Construction method of broccoli regenerated plant based on visual marker

Abstract

The invention belongs to the technical field of bioengineering, and relates to a construction method of a broccoli regenerated plant based on a visual marker, and the method comprises the following steps: transfecting agrobacterium tumefaciens containing a gene editing vector to a broccoli explant, and culturing; transferring the transfected broccoli explant to a callus induction culture medium, culturing, cutting off the callus, transferring to a bud induction culture medium, and culturing until buds are regenerated; and transferring the regenerated buds to a bud elongation culture medium, culturing until the buds are elongated, and then transferring to a rooting induction culture medium to obtain regenerated plants. Through agrobacterium tumefaciens-mediated genetic transformation, rapid identification of broccoli transformed tissues, efficient regeneration of calluses and stable acquisition of gene editing plants are realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene editing, and relates to a method for constructing Brassica oleracea var. italica regenerated plants based on a visual marker. BACKGROUND

[0002] Brassica oleracea var. italica is a high-value cruciferous vegetable rich in vitamins, minerals and bioactive compounds. In Brassica oleracea var. italica flowers, traditional Agrobacterium-mediated transformation usually takes hypocotyls or cotyledon petioles as explants, but is often restricted by problems such as low transformation efficiency, high false positive rate and dependence on antibiotic screening. In contrast, Agrobacterium rhizogenes usually exhibits higher infection and transformation efficiency in many crops. At present, in Brassica oleracea var. italica research, hairy root systems are mainly used to induce leaf-derived hairy roots to study the production of metabolic products, or to construct hypocotyl-induced composite plants for root function analysis. However, for many crops including Brassica oleracea var. italica, the low efficiency and long time-consuming of regenerating complete plants from transgenic hairy roots are still the main bottlenecks that limit the wider application of this system in genetic improvement and genome editing.

[0003] Therefore, the present application aims to establish a Brassica oleracea var. italica regeneration method based on a visual marker. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application aims to provide a method for constructing Brassica oleracea var. italica regenerated plants based on a visual marker.

[0005] To achieve the object of the present application, the following technical solutions are used:

[0006] In a first aspect, the present application provides a method for constructing Brassica oleracea var. italica regenerated plants based on a visual marker, which comprises:

[0007] (1) Agrobacterium tumefaciens containing a gene editing vector is transfected into Brassica oleracea var. italica explants, and cultured;

[0008] (2) The explants obtained in step (1) are transferred to callus induction medium, cultured, the callus is cut off, and transferred to bud induction medium, and cultured until bud regeneration;

[0009] (3) The regenerated buds are transferred to bud elongation medium, cultured until the buds elongate, and then transferred to rooting induction medium, to obtain regenerated plants.

[0010] The application introduces a color marker system into broccoli, realizes exogenous gene introduction and plant regeneration through Agrobacterium tumefaciens-mediated genetic transformation, and produces visible pigment accumulation in the transformed tissue, so as to realize the identification of transformation events under the condition of antibiotic-free screening. By optimizing the hormone ratio and medium composition, callus is efficiently induced and sprouts are differentiated, and complete gene editing regeneration plants are obtained. Unlike the traditional hairy root system, the method described in the application does not need to go through the hairy root stage to directly induce the formation of callus, the whole process is simple and easy to operate, short in cycle and high in regeneration rate, and a gene editing regeneration system suitable for broccoli is established.

[0011] Preferably, the callus induction medium comprises 6-benzylaminopurine (6-BA) 1-5 mg / L, naphthalene acetic acid (NAA) 0.1-0.5 mg / L, silver nitrate 2-4 mg / L, and temik 200-300 mg / L in terms of concentration, and sucrose 2.5-3.5% and a basic medium in terms of mass percentage.

[0012] The concentration of 6-benzylaminopurine can be selected from 1 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 2 mg / L, 2.2 mg / L, 2.5 mg / L, 2.8 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, etc., the concentration of naphthalene acetic acid can be selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, etc., the concentration of silver nitrate can be selected from 2 mg / L, 3 mg / L, 4 mg / L, etc., the concentration of temik can be selected from 200 mg / L, 250 mg / L, 300 mg / L, etc., and the mass percentage of sucrose can be selected from 2.5%, 3%, 3.5%, etc. Other specific point values within the above numerical ranges can also be selected, which will not be described here.

[0013] Preferably, the bud induction medium comprises 6-benzylaminopurine 2-5 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, silver nitrate 2-4 mg / L, and temik 200-300 mg / L in terms of concentration, and sucrose 2.5-3.5% and a basic medium in terms of mass percentage.

[0014] The concentration of 6-benzylaminopurine can be selected from 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, etc., the concentration of naphthalene acetic acid can be selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, etc., the concentration of silver nitrate can be selected from 2 mg / L, 3 mg / L, 4 mg / L, etc., the concentration of temik can be selected from 200 mg / L, 250 mg / L, 300 mg / L, etc., and the mass percentage of sucrose can be selected from 2.5%, 3%, 3.5%, etc. Other specific point values within the above numerical ranges can be selected, and thus will not be repeated here.

[0015] Preferably, the bud elongation medium comprises 6-benzylaminopurine 0.1-0.3 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, temik 50-150 mg / L, and sucrose 2.5-3.5% by mass percentage, and a basic medium.

[0016] The concentration of 6-benzylaminopurine can be selected from 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, etc., the concentration of naphthalene acetic acid can be selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, etc., the concentration of temik can be selected from 50 mg / L, 100 mg / L, 150 mg / L, etc., and the mass percentage of sucrose can be selected from 2.5%, 3%, 3.5%, etc. Other specific point values within the above numerical ranges can be selected, and thus will not be repeated here.

[0017] Preferably, the bud elongation medium comprises 6-benzylaminopurine 0.1-0.3 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, temik 50-150 mg / L, and sucrose 2.5-3.5% by mass percentage, and a basic medium.

[0018] The concentration of 6-benzylaminopurine can be selected from 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, etc., the concentration of naphthalene acetic acid can be selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, etc., the concentration of temik can be selected from 50 mg / L, 100 mg / L, 150 mg / L, etc., and the mass percentage of sucrose can be selected from 2.5%, 3%, 3.5%, etc. Other specific point values within the above numerical ranges can be selected, and thus will not be repeated here.

[0019] Preferably, the bud elongation medium comprises 6-benzylaminopurine 0.1-0.3 mg / L, naphthalene acetic acid 0.1-0.5 mg / L, temik 50-150 mg / L, and sucrose 2.5-3.5% by mass percentage, and a basic medium.

[0020] Preferably, the gene editing vector contains a gene of a guide RNA, a gene of a Cas enzyme and a reporter gene.

[0021] Preferably, the guide RNA comprises an sgRNA.

[0022] Preferably, the nucleotide sequence of the sgRNA comprises a sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0023] SEQ ID No. 1: TATGCATCCCCGAGGTTGGAGGG.

[0024] SEQ ID No. 2: AGTCGGTGGCTGAGGCAACTGGG.

[0025] SEQ ID No. 3: GTTAAACTGGAGGTTAGCTGAGG.

[0026] Preferably, the Cas enzyme comprises Cas9.

[0027] Preferably, the reporter gene comprises a RUBY gene or a GFP gene.

[0028] Preferably, the Agrobacterium tumefaciens comprises Agrobacterium tumefaciens K599 strain.

[0029] Preferably, the target gene of the sgRNA comprises BoARF7 (BolC2t07069H) gene.

[0030] Preferably, the broccoli explant of step (1) is a broccoli explant with cotyledon-bearing hypocotyl segment.

[0031] Preferably, the preparation method of the broccoli explant comprises: after disinfecting the broccoli seeds, transferring the broccoli seeds to a MRS solid medium containing sucrose, culturing, and obtaining the broccoli explant.

[0032] Preferably, the disinfection specifically comprises: sequentially treating the broccoli seeds with 70-80% ethanol aqueous solution for 2-4 min, 5-10 vt% sodium hypochlorite aqueous solution for 8-12 min, and then washing the broccoli seeds with sterile water for 2-4 times.

[0033] The mass percentage of ethanol can be selected as 70%, 72%, 74%, 76%, 78%, 80%, etc., the ethanol treatment time is 2 min, 2.2 min, 2.5 min, 2.8 min, 3 min, 3.2 min, 3.5 min, 3.8 min, 4 min, etc., the volume percentage of sodium hypochlorite in the sodium hypochlorite aqueous solution can be selected as 5 vt%, 6 vt%, 7 vt%, 8 vt%, 9 vt%, 10 vt%, etc., the time can be selected as 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 10.5 min, 11 min, 11.5 min, 12 min, etc., the number of sterile water washing is 2 times, 3 times, 4 times, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0034] Preferably, the single washing time of the sterile water is 2-4 min, for example, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0035] Preferably, the temperature of the culture is 22-28℃, and the time is 6-8 days.

[0036] The temperature can be selected as 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, etc., and the time is 6 days, 7 days, 8 days, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0037] Preferably, the step (1) of transfecting the Agrobacterium tumefaciens containing the gene editing vector to the broccoli explant specifically includes:

[0038] (1) Resuspend the Agrobacterium tumefaciens containing the gene editing vector in the medium containing acetosyringone to obtain a bacterial suspension;

[0039] (2) Immerse the broccoli explant in the bacterial suspension.

[0040] Preferably, the OD of the bacterial suspension is 0.6-0.8, for example, 0.6, 0.65, 0.7, 0.75, 0.8, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0041] Preferably, the time of immersing in the bacterial suspension in step (2) is 10-30 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0042] Preferably, the culturing in step (1) specifically comprises culturing in a medium containing 100-200 μM acetosyringone for 2-4 days.

[0043] The molar concentration of acetosyringone can be selected from 100 μM, 150 μM, 200 μM, etc., and the culturing time can be selected from 2 days, 3 days, 4 days, etc., and other specific point values within the above numerical range can also be selected, which will not be repeated here.

[0044] Preferably, the temperature of the culturing is independently 22-28℃, such as 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, etc., and other specific point values within the above numerical range can also be selected, which will not be repeated here.

[0045] Preferably, the culturing time in step (2) is 14-25 days, such as 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, etc., and other specific point values within the above numerical range can also be selected, which will not be repeated here.

[0046] Preferably, the culturing time in the bud elongation medium is 14-25 days, such as 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, etc., and other specific point values within the above numerical range can also be selected, which will not be repeated here.

[0047] Preferably, the culturing time in the rooting induction medium is 14-25 days, such as 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, etc., and other specific point values within the above numerical range can also be selected, which will not be repeated here.

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] 1. By introducing a reporter gene into the transformation vector, the explants infected by Agrobacterium produce obvious pigment accumulation, thereby distinguishing the transformed tissues with the naked eye, significantly simplifying the screening process and improving the screening accuracy.

[0050] 2. By optimizing the types and concentration ratios of hormones in the medium and the transformation process, the formation of callus and adventitious buds and the regeneration into complete plants can be achieved without inducing hairy roots, thereby simplifying the operation process and shortening the regeneration cycle.

[0051] 3. By carrying a CRISPR / Cas9 editing element vector, the editing of target genes is realized, and the mutation type is verified by PCR and sequencing. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a schematic of the gene editing vector.

[0053] Figure 2 is a plasmid map of the gene editing vector.

[0054] Figure 3 is a flowchart of the process of Agrobacterium rhizogenes-mediated callus induction and gene edited plant regeneration.

[0055] Figure 4 is a picture of the phenotype of Brassica oleracea WT (wild type) and RUBY plants obtained in Example 1.

[0056] Figure 5 is the induction result of callus induction medium containing different concentrations of 6-BA to induce callus formation.

[0057] Figure 6 is the callus induction frequency of callus induction medium containing different concentrations of 6-BA.

[0058] Figure 7 is the induction result of Brassica oleracea callus in Example 3.

[0059] Figure 8 is the PCR verification of Cas9 transgene (755 bp) using target-specific primers.

[0060] Figure 9 is a nanopore sequencing chromatogram. DETAILED DESCRIPTION

[0061] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0062]

[0063] Method for preparing Brassica oleracea explants:

[0064] Brassica oleracea seeds were subjected to surface sterilization treatment, sequentially treated with 75% (v / v) ethanol aqueous solution for 3 minutes, 10% (v / v) sodium hypochlorite (NaClO) aqueous solution for 10 minutes, and then washed with sterile distilled water for 3 times, each for 3 minutes. The sterilized seeds were dried on sterile filter paper and then transferred to MS basic medium containing 0.8% (w / v) agar and 3% (w / v) sucrose, with pH adjusted to 5.8. The culture was carried out at 25°C under a photoperiod of 16 hours light / 8 hours dark for 7 days to obtain sterile seedlings as the source of explants.

[0065] Construction of gene editing vector:

[0066] The structure of the first gene editing vector is as follows: Figure 1 As shown in the image. The RUBY chromogenic gene is used for transformation screening visualization, the Cas9 editing element enables targeted genome editing, and the plasmid map is shown below. Figure 2 As shown.

[0067] The second gene editing vector was a 35S::GFP overexpression empty vector.

[0068] Preparation of Agrobacterium tumefaciens suspension:

[0069] The vector was introduced into Agrobacterium tumefaciens strain K599, and a single colony was picked and inoculated into YT liquid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin. The culture was carried out at 28°C in a shaker until the OD600 reached 0.8. After centrifugation, the bacterial cells were collected and resuspended in MS infection medium containing 200 μM acetylsuccinone (AS) for later use.

[0070] Example 1

[0071] This embodiment provides a method for constructing regenerated broccoli plants, the method comprising:

[0072] (1) Hypocotyl segments with cotyledons were cut from 7-day-old sterile broccoli seedlings and immersed in a bacterial suspension containing the first gene editing vector for 30 minutes. The explants were blotted dry with sterile filter paper and then co-cultured in MS medium containing 200 µM AS under dark conditions for 3 days. After co-culture, the explants were transferred to callus induction medium (MS medium containing 1 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3 wt% sucrose and 300 mg / L termethin; pH 5.8) and cultured at 25 ℃ under a photoperiod of 16 hours light / 8 hours dark. After about two weeks, the callus tissue increased significantly.

[0073] (2) Cut off the enlarged callus with red (RUBY) color and transfer it to the bud induction medium (MS salt medium with 2 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3wt% sucrose and 300 mg / L termethin) and culture until bud regeneration.

[0074] (3) Cut off the regenerated buds and transfer them to bud elongation medium (MS salt medium containing 0.2 mg / L 6-BA, 0.1 mg / L NAA, 3 wt% sucrose and 100 mg / L termethin).

[0075] (4) After the shoots elongate, they are transferred to rooting induction medium (MS salt medium containing 0.2 mg / L IBA, 0.1 mg / L NAA, 3 wt% sucrose and 100 mg / L termethin) to promote rooting and form edited plants. The process of callus induction and gene-edited plant regeneration mediated by Agrobacterium rhizogenes is as follows: Figure 3 As shown, edit the images of the plant and the wild-type broccoli plant as follows. Figure 4 As shown.

[0076] Example 2

[0077] This embodiment provides a method for inducing broccoli callus, the method comprising: cutting a hypocotyl segment with cotyledons from a 7-day-old sterile broccoli seedling and immersing it in a bacterial suspension containing a first gene editing vector for 30 minutes. Explants were blotted dry with sterile filter paper and then co-cultured in MS medium containing 200 µM AS under dark conditions for 3 days. After co-culture, the explants were transferred to callus induction media containing different concentrations of 6-BA: 1 mg / L MS medium (containing 1 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3 wt% sucrose, and 300 mg / L termetine; pH 5.8), 2 mg / L MS medium (containing 2 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3 wt% sucrose, and 300 mg / L termetine; pH 5.8), and 5 mg / L MS medium (containing 2 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3 wt% sucrose, and 300 mg / L termetine; pH 5.8). 5.8), and cultured at 25 ℃ with a photocycle of 16 hours light / 8 hours dark. Results are as follows: Figure 5 As shown, the results of the induced frequency are as follows Figure 6 As shown.

[0078] Example 3

[0079] This embodiment provides a method for inducing broccoli callus. The method includes: cutting hypocotyl segments with cotyledons from 7-day-old sterile broccoli seedlings and immersing them in a bacterial suspension containing a second gene editing vector for 30 minutes. The explants are blotted dry with sterile filter paper and then co-cultured in MS medium containing 200 µM AS under dark conditions for 3 days. After co-culture, the explants are transferred to callus induction medium (MS medium containing 1 mg / L 6-BA, 0.1 mg / L NAA, 4 mg / L AgNO3, 3 wt% sucrose, and 300 mg / L termethin; pH 5.8) and cultured at 25 ℃ under a photoperiod of 16 hours light / 8 hours dark. The results are as follows: Figure 7 As shown, using GFP as a reporter gene also achieves the desired visualization effect.

[0080] Test Example 1

[0081] Genomic DNA was extracted from regenerated plants using the CTAB method. PCR was performed using primers Cas9-F and Cas9-R, yielding a 755 bp product. The results are shown below. Figure 8 As shown in Table 1, BolC2t07069H was targeted amplified using primers BolC2t07069H-F and BolC2t07069H-R, followed by nanopore sequencing. "ins" indicates insertion and "-" indicates deletion. Figure 8 As shown, the nanopore sequencing chromatogram is as follows: Figure 9 As shown.

[0082] Table 1

[0083]

[0084] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for constructing regenerated broccoli plants based on visual tags, characterized in that, The method includes: (1) Agrobacterium tumefaciens containing a gene-editing vector was transfected into broccoli explants and cultured; (2) Transfer the explants obtained in step (1) to the callus induction medium, culture them, cut off the callus, transfer it to the shoot induction medium, and culture it until the shoot regenerates; (3) Transfer the regenerated buds to the bud elongation medium and culture them until the buds elongate. Then transfer them to the rooting induction medium to obtain the regenerated plant.

2. The method for constructing broccoli regenerated plants based on visual markers according to claim 1, characterized in that, The callus induction culture medium comprises, by concentration, 1-5 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, 2-4 mg / L of silver nitrate, and 200-300 mg / L of termethin, and by mass percentage, 2.5-3.5% of sucrose and basal culture medium; Preferably, the bud induction medium comprises, by concentration, 2-5 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, 2-4 mg / L of silver nitrate, 200-300 mg / L of termethin, and by mass percentage, 2.5-3.5% of sucrose and basal medium.

3. The method for constructing broccoli regenerated plants based on visual markers according to claim 1 or 2, characterized in that, The bud elongation medium comprises, by concentration, 0.1-0.3 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, 50-150 mg / L of termethin, and by mass percentage, 2.5-3.5% of sucrose and basal medium; Preferably, the rooting induction medium comprises, by concentration, 0.1-0.3 mg / L indole-3-butyric acid, 0.1-0.5 mg / L naphthaleneacetic acid, 50-150 mg / L termethin, and by mass percentage, 2.5-3.5% sucrose and basal medium; Preferably, the basal culture medium includes MS medium.

4. The method for constructing broccoli regenerated plants based on visual markers according to any one of claims 1-3, characterized in that, The gene editing vector contains the guide RNA gene, the Cas enzyme gene, and the reporter gene; Preferably, the guide RNA includes sgRNA; Preferably, the nucleotide sequence of the sgRNA includes the sequence shown in SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3; Preferably, the Cas enzyme comprises Cas9; Preferably, the reporter gene includes the RUBY gene or the GFP gene; Preferably, the Agrobacterium tumefaciens includes Agrobacterium tumefaciens strain K599.

5. The method for constructing broccoli regenerated plants based on visual markers according to claim 4, characterized in that, The target genes of the sgRNA include the BoARF7 gene.

6. The method for constructing broccoli regenerated plants based on visual markers according to any one of claims 1-5, characterized in that, The broccoli explants in step (1) are broccoli explants with hypocotyl segments containing cotyledons.

7. The method for constructing broccoli regenerated plants based on visual markers according to any one of claims 1-6, characterized in that, The method for preparing broccoli explants includes: sterilizing broccoli seeds, transferring them to sucrose-containing MRS solid medium, culturing them, and obtaining broccoli explants.

8. The method for constructing broccoli regenerated plants based on visual markers according to claim 7, characterized in that, The disinfection process specifically includes: treating broccoli seeds sequentially with a 70-80% ethanol aqueous solution for 2-4 minutes, then with a 5-10% sodium hypochlorite aqueous solution for 8-12 minutes, and finally rinsing them with sterile water 2-4 times. Preferably, the culture temperature is 22-28℃ and the time is 6-8 days.

9. The method for constructing broccoli regenerated plants based on visual markers according to any one of claims 1-8, characterized in that, Step (1) of transfecting Agrobacterium tumefaciens containing the gene-editing vector into broccoli explants specifically includes: (1) Agrobacterium tumefaciens containing the gene editing vector was resuspended in a culture medium containing acetylsuccinone to obtain a bacterial suspension; (2) Immerse the broccoli explants in the bacterial suspension; Preferably, the OD of the bacterial suspension is 0.6-0.8; Preferably, the immersion time in the bacterial suspension in step (2) is 10-30 min.

10. The method for constructing broccoli regenerated plants based on visual markers according to any one of claims 1-9, wherein the culture in step (1) specifically includes culture in a medium containing 100-200 μM acetylsyringone for 2-4 days; Preferably, the culture temperature is independently 22-28°C; Preferably, the culture time in step (2) is 14-25 days; Preferably, the culture time in the bud elongation medium is 14-25 days; Preferably, the culture time in the rooting induction medium is 14-25 days.

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