Method for introducing foreign gene into gynostemma pentaphyllum

Abstract

This invention discloses a method for introducing exogenous genes into Gynostemma pentaphyllum, comprising: S1: Selecting Gynostemma pentaphyllum seeds with seed coats within 3 months of harvest, disinfecting, and culturing to obtain aseptic seedlings from seedlings, or selecting stem segments with axillary buds after leaf removal, disinfecting, and culturing using a subculture proliferation medium containing cephalosporin to obtain aseptic rooted seedlings; S2: Selecting the aseptic seedlings or aseptic cuttings obtained in S1, culturing in a rooting medium containing cephalosporin to obtain aseptic rooted seedlings; S3: Transforming plasmid Pcambia1301-PMI into Agrobacterium and culturing in a medium containing kanamycin and rifampin to obtain an Agrobacterium resuspension; S4: Infecting the aseptic rooted seedlings obtained in S2 with the Agrobacterium resuspension, and performing co-culture, antibacterial culture, and screening culture to obtain transgenic adventitious shoots. This invention can successfully introduce exogenous genes into Gynostemma pentaphyllum, obtaining Gynostemma pentaphyllum positive plants.

Description

Technical Field

[0001] This invention relates to the field of plant transgenic technology. More specifically, this invention relates to a method for introducing exogenous genes into Gynostemma pentaphyllum. Background Technology

[0002] Gynostemma pentaphyllum (Thunb.) Makino, a perennial herbaceous climbing vine belonging to the genus Gynostemma in the family Cucurbitaceae, is one of the few plant sources, besides those in the Araliaceae family, that possess ginsenoside structures. It has various functions, including nourishing and health-promoting effects, anti-cancer and anti-aging properties, strengthening the body, and improving lipid metabolism, earning it the nicknames "Southern Ginseng" and "Longevity Herb." Ginseng is a widely used herb with effects such as anti-fatigue, delaying aging, regulating the central nervous system, improving immunity, improving blood supply to the heart and brain, and inhibiting tumor cell growth. Most of its active ingredients come from dammarane-type tetracyclic triterpenes. Plants capable of producing dammarane-type saponins are mainly concentrated in the genus Panax, such as ginseng, Panax notoginseng, and American ginseng; however, their production cycle is generally 3-5 years, with a crop rotation cycle of at least 10 years. Obtaining large quantities of dammarane-type saponins and their secondary glycosides and aglycones has become a key focus of modern pharmaceutical research. Gynostemma pentaphyllum itself possesses a complete dammarane-type saponin synthesis pathway, with all Gynostemma pentaphyllum saponins having aglycone fraction of dammarane-type tetracyclic triterpenoids. Furthermore, Gynostemma pentaphyllum is easy to grow and does not exhibit the continuous cropping obstacles commonly found in ginseng family plants, making it a promising candidate for use as a bioreactor for dammarane-type saponin synthesis. Establishing an efficient genetic transformation system is a necessary step in constructing this bioreactor.

[0003] However, introducing exogenous genes into Gynostemma pentaphyllum is quite difficult, and existing technologies have repeatedly reported failures. For example, the master's thesis from Guangxi Medical University, "Preliminary Study on Cloning and Transformation of Panax notoginseng SE Gene into Tobacco and Gynostemma pentaphyllum," failed to achieve Gynostemma pentaphyllum gene transformation despite trying various bacterial soaking times and antibiotic concentrations. After re-testing the literature, the inventors found that transformation was indeed impossible. Therefore, after conducting numerous experiments, the inventors shifted their research focus to the seed harvesting stage and explant selection, discovering that seed germination parameters, explant induction parameters, rooting parameters, cephalosporin concentration, and mannose concentration all had crucial influences on the success of introduction. After adjustments, the introduction of exogenous genes was finally achieved. Summary of the Invention

[0004] One objective of this invention is to provide a method for introducing exogenous genes into Gynostemma pentaphyllum, which can successfully introduce exogenous genes into Gynostemma pentaphyllum and obtain Gynostemma pentaphyllum positive plants.

[0005] To achieve these objectives and other advantages of the present invention, the present invention provides a method for introducing exogenous genes into Gynostemma pentaphyllum, comprising: S1: selecting Gynostemma pentaphyllum seeds with seed coat within 3 months of harvest, disinfecting, and culturing to obtain seed-grown sterile tissue culture seedlings, or selecting stem segments with axillary buds after leaf removal, disinfecting, and culturing using a subculture proliferation medium to obtain cutting sterile tissue culture seedlings, wherein the subculture proliferation medium includes cephalosporin; S2: selecting the seed-grown sterile tissue culture seedlings or cutting sterile tissue culture seedlings obtained in S1, culturing in a rooting medium to obtain sterile rooted seedlings, wherein the rooting medium includes cephalosporin; S3: transferring plasmid Pcambia1301-PMI into Agrobacterium, and culturing in a medium containing kanamycin and rifampin to obtain Agrobacterium resuspension; S4: using the Agrobacterium resuspension to infect the sterile rooted seedlings obtained in S2, and performing co-culture, antibacterial culture, and screening culture to obtain transgenic adventitious shoots.

[0006] Furthermore, in S1, the method for disinfecting seeds or stem segments with axillary buds after leaf removal within 3 months of harvest includes: soaking in 75% alcohol and 0.1% mercuric chloride aqueous solution in sequence.

[0007] Further, in S1, the sterilized seeds are cultured using an induction medium to obtain sterile tissue culture seedlings. The induction medium includes MS medium, sucrose, and agar, with each liter of MS medium containing 28g of sucrose and 5.5g of agar. The sterilized seeds are then cultured in the dark for 7 days and 24 hours, followed by a light culture:dark culture ratio of 12h:12h, with a light intensity of 2000-2500 Lux.

[0008] Further, in S1, the subculture proliferation medium includes MS medium, sucrose, agar, 6-benzyladenine, α-naphthaleneacetic acid and cephalosporin, with each liter of MS medium containing 28g of sucrose, 5.5g of agar, 1.5mg of 6-benzyladenine, 0.02mg of α-naphthaleneacetic acid and 30-200mg of cephalosporin.

[0009] Furthermore, in S2, the rooting medium comprises 1 / 2 MS medium, sucrose, agar, indoleacetic acid, and cephalosporin, wherein each liter of 1 / 2 MS medium contains 28 g of sucrose, 5.5 g of agar, 0.2 mg of indoleacetic acid, and 30–200 mg of cephalosporin.

[0010] Further, in S3, Agrobacterium containing pCAMBIA1301-PMI was grown on YEP solid medium containing 50 mg / L kanamycin and 100 mg / L rifampin for 28–48 h. Single colonies were picked and placed on YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampin for shaking culture until the OD value reached 0.8–1.0. The colonies were then removed, centrifuged, and the cells were collected. The cells were then shaken and cultured until the OD value reached 0.4–0.6 to obtain Agrobacterium resuspension.

[0011] Furthermore, the co-culture medium includes MS medium, sucrose, agar, 6-benzyladenine, and α-naphthaleneacetic acid. Each liter of MS medium contains 28g of sucrose, 5.5g of agar, 1.5mg of 6-benzyladenine, and 0.02mg of α-naphthaleneacetic acid. The medium is co-cultured in the dark for 2–4 days.

[0012] Furthermore, the antibacterial culture medium includes MS medium, sucrose, agar, 6-benzyladenine, α-naphthylacetic acid, and cephalosporin. Each liter of MS medium contains 28g of sucrose, 5.5g of agar, 1.5mg of 6-benzyladenine, 0.02mg of α-naphthylacetic acid, and 30-200mg of cephalosporin. The medium is cultured under light for 5 days to inhibit bacterial growth.

[0013] Furthermore, the screening medium includes MS medium, sucrose, agar, 6-benzyladenine, α-naphthylacetic acid and cephalosporin. Each liter of MS medium contains 28g of sucrose, 5.5g of agar, 1.5mg of 6-benzyladenine, 0.02mg of α-naphthylacetic acid, and 30-200mg of cephalosporin. The ratio of mannose to sucrose is (3-4):(24-25).

[0014] The present invention has at least the following beneficial effects:

[0015] 1) Starting from the seed harvesting and explant selection of Gynostemma pentaphyllum, this invention adjusts the seed germination parameters, explant induction parameters, rooting parameters, cephalosporin concentration, and mannose concentration. It can not only successfully induce aseptic tissue culture seedlings of Gynostemma pentaphyllum using seeds with skin as explants, but also successfully induce aseptic tissue culture seedlings using stem segments with buds as explants. It also explores suitable bud proliferation culture medium and rooting culture medium, and successfully establishes two sets of rapid tissue culture propagation technology systems for Gynostemma pentaphyllum, laying the necessary technical foundation for the establishment of the genetic transformation system of Gynostemma pentaphyllum.

[0016] 2) This invention utilizes stem segments with axillary buds as transgenic recipient materials, infecting them with Agrobacterium tumefaciens containing expression vectors, and employing a 6-phosphate mannose isomerase positive selection marker system. The culture period is short; transformation efficiency can be detected 30-45 days after infection. A total of 288 stem segments with axillary buds participated in the infection experiment, ultimately yielding 85 plant samples for DNA testing. Of these, 59 were positive, resulting in a DNA positive rate of 69.41% and an estimated transformation rate of 20.49%. Of the 58 samples tested for chlorophenol red, 31 were positive, resulting in a chlorophenol red positive rate of 53.45% and an estimated transformation rate of 10.76%. Of the 35 samples tested for RT-PCR, 21 were positive, resulting in an RT-PCR positive rate of 60% and an estimated transformation rate of 7.29%. The results show that the transformation efficiency is relatively stable. This invention effectively overcomes the shortcomings of genetic transformation systems using antibiotics, herbicides, etc., as negative selection markers, which suffer from low transformation efficiency.

[0017] 3) This invention successfully introduced exogenous genes into Gynostemma pentaphyllum and transformed it into Gynostemma pentaphyllum positive plants, providing a certain technical reference for the field of genetic transformation research of Gynostemma pentaphyllum.

[0018] 4) During the subculture and rooting culture stages, a large number of endophytic bacteria appeared, which seriously affected the maintenance of sterile tissue culture seedlings. This invention successfully solved this problem by adding an appropriate concentration of cephalosporin to the subculture and rooting culture media, thus providing the necessary guarantee for the source of recipient materials in the subsequent genetic transformation experiments.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0020] Figure 1 The overall growth effect of tissue culture seedlings of Gynostemma pentaphyllum with peel 3 months after sowing in Experiment 1;

[0021] Figure 2 The effect of tissue culture seedling root growth on Gynostemma pentaphyllum seeds with bark 3 months after sowing in Experiment 1;

[0022] Figure 3 The image shows the effect of Gynostemma pentaphyllum stem segment explants with buds in Experiment 2 after 30 days of culture under sterilization conditions of 75% alcohol (30s) + 0.1% mercuric chloride (5min);

[0023] Figure 4 The image shows the effect of Gynostemma pentaphyllum stem segment explants with buds in Experiment 2 after 60 days of culture under sterilization conditions of 75% alcohol (30s) + 0.1% mercuric chloride (5min);

[0024] Figure 5The induction effect of pure long-stalked parts of Gynostemma pentaphyllum in Experiment 2 on MS medium culture for 30 days was measured.

[0025] Figure 6 The effect of induction of the long petiole part of Gynostemma pentaphyllum compound leaves in Experiment 2 on MS medium culture for 30 days with sucrose 28 g / L + agar 5.5 g / L + 6-BA 1.5 mg / L + NAA 0.02 mg / L;

[0026] Figure 7 The induction effect of the short-stalked single leaf part of Gynostemma pentaphyllum in Experiment 2 on MS + sucrose 28g / L + agar 5.5g / L + 6-BA 1.5mg / L + NAA 0.02mg / L for 30 days;

[0027] Figure 8 The effect of sessile single leaflets of Gynostemma pentaphyllum on induction in Experiment 2 when cultured in MS medium containing 28 g / L sucrose, 5.5 g / L agar, 1.5 mg / L 6-BA, and 0.02 mg / L NAA for 30 days.

[0028] Figure 9 The induction effect of Gynostemma pentaphyllum buds and stem segments in Experiment 2 on MS medium culture for 30 days was measured.

[0029] Figure 10 The overall plant effect of Experiment 3 (culture medium: 1 / 2 MS + 0.2 NAA mg / L);

[0030] Figure 11 The effect of the experiment on the roots of 3 plants (culture medium: 1 / 2 MS + 0.2 NAA mg / L);

[0031] Figure 12 The overall plant effect of Experiment 3 (culture medium: 1 / 2 MS + 0.2 IAA mg / L);

[0032] Figure 13 The effect of the experiment on the roots of 3 plants (culture medium: 1 / 2 MS + 0.2 IAA mg / L);

[0033] Figure 14 The overall plant effect of Experiment 3 (culture medium: 1 / 2 MS + 0.2 IBA mg / L);

[0034] Figure 15 The effect of the experiment on the roots of 3 plants (culture medium: 1 / 2 MS + 0.2 IBA mg / L). Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0036] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0037] Example 1:

[0038] 1) Obtaining aseptic tissue culture seedlings from seeds:

[0039] Fresh, mature fruits harvested from the end of December of the previous year to the beginning of January of the current year were selected. Seeds were stored at room temperature in a well-ventilated, cool place within three months of the harvest date and underwent pretreatment: removing pulp and impurities, selecting clean, plump fresh seeds with seed coats, rinsing with running tap water at room temperature for 12 hours, draining the surface moisture, and then performing surface sterilization: soaking in 75% alcohol for 30 seconds, rinsing with sterile water 3-4 times, soaking in 0.1% mercuric chloride for 5 minutes, rinsing with sterile water 5-6 times, absorbing surface moisture on sterile dry filter paper, and then inoculating in induction medium containing 28g sucrose and 5.5g agar per liter, pH=5.8-6.2 for a certain period of time to obtain aseptic tissue culture seedlings from the seeds.

[0040] 2) Obtaining aseptically rooted seedlings:

[0041] Take the robust, 1.5–3.0 cm tall sterile seedlings obtained in step 1) and transfer them to a rooting medium containing 28 g sucrose, 5.5 g agar, 0.2 mg indoleacetic acid (IAA), and pH 5.8–6.2 per liter of 1 / 2 MS medium to obtain sterile rooted seedlings; the rooting medium also contains 30–200 mg cephalosporin (Cef).

[0042] 3) Agrobacterium-mediated genetic transformation:

[0043] (1) Preparation of bacterial strain: Plasmid Pcambia1301-PMI was electroporated into Agrobacterium, and its GUS gene was replaced by the target gene. Agrobacterium LBA4404 containing pCAMBIA1301-PMI was grown on YEP solid medium containing 50 mg / L kanamycin and 100 mg / L rifampin at 28℃ for 28-48 h. Single colonies were picked and placed on 10 mL of YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampin and cultured overnight with shaking at 200 rpm at 28℃ until the OD value reached 0.8-1.0. The bacterial strain was then removed and retained for later use.

[0044] (2) Pre-culture: Select healthy, aseptic tissue culture seedlings with buds, 0.8-1.0 cm long, and stem segments with one axillary or terminal bud as recipient materials. Pre-culture them for 2-3 days in MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2.

[0045] (3) Preparation and infection of engineered Agrobacterium:

[0046] Preparation of engineered Agrobacterium: The Agrobacterium bacterial suspension containing the plasmid expression vector prepared in steps 3)(1) was inoculated into YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampicin, with a volume ratio of bacterial suspension to YEP liquid medium = 1:(50-75) and cultured overnight on a constant temperature shaker at 28℃ and 220 r / min until the OD value reached 0.8-1.0. The bacterial cells were collected by centrifugation at 4000 rpm for 5 min at room temperature, and then resuspended in a resuspension solution after being dispersed by a vortex mixer. The cells were cultured at 28℃ and 220 rpm until the OD value reached 0.4-0.6.

[0047] Infection and co-culture: On a clean bench, the pre-cultured material was placed in a prepared Agrobacterium resuspension with an OD value of 0.4-0.6 for 10 min. The explants were removed and the attached bacterial solution was aspirated onto sterile filter paper. The explants were then transferred to MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter and co-cultured at approximately 25°C in the dark for 2-4 days.

[0048] Antibacterial culture: Explants that have completed the co-culture period were inoculated into antibacterial medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), 30–200 mg cephalosporin (Cef), and pH 5.8–6.2 per liter of MS medium and cultured under light conditions at approximately 25°C for 5 days.

[0049] Screening culture: After the antibacterial culture period of the infected material is completed, it is transferred to MS medium containing 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine (6-BA), 0.02mg α-naphthaleneacetic acid (NAA), 30-200mg cephalosporin (Cef), mannose:sucrose = (3-4g):(24-25g), pH = 5.8-6.2 per liter for screening culture to obtain transgenic adventitious shoots.

[0050] 4) Chlorophenol Red Detection (CPR Detection) of Transgenic Plants: Leaves of transgenic plants were taken for chlorophenol red detection. The chlorophenol red detection reagent contained 2.5 g of mannose and 50 mg of chlorophenol red per liter of 1 / 2 MS medium, with a pH of 6.4-7.0. The blank control was not supplemented with callus tissue, and the negative control was uninfected plants. After 24 h of dark incubation at room temperature, the color change was observed, and the proportion of chlorophenol red positive plants was counted.

[0051] The chlorophenol red (CPR) detection method is simple and convenient, and the color change is easy to observe. Plants that turn rose-red on the CPR medium are negative, while those that turn orange or yellow are positive. A total of 288 stem segments with axillary buds participated in the infection experiment. Of the 58 plants tested for CPR, 31 were positive, with a positive rate of 53.45%. The estimated transformation rate using the CPR method was 10.76%.

[0052] Example 2:

[0053] 1) Obtaining aseptic tissue culture seedlings from cuttings:

[0054] Using stem segments with axillary buds from outdoor Gynostemma pentaphyllum cuttings (with leaves removed) as explants, clean the surface of the explants by brushing off dust and impurities, then rinse with tap water for 20-30 minutes. After draining the surface water, soak the explants in 75% alcohol for 30 seconds, rinse 3-4 times with sterile water, then soak in 0.1% mercuric chloride solution for 5 minutes, and rinse 5-6 times with sterile water. Then, culture the explants in MS medium containing 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine (6-BA), 0.02mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter to obtain sterile tissue culture seedlings. The subculture medium also includes 30-200mg cephalosporin (Cef).

[0055] 2) Obtaining aseptically rooted seedlings:

[0056] Take the robust, 1.5–3.0 cm tall sterile seedlings obtained in step 1) and transfer them to a rooting medium containing 28 g sucrose, 5.5 g agar, 0.2 mg indoleacetic acid (IAA), and pH 5.8–6.2 per liter of 1 / 2 MS medium to obtain sterile rooted seedlings; the rooting medium also contains 30–200 mg cephalosporin (Cef).

[0057] 3) Agrobacterium-mediated genetic transformation:

[0058] (1) Preparation of bacterial strain: Plasmid Pcambia1301-PMI was electroporated into Agrobacterium, and its GUS gene was replaced by the target gene. Agrobacterium LBA4404 containing pCAMBIA1301-PMI was grown on YEP solid medium containing 50 mg / L kanamycin and 100 mg / L rifampin at 28℃ for 28-48 h. Single colonies were picked and placed on 10 mL of YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampin and cultured overnight with shaking at 200 rpm at 28℃ until the OD value reached 0.8-1.0. The bacterial strain was then removed and retained for later use.

[0059] (2) Pre-culture: Select healthy, aseptic tissue culture seedlings with buds, 0.8-1.0 cm long, and bud-bearing stem segments with one axillary or terminal bud as recipient materials. Pre-culture them for 2-3 days in MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter.

[0060] (3) Preparation and infection of engineered Agrobacterium:

[0061] Preparation of engineered Agrobacterium: The Agrobacterium bacterial suspension containing the plasmid expression vector prepared in steps 3)(1) was inoculated into YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampicin, with a volume ratio of bacterial suspension to YEP liquid medium = 1:(50-75) and cultured overnight on a constant temperature shaker at 28℃ and 220 r / min until the OD value reached 0.8-1.0. The bacterial cells were collected by centrifugation at 4000 rpm for 5 min at room temperature, and then resuspended in a resuspension solution after being dispersed by a vortex mixer. The cells were cultured at 28℃ and 220 rpm until the OD value reached 0.4-0.6.

[0062] Infection and co-culture: On a clean bench, the pre-cultured material was placed in a prepared Agrobacterium resuspension with an OD value of 0.4-0.6 for 10 min. The explants were removed and the attached bacterial solution was aspirated on sterile filter paper. The explants were then transferred to MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter and co-cultured at around 25°C in the dark for 2-4 days.

[0063] Antibacterial culture: Explants that have completed the co-culture period were inoculated into antibacterial medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), 30–200 mg cephalosporin (Cef), and pH 5.8–6.2 per liter of MS medium and cultured under light conditions at approximately 25°C for 5 days.

[0064] Screening culture: After the antibacterial culture period of the infected material is completed, it is transferred to MS medium containing 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine (6-BA), 0.02mg α-naphthaleneacetic acid (NAA), 30-200mg cephalosporin (Cef), mannose:sucrose = (3-4g):(24-25g), pH = 5.8-6.2 per liter for screening culture to obtain transgenic adventitious shoots.

[0065] 4) PCR detection of transgenic adventitious shoots: Genomic DNA was extracted from leaves of the plants to be tested using the CTAB method for PCR detection. The primers were: PMI1F: 5'-CACTGCGTGATGTGATTGAGAG-3' (SEQ ID NO.1); PMI1R: 5'-CGGCTGGAGTAGGGAGACA-3' (SEQ ID NO.2). Plasmid Pcambia1301-PMI was used as a positive control, and uninfected Lonicera japonica callus tissue was used as a negative control. The PCR reaction system was: 6 μl of 2×Rapid Taq Master Mix, 0.8 μl of primer PMI1R1F (10 μM), and 40 ng of DNA, which was brought to 20 μl with ddH2O. The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min. Electrophoresis was performed using a 1.7% agarose gel under the following conditions: U = 120 V, electrophoresis time: 15 min. Images were then taken using a gel imaging system. A total of 288 stem segments with axillary buds participated in the infection experiment, and 85 plants were obtained for PCR testing. Of these, 59 were positive, resulting in a DNA positivity rate of 69.41%. The estimated transformation rate of DNA was 20.49%.

[0066] Example 3:

[0067] 1) Obtaining aseptic tissue culture seedlings from cuttings:

[0068] Using stem segments with axillary buds from outdoor Gynostemma pentaphyllum cuttings (with leaves removed) as explants, clean the surface of the explants by brushing off dust and impurities, then rinse with tap water for 20-30 minutes. After draining the surface water, soak the explants in 75% alcohol for 30 seconds, rinse 3-4 times with sterile water, then soak in 0.1% mercuric chloride solution for 5 minutes, and rinse 5-6 times with sterile water. Then, culture the explants in MS medium containing 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine (6-BA), 0.02mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter to obtain sterile tissue culture seedlings. The subculture medium also includes 30-200mg cephalosporin (Cef).

[0069] 2) Obtaining aseptically rooted seedlings:

[0070] Take the robust, 1.5–3.0 cm tall sterile seedlings obtained in step 1) and transfer them to a rooting medium containing 28 g sucrose, 5.5 g agar, 0.2 mg indoleacetic acid (IAA), and pH 5.8–6.2 per liter of 1 / 2 MS medium to obtain sterile rooted seedlings; the rooting medium also contains 30–200 mg cephalosporin (Cef).

[0071] 3) Agrobacterium-mediated genetic transformation:

[0072] (1) Preparation of bacterial strain: Plasmid Pcambia1301-PMI was electroporated into Agrobacterium, and its GUS gene was replaced by the target gene. Agrobacterium LBA4404 containing pCAMBIA1301-PMI was grown on YEP solid medium containing 50 mg / L kanamycin and 100 mg / L rifampin at 28℃ for 28-48 h. Single colonies were picked and placed on 10 mL of YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampin and cultured overnight with shaking at 200 rpm at 28℃ until the OD value reached 0.8-1.0. The bacterial strain was then removed and retained for later use.

[0073] (2) Pre-culture: Select healthy, aseptic tissue culture seedlings with buds, 0.8-1.0 cm long, and bud-bearing stem segments with one axillary or terminal bud as recipient materials. Pre-culture them for 2-3 days in MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter.

[0074] (3) Preparation and infection of engineered Agrobacterium:

[0075] Preparation of engineered Agrobacterium: The Agrobacterium bacterial suspension containing the plasmid expression vector prepared in steps 3)(1) was inoculated into YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampicin, with a volume ratio of bacterial suspension to YEP liquid medium = 1:(50-75) and cultured overnight on a constant temperature shaker at 28℃ and 220 r / min until the OD value reached 0.8-1.0. The bacterial cells were collected by centrifugation at 4000 rpm for 5 min at room temperature, and then resuspended in a resuspension solution after being dispersed by a vortex mixer. The cells were cultured at 28℃ and 220 rpm until the OD value reached 0.4-0.6.

[0076] Infection and co-culture: On a clean bench, the pre-cultured material was placed in a prepared Agrobacterium resuspension with an OD value of 0.4-0.6 for 10 min. The explants were removed and the attached bacterial solution was aspirated onto sterile filter paper. The explants were then transferred to MS medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), and pH 5.8-6.2 per liter and co-cultured at around 25°C in the dark for 2-4 days.

[0077] Antibacterial culture: Explants that have completed the co-culture period were inoculated into antibacterial medium containing 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine (6-BA), 0.02 mg α-naphthaleneacetic acid (NAA), 30–200 mg cephalosporin (Cef), and pH 5.8–6.2 per liter of MS medium and cultured under light conditions at approximately 25°C for 5 days.

[0078] Screening culture: After the antibacterial culture period of the infected material is completed, it is transferred to MS medium containing 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine (6-BA), 0.02mg α-naphthaleneacetic acid (NAA), 30-200mg cephalosporin (Cef), mannose:sucrose = (3-4g):(24-25g), pH = 5.8-6.2 per liter for screening culture to obtain transgenic adventitious shoots.

[0079] 4) RT-PCR detection of transgenic plants: Leaves of transgenic plants were further tested using RT-PCR. Total RNA was extracted from leaves of the plants to be tested using the TRIZOL method, and then reverse transcribed using the PrimeScript RT kit to obtain cDNA. The detection primers were: PMI1F: 5'-CACTGCGTGATGTGATTGAGAG-3' (SEQ ID NO.1); PMI1R: 5'-CGGCTGGAGTAGGGAGACA-3' (SEQ ID NO.2). Plasmid Pcambia1301-PMI was used as a positive control, and uninfected Lonicera japonica callus tissue was used as a negative control. The PCR reaction system was: 2×Rapid Taq Master Mix 6 μl, primer PMI1R1F (10 μM) 0.8 μl; DNA (16 ng / μl) 2.5 μl; and ddH2O was added to bring the total volume to 20 μl. The RT-PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min. Electrophoresis was performed on a 1.7% agarose gel under the following conditions: U = 130 V, electrophoresis time: 15 min. Afterwards, the gel was observed and photographed using a gel imaging system. A total of 288 stem segments with axillary buds participated in the infection experiment. Of the 35 plants tested for RT-PCR, 21 were positive, resulting in a positive rate of 60% and an estimated transformation rate of 7.29%. Several experiments are described in detail below.

[0080] Experiment 1: Seed germination rate experiment

[0081] Materials and sources: Fresh, ripe fruits were harvested from the end of December of the previous year to the beginning of January of the current year. After being air-dried in a well-ventilated and shady place, they were stored naturally without any treatment. Experiments were conducted within 3 months after harvest, and in the 7th and 8th months after harvest.

[0082] Methods: Gynostemma pentaphyllum seeds were rinsed with running tap water for 12 hours and then divided into two groups for treatment: Group 1: seed coat retained; Group 2: seed coat removed. The two groups of seeds were then subjected to the following nine sterilization treatments in a clean bench: 75% ethanol for 30 seconds + 0.1% HgCl2 (2 min, 3 min, 4 min, 5 min, 10 min, 12 min, 15 min). Afterwards, they were inoculated into MS medium (pH 5.8-6.2) with 28 g / L sucrose and 5.5 g / L agar and cultured in the dark for 7 days, then transferred to light. Germination occurred in approximately 10 days. Each treatment was replicated three times, with four bottles per replicate, and 10 seeds inoculated per bottle. The number of contaminants was recorded on day 7 post-inoculation, and the number of germinations was recorded on day 30 post-inoculation. The specific experimental design is shown in Table 1 below, and the results are shown in Table 2.

[0083] The above results demonstrate that Gynostemma pentaphyllum seeds, after being dried in a cool, ventilated place without any further treatment and stored naturally, can germinate when inoculated onto a culture medium within three months of harvesting, with seeds in their husks being the most suitable for germination. Seeds in their husks, after sterilization, begin to germinate after 10 days of culture on MS medium. However, seeds stored for more than six months, whether sterilized before sowing in a culture medium or directly sown in potted soil without sterilization, showed no germination for a long time. The growth effect of tissue-cultured seedlings of Gynostemma pentaphyllum seeds with husks after three months in a culture medium is shown below. Figure 1 .

[0084] Currently, in the tissue culture of Gynostemma pentaphyllum, young stem segments, stem tips, leaves, petioles and flower buds are often selected as explants. After appropriate disinfection, callus and adventitious shoot induction are carried out. However, there are no reports on the use of seed materials as explants. This experiment aims to obtain a large amount of transgenic recipient material and selects seeds as explants. After appropriate disinfection and sterilization, sterile seedlings are obtained.

[0085] The experimental results showed that seeds stored for six months failed to germinate for a long time, whether they were sterilized and sown in a culture medium or directly in the soil. This is consistent with the research results of Wu Xiaoyu et al. [Wu Xiaoyu, Peng Xiaolie, Liu Shibiao, et al. The promoting effect of short-term low temperature on the germination of Gynostemma pentaphyllum seeds [J]. Journal of Jishou University (Natural Science Edition), 2016, 37(4): 29-34.], which stated that "a certain storage time is conducive to breaking its own dormancy mechanism, but if the storage time is too long, the germination rate of the seeds will drop rapidly, or even stop germinating." However, within 3 months after harvesting fresh seeds, seeds with skin were sterilized by disinfection with 75% alcohol for 30s + 0.1% HgCl for 25min and then cultured in MS medium. After 30 days, the germination rate was high, reaching 45.80%. Sowing should be carried out as soon as possible within 3 months after harvesting fresh seeds to improve the germination rate and reduce losses.

[0086] Table 1. Experimental design of the effects of different sterilization conditions on the sterilization and germination of Gynostemma pentaphyllum seeds.

[0087]

[0088] Table 2. Germination rate of Gynostemma pentaphyllum seeds with and without seed coat under different sterilization conditions.

[0089]

[0090]

[0091] Note: 1. DPS 6.55 statistical software was used, and Duncan's multiple comparison method was used for analysis. The significance level was 1% for uppercase letters and 5% for lowercase letters.

[0092] 2. Treatments A1-A7 were sown within 3 months after collecting fresh seeds, treatments A8 and A10 were sown in the 7th month after collecting fresh seeds, and treatment A9 was sown in the 8th month after collecting fresh seeds.

[0093] Experiment 2: Induction of buds from explants of different parts of Gynostemma pentaphyllum

[0094] Experiment 2-1: Effects of sterilization conditions of 75% alcohol (30s) + 0.1% mercuric chloride (5min) on the contamination rate, survival rate and bud induction rate of different parts of Gynostemma pentaphyllum.

[0095] Method: After rinsing the surface of each explant of Gynostemma pentaphyllum with tap water to remove dust and impurities, the material surface was gently brushed with detergent solution. Then, in a clean bench, each part (leaf, short petiole, long petiole, stem segment without buds, and stem segment with buds) was sterilized separately using (70% alcohol for 30 seconds + 0.1% HgCl2 for 5 minutes). Finally, the material was transferred to a culture medium.

[0096] The explants were cultured in the dark using MS medium containing 1.5 mg / L 6BA and 0.02 mg / L NAA (pH 5.8-6.0). Each treatment was repeated three times, with four bottles per replicate, and eight explants were inoculated into each bottle. The number of contaminated plants and the number of viable plants were recorded on day 7 after inoculation, and the fertilization rate of shoots was calculated on day 30. The experimental results are shown in Table 3 below.

[0097] Table 3. Effects of sterilization conditions (75% alcohol (30s) + 0.1% mercuric chloride (5min)) on contamination rate, survival rate, and bud induction rate of different parts of Gynostemma pentaphyllum.

[0098]

[0099]

[0100] Note: Data in the same column was analyzed using DPS 6.55 software. Significance differences are indicated by different letters: lowercase letters represent a significance level of 0.05, and uppercase letters represent a significance level of 0.01. The same applies below. +: Average; ++: Good; +++: Best

[0101] The above data indicate that, among the five selected explant sites—leaf, short petiole, long petiole, stem segments without buds, and stem segments with buds—only the stem segments with buds could induce adventitious buds after disinfection with 75% alcohol and 0.1% mercuric chloride, achieving an induction rate of 59.18%. Furthermore, the newly induced adventitious buds were relatively robust, successfully yielding aseptic tissue culture seedlings. (See [reference needed]). Figure 3 and Figure 4 .

[0102] Experiment 2-2:

[0103] To explore the induction of adventitious buds in different parts of Gynostemma pentaphyllum, pure long-stalked (0.8-1.0 cm long), long-stalked compound leaves, single leaves with short petioles, sessile single leaves, and stem segments with buds from aseptic tissue culture seedlings obtained in Experiment 1 and Experiment 2-1 were used as raw materials for adventitious bud induction. They were inoculated into MS medium containing 28 g / L sucrose, 5.5 g / L agar, 1.5 mg / L 6-BA, and 0.02 mg / L NAA (pH 5.8-6.0). After 30 days of culture, the adventitious bud induction effect of each part is shown in the table below.

[0104] Table 4: Induction of adventitious buds from different parts of Gynostemma pentaphyllum

[0105]

[0106] The results in the table above and Figures 5-9 The results showed that, among the five selected sites, the adventitious bud induction rate was as follows: stem segment with bud (99.17%, highest) > long petiole with compound leaves (9.26%) > pure long petiole (8.33%) > single leaf with short petiole (0) and single leaf without petiole (0); the average number of buds produced by explants was as follows: long petiole with compound leaves (5.67 buds / plant) > stem segment with bud (4.50 buds / plant) > long petiole (1 bud / plant) > single leaf with short petiole (0) and single leaf without petiole (0). Although pure long petiole and long petiole with compound leaves could also induce adventitious buds, the induction rate was extremely low, less than 10%.

[0107] Experimental Example 2-3:

[0108] Based on the results of Experiment 2-2, further experiments were designed to explore suitable culture media for inducing adventitious buds with compound leaves and long petioles. Gynostemma pentaphyllum with compound leaves and long petioles (the base of the petiole vertically inserted into the culture medium) were inoculated into various culture media (pH 5.8-6.0). Their growth was observed 1-2 times per week, and the budding start date was recorded in detail. Data on the adventitious bud induction effect were collected 30 days after inoculation. Specific experimental design and results are shown in the following tables.

[0109] Table 5: Experimental design of the effect of different types of cytokinins on the induction of adventitious buds on long petioles of Gynostemma pentaphyllum compound leaves

[0110] Processing Number Types of culture media 1 MS(CK) 2 MS solution: 28 g / L sucrose, 5.5 g / L agar, 1.5 mg / L 6-BA, and 0.02 mg / L NAA. 3 1 / 2 MS + 28 g / L sucrose + 5.5 g / L agar + 0.2 mg / L NAA 4 MS + sucrose 28g / L + agar 5.5g / L + 6-BA 2.0mg / L + KT 0.3mg / L 5 <![CDATA[MS + 28 g / L sucrose + 5.5 g / L agar + 2.0 mg / L 6-BA + 0.3 mg / L GA3]]>

[0111] The effects of adventitious bud induction are shown in the table below:

[0112] Table 6: Comparison of the effects of different types of cytokinins on the induction of adventitious buds on long petioles of Gynostemma pentaphyllum compound leaves

[0113]

[0114] The results showed that Gynostemma pentaphyllum with compound leaves and long petioles was more effective in treating diseases on MS medium containing 28 g / L sucrose, 5.5 g / L agar, 1.5 mg / L 6-BA, and 0.02 mg / L NAA.

[0115] Adventitious shoots could be induced in 2.0 mg / L + GA3 0.3 mg / L, but the induction rates were low, at 9.26% and 3.92% respectively, both <10%.

[0116] As seen in the comprehensive experiments 2-1, 2-2, and 2-3, among different parts of Gynostemma pentaphyllum (pure long petiole, long petiole with compound leaves, single leaf with short petiole, sessile single leaf, and stem segment with bud), the adventitious bud induction rate was highest in the stem segment with bud, at 99.17%, with an average of 4.50 buds per explant. Although pure long petiole and long petiole with compound leaves could induce adventitious buds, the induction rate was extremely low, <10%, similar to the findings of Zhao Ruiqiang [Zhao Ruiqiang. Preliminary study on the cloning and transformation of Panax notoginseng SE gene into tobacco and Gynostemma pentaphyllum [D]. Guangxi Medical University, 2017:15], they are not suitable as subjects for genetic transformation. In later infection experiments, stem segments with buds should be selected as subjects.

[0117] Experiment 3: Comparative Experiment on the Rooting Effect of Gynostemma pentaphyllum

[0118] Methods: A basal medium of 1 / 2 MS medium (28 g / L sucrose + 5.5 g / L agar) was used, supplemented with IAA (0.2 mg / L), IBA (0.2 mg / L), and NAA (0.2 mg / L), respectively, at pH 5.8-6.0. Healthy subculture seedlings (2-3 cm long) of *Gynostemma pentaphyllum* were inoculated onto the three media and cultured under light in a culture room to explore the effects of different auxins on rooting. Three treatments were administered, each with three replicates, with 10 bottles per replicate and 3 explants per bottle. Rooting indicators (number of rooted plants, root length, root color, quality, etc.) were recorded and analyzed on days 10, 20, and 30 post-inoculation. Specific experimental results are as follows:

[0119] Table 7. Effects of different types of auxins on the rooting effect of Gynostemma pentaphyllum during rooting culture.

[0120]

[0121] Note: Data in the same column was analyzed using DPS 6.55 software. Significance differences are indicated by different letters: lowercase letters represent a significance level of 0.05, and uppercase letters represent a significance level of 0.01. The same applies below. +: Average; ++: Good; +++: Best

[0122] from Figures 10-15It is evident that the rooting effect of Gynostemma pentaphyllum varied significantly among the three culture media, as detailed in the remarks column of the table. After 30 days of culture, the rooting rate reached 100% in all three culture media (NAA 0.2 mg / L, IAA 0.2 mg / L, IBA 0.2 mg / L); the average number of roots was (NAA 0.2 mg / L, 55.28 roots / plant) > (IBA 0.2 mg / L, 31.09 roots / plant) > (IAA 0.2 mg / L, 17.78 roots / plant), with highly significant differences; the average root length was (IBA 0.2 mg / L, 1.94 cm) > (IAA 0.2 mg / L, 1.69 cm) > (NAA 0.2 mg / L, 1.44 cm); furthermore, the leaf expansion and robustness were approximately (IAA 0.2 mg / L) ≈

[0123] (IBA0.2mg / L)>(NAA0.2mg / L).

[0124] In summary, the most suitable culture medium for Gynostemma pentaphyllum to promote rooting and seedling growth among the three culture media is 1 / 2 MS + 28 g / L sucrose + 5.5 g / L agar + 0.2 mg / L IAA.

[0125] Experiment 4: Exploration of the optimal concentration of the antibacterial agent cephalosporin (Cef)

[0126] Methods: MS medium containing 28 g / L sucrose, 5.5 g / L agar, 1.5 mg / L 6-BA, and 0.02 mg / L NAA was used as the basal medium. Five treatments were selected using cephalosporin (Cef) at concentrations of 0, 50, 100, 150, and 200 mg / L, with a pH of 5.8–6.0. ​​Stem segments (1.0–1.5 cm long) with one axillary bud (or terminal bud) were cut from vigorous subcultured Gynostemma pentaphyllum seedlings and inoculated into the above medium with the morphological upper end facing upwards. Five treatments were performed. Each treatment was replicated three times, with six bottles inoculated per replicate. Each bottle contained eight explants. After one month of culture, growth (survival rate, plant height, number of buds, color, and other relevant indicators) were observed and recorded. The optimal cephalosporin concentration for antibacterial effect was investigated. Specific experimental results are as follows:

[0127] Table 8. Effects of different concentrations of the antibacterial agent cephalosporin (Cef) on the induction of adventitious buds infected by Agrobacterium tumefaciens in stem segments of Gynostemma pentaphyllum with one axillary bud (or terminal bud).

[0128]

[0129] The results in the table above show that with increasing cephalosporin concentration, the average number of shoots in the explants first increased and then decreased, exhibiting a parabolic trend. Under the cephalosporin concentration of 150 mg / L, the shoot induction rate was 100%, and the average number of shoots in the explants was higher than the other four treatments, at 5.00 shoots / plant, with a shoot height of 2.77 cm and a green color. Under the cephalosporin concentration of 200 mg / L, the shoot induction rate was 100%, and the average number of shoots in the explants was 4.66 shoots / plant, second only to the 150 mg / L treatment. Its shoot height was 3.58 cm, the best, and the shoots were green. Conclusion: Cephalosporin concentrations of 150-200 mg / L are the optimal antibacterial concentration.

[0130] Experiment 5: An exploratory experiment to screen mannose concentration

[0131] Methods: Stem segments (1.0-1.5 cm long) with one axillary bud (or terminal bud) were taken from healthy subcultured Gynostemma pentaphyllum seedlings and inoculated onto mannose selection medium with the morphological upper end facing upwards. The selection medium was prepared using MS medium with agar.

[0132] The basal culture medium consisted of 5.5 g / L 6-BA 1.5 mg / L 6-BA 0.02 mg / L NAA. Two rounds of screening were conducted. In the first round, eight treatments were performed using the basal culture medium supplemented with mannose / sucrose (0 / 28, 4 / 24, 8 / 20, 12 / 16, 16 / 12, 20 / 8, 24 / 4, 28 / 0) g / L. In the second round, five treatments were performed using the basal culture medium supplemented with mannose / sucrose (0 / 28, 1 / 27, 2 / 26, 3 / 25, 4 / 24) g / L. Each treatment was replicated three times, with six bottles inoculated per replicate. Each bottle contained eight explants. After inoculation, the explants were placed on culture racks in the culture room and observed 1-2 times per week. After one month of culture, growth (survival rate, plant height, number of buds, color, and other relevant indicators) was observed and recorded. The optimal mannose concentration for screening was investigated. Specific experimental results are shown in Tables 6 and 7 below.

[0133] Table 9. Effects of different concentrations of mannose on the induction effect of Gynostemma pentaphyllum buds in the first round.

[0134]

[0135] Table 10. Effects of different concentrations of mannose on the induction effect of Gynostemma pentaphyllum buds in the second round.

[0136]

[0137] Based on the survival rate and bud induction effect, it can be seen that among the eight treatments in the first round of screening, the mannose / sucrose ratio of 4 / 24 (g / L) was the most suitable for screening uninfected Gynostemma pentaphyllum adventitious buds. At this time, the cure rate was 0, the survival rate was 45.69%, the explant bud induction rate was 23.75%, the average number of buds per explant was 1.10, the average bud height was 0.28 cm, and the newly emerged buds were light green and of average quality.

[0138] In the second round of screening, among the five treatments, when the mannose / sucrose ratio was 2 / 26 (g / L), the callus emergence rate was 61.11%, the survival rate was 100%, the explant shoot induction rate was 86.12%, the average number of shoots per explant was 2.26, the average shoot height was 1.01 cm, and the newly emerging shoots were light green and of average quality. Based on the above experiments, it was found that there is a critical point between mannose concentrations of 2 g / L and 3 g / L that affects the relevant indicators of callus tissue and adventitious shoot induction infected by *Agrobacterium gypenosus*.

[0139] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for introducing exogenous genes into Gynostemma pentaphyllum, characterized in that, include: S1: Select Gynostemma pentaphyllum seeds with seed coat within 3 months after harvest, disinfect them, and culture them to obtain seed-grown sterile tissue culture seedlings, or select stem segments with axillary buds after removing leaves, disinfect them, and culture them using a subculture proliferation medium to obtain cutting sterile tissue culture seedlings, wherein the subculture proliferation medium contains cephalosporins. S2: Select the seedlings or cuttings obtained in S1 and culture them in a rooting medium to obtain sterile rooted seedlings. The rooting medium includes cephalosporins. S3: Plasmid Pcambia1301-PMI was transferred into Agrobacterium and cultured in a medium containing kanamycin and rifampin to obtain Agrobacterium resuspension; S4: The aseptic rooted seedlings obtained in S2 were infected with the Agrobacterium resuspension and then co-cultured, inhibited cultured and screened to obtain transgenic adventitious shoots; In S1, the subculture proliferation medium contains 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine, 0.02mg α-naphthylacetic acid, and 30-200mg cephalosporin per liter of MS medium; In S2, the rooting medium contains 28g sucrose, 5.5g agar, 0.2mg indoleacetic acid, and 30-200mg cephalosporin per liter of 1 / 2 MS medium; The co-culture medium contained 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine, and 0.02 mg α-naphthaleneacetic acid per liter of MS medium, and was co-cultured in the dark for 2-4 days. The antibacterial culture medium contained 28g sucrose, 5.5g agar, 1.5mg 6-benzyladenine, 0.02mg α-naphthylacetic acid, and 30-200mg cephalosporin per liter of MS medium, and was cultured under light for 5 days. The screening medium contained 28 g sucrose, 5.5 g agar, 1.5 mg 6-benzyladenine, 0.02 mg α-naphthylacetic acid, and 30-200 mg cephalosporin per liter of MS medium, with a mannose:sucrose ratio of (3-4 g):(24-25 g).

2. The method for introducing exogenous genes into Gynostemma pentaphyllum as described in claim 1, characterized in that, In S1, the method for disinfecting seeds or stem segments with axillary buds after leaf removal within 3 months of harvest includes soaking them in 75% alcohol and 0.1% mercuric chloride aqueous solution in sequence.

3. The method for introducing exogenous genes into Gynostemma pentaphyllum as described in claim 1, characterized in that, In S1, sterilized seeds are cultured in an induction medium to obtain sterile tissue culture seedlings. The induction medium contains 28g of sucrose and 5.5g of agar per liter of MS medium. The sterilized seeds are then cultured in the dark for 7 days and 24 hours, followed by a light culture:dark culture ratio of 12h:12h, with a light intensity of 2000~2500 Lux.

4. The method for introducing exogenous genes into Gynostemma pentaphyllum as described in claim 1, characterized in that, In S3, Agrobacterium containing pCAMBIA1301-PMI was grown on YEP solid medium containing 50 mg / L kanamycin and 100 mg / L rifampin for 28-48 h. Single colonies were picked and placed on YEP liquid medium containing 50 mg / L kanamycin and 100 mg / L rifampin and cultured with shaking until the OD value reached 0.8-1.

0. The colonies were then removed, centrifuged, and the cells were collected. The cells were cultured with shaking until the OD value reached 0.4-0.6 to obtain Agrobacterium resuspension.

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