Rapid suspension culture method and high-efficiency genetic transformation method of Rosa chinensis cell

Abstract

The application discloses a rapid suspension culture method and a high-efficiency genetic transformation method of Rosa chinensis cells, and relates to the technical field of plant cell culture. The rapid suspension culture method of Rosa chinensis cells comprises the following steps: (1) inducing a callus; (2) preparing a suspension cell; and (3) subculturing and storing the suspension cell. The high-efficiency genetic transformation method of Rosa chinensis cells comprises the following steps: (1) using the Rosa chinensis suspension cell obtained by the rapid suspension culture method of Rosa chinensis cells; (2) transforming Agrobacterium with a vector; (3) infecting the Rosa chinensis suspension cell with the Agrobacterium; (4) co-culturing and screening the transfected suspension cell; and (5) detecting the genetic transformation result. The application solves the problem that the prior art has not established a genetic transformation system of Rosa chinensis suspension cells, can rapidly culture a large amount of Rosa chinensis suspension cells in a short time, efficiently genetically transforms the Rosa chinensis suspension cells, and realizes gene function verification, subcellular localization, protein expression and synthetic biology research in the Rosa chinensis body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for rapid suspension culture and efficient genetic transformation of rose cells. Background Technology

[0002] The rose, belonging to the genus Rosa in the family Rosaceae, is one of the most important ornamental crops worldwide, widely used in cut flowers, potted plants, landscaping, and roadside beautification, with a history of application exceeding 5000 years. As the distribution center of the genus Rosa in the world, my country has seen the continuous flowering and fragrance of its 'Yueyuefen' and 'Xiangshui Yueji' varieties play a crucial role in the development of modern roses.

[0003] Roses are the world's leading cut flower, and my country has the largest rose production area globally, with a production value reaching 20 billion yuan in 2020. The rose genome is relatively small; the diploid ancient variety 'Yueyuefen' has a DNA content of approximately 1.2 pg / 2C, about four times that of the model plant Arabidopsis thaliana (0.3 pg / 2C). Modern roses have a short juvenile period, with each genotype generation lasting only about one year. Rose plants can bloom repeatedly, and newly sprouted branches after pruning can bloom in about 40 days, similar to the growth cycle of Arabidopsis thaliana. Roses possess extremely rich flower shapes, colors, and fragrances—unique traits that cannot be studied in other model plants. These rich phenotypic variations, combined with modern omics tools, make it relatively easy to establish associations between gene loci and phenotypes. Rose flowers are relatively large, facilitating observation; most genotypes are easily propagated asexually, blooming in about 40-50 days after cuttings, making it easy to obtain a large amount of homogeneous material in a short period. In particular, modern roses are mostly double-flowered with relatively uniform petal structure, have a short lifespan, and can be treated in various ways without causing substantial damage. Therefore, roses can be used as model plants for ornamental plant research.

[0004] Gene modification is widely used in molecular research and crop breeding. However, as a woody plant, roses not only have a long genetic transformation cycle (approximately 8-10 months) but also low genetic transformation efficiency. This long transformation cycle and low efficiency severely impact rose gene function research and molecular breeding efforts. Therefore, there is an urgent need to develop a rapid and efficient genetic transformation and validation system to improve the efficiency of rose genetic transformation and reduce the risk of stable transformation failure.

[0005] Currently, the genetic transformation of roses mainly employs Agrobacterium-mediated callus transformation. However, callus tissue, as a genetic transformation material, suffers from low transformation efficiency. Plant suspension cells, due to their good dispersibility, uniformity, and rapid cell proliferation, are excellent materials for genetic transformation. Highly efficient suspension cell genetic transformation systems have been reported for several crops, including sweet potato, rice, tobacco, and poplar, but the establishment and genetic transformation of rose suspension cell lines have not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a rapid suspension culture method for rose cells and a highly efficient genetic transformation method for rose suspension cells. This addresses the problem that the existing technology has not yet established a genetic transformation system for rose suspension cells and that the transformation efficiency of rose callus tissue as a genetic transformation material is low. This invention provides an effective way to rapidly verify gene function, subcellular localization, and verify protein interactions through rose cell culture, while also laying the foundation for genetic engineering modification of roses.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0008] A rapid suspension culture method for rose cells includes the following steps:

[0009] (1) Induction of callus: Select young rose leaves and inoculate them into induction medium for callus induction culture; transfer the induced callus to proliferation medium for proliferation culture.

[0010] (2) Preparation of suspension cells: Select the callus tissue that has been cultured for 1 month in step 1); inoculate the selected callus tissue into a gel-free proliferation medium; place the medium in a constant temperature shaking incubator for culture to obtain stable primary suspension cells;

[0011] (3) Subculture of suspension cells: Select the stable primary suspension cells obtained in step 2) and inoculate them onto gel-free proliferation medium; place the gel-free proliferation medium inoculated with suspension cells in a constant temperature shaking incubator for culture, and then obtain subcultured rose suspension cells.

[0012] (4) Preservation of subcultured suspension cells: The gel-free proliferation medium containing subcultured rose suspension cells obtained in step 3) is filtered to obtain subcultured rose suspension cells. The cells are then placed on a proliferation medium containing gel for subculture preservation.

[0013] Furthermore, the above-mentioned induction culture medium composition includes 4.40 g·L⁻¹ -1 -4.45g·L -1MS salt, 2.5 mg·L -1 -3.5 mg·L -1 2,4-D, 0.05 mg·L -1 KT, 30g·L -1 Glucose, 0.3% Gel.

[0014] Furthermore, the above-mentioned proliferation medium comprises 4.40 g·L⁻¹ -1 -4.45g·L -1 MS salt, 0.8 mg·L -1 -1.2 mg·L -1 2,4-D, 0.8 mg·L -1 -1.2 mg·L -1 6-BA, 6% Glucose, 0.3% Gel.

[0015] Furthermore, the conditions for inducing and culturing the callus tissue are 25°C in the dark for 2 weeks; the conditions for proliferation culture are 25°C in the dark.

[0016] Furthermore, the culture conditions for preparing suspension cells are 25°C, in the dark, with shaking culture at 130 rpm / min, the culture medium is changed weekly for 4 to 8 weeks, and large pieces of callus are removed weekly.

[0017] Based on the above-mentioned rapid suspension culture method for rose cells, this invention also provides a highly efficient genetic transformation method for rose suspension cells. This method uses rose suspension cells obtained by the above-mentioned rapid suspension culture method as transformation material, infects the transformation material with Agrobacterium strains transformed by plasmids, and then screens and cultures them to obtain successfully transformed rose suspension cells.

[0018] The above-mentioned efficient genetic transformation method for rose suspension cells includes the following steps:

[0019] (1) Transformation of Agrobacterium: The plasmid was transformed into the Agrobacterium strain, and the successfully transformed single colonies were selected, inoculated into LB liquid medium, centrifuged to remove the supernatant, and the positive Agrobacterium was collected.

[0020] (2) Soak the positive Agrobacterium positivity collected by centrifugation in MS resuspension solution to obtain the infection solution;

[0021] (3) Filter and collect the above-mentioned subcultured rose suspension cells;

[0022] (4) Place the rose suspension cells from step 3) into the infection solution from step 2) for infection to obtain transfected suspension cells.

[0023] (5) Co-culture: Take the transfected suspension cells from step 4) and place them in a co-culture medium for co-culture.

[0024] (6) Screening culture: Take the suspension cells from step 5) after co-culture and place them in selective proliferation medium for selective culture to obtain rose suspension cell clusters.

[0025] Further, the MS resuspension solution in step 2) comprises 4.40 g·L⁻¹ -1 -4.45g·L -1 MS salt, 8mM-12mMES, 8mM-12mM MgCl2, 3% Glucose, 180μM-220μM AS.

[0026] Further, the co-culture medium in step 5) comprises 4.40 g·L⁻¹ -1 -4.45g·L -1 MS salt, 0.8 mg·L -1 -1.2 mg·L -1 2,4-D, 0.8 mg·L -1 -1.2 mg·L -1 6-BA, 4.5% Glucose, 0.3% Gel.

[0027] Further, step 6) involves selecting a proliferation medium containing 4.40 g·L⁻¹. -1 -4.45g·L -1 MSsalt, 0.8 mg·L -1 -1.2 mg·L -1 2,4-D, 0.8 mg·L -1 -1.2 mg·L -1 6-BA, 4.5% Glucose, 0.3% Gel, 280mg·L -1 -300mg·L -1 Cef, 8 mg·L -1 -12mg·L -1 Hygromyci.

[0028] Further, the plasmid in step 4) is pCAMBIA1300; the Agrobacterium is EHA105.

[0029] Beneficial effects:

[0030] 1. This invention establishes a rapid suspension culture method for rose cells. The callus induction conditions of this method can efficiently induce rose callus. The obtained rose callus has the characteristics of vigorous growth and no browning, and can be subcultured and preserved for a long time. Suspension culture of the obtained rose callus can rapidly obtain a large number of rose suspension cells in 14 days.

[0031] 2. Based on the rapid suspension culture method for rose cells established in this invention, this invention also provides an efficient genetic transformation method for rose suspension cells. This method can efficiently transform rose cells and can be used to study the mechanisms and genetic variation patterns of rose growth, development, and differentiation.

[0032] 3. This invention establishes a rapid suspension culture method and an efficient genetic transformation method for rose cells. It not only has a short cycle but also high genetic transformation efficiency, solving the problem that rose cells cannot achieve protein overexpression. Stable transformed suspension cell lines can be obtained after 14 days of screening. It can also quickly realize gene function verification, subcellular localization, protein expression, and synthetic biology research on rose cells, thereby accelerating and deepening the research on rose color, fragrance, senescence, stress, etc. Attached Figure Description

[0033] Figure 1 The diagrams show the results of suspension culture cell preparation and preservation. A is an image of induced callus; B is an image of the prepared suspension cell line; and C is an image of the preserved suspension cell line.

[0034] Figure 2 The figure shows the observation results of the viability and status of the suspension cell line after 14 days of subculture.

[0035] Figure 3 The image shows the PCR electrophoresis results of positive colonies containing the GFP gene.

[0036] Figure 4 The fluorescence detection graph shows the results of genetic transformation of suspension cells;

[0037] Figure 5 Figure showing the genetic transformation efficiency, DNA level, and PCR electrophoresis results of the GFP gene;

[0038] Figure 6 Figure showing the genetic transformation efficiency, RNA level, and GFP gene PCR electrophoresis results;

[0039] Figure 7 The image shows the results of Western blotting detection of GFP protein expression. Detailed Implementation

[0040] The following examples further illustrate the rapid suspension culture and efficient genetic transformation method for rose cells provided by this invention. It should be noted that the following examples are only for further illustration of this invention and should not be construed as limiting the scope of protection of this invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the invention to implement it are still within the scope of protection of this invention.

[0041] In the following examples, the formulation of the induction medium is as follows:

[0042]

[0043] In the following examples, the formulation of the proliferation medium is as follows:

[0044]

[0045] In the following examples, the formulation of the gel-free proliferation medium is as follows:

[0046]

[0047]

[0048] In the following examples, the formulation of the LB liquid culture medium is as follows:

[0049]

[0050] In the following examples, the formulation of the LB solid culture medium is as follows:

[0051]

[0052] In the following embodiments, the MS resuspension solution is formulated as follows:

[0053]

[0054] In the following embodiments, the formulation of the co-culture medium is as follows:

[0055]

[0056]

[0057] In the following examples, the formulation of the selective proliferation medium is as follows:

[0058]

[0059] In the following examples, the MS salt was purchased from Caisson Labs, catalog number MSP09; 2,4-D (2,4-dichlorophenoxyacetic acid) was purchased from Coolaber, catalog number CC3511; KT (kinin, a cytokinin) was purchased from Coolaber, catalog number CK6721; Glucose was purchased from Shanghai Experimental, catalog number 10010518; Gel was purchased from SIGMA, catalog number P8169; 6-BA was purchased from Coolaber, catalog number CB2611; Kan+ was purchased from ACMEC, catalog number K12100; Rif+ was purchased from ACMEC, catalog number R95501; Tryptone was purchased from OXOID, catalog number LP0042; Yeast Extracts were purchased from OXOID (LP0021); NaCl from Shanghai Experimental Laboratory (10019318); AgarPowder from Chembase (B0018K0930001H); MES (2-morpholinoethanesulfonic acid) from Solarbio (M8010); MgCl2 from Shanghai Experimental Laboratory (1012818); AS from Meilunbio (2478-38-8); Cef (cephalosporin) from Coolaber (CC3251); Hygromycin from Roche (21362688); plasmid pCAMBIA1300 was preserved in our laboratory; Agrobacterium competent cells EHA105 were purchased from Coolaber (CC403).

[0060] In the examples, positive strains and GFP were identified, and the primers used for amplification are shown in Table 1:

[0061] Table 1 Primer sequences

[0062]

[0063]

[0064] Example

[0065] 1. Rapid suspension culture and efficient genetic transformation method for rose cells

[0066] 1) Callus induction: ① Select tender, flat leaves from rose tissue culture seedlings. Inoculate the selected tender rose leaves into the induction medium and induce callus formation at 25℃ in the dark. Callus will be induced in 2 weeks; ② Remove residual leaves and browned tissue. Transfer the induced callus to the proliferation medium and culture at 25℃ in the dark. The induced callus will appear as follows. Figure 1 -A is shown.

[0067] 2) Preparation of suspension cells: ① Select soft callus tissue cultured for 1 month in step 1); ② Inoculate the selected callus tissue into gel-free proliferation medium; ③ Place the gel-free proliferation medium containing the inoculated callus tissue in a constant temperature shaking incubator, and culture at 25℃ in the dark with shaking at 130 rpm / min. Change the medium weekly for 6 weeks, removing large pieces of callus weekly to obtain stable primary suspension cells. The primary suspension cells are shown below. Figure 1 -B is shown.

[0068] 3) Subculture and preservation of suspension cells: ① Select the stable primary suspension cells obtained in step 2) and re-inoculate them onto gel-free proliferation medium; ② Place the gel-free proliferation medium containing the suspension cells in a constant temperature shaking incubator and culture at 130 rpm / min for 14 days in the dark at 25°C to obtain suspension cells after 14 days of subculture; ③ Filter the gel-free proliferation medium containing the 14-day subcultured suspension cells obtained in step 2 using a 400-mesh filter cloth to obtain 14-day subcultured suspension cells. Place these cells onto a proliferation medium containing gel and subculture them at 25°C in the dark. The preserved suspension cells are shown below. Figure 1 As shown in -C, the observation results of the viability and status of the suspension cell line after 14 days of subculture are as follows: Figure 2 As shown.

[0069] 4) Transformation of Agrobacterium: ① Select the pCAMBIA1300 vector carrying the GFP tag as the target vector. Mix the pCAMBIA1300 vector with Agrobacterium competent cells EHA105 in a shaker tube. ② Place the shaker tube on ice for 5 min, flash freeze in liquid nitrogen for 5 min, and incubate at 37℃ for 5 min. ③ Add 500 μl of LB liquid medium to the shaker tube and incubate at 28℃ with shaking at 200 rpm for 3 h. ④ Centrifuge the shaker tube at 5000 rpm, discard 400 μl of supernatant, resuspend the cells in the remaining supernatant, spread on LB solid medium containing 50 mg / L Kan+ and 50 mg / L Rif+, and incubate in the dark at 28℃ for 2-3 days until colonies grow. ⑤ Pick single colonies and perform PCR identification. Primer sequences are shown in Table 1 as SEQ ID NO.1 and SEQ ID NO. As shown in NO.2, positive Agrobacterium was obtained, and the identification results are as follows. Figure 3 As shown.

[0070] 5) Preparation of infection solution: ① Incubate Agrobacterium strain EHA105 carrying the target vector in LB liquid medium until the OD600 is 0.8; ② Collect Agrobacterium cells by centrifugation and resuspend them in MS resuspension solution, and incubate until the OD600 value reaches 0.8. This liquid is the infection solution.

[0071] 6) Collection of suspension cultured cells: Collect suspension cells from step 3) after 14 days of subculture using a 400-mesh filter cloth;

[0072] 7) Transformation of suspension culture cells: Place the suspension cells collected in step 6) after 14 days of subculture into the infection medium in step 5), and shake them in a shaker at 180 rpm / min for 30 min in the dark at 28°C. Filter and collect the transfected suspension cells, and wash them twice with ddH2O to remove the infection medium.

[0073] 8) Co-culture: Take the transfected suspension cells from step 7) and place them in a co-culture medium. Co-culture them for 2-3 days in the dark at 25°C for subsequent experiments.

[0074] 9) Screening culture: Take the suspension cells from step 8) after co-culture and place them in selective proliferation medium for selective culture. New suspension cell clusters will be obtained after 2 weeks.

[0075] 10) Detection of transformation results: The transformation results of the suspended cell clusters obtained in step 9) were detected using the following four methods: ① Because plasmid pCAMBIA1300 carries a GFP tag, suspended culture cells 48 hours after transformation or 14 days after selection were observed under a fluorescence microscope. The observation results are as follows: Figure 4 As shown; ② DNA was extracted from transformed suspension cells, primers targeting the GFP sequence were designed (see SEQ ID NO.3 and SEQ ID NO.4 in Table 1), PCR amplification and gel electrophoresis were performed, and the results are shown in the figure. Figure 5 As shown; ③ RNA was extracted from the transformed suspension cell line, reverse transcribed to obtain cDNA of the GFP gene sequence, and then amplified by PCR or qRT-PCR. The results are shown in the figure. Figure 6 As shown; ④ Total protein was extracted from the transformed suspension cell line, and the target GFP protein was detected by Western blot. The results are shown in the figure. Figure 7 As shown.

[0076] 11) Results Analysis: Analyze the conversion detection results of step 10).

[0077] ① Fluorescence observation revealed that the suspended cell clusters emitted green fluorescence, indicating that GFP had been successfully inserted and expressed in the rose cells, achieving efficient transformation; ② DNA and RNA were extracted from 20 suspension culture cells selected from proliferation medium and subjected to PCR and gel electrophoresis. The results showed that GFP gene bands could be detected in all suspension cells, indicating successful efficient transformation; ③ Western blot detection of the GFP target protein showed the presence of the target GFP protein band, indicating successful efficient transformation.

[0078] The above embodiments are merely examples illustrating the explanation, specific implementation methods, and effects of the present invention, and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to the invention without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

Claims

1. A method for genetic transformation of Rosa hybrida suspension cells, characterized by, The method comprises the following steps: (1) transforming Agrobacterium with a vector: transforming a plasmid into an Agrobacterium strain, selecting a single colony with successful transformation, inoculating in LB liquid medium for culture, centrifuging to remove supernatant, and collecting positive Agrobacterium; (2) resuspend the positive Agrobacterium collected by centrifugation in step (1) with MS resuspension solution, so as to obtain an infection solution; the MS resuspension solution is: 4.40 g• L -1 -4.45 g• L -1 MS salt, 8 mM-12 mM MES, 8 mM-12 mM MgCl2, 3% glucose, 180 μM-220 μM AS; (3) filtering the Rosa hybridae suspension cells collected after 14 days of subculture using a 400-mesh filter cloth; (4) placing the Rosa hybridae suspension culture cells subcultured for 14 days in step (3) in the infection liquid obtained in step (2) for infection to obtain transfected suspension cells; the infection is specifically as follows: placing in a shaking bed at 28°C in dark conditions and shaking at 180 rpm / min for 30 min, filtering to collect the transfected suspension cells, washing twice with ddH2O, and washing away the infection liquid to obtain the transfected suspension cells; (5) Co-culture: the transfected suspension cells obtained in step (4) are placed in a co-culture medium at 25°C in the dark for 2-3 days; the co-culture medium comprises the following components: 4.40 g• L -1 -4.45 g• L -1 MS salt, 0.8 mg• L -1 -1.2 mg• L -1 2,4-D, 0.8 mg• L -1 -1.2 mg• L -1 6-BA, 4.5% Glucose, 0.3% Gel; (6) screening culture: take the suspension cells of step (5) at the end of co-culture, place in selection proliferation medium for selection culture, obtain Chinese rose suspension cell mass in 2 weeks; the components of the selection proliferation medium are: 4.40 g• L -1 -4.45 g• L -1 MSsalt, 0.8 mg• L -1 -1.2 mg• L -1 2,4-D, 0.8 mg• L -1 -1.2 mg• L -1 6-BA, 4.5% Glucose, 0.3% Gel, 280 mg• L -1 -300 mg• L -1 Cef, 8 mg• L -1 -12 mg• L -1 Hygromyci; The preparation method of the Rosa hybridae suspension cells is as follows: ① inducing callus: selecting Rosa hybridae young leaves, inoculating the selected Rosa hybridae young leaves into an induction medium for callus induction culture; transferring the induced callus into a proliferation medium for proliferation culture; ② preparing suspension cells: selecting the callus subcultured for 1 month in step ①; inoculating the selected callus into a gel-free proliferation medium; placing the medium in a constant-temperature shaking incubator for culture to obtain stable primary suspension cells; ③ subculturing suspension cells: inoculating the primary suspension cells obtained in step ② into a gel-free proliferation medium; placing the gel-free proliferation medium in a constant-temperature shaking incubator for culture to obtain subcultured Rosa hybridae suspension cells; ④ preserving subcultured suspension cells: filtering the gel-free proliferation medium containing the subcultured Rosa hybridae suspension cells obtained in step ③ to obtain the subcultured Rosa hybridae suspension cells, and placing the cells in a gel-containing proliferation medium for subculture preservation; The induction medium components are: 4.40 g• L -1 -4.45 g• L -1 MS salt, 2.5 mg• L -1 -3.5 mg• L -1 2,4-D, 0.05 mg• L -1 KT, 30 g• L -1 Glucose, 0.3% Gel; the proliferation medium is 4.40 g• L -1 -4.45 g• L -1 MS salt, 0.8 mg• L -1 -1.2 mg• L -1 2,4-D, 0.8 mg• L -1 -1.2 mg• L -1 6-BA, 6% Glucose, 0.3% Gel.

2. The genetic transformation method according to claim 1, characterized by, The callus induction culture in step ① is performed at 25°C in dark conditions for 2 weeks; and the proliferation culture is performed at 25°C in dark conditions.

3. The genetic transformation method according to claim 1 or 2, characterized by, The culture in step ② is performed at 25°C in dark conditions with shaking at 130 rpm / min, the medium is replaced once a week, and the culture lasts for 4 to 8 weeks, and large callus is removed every week.

4. The genetic transformation method according to claim 1, wherein, The plasmid in step (1) is pCAMBIA1300; and the Agrobacterium is EHA105.

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