An Infection Parameter Optimization Method for Improving VIGS Silencing Efficiency in Pepper Seedlings

Abstract

This invention discloses a method for optimizing infection parameters to improve the VIGS silencing efficiency of pepper seedlings, belonging to the technical field of VIGS silencing efficiency infection parameter optimization. The method includes the following steps: S1, graded screening; S2, pretreatment; S3, preparation of infection solution; S4, infection treatment; S5, moisturizing treatment. Through systematic optimization of the entire process—including seedling age-graded screening, leaf pretreatment, differentiated infection solution preparation, precise vacuum penetration infection, and gradient culture induction—the method achieves precise control throughout the entire process from plant selection and infection implementation to later culture. This completely solves the problems of low silencing efficiency, significant plant damage, and poor experimental reproducibility associated with traditional pepper VIGS infection methods. It can significantly improve the VIGS gene silencing efficiency and success rate of pepper seedlings of different ages, reduce seedling mortality after infection, and ensure stable and uniform silencing phenotypes. This provides a standardized and highly adaptable technical solution for rapid verification of gene function in pepper seedlings, greatly improving the reliability of experimental data and research efficiency.

Description

Technical Field

[0001] This invention relates to the field of VIGS silencing efficiency infection parameter optimization technology, and in particular to an infection parameter optimization method for improving VIGS silencing efficiency in chili seedlings. Background Technology

[0002] In chili pepper gene function research and molecular breeding, virus-induced gene silencing (VIGS) technology is a key means to rapidly verify the function of target genes. However, traditional chili pepper seedling VIGS infection methods generally suffer from problems such as a crude infection process and inconsistent parameter settings. They fail to differentiate between seedlings of different ages, often using uniform Agrobacterium concentrations, vacuum pressure, and infection durations. This easily leads to over-infection and damage to young plants, while older plants are insufficiently infected, directly resulting in low gene silencing efficiency and poor stability. Furthermore, traditional infection methods lack standardized leaf pretreatment procedures, scientific post-infection moisture and temperature control mechanisms, and are not paired with dedicated silencing enhancement methods. Chili pepper seedlings are prone to wound infection, wilting, and death after infection, severely impacting the success rate and data reliability of VIGS experiments.

[0003] The existing VIGS infection system for peppers still has shortcomings such as non-standard Agrobacterium activation, rough control of vacuum permeation conditions, and unreasonable culture environment regulation. The Agrobacterium resuspension is not subjected to precise ice bath and secondary activation, making it difficult to guarantee infection activity. There is no oscillation assistance during vacuum infection, making it difficult for Agrobacterium to uniformly invade leaf tissues. After infection, conventional light culture is carried out directly without considering the environmental requirements for seedling wound repair and silencing induction, and no compound silencing enhancer is used to improve the silencing effect. This results in poor reproducibility between different batches of experiments, which cannot meet the needs of efficient and stable VIGS gene silencing research in pepper seedlings, and restricts the progress of research on functional genes such as disease resistance and stress resistance in peppers. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for optimizing infection parameters to improve the VIGS silencing efficiency of chili seedlings, thus solving the problems mentioned in the background technology.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for optimizing infection parameters to improve the VIGS silencing efficiency of pepper seedlings, comprising the following steps: S1. Grading and screening: Pepper seedlings are graded and screened by age. Healthy and undamaged pepper seedlings at the cotyledon unfolding stage, 1 true leaf stage, and 2 true leaf stage are selected, while diseased, weak, and deformed plants are removed. S2. Pretreatment: Surface pretreatment of the leaves of the selected pepper seedlings; S3. Preparation of Infection Solution: Prepare corresponding OD solutions according to the different seedling ages specified in S1. 600 Agrobacterium resuspension infection solution with added acetylsuccin at a high concentration; S4. Infection treatment: Using a vacuum penetration infection method, according to the seedling age classification in S1, the corresponding vacuum negative pressure and infection time are matched to carry out targeted infection treatment on the leaves of pepper seedlings. S5. Moisturizing treatment: Immediately after infection, the pepper seedlings are kept in a sealed, high-humidity environment for a short period of time, followed by gradient cooling and temperature control culture to complete the repair of the infection wounds. Then, weak light directional induction culture is carried out, while a small amount of silencing enhancer is applied to the leaves.

[0006] Furthermore, the concentration of acetylsuccione in the Agrobacterium resuspension infection solution in S3 is 180-220 g / L. The infection parameters matched for different seedling ages in S4 are specifically: OD at the cotyledon expansion stage. 600 =0.7~0.9, vacuum negative pressure 0.05~0.07MPa, infection duration 25~35s, OD of 1 true leaf stage 600 =0.9~1.1, vacuum negative pressure 0.06~0.08MPa, infection duration 35~45s, OD at 2 true leaf stage 600 =1.1~1.3, vacuum negative pressure 0.07~0.09MPa, infection time 45~55s.

[0007] Furthermore, the S5 process involves a transparent, sealed humidity chamber for instantaneous high-humidity humidification, with relative humidity controlled at 88%~96% and a humidification treatment duration of 1.5~2.5 hours. The gradient cooling temperature control starts at 21~23℃ and decreases by 2℃ every 5~7 hours, successively decreasing to 19~21℃ and 17~19℃, with a total temperature control and repair duration of 22~26 hours.

[0008] Furthermore, in S5, the weak-light directional induction culture uses a cold light source, with the light intensity controlled at 25~55. The photoperiod was 9-11 hours of light / 13-15 hours of darkness, and the induction treatment duration was 44-52 hours. The silencing enhancer was an 8-12 mg / L linolenic acid aqueous solution, which was applied to the leaves by micro-drip method. The amount applied per plant was 45-55 μL, and the dripping location was on both sides of the veins on the underside of the leaf.

[0009] Furthermore, the S2 leaf surface pretreatment involves wiping with 72%~78% ethanol by volume. Sterile cotton swabs are used to wipe along the leaf veins in one direction, with the wiping time controlled at 5~8 seconds per plant. After wiping, the leaves are rinsed with sterile water 2~3 times. After rinsing, the leaves are placed in a sterile operating table to air dry for 1~2 minutes. The temperature inside the operating table is controlled at 20~22℃, and the relative humidity is controlled at 60%~70%. The leaf epidermis is kept intact during the pretreatment process.

[0010] Furthermore, the Agrobacterium resuspension infection solution in S3 was activated and cultured in LB liquid medium at a temperature of 27-29°C, a shaking speed of 110-130 r / min, and a culture time of 16-20 h. After the culture was completed, the bacterial cells were collected by centrifugation at 8000-10000 r / min for 10-15 min, resuspended in MS liquid medium to the set OD600 concentration, and then placed in an ice bath at 2-4°C for 18-22 min, and then transferred to a shaker at 27-29°C for activation for 13-17 min.

[0011] Furthermore, in step S4, the vacuum permeation and contamination are carried out in an adjustable vacuum sealed container. The vacuuming speed is controlled at 0.01~0.02MPa / min and remains constant after reaching the set negative pressure value. During the contamination process, a low-frequency oscillation device is turned on simultaneously with an oscillation frequency of 28~42Hz and an oscillation amplitude of 4~6mm. After the contamination is completed, the gas is slowly released at the same speed.

[0012] Furthermore, in the S5 process, the humidity is maintained by using sterile wet cotton balls to maintain the humidity in a closed high-humidity instantaneous moisturizing environment. The distance between the wet cotton balls and the pepper seedlings is 5-8 cm. During the gradient cooling process, the temperature deviation is controlled within ±0.5℃ and the humidity deviation is controlled within ±2%. After the cooling and repair process is completed, a slow-humidification ventilation treatment is carried out for 30-60 minutes.

[0013] Furthermore, the silencing enhancer is a compound system of linolenic acid and methyl jasmonic acid, with the concentration of methyl jasmonic acid being 4-6%. After compounding, use 180~220W power for ultrasonic treatment for 8~12 minutes. After ultrasonic treatment, let stand for 5~10 minutes, and then apply using a micro sprayer with the nozzle 10~15cm away from the leaf, spraying evenly on both sides of the leaf.

[0014] Furthermore, after the weak light directional induction culture in S5 is completed, a gradient light enhancement culture is performed, with the initial light intensity as the baseline, increasing by 13-17 ppm every 10-14 hours. Until the light intensity reaches 80~100 During gradient enhancement, the original light period remains unchanged.

[0015] (III) Beneficial Effects This invention provides a method for optimizing infection parameters to improve the VIGS silencing efficiency in pepper seedlings, which has the following beneficial effects: Through systematic optimization of the entire process, including seedling age grading and screening, leaf pretreatment, differentiated infection solution preparation, precise vacuum penetration infection, and gradient culture induction, precise control is achieved throughout the entire process from plant selection and infection to post-culture. This completely solves the problems of low silencing efficiency, large plant damage, and poor experimental reproducibility of traditional pepper VIGS infection methods. It can significantly improve the VIGS gene silencing efficiency and success rate of pepper seedlings of different ages, reduce seedling mortality after infection, and ensure stable and uniform silenced phenotypes. It provides a standardized and highly adaptable technical solution for rapid verification of gene function in pepper seedlings, greatly improving the reliability of experimental data and research efficiency.

[0016] This method involves precise age grading of chili seedlings at the cotyledon unfolding stage, one true leaf stage, and two true leaf stage, and removing diseased, weak, and deformed plants. Combined with standardized ethanol leaf surface pretreatment, this ensures the consistency and robustness of the experimental plants from the outset. One-way ethanol wiping, sterile water rinsing, and sterile air drying effectively kill surface bacteria while maintaining leaf epidermal integrity, reducing the probability of microbial contamination during infection. This prevents microbial interference with Agrobacterium infection and gene silencing, creating a clean and safe tissue foundation for efficient Agrobacterium infection. It also ensures uniform experimental conditions among different plants, improving the uniformity and success rate of the VIGS experiment from the initial preparation stage.

[0017] This method involves matching differentiated Agrobacterium OD values ​​to pepper seedlings of different ages. 600 By optimizing the concentration, vacuum negative pressure, and infection duration, and adding an appropriate concentration of acetylsuccinone to the infection solution, as well as optimizing the entire process of Agrobacterium activation, centrifugation, resuspension, and ice bath activation, and combining low-frequency oscillation and uniform gas extraction control during the vacuum infection process, the Agrobacterium infection solution can fully and evenly penetrate the pepper leaf tissue. This avoids tissue necrosis in young seedlings due to excessive concentration or negative pressure, and also prevents incomplete silencing in older seedlings due to insufficient infection intensity. Acetylsuccinone can effectively activate the infection activity of Agrobacterium, and low-frequency oscillation increases the contact area between the infection solution and the leaves, maximizing the infection effect and gene silencing efficiency of Agrobacterium from the core infection stage.

[0018] This method employs a closed, high-humidity instantaneous moisturizing and gradient cooling temperature control to repair wounds after infection, combined with slow-humidity ventilation. This provides the optimal healing environment for chili seedlings infected with wounds. The high-humidity environment prevents leaves from wilting due to water loss, while the gradient cooling reduces the metabolic intensity of the seedlings, minimizing nutrient consumption and infection risk. Precise control of temperature and humidity deviations ensures stable repair conditions, and slow-humidity ventilation avoids secondary stress on seedlings caused by sudden environmental changes. This significantly reduces seedling mortality after infection and ensures that plants enter the induction culture stage only after the wounds are fully healed, providing physiological support for stable expression of subsequent gene silencing. This method solves the technical problems of easy seedling death and poor repair after traditional infection.

[0019] This method employs a combined strategy of low-light directional induction culture, foliar application of a specialized silencing enhancer, and subsequent gradient light enhancement culture. The low-light environment avoids light stress on seedlings, facilitating the induction and transmission of silencing signals. Linolenic acid or a combination of linolenic acid and methyl jasmonate can significantly activate the plant's silencing pathway, further enhancing gene silencing efficiency and depth. Gradual light enhancement allows seedlings to gradually adapt to normal light conditions, maintaining normal plant growth while ensuring the silencing effect. Combined with precise application of the enhancer and control of light parameters, the gene silencing phenotype can appear earlier and last longer, achieving a simultaneous improvement in VIGS silencing efficiency, stability, and plant survival rate during the pepper seedling stage. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the infection parameter optimization method for improving the VIGS silencing efficiency of pepper seedlings according to the present invention. Detailed Implementation

[0021] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1 like Figure 1 As shown, according to one aspect of the present invention, a technical solution is provided: a method for optimizing infection parameters to improve the VIGS silencing efficiency of pepper seedlings, comprising the following steps: S1, screening pepper seedlings by seedling age grading, selecting robust plants at the cotyledon unfolding stage, the one true leaf stage, and the two true leaf stage, and removing diseased, weak, and deformed plants; S2, performing surface pretreatment on the leaves of the screened seedlings; S3, preparing corresponding OD according to different seedling ages. 600 The *Agrobacterium* resuspension infection solution was prepared with a concentration of acetylsuccinyl syringone. S4 employed vacuum permeation infection, with vacuum negative pressure and infection duration matched to seedling age for targeted infection. S5 involved sealed high-humidity instantaneous moisturization, gradient cooling temperature control culture to repair wounds, followed by weak light targeted induction culture and application of a silencing enhancer to the leaves. In practice, healthy pepper seedlings of suitable age were first selected, and the leaves were disinfected and pretreated. Then, an appropriate infection solution was prepared, and targeted infection was completed through vacuum permeation. After infection, moisturizing repair, temperature control culture, and weak light induction were performed sequentially, while an enhancer was applied. Through precise control at different levels throughout the entire process, the success rate of VIGS infection and gene silencing efficiency in pepper seedlings was effectively improved, and plant damage was reduced.

[0023] Specifically, the concentration of acetylsuccione in the Agrobacterium resuspension infection solution in S3 was 180-220 g / L. ; S4 neutron leaf expansion period OD 600 =0.7~0.9, vacuum negative pressure 0.05~0.07MPa, infection duration 25~35s, OD of 1 true leaf stage 600=0.9~1.1, vacuum negative pressure 0.06~0.08MPa, infection duration 35~45s, OD at 2 true leaf stage 600 =1.1~1.3, vacuum negative pressure 0.07~0.09MPa, infection time 45~55s; When using, acetylsyleugenone activates the infection activity of Agrobacterium. Gradient OD concentration, negative pressure and time are set for different seedling ages to adapt to the tolerance of plant tissues and infection needs. Through differentiated parameter matching, it can ensure that Agrobacterium can fully invade while avoiding plant infection damage, and greatly improve the uniformity of silencing of plants of different ages.

[0024] Specifically, the S5 uses a transparent, sealed humidity chamber for instantaneous high-humidity humidification, with a relative humidity of 88%~96% and a humidification time of 1.5~2.5 hours. The gradient cooling starts at 21~23℃ and decreases by 2℃ every 5~7 hours, successively decreasing to 19~21℃ and 17~19℃, with a total temperature control and repair time of 22~26 hours. During use, the high-humidity environment prevents leaves from wilting due to water loss, the gradient cooling reduces the metabolic stress on seedlings, promotes rapid healing of infected wounds, and the precise temperature and humidity control reduces wound infection and plant death, providing a healthy plant foundation for subsequent gene silencing induction.

[0025] Specifically, in S5, the weak-light directional induction culture uses a cold light source with a light intensity of 25~55. The photoperiod is 9-11 hours of light / 13-15 hours of darkness, with an induction time of 44-52 hours. The silencing enhancer is an 8-12 mg / L linolenic acid aqueous solution, applied by micro-drip method, 45-55 μL per plant, dripped onto both sides of the veins on the underside of the leaf. When using it, a low-light environment reduces seedling stress and facilitates the transduction of silencing signals. The linolenic acid enhancer can activate the plant's silencing pathway. Precise drip application ensures efficient absorption of the agent, significantly improving the depth and efficiency of gene silencing, without affecting the normal growth of the plant.

[0026] Specifically, the S2 leaf surface pretreatment involves unidirectional wiping with 72%~78% ethanol (volume fraction) for 5~8 seconds per leaf, followed by rinsing with sterile water 2~3 times, and air drying at 20~22℃ and 60%~70% humidity on a sterile operating table for 1~2 minutes to maintain the integrity of the leaf epidermis. During use, ethanol wiping kills bacteria on the leaf surface, unidirectional operation avoids epidermal damage, and sterile rinsing and air drying eliminate ethanol residue and bacterial contamination, providing a clean and safe leaf environment for Agrobacterium infection and reducing experimental failures caused by contamination.

[0027] Specifically, the Agrobacterium tumefaciens resuspension infection solution in S3 was activated in LB liquid medium at 27-29℃ and 110-130 rpm for 16-20 h, and the bacterial cells were collected by centrifugation at 8000-10000 rpm for 10-15 min, and then resuspended in MS medium to the set OD value. 600Activate in an ice bath at 2-4℃ for 18-22 minutes, and in a shaker at 27-29℃ for 13-17 minutes. When using it, standardized activation culture ensures the activity of Agrobacterium, centrifugation collection and ice bath activation enhance the infectivity, and precise parameter control keeps Agrobacterium in the best infectivity state, thereby improving the leaf infection rate and gene silencing effect.

[0028] Specifically, in S4, vacuum penetration infection is carried out in an adjustable vacuum-sealed container with a vacuuming speed of 0.01~0.02MPa / min and a constant negative pressure. Simultaneously, a low-frequency oscillation of 28~42Hz and 4~6mm is activated, and gas is slowly released at the same speed after infection. During use, uniform gas extraction and release avoid sudden damage to plant tissues. The low-frequency oscillation allows the infection solution to be evenly distributed on the leaf surface, improving the Agrobacterium invasion efficiency, making the infection more thorough and uniform, and further improving the VIGS silencing efficiency.

[0029] Specifically, the S5 system uses sterile wet cotton balls to maintain humidity in a closed, high-humidity instantaneous moisturizing environment. The distance between the wet cotton balls and the seedlings is 5-8 cm. The temperature and humidity deviations during gradient cooling are ±0.5℃ and ±2%, respectively. Slow-humidification ventilation is performed 30-60 minutes after repair. During use, the sterile wet cotton balls stably maintain humidity, and the precise control of temperature and humidity deviations ensures a stable repair environment. Slow-humidification ventilation avoids the stress of sudden environmental changes, effectively improving the wound healing rate and reducing the mortality rate of seedlings after infection.

[0030] Specifically, the silencing enhancer is a compound system of linolenic acid and methyl jasmonic acid, with a methyl jasmonic acid concentration of 4-6%. Ultrasound treatment at 180-220W for 8-12 minutes, followed by standing for 5-10 minutes, and then spraying evenly on both sides of the leaves from 10-15cm using a micro-sprayer. When using this product, the compound enhancer synergistically activates the silencing pathway, ultrasonic treatment improves the uniformity of the agent, and spraying ensures full absorption by the leaves. Compared with a single enhancer, this product further significantly improves gene silencing efficiency and persistence.

[0031] Specifically, after the weak light directional induction culture in S5 was completed, the light was increased by gradient, with the light intensity increased by 13-17 every 10-14 hours. Until 80~100 Maintaining the original photoperiod; during use, gradient light enhancement allows seedlings to gradually adapt to light intensity, ensuring normal photosynthesis while maintaining gene silencing effect, promoting healthy seedling growth, and achieving a dual guarantee of silencing effect and plant growth.

[0032] Example 2 This embodiment employs the infection parameter optimization method described in this application for improving VIGS silencing efficiency in pepper seedlings. The specific steps are as follows: all parameters are selected from the median values ​​within the defined weight ranges to ensure operational standardization and repeatability: S1. Seedling Age Grading and Screening: Select robust, disease-free, and deformed chili seedlings, and precisely select plants at three stages: cotyledon unfolding stage, 1 true leaf stage, and 2 true leaf stage. Select 50 plants from each stage and cultivate them under a uniform environment (temperature 25℃, relative humidity 70%, light intensity 60 μmol·m⁻²). -2 ・s -1 ),spare.

[0033] S2. Leaf surface pretreatment: Use 75% ethanol (volume fraction) and wipe along the veins of the pepper seedling leaves in one direction with a sterile cotton swab. Wipe each seedling for 6 seconds. After wiping, rinse twice with sterile water. Then place the seedlings in a sterile operating table to air dry for 1.5 minutes. The temperature in the operating table is controlled at 21℃ and the relative humidity is controlled at 65%. Keep the leaf epidermis intact throughout the process and avoid damage.

[0034] S3, Preparation of Agrobacterium resuspension infection solution: Prepare the corresponding OD according to the three seedling age grades in S1. 600 Agrobacterium tumefaciens was resuspended in the infection solution at a concentration that was high, and 200 μmol / L acetylsyl syringone was added to all infection solutions; among them, the OD at the cotyledon expansion stage... 600 =0.8, OD at the 1 true leaf stage 600 =1.0, OD at the 2 true leaf stage 600 =1.2.

[0035] Agrobacterium activation and resuspension procedure: Agrobacterium was activated and cultured on LB liquid medium at 28℃ with a shaking speed of 120 r / min for 18 h. After culture, the bacterial cells were collected by centrifugation at 9000 r / min for 12 min and resuspended on MS liquid medium to the corresponding OD. 600 Concentration; after resuspension, place the inoculum in an ice bath at 3°C ​​for 20 min, then transfer it to a shaker at 28°C for 15 min for activation, and set aside.

[0036] S4. Vacuum Infiltration Infection: Vacuum infiltration infection is performed using an adjustable vacuum sealed container. The vacuuming speed is controlled at 0.015 MPa / min, and the set negative pressure value is maintained at a constant value. At the same time, a low-frequency oscillation device is turned on with an oscillation frequency of 35 Hz and an oscillation amplitude of 5 mm. The corresponding infection parameters are matched according to the seedling age: 0.06 MPa vacuum negative pressure and 30 s infection time at the cotyledon unfolding stage; 0.07 MPa vacuum negative pressure and 40 s infection time at the 1 true leaf stage; and 0.08 MPa vacuum negative pressure and 50 s infection time at the 2 true leaf stage. After the infection is completed, the air is slowly released at the same rate to complete the infection treatment.

[0037] S5. Post-infection culture and induction: After infection, pepper seedlings were immediately placed in a transparent, sealed humidity chamber for short-term high-humidity humidification. Sterile wet cotton balls were used to maintain humidity, with a distance of 6.5 cm between the wet cotton balls and the pepper seedlings. The relative humidity was controlled at 92%, and the humidification treatment lasted for 2 hours. Subsequently, a gradient cooling temperature control culture was carried out, with an initial temperature of 22℃. The temperature was reduced by 2℃ every 6 hours, successively to 20℃ and 18℃, with a total temperature control and repair time of 24 hours. During the gradient cooling process, the temperature deviation was controlled within ±0.5℃, and the humidity deviation was controlled within ±2%. After the repair was completed, a slow-humidification ventilation treatment was carried out for 45 minutes.

[0038] After slow humidification and ventilation, weak light directional induction culture was carried out: a cold light source was used, and the light intensity was controlled at 40 μmol·m⁻². -2 ・s -1 The photoperiod was 10h light / 14h dark, and the induction treatment lasted for 48h. During the induction culture, a silencing enhancer was applied to the leaves using a micro-sprayer. The silencing enhancer was a compound system of linolenic acid and methyl jasmonic acid, with a linolenic acid concentration of 10mg / L and a methyl jasmonic acid concentration of 5μmol / L. The compound enhancer was ultrasonically treated with 200W power for 10min, and then allowed to stand for 8min after ultrasonic treatment. The distance between the nozzle and the leaf was 12cm, and the mixture was sprayed evenly on both sides of the leaf.

[0039] After the induction culture under weak light was completed, a gradient light enhancement culture was performed: with 40 μmol·m -2 ・s -1 The initial light intensity was increased by 15 μmol·m⁻² every 12 hours. -2 ・s -1 Until the light intensity reaches 90 μmol·m -2 ・s -1 During the gradient illumination period, the photoperiod of 10h light / 14h darkness was kept constant until a stable silent phenotype was observed.

[0040] II. Comparative Example (Traditional Infection Method, No Optimization) This comparative example uses the traditional VIGS infection method for chili seedlings without any parameter optimization. All other experimental conditions (chili variety, culture environment, Agrobacterium strain, target silenced gene) are completely consistent with the above. The specific steps are as follows: S1. Seedling selection: Only healthy chili seedlings are selected, without age grading. 150 seedlings are randomly selected, without removing diseased, weak, or deformed plants (only obviously withered plants are removed).

[0041] S2. Leaf pretreatment: The leaf surface is not wiped with ethanol, rinsed with sterile water and air-dried. The infection operation is carried out directly.

[0042] S3. Preparation of Agrobacterium infection solution: OD is prepared uniformly regardless of seedling age.600 Agrobacterium resuspension with a concentration of 1.0, without the addition of acetylsuccinyl syringone; Agrobacterium was activated in LB liquid medium at 28°C and 120 r / min for 18 h, collected by centrifugation, and directly resuspended in MS medium without ice bath or secondary activation treatment.

[0043] S4. Vacuum penetration infection: Regardless of seedling age, a uniform vacuum negative pressure of 0.07MPa and an infection time of 40s are used. The vacuuming speed and the venting speed are not controlled, and the low-frequency oscillation device is not turned on. The rest of the operation is the same as in Example 1.

[0044] S5. Post-infection culture and induction: After infection, without closed high-humidity instantaneous moisturization and gradient cooling temperature control repair, the culture was directly placed in a standard culture environment (temperature 25℃, relative humidity 70%, light intensity 60μmol·m). -2 ・s -1 Cultured under conventional light without weak light directional induction, without applying any silencing enhancers, and without gradient light enhancement, until the silencing phenotype is observed.

[0045] III. Performance Test and Comparison Results Under the same experimental conditions, the infection effects of the examples and comparative examples were tested uniformly. Three replicates were selected for each treatment group, with 50 plants in each replicate. The test indicators included VIGS silencing efficiency (the proportion of plants with the target gene silenced), seedling mortality rate after infection, duration of silencing phenotype, and experimental repeatability (coefficient of variation of silencing efficiency in the three replicate groups). The test results are shown in the table below:

[0046] IV. Comparative Conclusions Test results show that the infection parameter optimization method for improving VIGS silencing efficiency in pepper seedlings described in this application, through optimization of the entire process including seedling age grading screening, standardized leaf pretreatment, differentiated infection parameter matching, scientific Agrobacterium activation and post-infection culture induction, can significantly improve VIGS silencing efficiency in pepper seedlings compared to traditional infection methods, greatly reduce seedling mortality after infection, prolong the duration of silencing phenotype, and improve experimental repeatability. It effectively solves the problems of low silencing efficiency, large plant damage, and poor experimental stability of traditional methods, providing efficient and stable technical support for gene function verification in pepper seedlings.

[0047] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for optimizing infection parameters to improve VIGS silencing efficiency in chili seedlings, characterized in that, Includes the following steps: S1. Grading and screening: Pepper seedlings are graded and screened by age. Healthy and undamaged pepper seedlings at the cotyledon unfolding stage, 1 true leaf stage, and 2 true leaf stage are selected, while diseased, weak, and deformed plants are removed. S2. Pretreatment: Surface pretreatment of the leaves of the selected pepper seedlings; S3. Preparation of Infection Solution: Prepare corresponding OD solutions according to the different seedling ages specified in S1. 600 Agrobacterium resuspension infection solution with added acetylsuccin at a high concentration; S4. Infection treatment: Using a vacuum penetration infection method, according to the seedling age classification in S1, the corresponding vacuum negative pressure and infection time are matched to carry out targeted infection treatment on the leaves of pepper seedlings. S5. Moisturizing treatment: Immediately after infection, the pepper seedlings are kept in a sealed, high-humidity environment for a short period of time, followed by gradient cooling and temperature control culture to complete the repair of the infection wounds. Then, weak light directional induction culture is carried out, while a small amount of silencing enhancer is applied to the leaves.

2. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: The concentration of acetosyringone in the Agrobacterium resuspension infection solution in S3 was 180-220. The infection parameters matched for different seedling ages in S4 are specifically: OD at the cotyledon expansion stage. 600 =0.7~0.9, vacuum negative pressure 0.05~0.07MPa, infection duration 25~35s, OD of 1 true leaf stage 600 =0.9~1.1, vacuum negative pressure 0.06~0.08MPa, infection duration 35~45s, OD at 2 true leaf stage 600 =1.1~1.3, vacuum negative pressure 0.07~0.09MPa, infection time 45~55s.

3. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: The S5 uses a transparent sealed humidity chamber for instantaneous high-humidity humidification, with relative humidity controlled at 88%~96% and humidification treatment time of 1.5~2.5h. The gradient cooling temperature control starts at 21~23℃ and decreases by 2℃ every 5~7h, successively decreasing to 19~21℃ and 17~19℃, with a total temperature control and repair time of 22~26h.

4. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: In S5, the weak-light directional induction culture uses a cold light source, with the light intensity controlled at 25~55. The photoperiod was 9-11 hours of light / 13-15 hours of darkness, and the induction treatment duration was 44-52 hours. The silencing enhancer was an 8-12 mg / L linolenic acid aqueous solution, which was applied to the leaves by micro-drip method. The amount applied per plant was 45-55 μL, and the dripping location was on both sides of the veins on the underside of the leaf.

5. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: The S2 leaf surface pretreatment involves wiping with 72%~78% ethanol by volume. Sterile cotton swabs are used to wipe along the leaf veins in one direction, with the wiping time controlled at 5~8 seconds per plant. After wiping, the leaves are rinsed with sterile water 2~3 times. After rinsing, the leaves are placed in a sterile operating table to air dry for 1~2 minutes. The temperature inside the operating table is controlled at 20~22℃, and the relative humidity is controlled at 60%~70%. The leaf epidermis is kept intact during the pretreatment process.

6. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: The Agrobacterium resuspension infection solution in S3 was activated and cultured in LB liquid medium at a temperature of 27-29℃, a shaking speed of 110-130 r / min, and a culture time of 16-20 h. After the culture was completed, the bacterial cells were collected by centrifugation at 8000-10000 r / min for 10-15 min, and resuspended in MS liquid medium to the set OD600 concentration. After resuspension, the cells were placed in an ice bath at 2-4℃ for 18-22 min, and then transferred to a shaker at 27-29℃ for activation for 13-17 min.

7. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: In step S4, vacuum permeation and contamination are carried out in an adjustable vacuum sealed container. The vacuuming speed is controlled at 0.01~0.02MPa / min and is kept constant after reaching the set negative pressure value. During the contamination process, a low-frequency oscillation device is turned on simultaneously with an oscillation frequency of 28~42Hz and an oscillation amplitude of 4~6mm. After the contamination is completed, the gas is slowly released at the same speed.

8. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: In the S5, the humidity is maintained by using sterile wet cotton balls for the closed high-humidity instantaneous moisturization. The distance between the wet cotton balls and the pepper seedlings is 5-8cm. During the gradient cooling process, the temperature deviation is controlled within ±0.5℃ and the humidity deviation is controlled within ±2%. After the cooling and repair are completed, a slow-humidification ventilation treatment is carried out for 30-60 minutes.

9. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 4, characterized in that: The silencing enhancer is a compound system of linolenic acid and methyl jasmonic acid, with a methyl jasmonic acid concentration of 4-6%. After compounding, use 180~220W power for ultrasonic treatment for 8~12 minutes. After ultrasonic treatment, let stand for 5~10 minutes, and then apply using a micro sprayer with the nozzle 10~15cm away from the leaf, spraying evenly on both sides of the leaf.

10. The method for optimizing infection parameters to improve VIGS silencing efficiency in pepper seedlings according to claim 1, characterized in that: After the weak light directional induction culture in step S5 is completed, a gradient light enhancement culture is performed, with the initial light intensity used as a baseline, and the intensity increased by 13-17 ppm every 10-14 hours. Until the light intensity reaches 80~100 During gradient enhancement, the original light period remains unchanged.

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