A method for rapid proliferation and genetic transformation regeneration of curcuma wenyujin embryonic callus

Abstract

The application discloses a method for rapid proliferation and genetic transformation regeneration of Radix Morindae Officinalis embryogenic callus and belongs to the technical field of plant tissue culture. The method comprises the following steps: firstly, inoculating Radix Morindae Officinalis embryogenic callus into a liquid culture medium to carry out suspension culture to obtain Radix Morindae Officinalis embryogenic callus cell groups; then adding the callus cell groups into a bacterial infection liquid to carry out infection; and then transferring the callus cell groups to a differentiation culture medium to carry out culture to obtain Radix Morindae Officinalis transgenic regenerated plants. The suspension culture medium provided by the application can realize large-scale and rapid proliferation of Radix Morindae Officinalis embryogenic callus, and the embryogenic callus cell groups obtained by the proliferation have a good growth state. In the process of establishing the genetic transformation system of Radix Morindae Officinalis, the method of directly carrying out differentiation culture after co-culture is adopted, the differentiation culture medium does not add resistance screening, and an antibacterial agent, i.e., trimethoprim, is added, so that the transgenic plants are regenerated with high efficiency.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for rapid proliferation and genetic transformation and regeneration of embryogenic callus from Curcuma zedoaria. Background Technology

[0002] Curcuma wenyujin (YHChen & C.Ling) is one of the famous "Eight Treasures of Zhejiang" in Zhejiang Province, also known as "Wen Ezhu". It is a perennial herb belonging to the genus Curcuma in the ginger family and has a history of medicinal use for over 1500 years. Three traditional medicinal materials and processed slices derived from Curcuma wenyujin—Ezhu, sliced ​​Curcuma, and Curcuma—have significant effects in treating amenorrhea due to blood stasis, chest pain, and promoting qi circulation and relieving depression. They are known as qi-regulating herbs in the blood, and all are included in the *Pharmacopoeia of the People's Republic of China*. Modern medical research and clinical application have shown that the volatile oil extract of Curcuma wenyujin has significant efficacy in the prevention and treatment of malignant tumors, especially early-stage cervical cancer.

[0003] The volatile oils mainly consist of sesquiterpenes, which are secondary metabolites. Their content in Curcuma zedoaria is very low, especially since the traditional asexual propagation method still prevalent in Curcuma zedoaria cultivation has led to varietal degeneration and a significant reduction in both yield and active ingredient content. The active ingredient elemene accounts for only 4-10% of the dry weight, which is insufficient to meet market demand. Taking β-elemene, a small-molecule anticancer drug extracted from Curcuma zedoaria, as an example, although it can be obtained through artificial synthesis, microbial cell factories, and plant cell factories, extraction from Curcuma zedoaria remains the safest and most convenient method. This is because β-elemene obtained through heterologous production may have a different configuration than that extracted from the plant source, or may undergo further chemical modification, posing certain risks for clinical application. Curcuma zedoaria oil typically contains multiple active ingredients, and the efficacy of single-component heterologous synthesis is not ideal.

[0004] Currently, the production of plant secondary metabolites using tissue culture technology is widely studied and applied. Cell culture methods can not only synthesize the original natural products of plants, but also control the culture conditions, enabling continuous production of products. Regarding the cell culture of Curcuma zedoaria, Luo Hongmei and Dong Lixia et al. have initially established a suspension cell culture system for Curcuma zedoaria (Cyclocarya zedoaria cell suspension culture, volatile oil, polysaccharide separation and its polysaccharide biological activity study. 2003, 6; Suspension cell culture of Curcuma zedoaria from Hainan and its accumulation of volatile components study. Chinese Herbal Medicines, 2023, 46(4).), but due to the differences in raw material origin and culture environment, their research results cannot be successfully replicated in our laboratory. In addition, the establishment of large-scale plant cell factories not only requires expensive equipment and professional staff, but also has high requirements for workshop environmental conditions, making it difficult to popularize.

[0005] With the gradual elucidation of the synthetic pathways of effective components in Curcuma zedoaria, especially the molecular regulatory mechanisms of terpene biosynthesis, obtaining high-yielding Curcuma zedoaria plants through genetic improvement is a fundamental solution to the problem, in accordance with the requirements of the modernization of traditional Chinese medicine. Therefore, the establishment of a Curcuma zedoaria genetic transformation system is urgently needed. Patent document CN116463372A discloses the genetic transformation of Curcuma zedoaria leaves; patent document CN 111621517 B discloses the successful induction of hairy roots by infecting the base of Curcuma zedoaria tissue culture seedlings with Agrobacterium. However, there are still no reports on establishing a Curcuma zedoaria genetic transformation system using Agrobacterium infection of callus tissue.

[0006] Our research group previously established a method for inducing embryogenic callus and regenerating plants in *Curcuma wenyujin* (ZL202010522942.1), laying the foundation for the genetic transformation of *Curcuma wenyujin*. However, studies have found that the embryogenic callus induced by this method has a low proliferation rate during subsequent subcultures. Furthermore, after multiple subcultures, the embryogenic callus exhibits browning and hydration, leading to difficulties in genetic transformation and reduced differentiation and regeneration capacity. Therefore, how to achieve large-scale and rapid proliferation of *Curcuma wenyujin* embryogenic callus and successfully establish a genetic transformation system for *Curcuma wenyujin* is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a method suitable for the rapid proliferation and genetic transformation and regeneration of embryogenic callus of Curcuma zedoaria, so as to achieve large-scale rapid proliferation of embryogenic callus of Curcuma zedoaria, and the embryogenic callus obtained by proliferation can be efficiently differentiated and regenerated into transgenic plants after genetic transformation by Agrobacterium.

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

[0009] This invention provides a method for rapid proliferation and genetic transformation regeneration of embryogenic callus from Curcuma longa, comprising the following steps:

[0010] (1) The embryogenic callus of Curcuma zedoaria was inoculated into a liquid culture medium and suspended to obtain a cell mass of embryogenic callus of Curcuma zedoaria. The liquid culture medium was MS medium supplemented with 25-35 g / L sucrose, 0.2-0.4 mg / L 6-BA, and 0.5-1.5 mg / L 2,4-D.

[0011] (2) Add the embryogenic callus cell mass of Curcuma zedoaria to Agrobacterium infection solution for infection;

[0012] (3) The infected callus tissue was transferred to a co-culture medium for co-culture. The co-culture medium was MS medium supplemented with 25-35 g / L sucrose, 5-10 g / L agar, and 100-200 μmol / L acetylsuccinone.

[0013] (4) The co-cultured callus tissue was transferred to a differentiation medium to obtain transgenic regenerated plants of Curcuma longa. The differentiation medium was MS medium supplemented with 25-35 g / L sucrose, 5-10 g / L agar, 1-3 mg / L 6-BA, 1-3 mg / L NAA, and 150-250 mg / L termethin.

[0014] In step (1), the present invention obtains a large number of embryonic cell clusters of Curcuma zedoaria callus with good growth status by optimizing the suspension culture medium formula for suspension culture of Curcuma zedoaria callus embryonic callus, which are then used for subsequent genetic transformation.

[0015] The embryogenic callus of *Curcuma wenyuensis* was induced from the root base of *Curcuma wenyuensis* clump seedlings as explants, and the induction method can be found in patent document CN111616053B. The *Curcuma wenyuensis* came from the *Curcuma wenyuensis* GAP demonstration planting base in Ruian, Wenzhou.

[0016] Specifically, the method for preparing the embryogenic callus of *Curcuma wenyujin* includes: firstly, taking young shoots from the tubers of *Curcuma wenyujin* and inoculating them in a young shoot culture medium to obtain sterile seedlings of *Curcuma wenyujin*. The young shoot culture medium is MS medium supplemented with 0.15% PPM and 3.0 mg / L 6-BA. The culture is carried out in two steps. In the first step, the seedlings are placed at 25-28℃, under light for 6 hours and darkness for 18 hours, with a light intensity of 30-40 μmol / L. -2 .s -1 The culture was carried out for 10-14 days under the following conditions; the second step involved placing the specimen at 25-28℃ with a 12-hour light / 12-hour dark cycle and a light intensity of 50-60 μmol / m. -2 .s -1 The seedlings were cultured for 10-14 days under controlled conditions; then, the aseptic seedlings were transferred to a shoot induction medium (MS medium supplemented with 3.0 mg / L 6-BA) at 25-28°C, with a 12-hour light / 12-hour dark cycle and a light intensity of 50-60 μmol / L. -2 .s -1 Under certain conditions, sterile clustered seedlings were obtained after culturing for 25-30 days. The root base of the sterile clustered seedlings was then used as explants and inoculated into callus induction medium. The callus induction medium was MS medium supplemented with 0.5 mg / L 6-BA and 0.5 mg / L 2,4-D. The seedlings were cultured in the dark at 25-28℃ for 45-60 days to obtain embryogenic callus of *Curcuma longa*.

[0017] As a preferred method, the suspension culture conditions are: dark culture at 23±2℃ and 100-150rpm for 50-70 days, with fresh culture medium added every two weeks during this period.

[0018] Preferably, the liquid culture medium is MS medium supplemented with 30 g / L sucrose, 0.3 mg / L 6-BA, 1.0 mg / L 2,4-D, and a pH of 5.8.

[0019] In step (2), Agrobacterium infection solution is mixed with the embryogenic callus cell cluster of Curcuma longa to achieve Agrobacterium infection.

[0020] The method for preparing the Agrobacterium infection solution includes: transforming recombinant plasmids into Agrobacterium via chemical transformation, collecting bacterial cells after culture, resuspending them in a susceptible solution, and determining the OD value. 600 The concentration was maintained at 0.4-0.6, and 100-200 μmol / L of acetylsylgenone was added to induce culture for 0.5-1 h; the inoculum was MS medium supplemented with 25-35 g / L of sucrose.

[0021] Preferably, the formulation of the inoculum is MS medium (agar-free) + 30 g / L sucrose, pH 5.3. Agrobacterium is resuspended in the inoculum to prepare an Agrobacterium suspension, OD... 600 The concentration was maintained at around 0.5, and 150 μmol / L of acetylsalicylic acid was added. The mixture was then induced and cultured at 28°C and 150 rpm for 1 hour to obtain the Agrobacterium infection solution.

[0022] As a preferred method, the embryogenic callus cell clusters of Curcuma longa are added to Agrobacterium tumefaciens infection solution and infected at 60-100 rpm and room temperature for 25-30 min. Then, the callus tissue is placed on sterile filter paper and dried.

[0023] In step (3), the infected callus tissue is transferred to a co-culture medium for co-culture, and the target gene fragment is introduced into the embryogenic callus tissue cells of Curcuma longa using Agrobacterium tumefaciens to achieve genetic transformation.

[0024] As a preferred method, the co-culture conditions are: dark incubation at 25±2℃ for 2-3 days.

[0025] Preferably, the co-culture medium is MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 150 μmol / L acetylsylgenone, and a pH of 5.3.

[0026] As a preferred method, after co-culturing, the callus tissue is washed sequentially with sterile water and sterile water containing 200 mg / L termethin, and then air-dried.

[0027] In step (4), the present invention directly transfers the co-cultured callus to a differentiation culture medium without antibiotics for differentiation culture, thereby improving the differentiation and regeneration rate of the callus.

[0028] Preferably, the differentiation medium is MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 2 mg / L 6-BA, 1 mg / L NAA, and 200 mg / L termethin.

[0029] The culture conditions for differentiated and regenerated transgenic plants were 25-28℃, 12h light / 12h dark, and a light intensity of 50-60 μmol / m. -2 .s -1 Culture for 50-70 days under suitable conditions.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) The suspension culture medium provided by the present invention can realize the large-scale and rapid proliferation of embryogenic callus of Curcuma zedoaria, and the cell clusters of embryogenic callus obtained by proliferation are in good growth state, laying the foundation for the successful establishment of the genetic transformation system of Curcuma zedoaria.

[0032] (2) In the process of establishing the genetic transformation system of Curcuma zedoaria, the present invention adopts the method of co-culture followed by direct differentiation culture, and does not add resistance screening to the differentiation culture medium, but adds the antibacterial agent termethin to achieve efficient differentiation and regeneration of transgenic plants, and successfully establishes the genetic transformation system of Curcuma zedoaria. Attached Figure Description

[0033] Figure 1 Images of embryogenic callus tissue of Curcuma zedoaria obtained in Example 1 are shown. A and B are Curcuma zedoaria embryogenic callus tissue samples numbered 1 and 7, respectively.

[0034] Figure 2 The images show the suspension culture of embryogenic callus tissue from Curcuma zedoaria in Example 1. A and B correspond to Curcuma zedoaria embryogenic callus tissue samples numbered 1 and 7, respectively.

[0035] Figure 3 The images show the embryogenic callus cell clusters of Curcuma zedoaria after 60 days of suspension culture in Example 1. A and B correspond to Curcuma zedoaria embryogenic callus samples numbered 1 and 7, respectively.

[0036] Figure 4 This is an image of the co-cultured organisms after being infected with Agrobacterium in Example 2.

[0037] Figure 5 The image shows the results of differentiation culture for 2 weeks in Example 2.

[0038] Figure 6 The image shows the regenerated plants obtained from the differentiation culture in Example 2.

[0039] Figure 7The results of GUS staining of callus tissue after 2 weeks of differentiation culture in Example 2 are shown. A is the control group transfected with pCambia1304-35S empty vector, and B is the experimental group transfected with pCambia1304-35S::GUS.

[0040] Figure 8 The results of GUS staining of regenerated plants in Example 2 are shown. A is the control group transfected with the empty vector pCambia1304-35S, and B is the experimental group transfected with pCambia1304-35S::GUS.

[0041] Figure 9 The results show the PCR amplification of the reporter gene green fluorescent protein (GFP) and the vector resistance gene kanamycin (Kan) in the regenerated plants in Example 2.

[0042] Figure 10 The suspension culture of embryogenic callus of Curcuma zedoaria in Comparative Example 1 is shown. A represents embryogenic callus of Curcuma zedoaria, B represents suspension culture, and C represents the differentiation, browning, and death of embryogenic cell clusters of callus.

[0043] Figure 11 This is a comparison of the induction of embryogenic callus in *Curcuma longa* in Example 2.

[0044] Figure 12 This is an image of the embryogenic callus of Curcuma zedoaria in Comparative Example 3, cultured in a selective medium containing antibiotics.

[0045] Figure 13 This is an image of the embryogenic callus of Curcuma zedoaria in Comparative Example 3, cultured in a differentiation medium containing antibiotics. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0048] The Curcuma longa material used to induce embryogenic callus formation in the following examples was sourced from the Curcuma longa GAP demonstration planting base in Ruian, Wenzhou, Zhejiang Province.

[0049] MS medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; 6-BA (6-benzyladenine), CAS No.: 1214-39-7; 2,4-D (2,4-dichlorophenoxyacetic acid), CAS No.: 94-75-7; As (acetylsyringone), CAS No.: 2478-38-8; NAA (1-naphthaleneacetic acid), CAS No.: 86-87-3; termethin, CAS No.: 86482-18-0.

[0050] Example 1: Suspension culture of embryogenic callus from Curcuma longa

[0051] 1. Induction of embryogenic callus formation in Curcuma longa

[0052] The embryogenic callus of *Curcuma wenyus* was induced by the method described in patent document CN111616053B, "A method for inducing embryogenic callus and regenerating plants in *Curcuma wenyus*". Figure 1 As shown.

[0053] 2. Suspension culture

[0054] Take 3-5g of *Curcuma longa* embryogenic callus and inoculate it into a 250mL Erlenmeyer flask containing 100mL MS (agar-free) + 30g / L sucrose + 0.3mg / L 6-BA + 1.0mg / L 2,4-D, pH 5.8. Figure 2 As shown. Suspension culture was carried out in the dark under shaking conditions at 23℃ and 130rpm.

[0055] Fresh culture medium is added every two weeks. Specifically, after removing the culture flask from the shaker, let it stand for about 30 minutes until most of the embryogenic cell clusters of the *Curcuma longa* callus are deposited at the bottom of the flask. Then, slowly pour out about one-third of the clear culture medium from the top layer in the clean bench and add the same volume of fresh culture medium to the culture flask.

[0056] During this period, the culture flasks were gradually replaced with larger ones based on the proliferation of the cell clusters, and the cells were then cultured in smaller flasks. The specific volumes of the culture flasks were 250 mL, 500 mL, 1 L, and 2 L.

[0057] After 60 days, a large number of turmeric embryogenic callus cell clusters were obtained, such as Figure 3 As shown in the figure. Specific data is shown in Table 1.

[0058] Table 1. Inoculum Size and Final Yield for Suspension Culture

[0059] Sample number 1 7 Inoculation amount (g) 3.74 4.11 Final yield (g) 959.47 978.56

[0060] Example 2: Agrobacterium-mediated genetic transformation of embryogenic callus from Curcuma longa

[0061] Plasmids pBI121-35S::GFP and pCambia1304-35S::GUS, carrying the GFP and GUS reporter genes respectively, were transformed into Agrobacterium GV3101 via chemical transformation. Single clones of Agrobacterium were selected, and transgenic plants were obtained after shaking, infection, co-culture, differentiation and regeneration, and transgenic identification. The specific operational steps are as follows:

[0062] 1. Agrobacterium culture and preparation of inoculum

[0063] Select a single Agrobacterium colony and incubate it overnight at 28°C with shaking at 250 rpm in 15 mL of YEP liquid culture medium (containing 50 mg / L kanamycin and 50 mg / L streptomycin) until OD. 600 =0.8-1.5. Transfer the cultured bacterial solution to a 50 mL centrifuge tube, centrifuge at 4℃ and 4000 rpm for 10 min, and discard the supernatant. Add approximately 30 mL of the inoculum to the centrifuge tube, gently mix with a pipette tip to prepare an Agrobacterium suspension, OD... 600 Maintain the concentration at approximately 0.5, add 150 μmol / L acetylsyringone (As), and induce incubation at 28°C and 150 rpm for 1 hour. Infected culture formulation: MS (agar-free) + 30 g / L sucrose, pH 5.3.

[0064] 2. Infection

[0065] Select a cluster of *Curcuma longa* embryogenic callus cells obtained from suspension culture in Example 1 and place it into a 50 mL centrifuge tube. Add the inducing inoculum solution and infect the callus at room temperature for 25-30 min on a horizontal shaker at approximately 80 rpm. Discard the inoculum solution and place the infected callus tissue on sterile filter paper to dry in a laminar flow hood.

[0066] 3. Co-cultivation

[0067] The dried callus tissue was then transferred to a co-culture medium, such as... Figure 4 As shown, the culture was carried out in the dark at 25°C for 3 days. The co-culture medium formula was: MS + 30 g / L sucrose + 7 g / L agar + 150 μmol / L As, pH 5.3.

[0068] 4. Differentiation culture

[0069] The callus tissue obtained after co-culture was transferred to 50 mL centrifuge tubes, washed three times with sterile distilled water for approximately 5 minutes each time, and then washed 1-2 times with sterile water supplemented with 200 mg / L termethin. The tissues were then dried on sterile filter paper in a laminar flow hood and transferred to differentiation medium for differentiation and regeneration. The differentiation medium formula was: MS + 30 g / L sucrose + 7 g / L agar + 2 mg / L 6-BA + 1 mg / L NAA + 200 mg / L termethin. The differentiation culture conditions were 25℃, 12 h light / 12 h dark, and a light intensity of 50-60 μmol / L. - 2 .s -1 .

[0070] After 2 weeks of differentiation culture Figure 5 As shown, regenerated plants were obtained after approximately 60 days of differentiation culture, such as... Figure 6 As shown.

[0071] 5. Identification of transgenic plants

[0072] Two weeks after differentiation culture, a portion of the callus tissue was picked for GUS staining, and the results were as follows. Figure 7 As shown, the callus tissue in the experimental group transfected with pCambia1304-35S::GUS was stained blue, while the callus tissue in the control group transfected with the empty pCambia1304-35S vector showed no color change, indicating that Agrobacterium carrying the GUS reporter gene successfully infected the callus tissue of Curcuma zedoaria.

[0073] Sixty days after differentiation culture, GUS staining was also performed on the leaves of the transgenic regenerated plants, and the results were as follows: Figure 8 As shown, the GUS reporter gene was successfully expressed in the leaves of the experimental group, while the leaf color of the control group remained unchanged.

[0074] PCR amplification was used to detect whether the GFP reporter gene was successfully transfected into regenerated Tuberculia lychnophora plants. Specifically, primers were designed based on the sequence information of the vector 35S promoter, the reporter gene green fluorescent protein (GFP), and the vector resistance gene kanamycin (Kan). Genomic DNA from the regenerated plants was used as a template for PCR reaction.

[0075] The primer sequences used in the PCR reaction are as follows:

[0076] 35S-F: 5'-ATTGCCCAGCTATCTGTCACTTT-3';

[0077] GFP-R: 5'-CGTATGTTGCATCACCTTCACCC-3';

[0078] Kan-F: 5'-GGCTATGACTGGGCACAACA-3';

[0079] Kan-R: 5'-GATACCGTAAAGCACGAGGAA-3'.

[0080] The results are as follows Figure 9 As shown, the genetic transformation system of Curcuma longa was successfully established after PCR amplification and verification.

[0081] Comparative Example 1

[0082] This invention aims to obtain a sufficient number of embryogenic cell clusters from Curcuma zedoaria callus for subsequent genetic transformation experiments. This comparative example is based on the experimental results of the literature (Luo Hongmei. Study on the separation of volatile oil and polysaccharides and their biological activity in Curcuma zedoaria cell suspension culture. 2003, 6), selecting the culture conditions described in the literature that are most conducive to cell proliferation and maintain good growth. Specific culture conditions: MS (without agar, NH4+). + / NO3 - The solution was prepared by a 1:3 ratio (total nitrogen concentration of 40 mmol / L) + 15 g / L sucrose + 15 g / L glucose + 0.5 mg / L 6-BA + 2.0 mg / L 2,4-D, with an inoculum size of 50 g / L. The pH was 5.8, and the suspension culture was carried out at 25°C and 120 rpm in a shaker under a light-dark cycle (14 h / 10 h). Other conditions were the same as in Example 1.

[0083] After 4 weeks of culture, it was found that the embryonic cell clusters of callus differentiated, ceased proliferation, and gradually browned and died. See details below. Figure 10 .

[0084] Comparative Example 2

[0085] This comparative example refers to the method of inducing callus tissue from the tender roots of Curcuma zedoaria tissue culture seedlings in Hainan Province, as described in the literature (Dong Lixia et al. Study on suspension cell culture and accumulation of volatile components of Curcuma zedoaria from Hainan. Chinese Medicinal Herbs, 2023, 46(4).). The induction conditions were as follows: MS + 2 mg / L 2,4-D + 1.0 mg / L 6-BA + 30 g / L sucrose, 2.2 g / L plant gel, pH 5.8, 28℃, dark culture for 30 days.

[0086] The results are as follows Figure 11 As shown, the callus induction rate is low and the amount is small, which cannot meet the requirements for inoculation volume in the later suspension culture.

[0087] Comparative Example 3

[0088] The conventional transgenic process is "infection—co-culture—selection culture—differentiation culture". In this comparative example, during the establishment of the *Curcuma wenyujin* genetic transformation system, co-cultured *Curcuma wenyujin* callus tissue was cultured in selection and differentiation media supplemented with hygromycin B (HygB). Details are as follows:

[0089] In step 4 of Example 2, the *Curcuma longa* callus obtained after co-culture was washed with sterile water, dried, and transferred to a selective medium supplemented with hygromycin. The selective medium formulation was: 100 mL MS + 30 g / L sucrose + 7 g / L Agar + 0.3 mg / L 6-BA + 1.0 mg / L 2,4-D + 50 mg / L hygromycin, pH 5.8. The results were as follows... Figure 12 As shown, the callus tissue of Curcuma zedoaria exhibited browning and death in selective culture medium supplemented with hygromycin.

[0090] In step 4 of Example 2, the *Curcuma longa* callus obtained after co-culture was washed with sterile water, dried, and transferred to differentiation medium supplemented with hygromycin. The differentiation medium formula was: MS + 30 g / L sucrose + 7 g / L Agar + 2 mg / L 6-BA + 1 mg / L NAA + 50 mg / L hygromycin, pH 5.8. The results were as follows... Figure 13 As shown, the callus tissue of Curcuma longa exhibited browning and death in differentiation medium supplemented with hygromycin.

[0091] Therefore, this invention adopts a co-culture followed by direct differentiation culture method, and the differentiation culture medium does not contain the resistance selection marker HygB, but contains the antibacterial agent termethin.

Claims

1. A method for rapid proliferation and genetic transformation regeneration of embryogenic callus from Curcuma longa, characterized in that, Includes the following steps: (1) The embryogenic callus of Curcuma zedoaria was inoculated into a liquid culture medium and suspended to obtain the embryogenic callus cell cluster of Curcuma zedoaria. The liquid culture medium was MS medium supplemented with 25-35 g / L sucrose, 0.2-0.4 mg / L 6-BA and 0.5-1.5 mg / L 2,4-D. The suspension culture conditions were: 23±2℃, 100-150 rpm, and dark culture for 50-70 days, during which fresh culture medium was added every two weeks. (2) Add the embryogenic callus cell mass of Curcuma zedoaria to Agrobacterium infection solution for infection; The method for preparing the Agrobacterium infection solution includes: transforming recombinant plasmids into Agrobacterium via chemical transformation, collecting bacterial cells after culture, resuspending them in a susceptible solution, and determining the OD value. 600 The concentration was maintained at 0.4-0.6, and 100-200 μmol / L of acetylsyringone was added to induce culture for 0.5-1 h; the inoculum was MS medium supplemented with 25-35 g / L sucrose, pH 5.3; (3) The infected callus tissue was transferred to a co-culture medium for co-culture. The co-culture medium was MS medium with 25-35 g / L sucrose, 5-10 g / L agar, and 100-200 μmol / L acetylsylgenone added, and the pH value was 5.

3. The co-culture conditions were: dark culture at 25±2℃ for 2-3 days. (4) The co-cultured callus tissue was transferred to a differentiation medium to obtain transgenic regenerated plants of Curcuma longa. The differentiation medium was MS medium supplemented with 25-35 g / L sucrose, 5-10 g / L agar, 1-3 mg / L 6-BA, 1-3 mg / L NAA, and 150-250 mg / L termethin.

2. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, Embryogenic callus of Curcuma wenyujin was induced from the root base of Curcuma wenyujin seedlings as explants. The Curcuma wenyujin came from the GAP demonstration planting base of Curcuma wenyujin in Ruian, Wenzhou.

3. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, The liquid culture medium is MS medium supplemented with 30 g / L sucrose, 0.3 mg / L 6-BA, 1.0 mg / L 2,4-D, and a pH of 5.

8.

4. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, In step (2), the formulation of the infecting solution is MS medium with 30 g / L sucrose added, pH 5.3; Agrobacterium is resuspended in the infecting solution to prepare an Agrobacterium suspension, OD 600 The concentration was maintained at 0.5, and 150 μmol / L of acetylsalicylic acid was added. The mixture was induced and cultured at 28°C and 150 rpm for 1 hour to obtain the Agrobacterium infection solution.

5. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, In step (2), the callus is infected at 60-100 rpm and room temperature for 25-30 minutes, and then placed on sterile filter paper to dry.

6. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, In step (3), the co-culture medium is MS medium with 30 g / L sucrose, 7 g / L agar, 150 μmol / L acetylsuccinone added, and pH value of 5.

3.

7. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, In step (3), after co-culturing, the callus tissue is washed with sterile water and sterile water containing 200 mg / L termethin in sequence, and then air-dried.

8. The method for rapid proliferation and genetic transformation regeneration of *Curcuma longa* embryogenic callus as described in claim 1, characterized in that, In step (4), the differentiation medium is MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 2 mg / L 6-BA, 1 mg / L NAA, and 200 mg / L termethin.

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