rooting medium includes: MS macro-element mixture 4-5 g / L, MS... min Stock solution 4-6 mL / L, Fe-EDTA 8-12 mL / L, MS organic 8-12 mL / L, sucrose 15-25 g / L, gum powder 4-6 g / L, water.

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