A method for constructing transient genetic transformation of tea tree callus
By using a transient genetic transformation method for tea plant callus tissue and optimizing conditions such as suspension culture medium and co-culture time, the problem of low genetic transformation efficiency in tea plants was solved, and a highly efficient and stable genetic transformation effect was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-09
AI Technical Summary
The existing genetic transformation efficiency of tea trees is low, the reproducibility is poor, and in vitro regeneration is difficult. The main reason is the influence of the secondary metabolites of the tea tree itself on the transformation of Agrobacterium.
A transient genetic transformation method for tea tree callus was adopted, including callus suspension culture, Agrobacterium activation, preparation of Agrobacterium infection solution, Agrobacterium infection and GUS staining, and optimization of conditions such as suspension culture medium and co-culture time to improve transformation efficiency.
Efficient and stable genetic transformation of tea plant callus tissue was achieved, with a 100% success rate in GUS expression, laying the foundation for a stable genetic transformation system for tea plants.
Smart Images
Figure CN122168679A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, specifically to a method for constructing transient genetic transformation of tea tree callus. Background Technology
[0002] tea tree( Camellia sinensis (L.) O. Kuntze is a perennial woody plant belonging to the genus Camellia in the family Theaceae. It has a long history of cultivation and is mainly distributed in southwestern, southern, and eastern my country. Tea contains beneficial substances such as tea polyphenols, caffeine, and theanine, which have anti-cancer, antioxidant, and stimulating functions, making it popular among consumers.
[0003] Tea trees have characteristics such as a long growth cycle, self-incompatibility, and a short flowering period, which limit the progress of conventional breeding and make it difficult to achieve breakthroughs in tea tree breeding. Compared with traditional breeding, molecular breeding technology can solve the problems of long cultivation time and large workload in tea tree breeding, and genetic transformation technology is an important means to realize molecular breeding of tea trees.
[0004] Plant genetic transformation technology involves isolating target genes (DNA fragments) and transferring them into plant cells through direct introduction or bio-mediated methods to achieve expression. Common methods include Agrobacterium-mediated genetic transformation and gene gun bombardment transformation. While some progress has been made in constructing genetic transformation systems for tea plants, problems remain, such as low transformation efficiency, poor reproducibility, and difficulties in in vitro regeneration. The main reason for this is the influence of secondary metabolites from the tea plant itself on Agrobacterium-mediated transformation.
[0005] Agrobacterium-mediated transformation is a commonly used method for tea plant transformation, frequently employing *Agrobacterium tumefaciens* and *Agrobacterium rhizogenes*. Agrobacterium-mediated genetic transformation can be divided into the construction of plant expression vectors, the establishment of transformation systems, the transformation of the target gene, the screening and acquisition of transformants, and the molecular detection and acquisition of transgenic plants. *Agrobacterium tumefaciens* commonly uses the soaking method for genetic transformation. Taking tea seedling stem segments as transformation material as an example, the Agrobacterium transformation procedure is as follows: acquisition of sterile seedlings → collection of explants and preparation of *Agrobacterium* infection solution → *Agrobacterium* infection of explants → co-culture of explants and *Agrobacterium* → recovery culture → screening culture → rooting and transplanting. Besides *Agrobacterium tumefaciens*-mediated tea plant transgenic transformation, some researchers have also used *Agrobacterium rhizogenes* to infect tea plants and induce hairy roots. Xi Biao et al. used *Agrobacterium rhizogenes* to infect the leaves of sterile tea seedlings and ultimately induced hairy roots.
[0006] Currently, tea leaves, stem segments, cotyledons, somatic embryos, and callus tissue are commonly used as explant materials for infection. Among these, somatic embryos and callus tissue are the most frequently used explants in tea genetic transformation, and their transformation and regeneration effects are superior to other explants. During genetic transformation, embryonic cells can come into close contact with Agrobacterium and antibiotics, which improves transformation and screening efficiency and reduces the occurrence of chimeras induced by adventitious bud regeneration pathways. Therefore, they are considered ideal genetic transformation recipients.
[0007] Chinese patent application CN114208669A discloses a method for establishing a high-efficiency regeneration system using tea stem segments as explants. Through steps such as establishing a sterile system, inducing callus tissue, inducing bud differentiation from callus tissue, bud growth and development, bud strengthening, rooting, hardening, and transplanting, this method solves the problems of current stem segment asexual regeneration methods, which mostly rely on axillary bud proliferation, have poor reproducibility, and cannot meet the high-frequency regeneration requirements of genetic transformation. Theoretically, it can also...
[0008] Transgenic experiments involving Agrobacterium infection were conducted. This patent establishes a highly efficient system for tea stem segment tissue culture and plant regeneration, laying a foundation for research on the propagation and genetic transformation of superior tea varieties. Summary of the Invention
[0009] The technical problem to be solved by this invention is how to solve the problem of low conversion efficiency in existing tea tree genetic transformation, and to lay a foundation for a highly efficient and stable genetic transformation system for tea trees.
[0010] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a method for the transient transformation and construction of tea tree callus, comprising the following steps: (1) Callus suspension culture Select vigorous, white, and loose callus tissue, place it in liquid suspension culture medium, and culture it in suspension. (2) Agrobacterium activation Single colonies of Agrobacterium strain were picked, streaked on LB solid medium containing kanamycin, and incubated in the dark to obtain vigorous Agrobacterium colonies. (3) Preparation of Agrobacterium infection solution The Agrobacterium obtained in (2) was inoculated into MT liquid medium without antibiotics, and then acetosyringone (AS) was added and cultured in suspension. (4) Agrobacterium infection The tea tree callus obtained in (1) was placed in the Agrobacterium infection solution obtained in (3), vacuumed, and then placed on a shaker at room temperature for inoculation; after the inoculation was completed, the callus was inoculated into a co-culture medium and cultured in the dark. (5) GUS staining Remove the co-cultured callus tissue, wash it, then soak it in GUS staining solution, vacuum filter it, heat it in a water bath, and judge whether the transformation is successful based on the staining.
[0011] Preferably, in step (1), the callus is 'Longjing 43' embryogenic callus.
[0012] Preferably, in step (1), the conditions for suspension culture are: dark conditions, 23~25℃, 150~250 rpm; more preferably 24℃, 200 rpm.
[0013] Preferably, in step (1), the specific components of the liquid suspension culture medium can be any of the following formulations: Formula 1: MT + (0.5~2) mg / L 2,4-D + (0.05~2) mg / L 6-BA + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 2: MT + (0.5~2) mg / L 2,4-D + (0.05~2) mg / L KT + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 3: MT + (1.5~2.5) mg / L 2,4-D + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 4: MT + (2~2.5) g / L KCl + (38~42) g / L sucrose.
[0014] A further preferred formulation is: MT + 2 mg / L 2,4-D + 0.2 mg / L 6-BA + 2.2 g / L KCl + 40 g / L sucrose.
[0015] Preferably, in step (2), the concentration of kanamycin is 45-60 mg / L, more preferably 50 mg / L.
[0016] Preferably, in step (2), the conditions for dark culture are: 24~26℃, 1~3d; more preferably 25℃, 2d.
[0017] Preferably, in step (3), the concentration of acetylsuccinone is 90~110 μM; more preferably 100 μM.
[0018] Preferably, in step (3), the suspension culture conditions are: 24~26℃, 150~200 rpm for 1-2 h; more preferably, 25℃, 180 rpm for 1.5 h. Culture until OD 600 The value is in the range of 0.8 to 1.0.
[0019] Preferably, in step (4), the immersion conditions are 80~120 rpm for 15~25 min; more preferably 100 rpm for 20 min.
[0020] Preferably, in step (4), the composition of the co-culture medium is: MT + 90~110 µM AS + 20~40 g / L sucrose + 2~5% plant gel; more preferably, MT + 100 µM AS + 30 g / L sucrose + 3% plant gel.
[0021] Preferably, in step (4), the temperature for dark culture is 21~25℃; more preferably 23℃.
[0022] Preferably, in step (5), the water bath heating is: a dry constant temperature metal bath at 36~38℃ for 5~7 h; more preferably, at 37℃ for 6 h.
[0023] The beneficial effects of this invention are as follows: 1. This invention proposes a method for constructing transient genetic transformation of tea plant callus tissue. Using 'Longjing 43' callus tissue as the transformation recipient, the expression vector selected is pBI121 with a GUS tag, and the Agrobacterium competent cells are GV3101. The effects of suspension culture time, type of suspension culture medium, and co-culture time on GUS expression levels were investigated to screen efficient transient transformation conditions, laying the foundation for a stable genetic transformation system for tea plants. Through exploration and optimization of screening conditions, this invention achieves a 100% success rate in obtaining callus tissue with GUS blue spots.
[0024] 2. This invention proposes a method for transient genetic transformation of tea plant callus mediated by Agrobacterium, specifically comprising: culturing the callus in suspension for a certain period of time, subsequently using it as the recipient material for transformation; utilizing… 35S::GUS Agrobacterium strain GV3101 was used to infect callus tissue. The infected callus tissue was transferred to sterile filter paper and air-dried, then transferred to a co-culture medium for further culture. After co-culture, the callus tissue was identified using GUS histochemical staining, and positive callus tissue was isolated. This invention constructs a transient genetic transformation system for tea plant callus tissue, laying the foundation for a stable genetic transformation system in tea plants.
[0025] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description
[0026] Figure 1 This is a photograph of the tea tree callus tissue cultured in Example 1 of the present invention; Figure 2 The image shows the GUS staining results of tea tree callus tissue in different suspension culture media in Example 1 of this invention. Figure 3 This is a GUS staining result of tea tree callus tissue with different suspension times in Example 1 of the present invention; Figure 4 This is a GUS staining result of tea tree callus tissue at different co-cultivation times in Example 1 of the present invention. Detailed Implementation
[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.
[0028] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.
[0029] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.
[0030] Example 1: A method for the transient transformation and construction of tea plant callus tissue includes the following steps: (1) Callus suspension culture Vigorous, white, and loose callus tissue was selected and placed in liquid suspension culture medium. The culture was conducted in the dark at 24°C and 200 rpm. The callus tissue was 'Longjing 43' embryogenic callus tissue.
[0031] Table 1 Tea tree suspension culture medium
[0032] according to Figure 1 Except for culture medium 1, other bacteria were detected with a GU signal. The intensity of the blue light indicates the strength of the signal, with darker blue light indicating stronger signal.
[0033] (2) Agrobacterium activation and transformation Agrobacterium GV3101 competent cells were removed from a -80℃ freezer and thawed on ice. A plant expression vector plasmid, verified by sequencing, was added to the competent cells, gently mixed, and then incubated on ice for 30 min, frozen in liquid nitrogen for 1 min, and heat-shocked in a 37℃ water bath for 5 min. Subsequently, 800 μL of antibiotic-free LB broth was added, and the cells were incubated at 25℃ with shaking at 200 rpm for 2–4 h for recovery. The recovered bacterial culture was then evenly spread onto LB solid medium containing 50 mg / L kanamycin and incubated in the dark at 25℃ for 2 days to obtain vigorous positive single colonies.
[0034] (3) Preparation of Agrobacterium infection solution The cultured monoclonal Agrobacterium was scraped off with a scalpel and inoculated into untreated MT liquid medium. Then, 100 μM acetosyringone (AS) was added, and the culture was suspended and cultured at 25°C and 180 rpm for 1-2 h. Afterward, the OD of the bacterial culture was measured using a spectrophotometer. 600 Value, when OD 600 When the value is in the range of 0.8 to 1.0, it is used to infect tea tree callus tissue.
[0035] (4) Agrobacterium infection Tea tree callus was placed in Agrobacterium infection solution and vacuumed for 15 min at -0.8 atmospheres. It was then incubated on a shaker at room temperature and 100 rpm for 20 min. After infection, the Agrobacterium infection solution was discarded, and the callus was evenly spread on sterile filter paper, with residual bacterial solution blotted dry. The callus was then inoculated onto co-culture medium (MT + 100 µM AS + 30 g / L sucrose + 3% plant gel) and incubated in the dark at 23°C.
[0036] (5) GUS staining On a sterile laminar flow hood, the co-cultured callus tissue was removed and washed 3-5 times with sterile water (to avoid the influence of residual Agrobacterium on GUS staining results). It was then immersed in prepared GUS staining solution (purchased from Zhongke Ruitai Biotechnology Co., Ltd.), vacuum filtered for 10 min, and placed in a 37℃ dry constant temperature metal bath (water bath device) for 5-7 h. The stained tea tree callus tissue was then destained with 75% ethanol.
[0037] The success of the transformation can be determined by the staining results. A blue stain indicates a successful transformation, while the absence of a blue stain indicates a failed transformation.
[0038] (GUS staining is a detection method to determine whether the gus gene has been transferred into callus tissue. The appearance of GUS blue spots indicates that the gus gene has been transferred into the callus tissue, and the intensity of the blue spots indicates the strength of the transfer. This invention was repeated three times, and the transformation efficiency reached over 90%.)
[0039] The purpose of dyeing followed by decolorization: Some materials, such as stem segments and leaves, contain pigment and appear green, which can negatively impact the observation of staining results after GUS staining. In contrast, the blue spots that are positive for GUS staining are stable and do not fade in ethanol. Therefore, destaining with ethanol removes the pigment from the material itself, making it easier to observe the staining results. Figure 2-4 These are all images showing staining followed by destaining. The GUS staining results for tea tree callus tissue on different suspension culture media are shown below. Figure 2 As shown, the GUS staining results of tea tree callus tissue with different suspension times are as follows: Figure 3 As shown, the GUS staining results of tea tree callus tissue at different co-cultivation times are as follows: Figure 4 (As shown) result (1) Screening of suspension culture medium Based on GUS staining results, using medium No. 2 (MT + 2 mg / L 2,4-D + 0.2 mg / L 6-BA + 2.2 g / L KCl + 40 g / L sucrose) for suspension culture of tea tree callus was more conducive to the genetic transformation of Agrobacterium. Figure 1 ) (2) Optimization of hover time Callus tissue cultured in suspension for more than 20 days showed detectable GUS signals after transformation. Therefore, callus tissue cultured in suspension for more than 20 days was used as a control. Suspension times were set to 4 days, 7 days, 10 days, and 14 days. Based on the GUS staining results ( Figure 2 Tea tree callus tissue cultured in suspension for 10 days was suitable for Agrobacterium transformation.
[0040] (3) Screening of co-cultivation time Tea tree callus tissue infected with Agrobacterium was transferred to co-culture medium (MT + 100 µMAS + 30 g / L sucrose + 3% phytogel) after 20 days of suspension culture and incubated in the dark at 23°C for 3, 5, and 7 days, followed by GUS staining. The results showed that the callus tissue was lightly stained after 3 days of co-culture, and a strong GUS signal was only detected after 5 days. Therefore, the co-culture time for Agrobacterium transformation of tea tree callus tissue should be set to 5 days.
[0041] Example 2: The difference between this embodiment and Embodiment 1 is that: (1) At 23℃ and 250 rpm; (2) In the middle, 26℃, 1d; (3) The concentration of acetylsuccinone was 90 μM, and the suspension culture was carried out at 26℃ and 150 rpm for 1 h; (4) The composition of the co-culture medium is: MT + 90 µM AS + 20 g / L sucrose + 2% plant gel; the temperature for dark culture is 25℃. (5) In the process, a dry constant temperature metal bath at 38℃ is used for 5 hours.
[0042] Example 3: The difference between this embodiment and Embodiment 1 is that: (1) In the middle, 25℃, 150rpm; (2) In the middle, 24℃, 3d; (3) The concentration of acetylsuccinone was 110 μM, and the suspension culture was carried out at 24℃ and 200 rpm for 2 h. (4) The composition of the co-culture medium is: MT + 110 µM AS + 40 g / L sucrose + 5% plant gel; the temperature for dark culture is 21℃. (5) In the case of a dry constant temperature metal bath at 36℃ for 7 h.
[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for the transient transformation and construction of tea plant callus, characterized in that, Includes the following steps: (1) Callus suspension culture Select vigorous, white, and loose callus tissue, place it in liquid suspension culture medium, and culture it in suspension. (2) Agrobacterium activation Single colonies of Agrobacterium strain were picked, streaked on LB solid medium containing kanamycin, and incubated in the dark to obtain vigorous Agrobacterium colonies. (3) Preparation of Agrobacterium infection solution The Agrobacterium obtained in (2) was inoculated into MT liquid medium without antibiotics, and then acetylsuccione was added and cultured in suspension. (4) Agrobacterium infection The tea tree callus obtained in (1) was placed in the Agrobacterium infection solution obtained in (3), vacuumed, and then placed on a shaker at room temperature for inoculation; after the inoculation was completed, the callus was inoculated into a co-culture medium and cultured in the dark. (5) GUS staining Remove the co-cultured callus tissue, wash it, then soak it in GUS staining solution, vacuum filter it, heat it in a water bath, and judge whether the transformation is successful based on the staining.
2. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (1), the conditions for suspension culture are: dark conditions, 23~25℃, 150~250 rpm.
3. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (1), the specific components of the liquid suspension culture medium can be any of the following formulations: Formula 1: MT + (0.5~2) mg / L 2,4-D + (0.05~2) mg / L 6-BA + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 2: MT + (0.5~2) mg / L 2,4-D + (0.05~2) mg / L KT + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 3: MT + (1.5~2.5) mg / L 2,4-D + (2~2.5) g / L KCl + (38~42) g / L sucrose; Formula 4: MT + (2~2.5) g / L KCl + (38~42) g / L sucrose.
4. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (2), the concentration of kanamycin is 45~60 mg / L; the conditions for dark culture are: 24~26℃, 1~3d.
5. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (3), the concentration of acetylsuccinone is 90~110 μM; the suspension culture conditions are: 24~26℃, 150~200 rpm suspension culture for 1-2h.
6. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (4), the immersion conditions are 80~120 rpm for 15~25 min.
7. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (4), the co-culture medium consists of: MT + 90~110 µM AS + 20~40 g / L sucrose + 2~5% plant gel.
8. The method for transient transformation and construction of tea tree callus tissue according to claim 1, characterized in that, In step (4), the temperature for dark culture is 21~25℃.
9. The method for transient transformation and construction of tea plant callus tissue according to claim 1, characterized in that, In step (5), the water bath heating is specifically: a dry constant temperature metal bath at 36~38℃ for 5~7 hours.
10. The method for transient transformation and construction of tea plant callus tissue according to claim 9, characterized in that, In step (5), the water bath heating is specifically: a 37℃ dry constant temperature metal bath for 6 hours.