A genetic transformation method for *Clematis chinensis* callus.

Abstract

This invention belongs to the field of plant genetic transformation technology, specifically relating to a method for genetic transformation of callus tissue from *Clematis chinensis*. The method includes the following steps: inducing callus tissue from *Clematis chinensis* seeds, and utilizing transformation... PlCYP81Q38 Recombinant Agrobacterium tumefaciens was used to infect callus tissue. Following resistance selection, differentiation, and rooting culture, transgenic plants were obtained. This invention establishes for the first time a stable and efficient genetic transformation system for Clematis chinensis callus tissue, filling a gap in Clematis chinensis genetic transformation technology. By inducing Clematis chinensis callus tissue, further optimization and subculturing were performed. Through Agrobacterium-mediated genetic transformation, key genes in the HA biosynthesis pathway were overexpressed, resulting in a Clematis chinensis regeneration system with high lignan production. This provides an important technical tool for studying the gene function of Clematis chinensis or enhancing the activity of its biosynthetic pathway, promoting the development of molecular biology research on Clematis chinensis.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic transformation technology, specifically relating to a method for genetic transformation of callus tissue from Clematis armandii. Background Technology

[0002] Clematis chinensis ( Phryma leptostachya As a traditional medicinal plant, *Clematis chinensis* (also known as *Clematis armandii*) possesses the effects of dispelling wind and dampness, promoting blood circulation, and relieving pain. Its tetrahydrodifuran lignan compound, dioxane A, also exhibits excellent insecticidal activity. With in-depth research into its pharmacologically active components (such as lignans and flavonoids), *Clematis chinensis* shows promising application prospects in insecticidal applications. However, *Clematis chinensis* mainly relies on wild resources, and suffers from problems such as resource scarcity, long growth cycle, complex genetic background, and unstable content of active ingredients, which severely restricts its large-scale cultivation and industrial development.

[0003] Currently, tissue culture technology for Clematis chinensis has made some progress. Its hairy root induction system and CRISPR / Cas9 gene editing technology based on this system have been established, confirming that Agrobacterium-mediated transformation can be used for genetic manipulation. However, when conducting genetic transformation of Clematis chinensis callus using Agrobacterium-mediated transformation with existing technologies, the key parameters in each stage of callus induction, Agrobacterium-mediated transformation, and differentiation and regeneration are poorly matched, resulting in extremely low positive transformation efficiency and the inability to obtain complete transgenic regenerated plants. This severely restricts the progress of gene function research and molecular breeding of Clematis chinensis. Summary of the Invention

[0004] To address the problem in existing technologies for genetic transformation of Clematis chinensis callus using Agrobacterium-mediated transformation, where poor matching of key parameters in the induction, Agrobacterium-mediated transformation, and differentiation / regeneration of callus leads to extremely low positive transformation efficiency and the inability to obtain complete transgenic regenerated plants, severely hindering the research on Clematis chinensis gene function and the progress of molecular breeding, this invention provides a method for genetic transformation of Clematis chinensis callus. To achieve the above objective, this invention adopts the following technical solution.

[0005] This invention provides a method for genetic transformation of *Clematis chinensis* callus, comprising the following steps: Callus preparation: Seeds, leaves, stem segments, or root tips of *Clematis chinensis* were used as explants. After sterilization, the explants were placed in an induction medium containing 0.5 mg / L~2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg / L~2.0 mg / L 6-benzylaminopurine (6-BA), and 0.5 mg / L~2.0 mg / L α-naphthaleneacetic acid (NAA) for callus induction. The induced callus was subcultured every 15~20 days for a total of 1~3 times to obtain callus with stable growth. The induction medium was B5 medium.

[0006] Agrobacterium culture and activation: The overexpression vector of the target gene is transformed into Agrobacterium tumefaciens, and the resulting culture is screened to obtain an infection solution; wherein, the OD of the infection solution is... 600 The value is 0.4; where, the PlCYP81Q38 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Agrobacterium infection: The obtained callus tissue is immersed in the infection solution for infection, and after co-culture and sterilization treatment, transgenic callus tissue is obtained.

[0008] The transgenic callus tissue was induced by adventitious buds, rooted, and then transplanted to obtain transgenic regenerated plants.

[0009] This invention addresses the technical bottlenecks in the genetic transformation of *Clematis chinensis* callus by achieving a technological breakthrough through systematic optimization of key parameters at each stage: In the callus induction stage, seeds, leaves, stem segments, or root tips are used as explants, and B5 medium is employed with precise formulations of 0.5 mg / L to 2.0 mg / L of 2,4-D, 6-BA, and NAA hormones, combined with 1 to 3 subcultures, effectively solving the problems of difficult callus induction and unstable quality; In the genetic transformation stage, the target gene overexpression vector is transformed into *Agrobacterium tumefaciens* and the OD of the infection solution is controlled. 600 The values ​​ranged from 0.2 to 0.8, significantly improving the infection efficiency and positive transformation rate of Agrobacterium tumefaciens on callus tissue. During the plant regeneration stage, through stepwise regulation of adventitious bud induction and rooting culture, the differentiation of transgenic callus tissue into complete regenerated plants was successfully achieved. The synergistic adaptation of these three-stage parameters constructed a complete system for Agrobacterium-mediated genetic transformation of Clematis chinensis callus tissue, effectively overcoming the technical obstacles of low transformation efficiency and inability to obtain complete transgenic plants caused by poor parameter adaptation in existing technologies. This provides key technical support for the study of Clematis chinensis gene function and molecular breeding.

[0010] This invention includes an efficient Agrobacterium-mediated transformation process (controlling the OD of the infection solution). 600(Under the conditions of 0.4 and subsequent infection, co-culture, and sterilization), for the first time, complete transgenic regenerated plants were obtained from callus tissue of Clematis chinensis through differentiation induction and rooting culture. This breakthrough overcomes the bottleneck of existing technologies that can only rely on hairy root materials and cannot obtain complete plants, and provides a stable and heritable transformation system for the study of gene function and molecular breeding of Clematis chinensis.

[0011] Preferably, the infection time is 5 min to 15 min.

[0012] The co-culture medium is based on B5 medium, with 28 g / L to 32 g / L sucrose and 180 μmol / L to 220 μmol / L acetylsylgenone added.

[0013] Preferably, the co-cultivation process is as follows: The infected callus tissue was transferred to a co-culture medium and co-cultured at 24℃~26℃ in the dark for 0.5h~48h.

[0014] The co-culture medium is based on B5 medium, with 28 g / L to 32 g / L sucrose and 180 μmol / L to 220 μmol / L acetosyringone (AS) added.

[0015] Preferably, the B5 culture medium is prepared from raw materials with the following final concentration: B5 culture medium powder (Beijing Solarbio Science & Technology Co., Ltd., product number B8831) 3.21 g / L, sucrose 30.0 g / L, agar powder 8.0 g / L.

[0016] Preferably, the sterilization process is as follows: Rinse the co-cultured callus with sterile water containing 100 mg / L to 300 mg / L cefotaxime sodium (Cef) until sterile solution is precipitated, then rinse with sterile water 2 to 3 times.

[0017] Preferably, the adventitious bud induction process is as follows: The transgenic callus was transferred to an adventitious bud induction medium for adventitious bud induction culture until adventitious buds were induced.

[0018] The adventitious bud induction medium is based on B5 medium, with 1.8 mg / L to 2.2 mg / L of 6-benzylaminopurine (6-BA) and 0.8 mg / L to 1.2 mg / L of α-naphthaleneacetic acid (NAA) added.

[0019] The conditions for the induction and culture of adventitious buds are: temperature 25℃~28℃, 15h~17h light / 7h~9h darkness.

[0020] Preferably, the rooting culture process is as follows: The induced (differentiated) adventitious buds were transferred to a rooting medium for rooting culture.

[0021] The rooting medium is based on adventitious shoot culture, with the addition of 1.8 mg / L to 2.2 mg / L indole-3-butyric acid (IBA).

[0022] The conditions for rooting culture are: temperature 25℃~28℃, 15h~17h light / 7h~9h darkness.

[0023] Preferably, the explant disinfection process is as follows: Seed explants were disinfected sequentially with 70%–80% ethanol solution for 0.5–1.5 min and 3%–20% sodium hypochlorite solution for 5–20 min.

[0024] Leaves, stem segments, and root tip explants were disinfected sequentially with a 75% ethanol solution for 1 minute and a 10% sodium hypochlorite solution for 5 minutes.

[0025] After each disinfection step, wash continuously with sterile water 3 times, each time for 0.5 min to 2 min.

[0026] Preferably, the target gene includes PlCYP81Q38 Genes, the ones mentioned PlCYP81Q38 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0027] Preferably, the overexpression vector for the target gene is pCAMBIA1304. ＋ - PlCYP81Q38 Overexpression vector.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method for genetic transformation of *Clematis chinensis* callus. The method includes the following steps: using *Clematis chinensis* seeds, leaves, stem segments, and root tips as explants; disinfecting the explants and inducing callus formation; inducing callus differentiation culture; infecting the callus with *Agrobacterium*; co-culturing; sterilization; inducing adventitious buds; and rooting and seedling formation. Through the synergistic combination of three technical features—explant selection and precise construction of the callus induction system; optimized control of *Agrobacterium* infection parameters; and targeted restoration of the regeneration capacity of transformed callus—a dual breakthrough is achieved in improving the genetic transformation efficiency of *Clematis chinensis* callus and regenerating transgenic complete plants. Specifically, using sterilized seeds of *Clematis chinensis* as explants, callus induction was performed using B5 medium supplemented with a specific hormone combination of 2.0 mg / L 2,4-D, 2.0 mg / L 6-BA, and 1.0 mg / L NAA. Stable callus tissue was obtained after two subcultures. The equiconjugated ratio of 2,4-D and 6-BA in this formulation synergistically regulated the cell cycle, effectively maintaining the undifferentiated proliferative state of the callus tissue. The addition of NAA promoted callus densification, reduced cell decomposition and browning during subsequent *Agrobacterium* infection, and provided suitable recipient material. Simultaneously, recombinant *Agrobacterium* transformed with the target gene was cultured to OD... 600 =0.4% to prepare the infection solution. This concentration ensures sufficient bacterial density to improve T-DNA transfer efficiency, while avoiding browning and death of callus tissue due to excessive bacterial concentration. After infection, the callus tissue undergoes co-culture and sterilization processes, and finally completes the regeneration of the whole plant through adventitious bud induction and rooting culture. Each step is closely linked, forming a closed-loop technology system from seed explant to transgenic plant regeneration.

[0029] The inherent coordination mechanism of the above-mentioned technical features lies in the following: stable callus tissue is a prerequisite for efficient genetic transformation; suitable Agrobacterium infection parameters are a guarantee for positive transformation; and the establishment of a differentiation and regeneration system is the key to obtaining heritable transgenic plants. While existing hairy root transformation systems can obtain transgenic materials, they cannot regenerate complete plants through organogenesis, and callus transformation efficiency is low. This invention optimizes callus induction and subculture conditions, enabling the callus tissue of *Clematis chinensis* to possess good embryogenetic potential. Combined with precise control of Agrobacterium infection parameters, the transformed callus tissue retains its differentiation ability. Ultimately, complete transgenic regenerated plants are obtained through adventitious shoot induction and rooting culture. This systematically solves the technical bottlenecks of low genetic transformation efficiency and inability to obtain complete plants in existing *Clematis chinensis* technologies, providing a stable and reliable technical platform for gene function verification and molecular breeding of *Clematis chinensis*.

[0030] 2. This invention establishes for the first time a stable and efficient genetic transformation system for Clematis chinensis callus, filling a gap in the genetic transformation technology of Clematis chinensis. By inducing Clematis chinensis callus, further optimizing and subculturing it, and using Agrobacterium-mediated genetic transformation technology to overexpress key genes in the HA biosynthesis pathway, a Clematis chinensis regeneration system with high lignan production was obtained. This provides an important technical tool for studying the gene function of Clematis chinensis or enhancing the active components of the biosynthesis pathway, promoting the development of molecular biology research on Clematis chinensis.

[0031] This invention, based on the physiological characteristics of *Clematis chinensis*, was optimized through preliminary experiments, closely aligning with the growth and genetic transformation patterns of *Clematis chinensis*, overcoming the drawbacks of blindly applying parameters from other plants in existing technologies. It is simple to operate, with clear procedures for each step. The required equipment (such as a clean bench, constant temperature incubator, centrifuge, etc.) are standard equipment for plant tissue culture and genetic transformation, making it easy to promote and apply. It has high practicality; the established system can be directly applied to genetic improvement operations such as gene editing and exogenous gene introduction in *Clematis chinensis*, providing technical support for cultivating high-quality *Clematis chinensis* varieties. However, the time from explant disinfection to obtaining complete transgenic plants is relatively long, compared to model plants (such as tobacco and Arabidopsis thaliana), mainly limited by the growth and regeneration rate of callus tissue in *Clematis chinensis*. It also requires high technical skills from operators; strict control of operational details is needed in explant disinfection, *Agrobacterium* bacterial solution concentration control, callus subculturing, and screening, otherwise, the transformation efficiency may be affected. Attached Figure Description

[0032] Figure 1 This invention relates to the subculture of *Clematis chinensis* callus tissue; wherein: A: First subculture at 20 days; B: Second subculture at 30 days; C: Third subculture at 45 days.

[0033] Figure 2 This refers to the transplantation of regenerated *Clematis chinensis* plants in this invention; wherein: A: Before transplanting tissue culture seedlings; B: Tissue culture seedlings that have survived after hardening off.

[0034] Figure 3 This is the process flow of the genetic transformation system in this invention; wherein: A: Culture of Agrobacterium tumefaciens; B: Agrobacterium tumefaciens activation; C: Callus preparation; D: Agrobacterium tumefaciens infection; E: Co-culture and sterilization; F: Transgenic regenerated plant.

[0035] Figure 4This refers to the transgenic regenerated plant of Clematis armandii in this invention.

[0036] Figure 5 The results of GUS histochemical staining in this invention; wherein: A: Staining of callus tissue; B: Leaf staining; C: Root staining.

[0037] Figure 6 This is an electrophoresis image of a transgenic positive plant of *Clematis chinensis* in this invention, obtained by PCR detection. Wherein, M: 2000bp standard molecular weight DNA; WT: control group of regenerated plants; EV: transformed with pCAMBIA1304. ＋ Regenerated plants without load; OE- PlCYP81Q38 1-3: PlCYP81Q38 Transgenic regenerated plant lines overexpressing the gene.

[0038] Figure 7 This invention provides an RT-qPCR analysis of genes in the roots of transgenic regenerated plants of *Clematis chinensis*. Control: Transfer to pCAMBIA1304 ＋ Regenerated plants without load; OE- PlCYP81Q38 1-3: PlCYP81Q38 Transgenic regenerated plant lines overexpressing the gene; the bars in the figure represent the mean ± standard deviation (SD) of three replicates, and different lowercase letters indicate significant differences. P <0.05).

[0039] Figure 8 In this invention PlCYP81Q38 Content of lignan compounds in the roots of overexpressed transgenic regenerated plant lines; among which: A: OE- PlCYP81Q38 HPLC spectrum of lignans in the roots of the strain; B: OE- PlCYP81Q38 Content of lignan compounds in the roots of the plant strain; EV: Transfer to pCAMBIA1304 ＋ Regenerated plants from unloaded plants; 1: Acetyl styracil (LA); 2: 6-desmethoxy-LA; 3: P-I; 4: P-II; 5: HA. The bars in the figure represent the mean ± standard deviation (SD) of three replicates. An independent samples t-test was used, and "*" indicates a significant difference. P <0.05), "**" indicates a highly significant difference ( P <0.01), "***" indicates a highly significant difference ( P <0.001). Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1. Induction of aseptic seedlings of Clematis chinensis (1) Screening of seed disinfection conditions Place the required tweezers, filter paper, beaker, sterile water, etc. into the laminar flow hood, and sterilize the laminar flow hood for 30 minutes before use.

[0042] The herb used is *Clematis chinensis* (or *Clematis chinensis*). Phryma leptostachya Seeds and plants of *Clematis chinensis* (L.) were collected from the experimental field of the Institute of Pesticides, Northwest A&F University. The seeds were dried and stored at room temperature after collection.

[0043] Using *Clematis chinensis* seeds as the experimental material, disinfection condition screening experiments were conducted with different sodium hypochlorite concentrations and disinfection durations (Table 1). The optimal disinfection conditions suitable for *Clematis chinensis* seeds were ultimately determined. Details are as follows:

[0044] After rinsing the seeds of *Clematis chinensis* with 75% ethanol solution for 1 min, they were soaked in 20% sodium hypochlorite solution for 15 min. Following each disinfection step, the seeds were washed three times consecutively with sterile water for 1 min each time. After disinfection, excess moisture was blotted off with sterile filter paper, and the seeds were inoculated into 1 / 2 MS medium containing a final concentration of 2 mg / L GA. ​​Three replicates were set up, with 15 seeds inoculated onto each plate. After 7 days of dark incubation at 24℃±1℃, a 16h light / 8h dark incubation period was followed. The contamination rate was assessed after 20 days.

[0045] Table 1. Screening of Seed Disinfection Conditions (2) Aseptic seedling induction After sterilization, the seeds of Clematis chinensis were spread evenly on 1 / 2 MS medium supplemented with a final concentration of 2 mg / L GA. ​​After 7 days of dark culture at 24℃±1℃, the seeds were cultured under 16 hours of light and 8 hours of darkness to induce the generation of sterile Clematis chinensis seedlings.

[0046] 2. Screening of callus induction conditions for Clematis chinensis (1) Screening of explants In a clean bench, sterile *Clematis chinensis* seedlings with a height of 7-10 cm were cut with a scalpel to form three types of explants: leaves, stem segments, and root tips. Leaves were cut at the tip and base, and two incisions were made perpendicular to the midrib before inoculation with the leaf surface facing down. Stem segments and root tips were cut into 1 cm segments and inoculated flat. Sterilized seeds were cut transversely along the middle and inoculated with the cut side facing down. All explants were inoculated into B5 basal medium. After 21 days of culture, the callus induction rate of different explants was calculated, and the morphology, color, and texture of the callus tissue were observed and recorded.

[0047] Analysis of the data in Table 2 shows that root tip callus induction rate was the highest at 86.67%, followed by seed induction rate at 85.33%, and stem segment induction rate at 76.00%. There was no significant difference among the three. P >0.05), and both were significantly higher than those of leaf explants (34.67%). In terms of induction efficiency, root tip and seed explants showed significant advantages, but root tip-induced callus exhibited browning, a soft texture, and poor cell viability, which was unfavorable for subsequent differentiation and subculture. In contrast, seed-induced callus was bright in color, loosely structured, and showed no obvious browning or aging issues, demonstrating excellent proliferation and differentiation potential in subsequent culture. Considering callus induction efficiency, tissue morphology quality, and subsequent application potential, sterile seeds of *Clematis chinensis* were determined to be the optimal explant, suitable for optimizing subsequent callus induction systems and constructing genetic transformation systems.

[0048] Table 2. Callus differentiation of different explants (2) Screening of basal culture media To screen the optimal basal culture medium for callus induction from Clematis chinensis seeds, three types of culture media—B5 solid medium, MS solid medium, and 1 / 2 MS solid medium—were used in the experiment. All culture medium powders were purchased from Beijing Solarbio Science & Technology Co., Ltd., and the culture medium preparation methods are as follows: B5 solid medium (abbreviated as B5): Weigh 3.21g of B5 (medium) dry powder and 30g of sucrose, dissolve in 1L of distilled water, add 8g of agar powder to solidify, adjust the pH to 5.6, and autoclave at 121℃ for 15min.

[0049] MS solid medium (abbreviated as MS): Weigh 4.43g MS (medium) dry powder and 30g sucrose, dissolve in 1L distilled water, add 8g agar powder, adjust pH to 5.6, and autoclave at 121℃ for 15min.

[0050] 1 / 2MS solid medium (abbreviated as 1 / 2MS): Weigh 2.47g of 1 / 2MS (medium) dry powder and 30g of sucrose, dissolve in 1L of distilled water, add 8g of agar powder, adjust the pH to 5.6, and autoclave at 121℃ for 15min.

[0051] Five combinations of exogenous hormones, including 2,4-D (2,4-dichlorophenoxyacetic acid), 6-BA (6-benzylaminopurine), and NAA (α-naphthaleneacetic acid), were set up: Treatment 1: 2.0 mg / L 2,4-D + 2.0 mg / L 6-BA + 1.0 mg / L NAA.

[0052] Treatment 2: 0.5mg / L 2,4-D+2.0mg / L 6-BA+0.5mg / L NAA.

[0053] Treatment 3: 0.5 mg / L 2,4-D + 2.0 mg / L 6-BA + 1.0 mg / L NAA.

[0054] Treatment 4: 1.0 mg / L 2,4-D + 2.0 mg / L 6-BA + 0.5 mg / L NAA.

[0055] Treatment 5: 1.0 mg / L 2,4-D + 2.0 mg / L 6-BA + 1.0 mg / L NAA.

[0056] The five hormones were added to three basic solid culture media (including solid medium, MS solid medium, and 1 / 2 MS solid medium) to form different culture medium-hormone combination experimental groups. Each group was set up with three biological replicate plates, and 15 sterile seeds were inoculated on each plate. After 30 days of culture, the callus induction rate and germination rate of each group were counted. The optimal basic culture medium for seed callus induction was screened by comparison. The experimental results are shown in Table 3.

[0057] Table 3 shows that, under each hormone ratio treatment, the callus induction effect of B5 solid medium was generally better than that of MS solid medium and 1 / 2 MS solid medium. In treatment 1, B5 solid medium had the highest callus induction rate, reaching 91.33%, and a germination rate of 19.77%; the callus induction rates of MS solid medium and 1 / 2 MS solid medium were 85.22% and 83.11%, respectively, and the germination rates were 11.57% and 17.45%, respectively. In treatment 2, the callus induction rate of B5 solid medium was 87.14%, and the germination rate was 24.26%, both of which were significantly higher than those of MS solid medium (73.33% callus induction rate, 12.22% germination rate) and 1 / 2 MS solid medium (72.57% callus induction rate, 15.45% germination rate). Treatments 3 and 5 showed similar callus induction rates on the three basal media; treatment 4 had the worst overall callus induction effect, with a callus induction rate of only 61.67% on MS solid medium and a similarly low germination rate.

[0058] Based on the combined data of callus induction rate and germination rate across all hormone treatment groups, B5 solid medium exhibited higher callus induction and germination rates, which is more conducive to the induction and differentiation of seed callus tissue. Furthermore, the callus tissue was in good condition, making it suitable for establishing a subsequent regeneration system. Therefore, this invention selects B5 solid medium as the basal medium to provide high-quality recipient material for the subsequent construction of a complete regeneration system.

[0059] Table 3. Callus induction rate and budding rate under different hormone concentrations and culture medium combinations. (3) Screening of exogenous hormone ratios Based on the screened B5 solid culture medium, a concentration gradient combination experiment of three plant growth regulators, 2,4-D, 6-BA, and NAA, was set up, resulting in a total of 9 hormone ratio treatments (Table 4). Sterilized seeds were used as explants, with 3 biological replicates per group. Each plate was inoculated with 15 seeds and cultured at 25℃ in the dark for 30 days. After culture, the callus induction rate, browning rate, and germination rate of each group were recorded, and the callus growth morphology was observed to screen the optimal hormone ratio.

[0060] Table 4. Different Hormone Combinations and Ratios The culture results showed that after 10 days of culture, light yellow, loose callus granules began to appear at the cut surfaces of seeds in all treatments; after 30 days of culture, the callus tissue further proliferated, and some treatments formed dense clumps, with color and state varying depending on the hormone combination. Table 5 shows that different treatments had significant differences in their effects on callus induction rate, browning rate, and germination rate. Treatment 9 had the highest callus induction rate (91.33%), the lowest browning rate (10.00%), and a germination rate of 46.72%; treatment 6 was next, with a callus induction rate of 88.72%, a browning rate of 13.67%, and a germination rate of 56.78%; treatment 5 had a callus induction rate of 85.12%, a browning rate of 13.45%, and a germination rate of 43.33%. Overall, the callus induction rate increased with the concentration of 6-BA from 0.5 mg / L to 2.0 mg / L, but the germination rate fluctuated at excessively high concentrations. The concentration of 2,4-D significantly affected the callus browning rate; when 2,4-D was 2.0 mg / L, the browning rate was generally low in all treatments. A NAA concentration of 1.0 mg / L was more conducive to callus induction. Treatment 7 had the highest germination rate (76.78%), but also a relatively high browning rate (27.67%). Considering callus induction rate, browning control, and differentiation ability, treatment 9 maintained the lowest browning rate while ensuring a high induction rate, with moderate germination rate and good callus condition, making it the optimal hormone combination for seed callus induction.

[0061] Therefore, sterilized seed explants exhibited excellent callus induction effects under the conditions of 2.0 mg / L 2,4-D + 2.0 mg / L 6-BA + 1.0 mg / L NAA, which can be used for the establishment and optimization of subsequent regeneration systems.

[0062] Table 5. Seed callus differentiation under different hormone ratios (4) Screening of callus subculture period After determining the hormone conditions, the induced callus tissue was subcultured. After 30 days, the culture medium was changed, and subcultured again according to the initial culture conditions. Three plates were formed for each treatment, with 15 callus tissues per plate. The tissues were incubated in the dark at 25℃±2℃ with a relative humidity of approximately 50%, and observations were recorded until the callus tissue reached stable growth. At 20 days of culture (first subculture), Figure 1 In section A), a small amount of callus tissue is visible in the culture dish. It is small in size, loosely structured, and pale yellow or light white in color. At this stage, the callus tissue is in the adaptation and early proliferation phase. After 30 days of culture (second subculture), Figure 1 In section B), the individual callus tissue significantly increased in volume, with gradually denser edges. The callus exhibited enhanced metabolic activity and vigorous proliferation, demonstrating excellent overall growth. This period represents the most vigorous and morphologically optimal stage among the three subcultures. At 45 days of culture (the third subculture), Figure 1 In the C subculture, the callus tissue was fuller and denser, but its color deepened to yellowish-brown, and some showed signs of browning and aging, making it unsuitable for further experiments. Comparing the results of the three subcultures, the callus tissue from the second subculture (30 days) exhibited the strongest proliferative capacity and the most stable state, making it the best material for subsequent genetic transformation experiments.

[0063] Therefore, the callus tissue obtained from the second subculture was ultimately chosen for subsequent culture.

[0064] 3. Obtaining regenerated plants (1) Screening of hormone ratios for adventitious bud differentiation Among plant growth regulators, the synergistic effect of cytokinins and auxins plays a crucial regulatory role in the induction of callus and adventitious shoot differentiation in explants. Their effect depends not only on their individual concentrations but also on the ratio between them. This invention uses induced *Clematis chinensis* callus as material and B5 solid medium as the basal medium. Suitable hormone ratios were screened to induce adventitious shoot growth. Concentration gradients were set (as shown in Table 6) (6-BA: 1.00 mg / L, 2.00 mg / L; NAA: 0.50 mg / L, 1.00 mg / L, 2.00 mg / L), and cultured under light. Ten bottles were used for each treatment, with three plants per bottle. Growth was observed, and the shoot emergence rate was calculated after 30 days.

[0065] Table 6. Adventitious bud differentiation under different hormone ratios The results showed that different hormone ratios significantly affected the adventitious bud differentiation efficiency and bud quality of *Clematis chinensis* callus. Low-concentration hormone combinations resulted in no obvious browning of the callus, but a lower budding rate and fewer buds. Excessively high NAA concentrations exacerbated callus browning and inhibited normal adventitious bud growth. Based on a comprehensive analysis of budding rate, bud vigor, and callus health, treatment combination 4 showed the best results. Specifically, adding 2.0 mg / L 6-BA + 1.0 mg / L NAA to B5 medium resulted in the highest adventitious bud germination rate (93.33%), dense buds, robust growth, and no browning or aging of the callus.

[0066] Therefore, the combination of B5 + 2.0 mg / L 6-BA + 1.0 mg / L NAA was ultimately selected as the most suitable adventitious bud induction culture medium in this invention.

[0067] (2) Screening of adventitious shoot rooting medium Based on the crucial role of IBA in inducing root development and enhancing plant resistance, this invention, building upon the successful induction of adventitious buds in *Clematis chinensis*, further systematically investigated the effects of different IBA concentrations on the rooting efficiency of adventitious buds. Independent, robust adventitious buds larger than 2 cm were transferred to rooting media containing different concentrations of IBA for the experiment; IBA concentration gradients were set at 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L. Three adventitious buds were inoculated into each bottle, with five bottles inoculated for each treatment. Rooting was observed 30 days after inoculation, and the rooting rate was calculated.

[0068] The results, as shown in Table 7, indicate that when the IBA concentration is too low, the adventitious bud rooting rate is low and the root system is not fully developed; when the IBA concentration is too high, it inhibits root development, leading to a reduction in the number of roots and a decrease in the rooting rate. When the IBA concentration is 2.0 mg / L, the adventitious bud rooting rate is the highest, reaching 90.33%, with a large number of roots, suitable root length, and strong root vigor, resulting in the best overall rooting quality, which can meet the growth requirements for subsequent hardening and transplanting.

[0069] Therefore, the final choice was to add 2.0 mg / L IBA to the adventitious bud induction medium to improve the rooting rate and rooting quality of adventitious buds.

[0070] Table 7. Differentiation of adventitious shoots and roots under different hormone ratios (3) Hardening off and transplanting of regenerated plants When the rooted tissue culture seedlings grow to about 10cm in height and are close to the mouth of the culture bottle ( Figure 2If the conditions in section A are met, a seedling hardening and transplanting experiment can be conducted. The tissue culture bottle caps are partially open, and the seedlings are placed at room temperature for 2 days to harden them off, allowing the sterile seedlings to gradually adapt to the external temperature and humidity environment. The seedlings are then removed, the roots are washed to remove any residual culture medium, and they are transplanted into a pre-sterilized composite substrate (soil:vermiculite = 3:1). After transplanting, the substrate is covered with plastic film to retain moisture, and the seedlings are cultured under normal greenhouse conditions. During this period, the film is gradually uncovered for ventilation and hardening off, and water is sprayed daily to keep the substrate moist and ensure normal seedling growth.

[0071] The seedlings are growing well one week after transplanting. Figure 2 (B) The plants are vigorous, with upright and well-developed leaves, and no wilting, yellowing, or withering. The transplant survival rate can reach 90%. This indicates that the hardening-off transplanting scheme can effectively help the tissue culture seedlings of *Clematis chinensis* to complete a smooth transition from a sterile heterotrophic environment to a natural soil autotrophic environment. The regenerated plant transplanting system is stable and highly feasible.

[0072] 4. Preparation of Agrobacterium infection solution Total RNA was extracted from the root tissue of Clematis chinensis, and cDNA template was synthesized after reverse transcription. The target gene was amplified using specific primers (PlCYP81Q38-F / R, see Table 10). PlCYP81Q38 ( PlCYP81Q38 (Gene). The plant overexpression vector pCAMBIA1304 ＋ (Preserved by Professor Zhaonong Hu's research group at Northwest A&F University, see "Zhongjuan Sun, Yatian Li, Zheyuan Liu, Haoyu Zhang, Yakun Pei, and Zhaonong Hu. Functional Characterization of PlC4H1 and PlC3H1: Essential for Furofuran Lignan Biosynthesis in Phryma leptostachya, Journal of Agricultural and Food Chemistry, 2026, 74(2), 2098-2110.") Double digestion was performed. BamH I and Sac I) Post-connection PlCYP81Q38 Gene fragments were ligated, and the resulting product was transformed into *E. coli* DH5α competent cells. Positive monoclonal colonies were screened, amplified, and recombinant plasmids were extracted. The recombinant plasmid pCAMBIA1304 was then used... ＋ - PlCYP81Q38 (Overexpression vector of the target gene / containing) PlCYP81Q38 Gene overexpression vector / pCAMBIA1304 ＋ - PlCYP81Q38The overexpression vector was transduced into Agrobacterium tumefaciens GV3101 (pSoup-p19) competent cells to obtain transformed cells. PlCYP81Q38 Recombinant Agrobacterium GV3101 (pSoup-p19) of the gene.

[0073] in, PlCYP81Q38 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0074] A single colony of recombinant Agrobacterium was picked and inoculated into 1 mL of YEB liquid medium containing 50 mg / L kanamycin (Kan). The culture was activated by shaking at 28°C and 190 rpm. The activated culture was then transferred to a 50 mL sterile centrifuge tube and cultured at the same temperature and speed until the OD of the culture was reached. 600 After reaching 0.6, centrifuge at 5500 rpm for 6 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in resuspension solution (B5 solid medium + 100 μmol / L MES + 100 μmol / L MgCl2 + 200 μmol / L AS) and mix thoroughly. Adjust the OD of the bacterial solution. 600 To obtain the required concentration for the experiment, let it stand at 28°C in the dark for 3 hours to prepare the Agrobacterium infection solution for later use.

[0075] 5. Screening of Agrobacterium infection conditions (1) Screening of bacterial concentration Recombinant Agrobacterium was inoculated into YEB liquid medium supplemented with 50 mg / L Kan for large-scale culture, and the bacterial concentration was monitored in real time. OD was prepared accordingly. 600 Four groups of infection solutions (0.2, 0.4, 0.6, and 0.8) were used for callus infection experiments. Each group treated 15 callus tissues, with 3 biological replicates. The infection treatment, co-culture, and sterilization screening procedures are as follows:

[0076] Loose callus tissue was immersed in infection solutions of different concentrations and incubated with shaking at 190 r / min for 10 min. The callus tissue was then removed and placed on sterile filter paper to thoroughly absorb the surface bacterial solution until no liquid dripped. It was then transferred to a co-culture medium (B5 + 30 g / L sucrose + 200 μmol / L AS) lined with sterile filter paper and incubated in the dark at 25 ℃ for 24 h.

[0077] After co-culturing, the callus was repeatedly rinsed with sterile water supplemented with 200 mg / L cephalosporin (Cef) until no Agrobacterium precipitate was observed, followed by rinsing three times with sterile water. After blotting with sterile filter paper, the callus was transferred to resistance selection medium (B5 + 2.0 mg / L 6-BA + 1.0 mg / L NAA + 2.0 mg / L IBA + 200 mg / L Cef) and cultured at 27°C under 16 h light / 8 h dark conditions. After 30 days of culture, the positive conversion rate of callus regeneration into plants was calculated for each group, and the optimal concentration of the infecting bacterial solution was selected.

[0078] The results of the effects of different bacterial suspension concentrations on plant regeneration (Table 8) show that the conversion efficiency first increases and then decreases with increasing bacterial suspension concentration. When the bacterial suspension concentration is 0.2, the conversion rate is 4.45%; when the concentration is increased to 0.4, the conversion rate reaches the highest value of 15.56%; however, when the concentration is further increased to 0.6 and 0.8, more Agrobacterium precipitates around the plant tissue, and the conversion efficiency decreases significantly.

[0079] Therefore, OD was ultimately chosen. 600 =0.4 is the optimal infection concentration.

[0080] Table 8. Effects of different bacterial suspension concentrations on plant regeneration (2) Screening of infection duration and co-culture time Infection duration and co-culture duration directly affect Agrobacterium attachment efficiency and T-DNA transfer efficiency, and are key parameters determining callus-positive transformation efficiency. The OD values ​​of the Agrobacterium-fixed infection solution in this experiment... 600 =0.4, and three infection duration gradients (7min, 10min and 15min) and four co-culture duration gradients (0h, 12h, 24h and 48h) were set up in combination to carry out the experiment.

[0081] Loose callus tissue with good growth was selected and immersed in the prepared Agrobacterium infection solution. The callus was shaken at 190 r / min for 7 min, 10 min and 15 min respectively. After infection, the callus was removed and placed on sterile filter paper to fully absorb the residual bacterial solution on the surface until no droplets fell. The callus was then transferred to co-culture medium (B5 solid medium + 30 g / L sucrose + 200 μmol / L AS) lined with sterile filter paper and co-cultured at 25℃ in the dark for 0 h, 12 h, 24 h and 48 h respectively. Three biological replicates were set up for each group.

[0082] After co-culture, sterilization was performed: the callus tissue was repeatedly rinsed with sterile water containing 200 mg / L cefotaxime sodium (Cef) until no Agrobacterium precipitate was observed, and then rinsed with sterile pure water for 3 hours. After drying with sterile filter paper, the tissue was transferred to the resistance selection medium and cultured at 27°C under 16 h light / 8 h dark conditions. The positive conversion rate of each group was calculated, and the experimental results are shown in Table 9.

[0083] Table 9. Effects of infection duration and co-culture time on plant regeneration As shown in Table 9, regarding the infection duration, when the infection time was 7 min, the positive transformation rate gradually increased with the extension of co-culture time: no transformation was observed at 0 h (0%), rising to 2.96% at 12 h, 5.19% at 24 h, and 7.41% at 48 h, with an overall low level. At 10 min of infection, there was also no transformation at 0 h of co-culture, but the transformation rate rapidly increased to 19.99% after 12 h, reaching a peak of 28.15% at 24 h, and then decreasing to 17.78% at 48 h. At 15 min of infection, the transformation rate was extremely low at 0 h (0.74%), rising to 13.33% at 12 h, and decreasing to 7.41% and 1.48% at 24 h and 48 h, respectively. Regarding the co-culture time, at 0 h of co-culture, except for the 15 min treatment which showed extremely low transformation, no positive transformation was obtained in the others. In the absence of the co-culture process, a large number of Agrobacterium proliferated around the plant tissue, making effective transformation impossible. After 12 hours of co-culture, positive transformation was observed at all infection times, with the transformation rate at 10 minutes significantly higher than that at 7 minutes and 15 minutes. After 24 hours of co-culture, the highest positive transformation rate of 28.15% was achieved at 10 minutes, while the transformation rate began to decline at 15 minutes. After 48 hours of co-culture, the transformation rate at 7 minutes was 7.41%, but both the transformation rates at 10 minutes and 15 minutes showed a significant decrease compared to 24 hours, with the lowest rate observed at 15 minutes.

[0084] Therefore, the optimal infection conditions were ultimately selected as the combination of callus infection for 10 minutes and co-culture for 24 hours.

[0085] 6. Obtain transgenic regenerated plants Based on the optimization of the above infection conditions, the Agrobacterium tumefaciens-mediated genetic transformation system established in this invention can obtain transgenic regenerated plants in as little as three months. This system mainly includes five core steps (…). Figure 3 First, construct pCAMBIA1304 ＋ - PlCYP81Q38 The overexpression vector was transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) for recombinant Agrobacterium culture and activation, ensuring the strain was in a highly viable growth state. Simultaneously, plant callus tissue was prepared to ensure good recipient cell activity. The prepared callus tissue was then placed in a bacterial solution for Agrobacterium infection. Subsequently, T-DNA was successfully integrated into the plant genome through co-culture, and sterilization was promptly performed to inhibit excessive Agrobacterium growth. Finally, recipient cell differentiation and development were induced on a culture medium containing selection pressure, ultimately yielding complete recipient cells within three months. PlCYP81Q38 Overexpression (OE-) PlCYP81Q38 Transgenic regenerated plants ( Figure 4 ).

[0086] 7. Positive identification of transgenic regenerated plants (1) Identification of transgenic callus and regenerated plants by GUS histochemical staining Positive transgenic callus tissue obtained through infection with recombinant Agrobacterium and resistance screening was divided into two parts: one part of the callus tissue was directly used for GUS histochemical staining identification; the other part of the callus tissue was further induced to differentiate and root to obtain complete transgenic regenerated plants. After the regenerated plants had stabilized, they were hardened off and transplanted into a light incubator for acclimatization. The culture conditions were set as follows: temperature 25℃, light intensity 2000 lx, and photoperiod of 16 h light / 8 h dark. GUS staining was performed on the transgenic callus tissue to be tested, leaves of regenerated plants, and root tissues. After staining, chlorophyll was removed by decolorization with anhydrous ethanol.

[0087] The staining results showed that the callus, leaves, and root tissues of the transgenic material all turned blue, proving that the exogenous target gene was present. PlCYP81Q38 Expression has been achieved in transgenic materials of Clematis chinensis (Thuja orientalis). Figure 5 ).

[0088] (2) PCR molecular identification of exogenous gene integration To verify PlCYP81Q38 To determine whether the exogenous fragment was stably integrated into the genome of *Clematis chinensis*, three robust, independently transgenic regenerated plants were selected, and genomic DNA was extracted from root tissue for PCR amplification. The M13 universal upstream primer (M13-F) was used in combination with... PlCYP81Q38 Downstream specific primers (PlCYP81Q38-R) were used for amplification. Primer information is shown in Table 10.

[0089] Electrophoresis results ( Figure 6 Display: All OE- PlCYP81Q38 All transgenic regenerated plants amplified a specific target band of approximately 2000 bp; wild-type control plants and transgenic pCAMBIA1304 plants also showed similar results. + The absence of corresponding bands in the regenerated plants from empty plants confirms that... PlCYP81Q38 The exogenous gene has been successfully integrated into the genome of Clematis armandii.

[0090] Table 10 Primer sequences used for gene detection (3) RT-qPCR quantitative detection of gene transcription level Select OE- that are positive for GUS staining PlCYP81Q38 Quantitative detection of transgenic regenerated plant roots was performed using RT-qPCR technology. PlCYP81Q38 Relative gene expression level, in Pl5.8S rRNA As an internal reference gene correction.

[0091] Quantitative results ( Figure 7 This indicates that, compared to the control group, OE- PlCYP81Q38 In the roots of transgenic regenerated plants PlCYP81Q38 Gene transcription levels were significantly upregulated, with an average expression level increasing by approximately 7-fold, successfully obtaining... PlCYP81Q38 Stable overexpression transgenic lines.

[0092] (4) HPLC detection of lignan compound content in roots HPLC was used to detect the transfer of pCAMBIA1304. + Empty-load regenerated plants and OE- PlCYP81Q38 The lignan metabolite content in the roots of transgenic regenerated plants was determined using the following sample pretreatment and extraction method: Fresh roots of regenerated *Clematis chinensis* plants were washed, dried at 50℃, and then thoroughly ground into powder. 50 mg of the root powder was accurately weighed and placed in a 2 mL EP tube, and 1 mL of ethyl acetate was added and thoroughly mixed to prepare the extraction mixture. The mixture was ultrasonically extracted for 30 min, with the tube being inverted and mixed periodically during extraction to ensure complete extraction. After extraction, the mixture was centrifuged at 7000 rpm for 5 min to remove solid impurities and collect the supernatant. The collected supernatant was evaporated to dryness, and the residual substances were redissolved in 200 μL of acetonitrile to prepare the sample for subsequent HPLC component detection and analysis.

[0093] Metabolite detection results showed that PlCYP81Q38 Gene overexpression significantly increased the accumulation of lignans in the roots of *Clematis chinensis*. The accumulation levels of five characteristic lignan compounds—acetylclethodim (LA), 6-desmethoxy-LA, clethodim-I (P-I), clethodim-II (P-II), and hydroxylamine A (HA)—were significantly higher than in the control group. Figure 8 (A) Among them, the accumulation increase of 6-desmethoxy-LA is the most significant, OE- PlCYP81Q38 The content of this substance in the transgenic regenerated lines reached 4414.48 μg / g, which was 3.55 times higher than that in the control group; the content of P-Ⅰ was 2002.72 μg / g, which was 2.1 times that of the control group. Figure 8 (B in the text). The results indicate that overexpression... PlCYP81Q38 The gene can effectively promote the synthesis and accumulation of key lignan active ingredients in the roots of Clematis chinensis, and significantly increase the content of various characteristic lignans.

[0094] As can be seen from the above, this not only fully verifies the above... PlCYP81Q38 The precise function of the gene in the biosynthesis of lignans in Clematis chinensis also indirectly confirms the stability and feasibility of this transformation system.

[0095] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0096] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A method for genetic transformation of *Clematis chinensis* callus, characterized in that, Includes the following steps: Seeds, leaves, stem segments, or root tips of *Clematis chinensis* were used as explants. After sterilization, the explants were placed in an induction medium containing 0.5 mg / L–2.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L–2.0 mg / L 6-benzylaminopurine, and 0.5 mg / L–2.0 mg / L α-naphthaleneacetic acid to induce callus formation. The induced callus was then subcultured 1–3 times to obtain callus tissue. The induction medium was B5 medium. An overexpression vector of the target gene was transformed into Agrobacterium tumefaciens, and after screening and culturing, an infection solution was obtained; wherein, the OD of the infection solution was... 600 The value is 0.2~0.8; The obtained callus tissue was immersed in the infection solution for infection, and then after co-culture and sterilization treatment, transgenic callus tissue was obtained. The transgenic callus tissue was induced by adventitious buds, rooted, and then transplanted to obtain transgenic regenerated plants.

2. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The infection time is 5 min to 15 min.

3. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The co-cultivation process is as follows: The infected callus tissue was transferred to a co-culture medium and cultured in the dark at 24℃~26℃ for 0.5h~48h. The co-culture medium is based on B5 medium, with 28 g / L to 32 g / L sucrose and 180 μmol / L to 220 μmol / L acetylsylgenone added.

4. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The sterilization process is as follows: Rinse the co-cultured callus tissue with sterile water containing 100 mg / L to 300 mg / L cefotaxime sodium until sterile solution is precipitated, then rinse with sterile water 2 to 3 times.

5. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The process of adventitious bud induction is as follows: The transgenic callus was transferred to an adventitious shoot induction medium for adventitious shoot induction culture until adventitious shoots were induced. The adventitious bud induction medium is based on B5 medium, with 1.8 mg / L to 2.2 mg / L of 6-benzylaminopurine and 0.8 mg / L to 1.2 mg / L of α-naphthaleneacetic acid added. The conditions for the induction and culture of adventitious buds are: temperature 25℃~28℃, 15h~17h light / 7h~9h darkness.

6. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 5, characterized in that, The rooting culture process is as follows: The induced adventitious buds were transferred to a rooting medium for rooting culture. The rooting medium is based on adventitious shoot culture, with the addition of 1.8 mg / L to 2.2 mg / L indole-3-butyric acid; The conditions for rooting culture are: temperature 25℃~28℃, 15h~17h light / 7h~9h darkness.

7. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The explant disinfection process is as follows: Disinfect with 70%~80% alcohol by volume for 0.5min~1.5min, and then disinfect with 3%~20% sodium hypochlorite by volume for 5min~20min. After each disinfection step, wash continuously with sterile water 3 times, each time for 0.5 min to 2 min.

8. The method for genetic transformation of *Clematis chinensis* callus tissue according to claim 1, characterized in that, The target gene includes PlCYP81Q38 Genes, the ones mentioned PlCYP81Q38 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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