Bacillus-mediated genetic transformation method of schizonepeta and application thereof

Abstract

The application discloses a kind of genetic transformation methods of Agrobacterium-mediated schizonepeta, comprising the following steps: with schizonepeta stem section as explant, using the infection bacteria liquid containing recombinant Agrobacterium to infect schizonepeta stem section, and then co-culture is carried out on the medium of co-culture, and the explant after co-culture is obtained;The obtained explant after co-culture is cultured on the induction screening medium, and the schizonepeta resistant bud is obtained;Then schizonepeta resistant bud is cultured on rooting screening medium, and schizonepeta resistant plant is obtained;From schizonepeta resistant plant, the positive plant containing marker gene GFP is screened and verified by PCR, and the schizonepeta positive plant of genetic transformation is obtained, the method of the application provides technical support for gene introduction or knock-out of original plant and the like function research, is beneficial to the molecular mechanism of parsing labiatae plant secondary metabolism, and provides reference for important gene function analysis of schizonepeta.

Description

Technical Field

[0001] This invention relates to the field of plant gene genetic transformation, specifically to an Agrobacterium-mediated genetic transformation method for Nepeta cataria and its application. Background Technology

[0002] Agrobacterium tumefaciens is a soil-borne pathogenic microorganism that, under the induction of phenolic substances, chemotactically migrates to plant wounds, inserting its T-DNA into the plant chromosome to complete gene integration and achieve plant transformation. Agrobacterium-mediated plant transformation is currently the most widely used and best-understood genetic transformation method. It is simple to operate, inexpensive, and can infect most dicotyledonous plants, and a few monocotyledonous and gymnosperms (Gelvin, et al., 2010). The first batch of transformed plants successfully expressing exogenous target genes were obtained using the Agrobacterium-mediated method, making it the most common means of plant genetic transformation. In Agrobacterium tumefaciens cells, DNA is transferred into the target plant via the transfer of the Ti plasmid (tumour-inducing plasmid). The T-DNA is a DNA fragment cleaved from the Agrobacterium tumefaciens Ti plasmid and transferred into the plant cells during Agrobacterium infection. The process of Agrobacterium-mediated plant transformation includes steps such as recognition of injured plant sites, attachment of Agrobacterium, formation of transfer complexes, and integration of T-DNA.

[0003] Green fluorescent protein (GFP) is a fluorescent protein that emits green light under ultraviolet light, discovered in 1962 from the jellyfish *Echinochloa crus-galli* (Shimomura O, 1979). It has been widely adopted due to its high sensitivity, small molecular weight, ease of detection, and non-toxicity. In 1994, the GFP gene was successfully expressed in *Escherichia coli*, ushering in a new era for GFP research (Chafee I, 1994). Currently, GFP is widely used in plants, animals, and microorganisms, serving as a marker gene in plant genetic transformation experiments, for the construction of identification systems, and for the introduction of target genes.

[0004] Many factors influence Agrobacterium-mediated plant genetic transformation, including pre-culture time, co-culture time, Agrobacterium concentration, and infection time. In addition, explant type and growth status also affect transformation efficiency. While explant type and growth status have a significant impact on Agrobacterium-mediated plant transformation efficiency, many experiments focus on determining pre-culture time, co-culture time, Agrobacterium concentration, and infection time to construct genetic transformation systems. Pre-culture is not a necessary step for all plants (Guo M, et al., 2012), while co-culture is crucial for successful Agrobacterium infection.

[0005] Injured plant cells secrete certain phenolic compounds (such as acetylsuccinone, AS), which attract Agrobacterium to accumulate and attach to the injured tissue. Once Agrobacterium tumefaciens attaches to a plant cell, the Vir region gene receives the injury signal from the plant cell and initiates the expression of Vir A, a membrane-bound chemopreceptor protein. This protein, upon binding to the phenolic compound, triggers a series of cascaded protein phosphorylation reactions, promoting the expression of Vir B, a transmembrane channel protein. This transmembrane channel protein helps T-DNA complete the first step of transmembrane transport, entering the plant cell.

[0006] *Schizonepeta tenuifolia* Briq., commonly known as catnip, is an annual herb belonging to the genus *Schizonepeta* in the family Lamiaceae. Wild and cultivated species are found in Liaoning, Hebei, and Gansu provinces of my country (Flora of China, 1959-2004). First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is used to relieve exterior syndromes, promote rash eruption, and eliminate sores. Due to its significant clinical medicinal value, it is included in the *Chinese Pharmacopoeia* (2020) and has a long history of medicinal use in my country. The dried aerial parts and flower spikes of *Schizonepeta tenuifolia* are used medicinally to treat colds, headaches, measles, rubella, and early-stage sores. Both can also be charred and used medicinally, possessing astringent and hemostatic effects. Modern research shows that *Schizonepeta tenuifolia* is rich in chemical components, including terpenes, flavonoids, and other compounds. Its pharmacological activities are diverse, exhibiting antiviral (Shu Yachun et al., 2021), immunomodulatory (Yang Minghui et al., 2019), and antibacterial and anti-inflammatory (Zhu Meifang et al., 2018) biological activities. The modified Jingfang Baidu Powder combined with Huopu Xialing Decoction is used to treat "wind-cold-dampness syndrome" in clinical practice. It mainly consists of 19 herbs including Schizonepeta, Saposhnikovia, and Magnolia officinalis. The quality of Schizonepeta directly affects its clinical efficacy; therefore, research on the regulation of terpenoid compounds, the material basis for Schizonepeta's clinical effects, is of great significance for achieving precise regulation in its cultivation and production. Currently, research on Schizonepeta is relatively limited, mainly involving the determination of secondary metabolite content, analysis of physiological and biochemical indicators, and cloning and expression analysis of monoterpene metabolic genes. With the deepening research on key enzyme genes and regulatory factors in the terpene metabolic pathway of Schizonepeta, there is an urgent need to construct a genetic transformation system for Schizonepeta. Summary of the Invention

[0007] The purpose of this invention is to provide an Agrobacterium-mediated genetic transformation method for Nepeta cataria and its application, so as to solve the problem of how to carry out genetic transformation of Nepeta cataria in the background art.

[0008] To achieve the above objectives, the specific technical solution of the Agrobacterium-mediated genetic transformation method for Nepeta cataria and its application is as follows:

[0009] An Agrobacterium-mediated genetic transformation method for Nepeta cataria includes the following steps:

[0010] (1) Using catnip stem segments as explants, the catnip stem segments were pre-cultured on an antibiotic-free culture medium and then infected with an infection solution containing recombinant Agrobacterium. The infected catnip stem segments were then cultured on a co-culture culture medium to obtain co-cultured explants.

[0011] (2) The co-cultured explants were cultured on an induction selection medium to obtain resistant buds of Nepeta cataria;

[0012] (3) The resistant buds of Catnip were cultured on a rooting selection medium to obtain resistant Catnip plants;

[0013] (4) Screening positive plants containing the marker gene GFP from catnip resistant plants to obtain catnip positive plants that have completed genetic transformation.

[0014] Furthermore, in step (1), the recombinant Agrobacterium contains a recombinant expression vector carrying the marker gene GFP, and the infection solution is a liquid obtained by suspending the recombinant Agrobacterium in MS liquid culture medium.

[0015] The recombinant Agrobacterium is Agrobacterium GV3101 containing the recombinant expression vector.

[0016] Furthermore, the recombinant expression vector is pCAMBIA1305, obtained by inserting a GFP marker gene.

[0017] Furthermore, in step (1), the pre-culture time is 10 days, the co-culture time is 3 days, the co-culture conditions are darkness, the culture temperature is (24±1)℃, and the concentration of the infection solution can meet the OD requirements. 600 The value was 0.7, and the infection time was 15 minutes.

[0018] Furthermore, in step (1), both the pre-culture and co-culture media consist of a solvent and a solute. The solvent is 1 / 2 MS medium, and the solute and its concentration in the medium are 0.5 mg / L naphthaleneacetic acid (NAA) and 1.5 mg / L thidiazuron (TDZ), respectively, with a pH of 5.8.

[0019] Furthermore, in step (2), the induction screening medium consists of a solvent and a solute. The solvent is 1 / 2 MS medium, and the solute and its concentration in the induction screening medium are 0.5 mg / L naphthaleneacetic acid (NAA), 1.5 mg / L thiamethoxam (TDZ), and the corresponding concentration of antibiotics, respectively. The pH value is 5.8. The culture temperature is (24±1)℃, the light intensity is 3000 lx, and the photoperiod is 16 hours of light / 8 hours of darkness.

[0020] The antibiotics in the induction and screening medium were kanamycin 50 mg / L and carbenicillin 500 mg / L, respectively.

[0021] Furthermore, in step (3), the rooting selection medium consists of a solvent and a solute. The solvent is 1 / 2 MS medium, and the solute and its concentration in the rooting selection medium are 0.4 mg / L naphthaleneacetic acid (NAA) and the corresponding concentration of antibiotics, respectively. The pH value is 5.8. The culture temperature is (24±1)℃, the light intensity is 3000 lx, and the photoperiod is 16 hours of light / 8 hours of darkness.

[0022] The antibiotics in the rooting selection medium were kanamycin 50 mg / L and carbenicillin 500 mg / L, respectively.

[0023] Furthermore, the identification of positive plants in step (4) includes GFP fluorescent protein identification and DNA molecular identification.

[0024] In the above method, the 1 / 2MS medium is a medium obtained by halving the solutes other than sucrose in the MS medium.

[0025] Furthermore, the method also includes obtaining regenerated plants using the obtained Nepeta cataria plants that have undergone genetic transformation.

[0026] Specifically, regenerated plants can be obtained by culturing stem segments of genetically transformed Catnip chinensis positive plants. In particular, regenerated plants can be obtained by sequentially inducing regeneration buds and rooting culture on stem segments of genetically transformed Catnip chinensis positive plants.

[0027] The regenerated bud induction culture can be carried out in a regenerated bud induction medium, which is a medium obtained by adding NAA and TDZ to MS medium, wherein the NAA concentration is 0.5 mg / L and the TDZ concentration is 1.5 mg / L.

[0028] The rooting culture can be carried out in a rooting medium, which is a medium obtained by adding NAA to MS medium, wherein the NAA concentration is 0.4 mg / L.

[0029] Furthermore, the application of an Agrobacterium-mediated genetic transformation method for Nepeta cataria in the breeding of Nepeta cataria varieties / lines.

[0030] The beneficial effects of this invention are:

[0031] 1. This invention uses Nepeta cataria stem segments as explants. After pre-culturing, the plants are infected with bacterial solution, then co-cultured, and finally screened, rooted, and identified to obtain Nepeta cataria positive plants that have completed genetic transformation. This method lays a foundation for the study of Nepeta cataria genetic transformation, constructs a Nepeta cataria genetic transformation system, and provides technical support for the genetic transformation of Nepeta cataria varieties / strains. It also provides technical support for the functional study of gene introduction or knockout in the original plant, which is beneficial for elucidating the molecular mechanism of secondary metabolism in Lamiaceae plants and provides a reference for the functional analysis of important genes in Nepeta cataria. Detailed implementation method:

[0032] To better understand the present invention, specific examples are provided below for further illustration. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention; they are provided as a guide for those skilled in the art to make further improvements, and do not constitute a limitation on the invention in any way.

[0033] The experimental methods described in the following examples, unless otherwise specified, are conventional methods and conditions well known in the art, and can be performed according to the techniques or conditions described in the literature or the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0034] The following examples use Nepeta cataria as material and transform Agrobacterium tumefaciens GV3101 as the infecting strain with the pCAMBIA1305 plasmid (the plasmid carries the rifampicin resistance gene) containing the GUS gene sequence.

[0035] Preparation of culture medium:

[0036] (1) The primary culture medium was a sterile culture medium composed of a solvent and a solute. The solvent was water, and the solute and its concentration in the culture medium were 2.37 g / L for MS basic medium, 30 g / L for sucrose, 7 g / L for agar, and the pH value was 5.8.

[0037] (2) The subculture medium was a sterile medium composed of a solvent and a solute. The solvent was water, and the solute and its concentration in the medium were 2.37 g / L for MS basal medium, 30 g / L for sucrose, and 7 g / L for agar, with a pH of 5.8.

[0038] (3) The bud regeneration induction medium was a sterile medium composed of a solvent and a solute. The solvent was water, and the solute and its concentration in the medium were 2.37 or 4.74 g / L of MS basal medium, 0.1-2.0 mg / L of naphthaleneacetic acid (NAA), 0.5-2.5 mg / L of thiazidazole (TDZ), 0-8 mg / L of 6-benzylaminopurine (6-BA), 30 g / L of sucrose, and 5 g / L of agar. The pH value was 5.8.

[0039] In the optimal regeneration bud induction medium, MS 2.37 g / L, naphthaleneacetic acid (NAA) 0.5 mg / L, thidiazuron (TDZ) 1.5 mg / L, and 6-benzylaminopurine (6-BA) were less effective at inducing regeneration buds than thidiazuron (TDZ) at a concentration of 0 mg / L.

[0040] The screening medium containing kanamycin was obtained by adding kanamycin and carbenicillin to the optimal induction medium (NAA concentration of 0.5 mg / L and TDZ concentration of 1.5 mg / L), wherein the concentration of kanamycin was 50 mg / L and the concentration of carbenicillin was 500 mg / L.

[0041] (4) The rooting medium is a sterile medium composed of solvent and solute. The solvent is water, and the solute and its concentration in the medium are 2.37 or 4.74 g / L of MS basic medium, 0-0.5 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, 5 g / L of agar, and the pH value is 5.8.

[0042] The optimal rooting medium contains MS 2.37 g / L and naphthaleneacetic acid (NAA) 0.4 mg / L.

[0043] The kanamycin selection medium for rooting was obtained by adding kanamycin and carbenicillin to the optimal rooting medium (NAA concentration of 0.4 mg / L), where the concentration of kanamycin was 50 mg / L and the concentration of carbenicillin was 500 mg / L.

[0044] (5) Pre-culture medium, the same as the regeneration bud induction medium, wherein the concentration of NAA is 0.5 mg / L and the concentration of TDZ is 1.5 mg / L.

[0045] (6) Co-culture medium, same as pre-culture medium.

[0046] (7) The induction and screening medium was a sterile medium composed of solvent and solute. The solvent was water, and the solute and its concentration in the medium were 2.37 g / L MS basic medium, 0.5 mg / L naphthaleneacetic acid (NAA), 1.5 mg / L thidiazuron (TDZ), 30 g / L sucrose, 7 g / L agar, 50 mg / L kanamycin, and 500 mg / L carbenicillin. The pH value was 5.8.

[0047] (8) The rooting screening medium was a sterile medium composed of a solvent and a solute. The solvent was water, and the solutes and their concentrations in the medium were 2.37 g / L MS basic medium, 0.4 mg / L naphthaleneacetic acid, 30 g / L sucrose, 5 g / L agar, 50 mg / L kanamycin, and 500 mg / L carbenicillin, with a pH of 5.8.

[0048] (9) MS liquid culture medium is a sterile culture medium composed of solvent and solute. The solvent is water, and the solute and its concentration in the culture medium are 4.74 g / L of MS basic culture medium or 2.37 g / L of 1 / 2 MS basic culture medium.

[0049] The MS basal culture medium was a product of Qingdao High-tech Industrial Park Haibo Biotechnology (China) Co., Ltd.

[0050] Example 1

[0051] I. Obtaining Catnip Explants

[0052] 1. Primary culture of aseptic seedlings

[0053] Take whole and plump catnip seeds, soak them in warm water for 3 hours, then soak them in a 75% ethanol aqueous solution in a clean bench for 1 minute, rinse them with sterile water 3-4 times; then disinfect them with a 2% sodium hypochlorite solution for 30-40 minutes (the disinfection time depends on the degree of discoloration of the seed coat), rinse them with sterile water 3-4 times to obtain disinfected catnip seeds, place them on sterile dry filter paper to absorb the surface moisture, and set aside for later use;

[0054] Sterilized Nepeta cataria seeds were inoculated onto primary culture medium and cultured aseptically as seedlings under a light intensity of 3000 lx, a 16-hour light / 8-hour dark condition, at a temperature of (24±1)℃ for 35-40 days. During this stage, the aseptic seedlings showed good growth, with elongated internodes, axillary buds appearing between stems, and vigorous root development. The aseptic seedlings obtained from the primary culture were then propagated by stem segment. Stem segments were used as explants in a clean bench, and subcultured using the method described above. The resulting aseptic seedlings were used for subsequent Nepeta cataria stem segment regeneration experiments.

[0055] 2. Regeneration bud induction culture

[0056] In a clean bench, *Nepeta cataria* stem segments were cut into 0.5 cm long sections using a scalpel, retaining the axillary buds at the internodes. These segments were inoculated into a regeneration bud induction medium and cultured under normal light or dark conditions at a temperature of (24±1)℃. The NAA concentration in the regeneration bud induction medium was 0.5 mg / L, and the TDZ concentration was 1.5 mg / L. Five days after explant inoculation, the edges of the stem segment cuts began to swell, and the regeneration buds showed obvious growth.

[0057] II. Preparation of Plasmid Vectors and Infected Bacterial Fluids

[0058] Plasmids containing 35S:GUS (pCAMBIA1305) from DH5α *E. coli* preserved in the laboratory were amplified. Single colonies were isolated by streaking on LB agar plates (containing 50 mg / L kanamycin and 20 mg / L rifampin). The LB agar plates were incubated overnight at 37°C. Four single colonies were picked and dissolved in 20 μL of sterile water, and mixed by pipetting. Primers for the GUS sequence were designed using Primer Premier 5 software to amplify the bacterial culture by PCR. Electrophoresis analysis was performed on a 1.5% agarose gel.

[0059] Thaw GV3101 competent Agrobacterium cells stored at -80℃ at room temperature and place them on ice. Add 1 μg of plasmid DNA extracted in step 1 above to 100 μL of Agrobacterium and mix gently. Incubate the mixture sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min. Then add 500 μL of antibiotic-free LB liquid medium and incubate at 28℃ with shaking for 2-3 h. Spread the mixture onto LB solid medium (containing 50 μg / mL kanamycin and 20 μg / mL rifampin) and incubate upside down at 28℃ for 2-3 days.

[0060] The transformed Agrobacterium was picked and cultured in 10 mL LB medium (containing 50 μg / mL kanamycin and 20 μg / mL rifampin) at 28°C and 200 rpm for 12-16 h on a shaker. OD 600 Approximately 0.6-1.0. Take 6 mL of the bacterial culture from step 1 and place it in a fresh 200 mL LB medium (LB contains kanamycin and rifampicin resistance) for expansion culture, and incubate overnight at 28°C and 200 rpm. Centrifuge the expanded Agrobacterium culture at 25°C and 3000 rpm for 10 min in a high-speed centrifuge, and collect the precipitate.

[0061] III. Genetic Transformation of Catnip

[0062] S1. Kanamycin susceptibility test for schizonepeta tenuifolia

[0063] Sterile *Nepeta cataria* seedlings were used to cut stem segments with leaf axils in a clean bench. These segments were inoculated onto regeneration induction medium containing different concentrations of kanamycin (0, 50, and 100 mg / L) and cultured at (24±1)℃, 3000 lx, with a 16-hour light / 8-hour dark cycle. Each treatment involved 20 *Nepeta cataria* stem segments, and the treatment was repeated three times. The growth of the stem segments with leaf axils was observed, and the survival rate was calculated after one month.

[0064] Regeneration shoot induction rate (%) = Number of explants that induced regeneration shoots / Total number of inoculated explants × 100%.

[0065] Regeneration bud induction rate (%) = Total number of explants with differentiated adventitious buds / Total number of inoculated explants × 100%.

[0066] Table 1 shows the results of culturing with different kanamycin concentrations for 30 days. The results indicate that the growth of Nepeta cataria stem segments was completely inhibited and the stem segments died when the kanamycin concentration was 100 mg / L. When the kanamycin concentration was 50 mg / L, axillary buds on the stem segments elongated, but no adventitious buds differentiated. After 30 days, only 5% of the leaf-bearing axillary stem segments survived and no regenerated buds were formed. Therefore, the optimal kanamycin concentration for stem segment differentiation is 50 mg / L.

[0067] Table 1. Effects of kanamycin concentration on Nepeta cataria stem segments and regenerated buds

[0068]

[0069] S2. Stem segment pre-culture

[0070] Using sterile Nepeta cataria seedlings cultured for 30 days as material, Nepeta cataria stem segments were cut into 0.5cm long segments with a scalpel in a clean bench, retaining the axillary buds between the nodes, and inoculated into a pre-culture medium for pre-culture. The pre-culture time was set to 0 days, 5 days, 10 days and 20 days. The pre-cultured stem segments were obtained after the culture was completed.

[0071] Pretreated Nepeta cataria stem segments were subjected to bacterial inoculation, co-culture, screening culture, and root induction to obtain resistant seedlings that maintained a bright green color and normal growth. The yield of resistant seedlings after transformation culture under the same bacterial concentration, inoculation time, and co-culture time after 40 days was statistically analyzed to determine the optimal pre-culture time. The results are shown in Table 2.

[0072] Appropriate pre-culture promotes multiple cell divisions and vigorous growth in plant cells, placing the explants in a competent state where they readily accept exogenous DNA, thus facilitating Agrobacterium infection. Furthermore, phenolic substances secreted from plant wounds are released into the culture medium, guiding Agrobacterium to invade the wound site and promoting transformation. Compared to no pre-culture, a certain period of pre-culture significantly improves plant transformation efficiency. However, excessively long pre-culture leads to wound healing in the plant explants, reducing transformation efficiency and hindering Agrobacterium infection and transformation.

[0073] Statistical data shows significant differences in resistant bud transformation between different pre-culture durations. The transformation efficiency of *Nepeta cataria* stem segments without pre-culture was the lowest, with severe browning in the later stages, as antibiotics and other substances in the culture medium inhibited growth and promoted senescence and death. Pre-culture for 5 to 10 days resulted in a similar rate of resistant seedling acquisition, reaching a maximum of 45.5%, with the optimal pre-culture time being 10 days. Extending the pre-culture time to 20 days reduced the efficiency of resistant plant acquisition, and resulted in more stem segments browning and dying in the later stages, which was detrimental to genetic transformation.

[0074] Table 2. Effects of pre-culture time on genetic transformation of Nepeta cataria stem segments.

[0075]

[0076] S3. Stem segment infection

[0077] Following step S2, the *Nepeta cataria* stem segments underwent pre-culture, bacterial inoculation, co-culture, screening, rooting, hardening-off, and transplanting. The pre-culture period was set at 10 days. The stem segments were then inoculated using the bacterial solution obtained in step S2. OD values ​​of the bacterial solutions were prepared using sterile MS salt solution. 600 Values ​​of 0.5, 0.7, and 1.0 were used to infect the pre-cultured stem segments. The rate of resistant seedlings obtained after transformation culture at the same pre-culture time, infection time, and co-culture time after 40 days was statistically analyzed to determine the optimal bacterial concentration. The results are shown in Table 3.

[0078] Both excessively high and low Agrobacterium concentrations reduce the transformation rate. In this experiment, the transformation efficiency of Nepeta cataria stem segments showed a trend of first increasing and then decreasing with the concentration of the bacterial solution. If the bacterial solution concentration is too low, the number of Agrobacterium on the explants is too small, resulting in a lower transformation rate. If the bacterial solution concentration is too high, too many Agrobacterium will produce toxic substances that inhibit explant respiration and also affect their attachment at the explant wound site, thus reducing the transformation rate.

[0079] Statistical data shows that different bacterial concentrations result in varying effects on resistant bud transformation. OD 600 The transformation efficiency of *Nepeta cataria* stem segments infected with a low concentration of 0.5% bacterial solution was low, and the regenerated buds of *Nepeta cataria* failed to acquire resistance. OD 600 When the concentration was increased to 0.7, the resistant seedling acquisition rate was the highest, at 41.78%, which is the suitable bacterial suspension concentration. When the bacterial suspension concentration increased to OD...600 When the value is 1.0, the rate of obtaining resistant seedlings decreases. At the same time, the original antibiotic content in the screening medium is difficult to stably inhibit the growth of Agrobacterium, and colonies appear, inhibiting the normal growth and transformation of stem segments.

[0080] Table 3. Effects of bacterial concentration on genetic transformation of Catnip stem segments

[0081]

[0082] Infection times were set to 5 min, 15 min, and 30 min, respectively. Infected stem segments were obtained after infection. The rate of obtaining resistant seedlings after transformation culture was calculated 40 days later under the same pre-culture, infection solution concentration, and co-culture time. The optimal infection time was determined, and the results are shown in Table 4.

[0083] The effect of infection time varies among different plant species; for some plants, the transformation efficiency is unaffected by infection time. In this experiment, the optimal infection time was 15 minutes. If the time is too short, Agrobacterium will not have fully penetrated the wound; however, there is no significant difference in transformation efficiency between 15 and 30 minutes of infection time. Considering the time cost of operation, an infection time of 15 minutes should be chosen.

[0084] Table 4. Effects of bacterial infection time on genetic transformation of Catnip stem segments

[0085]

[0086] S4. Stem segment co-culture

[0087] The infected stem segments were transferred to a co-culture medium and co-cultured in a dark environment for 1 day, 3 days and 5 days, respectively, at a temperature of (24±1)℃. Transformed seedlings were obtained after the culture was completed. The results are shown in Table 5.

[0088] Co-culture significantly impacts the transformation rate of explants, as the transfer of Agrobacterium T-DNA sequences into plants primarily occurs during the co-culture period. If the co-culture time is too short, antibiotics in the selection medium kill bacteria at the plant wounds and inhibit the normal growth of untransformed plants. Conversely, if the co-culture time is too long, the massive proliferation of Agrobacterium poisons the plant recipient, leading to the formation of Agrobacterium colonies on the culture medium and inhibiting normal plant growth.

[0089] Statistical data shows significant differences in resistant bud transformation across different co-culture times. With a co-culture time of 1 day, the transformation efficiency of infected Catnip stem segments was low, failing to induce resistance in regenerated Catnip buds, and the plants succumbed to antibiotic resistance, turning brown and dying. A co-culture time of 3 days resulted in the highest resistant seedling acquisition rate (48.3%), representing the optimal co-culture time. When the co-culture time was extended to 5 days, the resistant seedling acquisition rate was lowest, and the antibiotic content in the selection medium was insufficient to stably inhibit Agrobacterium growth, leading to colony formation and inhibiting normal stem segment growth and transformation.

[0090] Table 5. Effects of co-cultivation time on genetic transformation of Nepeta cataria stem segments.

[0091]

[0092] S5. Screening of positively transformed seedlings

[0093] After co-culture, the stem segments were transferred to an induction and selection medium for regeneration bud induction culture. The culture temperature was (24±1)℃, the light source was a fluorescent lamp with a light intensity of 3000lx, and the light cycle was 16 hours of light / 8 hours of darkness. Every 5-7 days, the regenerated buds were taken out and placed in another fresh induction and selection medium to maintain the effects of plant hormones and antibiotics.

[0094] S6. Rooting Culture

[0095] Adventitious buds of *Nepeta cataria* with a length of 1.0-1.5 cm were separated by forceps in a clean bench and inoculated into rooting medium for rooting culture. The culture temperature was (24±1)℃, the light source was fluorescent lamp with a light intensity of 3000 lx, and the photoperiod was 16 hours light / 8 hours dark. The NAA concentration used in the rooting medium for adventitious roots was 0.4 mg / L. After 15 days of rooting culture, the adventitious roots grew to 1.2-1.5 cm, were relatively thick and short, and were covered with root hairs, which was conducive to the absorption of nutrients by the adventitious buds of *Nepeta cataria*.

[0096] S7. Seedling hardening and transplanting

[0097] When the adventitious roots of the robust, sterile seedlings reach 1.5-2.0 cm in length, place the culture bottles outdoors under natural light for 3 days to harden them off. Then, in a light-cured incubator, open the bottle caps and cover the surface of the culture medium with water. After 3 days of hardening off, remove the plants, wash away any remaining culture medium from the roots with running water, and transplant them into a soilless culture medium containing vermiculite and perlite in a 1:1 weight ratio. In the light-cured incubator, maintain a temperature of (24±1)℃, use fluorescent lamps with a light intensity of 3000 lx, a light cycle of 16 hours light / 8 hours darkness, and a relative humidity of 50-80%. After 15 days of cultivation, assess the transplant survival rate; the plants should be robust. After one month of cultivation, move them outdoors to a normal environment until the catnip flowers.

[0098] S8. Fluorescence detection

[0099] GFP fluorescence was observed in some of the resistant plants obtained in step S7 under an upright fluorescence microscope. Wild-type Nepeta cataria (WT) was used as a negative control. Under excitation light with a fluorescence wavelength of 488 nm, the WT wild-type plants showed no fluorescence, while the resistant plants showed green fluorescence, which indicated positive plants.

[0100] S9. PCR detection of resistant plants

[0101] Total DNA was extracted from some of the resistant plants obtained in step S7, and the expression of the target gene was detected by PCR amplification, with wild-type Nepeta cataria (WT) as a negative control. The primers used are shown in Table 6. Partial results of 1% agarose gel electrophoresis of the obtained PCR products are shown; the wild-type plants (negative control) showed no target band, while the positive plants amplified the target band. Based on the PCR results, the transgenic rate was calculated to be approximately 28.05%.

[0102] Table 6 Primer sequence information

[0103]

[0104] Example 2

[0105] Following the method in Example 1, the co-culture environment was changed from darkness and temperature (24±1)℃ to light intensity of 3000lx, light cycle of 16 hours light / 8 hours darkness, and temperature (24±1)℃, while other steps remained unchanged. The state of the catnip stem segments was observed and the final resistant bud acquisition rate was statistically analyzed.

[0106] In the dark environment, the lower end of the catnip stem segments swelled, the axillary buds were pale yellow, and callus tissue appeared at the wound site. Catnip stem segments without dark environment pre-culture showed no obvious changes, the axillary buds were green, and the transformation efficiency was lower than that of the dark environment pre-culture treatment.

[0107] Example 3

[0108] Following the method in Example 1, TDZ in all culture media was replaced with 6-BA. The hormone ratio in the regeneration bud induction medium, pre-culture medium, co-culture medium, and induction screening medium was set to 0.8 mg / L NAA and 2 mg / L 6-BA. All other steps remained unchanged. The regeneration bud induction rate, resistant bud acquisition rate, and rooting status of the regenerated plants during the culture and genetic transformation process were statistically analyzed, and the plant growth status was observed.

[0109] Experiments showed that replacing TDZ with 6-BA had no effect on the regeneration of Nepeta cataria stem segments, but the callus formed did not further differentiate into adventitious buds. Among various NAA and 6-BA combinations, the combination of 0.8 mg / L NAA and 2 mg / L 6-BA was optimal, resulting in good growth of Nepeta cataria stem segments. At 10 days, the stems showed significant swelling, and callus tissue appeared at the lower morphological end of the stem segments, with small adventitious buds. At 20 days, significant callus tissue was produced on the stems and petioles, and the leaves turned pale yellow. The NAA and 6-BA combination culture medium had no significant effect on the genetic transformation rate of Nepeta cataria and can be used as an alternative formula for stem segment genetic transformation.

[0110] Example 4

[0111] Following the method in Example 1, the hormone components in the regeneration bud induction medium, pre-culture medium, co-culture medium, and induction screening medium were set to NAA, TDZ, and 6-BA, with a specific ratio of 0.2 mg / L NAA, 1.0 mg / L TDZ, and 1.0 mg / L 6-BA. All other steps remained unchanged. The regeneration bud induction rate, resistant bud acquisition rate, and rooting status of the regenerated plants during the culture and genetic transformation process were statistically analyzed, and the plant growth status was observed.

[0112] Experiments showed that using NAA, 6-BA and TDZ culture media simultaneously had poor effects on inducing differentiation and regeneration of Catnip callus, but had no effect on the regeneration of Catnip stem segments and no significant effect on the genetic transformation efficiency of Catnip stem segments.

[0113] Example 5

[0114] Following the method in Example 1, the basic content of 1 / 2 MS in the regeneration bud induction medium, pre-culture medium, co-culture medium and induction screening medium was replaced with MS, while other steps remained unchanged. The regeneration bud induction rate, resistant bud acquisition rate and rooting status of Catnip regeneration plants during the culture process and genetic transformation process were statistically analyzed, and the plant growth status was observed.

[0115] Experiments showed that increasing the MS (monochromatographic medium) concentration increased the ion concentration in the medium, altering the growth status of regenerated buds and new leaves. Leaves in full-volume MS were translucent and brittle; the plants could grow and root normally, but hardening and transplanting were difficult, and the leaves could not withstand strong light and dry outdoor environments. In contrast, new leaves in half-volume MS medium were normal, allowing for rapid propagation and genetic transformation of *Nepeta cataria*, making it a better choice for hardening and transplanting.

[0116] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.

Claims

1. An Agrobacterium-mediated genetic transformation method for Nepeta cataria, characterized in that, Includes the following steps: (1) With catnip Schizonepeta tenuifolia Briq. The stem segments were used as explants. After pre-culturing on an antibiotic-free culture medium, the stem segments of Nepeta cataria were infected with an infection solution containing recombinant Agrobacterium. The infected Nepeta cataria stem segments were then cultured on a co-culture culture medium to obtain co-cultured explants. The pre-culture time was 10 days, and the co-culture time was 3 days. The co-culture conditions were darkness and a culture temperature of 24 ± 1℃. The concentration of the infection solution met the OD requirements. 600 The value was 0.7, and the infection time was 15 minutes; Both the pre-culture and co-culture media consist of a solvent and a solute. The solvent is 1 / 2 MS medium, and the solutes and their concentrations in the medium are 0.5 mg / L naphthaleneacetic acid (NAA) and 1.5 mg / L thidiazuron (TDZ), respectively, with a pH of 5.

8. (2) The co-cultured explants were cultured on an induction and selection medium to obtain resistant shoots of Nepeta cataria. The induction and selection medium consisted of a solvent and a solute. The solvent was 1 / 2 MS medium. The solute and its concentration in the induction and selection medium were 0.5 mg / L naphthaleneacetic acid (NAA), 1.5 mg / L thiazuron (TDZ), and the corresponding concentration of antibiotics, respectively. The pH value was 5.

8. The culture temperature was 24±1℃, the light intensity was 3000 lx, and the photoperiod was 16 hours of light / 8 hours of darkness. (3) The resistant shoots of Catnip were cultured on a rooting selection medium to obtain resistant Catnip plants. The rooting selection medium consisted of a solvent and a solute. The solvent was 1 / 2 MS medium. The solute and its concentration in the rooting selection medium were 0.4 mg / L naphthaleneacetic acid (NAA) and the corresponding concentration of antibiotics, respectively. The pH value was 5.

8. The culture temperature was 24±1℃, the light intensity was 3000 lx, and the photoperiod was 16 hours of light / 8 hours of darkness. (4) Screening positive plants containing the marker gene GFP from catnip resistant plants to obtain catnip positive plants that have completed genetic transformation.

2. The method for Agrobacterium-mediated genetic transformation of Nepeta cataria according to claim 1, characterized in that, In step (1), the recombinant Agrobacterium contains a recombinant expression vector carrying the marker gene GFP, and the infection solution is a liquid obtained by suspending the recombinant Agrobacterium in MS liquid culture medium.

3. The method for Agrobacterium-mediated genetic transformation of Nepeta cataria according to claim 2, characterized in that, The recombinant expression vector is pCAMBIA1305, obtained by inserting a GFP marker gene.

4. The method for Agrobacterium-mediated genetic transformation of Nepeta cataria according to claim 1, characterized in that, The identification of positive plants in step (4) includes GFP fluorescent protein identification and DNA molecular identification.

5. The method for Agrobacterium-mediated genetic transformation of Nepeta cataria according to claim 1, characterized in that, The method also includes obtaining regenerated plants from the genetically transformed Nepeta cataria plants.

6. The application of the Agrobacterium-mediated genetic transformation method for Nepeta cataria according to any one of claims 1-5 in the cultivation of Nepeta cataria varieties / lines.

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