Bacillus-mediated genetic transformation method of red mustard

Abstract

The application discloses a kind of Agrobacterium-mediated genetic transformation methods of red mustard cabbage, belong to the field of plant genetic engineering application.The method includes the steps of sterile seedling obtaining, explant pre-culture, infection, recovery culture, screening, rooting culture and seedling raising and transplanting;The application establishes a kind of light regulation under Agrobacterium-mediated genetic transformation system of red mustard cabbage by studying the influence of different light quality and light supply mode on the plant type of red mustard cabbage sterile seedling, different plant tissue culture coagulant formula, different light intensity irradiation recovery medium on explant wound browning and medium contamination, adventitious bud regeneration rate and transformation efficiency, simultaneously, the method is simple and efficient, high repeatability, short time required for transformation, and the transformation result is stable, and the positive rate of genetic transformation plant is about 6%, significantly better than existing mustard in vitro regeneration system and genetic transformation method, and it has important significance for Brassica plant gene function research and molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering applications, and in particular to an Agrobacterium-mediated genetic transformation method for red mustard greens. Background Technology

[0002] Chinese kale originated in South China and is the largest cultivated and produced specialty vegetable in the region. Its tender flower stalks and young leaves are edible, possessing a crisp texture, sweet flavor, and high nutritional value and health benefits, making it highly popular among consumers in South China.

[0003] Since the first transgenic Brassica plants were obtained using Agrobacterium-mediated transformation, tissue culture and high-frequency regeneration systems for Brassica crops have been gradually established. However, transgenic research based on these systems has mainly focused on the genetic transformation of crops such as Chinese cabbage, rapeseed, Chinese cabbage, and mustard greens. There are few reports of successful genetic transformation of Chinese kale, which not only lacks a systematic genetic transformation system but also neglects the influence of light environment on Chinese kale tissue culture. Furthermore, it suffers from problems such as difficult regeneration, low transformation efficiency, and poor experimental reproducibility. Therefore, there is an urgent need to establish a systematic, rapid, and efficient genetic transformation technology system for Chinese kale with suitable light environment. Summary of the Invention

[0004] The purpose of this invention is to provide an Agrobacterium-mediated genetic transformation method for Brassica oleracea to address the problems existing in the prior art. This invention utilizes different light qualities and light supply modes to regulate the plant type of aseptic Brassica oleracea seedlings, different plant tissue culture coagulant formulations, and the effects of different light intensities on the recovery medium irradiation of explant wound browning and culture medium contamination, adventitious bud regeneration rate, and transformation efficiency. This establishes a light-regulated Agrobacterium-mediated genetic transformation system for Brassica oleracea, laying the foundation for gene function research and molecular breeding in Brassica plants.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an Agrobacterium-mediated genetic transformation method for red mustard greens, comprising the following steps:

[0007] (1) Obtaining sterile seedlings: After disinfecting the seeds of red mustard greens, they were inoculated into the sowing medium, germinated, and then treated in the dark for 3 days and in white light for 2 days to obtain sterile seedlings.

[0008] (2) Pre-culture of explants: The cotyledons and hypocotyls of the sterile seedlings were cut off as explants and inoculated into a pre-culture medium in the dark.

[0009] (3) Infection of explants: Agrobacterium-mediated infection was used to infect pre-cultured explants;

[0010] (4) Co-culture of explants: Inoculated explants were inoculated into a co-culture medium for co-culture;

[0011] (5) Explant recovery culture: The co-cultured explants were transferred to recovery culture medium and cultured under a light intensity of 40±5 μmol·m⁻². -2 ·s -1 Cultivate in an LED white light environment for 20–25 days;

[0012] (6) Screening and rooting culture of explants: adventitious buds grown from the regenerated explants are cut off and transferred to the screening medium. After screening and culture for 20-30 days, intact green buds are peeled off and transferred to the screening and rooting medium. After culture for 20-30 days, resistant buds are obtained. The resistant buds are transferred to the rooting medium and cultured under light for 20-30 days to obtain tissue culture seedlings.

[0013] (7) Hardening and transplanting of tissue culture seedlings: The obtained tissue culture seedlings were hardened off by opening the cover, and then planted in the seedling substrate. They were then managed in a routine manner, and after positive testing, the genetically transformed positive plants of red mustard greens were obtained.

[0014] In steps (1) to (2) and steps (4) to (6), the solidifying agent used in the culture medium is 4 g·L⁻¹. -1 agar powder and 2.5 g·L -1 Plant-based gels.

[0015] Further, in step (1), the white light treatment specifically refers to a light intensity of 80 ± 5 μmol·m. -2 ·s -1 The LED white light has a red light to blue light intensity ratio of 2.2 and a red light to far-red light intensity ratio of 3.3; the sowing medium has a composition of 2.37 g·L⁻¹. -1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant-based gel, pH adjusted to 5.86.

[0016] Further, in step (2), the conditions for the light-protected pre-culture are: light-protected inverted pre-culture at 22°C for 2 days; the composition of the pre-culture medium and the co-culture medium in step (4) are both 4.74 g·L⁻¹. -1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant-based gel, adjusted to pH 5.86, sterilized, then 0.025 mg / L was added. -1 Indole-3-acetic acid, 2 mg·L -1 Trans-zeatin, 7 mg·L -1 AgNO3.

[0017] Further, in step (3), the specific steps of the infection are as follows: the expression vector containing the exogenous gene is transferred into Agrobacterium, the bacterial cells are resuspended in the infection medium, and cultured with shaking to obtain the Agrobacterium infection solution. The explants pre-cultured in step (2) are transferred to the Agrobacterium infection solution and infected for 10 min. The composition of the infection medium is: 4.74 g·L⁻¹. -1 MS medium, 30 g·L -1 Sucrose, 1.0 mg·L -1 Acetyleugenol, adjust pH to 5.85.

[0018] Further, in step (5), the explants co-cultured for 2 days are transferred to a recovery medium, the composition of which is 4.74 g·L⁻¹. -1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant-based gel, adjusted to pH 5.88, sterilized, then 0.025 mg / L was added. -1 Indole-3-acetic acid, 2 mg·L -1 Trans-zeatin, 7 mg·L -1 AgNO3, 300 mg·L -1 Termethin.

[0019] Furthermore, in step (6), the conditions for screening and rooting culture are as follows: a light intensity of 80±5 μmol·m -2 ·s -1 The cells were cultured under LED white light for 20–30 days, wherein the ratio of red light to blue light intensity in the LED white light was 2.2, and the ratio of red light to far-red light intensity was 3.3.

[0020] The screening medium composition was: 4.74 g·L⁻¹ -1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant gel, 500 μL·L -1 Plant tissue culture antibacterial agent, adjusted to pH 5.88, sterilized, then added at 0.05 mg / L. -1 Indole-3-acetic acid, 2 mg·L -1 Trans-zeatin, 7 mg·L -1 AgNO3, 300 mg·L -1 Termetidine, 4 mg / L -1 Glufosinate;

[0021] The composition of the screening rooting medium was: 4.74 g·L⁻¹-1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant gel, 500 μL·L -1 Plant tissue culture antibacterial agent, adjusted to pH 5.88, sterilized, then added at 0.3 mg / L. -1 Indole-3-acetic acid, 7 mg·L -1 AgNO3, 300 mg·L -1 mg·L -1 Termetidine, 6 mg / L -1 Glufosinate;

[0022] The rooting medium composition was: 4.74 g·L⁻¹ -1 MS medium, 30 g·L -1 Sucrose, 4 g·L -1 Agar powder, 2.5 g / L -1 Plant gel, 500 μL·L -1 Plant tissue culture antibacterial agent, adjusted to pH 5.88, sterilized, then added at 0.45 mg / L. -1 Indole-3-acetic acid, 7 mg·L -1 AgNO3, 300 mg·L -1 Termethin.

[0023] Further, in step (7), the conventionally managed light environment is a light intensity of 160 ± 5 μmol·m⁻². -2 ·s -1 The LED red and white light has a light intensity ratio of 1.7 for red light to blue light and a light intensity ratio of 21.3 for red light to far-red light.

[0024] The present invention discloses the following technical effects:

[0025] (1) This invention establishes a light-regulated Agrobacterium-mediated genetic transformation system for Brassica rapa by studying the effects of different light qualities and light supply modes on the plant type of sterile seedlings of Brassica rapa, different plant tissue culture coagulant formulations, different light intensities on the recovery medium for explant wound browning and medium contamination, adventitious bud regeneration rate and transformation efficiency. This lays the foundation for gene function research and molecular breeding of Brassica rapa plants.

[0026] (2) The genetic transformation system of *Brassica juncea* constructed in this invention combines PCR detection and GUS staining for dual detection to obtain transgenic plants. This invention focuses on the light environment during plant tissue culture, selecting agar powder and plant gel as medium solidifying agents. After inoculation, the plants are protected from light for 3 days to promote germination, followed by 3 days of dark treatment, and then 2 days of normal light under suitable light quality. This promotes the elongation of hypocotyls and the synthesis of photosynthetic pigments in cotyledons of sterile seedlings, significantly reduces fungal contamination of explants, and greatly improves the utilization rate of sterile seedlings. The recovery culture stage has a concentration of 40±5 μmol·m⁻¹. -2 ·s -1 The use of LED white light (with a red to blue light intensity ratio of 2.2 and a red to far-red light intensity ratio of 3.3) significantly improved the browning of explants and the contamination of the culture medium in *Brassica oleracea*, thereby increasing the regeneration rate and transformation efficiency of the explants. Furthermore, the entire process is simple, efficient, highly reproducible, and requires a short transformation time. The transformation results are stable, with a positive rate of approximately 6% for genetically transformed plants, significantly superior to existing in vitro regeneration systems and genetic transformation methods for *Brassica oleracea*. This has significant implications for gene function research and molecular breeding in *Brassica* species. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pCAMBIA3301-35S-MYC3 used in this invention;

[0029] Figure 2 This is a spectral diagram of the light environment during the genetic transformation process of red mustard greens;

[0030] Figure 3 This is a genetic transformation diagram of the transgenic red kale plants obtained by this invention; wherein, 1: sowing, 2: sterile seedlings after 3 days of dark treatment, 3: sterile seedlings after 2 days of light treatment, 4-5: pre-culture, 6-7: co-culture, 8-9: recovery culture, 10: selection culture, 11: selection and rooting culture, 12: rooting culture, 13: hardening off the seedlings, 14: bagging and acclimatization, 15-16: routine management of transgenic plants;

[0031] Figure 4 This is a graph showing the PCR detection results of the target gene in the transformed plants; where M: Marker, the left side of the graph shows the size of the marker band, 1: water, 2: wild type, 3: plasmid, and 4-14 are different transformed lines;

[0032] Figure 5 This is a comparison diagram of the sequencing results of the target gene in the transformed plants;

[0033] Figure 6 This is a diagram of the GUS staining results of the transformed plants, where 1: wild type, 2: transgenic red mustard greens. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] 1. Material Preparation

[0041] 1.1 Plant materials

[0042] The experiment was conducted in the Vegetable Cultivation and Physiology Laboratory, Molecular Laboratory, and Tissue Culture Room of the College of Horticulture, South China Agricultural University. The tested Chinese kale variety was the red-stemmed Chinese kale "Red Foot" preserved in the Vegetable Cultivation and Physiology Laboratory of the College of Horticulture, South China Agricultural University.

[0043] 1.2 Construction of Red Mustard Green Gene Expression Vector

[0044] (1) Primer design

[0045] Primers were designed using the MYC3 sequence (accession number: XM_013742789.1) and PrimerPremier 5 software (Table 1).

[0046] Table 1 Primer sequence list

[0047]

[0048]

[0049] (2) Gene cloning

[0050] Young leaves of *Gnaphalium affine* were ground into powder using liquid nitrogen. RNA was extracted using the SteadyPure Plant RNA Extraction Kit (Aikerui, Hunan), and cDNA was synthesized using the company's Evo M-MLV Reverse Transcription Premix Kit. Using the *Gnaphalium affine* cDNA as a template, PCR amplification was performed using overexpression vector primers. After purification, the product was cloned with the linearized vector pCAMBIA3301 (Dingguo) using the Hieff one-step cloning kit. Recombinant ligation was performed using the Plus One Step Cloning Kit (Yisheng, Shanghai). The vector was linearized by double restriction enzyme digestion with Xba I and Sma I. The ligated recombinant plasmid pCAMBIA3301-35S-MYC3 was transformed into Escherichia coli DH5α (Qingke, Beijing) using standard methods. Positive single colonies were obtained by culturing. After plasmid extraction, it was introduced into Agrobacterium GV3101 (Weidi, Shanghai) using the freeze-thaw method. Positive single colonies were obtained by culturing for subsequent infection. Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pCAMBIA3301-35S-MYC3 used in this invention.

[0051] 1.3 Culture medium: The culture medium used in the genetic transformation process is shown in Table 2.

[0052] Table 2 Culture medium formulation

[0053]

[0054]

[0055] 2. Experimental Methods

[0056] 2.1 Genetic transformation steps of red mustard greens

[0057] (1) Obtaining sterile vaccines

[0058] Select plump red mustard seeds into centrifuge tubes and perform surface sterilization in a clean bench according to the following steps: rinse 5 times with sterile water, soak in 75% (volume fraction) ethanol for 2 min, rinse 2 times with sterile water, soak in 5% (mass fraction) NaClO solution for 10 min, rinse 5 times with sterile water, filter with a sterile sieve, and blot dry with sterile filter paper; inoculate 20 seeds evenly into the sowing medium per bottle; place the inoculated sowing medium in a tissue culture room in the dark for 3 days to germinate, and then subject to different light treatments until the total PPFD is 80±5 μmol·m -2 ·s -1 (Table 3). Tissue culture room environmental conditions: 22±0.5℃, photoperiod 16h·d -1 ;

[0059] Table 3 Different light supply modes for sterile seedlings

[0060] Light processing W White Light 5D FR Far-infrared 5D UV 5D UV light D Dark Processing 3D DW Dark processing 3D, white light 2D WD White light 3D, dark processing 2D

[0061] Note: W represents LED white light, where the ratio of red light intensity to blue light intensity is 2.2, and the ratio of red light intensity to far-red light intensity is 3.3.

[0062] (2) Pre-culture of explants

[0063] In a clean bench, select aseptic seedlings of *Brassica rapa* with flattened cotyledons. Cut cotyledons with petioles (0.2–0.3 cm in length) and hypocotyls (0.5–0.9 cm in length; if the hypocotyl length of the aseptic seedling is suitable, multiple segments can be cut) as explants. Insert the wounds of the petioled cotyledons into pre-culture medium, and lay the hypocotyls flat on the surface of the pre-culture medium. Place the pre-culture medium in a 22°C tissue culture room in the dark and inverted for 2 days.

[0064] (3) Infection of explants

[0065] Select a single positive colony from step 1.2, shake it overnight, then transfer it to an Erlenmeyer flask and agitate for 5 hours. Centrifuge at 5000 rpm for 10 minutes and discard the supernatant. Resuspend the bacterial cells in infection medium and adjust the optical density (OD) value. 600 The concentration of the culture medium was 0.4–0.5, and the culture was carried out at 28°C with shaking at 200 rpm for 60 min to obtain activated Agrobacterium infection solution. The explants that had been cultured for 2 days were transferred to the Agrobacterium infection solution in a clean bench and incubated with shaking for 10 min.

[0066] (4) Co-culture of explants and bacterial culture

[0067] Filter the bacterial suspension using a sterile sieve. Place the infected explants on sterile filter paper to blot away excess bacterial suspension. Take another piece of appropriately sized sterile filter paper, lay it flat on the co-culture medium, and take 2.0 mL of the medium containing 0.05 mg·L⁻¹ of bacterial suspension. -1 Indole-3-acetic acid, 2 mg·L -1 anti-zeatin and 7 mg·L -1 Moisten the filter paper with AgNO3-infected medium; lay the explants flat on the co-culture medium, ensuring that the cut ends of the explants contact the filter paper, and co-culture in the dark for 2 days.

[0068] (5) Explant recovery culture

[0069] Explants co-cultured for 2 days were transferred to recovery medium. The wounds on the stalked cotyledons were inserted into the recovery medium, and the hypocotyls were placed in grooves drawn with sterile forceps, ensuring that the cut ends of the hypocotyls were in contact with the recovery medium. The medium was then placed in a culture medium with total PPFD of 80±5 μmol·m³. -2 ·s -1 The tissue culture was inverted and cultured under LED white light for 20–25 days. After transferring adventitious buds, the culture medium was replaced once to restore the tissue. The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The culture room environment was 22 ± 0.5℃, and the photoperiod was 16 h·d. -1 ;

[0070] (6) Screening and culture of explants

[0071] ① Adventitious buds were excised from explants in the recovery culture and transferred to selection medium, with a total PPFD of 80±5 μmol·m -2 ·s -1 Cultivate under LED white light for 20–30 days;

[0072] ② Remove a single, intact green shoot from a normally growing adventitious bud in the selection medium, transfer it to the selection and rooting medium, and place it in a medium with a total PPFD of 80±5 μmol·m -2 ·s -1 After culturing under LED white light for 20–30 days, green shoots with normal growth were obtained, namely resistant shoots;

[0073] The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The tissue culture room environment was 22±0.5℃, and the photoperiod was 16h·d. -1 ;

[0074] (7) Rooting culture of resistant buds

[0075] Resistant shoots were selected from the rooting medium, aged wounds were removed, and single, intact resistant shoots were separated and transferred to the rooting medium. The medium was then placed in an environment with a total PPFD concentration of 80 ± 5 μmol·m⁻¹.-2 ·s -1 Tissue culture seedlings were obtained by culturing under LED white light for 20–30 days. The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The tissue culture environment was maintained at 22 ± 0.5℃ with a photoperiod of 16 h·d. -1 ;

[0076] (8) Hardening off and transplanting of tissue culture seedlings

[0077] When the tissue culture seedlings have a large number of adventitious roots and are growing vigorously, open the lid, put on disposable plastic film gloves, and harden the seedlings for 7 days. After that, wash off all the culture medium from the roots and transplant them into a seedling substrate of peat moss and vermiculite in a 3:1 mass ratio. Cover them with plastic bags and let them recover for 7 days. Place them in a container with a total PPFD of 160±5 μmol·m -2 ·s -1 Routine management was performed under LED red and white light; the ratio of red light intensity to blue light intensity in the LED red and white light was 1.7, and the ratio of red light intensity to far-red light intensity was 21.3. The environmental conditions in the tissue culture room and cultivation room were 22±0.5℃, and the photoperiod was 16h·d. -1 ;

[0078] (9) Positive detection of regenerated plants

[0079] a) PCR detection

[0080] After the tissue culture seedlings grew into complete plants, leaves from the regenerated plants were taken, and DNA was extracted using the CTAB method. PCR amplification was then performed using pCAMBIA3301-35S-MYC3 plasmid as a positive control, wild-type red mustard DNA as a negative control, and sterile water as a blank control. After the PCR reaction, the PCR products were detected by 1% (mass fraction) agarose gel electrophoresis. PCR products with bands consistent with the positive plasmid were sequenced, and the sequencing results were compared with reference sequences using the BLAST network service tool in the NCBI database.

[0081] b) GUS staining

[0082] Based on the PCR results, new leaves from regenerated plants with consistent sequence alignment were placed in 2.0 mL centrifuge tubes. GUS staining working solution was added until the leaves were completely submerged. The tubes were then vacuum-sealed for 20 min and reacted at 37°C in the dark for 8 h. The leaves were washed three times with 95% (v / v) ethanol solution and stored in 95% (v / v) ethanol solution. Visually, the negative control appeared white, the positive control appeared blue, and the leaves of positive plants were blue or had a blue background on a white background.

[0083] Table 4 PCR reaction system

[0084]

[0085] Table 5 PCR reaction procedure

[0086]

[0087]

[0088] 3 Results and Analysis

[0089] 3.1 Effects of sterile seedling plant type on explant regeneration rate and transformation rate

[0090] The cultivation of sterile seedlings is an indispensable step in the genetic transformation of *Gynostemma pentaphyllum*. Different light supply modes can effectively regulate the plant type of sterile seedlings, facilitating the cultivation of robust seedlings and significantly impacting the efficiency of genetic transformation. After germination, different light qualities resulted in significant differences in the hypocotyls and cotyledons of sterile seedlings. The hypocotyls of sterile seedlings treated with both light and dark elongation were significantly elongated, while the cotyledons with petioles showed no significant difference from those under continuous light. Among these, the explants from sterile seedlings treated with 3 days of darkness followed by 2 days of light not only had petioles but also hypocotyls, resulting in the highest number of explants and greatly improving the utilization rate of sterile seedlings. Simultaneously, the regeneration rate of explants was significantly higher than other treatments, and the browning rate of explants after 20 days of recovery culture was the lowest. Two transgenic *Gynostemma pentaphyllum* plants were obtained in the later stages (Table 6). Therefore, after 3 days of seed germination followed by 3 days of darkness treatment, and then at 80±5 μmol·m⁻¹, a further treatment of the hypocotyls and cotyledons was recommended. -2 ·s -1 The plants were cultured under LED white light for 2 days, and this lighting mode was the most suitable for the genetic transformation of *Brassica oleracea*. The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3.

[0091] Table 6. Effects of different light treatments on regeneration rate and conversion rate

[0092]

[0093] Note: Browning rate = number of browned explants / total number of explants × 100%; Regeneration rate = number of differentiated adventitious buds / total number of explants × 100%.

[0094] Example 2

[0095] Screening coagulant composition

[0096] 1. Material Preparation

[0097] 1.1 Plant materials: Same as in Example 1;

[0098] 1.2 Culture medium

[0099] The coagulant composition is as follows: ① 8g agar powder; ② 5g plant gel (CP8581Z, Cooler Master, Beijing); ③ 4g agar powder and 2.5g plant gel; other components of the culture medium are the same as in Example 1.

[0100] 1.3 Agrobacterium: Same as in Example 1;

[0101] 1.4 Detection primers: Same as in Example 1;

[0102] 2. Experimental method: Same as in Example 1;

[0103] 3 Results and Analysis

[0104] Solidifying agents are an indispensable component of solid culture media, and the hardness and transparency of the media are affected by solidifying agents. Agar powder alone reduces the transparency of the culture medium, making it difficult to remove fungal contamination in time when sowing, resulting in low seed utilization, severe contamination of sterile seedlings, and difficulty in suppressing fungal contamination of explants in the later stages. Fungal contamination occurs at all stages, leading to a significant decrease in explant regeneration rate. Furthermore, the hardness of the solidifying agent makes it difficult to clean tissue culture seedlings even under suitable pH conditions, causing severe root damage and affecting the survival rate of regenerated plants after transplanting. When the solidifying agent is plant gel and the pH is 5.88±0.05, the culture medium has high transparency, high rooting rate of tissue culture seedlings, and simple root cleaning, but the solidification effect is poor, the culture medium becomes waterlogged, exacerbates vitrification of explant callus tissue, and has a low regeneration rate. When 4g of agar powder is used in combination with 2.5g of plant gel, under the condition that the pH of the culture medium remains unchanged, the solidification effect, culture medium hardness, and transparency are significantly better than those of a single plant solidifying agent. Fungal contamination of the culture medium is significantly reduced, and the regeneration and transformation rates are significantly improved. Two transgenic plants were obtained in the later stages (Table 7). Therefore, the combination of 4g agar powder and 2.5g plant gel is the optimal solidifying agent for solid culture medium during the genetic transformation of red mustard greens.

[0105] Table 7. Effects of different coagulants on regeneration rate and conversion rate

[0106]

[0107] Note: Contamination rate = Number of explants contaminated with fungi and bacteria / Total number of explants × 100%.

[0108] Example 3

[0109] Restore light intensity screening

[0110] 1. Material Preparation

[0111] 1.1 Plant materials: Same as in Example 1;

[0112] 1.2 Culture medium: The culture medium formulations for the genetic transformation process are shown in Table 8;

[0113] 1.3 Agrobacterium: Same as in Example 1;

[0114] 1.4 Detection primers: Same as in Example 1.

[0115] Table 8 Culture medium formulation

[0116]

[0117] 2. Experimental Methods

[0118] 2.1 Genetic transformation steps of red mustard greens

[0119] The seed culture medium, which had been inoculated and germinated for 3 days, was dark-treated for 3 days, and then placed in an environment with 80±5 μmol·m⁻² ... -2 ·s -1 The plants were grown under LED white light for 2 days. Sterile seedlings with flattened cotyledons and hypocotyls were used as explants and pre-cultured in the dark for 2 days. They were then infected with activated Agrobacterium infection solution with shaking for 10 min, co-cultured in the dark for 2 days, and then transferred to recovery medium. The wounds of the cotyledons were inserted into the recovery medium, and the hypocotyls were placed in grooves drawn with sterile forceps, ensuring that the cut ends of the hypocotyls were in contact with the recovery medium. Equal numbers of cotyledons and hypocotyls were placed in PPFD at concentrations of 20, 40, and 80 μmol·m³, respectively. -2 ·s -1 The culture medium was inverted and cultured under LED white light for 20-25 days. After the adventitious buds were transferred, the medium was replaced once to restore the growth. The remaining steps were the same as in Example 1.

[0120] The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The tissue culture room environment was 22±0.5℃, and the photoperiod was 16h·d. -1 ;

[0121] 3 Results and Analysis

[0122] Light intensity is a crucial factor affecting the differentiation, regeneration rate, and transformation rate of adventitious buds in explants during the recovery culture stage. Insufficient light intensity leads to the proliferation of Agrobacterium in the culture medium, resulting in severe explant contamination and making it difficult to inhibit Agrobacterium in the later stages, thus hindering explant regeneration. Excessive light intensity, on the other hand, causes severe browning of explant wounds after 20 days of recovery culture, inhibiting adventitious bud differentiation and significantly reducing regeneration and transformation rates; this treatment only yielded two transgenic red mustard greens. The total PPFD was 40±5 μmol·m⁻¹. -2 ·s -1 Under LED white light, explant browning and Agrobacterium growth were significantly inhibited, resulting in the highest explant regeneration and transformation rates, yielding a total of 6 transgenic red mustard greens. Therefore, the total PPFD was 40±5 μmol·m⁻¹. -2 ·s -1 The LED white light is the optimal light intensity for the recovery and cultivation stage. The ratio of red light intensity to blue light intensity in the LED white light is 2.2, and the ratio of red light intensity to far-red light intensity is 3.3.

[0123] Table 9. Effects of light intensity on regeneration rate and conversion rate

[0124]

[0125] Note: Contamination rate = (Number of browned explants + Number of straw-contaminated explants) / Total number of explants × 100%.

[0126] Example 4

[0127] According to Examples 1, 2, and 3, a genetic transformation system for red mustard greens was established: Seed culture medium inoculated and germinated for 3 days was grown in the dark for 3 days, and then placed in an 80±5 μmol·m⁻² ... -2 ·s -1 Cultured under LED white light for 2 days, using aseptic seedlings with flattened cotyledons, petioles, and hypocotyls as explants, pre-cultured in the dark and upside down for 2 days, then resuspended in infection medium and OD 600 The activated Agrobacterium infection solution was cultured at 0.4 to 0.5 μmol·m⁻¹ for 60 min at 28°C with shaking at 200 rpm, followed by shaking infection for 10 min. The mixture was then co-cultured in the dark, upright, for 2 days at 40 ± 5 μmol·m⁻¹. -2 ·s -1 Under LED white light, the seedlings were cultured and regenerated. After adventitious buds emerged, they were screened and cultured for 20 days. Single, intact green shoots were peeled from normally growing adventitious buds and transferred to a selection and rooting medium. The resulting resistant shoots were then transferred to a rooting medium and cultured. When the number of adventitious roots was large and robust, the seedlings were opened for hardening off. After transplanting and recovery, the seedlings were cultured under a total PPFD of 160±5 μmol·m⁻¹. -2 ·s -1 The LED red and white light was managed under standard procedures, with other operational details the same as in Example 1. The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The ratio of red to blue light intensity in the LED red and white light was 1.7, and the ratio of red to far-red light intensity was 21.3. The environmental conditions in the tissue culture room and cultivation room were 22±0.5℃, with a photoperiod of 16 h·d. -1 .

[0128] Figure 2 This is a spectral diagram of the light environment during the genetic transformation process of red mustard greens. Figure 3 This is a genetic transformation diagram of the transgenic red kale plants obtained by this invention; wherein, 1: sowing, 2: sterile seedlings after 3 days of dark treatment, 3: sterile seedlings after 2 days of light treatment, 4-5: pre-culture, 6-7: co-culture, 8-9: recovery culture, 10: selection culture, 11: selection and rooting culture, 12: rooting culture, 13: hardening off the seedlings, 14: bagging and acclimatization, 15-16: routine management of transgenic plants.

[0129] PCR detection was performed using pCAMBIA3301-35S-MYC3 plasmid as a positive control, wild-type red mustard greens as a negative control, and sterile water as a blank control. Figure 4 It can be seen that 11 strains amplified the target band, while the wild-type and sterile water strains did not amplify any bands. The PCR products were sequenced and analyzed, and the nucleotide sequences were searched and compared using the BLAST web service tool in the NCBI database, such as... Figure 5 As shown, 11 samples had a similarity of 99% to the reference sequence.

[0130] Using wild-type red mustard greens as a negative control, new leaves from 11 regenerated plants with highly similar sequences were collected in 2.0 mL centrifuge tubes, stained with GUS staining solution, and destained with 95% (v / v) ethanol solution. The staining of the leaves was observed; the negative control leaves were white, while the leaves of the 6 plants were blue or had blue on a white background, indicating that the 6 regenerated plants had been successfully transformed. Figure 6 This is a diagram of the GUS staining results of the transformed plants, where 1: wild type, 2: transgenic red mustard greens.

[0131] In summary, this invention establishes a genetic transformation system for red mustard greens: using 4g agar powder and 2.5g plant gel as solid culture medium solidifying agents, and selecting a medium that has been pre-germinated for 3 days, followed by dark treatment for 3 days, at 80±5μmol·m -2 ·s -1 Sterile seedlings with stalked cotyledons and hypocotyls cultured under LED white light for 2 days were used as explants. These seedlings were pre-cultured upside down in the dark for 2 days, and then resuspended using OD... 600 The activated Agrobacterium infection solution was cultured at 0.4 to 0.5 μmol / L on a shaker at 200 rpm for 40–60 min at 28 °C for 10 min with shaking. The culture was then co-cultured in the dark for 2 days under an illumination of 40 ± 5 μmol / L. -2 ·s -1 Under LED white light, the seedlings were cultured and regenerated. After adventitious buds emerged, they were selected and cultured for 20–30 days. Single, intact green buds were peeled off and transferred to a selection and rooting medium for 20 days. Resistant buds were then transferred to a rooting medium and cultured until a large number of robust adventitious roots were obtained. The seedlings were then hardened off by opening the bottle and, after transplanting and recovery, were placed in a 160±5 μmol·m⁻² ... -2 ·s -1 Routine management was performed under LED red and white light. Six transgenic red mustard greens were obtained from 100 explants, with a conversion rate of 6%. The ratio of red to blue light intensity in the LED white light was 2.2, and the ratio of red to far-red light intensity was 3.3. The ratio of red to blue light intensity in the LED red and white light was 1.7, and the ratio of red to far-red light intensity was 21.3. The environmental conditions in the tissue culture and cultivation rooms were 22±0.5℃, with a photoperiod of 16 h·d. -1 .

[0132] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An Agrobacterium-mediated genetic transformation method for red mustard greens, characterized in that, The steps are as follows: (1) Obtaining sterile seedlings: After disinfecting the seeds of *Caulis Rubus esculentus*, they were inoculated into the sowing medium, germinated, and then treated in the dark for 3 days. Sterile seedlings were obtained after 2 days of white light treatment; specifically, the white light treatment involved a light intensity of 80 ± 5 μmol·m⁻². -2 ·s -1 The LED white light has a light intensity ratio of 2.2 for red light to blue light and a light intensity ratio of 3.3 for red light to far-red light. The sowing medium consists of 2.37 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, and pH adjusted to 5.

86. (2) Pre-culture of explants: The cotyledons and hypocotyls of the sterile seedlings were cut off as explants and inoculated into a pre-culture medium in the dark. The conditions for the pre-culture in the dark were: inverted pre-culture at 22°C for 2 days in the dark. (3) Infection of explants: Agrobacterium-mediated infection was used to infect pre-cultured explants; (4) Co-culture of explants: Inoculated explants were inoculated into a co-culture medium for co-culture; (5) Explant recovery culture: The co-cultured explants were transferred to recovery culture medium and cultured under a light intensity of 40±5 μmol·m⁻². -2 ·s -1 The culture medium was incubated in an LED white light environment for 20–25 days. The composition of the recovery medium was: 4.74 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, pH adjusted to 5.88, sterilized and then 0.025 mg / L indole-3-acetic acid, 2 mg / L trans-zeatin, 7 mg / L AgNO3, and 300 mg / L termethin were added. (6) Screening and rooting culture of explants: Adventitious shoots growing from the regenerated explants were excised and transferred to a screening medium. After screening and culturing for 20–30 days, intact green shoots were peeled off and transferred to a screening and rooting medium. These shoots were then cultured for 20–30 days to obtain resistant shoots. The resistant shoots were then transferred to a rooting medium and cultured under light for 20–30 days to obtain tissue culture seedlings. The conditions for both screening and rooting culture were: a light intensity of 80 ± 5 μmol·m⁻¹. -2 ·s -1 The cells were cultured under LED white light for 20–30 days, wherein the ratio of red light to blue light intensity in the LED white light was 2.2, and the ratio of red light to far-red light intensity was 3.

3. The screening medium consisted of: 4.74 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, 500 μL / L plant tissue culture antibacterial agent, pH adjusted to 5.88, and after sterilization, 0.05 mg / L indole-3-acetic acid, 2 mg / L trans-zeatin, 7 mg / L AgNO3, 300 mg / L termethin, and 4 mg / L glufosinate were added. The screening rooting medium consisted of: 4.74 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, 500 μL / L plant tissue culture antibacterial agent, pH adjusted to 5.88, and after sterilization, 0.3 mg / L indole-3-acetic acid, 7 mg / L AgNO3, 300 mg / L termethin, and 6 mg / L glufosinate were added. The rooting medium consisted of: 4.74 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, 500 μL / L plant tissue culture antibacterial agent, pH adjusted to 5.88, and after sterilization, 0.45 mg / L indole-3-acetic acid, 7 mg / L AgNO3, and 300 mg / L termethin were added. (7) Hardening and transplanting of tissue culture seedlings: The obtained tissue culture seedlings were hardened off by opening the cover, and then planted in the seedling substrate. They were then managed in a routine manner, and after positive testing, the genetically transformed positive plants of red mustard greens were obtained. In steps (1) to (2) and steps (4) to (6), the solidifying agents used in the culture media are 4 g / L agar powder and 2.5 g / L plant gel. The composition of the pre-culture medium and the co-culture medium in step (4) is: 4.74 g / L MS medium, 30 g / L sucrose, 4 g / L agar powder, 2.5 g / L plant gel, pH adjusted to 5.86, sterilized and then 0.025 mg / L indole-3-acetic acid, 2 mg / L trans-zeatin, and 7 mg / L AgNO3 are added.

2. The method for Agrobacterium-mediated genetic transformation of red mustard greens according to claim 1, characterized in that, In step In step (3), the specific steps of the infection are as follows: the expression vector containing the exogenous gene is transferred into Agrobacterium, the bacterial cells are resuspended in the infection medium, and cultured by shaking to obtain the Agrobacterium infection solution. The explants pre-cultured in step (2) are transferred to the Agrobacterium infection solution and infected for 10 min. The composition of the infection medium is: 4.74 g / L MS medium, 30 g / L sucrose, 1.0 mg / L acetylsuccinone, and pH adjusted to 5.

85.

3. The method for Agrobacterium-mediated genetic transformation of red mustard greens according to claim 1, characterized in that, In step In step (5), the explants co-cultured for 2 days were transferred to the recovery medium.

4. The method for Agrobacterium-mediated genetic transformation of red mustard greens according to claim 1, characterized in that, In step (7), the conventionally managed light environment is a light intensity of 160 ± 5 μmol·m⁻². -2 ·s -1 The LED red and white light has a light intensity ratio of 1.7 for red light to blue light and a light intensity ratio of 21.3 for red light to far-red light.

Images