Method for transgenosis by using dendrobium huoshanense protocorm and application thereof

Abstract

The invention relates to the technical field of transgenosis, and discloses a method for transgenosis by using dendrobium huoshanense protocorm, which comprises the following steps: S1, induction culture of dendrobium huoshanense protocorm; s2, constructing a plant expression vector; s3, infecting and culturing the dendrobium huoshanense protocorm by agrobacterium tumefaciens; s4, screening and culturing the dendrobium huoshanense transgenic tissue and seedlings, the dendrobium huoshanense protocorm is used as a material, agrobacterium tumefaciens-mediated genetic transformation is realized on the basis of a dendrobium huoshanense protocorm tissue rapid propagation system, a stable and rapid genetic transformation system of dendrobium huoshanense is established, and creation of a new variety of dendrobium huoshanense is realized.

Description

Technical Field

[0001] The present invention relates to the field of transgenic technology, and more particularly to a method for performing transgenic production using protocorms of Dendrobium huoshanense and its application. Background Art

[0002] Huoshan Dendrobium, a precious medicinal herb of the genus Dendrobium in the Orchidaceae family, has held a vital position in traditional Chinese medicine since ancient times. Rich in polysaccharides, alkaloids, flavonoids, and other active ingredients, Huoshan Dendrobium boasts numerous health benefits, including immune-boosting, antioxidant, and anti-tumor properties. However, its limited natural reproductive capacity, coupled with its unique habitat, has led to a growing depletion of wild resources, making it unable to meet the growing market demand. While artificial cultivation has alleviated resource pressures to some extent, it still faces numerous challenges. For example, Huoshan Dendrobium's long growth cycle and demanding growing conditions make it susceptible to pests and diseases, and its limited genetic diversity severely restrict its industrial development. To improve the yield and stress resistance of Huoshan Dendrobium, researchers have been dedicated to genetic improvement and the selection of new varieties. Traditional breeding methods, such as hybridization, while capable of introducing new genetic traits to a certain extent, are cumbersome, time-consuming, and difficult to precisely control the introduction of target genes. Therefore, with the rapid development of biotechnology, transgenic technology has provided new possibilities for the genetic improvement of Huoshan Dendrobium.

[0003] Transgenic technology involves the artificial introduction of exogenous genes into plants, where they are stably expressed and inherited. This technology can be used to modify plant traits, such as enhancing stress resistance, increasing yield, and improving quality. However, research on transgenic technology for the Dendrobium genus in the Orchidaceae family started relatively late, and currently faces numerous technical bottlenecks, such as low transgenic efficiency, unstable exogenous gene expression, and abnormal growth of transgenic plants. To address these issues, the present invention aims to construct an efficient and stable transgenic technology system for Dendrobium huoshanense, and based on this, to create new varieties with specific traits. This technology system enables the introduction and efficient expression of exogenous genes in Dendrobium huoshanense, thereby accelerating the breeding process for new varieties, improving their yield and stress resistance, and laying a solid foundation for the industrialization and in-depth research of Dendrobium huoshanense. Furthermore, the implementation of this invention will help promote the development and application of plant transgenic technology, providing valuable insights for the genetic improvement of other precious Chinese medicinal materials. Currently, no reports on transgenic protocorms of Dendrobium huoshanense have been found. Summary of the Invention

[0004] The present invention provides a method for genetically modifying the protocorms of Dendrobium huoshanense. The protocorms of Dendrobium huoshanense are used as materials. On the basis of the rapid propagation system of Dendrobium huoshanense tissue, Agrobacterium-mediated genetic transformation is realized, a stable and rapid genetic transformation system of Dendrobium huoshanense is established, and germplasm creation of Dendrobium huoshanense is realized.

[0005] The present invention provides a method for genetically modifying a Dendrobium huoshanense protocorm, comprising the following steps:

[0006] S1: Induction culture of protocorms of Dendrobium huoshanense;

[0007] S2: Construction of plant expression vector;

[0008] S3: Agrobacterium infection and culture of protocorms of Dendrobium huoshanense;

[0009] S4: Screening and cultivation of transgenic tissues and seedlings of Dendrobium huoshanense.

[0010] Preferably, the method for inducing the culture of the protocorms of Dendrobium huoshanense in S1 is as follows: the sterilized Dendrobium huoshanense seeds are sown in piles on an induction medium, and after about half a month of induction, the protocorms with vigorous growth are selected as genetic transformation recipients.

[0011] Preferably, the induction medium is MS+1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.5 g / L activated carbon and 7 g / L agar, pH=5.8.

[0012] Preferably: a method for preparing an induction culture medium, comprising the following steps: preparing a composite antioxidant solution of ascorbic acid, glutathione, and α-tocopherol mixed in a mass ratio of 2:1:1; preparing an enzyme separation carrier of a catalase gelatin microcapsule carrier and a superoxide dismutase sodium alginate gel carrier; preparing an organic acid stabilized carrier complex of malic acid, succinic acid combined with chitosan and pectin and adding dipotassium hydrogen phosphate and sodium bicarbonate buffering protective agents; preparing a chelated stabilizing synergistic solution of phytic acid, tea polyphenols, sodium citrate chelating agent mixed with lecithin and gum arabic stabilizer; and sequentially adding the above components to MS basal culture medium in a volume ratio of basal culture medium: chelated stabilizing solution: composite antioxidant solution: enzyme system carrier: organic acid carrier = 100:5:3:2:2 and mixing.

[0013] Preferably, the total concentration of the compound antioxidant solution is 50 mg / L.

[0014] Preferably, the diameter of the catalase gelatin microcapsule carrier microspheres is 50-100 μm, and the embedding efficiency reaches more than 85%.

[0015] Preferably, the diameter of the gel balls of the superoxide dismutase sodium alginate gel carrier is 100-200 μm.

[0016] Preferably, the final concentrations of dipotassium hydrogen phosphate and sodium bicarbonate in the organic acid-stabilized carrier complex are 5 mM and 3 mM, respectively, and the pH is adjusted to 6.0-6.5.

[0017] Preferably, the total concentration of the chelating agent solution is 30 mg / L, and the total concentration of the stabilizer solution is 20 mg / L.

[0018] Preferably, the chelating agent solution and the stabilizing agent solution are mixed in a volume ratio of 2:1 to form a chelating stabilizing synergistic solution.

[0019] Preferably, the MS basal medium contains 1.5 mg / L 6-benzylaminopurine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.5 g / L activated carbon and 7 g / L agar, with a pH of 5.8.

[0020] Preferably, the order of adding the components is: first add the chelating stabilization synergistic solution, then add the compounded antioxidant solution, the enzyme separation carrier and the organic acid stabilization carrier complex in sequence.

[0021] Preferably: application of the method for preparing the induction culture medium in plant transgenic culture.

[0022] Preferably, the method for constructing the plant expression vector in S2 is: downloading the green fluorescent protein sequence GenBank: KF206153.1 from the NCBI database and performing gene synthesis;

[0023] The GFP gene fragment was connected to the plant expression vector pCAMBIA1300 by homologous recombination to construct the recombinant vector pCAMBIA1300-GFP.

[0024] The PCR amplification primers used for vector construction are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0025] SEQ ID NO: 1

[0026] OE-GFP-F:

[0027] 5'-GGAGAGGACAGGGTACCCGGGGATCCTGAGCAAGGGCGAGGAGC-3'.

[0028] SEQ ID NO: 2

[0029] OE-GFP-R:

[0030] 5'-ACCATGGTACTAGTGTCGACTCTAGATTACTTGTACAGCTCGTCCA-3'.

[0031] Preferably, the method of infection and co-cultivation in S3 is: introducing the recombinant vector pCAMBIA1300-GFP into Agrobacterium tumefaciens, activating the transformed Agrobacterium by the plate streak method, picking a single colony and shaking the bacteria, resuspending the obtained bacterial liquid with a solution to obtain an infection solution, immersing the Huoshan Dendrobium protocorm of S1 in the infection solution, slowly shaking for 25 minutes, and then inoculating it into the co-cultivation medium for culture.

[0032] Preferably, the impregnation liquid is YEP liquid medium + 20g / L sucrose.

[0033] Preferably, the co-cultivation medium is MS+1.5 mg / L 6-benzylaminopurine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.01942 g / L acetosyringone and 7 g / L agar, pH=5.8.

[0034] Preferably, the method for screening and culturing transgenic tissues and seedlings in S4 is: washing the protocorms of Dendrobium huoshanense in S3 with sterile water for more than 10 times, and then spreading them flat on an adventitious bud induction medium containing antibiotic screening; after germination, transplanting the germinated seedlings onto an adventitious root induction medium containing antibiotic screening to form complete Dendrobium huoshanense seedlings.

[0035] Preferably, the adventitious bud induction medium is MS+1.5 mg / L 6-benzylaminopurine, 0.25 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8.

[0036] Preferably, the adventitious root induction medium is MS+1.5 mg / L 6-benzylaminopurine, 0.25 mg / L naphthaleneacetic acid, 20 g / L sucrose and 7 g / L agar, pH=5.8.

[0037] The invention discloses an application of a method for genetically modifying the protocorm of Dendrobium huoshanense, and the application of the method to creating new varieties of Dendrobium huoshanense.

[0038] Beneficial technical effects of the present invention:

[0039] The present invention uses the protocorm of Dendrobium huoshanense as material, realizes Agrobacterium-mediated genetic transformation on the basis of the rapid propagation system of the protocorm tissue of Dendrobium huoshanense, establishes a stable and rapid genetic transformation system of Dendrobium huoshanense, and realizes the creation of a new variety of Dendrobium huoshanense.

[0040] The preparation method of the induction culture medium provided by the present invention realizes efficient scavenging and multi-level protection of reactive oxygen free radicals in the genetic modification process by constructing a four-fold synergistic protection system of antioxidant compound, enzymatic preservation, metabolic regulation and chelation stabilization, thereby solving technical problems such as short protection time of traditional culture medium, low multi-component synergistic efficiency, and poor component stability, significantly improving the genetic modification success rate and plant quality, and prolonging the maintenance time of the vitality of the genetically modified plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Diagram of the induction and genetic transformation process of the Huoshan Dendrobium protocorm proposed in the present invention

[0042] Figure 2 The pCAMBIA1300-GFP recombinant plasmid proposed in the present invention;

[0043] Figure 3 This is a diagram showing the PCR detection results of the GFP gene transformed into the Huoshan Dendrobium proposed in the present invention;

[0044] Figure 4 The present invention proposes to use GFP tags to detect the overexpression of GFP gene in Huoshan Dendrobium. DETAILED DESCRIPTION

[0045] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that these embodiments are discussed solely to enable those skilled in the art to better understand and implement the subject matter described herein, and that the functions and arrangements of the elements discussed may be varied without departing from the scope of this specification. Various examples may omit, substitute, or add various processes or components as needed. Furthermore, features described in some examples may be combined in other examples.

[0046] At least one embodiment of the present invention discloses a method for genetically modifying a Dendrobium huoshanense protocorm, comprising the following steps:

[0047] 1. Induction culture of protocorms of Dendrobium huoshanense

[0048] Healthy, plump pods of Dendrobium huoshanense were selected and rinsed with tap water. They were then disinfected by soaking in 75% ethanol for 15 seconds, rinsed 2-3 times with sterile water, and then soaked in 8% NaClO solution for 5-10 minutes. They were then rinsed 3-5 times with sterile water. Finally, any residual surface moisture was blotted with sterile filter paper. The disinfected seeds were sown in piles on a protocorm induction medium and incubated in a 25°C incubator with a 12h / 12h light / dark cycle for 20 days. After protocorms formed, vigorously growing protocorms were selected for subsequent experiments.

[0049] The induction medium of Dendrobium huoshanense protocorm is MS+1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.5 g / L activated carbon and 7 g / L agar, pH=5.8.

[0050] 2. Construction of plant expression vector

[0051] The green fluorescent protein (GFP) sequence was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) according to GenBank: KF206153.1 and submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis. Homologous recombination primers were designed, and the GFP gene fragment was ligated into the plant expression vector pCAMBIA1300 under the action of homologous recombination enzymes to form the recombinant plant expression vector pCAMBIA1300-GFP.

[0052] The homologous recombination primers are:

[0053] SEQ ID NO: 1

[0054] OE-GFP-F:

[0055] 5'-GGAGAGGACAGGGTACCCGGGGATCCTGAGCAAGGGCGAGGAGC-3'.

[0056] SEQ ID NO: 2

[0057] OE-GFP-R:

[0058] 5'-ACCATGGTACTAGTGTCGACTCTAGATTACTTGTACAGCTCGTCCA-3'.

[0059] The recombinant plant expression vector pCAMBIA1300-GFP was transformed into Agrobacterium tumefaciens GV3101 using liquid nitrogen freeze-thaw. The specific steps are as follows: add the recombinant vector pCAMBIA1300-GFP to the competent Agrobacterium tumefaciens GV3101, mix thoroughly, and place on ice for 5 minutes. Then, quickly freeze in liquid nitrogen for 5 minutes, bathe in a 37°C water bath for 5 minutes, and then place on ice for 2 minutes. Add 300 μL of YEP liquid medium and incubate at 28°C with shaking for 2 hours. Harvest the cells by centrifugation at 4000 rpm for one minute. 50 μL of the culture was plated on a YEP plate containing kanamycin and rifampicin. After incubation at 28°C for 3 days, single colonies were selected for PCR verification.

[0060] 3. Infection and co-cultivation of Agrobacterium

[0061] Streak a culture of Agrobacterium containing pCAMBIA1300-GFP and select a single colony for shaking. When the OD value reaches 0.8-1.0, resuspend the pellet in the prepared infection solution and dilute to an OD600 of approximately 0.5. Soak the protocorms of Dendrobium huoshanense obtained in step 1 in the infection solution and incubate at 28°C with shaking for approximately 10 minutes. Remove the protocorms, blot the surface liquid with sterile paper, transfer to co-cultivation medium, and incubate in the dark at 28°C for 3 days.

[0062] The co-culture medium used was formulated as: MS + 1.5 mg / L 6-benzylaminopurine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.01942 g / L acetosyringone, and 7 g / L agar, with a pH of 5.8.

[0063] 4. Screening of transgenic positive protocorms

[0064] After co-cultivation, the protocorms of Dendrobium huoshanense were rinsed with sterile water to remove surface bacteria, then repeatedly rinsed with 200 mg / L carbenicillin-containing water. Any remaining liquid was blotted off with sterile filter paper. The protocorms were then transferred to screening medium and incubated at 25°C with a 12h / 12h light / dark cycle to identify transgenic protocorms with hygromycin resistance.

[0065] The screening culture medium formula used is: MS+1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 60 μg / L hygromycin B, 30 g / L sucrose, 0.5 g / L activated carbon, 7 g / L agar, pH=5.8.

[0066] 5. Induction of adventitious buds

[0067] Under sterile conditions, the transgenic positive protocorms of Dendrobium huoshanense selected in step 4 were evenly spread on adventitious bud induction medium containing 60 μg / L hygromycin B to induce adventitious buds. The culture was maintained at 25°C with a 12h / 12h light / dark cycle.

[0068] The formula of the adventitious bud induction medium used was: MS+1.5 mg / L 6-benzylaminoadenine, 0.25 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, pH=5.8.

[0069] 6. Induction of adventitious roots

[0070] Under sterile conditions, transfer the germinated Dendrobium huoshanense seedlings from step 5 to an adventitious root induction medium containing 60 μg / L hygromycin B and culture at 25°C with a 12 h / 12 ​​h light / dark cycle. Induction on the adventitious root induction medium will result in the formation of complete Dendrobium huoshanense seedlings.

[0071] The adventitious root induction medium used was formulated as: MS+1.5 mg / L 6-benzylaminopurine, 0.25 mg / L naphthaleneacetic acid, 20 g / L sucrose, and 7 g / L agar, with a pH of 5.8.

[0072] 7. Molecular identification of positive transgenic Dendrobium Huoshanense

[0073] Total DNA was extracted from leaves of the regenerated D. huoshanense seedlings using a genomic plant DNA purification kit. Specific primers for the GFP gene, JD-GFP-F (5'-ATGGTGAGCAAGGGCGAGGA-3') and JD-GFP-R (5'-AGTTACTTGTACAGCTCGTCCATGC-3'), were designed using Primer Primer5 primer design software. Molecular identification of the transgenic D. huoshanense seedlings was performed by polymerase chain reaction (PCR). PCR amplification was performed using TaKaRa Taq enzyme, using the extracted total DNA as a template. The amplification system consisted of 50 μL of 10× PCR buffer (5 μL), 4 μL of dNTP mix, 1.0 μL each of forward and reverse primers, 1 μL of DNA template, 1.0 μL of TaKaRa Taq, and 38 μL of ddH2O. The reaction procedure was 98°C for 5 minutes, 98°C for 10 seconds, 55°C for 50 seconds, and 72°C for 50 seconds, followed by 35 cycles, and a 10-minute post-extension at 72°C. The amplified PCR products were verified by 1% agarose gel electrophoresis. The pCAMBIA1300-GFP recombinant plasmid served as a positive control, while uninfected Dendrobium huoshanense seedlings and the empty pCAMBIA1300 plasmid served as negative controls.

[0074] 8. Fluorescence identification of positive transgenic Dendrobium Huoshanense

[0075] Fluorescence identification of transgenic seedlings is a method based on fluorescent labeling technology and is commonly used to confirm the successful introduction and expression of exogenous genes in transgenic plants. In the transgenic process of Dendrobium Huoshanense, the green fluorescent protein gene (GFP) was inserted. Therefore, using a plant live imaging system, we observed the presence and intensity of fluorescence in transgenic seedlings to determine whether the GFP gene was successfully expressed in the plants.

[0076] Figure 1 The genetic transformation system of Huoshan Dendrobium was established. Figure 1 As shown in A, the seeds of Dendrobium huoshanense were used as explants to induce the protocorms of Dendrobium huoshanense on the induction medium ( Figure 1 B), and the target gene was introduced into the protocorm of Dendrobium huoshanense using Agrobacterium infection ( Figure 1 C) and induced budding on regeneration medium containing corresponding selection pressure ( Figure 1D) and roots formed a complete transgenic seedling of Dendrobium huoshanense ( Figure 1 E) These intact resistant plants were removed from the agar and transplanted into a greenhouse to serve as identification materials, thereby forming transgenic strains of Dendrobium huoshanense.

[0077] Figure 2 For the construction of plant expression vector, homologous recombination primers were designed with reference to the sequence of GFP gene that has been sequenced and verified:

[0078] OE-GFP-F (5'-GGAGAGGACAGGGTACCCGGGGATCCTGAGCAAGGGCGAGGAGC-3'), OE-GFP-R (5'-ACCATGGTACTAGTGTCGACTCTAGATTACTTGTACAGCTCGTCCA-3'), under the action of homologous recombinase, e.g. Figure 2 The GFP gene was integrated behind the 35S promoter as shown.

[0079] Figure 3 To obtain the molecular identification results of the transgenic lines of Dendrobium huoshanense, leaves of Dendrobium huoshanense seedlings were taken and DNA was extracted. The GFP gene identification primers JD-GFP-F: (5'-ATGGTGAGCAAGGGCGAGGA-3'); JD-GFP-R: (5'-AGTTACTTGTACAGCTCGTCCATGC-3') were designed. The transgenic plants of Dendrobium huoshanense were identified by specific PCR reaction. Figure 3 As shown, a total of 8 transgenic seedlings of Dendrobium huoshanense were identified.

[0080] Figure 4 In order to detect the transgenic seedlings of Dendrobium huoshanense using GFP tag, we introduced GFP tag in the process of constructing the vector, so that the expression of GFP protein in the seedlings and tissues of Dendrobium huoshanense can be detected by plant living imaging system. Figure 4 As shown, strong GFP green fluorescence can be detected using the plant living imaging system, further indicating that the GFP gene has been successfully introduced into the Huoshan Dendrobium and the corresponding GFP protein is expressed.

[0081] At least one embodiment of the present invention discloses an application of a method for genetically modifying the protocorm of Dendrobium huoshanense, and the method is applied to creating new varieties of Dendrobium huoshanense.

[0082] At least one embodiment of the present invention discloses a method for preparing an induction culture medium, comprising the following steps:

[0083] Step 1: Preparation of basal culture medium;

[0084] Prepare the basal culture medium according to the standard formula of MS culture medium, add 1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.5 g / L activated carbon and 7 g / L agar in that order, adjust the pH to 5.8 with sodium hydroxide or hydrochloric acid, and sterilize at high temperature and high pressure until ready for use.

[0085] Step 2: Preparation of antioxidant compound system;

[0086] A multi-antioxidant complex solution is prepared by mixing ascorbic acid, glutathione, and α-tocopherol in a mass ratio of 2:1:1 and adding sterile distilled water to a total concentration of 50 mg / L of the complex antioxidant stock solution. This ratio is determined based on the complementary scavenging activities of the different antioxidants: ascorbic acid primarily scavenges hydroxyl free radicals, glutathione scavenges hydrogen peroxide, and α-tocopherol protects cell membranes from lipid peroxidation damage. The three antioxidants work synergistically to comprehensively scavenge multiple reactive oxygen free radicals, resulting in a complex antioxidant solution with broad-spectrum antioxidant activity.

[0087] Step 3: Preparation of enzyme separation carrier;

[0088] Preparation of an enzyme separation carrier with sequential activation function, specifically comprising:

[0089] (1) Preparation of catalase carrier: catalase was dissolved in phosphate buffer containing coenzyme A, and enzyme carrier microspheres were prepared using gelatin microencapsulation technology. The diameter of the microspheres was controlled to be 50-100 μm, and the encapsulation efficiency reached more than 85%;

[0090] (2) Preparation of superoxide dismutase carrier: Superoxide dismutase was dissolved in a Tris buffer containing nicotinamide adenine dinucleotide, and enzyme carrier gel balls were prepared using sodium alginate gel embedding technology. The diameter of the gel balls was controlled at 100-200 μm. This carrier technology achieves the time-phased release of the enzyme system by the difference in degradation rates of different carrier materials. The gelatin microcapsules rapidly degraded and released catalase in the early stage of culture, and the sodium alginate gel slowly degraded and released superoxide dismutase in the middle and late stages of culture, avoiding competitive inhibition between enzymes and obtaining an enzyme separation carrier with time-sequential activation function.

[0091] Step 4: Preparation of organic acid stabilized carrier;

[0092] The method of preparing an organic acid-stabilized carrier complex comprises:

[0093] (1) Carrier pretreatment: chitosan and pectin were mixed in a mass ratio of 1:1 and dissolved in dilute acetic acid solution to form a carrier solution;

[0094] (2) Complex formation: Malic acid and succinic acid were mixed in a mass ratio of 1:1 and added dropwise to the carrier solution to form an organic acid-polysaccharide complex under continuous stirring;

[0095] (3) Stabilization treatment: Dipotassium hydrogen phosphate and sodium bicarbonate are added to the complex as buffering agents to a final concentration of 5 mM and 3 mM, respectively, and the pH is adjusted to 6.0-6.5. This carrier technology, through the encapsulation and protection of the polysaccharide carrier and the microenvironmental regulation of the buffer system, enables the organic acid to maintain structural stability and biological activity under conditions of pH fluctuation and temperature changes, achieving slow release and continuous supply of organic acid, and obtaining an organic acid carrier complex with long-term stability.

[0096] Step 5: Construction of chelation stabilization system;

[0097] The preparation of the chelating stable synergistic solution comprises:

[0098] (1) Chelating agent preparation: phytic acid, tea polyphenols, and sodium citrate were mixed in a mass ratio of 1:2:1 and dissolved in deionized water to prepare a chelating agent solution with a total concentration of 30 mg / L;

[0099] (2) Stabilizer preparation: Lecithin and gum arabic were mixed in a mass ratio of 1:1 and dissolved in warm water to prepare a stabilizer solution with a total concentration of 20 mg / L;

[0100] (3) Synergistic system construction: The chelating agent solution and the stabilizer solution are mixed in a volume ratio of 2:1 and thoroughly mixed under magnetic stirring to form a chelating stabilized synergistic solution. This chelating stabilization technology draws on mature experience in the field of food preservation. The chelating agent forms a stable complex with metal ions such as iron and copper in the culture medium, blocking the Fenton reaction and Haber-Weiss reaction catalyzed by the metal ions. The stabilizer maintains the active conformation of the antioxidant by forming a protective film. The two work synergistically to form a long-lasting and stable antioxidant protection environment, obtaining a synergistic solution with the dual functions of metal ion chelation and antioxidant stabilization.

[0101] Step 6: Integration of multiple collaborative systems;

[0102] The functional components are integrated in a certain order and proportion to form the final culture medium, which specifically includes:

[0103] (1) Order of component addition: First, add the chelating stabilization synergistic solution to the basal medium, mix thoroughly, and then add the composite antioxidant solution, enzyme separation carrier, and organic acid stabilization carrier complex in sequence;

[0104] (2) Proportion control: The volume ratio of each component in the final culture medium is basal medium: chelating stabilizing solution: compound antioxidant solution: enzyme carrier: organic acid carrier = 100:5:3:2:2;

[0105] (3) Homogenization: Use low-speed stirring to fully mix the components to avoid damaging the carrier structure, and finally adjust the pH to 5.8±0.1. This integrated technology ensures the uniform distribution and stable presence of each functional component in the culture medium through precise addition sequence and ratio control. The chelation stabilization system first establishes a protective environment. The antioxidant provides immediate protection, the enzyme carrier provides continuous protection, and the organic acid carrier provides metabolic regulation. The four protection methods work synergistically to obtain a transgenic induction culture medium with multi-level synergistic protection functions.

[0106] This embodiment achieves the following significant technical effects by building a four-layer coordinated protection system:

[0107] Effect 1: Multi-level coordinated protection effect;

[0108] Through the synergistic effect of four methods: antioxidant compounding, enzymatic preservation, metabolic regulation and chelation stabilization, all-round protection from chemical removal to enzymatic protection to metabolic regulation is achieved, effectively eliminating various active oxygen free radicals produced during the genetic modification process, significantly reducing the damage to plant cells caused by oxidative stress, and solving problems such as browning and growth stagnation of genetically modified plants.

[0109] Effect 2: Long-lasting and stable protection effect;

[0110] Through enzyme system sequential activation technology and organic acid stabilization carrier technology, the controlled release and continuous supply of protective ingredients can be achieved, the protection time can be extended, the defect of short protection time of traditional single antioxidant method can be overcome, and continuous protection can be ensured throughout the transgenic cultivation process.

[0111] Effect three: efficient synergistic enhancement effect;

[0112] Through carrier separation and sequential activation technology, the competitive inhibition problem in the multi-enzyme system is solved, ensuring that each protective component can exert its maximum effectiveness at the most appropriate time, avoiding mutual interference, and achieving synergistic enhancement of the protection effect rather than simple superposition.

[0113] Effect 4: Environmental adaptation and stabilization effect;

[0114] Through chelation stabilization technology and microenvironment regulation, the catalytic oxidation of metal ions is effectively controlled, the stability of active ingredients under environmental conditions such as pH fluctuations and temperature changes is protected, and the reliable performance of the culture medium under different environmental conditions is ensured.

[0115] Effect 5: Transgenic efficiency improvement effect;

[0116] Through the synergistic effect of multiple protection methods, the success rate of transgenic plants and plant quality are significantly improved, the vitality of transgenic plants is prolonged, and efficient and reliable technical support is provided for the transgenic cultivation of precious Chinese medicinal materials and other plants.

[0117] Experimental verification

[0118] Experiment 1: Verification of the effect of multi-level collaborative protection

[0119] Experimental purpose: To verify the scavenging effect and multi-level protection capability of the quadruple synergistic protection system on reactive oxygen free radicals.

[0120] Experimental Materials:

[0121] Dendrobium huoshanense protocorm; culture medium of the present invention (containing a quadruple synergistic protection system); control culture medium 1 (traditional MS culture medium + a single antioxidant); control culture medium 2 (MS culture medium + dual antioxidants); reactive oxygen species detection kit; cell viability detection kit.

[0122] Experimental steps:

[0123] 1. The protocorms of Dendrobium huoshanense were inoculated into the culture medium of the present invention and two control culture media, with 30 samples in each group.

[0124] 2. Take samples for testing on the 1st, 3rd, 7th, 14th and 21st days of culture.

[0125] 3. Use DCFH-DA fluorescent probe to detect the level of intracellular reactive oxygen species.

[0126] 4. Use MTT method to detect cell viability.

[0127] 5. Observe and record the browning degree and growth status of the plants.

[0128] 6. Statistical analysis of data differences among groups.

[0129] Experimental results:

[0130]

[0131] Result analysis: The culture medium of the present invention showed significant multi-level synergistic protection effects throughout the entire culture period. The active oxygen level was always kept at a low level, the cell viability was maintained at more than 90%, and the browning rate was controlled within 6.2%, which was significantly better than the control group.

[0132] Experiment 2: Verification of long-term and stable protection effect

[0133] Experimental purpose: To verify the long-term protective ability of enzyme sequential activation technology and organic acid stabilized carrier technology.

[0134] Experimental materials: Huoshan Dendrobium protocorm; culture medium of the present invention; control culture medium (traditional culture medium, protective components directly added); enzyme activity detection kit; organic acid content detection kit; high performance liquid chromatography.

[0135] Experimental steps:

[0136] 1. Prepare 500 mL each of the culture medium of the present invention and the control culture medium.

[0137] 2. Take samples at 0, 6, 12, 24, 48, and 72 hours after culture medium preparation.

[0138] 3. Detect the activities of catalase and superoxide dismutase in the culture medium.

[0139] 4. Detect the content of malic acid and succinic acid in the culture medium.

[0140] 5. Detect the retention rate of antioxidants (ascorbic acid, glutathione).

[0141] 6. Analyze the stability change trend of each component.

[0142] Experimental results:

[0143]

[0144] Result analysis: The culture medium of the present invention maintains more than 85% of the stability of the active ingredients after 72 hours through carrier protection and timed release technology, while the active ingredients of the control culture medium are greatly degraded after 48 hours, demonstrating the significant effect of long-term stable protection.

[0145] Experiment 3: Verification experiment of efficient synergistic enhancement effect

[0146] Experimental purpose: To verify the effectiveness of vector separation and sequential activation technology in resolving competitive inhibition of multi-enzyme systems.

[0147] Experimental materials: catalase and superoxide dismutase; gelatin microcapsules and sodium alginate carrier materials; enzyme activity detection reagents; spectrophotometer; constant temperature incubator.

[0148] Experimental steps:

[0149] 1. Four enzyme systems were prepared: Group A (two enzymes were directly mixed), Group B (two enzymes were separately embedded in carriers), Group C (catalase alone), and Group D (superoxide dismutase alone).

[0150] 2. Culture under the same conditions for 0, 2, 4, 8, 12, and 24 hours.

[0151] 3. Detect the activity changes of the two enzymes in each group respectively.

[0152] 4. Calculate the enzyme activity retention rate and synergistic effect coefficient.

[0153] 5. Analyze the effect of carrier separation on enzyme interactions.

[0154] Experimental results:

[0155]

[0156] Analysis of results: The carrier separation technology effectively solved the competitive inhibition problem in the multi-enzyme system. After 24 hours, the enzyme activity retention rate of the carrier separation group remained above 84%, while that of the direct mixing group dropped below 35%, and the synergistic effect coefficient increased by 2.4 times.

[0157] Experiment 4: Verification of the stabilization effect of environmental adaptation

[0158] Experimental purpose: To verify the control effect and environmental adaptability of chelation stabilization technology on metal ion catalytic oxidation.

[0159] Experimental materials: culture medium of the present invention (containing chelating stabilization system); control culture medium (without chelating agent); metal ion solution (Fe 3+ 、Cu 2+ 、Mn 2+ ); pH buffer; temperature control equipment; antioxidant stability test reagent.

[0160] Experimental steps:

[0161] 1. Prepare culture medium containing different metal ion concentrations (0, 5, 10, 20, 50 μM).

[0162] 2. Set different pH conditions (5.0, 5.5, 6.0, 6.5, 7.0).

[0163] 3. Set different temperature conditions (20, 25, 30, 35℃).

[0164] 4. The stability of the antioxidants was tested after 24 h of incubation under various conditions.

[0165] 5. Detect metal ion chelation rate and free radical generation.

[0166] 6. Analyze the impact of environmental factors on protection effects.

[0167] Experimental results:

[0168]

[0169] Analysis of results: Chelation stabilization technology demonstrated excellent metal ion chelation ability (>80%) and antioxidant protection effect under various environmental conditions. Even under high temperature and extreme pH conditions, it could still maintain an overall protection efficiency of more than 79%, which was significantly better than the 20-42% of the control group.

[0170] Experiment 5: Verification of the effect of improving transgenic efficiency

[0171] Experimental purpose: To verify the effect of the culture medium of the present invention on improving the success rate of plant genetic modification and plant quality.

[0172] Experimental materials: 200 protocorms of Dendrobium huoshanense; Agrobacterium transformation vector (containing target gene); culture medium of the present invention; traditional transgenic culture medium; PCR detection kit; fluorescence quantitative PCR instrument; and plant growth index measurement equipment.

[0173] Experimental steps: 1. Randomly divide the protocorms of Dendrobium huoshanense into two groups, with 100 in each group.

[0174] 2. Use Agrobacterium-mediated transgenic treatment.

[0175] 3. Cultivate in the culture medium of the present invention and traditional culture medium respectively.

[0176] 4. Calculate the transgenic success rate after 30 days of culture.

[0177] 5. Detect the growth indicators and gene expression levels of transgenic plants.

[0178] 6. Track the duration of plant vitality (60 days of cultivation).

[0179] Experimental results:

[0180] Evaluation indicators Culture medium of the present invention Traditional culture medium Improvement rate (%) Transgenic success rate (%) 78.5±.4.2 52.3±.5.8 +50.1 Plant survival rate (%) 92.1±3.1 68.7±6.2 +34.1 Average plant height (cm) 3.85±.0.32 2.41±.0.45 +59.8 Average number of leaves 8.2±1.1 5.6±1.4 +46.4 Root system development score 8.7±0.8 6.1±1.2 +42.6 Target gene expression level (relative value) 2.85±.0.28 1.92±0.35 +48.4 Chlorophyll content (mg / g) 1.82±0.15 1.24±0.21 +46.8 Soluble protein content (mg / g) 15.8±1.2 10.9±1.8 +44.9 60-day survival rate (%) 85.2±3.8 45.1±7.5 +89.0

[0181] Comprehensive effect comparison table:

[0182] Cultivation time Comprehensive score of the culture medium of the present invention Comprehensive score of traditional culture medium Advantage multiple 10 days 8.9±0.5 6.2±0.8 1.44 20 days 9.1±0.4 5.8±0.9 1.57 30 days 9.3±0.3 5.1±1.1 1.82 45 days 9.0±0.4 4.2±1.3 2.14 60 days 8.8±0.5 3.5±1.5 2.51

[0183] Analysis of the results showed that the culture medium of the present invention significantly improved the success rate of genetic modification of Dendrobium huoshanense (increased by 50.1%) and plant quality, with various growth indicators improved by more than 40%, and the 60-day survival rate increased by 89%. The comprehensive effect was more obvious in the later stage of cultivation, proving the significant effect of the quadruple synergistic protection system on improving the efficiency of genetic modification.

[0184] The above describes an embodiment of the present invention, but this embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Ordinary technicians in this field can also make more forms of equivalent embodiments based on the inspiration of this embodiment, all of which are protected by this embodiment.

Claims

1. A method for genetically modifying the protocorm of Dendrobium huoshanense, characterized in that: The steps include: S1: Induction culture of protocorms of Dendrobium huoshanense; S2: Construction of plant expression vector; S3: Agrobacterium infection and culture of protocorms of Dendrobium huoshanense; S4: Screening and cultivation of transgenic tissues and seedlings of Dendrobium huoshanense.

2. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 1, characterized in that: The method for inducing the protocorms of Dendrobium huoshanense in S1 is as follows: the sterilized Dendrobium huoshanense seeds are sown in piles on the induction culture medium; after about half a month of induction, the protocorms with vigorous growth are selected as the genetic transformation recipients.

3. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 2, characterized in that: The induction culture medium is MS+1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.5 g / L activated carbon and 7 g / L agar, with a pH of 5.

8.

4. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 1, wherein: The method for constructing the plant expression vector in S2 is as follows: download the green fluorescent protein sequence GenBank: KF206153.1 from the NCBI database and perform gene synthesis; The GFP gene fragment was connected to the plant expression vector pCAMBIA1300 by homologous recombination to construct the recombinant vector pCAMBIA1300-GFP. The PCR amplification primers used for vector construction are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

5. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 1, characterized in that: The infection and co-cultivation method in S3 is as follows: the recombinant vector pCAMBIA1300-GFP is introduced into Agrobacterium tumefaciens, the transformed Agrobacterium is activated by the plate streak method, and a single colony is picked and shaken. The obtained bacterial liquid is resuspended in a solution to obtain an infection solution, and the protocorms of Dendrobium huoshanense from S1 are immersed in the infection solution, slowly shaken for 25 minutes, and then inoculated onto the co-cultivation medium for culture.

6. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 5, characterized in that: The impregnation solution is YEP liquid medium + 20g / L sucrose.

7. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 5, characterized in that: The co-culture medium is MS+1.5 mg / L 6-benzylaminoadenine, 0.35 mg / L naphthaleneacetic acid, 30 g / L sucrose, 0.01942 g / L acetosyringone and 7 g / L agar, with a pH of 5.

8.

8. The method for genetically modifying Dendrobium huoshanense protocorms according to claim 1, characterized in that: The method for screening and culturing transgenic tissues and seedlings in S4 is as follows: the protocorms of Dendrobium huoshanense in S3 are rinsed with sterile water for more than 10 times, and then spread on an adventitious bud induction medium containing antibiotic selection; after germination, the germinated seedlings are transplanted on an adventitious root induction medium containing antibiotic selection to form complete Dendrobium huoshanense seedlings.

9. The method for genetically modifying the protocorm of Dendrobium huoshanense according to claim 1, characterized in that: The adventitious bud induction medium comprises MS+1.5 mg / L 6-benzylaminoadenine, 0.25 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, with a pH of 5.

8.

10. An application of the method for genetically modifying Dendrobium huoshanense protocorms according to any one of claims 1 to 9, characterized in that: This method was applied to create new varieties of Huoshan Dendrobium.

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