Genetic transformation method of dendrobium officinale

Abstract

The invention discloses an efficient dendrobium officinale genetic transformation method, and belongs to the technical field of plant genetic engineering. According to the method, the third to fifth stem sections of dendrobium officinale tissue culture seedlings are used as explants, and the transformation efficiency is remarkably improved by optimizing an agrobacterium infection system and combining physical damage (peeling and pricking) and vacuum infiltration technologies. The method specifically comprises the following steps: (1) pre-culturing an explant; (2) construction of agrobacterium engineering bacteria; (3) infection and co-culture; (4) resistance screening; (5) identifying positive plants and culturing strong seedlings; and (6) rooting and transplanting. TMT is adopted as a bacteriostatic agent, the pollution rate is controlled to be 4% or below, the positive conversion rate reaches 68.9%, and the positive conversion rate is nearly doubled compared with that of a traditional method; meanwhile, the culture period is shortened from 6-7 months to 4-5 months through a stem direct differentiation technology. The method is simple and convenient to operate and low in cost, and an efficient and reliable technical platform is provided for gene function research and molecular breeding of dendrobium officinale.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a method for genetic transformation of Dendrobium officinale. Background Technology

[0002] Dendrobium officinale, a perennial epiphytic herb belonging to the genus Dendrobium in the family Orchidaceae, is a traditional and precious Chinese medicinal herb. Its stems are rich in polysaccharides, alkaloids, amino acids, and various trace elements, possessing pharmacological activities such as enhancing immunity, anti-oxidation, anti-tumor effects, and lowering blood sugar. It has broad application prospects in traditional Chinese medicine preparations, health products, and cosmetics. With increasing market demand, wild Dendrobium officinale resources have drastically decreased due to over-harvesting and are now listed as a national second-class protected plant. Although artificial cultivation has achieved large-scale production, it still faces problems such as varietal degeneration, poor resistance to pests and diseases, and unstable content of active ingredients. Therefore, using genetic engineering technology to genetically improve Dendrobium officinale and cultivate high-yielding, high-quality, and stress-resistant new varieties has become an important way to solve the industry's bottlenecks.

[0003] Orchids have thick cell walls and long regeneration cycles, and most varieties lack mature tissue culture and genetic transformation systems. As a monocotyledonous plant, Dendrobium officinale generally has a lower genetic transformation efficiency than model plants (such as Arabidopsis thaliana and tobacco), mainly due to the following factors: (1) Traditionally, protocorms are used as transformation recipients, but protocorms are prone to browning, resulting in low callus differentiation rates. (2) Monocotyledonous plants lack signaling molecules (such as phenolic compounds) that Agrobacterium can recognize, leading to low T-DNA transfer efficiency. (3) When screening with commonly used antibiotics, non-transgenic cells may produce false positives due to endogenous resistance or slow growth. Currently developed genetic transformation methods for Dendrobium officinale mainly use protocorms as recipient materials, and their genetic transformation cycle is very long, usually taking more than 6 months to obtain positive plants.

[0004] Therefore, developing more efficient genetic transformation methods is of great significance for verifying the function of key genes, conducting basic research on molecular breeding, and accelerating the process of improving Dendrobium officinale varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a genetic transformation method for Dendrobium officinale, which is simple to operate and low in cost, providing an efficient and reliable technical platform for gene function research and molecular breeding of Dendrobium officinale.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A genetic transformation method for Dendrobium officinale, comprising the following steps:

[0008] (1) Explant preparation: Select stem segments of Dendrobium officinale tissue culture seedlings and pre-culture them in MS basal medium containing 100-200 μmol / L acetylsyringone for 48-72 h to obtain explants for later use;

[0009] (2) Construction of Agrobacterium engineered strain: The recombinant plasmid was transformed into Agrobacterium GV3101 and cultured in YEP medium for 12-16 h; then the culture was expanded to OD. 600 =0.6-0.8, resuspend the bacterial cells in MS basal medium containing 100-200 μmol / L acetylsyringone, and let stand to obtain bacterial suspension;

[0010] (3) Infection and co-culture: Peel the epidermis of the stem segment and make holes, immerse it in the bacterial solution obtained in step (2), and shake to infect for 10-30 min; then perform vacuum infiltration infection for 5-15 min, drain the liquid, and then transfer it to MS basal medium containing 100-200 μmol / L acetylsyringone, and co-culture on plates at 25-30℃ for 48-72 h;

[0011] (4) Resistance screening: Cultured in sterile medium for 2 weeks, then transferred to resistance screening medium. The medium was changed every 2 weeks until the resistant shoots grew to 2-3 cm.

[0012] (5) Identification and transplanting of positive plants: Transfer the resistant buds to the seedling culture medium and culture for 3-4 weeks to identify transgenic positive plants;

[0013] (6) Rooting and transplanting: Transplant the positive plants after culturing them in the rooting medium for 6-8 weeks.

[0014] In this invention, the stem segment selected in step (1) is the stem segment of the 3rd to 5th nodes.

[0015] In this invention, the MS basal culture medium in steps (1)-(3) further contains 3.0-5.0 mg / L 6-BA and 0.1-0.5 mg / L NAA.

[0016] In this invention, the YEP culture medium in step (2) further contains 50 mg / L kanamycin and 25 mg / L rifampicin.

[0017] In this invention, further, in step (2), the bacterial cells are resuspended and then left to stand at 28°C for 3 hours.

[0018] In this invention, further, the oscillation and impregnation speed in step (3) is 100 rpm / min, the oscillation and impregnation time is 20 min, and the vacuum penetration time is 10 min.

[0019] In this invention, further, the sterilization culture medium in step (4) is: MS-based culture medium, which also includes 4.0 mg / L 6-BA, 0.2 mg / L NAA and 400 mg / L TMT; the resistance screening culture medium is: MS-based culture medium, which also includes 4.0 mg / L 6-BA, 0.2 mg / L NAA, 400 mg / L TMT and 25 mg / L hygromycin B (Hyg).

[0020] In this invention, the seedling culture medium in step (5) is further: MS-based culture medium, which also includes 400 mg / L termethin (TMT), 20 mg / L hygromycin B (Hyg), 10% potato and 10% banana.

[0021] In this invention, the rooting medium in step (6) is further: MS as the base medium, and also includes 0.2 mg / L 6-BA, 0.2 mg / L NAA, 200 mg / L TMT and 20% banana.

[0022] In summary, due to the adoption of the above technical solutions, the present invention has at least the following beneficial effects:

[0023] Dendrobium officinale, a rare orchid with significant economic and medicinal value, is of great importance for the protection of its germplasm resources, the discovery of its superior traits, and the creation of new varieties. However, it has long been constrained by its slow growth, long breeding cycle, and the low efficiency of traditional breeding methods. This invention overcomes these obstacles by providing a highly efficient, stable, and scalable genetic transformation method for Dendrobium officinale, with the following outstanding advantages:

[0024] (1) High efficiency transformation and shortened cycle: By optimizing the stem segment recipient material (3rd-5th stem segment) and combining the synergistic infection technology of peeling and puncturing and vacuum penetration, the T-DNA transfer efficiency of Agrobacterium is significantly improved, and the positive transformation rate is increased to 68.9%, which is nearly double that of the traditional protocorm recipient (36.2%). At the same time, the overall culture cycle is shortened from 6-7 months to 4-5 months, which greatly accelerates the breeding process.

[0025] (2) Low contamination rate and high stability: By using TMT to replace traditional cephalosporin and other antibacterial agents, the contamination rate of explants is controlled below 4%, while the survival rate of explants is increased to over 90%, effectively reducing the risk of experimental failure and improving the stability and reproducibility of the transformation system. TMT is the first to achieve a synergistic improvement in Agrobacterium inhibition and plant survival rate, an effect that far exceeds the expectations of those skilled in the art.

[0026] (3) Simple operation and optimized cost: The stem segment recipient material can be directly differentiated into seedlings, eliminating the original bulb induction step and simplifying the operation process; at the same time, the addition of potato and banana homogenate to the culture medium to replace expensive coconut milk reduces production costs by more than 35%, making it more suitable for large-scale application.

[0027] (4) Reliable molecular verification: Through molecular detection methods such as PCR and Southern blot, it was verified that the integration of exogenous genes in transgenic plants was stable and the expression level of target genes was significantly improved, laying a solid foundation for subsequent functional research and variety improvement.

[0028] This invention not only solves the industry problems of low genetic transformation efficiency, long cycle and high false positive rate of Dendrobium officinale, but also provides an efficient and reliable technical system for gene function research and molecular breeding of orchid plants, and has important scientific research and industrial application value. Attached Figure Description

[0029] Figure 1 A schematic diagram of some steps in the standard process for high-efficiency genetic transformation of Dendrobium officinale;

[0030] Figure 2 The standard electrophoresis results for PCR identification of transgenic positive plants of Dendrobium officinale are shown in the figure. Detailed Implementation

[0031] The following embodiments can help those skilled in the art to more fully understand the present invention, but should not be construed as limiting the present invention in any way.

[0032] Example: Standardized Procedure for High-Efficiency Genetic Transformation of Dendrobium officinale

[0033] This embodiment provides a standard procedure for efficient genetic transformation of Dendrobium officinale, such as... Figure 1 As shown, the specific steps include the following:

[0034] 1. Explant preparation:

[0035] (1) Select the 4th stem segment (containing 1 stem node) of 10-12 month old Dendrobium officinale tissue culture seedlings and cut it into 5 mm long segments under sterile conditions;

[0036] (2) Pre-cultured in MS medium containing 150 μmol / L acetylsylgenone + 4.0 mg / L 6-BA + 0.2 mg / L NAA, and incubated in the dark at 25°C for 48 h.

[0037] 2. Construction of Agrobacterium-mediated transformation:

[0038] (1) The CDS sequence of the target gene DcSWEET14 (LOC110102373, 798 bp) was constructed into the pCAMBIA1300-GFP vector using KpnⅠ and SalⅠ as restriction sites;

[0039] (2) The constructed recombinant plasmid was transformed into Agrobacterium strain GV3101 (this strain is a standard Agrobacterium engineered strain, which is a biological material that can be legally obtained by the public) and spread on YEP solid medium containing 50 mg / L kanamycin (Kan) and 25 mg / L rifampicin (Rif), and incubated upside down at 28°C for 48 h;

[0040] (3) Pick a single colony and inoculate it into 3 ml of YEP liquid medium with the same formula, and culture it at 28℃ and 200 rpm for 14 h with shaking;

[0041] (4) Take 1 ml of bacterial culture and transfer it to 50 ml of YEP medium with the same formula, and culture until OD. 600 =0.7, centrifuge at 4000 rpm for 10 min to collect bacterial cells;

[0042] (5) Resuspend in MS medium with 150 μmol / L acetylsalicylic acid + 4.0 mg / L 6-BA + 0.2 mg / L NAA and let stand at 28°C for 3 h to induce Vir gene expression.

[0043] 3. Infection and co-cultivation:

[0044] (1) Peel off the epidermis of the stem segment 1-2 mm (exposing the meristematic tissue) and make a hole with a sterile needle (0.5 mm deep);

[0045] (2) Immerse in bacterial solution, shake at 100 rpm for 20 min, and then permeate under vacuum (-0.08 MPa) for 10 min;

[0046] (3) After draining, transfer to MS solid medium containing 150 μmol / L acetylsyringone and co-culture at 28℃ for 48 h (e.g. Figure 1 A). Keep the surface of the culture medium moist during co-culture to facilitate sufficient contact between Agrobacterium and explants.

[0047] 4. Resistance screening:

[0048] (1) After co-culturing, transfer to sterile medium (MS + 4.0 mg / L 6-BA + 0.2 mg / L NAA + 400 mg / L LTMT) Figure 1B) The cells were cultured for 2 weeks at 25±1℃, light intensity of 1500 lx, and light duration of 12 h / d to eliminate residual Agrobacterium and promote the recovery and growth of transformed cells.

[0049] (2) After sterilization culture, the explants were transferred to resistance selection medium (MS + 4.0 mg / L 6-BA + 0.2 mg / L NAA + 400 mg / L TMT + 25 mg / L Hyg), and the medium was changed every 2 weeks. The culture was continued for 6 weeks until the resistant shoots grew to 2-3 cm. Figure 1 C). During this stage, untransformed cells gradually die due to antibiotic selection pressure, and only successfully transformed cells can survive and form resistant buds.

[0050] 5. Positive Identification and Seedling Strengthening:

[0051] (1) Resistant buds were transferred to seedling culture medium (MS + 400 mg / L TMT + 20 mg / L Hyg + 10% (w / v) potato homogenate + 10% (w / v) banana homogenate) and cultured for 4 weeks. Figure 1 D);

[0052] (2) PCR identification:

[0053] Genomic DNA was extracted from young leaves of vigorous seedlings and subjected to the following PCR identification:

[0054] Primers: F: 5′-ATGGCAGCAGGTGGCATCTC-3′, R:5′-AGGCGATTAAGTTGGGTAACGC-3′;

[0055] PCR reaction system (20 μL): 10 μL of 2×Taq DNA Polymerase PCR Mix, 1 μL each of primers F and R, 1 μL of template DNA, and 7 μL of ddH2O;

[0056] Program: 94℃ for 3 min; 33×(94℃ 30 s, 63℃ 30 s, 72℃ 2 min); 72℃ for 5 min;

[0057] Result: As Figure 2 As shown:

[0058] Meaning of the band: The appearance of a clear and sharp band near the expected molecular weight (1899 bp) indicates that the target gene fragment has been successfully integrated into the genomic DNA of the plant.

[0059] Positive rate calculation: Total samples tested: 23 plants (2-23); Positive samples: 16 (4, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23); Negative / false positive samples: 7 (2, 3, 5, 7, 12, 21, plus the explicit negative control 1 not included in the sample count). The positive rate of this batch = 16 / 23 ≈ 69.6%; further confirming the high positive rate of this genetic transformation method.

[0060] Summarize: Figure 2 The image quality is good, and the bands are clear and distinguishable. The negative control (1) showed no band, effectively eliminating false positives. A total of 16 plants (4, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23) amplified specific bands at the expected 1899 bp position and were identified as transgenic positive plants, with a positive rate of 69.6% (for this batch). This figure intuitively demonstrates that the genetic transformation method described in this invention can efficiently introduce the target gene into Dendrobium officinale to obtain the expected transgenic plants.

[0061] 6. Rooting and Transplanting:

[0062] Plants that tested positive by PCR were transferred to rooting medium (MS + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 200 mg / L TMT + 20% (w / v) banana homogenate) and cultured for 6 weeks before transplanting (e.g., ...). Figure 1 E).

[0063] Comparative experiment:

[0064] I. To verify the superiority of the method of the present invention, a comparative experiment was designed to compare the genetic transformation effects of using stem segments as recipient materials and using protocorms as recipient materials.

[0065] Experimental Design:

[0066] The following different receptor materials were selected as explants:

[0067] Stem segment group: Take 500 stem segments (3rd-5th sections) from Dendrobium officinale tissue culture seedlings.

[0068] Original corm group: 500 Dendrobium officinale original corms were taken.

[0069] Cultivation conditions:

[0070] Apart from the different materials used, the other treatment methods were the same. Both groups were cultured at a temperature of 25±1℃, a light intensity of 1500 lx, and a light duration of 12 h / d, using the same Agrobacterium strain and vector system.

[0071] Results analysis:

[0072] Table 1 Comparison of genetic transformation-related indicators for different recipient materials

[0073] index stem segment group Protocorm section Material quantity 500 500 Training time 4-5 months 6-7 months Positive rate 68.9% 36.2% Pollution rate 4.0% 8.0% Explant survival rate 92.5% 78.1% Transplant survival rate 90.3% 82.2%

[0074] The results showed that the method of this invention (stem segment group) was significantly superior to the traditional method (protocorm group) in key indicators such as culture period, positive rate, and contamination rate. In particular, the positive rate increased from 36.2% to 68.9%, nearly doubling; the culture period was shortened from 6-7 months to 4-5 months, a reduction of about 30%; at the same time, the contamination rate decreased from 8% to 4%, and the survival rate of explants and transplant survival rate were also significantly improved.

[0075] II. PCR identification results:

[0076] PCR identification was performed on 23 randomly selected resistant plants using specific primers. The results showed that 16 plants were able to amplify a specific band of 1899 bp. Figure 2 The positive rate for samples 4, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, and 23 was 69.6%, which is basically consistent with the overall positive rate (68.9%) (slight fluctuations are normal in a single PCR identification with a small sample size). Samples without detected bands (samples 1-3, 5, 7, 12, and 21) were false positive plants.

[0077] Third, the applicant also conducted comparative experiments on the effects of antibacterial agents:

[0078] 1. Experimental Materials and Methods:

[0079] Explants: stem segments of Dendrobium officinale tissue culture seedlings, 3rd-5th nodes (pretreatment method is the same as step (1) of claim 1).

[0080] Antibacterial agent control group:

[0081] Experimental group: sterile culture medium containing 400 mg / L Timentin (TMT) (formulation as claimed in claim 7);

[0082] Control group 1: Sterile culture medium containing 500 mg / L cephalosporin (replacing 400 mg / L termethin, other components are the same, the same below);

[0083] Control group 2: Sterile culture medium containing 100 mg / L gentamicin;

[0084] Control group 3: Sterilized culture medium containing 0.25% (v / v) plant growth regulator;

[0085] Control group 4: Sterile culture medium containing 0.5 mL / L isothiazolinone;

[0086] Control group 5: Sterile culture medium containing 0.15% (v / v) sodium hypochlorite.

[0087] Agrobacterium infection: Constructing the GV3101 engineered bacteria (OD) according to steps (2)-(3) of claim 1. 600 =0.6) and infect the stem segment.

[0088] Culture conditions: After co-culture, the explants were transferred to the above-mentioned antibacterial medium and cultured at 25°C under light for 2 weeks. Each group had 300 explants.

[0089] Testing indicators:

[0090] Contamination rate: The percentage of explant contamination caused by Agrobacterium residue;

[0091] Explant survival rate: Calculate the percentage of uncontaminated explants that maintain growth viability.

[0092] 2. Experimental Results:

[0093] The experimental results are shown in Table 2 below:

[0094] Table 2. Effects of different antibacterial agents

[0095] Antibacterial agents and concentrations Pollution rate (%) Explant survival rate (%) TMT 400 mg / L 4.0 92.5 Cephalosporin 500 mg / L 12.6 71.3 Gentamicin 100 mg / L 18.3 63.2 Plant growth regulator 0.25% 8.5 85.4 Isothiazolinone 0.5 mL / L 6.7 78.6 Sodium hypochlorite 0.15% 22.4 45.1

[0096] The results in the table show that the contamination rate in the TMT group (4.0%) was significantly lower than that in all control groups (cephalosporin 12.6%, gentamicin 18.3%, sodium hypochlorite 22.4%), and 40% lower than the optimal control group (isothiazolinone 6.7%). While Plant Growth Enhancer showed good antibacterial effect (8.5%), it required a high concentration (0.25%), making it more expensive than TMT. The explant survival rate in the TMT group (92.5%) was significantly higher than in other groups (cephalosporin 71.3%, gentamicin 63.2%, sodium hypochlorite 45.1%). Although sodium hypochlorite has a broad-spectrum antibacterial effect, it resulted in a low survival rate (45.1%), making it unsuitable for genetic transformation.

[0097] It is evident that TMT is the only antibacterial agent that simultaneously achieves a contamination rate of ≤5% and a survival rate of ≥90%, while other groups all exhibit significant shortcomings (e.g., isothiazolinone survival rate is less than 80%, and plant growth regulator contamination rate is >8%). Traditional open tissue culture antibacterial agents (such as plant growth regulator and sodium hypochlorite) perform poorly in genetic transformation systems, demonstrating that the cross-disciplinary advantages of TMT are not readily apparent. TMT achieves for the first time a synergistic improvement in Agrobacterium inhibition and plant survival rate, an effect far exceeding the expectations of those skilled in the art.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for genetic transformation of Dendrobium officinale, characterized in that, The method includes the following steps: (1) Explant preparation: Select stem segments of Dendrobium officinale tissue culture seedlings and pre-culture them in MS basal medium containing 100-200 μmol / L acetylsyringone for 48-72 h to obtain explants for later use; (2) Construction of Agrobacterium engineered strain: The recombinant plasmid was transformed into Agrobacterium GV3101 and cultured in YEP medium for 12-16 h; then the culture was expanded to OD. 600 =0.6-0.8, resuspend the bacterial cells in MS basal medium containing 100-200 μmol / L acetylsyringone, and let stand to obtain bacterial suspension; (3) Infection and co-culture: Peel the epidermis of the stem segment and make holes, immerse it in the bacterial solution obtained in step (2), and shake to infect for 10-30 min; then perform vacuum infiltration infection for 5-15 min, drain the liquid, and then transfer it to MS basal medium containing 100-200 μmol / L acetylsyringone, and co-culture on plates at 25-30℃ for 48-72 h; (4) Resistance screening: Cultured in sterile medium for 2 weeks, then transferred to resistance screening medium. The medium was changed every 2 weeks until the resistant shoots grew to 2-3 cm. (5) Identification and transplanting of positive plants: Transfer the resistant buds to the seedling culture medium and culture for 3-4 weeks to identify transgenic positive plants; (6) Rooting and transplanting: Transplant the positive plants after culturing them in the rooting medium for 6-8 weeks.

2. The method according to claim 1, characterized in that, The stem segment selected in step (1) is the 3rd to 5th stem segment.

3. The method according to claim 1, characterized in that, The MS basal culture medium in steps (1)-(3) contains 3.0-5.0 mg / L 6-BA and 0.1-0.5 mg / L NAA.

4. The method according to claim 1, characterized in that, In step (2), the YEP medium contains 50 mg / L kanamycin and 25 mg / L rifampicin.

5. The method according to claim 1, characterized in that, In step (2), the bacterial cells are resuspended and then left to stand at 28°C for 3 hours.

6. The method according to claim 1, characterized in that, In step (3), the oscillation and impregnation speed is 100 rpm / min, the oscillation and impregnation time is 20 min, and the vacuum penetration time is 10 min.

7. The method according to claim 1, characterized in that, The sterilization medium in step (4) is MS-based medium, which also includes 4.0 mg / L 6-BA, 0.2 mg / L NAA and 400 mg / L TMT; the resistance screening medium is MS-based medium, which also includes 4.0 mg / L 6-BA, 0.2 mg / L NAA, 400 mg / L TMT and 25 mg / L hygromycin B (Hyg).

8. The method according to claim 1, characterized in that, The seedling culture medium in step (5) is: MS-based medium, which also includes 400 mg / L termethin (TMT), 20 mg / L hygromycin B (Hyg), 10% potato and 10% banana.

9. The method according to claim 1, characterized in that, The rooting medium in step (6) is MS-based medium, which also includes 0.2 mg / L 6-BA, 0.2 mg / L NAA, 200 mg / L TMT and 20% banana.

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