A method for in vitro regeneration and genetic transformation of liquidambar formosana

Abstract

The application discloses a liquidambar formosanum regeneration and genetic transformation method, and belongs to the technical field of plant genetic transformation. The regeneration takes leaves as explants, and is subjected to induction differentiation, elongation and rooting culture, and the rooting rate reaches 90.6%. The genetic transformation adopts a leaf disc method mediated by agrobacterium tumefaciens: taking leaves as the most suitable acceptor, the bacterial liquid OD 600 =0.5 is infected for 20 min, the kanamycin screening concentration is 50 mg / L, after co-culture, screening culture and rooting culture, the transformation efficiency of positive regenerated plants reaches 62.2%, which is 7 times higher than that of petioles. The related genes for regulating anthocyanin synthesis are used to verify the system, and transgenic plants with a red phenotype are obtained. The application provides an efficient and stable technical platform for genetic improvement of liquidambar formosanum.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic transformation technology, specifically relating to a method for in vitro regeneration and genetic transformation of Liquidambar formosana. Background Technology

[0002] Sweetgum (Liquidambar formosana Hance.) belongs to the genus Liquidambar in the family Hamamelidaceae. It is an important native afforestation and landscape ecological tree species in southern my country. Due to its rich autumn foliage colors, high ornamental value, strong ecological adaptability, and tolerance to drought and poor soil, it has received widespread attention in recent years for land greening, carbon sequestration afforestation, and urban and rural landscape construction.

[0003] Currently, research on Liquidambar formosana mainly focuses on resource distribution surveys, breeding of superior varieties, population genetics and evolution, and ecological monitoring. Studies on leaf color changes are largely limited to phenotypic observations and physiological and biochemical index analysis, such as changes in pigment content and photosynthesis. Due to a lack of genetic information and a stable genetic transformation system, the exploration and utilization of Liquidambar formosana leaf color-related gene resources are severely constrained. Existing new variety breeding still relies primarily on natural selection and hybridization, which suffers from long breeding cycles, limited leaf color variation types, and unclear genetic backgrounds, making it difficult to meet the industry's demand for superior new varieties with stable colorful leaf traits and strong adaptability.

[0004] The establishment of in vitro regeneration systems is a prerequisite and foundation for conducting genetic transformation research. Currently, there are some studies on in vitro regeneration of Liquidambar species both domestically and internationally. For example, using cotyledons, cotyledonary nodes, and hypocotyls of hybrid Liquidambar as explants, adventitious bud differentiation can be induced on WPM medium containing TDZ and NAA, with a rooting rate reaching 100%. However, existing Liquidambar regeneration systems generally suffer from low regeneration efficiency, poor stability, and strong dependence on genotype. Studies have shown significant differences in adventitious bud differentiation rates among different genotypes; some genotypes have a maximum regeneration rate of up to 90%, while others have a maximum adventitious bud differentiation rate of only 32.6%. Furthermore, although TDZ can effectively promote the induction of adventitious buds, excessively high concentrations can easily hinder adventitious bud elongation, affecting the normal development of subsequent plants. These factors collectively restrict the improvement and standardization of Liquidambar in vitro regeneration systems.

[0005] In genetic transformation, Agrobacterium tumefaciens-mediated transformation is one of the most commonly used methods in woody plant genetic transformation research due to its relatively simple operation and stable integration of exogenous genes. Researchers have conducted preliminary explorations of Agrobacterium tumefaciens-mediated genetic transformation conditions using Liquidambar formosana leaves as explants, obtaining a certain number of transgenic plants. Other studies have established a Liquidambar formosana genetic transformation system based on a mannose selection system, obtaining 134 transgenic positive plants. However, existing Liquidambar formosana genetic transformation systems still face a series of technical problems that urgently need to be solved. First, the transformation efficiency is generally low. Studies have shown that when using leaves as recipient materials for genetic transformation, the transformation efficiency is low and chimeras are easily produced, and the organogenesis culture procedure is complex and has a long culture cycle. Second, the recipient system is highly dependent on genotype; the differentiation ability of leaves with different genotypes varies significantly, making it difficult to achieve stable transformation output. Third, the selection pressure is difficult to balance: kanamycin has a significant inhibitory effect on Liquidambar formosana leaf regeneration; the regeneration ability of explants directly transferred to the selective medium after co-culture decreases sharply, but too low an antibiotic concentration is insufficient to effectively inhibit the growth of untransformed tissues. In addition, incomplete sterilization leading to excessive growth of Agrobacterium often affects the normal differentiation of explants.

[0006] In summary, there is currently a lack of an efficient and stable in vitro regeneration system for Liquidambar formosana and a genetic transformation method based on Agrobacterium tumefaciens. Existing technical solutions have failed to address the technical problems in Liquidambar formosana genetic transformation, such as low transformation efficiency, strong genotype dependence, and poor reproducibility. They also fail to provide systematically optimized culture medium formulations for each stage and a complete transformation procedure. Therefore, there is an urgent need in this field to establish an efficient, stable, and reproducible in vitro regeneration and genetic transformation method for Liquidambar formosana to overcome the shortcomings of existing technologies and provide technical support for functional gene verification and molecular breeding of Liquidambar formosana. Summary of the Invention

[0007] This invention aims to overcome the technical defects of existing in vitro regeneration and genetic transformation systems of Liquidambar formosana, such as low regeneration efficiency, low transformation efficiency, strong dependence on genotype, and poor reproducibility, and to provide an efficient, stable, and reproducible in vitro regeneration and genetic transformation method of Liquidambar formosana and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for in vitro regeneration of Liquidambar formosana includes the following steps:

[0010] (1) Leaves of Liquidambar formosana tissue culture seedlings were used as explants and inoculated into callus induction and adventitious shoot differentiation medium to induce callus and adventitious shoot differentiation and obtain adventitious shoots; the callus induction and adventitious shoot differentiation medium was based on WPM medium, with 1.0 mg / L of 6-BA, 0.1 mg / L of NAA and 0.2 mg / L of TDZ added.

[0011] (2) The adventitious shoots obtained in step (1) are transferred to the adventitious shoot elongation medium for elongation culture; the adventitious shoot elongation medium is based on WPM medium, with 0.5 mg / L of 6-BA, 0.2 mg / L of NAA and 0.1 mg / L of KT added.

[0012] (3) The elongated adventitious buds were transferred to the rooting medium for rooting culture to obtain complete regenerated plants; the rooting medium was based on WPM and supplemented with 1.0 mg / L IBA.

[0013] In some embodiments, in step (3), 0.8 g / L of activated carbon is also added to the rooting medium.

[0014] In some embodiments, in step (1), the leaf explant is cut into small pieces of 0.5 cm × 0.5 cm.

[0015] A method for genetic transformation of Liquidambar formosana includes the following steps:

[0016] (1) Using leaves of Liquidambar formosana tissue culture seedlings as explants, the leaves were immersed in Agrobacterium tumefaciens containing the target gene for infection. The OD of the Agrobacterium tumefaciens solution was... 600 The value is 0.4-0.6, and the infection time is 15-25 minutes;

[0017] (2) Co-culture the infected leaf explants;

[0018] (3) Sterilize the leaves after co-culture;

[0019] (4) After sterilization, the leaves were transferred to a medium for callus induction and adventitious shoot differentiation to screen for resistant shoots and obtain resistant shoots.

[0020] The medium for callus induction and adventitious shoot differentiation used for transformation was based on WPM medium, with the addition of 1.0 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mg / L TDZ, as well as screening agents and antibacterial agents.

[0021] (5) The obtained resistant shoots were transferred to the adventitious shoot elongation medium for transformation and elongation culture;

[0022] The adventitious shoot elongation medium for transformation is based on WPM medium, with the addition of 0.5 mg / L 6-BA, 0.2 mg / L NAA, 0.1 mg / L KT, as well as screening agents and antibacterial agents;

[0023] (6) The elongated resistant shoots were transferred to a rooting medium for transformation and rooting culture to obtain transgenic plants;

[0024] The rooting medium for transformation is based on WPM, with the addition of 1.0 mg / L IBA, as well as screening agents and antibacterial agents.

[0025] In some embodiments, the screening agent is Kan, and the antibacterial agent is Cef and Tim.

[0026] In some embodiments, in step (4), the concentration of Kan is 50 mg / L, the concentration of Cef is 200 mg / L, and the concentration of Tim is 150 mg / L; in steps (5) and (6), the concentration of Kan is 30 mg / L, the concentration of Cef is 200 mg / L, and the concentration of Tim is 150 mg / L.

[0027] In some embodiments, in step (6), 0.8 g / L of activated carbon is also added to the rooting medium for transformation.

[0028] In some embodiments, in step (1), the OD of the Agrobacterium tumefaciens bacterial solution... 600 The value was 0.5, and the infection time was 20 minutes.

[0029] In some embodiments, in step (2), the co-culture conditions are as follows: the infected leaf explants are placed in WPM medium containing 100 μM AS and cultured in the dark at 25±1℃ for 2-3 days.

[0030] The application of any of the in vitro regeneration methods or genetic transformation methods described herein in the breeding of Liquidambar formosana is characterized in that the application includes rapid propagation through tissue culture or obtaining new Liquidambar formosana varieties with target traits by introducing exogenous genes.

[0031] Compared with the prior art, the beneficial effects of this application are as follows:

[0032] This invention establishes a highly efficient and stable in vitro regeneration and genetic transformation system for Liquidambar formosana by systematically optimizing hormone combinations and transformation conditions in stages. In terms of regeneration, the callus induction stage utilizes a synergistic triple hormone formulation of 1.0 mg / L 6-BA, 0.1 mg / L NAA, and 0.2 mg / L TDZ, achieving a differentiation rate of 83.6% and an average of 3.85 buds per explant. In the elongation stage, reducing the 6-BA concentration and introducing 0.1 mg / L KT effectively overcomes the dwarfing of bud clusters caused by TDZ, achieving a bud elongation rate of 88.6%. In the rooting stage, the addition of 0.8 g / L activated carbon results in a rooting rate of 90.6% and a well-developed root system. This three-step regeneration system provides a reliable platform for the large-scale rapid propagation and genetic transformation of Liquidambar formosana.

[0033] In terms of genetic transformation, this invention uses leaves as recipients and determines the optimal infection conditions (OD). 600 =0.5, infection time 20 minutes) and a segmented screening scheme (Kan 50 mg / L during differentiation stage, Kan 30 mg / L during elongation and rooting stages), while using a combination of Cef and Tim to effectively control Agrobacterium contamination. Seven independent batches of experiments verified that the average transformation efficiency of leaves reached 62.2%, approximately seven times higher than the petiole transformation efficiency (8.88%), achieving unexpected technical results. Using this system, transgenic Liquidambar formosana exhibiting a red phenotype throughout the plant was successfully obtained, directly demonstrating its application value in functional gene verification and the breeding of new colorful-leaved varieties, providing key technical support for the targeted genetic improvement of Liquidambar formosana.

[0034] In summary, the in vitro regeneration system and genetic transformation method for Liquidambar formosana established in this invention can be applied to gene function research or molecular breeding of Liquidambar formosana, including but not limited to: obtaining new Liquidambar formosana varieties with target traits (such as colorful leaves, stress resistance, and fast growth) by introducing exogenous genes or performing gene editing. This invention effectively overcomes the shortcomings of existing technologies, such as low regeneration efficiency, low transformation efficiency, strong genotype dependence, and poor reproducibility, providing key technical support for functional gene research and molecular breeding of Liquidambar formosana, and has good application value and prospects. Attached Figure Description

[0035] Figure 1 This describes the process of establishing an in vitro regeneration system for Liquidambar formosana, in which AD is the induction of callus and adventitious bud differentiation from leaf explants; EF is the adventitious bud elongation culture; and GH is the rooting culture to form a complete plant.

[0036] Figure 2 The results of screening different explants as genetic transformation recipients are shown. AB represents the callus differentiation of leaf explants; CD represents the callus differentiation of petiole explants; and EF represents the callus differentiation of stem segment explants.

[0037] Figure 3 This refers to the process by which resistant buds regenerate into complete plants during genetic transformation.

[0038] Figure 4 This is an electrophoresis image of PCR detection of transgenic plants, where M is the DNA molecular weight marker; 1-2 and 8 are lines that failed to transform; 3-7 are transgenic positive lines; 9 is a wild-type plant (negative control); and 10 is plasmid DNA (positive control).

[0039] Figure 5 The diagram shows the genetic transformation process of the red phenotype transgenic plant. AC represents the induction of callus differentiation from leaf explants; D represents the adventitious shoot elongation culture; EF represents the comparison of rooting and phenotype between wild-type and transgenic Liquidambar formosana plants, with E representing wild-type plants and F representing transgenic plants, whose roots and aboveground parts both exhibit a red phenotype. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0041] Example 1

[0042] 1.1 Experimental Materials

[0043] Tissue culture seedlings of Liquidambar formosana Hance. were used as plant material, and healthy young leaves were taken as explants.

[0044] 1.2 Preparation of basal culture medium

[0045] WPM basal medium: Weigh 2.41 g / L L449 powder, 20 g / L sucrose, add 2.5 g / L Phyagel plant gel, and adjust the pH to 5.80-5.85. Autoclave the medium at 121℃ for 20 min before use.

[0046] 1.3 Optimization of callus induction and adventitious shoot differentiation culture medium

[0047] Using WPM as the basal medium, and referencing the previously screened WPM-DI-6 (WPM + 0.2 mg / L TDZ + 0.1 mg / L NAA) and WPM-DI-10 (WPM + 0.8 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L TDZ), the concentrations of 6-BA, TDZ, and NAA were further adjusted, resulting in a total of 7 treatments (Table 1). *Liquidambar formosana* leaf explants were inoculated into each treatment medium, with 50 explants per treatment and three replicates. After 30 days of culture, callus induction rate, adventitious shoot differentiation rate, and average number of shoots were recorded.

[0048] The results showed that the adventitious bud differentiation rate of WPM-DI-6 (containing only TDZ) was 68.4±1.93%, with an average number of buds of 1.98±0.85. After adding 6-BA (WPM-DI-10), the differentiation rate increased to 75.3±1.85%, and the average number of buds increased to 2.85±1.05. When the concentration of 6-BA was increased from 0.8 mg / L to 1.0 mg / L (WPM-DI-10-1), the adventitious bud differentiation rate further increased to 83.6±2.83%, with an average number of buds of 3.85±1.35. When the concentration was further increased to 1.5 mg / L (WPM-DI-10-2), the differentiation rate decreased to 71.3±1.36%, and the average number of buds decreased to 1.68±1.08. When TDZ was decreased from 0.2 mg / L to 0.1 mg / L (WPM-DI-10-3), the differentiation rate decreased to 73.1±0.89%; when NAA was increased to 0.2 mg / L (WPM-DI-10-4) or decreased to 0.05 mg / L (WPM-DI-10-5), the differentiation rates were 72.2±1.26% and 77.6±1.89%, respectively. In summary, WPM-DI-10-1 (WPM + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L TDZ) was the optimal formulation, achieving a callus induction rate of 93.2±2.45%, the highest adventitious shoot differentiation rate, and dense, robust shoots (e.g., ...). Figure 1 (As shown in AD).

[0049] Table 1. Effects of different hormone combinations on callus induction and adventitious shoot differentiation

[0050]

[0051] 1.4 Optimization of Adventitious Bud Elongation Culture Medium

[0052] Using WPM as the basal medium, three basic formulations (WPM-SE-3, WPM-SE-4, and WPM-SE-5) were selected based on literature recommendations. The concentrations of 6-BA, NAA, and KT were further adjusted, resulting in a total of seven treatments (Table 2). Adventitious shoots reaching 0.5–1.0 cm in length were excised and transferred to the respective treatment media, with 3–5 shoot clusters inoculated per bottle. Each treatment was replicated three times. After 30 days of culture, shoot elongation rate (shoot length ≥1 cm was considered elongation) was recorded, and growth status was observed.

[0053] The results showed that WPM-SE-4 (WPM + 0.5 mg / L 6-BA + 0.2 mg / L NAA) without KT had a shoot elongation rate of 83.2±3.3%; WPM-SE-3 (containing 1.0 mg / L 6-BA, 0.1 mg / L NAA, and 0.1 mg / L KT) had an elongation rate of 81.4±3.0%; and WPM-SE-5 (0.75 mg / L 6-BA + 0.05 mg / L NAA) had a lower elongation rate of 74.4±3.0%, with a significant tendency towards clustering. Adding KT to WPM-SE-5: adding 0.2 mg / L KT (WPM-SE-5-1) reduced the elongation rate to 71.6±2.8%, resulting in stunted shoots; adding 0.1 mg / L KT (WPM-SE-5-2) increased the elongation rate to 76.9±3.1%, improving growth. When 6-BA was reduced to 0.5 mg / L, NAA was increased to 0.2 mg / L, and 0.2 mg / L KT (WPM-SE-5-3) was added, the elongation rate was 73.6±2.4%, indicating moderate growth vigor. Under the same conditions, when 0.1 mg / L KT (WPM-SE-5-4) was added, the shoot elongation rate significantly increased to 88.6±2.3%, the shoots were robust, and the leaves were fully expanded (e.g., ...). Figure 1 (As shown in EF). Therefore, WPM-SE-5-4 (WPM + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L KT) was determined to be the optimal medium for shoot elongation.

[0054] Table 2 Effects of different hormone combinations on adventitious shoot elongation

[0055]

[0056] 1.5 Optimization of Rooting Culture Medium

[0057] Using WPM as the basal medium, WPM-R-1 (WPM + 1.0 mg / L IBA) and WPM-R-2 (1 / 2 WPM + 0.5 mg / L IBA + 0.1 mg / L NAA) were screened based on literature. The IBA and NAA concentrations were further adjusted, and activated charcoal (AC) was added, resulting in a total of five treatments (Table 3). Adventitious shoots with a height of 2–3 cm after elongation were cut and transferred to the respective treatment media, with one shoot inoculated per bottle and three replicates per treatment. Rooting rate, average number of roots, and average root length were recorded after 40 days of culture.

[0058] The results showed that WPM-R-1 had a rooting rate of 88.9±2.3%, an average of 4.2±0.35 roots, and an average root length of 3.8±0.28 cm, indicating a well-developed root system. WPM-R-2 (1 / 2 WPM) had a lower rooting rate (78.5±1.85%). Adding different concentrations of NAA to WPM-R-1: adding 0.5 mg / L NAA (WPM-R-1-1) or 0.1 mg / L NAA (WPM-R-1-2) reduced the rooting rate to 75.5±2.6% and 73.9±1.8%, respectively, and significantly shortened the average root length (2.9±0.26 cm and 1.7±0.22 cm, respectively). After adding 0.8 g / L activated carbon (WPM-R-1-3) to WPM-R-1, the rooting rate increased to 90.6±3.4%, the average number of roots increased to 4.8±0.42, and the average root length increased to 4.2±0.35 cm (e.g., Figure 1 (As shown in GH). Therefore, WPM-R-1-3 (WPM + 1.0 mg / L IBA + 0.8 g / L AC) was determined to be the optimal rooting medium.

[0059] Table 3 Effects of different hormone combinations on rooting

[0060]

[0061] 1.6 Establishment of a complete regeneration system

[0062] Based on the above optimization results, using leaves from tissue-cultured Liquidambar formosana seedlings as explants, an in vitro regeneration system was established according to the following steps:

[0063] (1) Callus induction and adventitious shoot differentiation: The leaves were cut into 0.5 cm × 0.5 cm pieces and inoculated into the optimal callus induction and adventitious shoot differentiation medium (WPM + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / LTDZ). After 30 days of culture, the callus induction rate reached 93.2%, the adventitious shoot differentiation rate reached 83.6%, and the average number of shoots was 3.85 per explant.

[0064] (2) Adventitious bud elongation: Adventitious buds of 0.5~1.0 cm were cut off and transferred to the optimal bud elongation medium (WPM + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L KT) and cultured for 30 days. The bud elongation rate reached 88.6% and the buds were robust.

[0065] (3) Rooting culture: The buds that have grown to 2-3 cm after elongation are transferred to the optimal rooting medium (WPM + 1.0 mg / L IBA + 0.8 g / L AC) and cultured for 40 days. The rooting rate reaches 90.6%, with an average of 4.8 roots and an average root length of 4.2 cm, forming complete regenerated plants.

[0066] The cultivation conditions for each stage are as follows: temperature 25±1℃, light intensity 2000-3000 lux, and photoperiod of 16 hours of light / 8 hours of darkness.

[0067] Example 2: Genetic transformation of Liquidambar formosana and acquisition of transgenic plants

[0068] 2.1 Screening for the optimal transformation receptor

[0069] Three types of explants were used from tissue-cultured seedlings of *Liquidambar formosana*: leaves, petioles, and stem segments. The treatment methods were as follows: leaves were cut into 0.5 cm × 0.5 cm pieces; petioles were cut into 0.5–1.0 cm long segments; and stem segments were cut into 0.5–1.0 cm long segments (with one internode). The treated explants were then cultured in *Agrobacterium tumefaciens* solution (OD200). 600 After inoculating with 0.5 mg / L Kana solution for 15 min and co-culturing for 2 days, the samples were transferred to a selection medium containing 30 mg / L Kana, 200 mg / L Cef, and 150 mg / L Tim. After 40 days of light-induced culture, the differentiation rate of resistant shoots was calculated. Results are as follows: Figure 2 As shown: the callus tissue of the leaf explant is well differentiated. Figure 2 AB), poor petiole differentiation ( Figure 2 CD), although the stem segments have a high differentiation rate, there is a risk of false positives. Figure 2 EF).

[0070] Specific data: The differentiation rate of resistant buds in leaves was 63.1±2.2%, with an average of 3.78±1.12 adventitious buds per explant. The buds were robust and densely distributed. The differentiation rate of resistant buds in stem segments was 77.5±2.4%, but most buds sprouted from the cut or axillary buds, resulting in a high risk of false positives. The differentiation rate of resistant buds in petioles was only 38.7±1.9%, with few buds. Therefore, leaves were determined to be the most suitable transformation recipients (Table 4).

[0071] Table 4 Comparison of genetic transformation effects of three explants

[0072]

[0073] Note: Resistance bud differentiation rate = number of explants producing resistance adventitious buds / total number of infected explants × 100%; False positive rate = total number of resistance buds / number of plants that are positive in resistance screening but negative in molecular testing × 100%.

[0074] 2.2 Determination of Kanamycin Screening Concentration

[0075] Explants of Liquidambar formosana leaves were inoculated onto callus induction and adventitious shoot differentiation media containing different concentrations of kanamycin (0, 25, 50, 75, 100 mg / L), with 30 explants treated at each concentration, and the treatment was repeated three times. The growth of the explants was observed after 30 days of culture.

[0076] The results showed that at 0 mg / L kanamycin, explants grew well and adventitious buds differentiated normally; at 25 mg / L, explants could still differentiate, but untransformed tissue was difficult to effectively inhibit differentiation; at 50 mg / L, explant growth was somewhat inhibited, but untransformed tissue was significantly reduced, and adventitious buds could still differentiate; at 75 mg / L and above, explants showed severe browning and even death. Therefore, 50 mg / L was determined to be the appropriate screening concentration for kanamycin.

[0077] 2.3 Optimization of Agrobacterium tumefaciens bacterial concentration

[0078] Set OD 600 Three gradients of pH values ​​(0.4, 0.5, and 0.6) were used to infect *Liquidambar formosana* leaves for 15 min, followed by 2 days of co-culture before transfer to selection medium. Each treatment contained 30 explants, replicated three times. Survival rate, callus induction rate, and resistant shoot differentiation rate were assessed after 40 days.

[0079] Result: OD 600 When the OD ratio was 0.5, the resistant bud differentiation rate was the highest (61.8±2.3%), the callus induction rate was 81.3±1.8%, and the survival rate was 85.6±2.1%; 600 When the concentration was 0.6, the survival rate decreased to 74.8±1.2%, and the resistant bud differentiation rate decreased to 48.2±1.7%. Therefore, the optimal bacterial concentration was determined to be OD0.6. 600 =0.5.

[0080] 2.4 Optimization of Infection Time

[0081] The infection time was set to 10, 20, and 25 minutes, at OD 600 The leaves of *Liquidambar formosana* were infected with a bacterial suspension containing 0.5 g / mL and co-cultured for 2 days before being transferred to selection medium. Each treatment contained 30 explants, with three replicates. Relevant indicators were analyzed after 40 days.

[0082] Results: At 20 min of infection, the resistant bud differentiation rate was the highest (65.5±2.2%), the callus induction rate was 77.3±1.4%, and the survival rate was 81.6±1.8%. At 25 min of infection, both the survival rate and differentiation rate decreased significantly. Therefore, the optimal infection time was determined to be 20 min.

[0083] 2.5 Genetic Transformation Procedure

[0084] (1) Preparation of Agrobacterium culture: Agrobacterium tumefaciens strain EHA105 containing the target gene (taking the transcription factor gene that regulates anthocyanin synthesis as an example) was inoculated into LB medium containing the corresponding antibiotic and cultured at 28°C with shaking until OD. 600 =0.5, centrifuge to collect bacterial cells, and resuspend in WPM liquid medium containing 100 μM acetylsuccinone.

[0085] (2) Infection and co-culture: Take tender leaves of Liquidambar formosana tissue culture seedlings, cut them into small pieces of 0.5 cm × 0.5 cm, and immerse them in the above bacterial solution for 20 min. After taking them out, wipe off the surface bacterial solution and transfer them to WPM solid medium containing 100 μM acetylsyl syringone. Incubate in the dark at 25℃ for 2-3 days.

[0086] (3) Sterilization and selection culture: After co-culture, the leaves were soaked in sterile water containing 200 mg / L cephalosporin for 10 min, washed 3 times, dried, and then transferred to callus induction and adventitious shoot differentiation medium (WPM + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L TDZ + 200 mg / L Cef + 150 mg / L Tim + 50 mg / L Kana) for light culture. Subculture was performed every 2 weeks. Figure 3 As shown, resistant buds gradually differentiate ( Figure 3 A) Elongation ( Figure 3 B) and rooting to form a complete plant ( Figure 3 CD).

[0087] (4) Resistance bud elongation: Cut off the resistance buds and transfer them to the adventitious bud elongation medium for transformation (WPM + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L KT + 200 mg / L Cef + 150 mg / L Tim + 30 mg / L Kana), and subculture once every 2 weeks.

[0088] (5) Rooting culture: The elongated resistant shoots were transferred to the rooting medium for transformation (WPM + 1.0 mg / L IBA + 0.8 g / L AC + 200 mg / L Cef + 150 mg / L Tim + 30 mg / L Kana) to obtain complete transgenic plants.

[0089] 2.6 Molecular identification of transgenic plants

[0090] Genomic DNA was extracted from resistant plants. Untransformed plants were used as negative controls, and plasmid DNA was used as a positive control. PCR amplification was performed using primers specific to the kanamycin resistance gene (Kana). Primer sequences: upstream 5'-TTCCTGATTAACCACAAACC-3', downstream 5'-CGGTTCGTTGGCAATACTCC-3'. PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; final extension at 72℃ for 5 min.

[0091] Electrophoresis results as follows Figure 4 As shown: M is the DNA molecular weight marker; lanes 1-2 and 8 are lines that failed to transform; lanes 3-7 are transgenic positive lines (amplified with the target band of the same size as the positive control, approximately 500 bp); lane 9 is a wild-type plant (negative control, no band); lane 10 is plasmid DNA (positive control). The results confirm that the exogenous gene has been integrated into the Liquidambar formosana genome.

[0092] 2.7 Conversion Efficiency Statistics

[0093] Seven independent transformation experiments were conducted using leaves and petioles as explants under the optimal conditions described above. The number of resistant plants was counted, and positive plants were confirmed by PCR. The results showed that the average transformation efficiency of leaves was 62.2%, which was about 7 times higher than that of petioles (8.88%) (Table 5).

[0094] Table 5 Conversion Efficiency

[0095]

[0096] Note: Transformation efficiency = Explant plant coefficient of positive regenerated plants / Total number of infected explants × 100%.

[0097] 2.8 Obtaining Red Phenotype Transgenic Plants

[0098] Using the genetic transformation system established in this invention, transcription factor genes that promote anthocyanin synthesis were introduced into *Liquidambar formosana* for overexpression. Transformation was performed according to the above-described procedure, and the resulting resistant buds were screened, elongated, and rooted to obtain fully regenerated plants. Figure 5As shown: Callus differentiation induced by leaf explants ( Figure 5 AC), adventitious bud elongation culture ( Figure 5 D), after rooting, the wild-type plant is green ( Figure 5 E), while the roots and above-ground parts of the transgenic plants all showed a distinct red phenotype ( Figure 5 F). This result indicates that the genetic transformation system established in this invention can be efficiently applied to the verification of gene function and the breeding of new colored varieties of Liquidambar formosana.

[0099] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for in vitro regeneration of Liquidambar formosana, characterized in that, Includes the following steps: (1) Leaves of Liquidambar formosana tissue culture seedlings were used as explants and inoculated into callus induction and adventitious shoot differentiation medium to induce callus and adventitious shoot differentiation and obtain adventitious shoots; the callus induction and adventitious shoot differentiation medium was based on WPM medium, with 1.0 mg / L of 6-BA, 0.1 mg / L of NAA and 0.2 mg / L of TDZ added. (2) The adventitious shoots obtained in step (1) are transferred to the adventitious shoot elongation medium for elongation culture; the adventitious shoot elongation medium is based on WPM medium, with 0.5 mg / L of 6-BA, 0.2 mg / L of NAA and 0.1 mg / L of KT added. (3) The elongated adventitious buds were transferred to the rooting medium for rooting culture to obtain complete regenerated plants; the rooting medium was based on WPM and supplemented with 1.0 mg / L IBA.

2. The in vitro regeneration method according to claim 1, characterized in that, In step (3), 0.8 g / L of activated carbon is also added to the rooting medium.

3. The in vitro regeneration method according to claim 1, characterized in that, In step (1), the leaf explant is cut into small pieces of 0.5 cm × 0.5 cm.

4. A method for genetic transformation of Liquidambar formosana, characterized in that, Includes the following steps: (1) Using leaves of Liquidambar formosana tissue culture seedlings as explants, the leaves were immersed in Agrobacterium tumefaciens containing the target gene for infection. The OD of the Agrobacterium tumefaciens solution was... 600 The value is 0.4-0.6, and the infection time is 15-25 minutes; (2) Co-culture the infected leaf explants; (3) Sterilize the leaves after co-culture; (4) After sterilization, the leaves were transferred to a medium for callus induction and adventitious shoot differentiation to screen for resistant shoots and obtain resistant shoots. The medium for callus induction and adventitious shoot differentiation used for transformation was based on WPM medium, with the addition of 1.0 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mg / L TDZ, as well as screening agents and antibacterial agents. (5) The obtained resistant shoots were transferred to the adventitious shoot elongation medium for transformation and elongation culture; The adventitious shoot elongation medium for transformation is based on WPM medium, with the addition of 0.5 mg / L 6-BA, 0.2 mg / L NAA, 0.1 mg / L KT, as well as screening agents and antibacterial agents; (6) The elongated resistant shoots were transferred to a rooting medium for transformation and rooting culture to obtain transgenic plants; The rooting medium for transformation is based on WPM, with the addition of 1.0 mg / L IBA, as well as screening agents and antibacterial agents.

5. The genetic transformation method according to claim 4, characterized in that, The screening agent is Kan, and the antibacterial agents are Cef and Tim.

6. The genetic transformation method according to claim 5, characterized in that, In step (4), the concentration of Kan is 50 mg / L, the concentration of Cef is 200 mg / L, and the concentration of Tim is 150 mg / L; in steps (5) and (6), the concentration of Kan is 30 mg / L, the concentration of Cef is 200 mg / L, and the concentration of Tim is 150 mg / L.

7. The genetic transformation method according to claim 4, characterized in that, In step (6), 0.8 g / L of activated carbon is added to the rooting medium for transformation.

8. The genetic transformation method according to claim 4, characterized in that, In step (1), the OD of the Agrobacterium tumefaciens bacterial solution... 600 The value was 0.5, and the infection time was 20 minutes.

9. The genetic transformation method according to claim 4, characterized in that, In step (2), the co-culture conditions are as follows: the infected leaf explants are placed in WPM medium containing 100 μM AS and cultured in the dark at 25±1℃ for 2-3 days.

10. The application of the in vitro regeneration method as described in any one of claims 1-3, or the genetic transformation method as described in any one of claims 4-9, in the breeding of Liquidambar formosana, characterized in that, The applications include rapid propagation through tissue culture or obtaining new varieties of Liquidambar formosana with target traits by introducing exogenous genes.

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