Agrobacterium-mediated genetic transformation method of pepper and application thereof

Abstract

The present application belongs to the field of plant tissue culture and bioengineering, and particularly relates to a method for genetic transformation of pepper mediated by Agrobacterium and application thereof. The method comprises the following steps: S1, using cotyledon and hypocotyl stem segment of pepper seedling as explants; S2, directly using Agrobacterium liquid with OD600 of 0.3-0.6 to infect the explants without pre-culture; S3, transferring the explants to callus induction medium after co-culture; S4, transferring the explants to bud emergence medium after green bud spots appear on the explants; S5, cutting the bud and transferring it to rooting medium after the regenerated bud elongates; and S6, performing transgenic detection on the transgenic plants after rooting. The present application provides a method for genetic transformation of pepper mediated by Agrobacterium, and the effective transformation efficiency reaches more than 5%, which breaks through an important bottleneck of genetic transformation of pepper. Through the transformation system, gene editing of pepper can be performed to obtain edited plants, which has great application value.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture and bioengineering, specifically relating to Agrobacterium-mediated genetic transformation of peppers and its applications. Background Technology

[0002] Chili peppers are one of the most important vegetables globally. Since Gunay first conducted chili pepper tissue culture work in 1978, numerous research reports have been published exploring the establishment of in vitro regeneration systems for chili peppers. However, chili pepper in vitro regeneration technology mainly focuses on the bud induction stage, and problems such as difficulty in bud elongation and rooting still exist. For example, CN109258469A used MS + 3.0 mg / L 6-BA + 0.1 mg / L IAA as the adventitious bud induction medium, and GA3 was added to the adventitious bud induction medium for bud elongation. CN108338073A also used GA3 to induce bud elongation. However, the effect of GA-induced adventitious bud elongation in chili pepper in vitro regeneration is not satisfactory, with low bud elongation efficiency and genotype dependence.

[0003] The technology for obtaining regenerated chili plants through tissue culture is difficult to master, making it difficult to achieve genetic transformation of chili using DNA recombination technology.

[0004] Establishing efficient in vitro regeneration and genetic transformation systems is fundamental for the efficient genetic improvement of crops using genetic engineering techniques. However, significant breakthroughs have not yet been achieved in establishing in vitro regeneration and genetic transformation systems for chili peppers, severely limiting the application of genetic engineering and gene editing in chili pepper genetic improvement.

[0005] Recently, significant progress has been made in gene editing of peppers. Zhao et al. (2024) established an efficient gene editing technology in peppers without transgenic components using a CRISPR / Cas nuclease transient delivery system mediated by tomato spotted wilt virus (TSWV) (Zhao C, Lou H, Liu Q, Pei S, Liao Q, Li Z (2024) Efficient and transformation-free genome editing in pepper enabled by RNA virus-mediated delivery of CRISPR / Cas9. J Integr Plant Biol 66:2079-2082). However, because this system relies on tissue culture regeneration of virus-mediated edited cells, obtaining edited plants with stable inheritance of the target gene remains challenging. To date, stable expression of transgenes in peppers remains a major challenge, a prominent bottleneck in pepper functional gene research and genetic improvement, necessitating the establishment of an efficient and stable in vitro regeneration and genetic transformation system for peppers.

[0006] To establish a stable transformation system in chili peppers, the inventors first attempted the maturation method they used in tomatoes, which utilizes a combination of zeatin and auxin hormones and employs a two-step regeneration process. In the second regeneration step, the cytokinin level was significantly reduced (Ouyang B, Chen YH, Li HX, Qian CJ, Huang SL, Ye ZB (2005) Transformation of tomato with osmotin and chitinase genes and their resistance to Fusarium wilt. J Hortic Sci Biotech 80:517-522). Following the tomato genetic transformation protocol, a dwarf pepper variety, MiniPep (Shi C, Shen X, Zhang Z, Zhou Y, Chen R, Luo J, Tang Y, Lu Y, Li F, Ouyang B (2022) Conserved role of fructokinase-like protein 1 in chloroplast development revealed by a seedling-lethal albino mutant of pepper. Hortic Res 9:uhab084), was tested. However, the frequency of positive transgenic plants obtained was extremely low; only a very small number of transgenic plants could be obtained from several hundred cotyledonary explants, making practical application difficult. Therefore, it is necessary to develop an effective pepper genetic transformation system. Summary of the Invention

[0007] This invention uses the ability of chili peppers to develop lateral branches as an indicator of regeneration capacity. Through exploration and optimization of relevant conditions, a genetic transformation system for chili peppers was finally obtained, thus completing this invention.

[0008] This invention discloses an Agrobacterium-mediated genetic transformation method for peppers, which includes the following steps:

[0009] S1 uses cotyledons and hypocotyl stem segments of pepper seedlings as explants;

[0010] S2 explants were directly inoculated into a suspension of Agrobacterium with an OD600 of 0.3-0.8 without pre-culturing. The explants were gently shaken to ensure full contact between the bacterial solution and the explants. The explants were then placed in a vacuum device and treated under a negative pressure of 0.4-0.8 MPa for 2-8 minutes. Afterward, excess bacterial solution was discarded and aspirated from the explants.

[0011] After co-culturing S3 explants for 1-4 days (preferably 2 days), they are transferred to callus induction medium. The induction medium is based on MS medium and supplemented with 1-3 mg / L zeatin, 0.05-0.15 g / L IAA, 300-400 mg / L termethin, 70-80 mg / L kanamycin sulfate, 3-5 mg / L silver nitrate, 7-8 g / L agar, and 25-35 g / L sucrose.

[0012] S4 When green buds appear on the explants, budding culture is carried out. The induction medium is changed to: MS medium as the base medium, with the following added: 0.2-0.8 mg / L zeatin, 0.15-0.25 g / L IAA, 300-400 mg / L termethin, 70-80 mg / L kanamycin sulfate, 3-5 mg / L silver nitrate, 80-120 mg / L activated carbon, 7-8 g / L agar, and 25-35 g / L sucrose.

[0013] After the regenerated shoots of S5 have elongated, cut them off and transfer them to the rooting medium.

[0014] Transgenic testing was performed on S6 after it rooted.

[0015] Preferably, 10-14 day old chili seedlings are used in step S1.

[0016] Specifically, the Agrobacterium target gene expression vector or gene editing vector mentioned in step S2 preferably also includes a reporter gene that facilitates the screening of transgenic plants, such as RUBY expressing betalains or GFP expressing green fluorescent protein.

[0017] More specifically, in step S2, the explants are directly inoculated into Agrobacterium bacterial suspension with an OD600 of 0.3-0.8 without pre-culturing. The suspension is slowly shaken to ensure full contact between the bacterial suspension and the explants. The explants are then placed in a vacuum device and treated under negative pressure of 0.6 MPa for 5-6 minutes. Afterward, excess bacterial suspension on the explants is discarded and aspirated.

[0018] Specifically, after co-culturing S3 explants for 1-3 days (e.g., 2 days), they are transferred to callus induction medium with the following formulation: 4.4 g / L MS basal medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 1.5-2.5 mg / L zeatin, 0.08-0.12 g / L IAA, 340-380 mg / L termethin, 74-76 mg / L kanamycin sulfate, and 3.5-4.5 mg / L silver nitrate.

[0019] Preferably, in step S4, when green buds appear on the explants, they are transferred to a budding medium for culture, the formulation of which is: 4-5 g / L (preferably 4.3-4.5) MS medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 0.2-0.6 mg / L zeatin, 0.18-0.22 g / L IAA, 300-380 mg / L termethin, 74-76 mg / L kanamycin sulfate, and 3.5-4.5 mg / L silver nitrate.

[0020] Specifically, the rooting medium in step S5 is formulated with MS medium as the base medium, supplemented with 7-8 g / L agar, 25-35 g / L sucrose, 300-400 mg / L termethin and 1.5-2.5 mg / L IBA.

[0021] Preferably, the rooting medium in step S5 contains 4.4 g / L MS medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 340-380 mg / L termethin and 1.8-2.2 mg / L IBA.

[0022] Choose any location, and after the S7 seedlings have rooted, harden them off and transplant them. This can be done using conventional methods.

[0023] The present invention also provides the use of the method in chili genetic transformation or gene editing.

[0024] The advantages of this invention are as follows: by screening different reporter genes, fluorescent markers such as RUBY were found to be suitable as reporter genes for pepper transformation; studies on negative pressure treatment and no pre-culture treatment were conducted, revealing that simultaneous negative pressure and no pre-culture treatment significantly improved the transformation efficiency of cotyledon explants; the optimal antibiotic concentration and optimal explants were also investigated, ultimately providing an Agrobacterium-mediated genetic transformation method for pepper with an effective transformation efficiency of over 5%, breaking through a major bottleneck in pepper genetic transformation. This transformation system can be used for pepper gene editing to obtain edited plants. Attached Figure Description

[0025] Figure 1 The schematic diagram illustrates the carrier structure used in this study.

[0026] Figure 2 eGFP-transformed callus and plants were observed under bright field and GFP channel conditions. Cotyledonary explants were cultured in callus induction medium (CIM) for 30 days. Transgenic plants were transplanted into nutrient pots containing vermiculite substrate and grown for one month.

[0027] Figure 3The RUBY phenotype is shown in T0 generation callus and transgenic plants. Transformed cotyledonary and hypocotyl explants were cultured in CIM for 40 days (left) and subcultured in budding medium (SIM) for 50 days. Explants within the dashed boxes are shown in magnified views. The right side shows one regenerated bud and three rooted plants exhibiting the RUBY phenotype.

[0028] Figure 4 The RUBY phenotype is shown in T1 generation transgenic plants. Seedlings 7 days after germination show segregation of the RUBY phenotype. Two-month-old plants show the RUBY phenotype, which is particularly evident in fruits and flowers.

[0029] Figure 5 This shows the effect of negative pressure treatment and pre-incubation time on transformation efficiency. V, -0.6 MPa negative pressure treatment; P, pre-incubation for 1 day; NV, no negative pressure; NP, no pre-incubation. Values ​​are expressed as mean ± standard error (n=3) and were arcsine transformed before ANOVA and Duncan's method for multiple comparisons. Different letters above the bars indicate significance at P < 0.05.

[0030] Figure 6 A flowchart illustrating the genetic transformation of peppers. RIM, rooting medium.

[0031] Figure 7 This image shows albino shoots produced by CRISPR / Cas9-mediated CaPDS gene editing. The top image shows the structure of the pepper CaPDS gene, the target editing site, and the PAM sequence. Sanger sequencing confirmed a single-base deletion variant upstream of the PAM. The albino plants are shown from the side (left) and top (right). WT, wild-type control. Detailed Implementation

[0032] The present invention will be described below through specific embodiments to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0033] Example 1: Selection of reporter genes

[0034] In plant genetic transformation, the application of marker genes provides researchers with a direct way to track and confirm transgenic events. To facilitate the observation of transformation events, we constructed three reporter gene transgenic vectors (…). Figure 1 Electroporation was performed on Agrobacterium GV3101 to compare the effectiveness of red fluorescent protein DsRed, green fluorescent protein GFP, and betalain reporter system RUBY.

[0035] Sowing of sterile seedlings: Take about 200 chili seeds, soak them in distilled water for 3 hours, and then surface sterilize them with 75% alcohol for 1-2 minutes in a clean bench. After discarding the alcohol, use 50% 84 disinfectant solution to seal the seeds in a sterile Erlenmeyer flask and shake to sterilize for 15 minutes. Discard the 84 disinfectant solution and rinse the seeds 3-4 times with sterile water. Sow the seeds in a 1 / 2 MS culture flask and incubate them in the dark at 28℃ for 5-8 days to promote germination. After germination, transfer the seeds to a tissue culture room for 6-10 days of light culture until the two cotyledons unfold.

[0036] Pre-culture: Select seedlings with two fully expanded cotyledons, cut off the cotyledons from the petiole, and cut the cotyledons and hypocotyls into 2-3 segments with a blade. Then, spread the cut explants flat on the culture medium covered with sterile filter paper, seal the culture dish with sealing film to prevent bacterial and fungal invasion, and place the culture dish in the tissue culture room for dark incubation for 1 day.

[0037] Agrobacterium activation and culture: Single colonies of Agrobacterium GV3101 containing the target vector were picked and inoculated onto solid LB agar plates with the appropriate resistance. After incubation in the dark at 28°C for 2 days, fresh cells were picked and transferred to 1 mL of LB liquid medium containing 50 mg / L kanamycin and 35 mg / L rifampin. The culture was incubated at 200 rpm and 28°C for 8-10 h. 100 μL of the bacterial culture was then transferred to 10 mL of LB liquid medium containing 50 mg / L kanamycin for expansion culture. The culture was incubated at 200 rpm and 28°C for 14-20 h until the OD600 of the bacterial culture was approximately 1.0. The bacterial culture was collected by centrifugation at 5000-8000 rpm for 2 min, the supernatant was discarded, and the cells were then resuspended in 0.2 MS liquid medium and diluted to OD600 = 0.3-0.6 for later use.

[0038] Infection: Pour a 0.2MS suspension containing Agrobacterium into a sterile culture flask, and transfer cotyledonary explants cultured in the dark for 1 day into the 0.2MS medium for infection. Gently shake for 5 minutes to ensure full contact between the bacterial suspension and the explants. After infection, pour out the bacterial suspension, blot dry the explants with sterile filter paper, and place the infected explants back onto KCMS medium for co-culturing in the dark for 2 days.

[0039] Regeneration and subculture: After co-culture, the explants were transferred to callus induction medium (CIM) with 20-25 explants per dish and placed in the tissue culture room. After a few weeks, when obvious green buds appeared on the explants, the explants were transferred to budding medium (SIM) and placed in the tissue culture room. After 1-2 months, when the regenerated buds elongated, the buds were cut off and transferred to rooting medium (RIM). After rooting, transgenic detection was performed, followed by hardening and transplanting.

[0040] Reporter gene observation: For DsRed and GFP, expression was detected in regenerated callus, shoots, and roots during tissue culture using a handheld fluorescence detector (LUYOR-3415RG, Luyang, Shanghai). For RUBY, expression was observed visually. We found that obvious fluorescence was difficult to observe in DsRed-transformed callus, while green fluorescence could be detected in GFP-transformed callus, although it was difficult to observe in regenerated shoots. Using a portable fluorescence detector, potential GFP signals could be observed in the roots of transformed regenerated plants. Figure 2 The study showcased callus and rooted seedlings from cotyledonary explants. RUBY, as a visually observable reporter system, exhibited a red phenotype in transformed callus tissue and in the seedlings, leaves, roots, and flowers of regenerated plants, indicating that RUBY is a suitable reporter gene for visual observation of pepper transformation. Figure 3 (This shows transgenic callus, buds, and rooted seedlings from cotyledons and hypocotyl explants).

[0041] Furthermore, we investigated the genetics of the transgenic plant progeny and found that all T0 plants produced offspring with the RUBY phenotype. In some transgenic lines, the RUBY phenotype segregation conformed to a Mendelian segregation ratio of 3:1. Figure 4 (A offspring of a hypocotyl explant is shown), indicating that the transgene can be stably inherited by offspring.

[0042] Example 2: Determination of Pretreatment

[0043] Pre-culture treatment: Select sterile pepper seedlings with two fully expanded cotyledons, cut off the cotyledons at the petiole, and cut the cotyledons and hypocotyls into 2-3 segments each with a blade. Place the cut explants flat on a culture medium lined with sterile filter paper, and seal the petri dish with sealing film to prevent bacterial and fungal invasion. Incubate the petri dishes in the dark in a tissue culture room for 1 day or use them directly for infection (without pre-culture).

[0044] Infection: A 0.2 MS suspension containing Agrobacterium was poured into a culture flask, and pre-cultured explants (without pre-culture or with 1 day of pre-culture) were immersed in 0.2 MS liquid medium for infection. The flask was gently shaken for 5 minutes to ensure full contact between the bacterial suspension and the explants, and then placed in a vacuum apparatus under 0.6 MPa negative pressure for 5 minutes. After infection, the bacterial suspension was poured out, and the explants were blotted dry with sterile filter paper. The infected explants were then returned to KCMS medium and co-cultured in the dark for 2 days. The transformation efficiency was measured by the ratio of explants exhibiting the RUBY phenotype to the total number of explants. We investigated the effects of negative pressure treatment and no pre-culture on transformation efficiency. Explants were placed flat on culture medium lined with sterile filter paper and cultured in the dark in a tissue culture room for 1 day (pre-culture), while direct infection without pre-culture was designated as NP. Explants were then placed in 0.2 MS liquid medium resuspending Agrobacterium for infection and subjected to a vacuum device at 0.6 MPa for 5 min (V), while no vacuum treatment was designated as NV. Four treatment combinations were set up: NP+NV, NP+V, P+NV, and P+V. It was found that combining no pre-culture with vacuum treatment (NP+V) significantly improved the transformation efficiency of cotyledonary explants. Figure 5 The statistical results of cotyledon explants are shown, with the x-axis combinations as shown in the text above: NP+NV (no pre-culture + no negative pressure treatment), NP+V (no pre-culture + negative pressure treatment), P+NV (with pre-culture + no negative pressure treatment), P+V (with pre-culture + negative pressure treatment). However, this effect was not observed in hypocotyl explants.

[0045] Example 3: Determination of the chili pepper conversion screening system

[0046] To determine the optimal kanamycin resistance screening concentration for transgenic pepper regeneration, this study explored the regeneration capacity of cotyledonary explants at different concentrations of kanamycin (Kan). Using pepper cotyledonary explants as the research material, the effects of different Kan concentrations (50, 75, 100, and 125 mg / L) on callus formation and shoot regeneration capacity of untransformed explants were compared. The study found that a Kan concentration of 75 mg / L was most suitable for screening transgenic peppers. After culturing pepper cotyledonary explants on regeneration media with different Kan concentrations for 20 days, callus formation and shoot emergence were observed. On a culture medium without Kan, all cotyledonary explants of pepper formed callus, with some regenerating buds appearing at the cut surfaces. At a Kan concentration of 50 mg / L, white callus and a small amount of green regenerating bud-like tissue were observed at the cotyledonary cut surfaces. However, at a concentration of 75 mg / L, only a small amount of callus appeared at the edges of the cotyledonary explants, with no obvious green bud formation, and a phenotype of edge chlorosis and yellowing was observed at the cotyledonary cut surfaces. When the Kan concentration was higher than 75 mg / L, no obvious callus formation was observed at the cotyledonary cut surfaces, and the cut surfaces showed significant yellowing. Since higher concentrations of Kan inhibit the regeneration of both transgenic and non-transgenic cells, while lower concentrations are ineffective in inhibiting regeneration, this study determined 75 mg / L as the optimal screening concentration. A suitable Kan concentration will provide screening pressure for the regeneration of transgenic peppers, improving the efficiency and accuracy of transgenic experiments.

[0047] Example 4: Determination of the chili pepper conversion system

[0048] Through the optimization of multiple factors, we have successfully developed an effective chili pepper conversion system. Figure 6 The following is an example of the optimal implementation.

[0049] In a preferred embodiment, the method is as follows:

[0050] S1 uses 12-day-old seedlings to prepare cotyledon stem segments as explants.

[0051] S2 explants were directly inoculated into an Agrobacterium suspension with an OD600 of 0.6 (the overexpression vector of RUBY and the knockout vector of CaPDS were tested separately), without pre-culturing. The explants were gently shaken for 5 minutes to ensure full contact between the bacterial suspension and the explants, and then placed in a vacuum apparatus for 0.6 MPa negative pressure treatment for 5 minutes. Afterward, excess bacterial suspension on the explants was discarded and aspirated.

[0052] After co-culturing S3 explants for 2 days, they were transferred to callus induction medium (CIM) for culture. The CIM formulation was: 4.4 g / L MS basal medium, 7.4 g / L agar, 30 g / L sucrose, 2 mg / L zeatin, 0.1 g / L IAA, 360 mg / L termethin, 75 mg / L kanamycin sulfate, and 4 mg / L silver nitrate.

[0053] When obvious green buds appear on the explants, reduce the zeatin concentration to 0.5 mg / L, replace IAA with 0.17 mg / L gibberellin (GA3), and add 100 mg / L activated charcoal.

[0054] After the regenerated shoots of S5 have elongated, the shoots are cut off and transferred to rooting medium (RIM). The RIM formula contains 4.4 g / L MS basal medium, 7.4 g / L agar, 30 g / L sucrose, 360 mg / L termethin and 2 mg / L IBA.

[0055] After the S6 seedlings have rooted, they undergo transgenic testing, hardening-off, and transplanting.

[0056] Using our transformation system, the effective transformation efficiency is defined as the ratio of the total number of explants with the RUBY phenotype to the total number of explants. The effective transformation efficiency reaches more than 5% (Table 1), which has practical value.

[0057] Table 1

[0058] Number of explants RUBY explant number RUBY bud number Effective conversion rate (%) 1 35 25 2 5.7 2 29 20 1 3.5 3 38 27 2 5.3

[0059] When we applied this transformation system to gene editing vectors, cotyledonary explants yielded albino leaves and albino regenerated shoots. Target amplification and sequencing of the albino regenerated shoots showed sequence editing at the target sites, indicating that this transformation system can also be applied to gene editing in chili peppers. Figure 7 ).

Claims

1. An Agrobacterium-mediated genetic transformation method for peppers, characterized in that, Includes the following steps: S1 uses cotyledons and hypocotyl stem segments of pepper seedlings as explants; S2 explants were directly inoculated into an Agrobacterium suspension with an OD600 of 0.3-0.6 without pre-culturing. The explants were gently shaken to ensure full contact between the bacterial suspension and the explants. The explants were then placed in a vacuum device and treated under a negative pressure of 0.4-0.8 MPa for 2-8 minutes. Afterward, excess bacterial suspension on the explants was discarded and aspirated. After co-culturing S3 explants for 1-3 days, they were transferred to callus induction medium. The induction medium was based on MS medium, supplemented with 1-3 mg / L zeatin, 0.05-0.15 g / L IAA, 300-400 mg / L termethin, 70-80 mg / L kanamycin sulfate, 3-5 mg / L silver nitrate, 7-8 g / L agar, and 25-35 g / L sucrose. After green buds appeared on the explants, S4 were transferred to budding medium for culture. The induction medium was changed to: MS medium as the base medium, with the following added: 0.2-0.8 mg / L zeatin, 0.15-0.25 g / L IAA, 300-400 mg / L termethin, 70-80 mg / L kanamycin sulfate, 3-5 mg / L silver nitrate, 80-150 mg / L activated charcoal, 7-8 g / L agar, and 25-35 g / L sucrose. After the regenerated shoots of S5 have elongated, cut them off and transfer them to the rooting medium. Transgenic testing was performed on S6 after it rooted.

2. The method as described in claim 1, characterized in that, In step S1, 10-14 day old chili seedlings are used.

3. The method as described in claim 1, characterized in that, The Agrobacterium target gene expression vector or gene editing vector mentioned in step S2 preferably also includes a reporter gene that facilitates the screening of transgenic plants, such as RUBY expressing betaine or GFP expressing green fluorescent protein.

4. The method as described in claim 1, characterized in that, In step S2, the explants are not pre-cultured and are directly infected with Agrobacterium suspension with an OD600 of 0.30-0.

65. The explants are slowly shaken to ensure full contact between the bacterial suspension and the explants, and then placed in a vacuum device under negative pressure of 0.6 MPa for 5-6 minutes. After that, the excess bacterial suspension on the explants is discarded and aspirated.

5. The method as described in claim 1, characterized in that, After co-culturing S3 explants for 1-3 days (preferably 2 days), they are transferred to callus induction medium with the following formulation: 4-5 g / L MS basal medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 1.5-2.5 mg / L zeatin, 0.08-0.12 g / L IAA, 340-380 mg / L termethin, 74-76 mg / L kanamycin sulfate, and 3.5-4.5 mg / L silver nitrate.

6. The method as described in claim 1, characterized in that, In step S4, when green buds appear on the explants, the explants are transferred to a budding medium with the following formula: 4.3-4.5 g / L MS basal medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 0.2-0.6 mg / L zeatin, 0.18-0.22 g / L IAA, 340-380 mg / L termethin, 74-76 mg / L kanamycin sulfate, and 3.5-4.5 mg / L silver nitrate.

7. The method as described in claim 1, characterized in that, The rooting medium in step S5 is based on MS medium, with the addition of 7-8 g / L agar, 25-35 g / L sucrose, 320-400 mg / L termethin, and 1.5-2.5 mg / L IBA.

8. The method as described in claim 7, characterized in that, The rooting medium in step S5 contains 4-5 g / L (preferably 4.3-4.5) MS basal medium, 7.2-7.6 g / L agar, 28-32 g / L sucrose, 300-380 mg / L termethin, and 1.8-2.2 mg / L IBA.

9. The method according to any one of claims 1 to 7, characterized in that, It also includes the following steps: After S7 has rooted, it should be hardened off and transplanted.

10. Use of the method according to any one of claims 1-9 in chili genetic transformation or gene editing.

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