Agrobacterium tumefaciens-mediated andrographis paniculata instantaneous transformation method

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an agrobacterium tumefaciens-mediated andrographis paniculata instantaneous transformation method. The invention provides an agrobacterium tumefaciens-mediated andrographis paniculata transient transformation technology which can realize high-efficiency expression of exogenous genes in andrographis paniculata plants and makes up for the shortages in the field. A general agrobacterium tumefaciens instantaneous transformation technology needs explant shearing, infection after sterile co-culture and continuous culture observation, the operation is complicated, the disinfection requirement is high, meanwhile, gene expression peaks are only maintained for about 5 days, the subsequent expression effect disappears rapidly, and longer-acting gene function observation cannot be met. The technology can effectively solve the problems, strict sterile conditions and tissue culture operation are not needed in the infection stage, follow-up maintenance and observation are also extremely simple and convenient, and the transgenic effect as long as 30 days can be kept only by simply planting the completely infected seedlings in wet nutrient soil.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a method for transient transformation of Andrographis paniculata mediated by Agrobacterium. BACKGROUND

[0002] Plant genetic transformation technology is an important technical means for studying plant biology and promoting plant excellent trait improvement and precision breeding. According to the principle of action, it can be divided into direct genetic transformation technology and indirect genetic transformation technology. The direct genetic transformation technology refers to the transformation technology for directly delivering the target gene into the plant body by using electroporation, gene gun and nano material wrapping, etc. The direct genetic transformation technology often has the shortcomings of high cost, unstable transgenic effect, etc. The indirect genetic transformation technology uses organisms (Agrobacterium, virus, etc.) as carriers for gene delivery. In the current research on the establishment of genetic transformation system for different plant species, the Agrobacterium transformation method is the most commonly used and effective one, which has the advantages of high transformation efficiency and low consumption cost.

[0003] According to whether the transgenic effect can be inherited to the next generation, the genetic transformation technology can be divided into stable transformation technology and transient transformation technology. The stable transformation technology often requires that the plant itself has high in vitro tissue regeneration ability, or needs to use plant tissue culture technology, and has a longer culture time and more complicated maintenance operation, and is subject to the dependence of the genotype of the species on the explant. Therefore, in the plant species for which the stable transformation system has not been established, the application of the transient transformation system also has great research value. Compared with the stable transformation system, the transient transformation system does not need to integrate the exogenous gene into the plant genome, and often has simpler operation, shorter culture time, higher transformation efficiency, and easier observation and verification of the function of the target gene.

[0004] The transient transgenic technology can introduce the exogenous gene into the target cell and perform high-level expression or silencing of the exogenous gene in a short time, and can be used to complete the plant functional genomics research without the regeneration process of the transformed cells, greatly shortening the research period. It is a powerful way to study and explore the function of plant genes without mature regeneration system, and can be effectively applied in the fields of promoter and transcription factor analysis, protein subcellular localization and interaction, etc.

[0005] Agrobacterium-mediated transformation is a method that takes advantage of the fact that Agrobacterium will insert a piece of DNA from its circular Ti plasmid into the genome of the infected plant. By purposefully modifying the Ti plasmid and infecting the corresponding plant material, the effect of transgenic regulation of plant traits or component content can be achieved. However, when using this technique for transient transformation, the plant material often needs to be sterile and cut. The Agrobacterium infection stage requires a clean environment, and the subsequent material culture also needs to be carried out on a sterile medium, with strict requirements for the experimental environment and operation. Some transformation techniques also choose to use a medium containing plant growth hormones for additional dark culture of the material before Agrobacterium infection, in order to promote the differentiation of the explant to a more easily transformed state, further increasing the complexity of the transformation technique.

[0006] Due to the differences between different plant species, each plant adapted to a higher transformation efficiency system technology based on the same Agrobacterium infection characteristics will have great differences, and genotype-dependent problems will also occur between the same plant, which requires screening of Agrobacterium strains, bacterial liquid concentration, the addition of active substances to promote Agrobacterium invasion, and the state of the target plant material during the transgenic process, or assisted by ultrasonic minimally invasive, vacuum, and other operations to promote the infiltration of Agrobacterium bacterial solution, in order to achieve significant effects on specific plant transgenesis and improve transgenic efficiency, which is more conducive to the completion of the regulation of the traits and component synthesis of the target plant. At present, transient transgenic technology has been successfully applied in a variety of medicinal plants, such as lilies (He S, Li H Y, Ma Y, et al. Isolation and culture of lily protoplasts and transient transformation [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2025, 51(01): 67.), hemp seeds (Sorokin A, Yadav N S, Gaudet D, et al. Transient expression of the β-glucuronidase gene in Cannabis sativa varieties [J]. Plant Signal Behav, 2020, 15(8): 1780037.), etc. The application of genetic transformation technology in medicinal plants can play a good role in promoting the research fields of medicinal plant quality improvement, functional gene analysis, and molecular breeding, which can not only be used to regulate the synthesis of secondary metabolites of medicinal plants, improve the content of active ingredients, and enhance stress resistance, but also effectively help to shorten the breeding period and improve the yield of medicinal materials.For example, in *Salvia miltiorrhiza* (Hao X, Pu Z, Cao G, et al. Tanshinone and salvianolic acid biosynthesis are regulated by SmMYB98 in *Salvia miltiorrhiza* hairy roots [J]. Journal of advanced research, 2020, 23:1-12.) and *Artemisia annua* (Fu X, He Y, Li L, et al. Overexpression of blue light receptor AaCRY1 improves artemisinin content in *Artemisia annua* L. [J]. Biotechnology and Applied Biochemistry, 2021, 68(2): 338-344.), the expression levels of genes synthesizing active ingredients can be significantly increased by using an Agrobacterium-mediated transgenic system to regulate the expression of these genes, thereby improving the quality of the medicinal materials.

[0007] Andrographis paniculata Andrographis paniculata (Burm. f.) Nees is an annual or perennial herb belonging to the genus Andrographis in the family Acanthaceae. Its dried aerial parts are an important and commonly used medicinal material in Lingnan (southern China), with andrographolide (AD) as the main medicinal component. AD possesses various pharmacological activities, including anti-inflammatory, antibacterial, antiviral, and antitumor effects. It is not only a major raw material for traditional Chinese medicines such as Qinghuo Zhimai tablets, Xiaoyan Lidan tablets, and Andrographolide tablets, but AD derivatives, which significantly reduce inflammatory responses and alleviate respiratory symptoms, have also been listed as candidate drugs for anti-epidemic treatment. Due to its strict self-pollination characteristics, long-term self-pollination easily leads to a gradual decline in the viability of Andrographis paniculata offspring. Furthermore, there are currently no good Andrographis paniculata breeding bases in China, and significant varietal differences and mixing exist in the germplasm and growth traits of Andrographis paniculata in different regions, resulting in unstable yields and medicinal components. This affects the actual medicinal effects of Andrographis paniculata and is extremely unfavorable for large-scale industrial pharmaceutical production.

[0008] Since the complete genome of A. paniculata was assembled and annotated in 2018 (Sun W, Leng L, Yin Q G, Xu M M, Huang M K, Xu Z C, Zhang Y J, Yao H, Wang C X, Xiong C, Chen S, Jiang C H, Xie N, Zheng X L, Wang Y, Song C, Peter R J, Chen S L. The genome of the medicinal plant Andrographis paniculata genome provides insight into biosynthesis of the bioactive diterpenoid neoandrographolide[J]. Plant Journal, 2019, 97(5): 841-857.), the researches on the molecular level of A. paniculata are increasing. The researches on the resource and genetic diversity of A. paniculata, multi-omics researches and germplasm innovation have good prospects.At present, there are a large number of literatures on cloning and functional research, expression localization of enzyme genes related to AD synthesis pathway [(Srinath M, Shailaja A, Bindu BBV, Giri CC. Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees. Mol Biotechnol. 2021, 63(2): 109-124.), (Qi Mengdie, Wang Jian, Ma Xiaojing, et al. Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata [J]. Chinese Journal of Chinese Medicine, 2018, 43(2): 7.), (Shailaja A, Srinath M, Bindu BVB, Giri CC. Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway, in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees. Physiol Mol Biol Plants. 2021, 27(2): 223-235.), (Zhang Long, Xu Shiqiang, Li Jingyu, et al. Genome-wide identification and expression analysis of TCP gene family in Andrographis paniculata under abiotic stress [J]. Chinese Journal of Chinese Medicine, 2024, 49(02): 379-388.)], but most of them only reach the judgment of tissue expression difference and prokaryotic expression observation, and because the effective Andrographis paniculata transgenic system has not been built, the further exploration of gene function in Andrographis paniculata plants is limited.

[0009] Therefore, under the condition that the genetic transformation system of medicinal plant Andrographis paniculata is not perfect and the gene function research is limited, developing a transient transgenic technology suitable for Andrographis paniculata has very important application value. SUMMARY

[0010] The purpose of the present application is to solve the existing problems, and provide an Agrobacterium-mediated Andrographis paniculata transient transformation method.

[0011] The application is realized by the following technical scheme: The application discloses a method for transient transformation of Andrographis paniculata by Agrobacterium-mediated transformation. S1, culture of A. paniculata sterile seedlings The A. paniculata seeds are inoculated on the surface of the culture medium by using sterile tweezers, and the inoculated culture bottle is placed on a tissue culture rack for culture. S2, Agrobacterium infection S201, 15-day-old A. paniculata seedlings with just germinated first pair of true leaves, good and consistent growth are selected, and after being completely peeled off from the tissue culture bottle, the root system residual culture medium is washed and placed in a beaker; S202, Agrobacterium infection liquid is added to the beaker to completely immerse the material, and the beaker is placed in a vacuum dryer; a syringe is used for multiple times of air suction and pressure reduction treatment, a digital differential pressure gauge is connected in the middle to display that the internal and external air pressure difference is 25-35 kPa, and the low-pressure environment is maintained for 5 min. S203, after the infection is completed, the air pressure valve is carefully opened, air slowly enters the vacuum dryer through the conduit to restore the air pressure, the cover of the dryer is opened, the plant material is taken out, the residual bacterial liquid is absorbed by using a water absorption paper, the plant material is transplanted in the moist nutrient soil, and then placed in a constant temperature and humidity incubator for culture.

[0012] Further, the A. paniculata seeds in step S1 need to be pretreated before inoculation, specifically, the selected seeds are loaded into a 1.5 mL centrifugal tube with a cover, 1 mL of double-distilled water is added for soaking for 16 h, after the liquid is sucked in a super-clean bench, 1 mL of 5% H2O2 is added for soaking for 20 min, the seeds are washed 2-3 times by using double-distilled water, and then 1 mL of double-distilled water is added for standby use.

[0013] Further, the culture condition in step S1 is that the culture is carried out under room temperature for 16 h, 8 h of light and dark conditions.

[0014] Further, the preparation method of the culture medium in step S1 is as follows: the sterile seedling culture medium uses MS basic culture medium, the sucrose concentration is 3%, the plant gel concentration is 0.6%, the pH value is adjusted to 5.8-6.0, after high-pressure sterilization at 115 DEG C for 30 min, Gibberellin A3 (GA3) is added to the concentration of 0.5 mg / L in the super-clean bench and is divided into tissue culture bottles, and after cooling and solidification, the culture medium is standby used.

[0015] Further, the preparation of the Agrobacterium infection liquid in step S201 comprises the following steps: (1) Take a clean, sterile shaking tube, add 5 mL of LB liquid medium and 150 μL of pRI 101-eGFP-GV3101 Agrobacterium tumefaciens in a laminar flow hood, add the antibiotics kanamycin and rifampin to a concentration of 50 mg / L, and shake at 200 r / min for 1 day at 28℃. Take 2 mL of the bacterial culture and add it to a clean conical flask containing 200 mL of LB liquid medium, and shake at 200 rpm for 18 h at 28℃. (2) Centrifuge the bacterial culture at 3000 g for 15 min at room temperature, discard the supernatant, add 1 / 2 MS solution (i.e., resuspension solution) to fully resuspend the bacterial block, so that Agrobacterium is suspended in the liquid, and adjust the OD. 600 A value of 1.0 indicates the presence of Agrobacterium infection solution. (3) Add acetosyringone (AS) to the inoculum to a concentration of 200 μmol / L, and use after standing at room temperature for 3 h.

[0016] Furthermore, during the incubation in the constant temperature and humidity incubator described in step S203, the temperature is controlled at 24°C and the humidity at 70%.

[0017] The present invention has the following advantages over the prior art: 1. This invention provides an Agrobacterium-mediated transient transformation technology for Andrographis paniculata, enabling efficient expression of exogenous genes in Andrographis paniculata plants, thus filling a gap in this field. Conventional Agrobacterium-mediated transient transformation technology requires cutting explants, aseptic co-culturing, infection, and further culture and observation. This process is complex and requires strict sterilization. Furthermore, the peak gene expression usually lasts only about 5 days, after which the expression effect rapidly disappears, failing to meet the requirements for longer-term gene function observation. This technology effectively solves these problems. Not only does it eliminate the need for strict aseptic conditions and tissue culture operations during the infection stage, but subsequent maintenance and observation are also extremely simple. Simply planting the intact infected seedlings in moist nutrient soil can maintain the transgenic effect for up to 30 days.

[0018] 2. This invention is simple to operate and convenient to use, using whole seedlings as conversion materials without the need for material cutting or other work; 3. This invention does not have strict cleanliness requirements during the infection and growth stages, nor does it require aseptic culture. It only requires planting infected seedlings in moist nutrient soil for cultivation and observation. Compared with most transient transgenic technologies, it eliminates the cumbersome tissue culture operation. 4. The transient transgenic technology has a typical transgenic effect that lasts from 3 to 5 days. However, the transient transformation technology of this invention for Andrographis paniculata seedlings has a longer effect that can last for about 1 month. Fluorescence and DNA identification positivity can be observed in the cotyledons and the first pair of new true leaves. This provides a feasible technology for gene function observation that requires a longer follow-up period. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the instantaneous transformation and infection operation of Andrographis paniculata according to the present invention; Figure 2 This is a diagram showing the results of the transient transformation identification of Andrographis paniculata (one month after transplantation) according to the present invention. Figure 3 The image shows the results of re-identification of Andrographis paniculata plants that have undergone transient positive transformation (two months after transplantation). Detailed Implementation

[0020] To further explain the present invention, the following specific embodiments are described.

[0021] 1. Materials The *Andrographis paniculata* seedlings used in this invention were cultivated from seeds collected on October 18, 2009, from Dingtang Village, Dawan Township, Yingde County, Guangdong Province (original data for this batch of seeds: moisture content of 10.3% was measured upon collection in 2009; thousand-seed weight was 1.2145 g based on 13% moisture content; germination rate was 88.2% in 2009 / 2010; seeds were stored at 4-6℃. Germination rate was tested again in 2024 and was 89.7%). 15-day-old aseptic tissue culture seedlings of *Andrographis paniculata* were used as plant material, containing pRI 101- EGFP The plasmid (developed by our laboratory based on pCAMBIA-2300-35S- donated by Professor Ma Yimian of the Chinese Academy of Medical Sciences) EGFP The recombinant plasmid was constructed and preserved using pRI 101-AN purchased from TaKaRa Dalian Takara Bio Co., Ltd. The preparation method of the recombinant plasmid was based on the method in the paper published by the research group of the inventors of this application (Chen Xiuzhen, Liang Lingling, Li Junren, Tang Xiaoting, Zhan Ruoting, He Rui. Construction of plant expression vector carrying LATERAL SUPPRESSOR(LS)-EGFP fusion gene [J]. Molecular Plant Breeding, 2015, 13(12):2843-2848.). That is, Agrobacterium tumefaciens carrying the enhanced green fluorescent protein (eGFP) reporter gene and kanamycin resistance marker GV3101(pSoup) was used as the transformation strain.

[0022] 2. Main Equipment Vacuum dryer (210 mm inner diameter, equipped with a flexible tubing and a 50 mL syringe), digital differential pressure gauge, constant temperature and humidity incubator, high temperature and high pressure sterilizer, handheld fluorescent excitation light source, PCR amplification instrument, electrophoresis and gel imaging instrument, etc.

[0023] Example 1: An Agrobacterium-mediated transient transformation method for Andrographis paniculata, comprising the following steps: S1, Sterile seedling culture of Andrographis paniculata (1) Preparation of culture medium The sterile seedling culture medium uses MS basic medium, the sucrose concentration is 3%, the plant gel concentration is 0.6%, the pH value is adjusted to 5.8-6.0, after high-pressure sterilization at 115°C for 30 min, Gibberellin A3 (GA3) is added to a concentration of 0.5 mg / L in the super-clean bench and is then divided into culture bottles, and is used after cooling and solidification.

[0024] (2) Seeding and culture 50 selected seeds are placed in a 1.5 mL centrifuge tube with a cover, 1 mL of double-distilled water is added for soaking for 16 h, after the liquid is sucked off in the super-clean bench, 1 mL of 5% H2O2 is added for soaking for 20 min, the seeds are washed with double-distilled water for 2-3 times, and then 1 mL of double-distilled water is added for standby.

[0025] The sterile forceps is used to inoculate the Andrographis paniculata seeds on the surface of the culture medium, and the inoculated culture bottle is placed on the culture shelf, and is cultured under the condition of room temperature, 16 h light and 8 h darkness.

[0026] S2, Preparation of Agrobacterium infection solution A clean sterile shake flask is taken, 5 mL of LB liquid medium and 150 μL of pRI 101-eGFP-GV3101 Agrobacterium liquid are added in the super-clean bench, and the concentrations of the antibiotics kanamycin and rifampicin are both 50 mg / L, and the shake culture is carried out at 28°C and 200 r / min for 1 d, 2 mL of bacterial liquid is taken and added into a clean conical flask containing 200 mL of LB liquid medium, and the shake culture is carried out at 28°C and 200 rpm for 18 h.

[0027] The bacterial liquid is centrifuged at a speed of 3000 g for 15 min at room temperature, the supernatant is discarded, 1 / 2 MS liquid (i.e. resuspension liquid) is added to resuspend the bacterial block, so that the Agrobacterium is suspended in the liquid, the OD value is adjusted to 1.0, and the Agrobacterium infection solution is obtained. 600

[0028] Acetosyringone (AS) is added to the infection solution to a concentration of 200 μmol / L, and the infection solution is used after being placed at room temperature for 3 h.

[0029] S3, Agrobacterium infection 15 d Andrographis paniculata seedlings with just germinated first pair of true leaves, good growth and uniformity are selected, are completely peeled off from the culture bottle, and the residual culture medium of the roots is washed, and is placed in a 50 mL beaker.

[0030] ​Into the beaker 30 mL of Agrobacterium infection solution to completely submerge the material, and the beaker is placed in a vacuum desiccator, using a 50 mL syringe for multiple suction to reduce pressure, in between connected digital differential pressure gauge shows the inside and outside air pressure difference is 25~30 kPa, maintain this low pressure environment for 5 min.

[0031] After the infection is completed, the air pressure valve is carefully opened, and air is slowly introduced into the vacuum desiccator through the conduit to restore the air pressure, and then the desiccator cover is opened to remove the plant material, and the residual bacterial solution is absorbed using a water absorption paper, and the plant material is transplanted in moist nutrient soil and placed in a constant temperature and humidity incubator with a temperature of 24°C and a humidity of 70%.

[0032] Example 2: Identification of transient transformation of Andrographis paniculata (one month after transplantation) Using 20 15-day-old Andrographis paniculata seedlings, i.e., cotyledons are unfolded and the first pair of true leaves are in the initial budding state, as the material for infection, the enhanced green fluorescent protein gene eGFP was used as the reporter gene, and after transfection, the culture was transplanted for one month, and a handheld fluorescent excitation light source was used for identification and observation, as shown in Figure 2 As shown, the control group plants showed a whole plant purple red under blue light, and the positive plants of the transgenic group showed obvious fluorescence at the leaf veins under the illumination of this wavelength. The true leaves of the test plants showing fluorescence were cut and the DNA was extracted for identification, and 11 plants had bands consistent with the size of the eGFP fragment in the positive plasmid. Combined with the results of PCR amplification and fluorescence coloration, it can be determined that the eGFP gene was successfully introduced and expressed in the 11 Andrographis paniculata seedlings, proving that the application of this technology successfully introduced the exogenous gene into the Andrographis paniculata plants, and the positive rate reached 55%.

[0033] Figure 2 Medium: a, Andrographis paniculata sterile seedlings were grown for 15 d, and the first pair of true leaves were in the initial budding state. b, Control group and part of the fluorescent positive plants under natural light and fluorescent excitation light source. c, DNA identification of fluorescent positive plants, where M: DL2000 DNA Marker; 1: ddH2O blank control; 2: pRI 101-eGFP positive plasmid; 3~5: no Agrobacterium introduction plants (ck); 6~19: fluorescent plants of the introduction group.

[0034] Example 3: Re-identification of positive plants of Andrographis paniculata transient transformation (two months after transplantation) After two months of culture, the new second pair of true leaves of the positive plants identified in the first identification were cut and the DNA was extracted for identification, as shown in Figure 3 As shown, some of the corresponding bands were visible but the fluorescence was weak, proving that the transient transgenic effect achieved by this technology was mainly expressed on the first pair of true leaves, which could be maintained for one month, and thereafter the transgenic effect of the newly grown leaves would decrease or completely disappear.

[0035] This technology uses 15 d Andrographis paniculata seedlings as plant material, and the bacterial solution concentration (OD600 GV3101 Agrobacterium tumefaciens resuspension solution with a value of 1.0 as the infection solution, and the infection for 5 min can obviously achieve the transient transfection effect in Andrographis plants, and the introduction of phenolic substance AS can improve the infection ability of Agrobacterium, and a syringe and a vacuum dryer are used for air extraction and pressure reduction treatment to achieve the effect of causing a micro-wound invisible to the naked eye on the surface of plant materials to accelerate the invasion of Agrobacterium into the tissue.

[0036] Figure 3 M: DL2000 DNA Marker; 1: pRI 101-eGFP plasmid (positive control); 2: ddH2O (blank control); 3~14: original positive identification plants. The above specific embodiments further specifically describe the purposes, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for Agrobacterium-mediated transient transformation of Andrographis paniculata, characterized by, It comprises the following steps: S1, sterile culture of andrographis seedlings The andrographis seed is inoculated on the surface of the culture medium using sterile forceps, and the inoculated culture bottle is placed on a tissue culture rack for culture; S2, agrobacterium infection S201, 15 d andrographis seedlings with just germinated first pair of true leaves, good and consistent growth are selected, peeled off from the tissue culture bottle, washed to remove residual culture medium, and placed in a beaker; S202, agrobacterium infection liquid is added to the beaker to completely submerge the material, and the beaker is placed in a vacuum dryer, multiple vacuum reduction treatments are performed using a syringe, a digital differential pressure gauge is connected in between to display the internal and external air pressure difference as 25~35 kPa, and the low pressure environment is maintained for 5 min; S203, after the infection is completed, the air pressure valve is carefully opened, air slowly enters the vacuum dryer through the conduit to restore the air pressure, the cover of the dryer is opened to take out the plant material, and the plant material is transplanted in the moist nutrient soil and then placed in a constant temperature and humidity incubator for culture.

2. The Agrobacterium-mediated transient transformation method of Andrographis paniculata according to claim 1, wherein, The andrographis seed in step S1 needs to be pretreated before inoculation, specifically: the selected seed is loaded into a 1.5 mL centrifuge tube with a cover, 1 mL of double distilled water is added for soaking for 16 h, after the liquid is sucked in the clean bench, 1 mL of 5% H2O2 is added for soaking for 20 min, the seed is washed 2~3 times with double distilled water, and 1 mL of double distilled water is added for standby.

3. The Agrobacterium-mediated transient transformation method of Andrographis paniculata according to claim 1, wherein, The culture condition in step S1 is: culture at room temperature for 16 h with light for 8 h in dark condition.

4. The Agrobacterium-mediated transient transformation method of Andrographis paniculata according to claim 1, wherein, The preparation method of the culture medium in step S1 is: the sterile seed culture medium uses MS basic medium, the sucrose concentration is 3%, the plant gel concentration is 0.6%, the pH value is adjusted to 5.8~6.0, after high pressure sterilization at 115℃ for 30 min, gibberellin is added to the concentration of 0.5 mg / L in the clean bench and is divided into tissue culture bottles, and after cooling and solidification, it is ready for use.

5. The Agrobacterium-mediated transient transformation method of Andrographis paniculata according to claim 1, wherein, The preparation of the agrobacterium infection liquid in step S201 comprises the following steps: (1) a clean and sterile shake flask is taken, 5 mL of LB liquid medium and 150 μL of pRI 101-eGFP-GV3101 agrobacterium liquid are added in the clean bench, the concentrations of antibiotics kanamycin and rifampicin are both 50 mg / L, and the liquid is cultured at 28℃ with 200 r / min for 1 d, 2 mL of the liquid is taken and added into a clean conical flask containing 200 mL of LB liquid medium, and the liquid is cultured at 28℃ with 200 rpm for 18 h; (2) centrifuge the bacteria liquid at 3000 g for 15 min at room temperature, discard the supernatant, add 1 / 2 MS liquid to resuspend the bacteria block, adjust the OD value of the bacteria suspension to 1.0 to obtain the bacteria infection liquid; 600 value of the bacteria suspension to 1.0 to obtain the bacteria infection liquid; (3) acetyl eugenol is added to the infection liquid to a concentration of 200 μmol / L, and the liquid is placed at room temperature for 3 h and then used.

6. The Agrobacterium-mediated transient transformation method of Andrographis paniculata according to claim 1, wherein, The temperature is controlled at 24℃ and the humidity is controlled at 70% when the plant material is cultured in the constant temperature and humidity incubator in step S203.

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