Method for obtaining transgenic or gene-edited plant

Abstract

The invention discloses a method for obtaining a transgenic or gene-edited plant, which comprises the following steps of: firstly, culturing a plant seedling, and obtaining a plant to be infected when the stem length is greater than 1cm; the method comprises the following steps: transforming a target vector into agrobacterium, selecting a monoclonal bacterial colony with the target vector, preparing an agrobacterium solution, cutting a wound on a stem of a plant to be infected, externally applying the activated agrobacterium solution to the wound for infection, then carrying out illumination culture, removing a growing point after a period of time, and continuing to culture. And obtaining a transgenic plant or a gene editing plant when callus tissue grows on the wound and a new bud containing the target gene is obtained. According to the method, a wound is generated on a stem of a plant, growing point removal operation is carried out after true leaves grow out, and wound calluses are issued under the stimulation of hormone of the plant to breed into buds. The method is not limited to tissue culture and sterile operation, does not need root regeneration buds, does not need external application of hormone and does not need root regeneration induction, the operation steps are simple, and transgenic or gene edited plants can be obtained in a short time.

Description

Technical Field

[0001] This invention relates to the field of genetic transformation, and more specifically to a method for obtaining transgenic or gene-edited plants. Background Technology

[0002] Plant genetic transformation is a core technology in plant genetic engineering. Many species have established stable genetic transformation systems, among which Agrobacterium-mediated transformation is widely used due to its low cost and stable characteristics. For example, in tissue culture, Agrobacterium tumefaciens is used to infect callus tissue, hypocotyls, stem segments, and leaves, and different plant hormone ratios are used to induce transgenic shoots and roots, thereby obtaining regenerated transgenic plants. This has been reported in rice, wheat, corn, cotton, and other plants. The CDB (cut-dip-budding) delivery system provides a novel soil-based genetic transformation method that does not require aseptic operation. This method uses Agrobacterium rhizogenes to transform sweet potato, dandelion, and other plants to obtain transgenic roots, which are then cultured to obtain transgenic plants. This method is simple to operate and does not require aseptic operation, but it requires the plant itself to have the ability to regenerate shoots from the roots. Invention patent application CN 113930441 A discloses a method for obtaining transgenic or gene-edited plant bodies. This method utilizes a non-tissue culture approach to obtain transgenic plants by infecting any part of a sweet potato with Agrobacterium rhizogenes carrying a vector, culturing the infected sweet potato tissue until hairy roots develop, and then screening for transgenic or gene-edited roots for propagation to obtain transgenic or gene-edited sweet potatoes. However, sweet potatoes are developed directly from roots, a capability that most plants do not possess. For most plants, obtaining transgenic roots through infection with Agrobacterium rhizogenes and then inducing bud regeneration through these transgenic roots presents a significant challenge. Invention patent application CN 118272428A discloses a method for efficient genetic transformation and gene editing of Brassica crops mediated by Agrobacterium rhizogenes. The method establishes a genetic transformation and gene editing system for Brassica crops by using three growth and development regulatory factors, ZmWUS2, AtIPT, and AtPLT5, mediated by Agrobacterium rhizogenes. The method induces callus formation in explants through these three growth and development regulatory factors, directly forming shoots, thus enabling effective transformation of species with poor root regeneration ability. However, this method is still based on tissue culture, requires a sterile environment, and is relatively complex and cumbersome to operate. Summary of the Invention

[0003] The purpose of this invention is to provide a method for obtaining transgenic or gene-edited plants. This method is not limited to tissue culture and aseptic operation, does not require the use of root regeneration shoots, and has simple operation steps, which can obtain transgenic or gene-edited plants in a short time.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A method for obtaining transgenic or gene-edited plants includes the following steps:

[0006] Step 1: Sow plant seeds, and after the seeds germinate and grow stems, you will get the plants to be infected;

[0007] Step 2: Transform the transgenic or gene-edited target vector into Agrobacterium, select monoclonal colonies carrying the target vector, and prepare Agrobacterium bacterial suspension;

[0008] Step 3: Make a wound on the stem of the plant to be infected, and infect the wound with activated Agrobacterium tumefaciens solution;

[0009] Step 4: After the infection is complete, carry out light cultivation, and remove the growing point after true leaves have grown.

[0010] Step 5: Continue culturing and maintain the removal of the growth point until new shoots containing the gene corresponding to the target vector grow from the wound, thus obtaining a transgenic or gene-edited plant.

[0011] Preferably, the plant to be infected is a plant that can produce callus tissue and sprout new shoots from wounds on its stem.

[0012] Preferably, the plant to be infected is a plant of the Brassica genus or a plant of the Solanum genus.

[0013] Preferably, the Brassica plant is Chinese cabbage, broccoli, or rapeseed; and the Solanum plant is eggplant.

[0014] Preferably, in step 4, for Brassica plants, the growing point is removed after the true leaves have grown, while the cotyledons are retained. For Solanaceae plants such as eggplant, if the cotyledons cannot be retained when removing the growing point, the cotyledons are removed together with the growing point.

[0015] Preferably, in step 3, multiple wounds are made on the neck of the plant using a blade, with the wound depth not exceeding half the diameter of the stem. Cotton soaked in activated Agrobacterium tumefaciens solution is wrapped around the wounds for a certain period of time, at room temperature and humidity of 65-80%. In step 4, the cotton is removed and the plant is cultured under light for 16 hours in the light and 8 hours in the dark, at room temperature. In step 5, the plant is cultured again under light for 16 hours in the light and 8 hours in the dark, at room temperature.

[0016] Preferably, the Agrobacterium is Agrobacterium rhizogenes or Agrobacterium tumefaciens.

[0017] Preferably, the Agrobacterium rhizogenes is K599, AR.1193, AR.A4, AR.Qual, C58C1 or MSU440; the Agrobacterium tumefaciens is GV3101, GV2260, EHA105, AGL1, LBA4404 or EHA101.

[0018] This invention offers the following advantages: By infecting stem wounds with Agrobacterium and performing a growth point removal procedure, callus tissue can grow from the wound, resulting in new shoots containing the target gene, thereby obtaining transgenic or gene-edited plants. This method can be applied to soil-grown seedlings, eliminating the need for aseptic techniques and greatly simplifying the process. It can also be used to obtain transgenic or gene-edited plants from tissue-cultured seedlings. The method has a short plant genetic transformation cycle; for Chinese cabbage, only 25-40 days are needed to obtain transgenic red Chinese cabbage or gene-edited albino Chinese cabbage, significantly shortening the time of 60-90 days compared to the method in CN 118272428 A. Furthermore, this invention utilizes growth and development factors and the visualized gene RUBY to directly observe red transgenic seedlings, and uses growth and development factors and a PDS-targeting knockout vector to directly obtain albino seedlings. Attached Figure Description

[0019] Figure 1 For carrier information diagram;

[0020] Figure 2 A photo showing cotton balls soaked in a bacterial solution being used to treat a cabbage wound;

[0021] Figure 3 A photo of a cabbage that has grown two true leaves after being infected;

[0022] Figure 4 Photos showing the condition of the cabbage after removing the sprout, one cotyledon, and the wound on the stem;

[0023] Figure 5 A photo of red genetically modified seedlings growing from a wound on a cabbage;

[0024] Figure 6 A photo of genetically modified cabbage sprouts growing;

[0025] Figure 7 Electrophoresis diagram of transgenic Chinese cabbage sprouts for PCR verification;

[0026] Figure 8 Infographic of the pB-RBpttCas12a-PDS-WIP gene editing vector;

[0027] Figure 9 A diagram showing the PDS target sites in Chinese cabbage;

[0028] Figure 10A comparison photo of albino Chinese cabbage plants after PDS gene editing and normal plants;

[0029] Figure 11 Electrophoresis image for PCR verification of PDS albino seedlings of Chinese cabbage;

[0030] Figure 12 Image showing the Sanger sequencing results of albino Chinese cabbage plant No. 2;

[0031] Figure 13 A photo of a broccoli wound infected with a cotton ball containing bacterial solution;

[0032] Figure 14 A photo of broccoli that has grown two true leaves after being infected;

[0033] Figure 15 Photos showing the condition of the broccoli buds, one cotyledon, and the wound on the stem after removal;

[0034] Figure 16 A photo of red genetically modified shoots growing from a broccoli wound;

[0035] Figure 17 A photo of genetically modified broccoli shoots growing;

[0036] Figure 18 Electrophoresis diagram of broccoli transgenic regenerated seedlings for further PCR verification;

[0037] Figure 19 Photos of rapeseed seedlings awaiting infection;

[0038] Figure 20 A photo showing two true leaves grown on rapeseed after infection;

[0039] Figure 21 A photo showing the rapeseed after removing the buds and one cotyledon;

[0040] Figure 22 A photograph showing the differentiation and growth of transgenic shoots during wound healing in rapeseed.

[0041] Figure 23 A photo of a red genetically modified rapeseed seedling growing into a new plant;

[0042] Figure 24 Photos of eggplant seedlings awaiting infection;

[0043] Figure 25 A photo showing the use of cotton balls soaked in a bacterial solution to treat an eggplant wound;

[0044] Figure 26 Photos showing the condition of the eggplant after the removal of buds and cotyledons, as well as the wounds on the stem;

[0045] Figure 27A photo of a new shoot growing from a wound on an eggplant;

[0046] Figure 28 Electrophoresis diagram for PCR verification of transgenic eggplant shoots;

[0047] Figure 29 The images show the growth status of Chinese cabbage sprouts under different treatments. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings.

[0049] Example 1: Obtaining Transgenic Chinese Cabbage Grown in Soil

[0050] Step 1: Cultivation of Chinese cabbage seedlings

[0051] 1.1 Select vermiculite and nutrient soil and mix them evenly in a 2:1 ratio. Use small pots to hold the potting soil.

[0052] 1.2 Sow the cabbage seeds and place them in a seedling tray. Incubate them in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). Once the seeds germinate and the stems reach a length of 1 cm, they are used for infection to obtain plants to be infected.

[0053] Step 2: Preparation of Agrobacterium tumefaciens bacterial culture

[0054] 2.1 The WIP-RUBY target vector ( Figure 1 Transformed Agrobacterium rhizogenes K599, identified by PCR, clones carrying the WIP-RUBY target vector were shaken, and cultured with an equal volume of 50% glycerol (glycerol and water were prepared at a volume ratio of 1:1) for preservation.

[0055] 2.2 Add 200 μL of K599 Agrobacterium tumefaciens containing the WIP-RUBY plasmid to 50 ml of TY liquid medium (50 mg / L Strep, 50 mg / L Kan) and incubate at 28°C in a shaker (200 rpm) for 16 hours.

[0056] 2.3 Centrifuge the above-cultured K599 Agrobacterium containing the WIP-RUBY plasmid at 5000 rpm for 8 min, remove the supernatant, collect the precipitate, and then resuspend the bacterial culture in 1 / 2 MS liquid medium, adjusting the OD600 to 0.8~1.0.

[0057] 2.4 Add acetylsuccione (AS, 100 mmol / L) to the above bacterial culture and incubate at 28℃ for 30 min.

[0058] 2.5 Pour the activated bacterial solution into sterile cotton for later use.

[0059] Step 3: Infecting cabbage seedlings

[0060] 3.1 Take out the cabbage seedlings cultivated in step 1.2 and make multiple cuts on the stem of the seedlings with a blade (the depth of the cuts should not exceed 1 / 2 of the stem diameter).

[0061] 3.2 Wrap the wound area of ​​the cabbage stem with cotton soaked in Agrobacterium solution prepared in step 2.5. Figure 2 Place the cabbage seedlings with the wounds wrapped in a dark place for 48 hours. During the dark culture period, cover the seedling tray and maintain the humidity between 65% and 80%.

[0062] Step 4: Light cultivation and bud removal of infected cabbage seedlings

[0063] 4.1 Transfer the cabbage seedlings that have completed the dark culture in step 3.2 to a culture room at 22-26℃ (16 hours of light and 8 hours of darkness) for cultivation. Remove the cotton. When the seedlings have grown two true leaves, remove the growing point (e.g., Figure 3 (As shown).

[0064] 4.2 Using a blade, make a 45° angled cut along the true leaf to remove the seedling bud (i.e., the growing point) along with one cotyledon, resulting in a cabbage seedling retaining one cotyledon. At this point, a large amount of white callus has formed at the wound. Figure 4 ).

[0065] Step 5: Cultivating and growing new shoots in cabbage seedlings after removing the sprouts.

[0066] 5.1 After removing the sprouts in step 4.2, the cabbage seedlings continue to be cultured in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). If the sprouts are not completely removed, use a blade to remove more sprouts.

[0067] 5.2 After approximately 4 more days of continued cultivation, red transgenic shoots began to appear. Figure 5 ), and the red buds grew one after another ( Figure 6 ).

[0068] Step 6: PCR identification of red cabbage sprouts

[0069] 6.1 Extract DNA from the new red shoots of Chinese cabbage and use it as a template for PCR amplification using primers RUBY-F (CAGACCACCAAGCTGC) and RUBY-R (GGAGGTTGAGGCTGGT).

[0070] 6.2 Electrophoresis: Marker (lane 1), red cabbage plants (1-5) showed PCR bands of the same size as the expected band (500 bp), while wild-type (6) showed no PCR band. Figure 7 This indicates that the RUBY vector was integrated into the genome of the five Chinese cabbage red shoots mentioned above.

[0071] The above experimental results show that the method provided by this invention can be used to quickly achieve genetic modification of Chinese cabbage.

[0072] Example 2: Obtaining Gene-Edited Chinese Cabbage Through Soil Culture

[0073] Step 1: Using the CRISPR / Cas12a gene editing system ( Figure 8 Gene editing in Chinese cabbage was performed using WIP growth and development factors, targeting the PDS gene with specific target sites. Target site information is as follows: Figure 9 As shown, the PDS knockout vector pB-RBpttCas12a-PDS-WIP was constructed. Agrobacterium tumefaciens culture containing pB-RBpttCas12a-PDS-WIP was prepared according to step 2 in Example 1.

[0074] Step 2: Cultivate Chinese cabbage seedlings according to Step 1 in Example 1.

[0075] Step 3: Following Step 3 in Example 1, infect Chinese cabbage seedlings with Agrobacterium tumefaciens containing pB-RBpttCas12a-PDS-WIP.

[0076] Step 4: Following step 4 in Example 1, remove the buds from the infected cabbage seedlings.

[0077] Step 5: Following step 5 in Example 1, cultivate the de-budded cabbage seedlings to obtain albino seedlings. Figure 10 ).

[0078] Step 6: Extract genomic DNA from albino seedlings and design primer pairs (PDS1-F, PDS1-R and PDS2-F, PDS2-R) for PCR identification. Figure 11 Sanger sequencing was performed on site 2, and the results showed that gene editing did indeed occur between the PDS target sites, achieving fragment deletion. Figure 12 ).

[0079] The experimental results above demonstrate that the method provided in this experiment can be used to quickly achieve gene editing in cabbage.

[0080] Example 3: Obtaining Transgenic Broccoli from Soil

[0081] Step 1: Broccoli Seedling Cultivation

[0082] 1.1 Mix vermiculite and potting soil evenly in a 2:1 ratio and plant in seedling pots.

[0083] 1.2 After sowing broccoli seeds, place the seedling trays in the seedling pans and cultivate them in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). Once the cotyledons have fully expanded, they can be used for infection.

[0084] Step 2: Preparation of Agrobacterium tumefaciens bacterial culture

[0085] The K599 Agrobacterium rhizogenes infection solution containing the WIP-RUBY target vector was prepared according to step 2 in Example 1. The difference from the above steps was that the bacterial solution was resuspended in 1 / 2 MS liquid medium, and the OD600 was adjusted to 0.3-0.4.

[0086] Step 3: Infect broccoli seedlings

[0087] 3.1 Take out the broccoli seedlings cultivated in step 1 and make multiple cuts on the stem of the seedlings with a blade (the depth of the cuts should not exceed 1 / 2 of the stem diameter).

[0088] 3.2 Wrap the wound on the broccoli stem with the cotton soaked in Agrobacterium tumefaciens prepared in step 2. Figure 13 Place the broccoli seedlings with the wound wrapped in a dark place for 48 hours. During the dark culture period, cover the seedling tray and maintain the humidity between 65% and 80%.

[0089] Step 4: Light cultivation and bud removal of infected broccoli seedlings

[0090] 4.1 Transfer the broccoli seedlings that have completed the dark culture in step 3.2 to a culture room at 22-26℃ (16 hours of light and 8 hours of darkness) for cultivation. Remove the cotton. When the seedlings have grown two true leaves ( Figure 14 ) can be debudded (i.e., the growing point).

[0091] 4.2 Use a blade to completely remove the growing point of the seedling, leaving the cotyledons intact, to obtain broccoli seedlings without a growing point. Figure 15 If lateral buds grow on both sides of the cotyledons, use a blade to remove them.

[0092] Step 5: Cultivating broccoli seedlings after removing the buds and allowing them to sprout new buds.

[0093] 5.1 Place the broccoli seedlings (after removing the buds in step 4.2) in a 22-26℃ incubator (16 hours light, 8 hours dark). Red transgenic buds will appear after approximately 20 days. Figure 16 ), and the red buds grew one after another ( Figure 17 ).

[0094] Step 6: PCR identification of red broccoli shoots

[0095] 6.1 Extract DNA from the new red shoots of broccoli and use it as a template for PCR amplification using primers RUBY-F (CAGACCACCAAGCTGC) and RUBY-R (GGAGGTTGAGGCTGGT).

[0096] 6.2 Electrophoresis, Marker (lane 1): Wild-type (#1) showed no PCR band; red broccoli plants (#2-#6) showed PCR bands of the same size (500 bp) as expected. Figure 18 This indicates that the RUBY vector was integrated into the genomes of the five red broccoli plants mentioned above.

[0097] The above experimental results demonstrate that the method provided in this experiment can be used to quickly achieve genetic modification of broccoli.

[0098] Example 4: Obtaining Transgenic Rapeseed Cultivated in Soil

[0099] Step 1: Rapeseed seedling cultivation

[0100] 1.1 Mix vermiculite and potting soil evenly in a 2:1 ratio and plant in seedling pots.

[0101] 1.2 After sowing rapeseed seeds, place seedling trays in seedling pans and cultivate in a 22-26℃ incubator (16 hours light, 8 hours dark). Once the cotyledons are fully expanded, the seeds can be used for infection. Figure 19 ).

[0102] Step 2: Preparation of Agrobacterium tumefaciens bacterial culture

[0103] Prepare K599 Agrobacterium rhizogenes infection solution containing the WIP-RUBY target vector according to step 2 in Example 1.

[0104] Step 3: Infect rapeseed seedlings

[0105] 3.1 Take out the rapeseed seedlings cultivated in step 1 and make multiple wounds on the stem of the seedlings with a blade (the depth of the wounds should not exceed 1 / 2 of the stem diameter).

[0106] 3.2 Wrap the wound area of ​​the rapeseed stem with cotton soaked in Agrobacterium tumefaciens prepared in step 2, and place the wrapped rapeseed seedlings in the dark for 48 hours. During the dark culture, cover the seedling tray and maintain the humidity between 65% and 80%.

[0107] Step 4: Light cultivation and bud removal of infected rapeseed seedlings

[0108] 4.1 Transfer the rapeseed seedlings that have completed the dark culture in step 3.2 to a culture room at 22-26℃ (16 hours of light and 8 hours of darkness) for cultivation. Remove the cotton. When the seedlings have grown two true leaves ( Figure 20 ) can be debudded.

[0109] 4.2 Use a blade to remove the seedling buds completely, obtaining rapeseed seedlings without buds. Figure 21 If subsequent cotyledon lateral buds grow, use a blade to continue removing the lateral buds.

[0110] Step 5: Cultivating rapeseed seedlings and promoting new shoot growth after removing the buds

[0111] 5.1 Place the rapeseed seedlings (after debudding in step 4.2) in a 22-26℃ incubation room (16 hours light, 8 hours dark). Red transgenic shoots will appear after approximately 20 days. Figure 22 ), and the red buds grew one after another ( Figure 23 ).

[0112] The above experimental results demonstrate that the method provided in this experiment can be used to quickly achieve the genetic modification of rapeseed.

[0113] Example 5: Obtaining Transgenic Eggplant from Soil

[0114] Step 1: Eggplant seedling cultivation

[0115] 1.1 Select vermiculite and nutrient soil and mix them evenly in a 2:1 ratio. Use small pots to hold the potting soil.

[0116] 1.2 Sow eggplant seeds and place them in seedling trays. Incubate in a 22-26℃ incubator (16 hours light, 8 hours dark). Once the cotyledons are fully expanded, obtain the plants to be infected. Figure 24 ).

[0117] Step 2: Preparation of Agrobacterium tumefaciens bacterial culture

[0118] The K599 Agrobacterium rhizogenes infection solution containing the WIP-RUBY target vector was prepared according to step 2 in Example 1. The difference from the above steps was that the bacterial solution was resuspended in 1 / 2 MS liquid medium, and the OD600 was adjusted to 0.6-0.8.

[0119] Step 3: Infect eggplant seedlings

[0120] 3.1 Take out the eggplant seedlings cultivated in step 1.2 and make multiple cuts on the stem of the seedlings with a blade (the depth of the cuts should not exceed 1 / 2 of the stem diameter).

[0121] 3.2 Wrap the wound area of ​​the eggplant stem with cotton soaked in Agrobacterium solution prepared in step 2.5. Figure 25 Place the eggplant seedlings with the wound wrapped in a dark place for 48 hours. During the dark culture period, cover the seedling tray and maintain the humidity between 65% and 80%.

[0122] Step 4: Light cultivation and bud removal of infected eggplant seedlings

[0123] 4.1 Transfer the eggplant seedlings that have completed the dark culture in step 3.2 to a culture room at 22-26℃ (16h light, 8h dark) for culture, remove the cotton, and remove the buds when the seedlings have grown two true leaves.

[0124] 4.2 Using a blade, make a 45° angled cut along the true leaves to remove the seedling's growing point along with the cotyledons, resulting in an eggplant seedling with the bud removed. At this point, a large amount of white callus has formed on the wound. Figure 26 ).

[0125] Step 5: Cultivating eggplant seedlings after removing buds and encouraging new bud growth.

[0126] 5.1 After removing the buds in step 4.2, the eggplant seedlings continue to be cultured in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). If the buds are not completely removed, use a blade to remove more buds.

[0127] 5.2 After approximately 4 more days of continued cultivation, the regenerated shoots began to appear. Figure 27 ), and the red buds grew one after another ( Figure 6 ).

[0128] Step 6: PCR identification of regenerated eggplant shoots

[0129] 6.1 Extract DNA from eggplant buds and use it as a template for PCR amplification with primers RUBY-F (CAGACCACCAAGCTGC) and RUBY-R (GGAGGTTGAGGCTGGT).

[0130] 6.2 Electrophoresis: Marker (lane 1), eggplant plants (1-2) showed PCR bands of the same size as the expected band (500 bp), negative control (3) showed no PCR band, positive control (4) showed no PCR band. Figure 28 This indicates that the RUBY vector was integrated into the genome of the two eggplant regenerated shoots. RUBY expression may not be obvious in the two plants, as it is not shown in red.

[0131] The above experimental results show that the method provided by this invention can be used to quickly achieve the genetic modification of eggplant.

[0132] Comparative Example 1: Effects of different treatments on the acquisition of transgenic Chinese cabbage seedlings

[0133] Step 1: Cultivation of Chinese cabbage seedlings

[0134] 1.1 Select vermiculite and nutrient soil and mix them evenly in a 2:1 ratio. Use small pots to hold the potting soil.

[0135] 1.2 Sow Chinese cabbage seeds and place them in a seedling tray. Incubate them in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). Once the cotyledons are fully expanded, use them for infection to obtain plants to be infected.

[0136] Step 2: Preparation of Infection Solution

[0137] 2.1 Preparation of sterile aqueous blank control infection solution (CK) without any target carrier and Agrobacterium:

[0138] 2.1.1 Sterilize distilled water in an autoclave to obtain sterile water;

[0139] 2.1.2 Pour the sterilized water into sterile cotton and set aside.

[0140] 2.2 Preparation of Agrobacterium rhizogenes K599 infection solution with RUBY target vector:

[0141] 2.2.1 The RUBY target vector was transformed into Agrobacterium rhizogenes K599. After PCR identification, the clones carrying the RUBY target vector were shaken and incubated with an equal volume of 50% glycerol (glycerol and water were prepared at a volume ratio of 1:1) for later use.

[0142] 2.2.2 Add 200 μL of K599 Agrobacterium tumefaciens containing the RUBY plasmid to 50 ml of TY liquid medium (50 mg / L Strep, 50 mg / L Kan), and incubate at 28°C in a shaker (200 rpm) for 16 hours.

[0143] 2.2.3 Centrifuge the above-cultured K599 Agrobacterium containing RUBY plasmid at 5000 rpm for 8 min, remove the supernatant, collect the precipitate, and then resuspend the bacterial culture in 1 / 2 MS liquid medium, adjusting the OD600 to 0.8~1.0;

[0144] 2.2.4 Add acetylsuccinone (AS, 100 mmol / L) to the above bacterial culture and incubate at 28℃ for 30 min;

[0145] 2.2.5 Pour the activated bacterial solution into sterile cotton for later use.

[0146] 2.3 Preparation of Agrobacterium rhizogenes K599 infection medium with WIP-RUBY target vector:

[0147] 2.3.1 The WIP-RUBY target vector was transformed into Agrobacterium rhizogenes K599. After PCR identification, the clones carrying the WIP-RUBY target vector were shaken and incubated with an equal volume of 50% glycerol (glycerol and water were prepared at a volume ratio of 1:1) for later use.

[0148] 2.3.2 Add 200 μL of K599 Agrobacterium tumefaciens containing the WIP-RUBY plasmid to 50 ml of TY liquid medium (50 mg / L Strep, 50 mg / L Kan), and incubate at 28°C in a shaker (200 rpm) for 16 hours.

[0149] 2.3.3 Centrifuge the above-cultured K599 Agrobacterium containing the WIP-RUBY plasmid at 5000 rpm for 8 min, remove the supernatant, collect the precipitate, and then resuspend the bacterial culture in 1 / 2 MS liquid medium, adjusting the OD600 to 0.8~1.0;

[0150] 2.3.4 Add acetylsuccinone (AS, 100 mmol / L) to the above bacterial culture and incubate at 28℃ for 30 min to activate it;

[0151] 2.3.5 Pour the activated bacterial solution into sterile cotton for later use.

[0152] 2.4 Preparation of Agrobacterium tumefaciens EHA105 infection medium with WIP-RUBY target vector:

[0153] 2.4.1 The WIP-RUBY target vector was transformed into Agrobacterium tumefaciens EHA105. After PCR identification, the clones carrying the WIP-RUBY target vector were shaken and incubated with an equal volume of 50% glycerol (glycerol and water were prepared at a volume ratio of 1:1) for later use.

[0154] 2.4.2 Add 200 μL of Agrobacterium tumefaciens EHA105 containing the WIP-RUBY plasmid to 50 ml of TY liquid medium (50 mg / L Strep, 50 mg / L Kan), and incubate at 28°C in a shaker (200 rpm) for 16 hours.

[0155] 2.4.3 Centrifuge the above-cultured EH105 Agrobacterium containing the WIP-RUBY plasmid at 5000 rpm for 8 min, remove the supernatant, collect the precipitate, and then resuspend the bacterial culture in 1 / 2 MS liquid medium, adjusting the OD600 to 0.8~1.0;

[0156] 2.4.4 Add acetylsuccinone (AS, 100 mmol / L) to the above bacterial culture and incubate at 28℃ for 30 min to activate it;

[0157] 2.4.5 Pour the activated bacterial solution into sterile cotton for later use.

[0158] Step 3: Infecting cabbage seedlings

[0159] 3.1 Take out the cabbage seedlings cultivated in step 1.2 and make multiple cuts on the stem of the seedlings with a blade (the depth of the cuts should not exceed 1 / 2 of the stem diameter).

[0160] 3.2 Use the cotton soaked in the infection solution prepared in steps 2.1.2, 2.2.5, 2.3.5, and 2.4.5 to wrap the wound area of ​​the cabbage stem. Place the wrapped cabbage seedlings in the dark for 48 hours. During the dark culture period, cover the seedling tray and maintain the humidity between 65% and 80%.

[0161] Step 4: Light cultivation and bud removal of infected cabbage seedlings

[0162] 4.1 Transfer the cabbage seedlings that have completed the dark culture in step 3.2 to a culture room at 22-26℃ (16h light, 8h dark) for culture. Remove the cotton. For some of the WIP-K599-infected seedlings, retain the buds. For the remaining seedlings, remove the buds when the seedlings have grown two true leaves.

[0163] 4.2 Using a blade, cut at an angle of about 45° to the ground along the true leaf to remove the seedling bud along with a cotyledon, resulting in a cabbage seedling with one cotyledon remaining. At this point, white callus has already formed on the wound.

[0164] Step 5: Cultivating and growing new shoots in cabbage seedlings after removing the sprouts.

[0165] 5.1 After removing the sprouts in step 4.2, the cabbage seedlings continue to be cultured in a 22-26℃ incubator (16 hours of light and 8 hours of darkness). If the sprouts are not completely removed, use a blade to remove more sprouts.

[0166] 5.2 After about 4 more days of cultivation, red transgenic shoots began to appear, and the red shoots continued to grow.

[0167] Step 6 Experimental Results ( Figure 29 ):

[0168] No new buds grew on the wounds after the cabbage was infected with sterile water solution and the sprouts were removed.

[0169] After K599 (RUBY) infection solution was used to infect Chinese cabbage and remove the sprouts, transgenic sprouts were generated.

[0170] After K599 (WIP-RUBY) infection solution was used to infect Chinese cabbage and remove the sprouts, a large number of transgenic sprouts were generated.

[0171] When K599 (WIP-RUBY) infection solution is used to infect Chinese cabbage without removing the buds (i.e. without removing the growing point), no new buds will grow at the wound site, but white roots will form.

[0172] After EHA105 (WIP-RUBY) infection solution was used to infect Chinese cabbage with debudded leaves, transgenic sprouts were generated.

[0173] 6.1 The experimental results comparing the sprouting of Chinese cabbage infected with sterile water (CK) and Agrobacterium rhizogenes K599 carrying the RUBY target vector showed that Agrobacterium infection is the key to the generation of transgenic sprouts in Chinese cabbage.

[0174] 6.2 The experimental results comparing the results of both Agrobacterium rhizogenes K599 carrying the RUBY target vector and Agrobacterium rhizogenes K599 carrying the WIP-RUBY target vector infecting Chinese cabbage with bud degeneration showed that the WIP growth regulator had a significant effect on improving the transgenic seedling generation rate.

[0175] 6.3 The experimental results of infecting Chinese cabbage with Agrobacterium rhizogenes K599 carrying the WIP-RUBY target vector with and without bud removal (removal of the growing point and without removal of the growing point) showed that bud removal (removal of the growing point) is the key to the generation of transgenic buds in Chinese cabbage.

[0176] 6.4 The experimental results of Agrobacterium tumefaciens EHA105 carrying the WIP-RUBY target vector and Agrobacterium rhizogenes K599 carrying the WIP-RUBY target vector infecting Chinese cabbage and removing the buds (removing the growing point) showed that transgenic seedlings can also be obtained by using Agrobacterium tumefaciens through this method.

Claims

1. A method for obtaining transgenic or gene-edited plants, characterized in that... Includes the following steps: Step 1: Sow plant seeds, and after the seeds germinate and grow stems, you will get the plants to be infected; Step 2: Transform the transgenic or gene-edited target vector into Agrobacterium, select monoclonal colonies carrying the target vector, and prepare Agrobacterium bacterial suspension; Step 3: Make a wound on the stem of the plant to be infected, and infect the wound with activated Agrobacterium tumefaciens solution; Step 4: After the infection is complete, carry out light cultivation, and remove the growing point after the true leaves grow. Step 5: Continue culturing and maintain the removal of the growth point until new shoots containing the gene corresponding to the target vector grow from the wound, thus obtaining a transgenic or gene-edited plant.

2. The method for obtaining transgenic or gene-edited plants as described in claim 1, characterized in that: The plants to be infected are those that can produce callus tissue from wounds on their stems.

3. The method for obtaining transgenic or gene-edited plants as described in claim 1, characterized in that: The plants to be infected are either Brassica plants or Solanum plants.

4. The method for obtaining transgenic or gene-edited plants as described in claim 3, characterized in that: The Brassica species are Chinese cabbage, broccoli, or rapeseed; the Solanum species are eggplant.

5. The method for obtaining transgenic or gene-edited plants as described in claim 1, 2, 3, or 4, characterized in that: In step 4, after the true leaves have grown, the growing point is removed. If the cotyledons can be retained while the growing point is completely removed, the cotyledons are retained. If the cotyledons cannot be retained after the growing point is completely removed, the cotyledons are removed together with the growing point.

6. The method for obtaining transgenic or gene-edited plants as described in claim 1, characterized in that: In step 3, multiple wounds are made on the stem of the plant using a blade, with the depth of the wounds not exceeding half the diameter of the stem. Cotton soaked in activated Agrobacterium bacterial solution is wrapped around the wounds for a certain period of time to infect the plant at room temperature and humidity of 65-80%. In step 4, the cotton is removed and the plant is cultured under light at room temperature. In step 5, the plant is cultured under light again at room temperature.

7. The method for obtaining transgenic or gene-edited plants as described in claim 1, characterized in that: The Agrobacterium is Agrobacterium rhizogenes or Agrobacterium tumefaciens.

8. The method for obtaining transgenic or gene-edited plants as described in claim 7, characterized in that: The Agrobacterium rhizogenes is K599, AR.1193, AR.A4, AR.Qual, C58C1 or MSU440; the Agrobacterium tumefaciens is GV3101, GV2260, EHA105, AGL1, LBA4404 or EHA101.

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