High-efficiency genetic transformation method of pinellia ternata

Abstract

The application discloses a high-efficiency genetic transformation method of pinellia ternate, and belongs to the technical field of plant tissue culture and genetic transformation, and comprises the following steps: (1) preparing a genetic transformation receptor; (2) preparing an infection solution; (3) infection; (4) co-culture; (5) screening culture; and (6) seedling culture. The method is simple in process and convenient in operation, and can obtain pinellia ternate transgenic plants with high genetic transformation rate.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture and genetic transformation technology, specifically relating to an Agrobacterium-mediated method for efficient genetic transformation of Pinellia ternata. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pinellia ternata (Thunb.) Breit. is an important medicinal plant in my country. Its dried tuber has the effects of resolving phlegm, relieving nausea and vomiting, and dissipating lumps and nodules. It is a commonly used bulk Chinese medicinal material. Modern pharmacological studies have shown that Pinellia ternata has anti-peptic ulcer, antihypertensive, anti-aging, and anti-tumor effects. Due to its wide distribution, Pinellia ternata easily forms populations with different resistances and medicinal values ​​in different environments. The differences between different populations gradually evolve into different genotypes of Pinellia ternata germplasm, providing a rich germplasm resource base for the breeding of superior Pinellia ternata varieties. Based on genetic transformation technology, the functional genes of Pinellia ternata can be studied to promote the breeding of superior Pinellia ternata varieties. Agrobacterium-mediated genetic transformation technology is very mature in crops such as wheat, tomato, and cotton, and can obtain high transformation efficiency. However, there are few reports on the genetic transformation research of Pinellia ternata, and most studies stop at obtaining transformed Pinellia ternata callus tissue. In studies that can obtain transformed Pinellia ternata seedlings, the transformation efficiency is only 4%. The lack of an efficient genetic transformation system for Pinellia ternata has severely limited the research on its functional genes and trait improvement. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly efficient genetic transformation method for Pinellia ternata. The method provided by this invention has a short cultivation cycle, with seedlings emerging within 90 days. Furthermore, the positive rate of callus tissue after screening can reach 80%, and all positive callus tissues can grow into seedlings, resulting in high transformation efficiency.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, a highly efficient genetic transformation method for Pinellia ternata includes the following steps:

[0007] (1) Preparation of genetic transformation recipients: The buds of Pinellia tuber or bulbils were cultured in 1 / 2MS medium to obtain sterile seedlings. The sterile seedlings were cut and pre-cultured to obtain genetic transformation recipients.

[0008] (2) Preparation of infection solution: Agrobacterium cells containing the target gene are suspended in an infection culture medium to prepare Agrobacterium infection solution;

[0009] (3) Infection: The genetic transformation receptor in (1) was infected with the Agrobacterium infection solution in (2);

[0010] (4) Co-culture: After removing the residual infection solution on the surface of the genetic transformation recipient after infection in (3), co-culture them with MS medium;

[0011] (5) Screening culture: The genetic transformation recipients after (4) co-culture were transferred to the screening medium for screening culture to obtain resistant callus tissue;

[0012] (6) Seedling culture: The resistant callus tissue from (5) was subcultured to obtain transformed Pinellia ternata tissue culture seedlings.

[0013] The beneficial effects of this invention are as follows:

[0014] The efficient genetic transformation method for Pinellia ternata of this invention comprises six parts: preparation of the genetic transformation recipient, preparation of the infection solution, infection, co-culture, screening culture, and seedling culture. The process is simple, convenient, and uses readily available culture media. Optimized parameters in each step enable the rapid acquisition of genetically transformed Pinellia ternata tissue culture seedlings. The cultivation cycle is short, with seedlings emerging within 90 days. Furthermore, the positive rate of callus tissue after screening can reach 80%, and all positive calluses can grow into seedlings, demonstrating high transformation efficiency. This invention utilizes Agrobacterium-mediated transformation to introduce the target gene into the Pinellia ternata genetic transformation recipient, obtaining resistant callus tissue of Pinellia ternata, which is then cultured into transformed Pinellia ternata tissue culture seedlings. This is of great significance for subsequent breeding of superior Pinellia ternata varieties, research on functional genes of Pinellia ternata, and the establishment of a Pinellia ternata genetic transformation system. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The red portion of the betalain gene (marker gene) expression phenotype is the genetic transformation receptor obtained in Example 1 of this invention.

[0017] Figure 2 This refers to the resistant callus tissue obtained in Example 1 of the present invention;

[0018] Figure 3 The transformed Pinellia ternata tissue culture seedlings obtained in Example 1 of this invention. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] Given that an efficient genetic transformation system for Pinellia ternata has not yet been established, the research on functional genes and trait improvement of Pinellia ternata is greatly limited. Therefore, this invention proposes an efficient genetic transformation method for Pinellia ternata.

[0022] A typical embodiment of the present invention provides a method for efficient genetic transformation of Pinellia ternata, comprising the following steps:

[0023] (1) Preparation of genetic transformation recipients: The buds of Pinellia tuber or bulbils were cultured in 1 / 2MS medium to obtain sterile seedlings. The sterile seedlings were cut and pre-cultured to obtain genetic transformation recipients.

[0024] (2) Preparation of infection solution: Agrobacterium cells containing the target gene are suspended in an infection culture medium to prepare Agrobacterium infection solution;

[0025] (3) Infection: The genetic transformation receptor in (1) was infected with the Agrobacterium infection solution in (2);

[0026] (4) Co-culture: After removing the residual infection solution on the surface of the genetic transformation recipient after infection in (3), co-culture them with MS medium;

[0027] (5) Screening culture: The genetic transformation recipients after (4) co-culture were transferred to the screening medium for screening culture to obtain resistant callus tissue;

[0028] (6) Seedling culture: The resistant callus tissue from (5) was subcultured to obtain transformed Pinellia ternata tissue culture seedlings.

[0029] In some embodiments of this implementation, (1) the preparation of the genetic transformation recipient is specifically as follows: select healthy Pinellia ternata with good growth, dig out the tubers and bulbils, carefully cut off the buds with a scalpel, disinfect them, and culture them in 1 / 2MS medium for 20-30 days to obtain sterile seedlings.

[0030] Preferably, the sterile seedling culture conditions are: light intensity 2000-6000 Lx, 12-14 h / d, temperature 22-27℃, and humidity 60±5%.

[0031] In some embodiments of this implementation, (1) the cutting of sterile seedlings specifically involves cutting the leaves of sterile seedlings into squares with a side length of 0.3 to 0.7 cm, cutting the petioles into lengths of 0.3 to 0.7 cm, and then pre-culturing them.

[0032] Preferably, the pre-culture is as follows: leaves or petioles are cultured in MS medium under light of 2000-6000 Lx for 7-10 days. When the leaves or petioles are slightly raised, they are transferred to a dark room for 2-4 days to obtain the genetic transformation recipient.

[0033] More preferably, the MS culture medium consists of: MS + 6-BA 1.0–3.0 mg / L and NAA 0.25–0.75 mg / L.

[0034] In some embodiments of this implementation, (2) the method for preparing Agrobacterium cells containing the target gene is as follows: Agrobacterium bacterial solution containing the target gene is added to a culture medium containing hygromycin and rifampin and cultured for 24-36 hours; the bacterial solution is centrifuged, the supernatant is discarded, and Agrobacterium cells containing the target gene are obtained.

[0035] Preferably, the culture is carried out in 1-3 ml of culture medium for 12-18 h, and then transferred to 10-15 ml of the same culture medium for 12-18 h.

[0036] Preferably, the culture medium is LB medium or YEB liquid medium.

[0037] Preferably, the bacterial culture conditions are 25–30℃ and 180 r / min.

[0038] Preferably, the centrifugation conditions are: 8000-12000 r / min, centrifugation for 10-15 min.

[0039] In some embodiments of this implementation, in (2), the infection medium is MGL medium.

[0040] The specific preparation method of the Agrobacterium infection solution is as follows: Agrobacterium containing the target gene is suspended in MGL infection medium to make the OD600 value of the infection solution 0.5-0.7, acetylsyleugenone AS is added to the infection solution, and the infection solution is incubated in an incubator at 25-30℃ at 170-190r / min for 10-20min.

[0041] Preferably, the AS concentration is 90–110 μM.

[0042] In some embodiments of this implementation, in (3), the infection specifically involves: transferring the genetic transformation recipient into the Agrobacterium infection solution, sonicating for 20–60 s, and then transferring it to a shaker at 25–30 °C and a rotation speed of 170–190 r / min for 10–15 min.

[0043] In some embodiments of this implementation, in (4), the co-culture specifically involves: transferring the genetic transformation recipient explant to MS medium and culturing in a dark room for 3 to 5 days at a temperature of 22 to 27°C and a humidity of 60 ± 5%.

[0044] Preferably, a sterile filter paper is placed on the culture medium during incubation.

[0045] Preferably, the MS culture medium consists of: MS + 6-BA 1.0–3.0 mg / L and NAA 0.25–0.75 mg / L.

[0046] In some embodiments of this implementation, (5) the genetic transformation recipient is pre-cultured before screening culture, specifically: the genetic transformation recipient is transferred to MS medium and cultured for 7-10 days; preferably, the MS medium composition is: MS+6-BA 1.0-3.0 mg / L, NAA 0.25-0.75 mg / L, cephalosporin 200-350 mg / L; preferably, the culture conditions are: light 2000-6000 Lx, 12-14 h / d, temperature 22-27℃, humidity 60±5%.

[0047] Preferably, after pre-culturing, the genetic transformation recipient containing the target gene is transferred to MS medium and cultured for 20–30 days to obtain resistant callus tissue with high expression of the target gene; preferably, the culture conditions are: light intensity 2000–6000 Lx, 12–14 h / d, temperature 22–27 °C, humidity 60 ± 5%; preferably, the MS medium composition is: MS + 6-BA 1.0–3.0 mg / L, NAA 0.25–0.75 mg / L, cephalosporin 200–350 mg / L, and hygromycin 8–12 mg / L.

[0048] In some embodiments of this implementation, in (6), the subculture specifically involves: transferring the resistant callus to MS medium and culturing it for 20 to 30 days under the following conditions: light intensity of 2000 to 6000 Lx, 12 to 14 h / d, temperature of 22 to 27°C, and humidity of 60 ± 5%, thereby obtaining the resistant callus.

[0049] Preferably, the MS culture medium consists of: MS + 6-BA 2.0-3.0 mg / L + NAA 0.5-0.75 mg / L.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] A High-Efficiency Genetic Transformation Method for Pinellia ternata

[0053] (1) Preparation of genetic transformation receptors

[0054] Select healthy Pinellia ternata with good growth, dig up the tubers and bulbils, carefully cut off the buds with a scalpel, rinse with tap water for 30 minutes, absorb the water with filter paper, sterilize with 75% ethanol for 45 seconds, disinfect with 0.1% mercuric chloride for 8 minutes, rinse 5 times with sterile water, and culture in 1 / 2 MS medium for 20 days at a temperature of 25±2℃, humidity of 60±5%, and light intensity of 5000 Lx to obtain sterile seedlings.

[0055] The sterile seedlings were cut into squares with sides of about 0.5 cm and petioles cut into 0.5 cm lengths. They were cultured on MS medium containing 2.0 mg / L 6-BA and 0.5 mg / L NAA for 7 days at a temperature of 25±2℃, a humidity of 60±5%, and a light intensity of 5000 Lx for 14 h / day, until the leaves or petioles became slightly convex. They were then transferred to a dark room at a temperature of 25±2℃ and a humidity of 60±5% for 2 days to obtain the genetic transformation recipient.

[0056] (2) Preparation of infection solution

[0057] Collect Agrobacterium cells: Add 20 μL of Agrobacterium culture containing the target gene to 1 ml of liquid medium containing 75 mg / L hygromycin, 100 mg / LLB rifampicin, or YEB, and incubate at 28℃ and 180 r / min for 12–18 h. Then transfer to 10–15 ml of the same medium and incubate for 12–18 h. Divide the culture into 2 ml sterile centrifuge tubes, centrifuge at 10000 r / min for 10 min, and discard the supernatant. Add 1 ml of sterile water, shake until mixed, and centrifuge again at 10000 r / min for 10 min. Discard the supernatant to obtain Agrobacterium cells containing the target gene.

[0058] Preparation of infection solution: Agrobacterium containing the target gene was suspended in MGL culture medium to make the OD600 value of the infection solution 0.6. AS (acetylsyleugenol) was added to the infection solution to make the concentration 100 μM. The infection solution was incubated in a 28℃ incubator at 180 r / min for 15 min.

[0059] MGL culture medium formulation

[0060]

[0061] (3) Infection

[0062] The genetic transformation receptor of (1) was transferred into the infection solution of (2), sonicated for 30s, and then transferred to a shaker at 28℃ and 180r / min for 15min.

[0063] (4) Co-cultivation

[0064] The genetic transformation recipients infected in (3) were removed, placed on sterile filter paper, the bacterial solution was blotted dry, and then placed in MS medium containing 2.0 mg / L 6-BA and 0.5 mg / L NAA for 3 days in the dark. A sterile filter paper was placed on the medium, and the temperature was 25±2℃ and the humidity was 60±5%.

[0065] (5) Screening and Cultivation

[0066] The genetic transformation recipients infected in (4) were transferred to MS medium containing 300 mg / L cephalosporin, 2.0 mg / L 6-BA and 0.5 mg / L NAA and cultured for 10 days under the same conditions as the sterile seedling culture.

[0067] The genetic transformation receptor expressing the target gene was transfected into MS medium containing cephalosporin 300 mg / L, hygromycin 10 mg / L, 6-BA 2.0 mg / L, and NAA 0.5 mg / L and cultured for 30 days under conditions identical to those for aseptic seedling culture, resulting in resistant callus tissue with high expression of the target gene.

[0068] (6) Seedling cultivation

[0069] The resistant callus tissue in (5) was transferred to MS medium containing antibiotics, 6-BA 2.0 mg / L and NAA 0.5 mg / L and cultured for 30 days under the same conditions as the sterile seedling culture conditions to obtain Hyg-resistant transformed Pinellia ternata tissue culture seedlings.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A highly efficient genetic transformation method for Pinellia ternata, characterized in that, Includes the following steps: (1) Preparation of genetic transformation recipients: The buds of Pinellia tuber or bulbils were cultured in 1 / 2MS medium to obtain sterile seedlings. The sterile seedlings were cut and pre-cultured to obtain genetic transformation recipients. (2) Preparation of infection solution: Agrobacterium cells containing the target gene are suspended in an infection culture medium to prepare Agrobacterium infection solution; (3) Infection: The genetic transformation receptor in (1) was infected with the Agrobacterium infection solution in (2); (4) Co-culture: After removing the residual infection solution on the surface of the genetic transformation recipient after infection in (3), co-culture them with MS medium; (5) Screening culture: The genetic transformation recipients after (4) co-culture were transferred to the screening medium for screening culture to obtain resistant callus tissue; (6) Seedling culture: The resistant callus tissue from (5) was subcultured to obtain transformed Pinellia ternata tissue culture seedlings; The pre-culture process is as follows: leaves or petioles are cultured in MS medium under light of 2000-6000 Lx for 7-10 days. When the leaves or petioles are slightly raised, they are transferred to a dark room for 2-4 days to obtain the genetic transformation recipient. The MS medium consisted of: MS + 6-BA 1.0~3.0 mg / L and NAA 0.25~0.75 mg / L. (2) In this context, the infection medium is MGL medium; The specific preparation method of the Agrobacterium infection solution is as follows: Agrobacterium containing the target gene is suspended in MGL infection medium to make the OD600 value of the infection solution 0.5~0.7, acetylsyl syringone AS is added to the infection solution, and the infection solution is incubated in an incubator at 25~30℃ at 170~190 r / min for 10~20 min. The AS concentration is 90~100μM; (5) Before screening and culturing, the genetic transformation recipients are pre-cultured, specifically as follows: the genetic transformation recipients are transferred to MS medium and cultured for 7-10 days; the composition of the MS medium is: MS + 6-BA 1.0-3.0 mg / L, NAA 0.25-0.75 mg / L, cephalosporin 200-350 mg / L; the culture conditions are: light intensity 2000-6000 Lx, 12-14 h / d, temperature 22-27℃, humidity 60±5%; After pre-culture, the genetic transformation recipient containing the target gene was transferred to MS medium and cultured for 20-30 days to obtain resistant callus tissue with high expression of the target gene. The culture conditions were: light intensity 2000-6000 Lx, 12-14 h / d, temperature 22-27℃, and humidity 60±5%. The MS medium consisted of: MS + 6-BA 1.0-3.0 mg / L, NAA 0.25-0.75 mg / L, cephalosporin 200-350 mg / L, and hygromycin 8-12 mg / L.

2. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, In (1), the preparation of the genetic transformation recipient is specifically as follows: select healthy Pinellia ternata with good growth, dig out the tubers and bulbils, carefully cut off the buds with a scalpel, disinfect them, and culture them in 1 / 2MS medium for 20-30 days to obtain sterile seedlings; The aseptic seedling culture conditions are as follows: light intensity 2000~6000 Lx, 12~14h / d, temperature 22~27℃, humidity 60±5%.

3. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, In (1), the process of cutting the sterile seedlings specifically involves cutting the leaves of the sterile seedlings into squares with a side length of 0.3 to 0.7 cm and cutting the petioles into lengths of 0.3 to 0.7 cm, followed by pre-culturing. The MS medium consisted of: MS + 6-BA 1.0~3.0 mg / L and NAA 0.25~0.75 mg / L.

4. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, (2) The method for preparing Agrobacterium cells containing the target gene is as follows: Agrobacterium bacterial solution containing the target gene is added to a culture medium containing hygromycin and rifampin and cultured for 24-36 h; the bacterial solution is centrifuged, the supernatant is discarded, and Agrobacterium cells containing the target gene are obtained; The culture involves culturing in 1-3 ml of culture medium for 12-18 hours, then transferring to 10-15 ml of the same culture medium and culturing for another 12-18 hours. The culture medium is LB medium or YEB liquid medium; The bacterial culture conditions are 25-30℃ and 180 r / min; Centrifugation conditions: 8000~12000 r / min, centrifugation for 10~15 min.

5. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, In (3), the infection specifically involves: transferring the genetic transformation recipient into the Agrobacterium infection solution and sonicating it for 20-60 s; then transferring it to a shaker at 25-30℃ and a rotation speed of 170-190 r / min and culturing it for 10-15 min.

6. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, In (4), the co-culture specifically involves: transferring the genetic transformation recipient explants to MS medium and culturing in the dark for 3-5 days at a temperature of 22-27℃ and a humidity of 60±5%; During incubation, place a sterile filter paper on the culture medium; The MS medium consisted of: MS + 6-BA 1.0~3.0 mg / L and NAA 0.25~0.75 mg / L.

7. The efficient genetic transformation method for Pinellia ternata according to claim 1, characterized in that, In (6), the subculture is specifically as follows: the resistant callus is transferred to MS medium and cultured for 20-30 days. The culture conditions are: light intensity 2000-6000 Lx, 12-14 h / d, temperature 22-27℃, humidity 60±5%, and the resistant callus is obtained. The MS medium composition is: MS + 6-BA 2.0-3.0 mg / L + NAA 0.5-0.75 mg / L.

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