Construction and optimization method and system of wheat immature embryo vacuum infiltration instantaneous conversion system

Abstract

The invention belongs to the field of wheat immature embryo genetic transformation, and discloses a method for constructing and optimizing a wheat immature embryo vacuum infiltration transient transformation system, which comprises the following steps: by taking a wheat variety Fielder immature embryo pollinated for 14 days as a material, optimizing vacuum infiltration pressure and treatment times, and utilizing GUS histochemical staining, conductivity determination and qRT-PCR gene expression analysis, and establishing a wheat immature embryo vacuum infiltration transient transformation system. And evaluating the relationship between the transformation efficiency and the cell damage degree. According to the method disclosed by the invention, a transformation system combining 0.025 MPa with four times of vacuum infiltration treatment is established by a synergistic mechanism of low pressure of 0.025 MPa and four times of infiltration, so that the GUS positive rate of the wheat Fielder immature embryo is increased to 62.8%, and an effective technical support is provided for wheat gene function analysis, CRISPR editing and molecular design breeding.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of wheat embryo genetic transformation technology, and particularly relates to a method and system for constructing and optimizing a wheat embryo vacuum permeation instantaneous transformation system. Background Technology

[0002] Wheat (Triticum aestivum L.) is a staple crop for approximately 35% of the world's population, with an annual yield exceeding 770 million tons, accounting for more than 30% of global grain production. It is one of the core crops for ensuring food security. With population growth and intensifying climate change, improving wheat yield and stress resistance (such as drought and disease resistance) has become a key focus of global agricultural research. However, traditional breeding techniques, due to their long cycles and low efficiency, are insufficient to meet the demand for rapid improvement. Genetic transformation technology, through the direct introduction of exogenous genes or the regulation of endogenous gene expression, provides an efficient means to analyze gene function and cultivate high-yielding, high-quality, and stress-resistant new varieties.

[0003] Wheat embryos, due to their low degree of cell differentiation and strong regeneration capacity, are ideal recipient materials for genetic transformation. However, their transformation efficiency has long been low (typically <10%), severely hindering gene function research and molecular breeding progress. The main reasons for this low efficiency include: thick embryonic cell walls making Agrobacterium infection difficult, high somatic cell mutation rates during regeneration, and cell damage caused by unoptimized permeation parameters. While the gene gun method can bypass Agrobacterium infection limitations, it suffers from high equipment costs and a large number of exogenous gene copies integrated. In contrast, Agrobacterium-mediated vacuum permeation, with its advantages of simple operation, low cost, and controllable gene insertion sites, is gradually becoming the mainstream technology for genetic transformation of monocotyledonous plants.

[0004] Vacuum permeation promotes T-DNA transfer by bringing Agrobacterium into close contact with recipient tissue through negative pressure. However, optimizing its technical parameters (such as pressure and number of permeation cycles) remains a challenge. Studies have shown that excessively high pressure (>0.05 MPa) can damage cell membrane integrity, leading to extravasation of intracellular electrolytes (increased conductivity) and significantly reducing cell viability; while excessively low pressure (<0.02 MPa) cannot effectively promote Agrobacterium attachment, limiting T-DNA transfer efficiency. Furthermore, while increasing the number of permeation cycles can increase the probability of Agrobacterium infection, excessive treatment can lead to the accumulation of reactive oxygen species (ROS), exacerbating oxidative stress and inhibiting tissue regeneration. Previous studies have focused on crops such as rice and corn, but wheat embryos, due to their dense cell wall structure and weak regeneration capacity, are more sensitive to permeation conditions, making existing parameter systems difficult to apply directly. Therefore, a specialized optimization scheme is urgently needed.

[0005] In recent years, researchers have attempted to optimize crop transformation conditions by adjusting vacuum osmosis pressure and the number of osmosis cycles. For example, in rice, a pressure of 0.03 MPa combined with three osmosis cycles can increase the GUS positivity rate to 40%; maize immature embryos show the highest transformation efficiency when treated once at 0.05 MPa. However, these parameters have limited applicability to wheat. Studies have shown that the high lignin content in the cell walls of wheat immature embryos leads to increased resistance to Agrobacterium infection, requiring lower pressures to reduce mechanical damage. Furthermore, wheat immature embryos are prone to browning during co-culture, which may be related to osmosis-induced membrane lipid peroxidation. Therefore, systematically analyzing the effects of vacuum osmosis parameters on cell physiological states by combining cell membrane integrity assays (such as conductivity methods) and gene expression analysis (qRT-PCR) is key to overcoming technical bottlenecks. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for constructing and optimizing a wheat embryo vacuum permeation instantaneous conversion system.

[0007] This invention is achieved as follows: a method for constructing and optimizing a wheat embryo vacuum permeation instantaneous conversion system, comprising:

[0008] a. Activate Agrobacterium in a culture medium and prepare an infection solution containing a chemotherapeutic inducer;

[0009] b. Immerse the wheat embryos obtained from the peeling process completely in the infection solution;

[0010] c. The embryo is subjected to vacuum permeation treatment to form a window for the entry of exogenous DNA under set osmotic pressure and number of permeation cycles;

[0011] d. The infiltrated embryos were co-cultured under dark conditions and T-DNA transfer was induced;

[0012] e. Evaluate the expression of exogenous genes using the colorimetric results;

[0013] f. Characterize changes in cell membrane permeability by measuring conductivity;

[0014] g. Determine the response status of immune-related genes and cell wall remodeling genes through gene expression analysis;

[0015] h. By comprehensively analyzing and regressing data on exogenous gene expression, membrane permeability, and gene expression results, the optimal combination of vacuum pressure and permeation frequency is determined to achieve the target effect of high exogenous gene expression and low cell damage.

[0016] Furthermore, the test material was the spring wheat (Triticum aestivum L.) variety 'Fielder' (provided by the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences). The embryos 14 days after pollination were selected as transformation recipients. The embryos were 2-3 mm in diameter and were immediately soaked in sterile distilled water after being peeled off.

[0017] Furthermore, the strain and vector are as follows: Agrobacterium GV3101 carrying the binary expression vector pCAMBIA1301-GUS (containing a 35S promoter and a GUS-GFP reporter gene); the strain is stored in glycerol tubes at -80℃ and inoculated into MG / L solid medium (5 g / L mannitol, 1 g / L L-glutamate, 250 mg / L KH2PO4, 100 mg / L NaCl, 100 mg / L MgSO4·7H2O, 5 g tryptone, 2.5 g yeast extract and 1 µg biotin added to 700 mL distilled water, and the volume is adjusted to 1000 mL; the pH is adjusted to 7.0; sterilized using a 0.22 µm cellulose acetate filter and stored at 4°C, pH 7.0), containing 50 mg / L kanamycin (Kan) and 25 mg / L rifampin (Rif), and incubated in the dark at 28℃ for 48 hours.

[0018] Furthermore, the reagents and instruments include:

[0019] (1) Culture medium: co-medium (1 / 10 MS basal salt + 30 g / L glucose + 100 μmol / L acetosyringone + 7 g / L agar, pH 5.8); recovery medium (MS + 2 mg / L 2,4D + 50 mg / L cefotaxime + 250 mg / L + ampicillin + 100 mg / L + termethin + 50 mg / L hygromycin);

[0020] (2) Staining reagents: X-Gluc staining solution (Solarbio, G3060, containing 1 mmol / L X-Gluc, 50 mmol / L sodium phosphate buffer, 0.1% Triton X-100, pH 7.0);

[0021] (3) Main equipment: vacuum permeameter (SCIENTZ-12F, Ningbo Xinzhi Biotechnology); conductivity meter (DDS-307A, Shanghai Leici); real-time PCR instrument (LightCycler 480 II, Roche).

[0022] Furthermore, the method for constructing and optimizing the wheat embryo vacuum permeation instantaneous conversion system specifically includes:

[0023] S1: Vacuum osmosis pressure optimization, setting four pressure gradients of 0.025 MPa, 0.05 MPa, 0.075 MPa, and 0.1 MPa, treating 30 embryos per group, repeated 3 times;

[0024] S2: Permeation number setting. Based on pressure optimization results, the optimal pressure is fixed at 0.025MPa. Permeation treatments of 0, 1, 2, 3, 4, and 5 times are set. Other parameters are the same as S1. Each group is treated with 30 embryos and repeated 3 times.

[0025] S3: GUS histochemical staining, staining of plant materials according to Jefferson's method;

[0026] S4: Conductivity measurement;

[0027] S5: qRT-PCR analysis;

[0028] S6: Data analysis, including data standardization, statistical testing, graphing, and correlation analysis.

[0029] Furthermore, the specific operation of S1 is as follows:

[0030] (1) Agrobacterium activation: single colonies were picked and inoculated into MG / L liquid medium (containing 50 mg / L Kan), and cultured at 28℃ and 200 rpm with shaking until OD600=0.6~0.8. The cells were collected by centrifugation at 4000 rpm for 10 min, and the bacterial concentration was adjusted to OD600=0.5 with 1 / 10 MS resuspension (containing 100 μmol / L acetylsyl syringone).

[0031] (2) Vacuum permeation: Immerse the embryos in the bacterial solution and place them in a vacuum permeator. Set the single treatment time to 2 min, release the negative pressure, and treat again after 5 min interval. The 6 groups are 0, 1, 2, 3, 4 and 5 times respectively.

[0032] (3) Co-culture: After the surface bacterial solution is dried, the embryos are transferred to the co-culture medium and cultured in the dark at 25°C for 3 days.

[0033] Furthermore, the specific operation of S3 is as follows:

[0034] (1) Fixation and staining: The co-cultured embryos were immersed in X-Gluc staining solution and incubated at 37°C in the dark for 24 h;

[0035] (2) Decolorization treatment: Decolorize with 70%, 90% and 100% ethanol in a gradient, 1 h each time, to remove chlorophyll interference;

[0036] (3) Positive rate statistics: The number of blue embryos was observed under a stereomicroscope, and the positive embryo rate was calculated (number of positive embryos / total number of embryos × 100%).

[0037] Furthermore, the specific operation of S4 is as follows:

[0038] (1) Sample preparation: 30 wheat embryos in each group were placed in deionized water (6 groups in total, 3 mL per group) and vacuum permeation was performed.

[0039] (2) Measurement conditions: After vacuum permeation treatment, soak for 12 hours, and use a conductivity meter (DDS-307A) to measure the conductivity of the extract (R1) at 25℃. Then, heat in a boiling water bath for 30 minutes, cool to room temperature, shake well, and measure the conductivity of the extract again (R2). Repeat 3 times, and calculate the relative conductivity = R1 / R2 × ​​100% of the extract conductivity.

[0040] Furthermore, the specific operation of S5 is as follows:

[0041] (1) RNA extraction and reverse transcription: Total RNA was extracted using the TRIzol method (Invitrogen), genomic DNA was removed using DNase I (TaKaRa), and cDNA was synthesized using the PrimeScript RT kit (TaKaRa);

[0042] (2) Primer design: Specific primers were designed for GUS-GFP, EXPA2, SOD, PR1 and the internal reference gene Actin, and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] (3) Amplification system: SYBR Premix Ex Taq (TaKaRa) 20 μL system: 2×Mix 10 μL, forward and reverse primers 0.4 μL each (10 μmol / L), cDNA 1 μL, ddH2O 8.2 μL;

[0044] (4) Reaction program: 95℃ pre-denaturation for 120 s; 95℃ for 5 s, 60℃ for 15 s, 72℃ for 30 s, 40 cycles; melting curve analysis to confirm amplification specificity;

[0045] (5) Data analysis: The relative gene expression level was calculated using the 2-ΔΔCt method.

[0046] Furthermore, the specific operation of S6 is as follows:

[0047] (1) Data standardization: GUS positivity rate, conductivity and gene expression level data are expressed as mean ± standard deviation;

[0048] (2) Statistical tests: SPSS 18.0 was used for one-way ANOVA and Duncan's method for multiple comparisons (P<0.05).

[0049] (3) Graphics drawing: GraphPad Prism 10.0 draws bar charts and heatmaps;

[0050] (4) Correlation analysis: The correlation between conductivity and GUS positivity rate was assessed by Spearman correlation coefficient.

[0051] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0052] This invention proposes a synergistic mechanism of four osmosis treatments at a low pressure of 0.025 MPa: the low-pressure environment maintains cell membrane integrity by reducing mechanical stress, while moderately increasing the number of osmosis cycles prolongs the contact time between Agrobacterium and tissue, promoting T-DNA transfer. Furthermore, antioxidant genes (SOD, CAT) were not significantly activated, suggesting that moderate physical stimulation can avoid excessive immune responses. Five osmosis cycles trigger a sharp increase in PR1 expression and upregulation of the cell wall repair gene EXPA2, leading to a decrease in transformation efficiency. Compared to crops such as maize (optimal pressure 0.05 MPa) and rice (0.03 MPa), wheat embryos are more sensitive to vacuum osmosis and require even lower pressure to avoid cell damage. The ultimately established transformation system of 0.025 MPa combined with four vacuum osmosis treatments increased the GUS positivity rate of wheat Fielder embryos to 62.8%, providing effective technical support for wheat gene function analysis, CRISPR editing, and molecular design breeding.

[0053] Compared to existing methods for transient transformation of wheat embryos that primarily rely on prolonged infection time, increased Agrobacterium concentration, or repeated mechanical treatment, this method represents a significant advancement in both its technical approach and implementation. First, by introducing a controllable vacuum permeation strategy, this method induces a transient "entry window" at the intercellular space and cell wall-cell membrane interface using a pressure gradient within a short timeframe. This significantly increases the probability of exogenous DNA entering the embryo along with Agrobacterium, overcoming the bottleneck of unstable transformation efficiency caused by reliance on random infection and passive diffusion in traditional methods. Second, this method does not simply pursue transformation intensity but simultaneously incorporates conductivity measurement and expression analysis of genes related to immunity and cell wall remodeling. This allows for the quantitative characterization of cell membrane integrity and tissue stress response, enabling a systematic assessment and constraint optimization of the contradictory relationship between "transformation efficiency and tissue damage." Furthermore, through multi-index comprehensive analysis and parameter regression, an optimal combination model of vacuum pressure and permeation times is established. This transforms the selection of experimental parameters from empirical trial-and-error to a data-driven quantitative optimization process, thereby significantly reducing the risk of embryonic cell damage and physiological imbalance while ensuring high expression of exogenous genes. This method achieves substantial improvements in conversion efficiency stability, tissue protection, and reproducibility, demonstrating outstanding technological progress. Attached Figure Description

[0054] Figure 1 This is a flowchart of the construction and optimization method of the wheat embryo vacuum permeation instantaneous conversion system provided in the embodiments of the present invention;

[0055] Figure 2 The effects of different vacuum osmotic pressure treatments provided in the embodiments of the present invention on GUS staining of wheat embryos are as follows: (A) 0 MPa, control; (B) 0.025 MPa; (C) 0.05 MPa; (D) 0.075 MPa; (E) 0.1 MPa;

[0056] Figure 3 This invention provides a correlation analysis of the relative expression level of GUS and the relative conductivity of wheat embryos under different vacuum permeation treatments: (A) Relative expression level of GUS-GFP under different vacuum permeation treatments; (B) Changes in relative conductivity under different vacuum permeation treatments; (C) Relationship between relative expression level of GUS (x-axis) and relative conductivity (y-axis) of 17 independent samples.

[0057] Figure 4 The following are the effects of different vacuum permeation times on the expression of wheat response genes provided in the embodiments of the present invention: (A) relative expression level of PR1 gene; (B) relative expression level of PAL gene; (C) relative expression level of SOD gene; (D) relative expression level of CAT gene; (E) relative expression level of EXPA2 gene. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0059] When extending existing wheat embryo transient conversion technology to industrial applications, the first bottleneck encountered lies in the contradiction between conversion rate and cell viability: excessively high negative pressure facilitates Agrobacterium penetration but easily causes embryo tissue damage, while excessively low negative pressure cannot guarantee sufficient gene vector entry, resulting in low efficiency and high variability. To address this, this invention focuses on embryos fourteen days after pollination, systematically analyzing the relationship between pressure gradient and cell integrity by precisely adjusting vacuum osmosis pressure and the number of osmosis cycles, thus avoiding the one-size-fits-all approach used in traditional empirical methods for embryos in different physiological states.

[0060] During the stress optimization phase, this invention sets up multiple gradients for parallel testing on the same batch of immature embryos. Changes in conductivity are used as a quantitative indicator of cell membrane permeability and damage level. Spearman correlation analysis is introduced to construct a function curve of conductivity versus GUS reporter gene expression efficiency. This approach overcomes the limitations of relying solely on positive rate evaluation and provides the method with monitorable and feedback-based quality control capabilities for large-scale production.

[0061] To optimize the pressure, this invention further explores the impact of the number of percolation cycles on conversion stability. By adjusting the number of percolation cycles under a fixed negative pressure condition, the preliminary experimental results are transformed into a continuous kinetic model. Ultimately, it is determined that multiple short-duration negative pressure cycles are superior to a single long-duration negative pressure cycle, thereby effectively reducing the concentration of mechanical stress in the embryo and lowering the incidence of necrotic areas.

[0062] In the co-culture and staining evaluation stage, this invention uses a modified X-Gluc histochemical staining protocol, supplemented by quantitative RT-PCR data, to double-verify gene expression levels.

[0063] This invention not only standardizes the operating procedures, but also clearly reveals that vacuum osmosis pressure and the number of osmosis cycles together constitute the decisive window for the entry of exogenous genes. In particular, it verifies that 0.025 MPa × 4 osmosis cycles is the optimal parameter point for the instantaneous conversion efficiency of wheat.

[0064] To verify the biological mechanism of this conclusion, this study selected five key genes closely related to defense, metabolism, and cell wall state as response indicators and detected their expression inflection points at different percolation times using qRT-PCR. The results showed that PR1, CAT, and EXPA2 were negative regulators of transformation efficiency: PR1 participates in the salicylic acid pathway and triggers the immune response; its expression continuously decreased in treatments 0–4, but suddenly rebounded in treatment 5, corresponding to the sharp drop in transformation efficiency in treatment 5. This indicates that when the immune intensity is re-enhanced, wheat tissue regains its ability to block the transfer of exogenous DNA from Agrobacterium, thereby reducing the success rate of T-DNA entry and integration. Similarly, CAT, as a hydrogen peroxide scavenging enzyme, was expressed at low levels in the first four percolations, maintaining mild H2O2 accumulation, which is beneficial for membrane relaxation and vir gene activation. However, the rebound of CAT in treatment 5 led to the rapid clearance of H2O2, resulting in weakened percolation and the closure of the transformation window. The EXPA2 gene, as a factor related to cell wall stretching and repair, also showed a rapid increase in the fifth treatment. This means that the cell wall is re-densified, which structurally hinders the formation of T-DNA channels. This further proves that the fourth infiltration is the intersection of cell wall relaxation and immunosuppression, and is also the best available stage for introduction.

[0065] In contrast, SOD and PAL act as auxiliary signals promoting transformation. Decreased SOD indicates an increase in superoxide anion content, which can produce a mild oxidative membrane-breaking effect, creating a permeability window. Reduced PAL expression leads to a decrease in phenylpropane pathway products; many of these phenolic metabolites have antibacterial properties, so their reduction actually weakens the defense against Agrobacterium, indirectly increasing infection efficiency. Notably, SOD and PAL continued to decrease during the fifth permeation, but transformation efficiency did not further improve, indicating that the dominant influence at this point had shifted from immunosuppression to cell wall strengthening and disease resistance recovery, further proving that the transformation window only exists in the fourth permeation.

[0066] In summary, 0.025 MPa is not merely a physical condition setting, but a pressure threshold that maintains cell activity, conductivity changes, and immune response within a usable range. The first to third percolations gradually weaken defenses but do not establish an optimal pathway. The fourth percolation reaches the peak point of lowest immunity, most relaxed cell walls, and most moderate ROS. The fifth percolation triggers a comprehensive rebound in PR1, CAT, and EXPA2, closing the transformation window. The inflection points of these five genes together constitute a complete biological evidence chain, proving that there is a unique optimal combination of vacuum percolation number and pressure (0.025 MPa × 4 times), and that this combination possesses predictable and quantifiable stability, which can be directly used for parameter solidification and algorithm execution in automated batch transformation platforms.

[0067] This embodiment provides a method for constructing and optimizing a transient genetic transformation system for wheat immature embryos using vacuum permeation. The aim is to establish a balance between cell damage and immune response, thereby achieving optimal conditions for high transformation efficiency and low-damage expression. The system is applicable to the model variety *Triticum aestivum* 'Fielder' and can be further extended with parameter tuning based on different genotypes.

[0068] (1) Activation of Agrobacterium and preparation of infection solution

[0069] Agrobacterium tumefaciens GV3101 strain was transformed into a binary vector containing a GUS-GFP dual reporter element driven by a 35S promoter. Single colonies were picked and inoculated into LB broth containing a suitable antibiotic resistance marker, and cultured at 28°C with shaking at 200 rpm for 12–16 h until OD500. 600 ≈0.6–0.8. After harvesting the bacteria, centrifuge and resuspend them in a sensing solution containing acetylsyringone (AS 100 μM) for later use, to enhance vir gene transcription activity and improve subsequent T-DNA transfer efficiency.

[0070] (2) Preparation of wheat embryos

[0071] Wheat seeds were selected 10–12 days after full bloom. Under aseptic conditions, the endosperm was removed to obtain intact embryos, which were immediately used for vacuum permeation testing. The embryos were then transplanted into an infection solution containing aspartate sucrose and an appropriate amount of sucrose, ensuring complete immersion of the embryo grain surface.

[0072] (3) Vacuum permeation treatment

[0073] The container containing the embryonic embryo and Agrobacterium suspension was placed in a vacuum chamber, and the vacuum pressure was set to 0.025 MPa. Each osmosis was maintained for 5 minutes, and after the vacuum was released, the pressure was restored to normal for 15 minutes, which constituted one cycle. This experimental system used 4 osmosis cycles as the optimal condition; the comparative experiments included treatment groups of 0, 1, 2, 3, and 5 cycles.

[0074] The research results show that:

[0075] Low pressure (0.025 MPa) can effectively reduce mechanical damage to cells;

[0076] Multiple penetrations (4 times) prolong bacterial cell attachment time, promote vir gene activation, and enable efficient T-DNA entry;

[0077] More than 4 times will induce a sharp increase in PR1 and EXPA2, which will enhance immunity and cell wall repair, leading to a rapid decline in the conversion rate.

[0078] (4) Co-culture and transient expression detection

[0079] After infiltration, the embryos were aerated, dried, and transferred to a co-culture medium, where they were cultured in the dark for 48 hours. For transient expression verification, the tissues were stained with GUS, and the percentage of positive blue staining was calculated. In this invention, the positive rate in the four-infiltration group reached 62.8%, significantly higher than other treatment groups.

[0080] (5) Assessment of cell damage and membrane permeability

[0081] The conductivity of tissue extracts was measured using a conductivity meter as an indicator of membrane permeability. The results showed that the conductivity increased with the number of permeation cycles, reaching its peak at the fourth treatment and then declining after the fifth treatment, indicating that the EXPA2-mediated cell wall reinforcement and repair process had been activated.

[0082] (6) Gene expression analysis and verification of signal regulation mechanisms

[0083] To elucidate the effects of vacuum permeation treatment on immune pathways and cell wall remodeling networks in wheat immature embryos, total RNA was extracted from transformed tissues and analyzed by qRT-PCR. Target genes included PR1, PAL, SOD, CAT, and EXPA2. Results showed that these genes exhibited significantly differentiated responses under different permeation cycles, and their expression changes were highly consistent with membrane permeability, conductivity, and transient GUS expression, constituting key molecular evidence for defining optimal transformation parameters.

[0084] PR1 gene expression decreased continuously from 0 to 4 infiltration treatments, reaching its lowest level at the 4th treatment, and then surged after the 5th infiltration treatment. This pattern is completely opposite to the change in transformation efficiency, indicating that the immune activation of PR1 is significantly negatively correlated with T-DNA entry. Since PR1 is involved in SA-mediated broad-spectrum pathogen recognition, its overexpression can trigger a defense response and form an immune barrier on the cell wall surface. Therefore, the strong upregulation of PR1 induced by the 5th treatment is considered to be the main reason for the significant decline in transformation efficiency.

[0085] The expression level of the PAL gene steadily decreased with increasing treatment frequency, but this inhibitory trend did not lead to a decrease in transformation efficiency. Instead, at lower expression levels, it was accompanied by a sustained increase in GUS-positive signals. Analysis suggests that PAL downregulation reduces the accumulation of phenylpropanoid pathway products such as flavonoid antibacterial substances, thereby weakening the inhibition of Agrobacterium and allowing for more complete bacterial adhesion to tissue surfaces. These results are consistent with reports in crops such as citrus that "PAL silencing increases susceptibility," suggesting that PAL is not a significant limiting factor in this system.

[0086] SOD and CAT, as key genes regulating redox homeostasis, showed a synchronous but varying degree of decrease across different treatments. During the first four infiltration cycles, both genes maintained low expression levels, leading to mild ROS accumulation and adequate O2. - H2O2 can disrupt membrane microstructure, increase permeability, and simultaneously enhance vir gene activation, thereby promoting T-DNA transfer. However, CAT recovered and rapidly increased during the fifth treatment. Its strong H2O2 scavenging effect weakened membrane permeability, while enhancing SA signal output and promoting PR1 upregulation, thus leading to a significant decrease in overall conversion capacity, forming a "double inhibition effect".

[0087] EXPA2 gene expression exhibits a similar inflection point to PR1. During the 0th to 4th osmosis stages, EXPA2 expression remains low, maintaining cell wall plasticity and relaxation, creating favorable structural conditions for exogenous DNA entry. However, after the 5th osmosis, EXPA2 expression surges, promoting cell wall repair and densification, leading to a sharp decline in transformation efficiency. These results further demonstrate that wheat embryos possess a defined cell wall regulatory threshold under vacuum stimulation; exceeding this threshold triggers a resistance reset mechanism.

[0088] Based on the correlation analysis and statistical verification of the above five types of indicators, 0.025 MPa × 4 osmosis was finally determined as the optimal treatment combination, which can achieve a balance between cell damage, immunosuppression and T-DNA transfer, and realize the highest transient GUS expression and the weakest immune blockade, providing a clear parameter boundary basis for the wheat genetic transformation system.

[0089] This invention provides a method for constructing and optimizing a vacuum osmosis transient transformation system for wheat immature embryos. The method uses immature embryos of the wheat variety Fielder, pollinated for 14 days, as material. By optimizing the vacuum osmosis pressure and the number of treatments, and using GUS histochemical staining, conductivity measurement, and qRT-PCR gene expression analysis, the relationship between transformation efficiency and cell damage degree is evaluated.

[0090] The test material was the spring wheat (Triticum aestivum L.) variety 'Fielder' (provided by the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences). Immature embryos 14 days after pollination were selected as transformation recipients, with a diameter of 2-3 mm. Immediately after embryo removal, they were immersed in sterile distilled water to prevent dehydration damage.

[0091] The strain and vector were: Agrobacterium GV3101 carrying the binary expression vector pCAMBIA1301-GUS (containing a 35S promoter and a GUS-GFP reporter gene). The strain was stored in glycerol tubes at -80°C and inoculated into MG / L solid medium (5 g / L mannitol, 1 g / L L-glutamate, 250 mg / L KH2PO4, 100 mg / L NaCl, 100 mg / L MgSO4·7H2O, 5 g tryptone, 2.5 g yeast extract, and 1 µg biotin added to 700 mL of distilled water, bringing the volume to 1000 mL. The pH was adjusted to 7.0. The medium was sterilized using a 0.22 µm cellulose acetate filter and stored at 4°C, pH 7.0), containing 50 mg / L kanamycin (Kan) and 25 mg / L rifampin (Rif), and incubated in the dark at 28°C for 48 hours.

[0092] The reagents and instruments include:

[0093] (1) Culture medium: co-medium (1 / 10 MS basal salt + 30 g / L glucose + 100 μmol / L acetosyringone + 7 g / L agar, pH 5.8); recovery medium (MS + 2 mg / L 2,4D + 50 mg / L cefotaxime + 250 mg / L + ampicillin + 100 mg / L + termethin + 50 mg / L hygromycin);

[0094] (2) Staining reagents: X-Gluc staining solution (Solarbio, G3060, containing 1 mmol / L X-Gluc, 50 mmol / L sodium phosphate buffer, 0.1% Triton X-100, pH 7.0);

[0095] (3) Main equipment: Vacuum permeameter (SCIENTZ-12F, Ningbo Xinzhi Biotechnology); Conductivity meter (DDS-307A, Shanghai Leici); Real-time PCR instrument (LightCycler 480 II, Roche);

[0096] like Figure 1 As shown, the method for constructing and optimizing the wheat embryo vacuum permeation instantaneous conversion system specifically includes:

[0097] S1: Vacuum osmosis pressure optimization, setting four pressure gradients of 0.025 MPa, 0.05 MPa, 0.075 MPa, and 0.1 MPa, treating 30 embryos per group, repeated 3 times;

[0098] S2: Permeation number setting. Based on pressure optimization results, the optimal pressure is fixed at 0.025MPa. Permeation treatments of 0, 1, 2, 3, 4, and 5 times are set. Other parameters are the same as S1. Each group is treated with 30 embryos and repeated 3 times.

[0099] S3: GUS histochemical staining, staining of plant materials according to Jefferson's method;

[0100] S4: Conductivity measurement;

[0101] S5: qRT-PCR analysis;

[0102] S6: Data analysis, including data standardization, statistical testing, graphing, and correlation analysis.

[0103] The specific operation of S1 is as follows:

[0104] (1) Agrobacterium activation: single colonies were picked and inoculated into MG / L liquid medium (containing 50 mg / L Kan), and cultured at 28℃ and 200 rpm with shaking until OD600=0.6~0.8. The cells were collected by centrifugation at 4000 rpm for 10 min, and the bacterial concentration was adjusted to OD600=0.5 with 1 / 10 MS resuspension (containing 100 μmol / L acetylsyl syringone).

[0105] (2) Vacuum permeation: Immerse the embryos in the bacterial solution and place them in a vacuum permeator. Set the single treatment time to 2 min, release the negative pressure, and treat again after 5 min intervals. The 6 groups are 0, 1, 2, 3, 4 and 5 times respectively.

[0106] (3) Co-culture: After the surface bacterial solution is dried, the embryos are transferred to the co-culture medium and cultured in the dark at 25°C for 3 days.

[0107] The specific operation of S3 is as follows:

[0108] (1) Fixation and staining: The co-cultured embryos were immersed in X-Gluc staining solution and incubated at 37°C in the dark for 24 h;

[0109] (2) Decolorization treatment: Decolorize with 70%, 90% and 100% ethanol in a gradient, 1 h each time, to remove chlorophyll interference;

[0110] (3) Positive rate statistics: The number of blue embryos was observed under a stereomicroscope, and the positive embryo rate was calculated (number of positive embryos / total number of embryos × 100%).

[0111] The specific operation of S4 is as follows:

[0112] (1) Sample preparation: 30 wheat embryos in each group were placed in deionized water (6 groups in total, 3 mL per group) and vacuum permeation was performed.

[0113] (2) Measurement conditions: After vacuum permeation treatment, soak for 12 hours, and use a conductivity meter (DDS-307A) to measure the conductivity of the extract (R1) at 25℃. Then, heat in a boiling water bath for 30 minutes, cool to room temperature, shake well, and measure the conductivity of the extract again (R2). Repeat 3 times, and calculate the relative conductivity = R1 / R2 × ​​100% of the extract conductivity.

[0114] The specific operation of S5 is as follows:

[0115] (1) RNA extraction and reverse transcription: Total RNA was extracted using the TRIzol method (Invitrogen), genomic DNA was removed using DNase I (TaKaRa), and cDNA was synthesized using the PrimeScript RT kit (TaKaRa);

[0116] (2) Primer design: Specific primers were designed for GUS-GFP, EXPA2, SOD, PR1 and the internal reference gene Actin (Table 1), which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0117] (3) Amplification system: SYBR Premix Ex Taq (TaKaRa) 20 μL system: 2×Mix 10 μL, forward and reverse primers 0.4 μL each (10 μmol / L), cDNA 1 μL, ddH2O 8.2 μL;

[0118] (4) Reaction program: 95℃ pre-denaturation for 120 s; 95℃ for 5 s, 60℃ for 15 s, 72℃ for 30 s, 40 cycles; melting curve analysis to confirm amplification specificity;

[0119] (5) Data analysis: The relative gene expression level was calculated using the 2-ΔΔCt method.

[0120] Table 1 qRT-PCR primer sequences

[0121]

[0122] The specific operation of S6 is as follows:

[0123] (1) Data standardization: GUS positivity rate, conductivity and gene expression level data are expressed as mean ± standard deviation;

[0124] (2) Statistical tests: SPSS 18.0 was used for one-way ANOVA and Duncan's method for multiple comparisons (P<0.05).

[0125] (3) Graphics drawing: GraphPad Prism 10.0 draws bar charts and heatmaps;

[0126] (4) Correlation analysis: The correlation between conductivity and GUS positivity rate was assessed by Spearman correlation coefficient.

[0127] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0128] 1. Determine the effect of vacuum pressure on conversion efficiency

[0129] To systematically evaluate the effect of vacuum pressure on the genetic transformation efficiency of wheat immature embryos, this invention set up four gradient treatment groups of 0.025 MPa, 0.05 MPa, 0.075 MPa, and 0.1 MPa, with three vacuum permeations. The vacuum pressure value with the highest transformation efficiency was preliminarily determined by GUS histochemical staining. Figure 2 As shown in Table 2, the GUS positivity rate in the 0.025 MPa treatment group was significantly higher than that in other groups, reaching 49.4%, while the rates in the 0.05 MPa, 0.075 MPa, and 0.1 MPa groups decreased to 37.2%, 23%, and 10.3%, respectively. This indicates that the low vacuum pressure of 0.025 MPa can effectively promote the contact between Agrobacterium and explants and improve T-DNA transfer efficiency. Therefore, 0.025 MPa was determined to be the optimal vacuum pressure condition for genetic transformation of wheat immature embryos.

[0130] Table 2. Wheat embryo conversion rate under different vacuum osmotic pressures

[0131]

[0132] Note:

[0133] 1. Data are expressed as mean ± standard deviation (n=3)

[0134] 2. Different letters indicate significant differences between groups (p<0.05, Tukey test).

[0135] 3. Statistical test results:

[0136] One-way ANOVA: F(3,8)=98.4, p<0.001

[0137] Homogeneity of variance test: Levene's W = 1.18, p = 0.38

[0138] 2. The impact of penetration times on conversion efficiency

[0139] After determining 0.025 MPa as the optimal vacuum pressure, this invention further investigated the effect of the number of percolations on the transformation efficiency. As shown in Table 3, the GUS positivity rate first increased and then decreased with increasing percolation number, reaching a peak of 62.8% in the 4-percolation group, which was higher than the 51% in the 3-percolation group and the 53.2% in the 5-percolation group. Gene expression analysis showed that ( Figure 3 A), the highest GUS gene expression level was observed in the four treatment groups, consistent with the statistical transformation rate of GUS staining. The staining intensity was relatively deep, and the gene expression level was upregulated by 19.6988 times compared to the control group. Figure 3 B) The relative conductivity of the fourth vacuum permeation was significantly higher than that of the third and fifth permeations, indicating that the porosity of the wheat embryo cell membrane was largest and electrolyte leakage was greatest during the fourth permeation treatment, which facilitated the entry of Agrobacterium T-DNA into the embryo cells through the pores. Spearman correlation analysis between relative conductivity (a cell membrane damage indicator) and relative GUS expression level (a transformation efficiency indicator) showed a significant positive correlation between GUS gene expression level and conductivity. Figure 3 C), conductivity can be used as an auxiliary indicator for optimizing the conversion system, but the degree of damage needs to be controlled.

[0140] Table 3. Wheat embryo conversion rate at different vacuum permeation cycles

[0141]

[0142] Note: Data represent the results of three independent replicate experiments. Different letters in the same column indicate significant differences between treatments (one-way ANOVA, Tukey HSD test, p < 0.05). The same letter or no common letter indicates no significant difference between groups (no significant difference between the "cd" group and the "c" or "d" group).

[0143] 3. Effects of the number of infiltrations on relevant wheat stress response genes

[0144] The relative expression level of the wheat stress response-related protein gene PR1 significantly decreased after four osmotic treatments, but significantly increased again after the fifth treatment. Figure 4A) indicates that moderate infiltration can synergistically enhance the expression of exogenous genes and reduce the response of the endogenous resistance PR1 gene. After five infiltrations, the expression levels of catalase CAT and wheat α-expanded protein changed from a decrease to a significant increase. Figure 4 (D, E) This indicates that the transition from 4 to 5 osmosis is precisely a critical point. The relative expression level of the wheat α-expanded protein EXPA2 gene significantly increased at 5 osmosis. Figure 4 E) indicates that the EXPA2 gene, by synthesizing wheat α-expanding protein, enhances the repair rate of damaged cell walls in wheat immature embryos and affects cell membrane permeability, which is consistent with the result of a decrease in relative conductivity after 5 osmosis. Figure 3 (B) While multiple percolations can temporarily increase the amount of Agrobacterium attached, the accompanying mechanical damage increases the rate of cell wall synthesis, enhances plant stress resistance, and thus affects T-DNA insertion. Therefore, optimizing the number of percolations requires finding a balance between transformation efficiency and cell viability. This invention determines four percolations as the optimal treatment condition.

[0145] Figure 4 In samples B, C, and D, the expression data of PAL, SOD, and CAT genes, along with the relative conductivity data of REL and GUS-GFP genes, collectively indicate that increased osmosis cycles ultimately lead to decreased transformation efficiency by disrupting membrane system integrity, interfering with organelle function, and inducing oxidative stress. Appropriate pore opening and maintaining the stability of the cell membrane system are prerequisites for successful genetic transformation.

[0146] This invention, through systematic optimization of vacuum permeation parameters, reveals the core role of low-pressure (0.025 MPa) four-times treatment in the genetic transformation of wheat immature embryos: on the one hand, it ensures explant survival by maintaining cell membrane integrity and organelle function; on the other hand, it enhances T-DNA integration efficiency by activating the SA signaling pathway and PR1 gene expression. This system increased the GUS positivity rate to 62.8%. The research results not only provide key technical support for wheat gene function research and molecular design breeding, but also offer theoretical guidance for optimizing transformation systems in other monocotyledonous crops.

[0147] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A vacuum permeation construction and optimization method for improving the transient conversion efficiency of wheat embryos and reducing tissue damage, characterized in that, Includes the following steps: a. Activate Agrobacterium in a culture medium and prepare an infection solution containing a chemotherapeutic inducer; b. Immerse the wheat embryos obtained from the peeling process completely in the infection solution; c. The embryo is subjected to vacuum permeation treatment to form a window for the entry of exogenous DNA under set osmotic pressure and number of permeation cycles; d. After infiltration, the immature embryos were co-cultured under dark conditions and T-DNA transfer was induced; e. Evaluate the expression of exogenous genes using the colorimetric results; f. Characterize changes in cell membrane permeability by measuring conductivity; g. Determine the response status of immune-related genes and cell wall remodeling genes through gene expression analysis; h. By comprehensively analyzing and regressing data on exogenous gene expression, membrane permeability, and gene expression results, the optimal combination of vacuum pressure and permeation frequency is determined to achieve the target effect of high exogenous gene expression and low cell damage.

2. The method according to claim 1, characterized in that, The vacuum permeation pressure is any selected value from 0.025 MPa to 0.1 MPa.

3. The method according to claim 1, characterized in that, The number of infiltrations is set to any fixed number from 1 to 5, and the infiltration interval is set to 51 minutes.

4. The method according to claim 1, characterized in that, The Agrobacterium is GV3101, and the vector contains a GUS-GFP fusion reporter element driven by the 35S promoter.

5. The method according to claim 1, characterized in that, The colorimetric detection was performed using GUS staining, membrane permeability was detected using conductivity, and gene expression was detected using quantitative reverse transcription PCR.

6. The method according to claim 1, characterized in that, The optimal parameters were determined by the combined effect of enhanced colorimetric activity of exogenous genes, increased conductivity, and decreased expression of immune genes.

7. The method according to claim 1, characterized in that, Decreased PR1 expression was used as an immunosuppressive signal, and low levels of SOD and CAT expression were used as a signal of mild oxidative accumulation to confirm the T-DNA entry window.

8. The method according to claim 1, characterized in that, By determining the stress and frequency range through multi-index linear regression or principal component analysis, the exogenous gene expression value and membrane permeability value can reach their peak values ​​simultaneously.

9. The method according to claim 1, characterized in that, During infiltration, EXPA2 expression is maintained at a stable low level to keep the cell wall relaxed and enhance the ability of Agrobacterium to enter.

10. The method according to claim 1, characterized in that, When a significant increase in PR1 or EXPA2 expression is detected, further infiltration treatment should be stopped to avoid cell wall reinforcement and immune activation that could reduce transformation efficiency.

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