A method for the instantaneous conversion of a dyeing solution and camphor trees.

Abstract

This invention discloses an inoculum and a method for the instantaneous transformation of camphor trees, relating to the field of instantaneous transformation technology of camphor trees. The method for the instantaneous transformation of camphor trees is characterized by: camphor tree cultivation, taking camphor leaves; the camphor leaves being washed and then inoculated with the aforementioned inoculum. The viability of Agrobacterium varies significantly at different growth stages. The amplified bacterial solutions at different growth stages are centrifuged, and the concentration of the inoculum is adjusted using a lysate. After inoculating the camphor leaves, they are placed in an incubator for co-cultivation, and the Ca2+ level is measured. 2+ The fluorescence intensity analysis clarified key technologies such as suitable infection methods, Agrobacterium state, infection solution concentration, and co-culture time for camphor trees, which is of great value for promoting the smooth development of molecular biology research and molecular breeding of camphor trees.

Description

Technical Field

[0001] This invention relates to the field of camphor tree instantaneous conversion technology, specifically to a staining solution and a method for instantaneous conversion of camphor tree. Background Technology

[0002] Camphor tree (Cinnamomum camphora) is an excellent evergreen broad-leaved tree in subtropical regions. It serves not only as a street tree and landscaping tree but also has various economic uses, including medicinal purposes, timber production, and the extraction of essential oils. Currently, a preliminary regeneration system for camphor tree has been established; however, progress in its molecular research has been very slow. Although there are reports on Agrobacterium infection of camphor callus tissue, these studies suffer from poor stability and lengthy, time-consuming transformation processes. Therefore, a mature and efficient genetic transformation system for camphor tree has not yet been established. This limits the ability to deeply elucidate the formation mechanisms of many desirable traits of camphor tree from a molecular biological perspective, identify related functional genes, and improve camphor tree from a molecular breeding perspective to enhance its medicinal and essential oil content and expand its ecological adaptability.

[0003] Transient transformation technology refers to the introduction of a target gene into recipient cells to establish a temporary, highly efficient expression system, thereby enabling the target gene to be expressed in large quantities within a relatively short period of time. When using plant transient technology to express recombinant proteins, the transformed gene does not need to be integrated into the plant genome, thus saving the lengthy processes of genetic transformation and selection, and achieving target protein expression in a short period of time. Therefore, compared with stable expression, transient transformation has the advantages of simplicity, speed, short cycle, high efficiency, and strong biosafety.

[0004] Based on the current status of genetic transformation of camphor trees and the advantages of instantaneous transformation technology, this invention provides an inoculum and a method for instantaneous transformation of camphor trees. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for the instantaneous conversion of a dyeing solution and camphor trees, solving at least one technical problem mentioned in the background art.

[0007] (II) Technical Solution

[0008] The technical solution adopted in this invention is:

[0009] On one hand, the present invention provides a staining solution, the preparation method of which includes:

[0010] Will contain Ca 2+ Agrobacterium GV3101 with the fluorescent indicator protein gene was rejuvenated by streaking on LB plates containing kanamycin, rifampin, and streptomycin;

[0011] Single colonies were picked and activated in LB liquid medium containing kanamycin, rifampin and streptomycin until the bacterial culture became turbid;

[0012] Aspirate bacterial culture into culture medium to amplify to OD. 600 The value is 0.3-1.1;

[0013] After centrifugation, the bacterial cells were collected and dissolved to the infective concentration using a lysate containing sucrose, Silwet-77, and MES.

[0014] Preferably, the LB plate contains 50 mg·L⁻¹ -1 Kanamycin, 25 mg / L -1 Rifampin and 25 mg / L -1 Streptomycin.

[0015] Preferably, the centrifugation is performed at 4000-6000 rpm for 10-15 minutes.

[0016] Preferably, a single colony is activated in LB liquid medium for 18 hours until the bacterial solution becomes turbid.

[0017] Preferably, the amount of sucrose added is 5 g·L. -1 The addition amount of Silwet-77 is 0.04% and the addition amount of MES lysate is 0.1M, and the pH of the MES lysate is 5.7.

[0018] On the other hand, the present invention also provides a method for instantaneous conversion of camphor trees, the method comprising:

[0019] The above-mentioned infiltration solution was used;

[0020] Cultivation of camphor trees, using camphor leaves;

[0021] After the camphor leaves were cleaned, they were infected with the above-mentioned inoculum and co-cultured.

[0022] Preferably, the leaf infection method is injection infection or immersion infection.

[0023] Preferably, the bacterial culture is amplified to OD in a culture medium. 600 It is 0.5-0.7.

[0024] Preferably, the concentration OD of the inoculum is... 600 It is 0.7-0.8.

[0025] Preferably, the co-cultivation time is 24-48 hours.

[0026] (III) Beneficial Effects

[0027] This invention provides a method for the instantaneous conversion of a dyeing solution and camphor trees, which has the following advantages compared with the prior art:

[0028] 1. The infection solution and instantaneous transformation method for camphor trees provided in this invention show significant differences in the viability of Agrobacterium at different growth stages. The amplified bacterial solutions at different growth stages are centrifuged, and the concentration of the infection solution is adjusted using a lysate. After infecting camphor leaves, they are co-cultured in an incubator, and the Ca2+ activity is measured. 2+ The fluorescence intensity analysis clarified key technologies such as suitable infection methods, Agrobacterium state, infection solution concentration, and co-culture time for camphor trees, which is of great value for promoting the smooth development of molecular biology research and molecular breeding of camphor trees. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0030] Figure 1 Before and after comparison images. Note: A: Untreated; B: Scratches; C: Injection infection; D: Immersion infection;

[0031] Figure 2 Fluorescence images of camphor leaves under different infection methods. Note: A: Injection infection; B: Immersion infection;

[0032] Figure 3 Fluorescence intensity of camphor leaves under different infection methods. Note: Different lowercase letters indicate significant differences at the P<0.05 level;

[0033] Figure 4 Fluorescence images of camphor tree leaves at different co-culture times. Note: A: Uninfected; B: Infected for 12 h; C: Infected for 24 h; D: Infected for 48 h; E: Infected for 60 h; F: Infected for 72 h.

[0034] Figure 5 Fluorescence intensity of camphor leaves at different co-culture times. Note: Different lowercase letters indicate significant differences at the P<0.05 level;

[0035] Figure 6 Fluorescence intensity of camphor leaves after infection with different concentrations of inoculum. Note: Different lowercase letters indicate significant differences at the P<0.05 level;

[0036] Figure 7 Fluorescence intensity of camphor leaves infected with Agrobacterium in different states. Note: Different lowercase letters indicate significant differences at the P<0.05 level;

[0037] Figure 8 camphor leaf Ca 2+ Fluorescence images;

[0038] Figure 9 camphor leaf Ca 2+Fluorescence intensity. Note: Different lowercase letters indicate significant differences at the P<0.05 level. Specific implementation methods

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. 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.

[0040] To better understand the above technical solution, the following will provide a detailed description of the technical solution in conjunction with the accompanying drawings and specific implementation methods:

[0041] In a first aspect, embodiments of the present invention provide an infiltration solution, the preparation method of which includes:

[0042] Will contain Ca 2+ Agrobacterium GV3101 with the fluorescent indicator protein gene was rejuvenated by streaking on LB plates containing kanamycin, rifampin, and streptomycin;

[0043] Single colonies were picked and activated in LB liquid medium containing kanamycin, rifampin and streptomycin until the bacterial culture became turbid;

[0044] Aspirate bacterial culture into culture medium to amplify to OD. 600 The value is 0.3-1.1;

[0045] After centrifugation, the bacterial cells were collected and dissolved to the infective concentration using a lysate containing sucrose, Silwet-77, and MES.

[0046] In a first aspect, embodiments of the present invention also provide a method for the instantaneous transformation of camphor trees, the method comprising:

[0047] The above-mentioned infiltration solution was used;

[0048] Cultivation of camphor trees, using camphor leaves;

[0049] After the camphor leaves were cleaned, they were infected with the above-mentioned inoculum and co-cultured.

[0050] The following detailed explanation is provided with reference to specific embodiments:

[0051] Camphor cultivation

[0052] Select 2-3 year old camphor seedlings and cultivate them in an incubator under the following conditions: light intensity of 100-200 μmol / m². -2 s -1The photoperiod was 14 hours of light / 10 hours of darkness, the temperature was 28℃ (light) / 25℃ (dark), and the humidity was 60-70%. After acclimatization for 7-15 days, the 4th to 8th leaves from top to bottom were selected for the instantaneous conversion experiment.

[0053] Preparation of Infection Solution

[0054] Will contain Ca 2+ Agrobacterium GV3101 containing the fluorescent indicator protein gene (GCaMP3) was subjected to a solution of 50 mg·L⁻¹. -1 Kanamycin, 25 mg / L -1 Rifampin, 25 mg·L -1 Streptomycin was streaked onto LB agar plates for rejuvenation. Single colonies were picked and activated in 10 ml of LB liquid medium containing the same antibiotic for 18 hours until the culture became distinctly turbid. 200 μL of the bacterial culture was then transferred to 20 ml of LB medium for amplification. After centrifugation at 4000-6000 rpm for 10-15 min, the bacterial cells were collected and cultured with a solution containing 5 g / L of streptomycin. -1 Sucrose, 0.04% Silwet-77, and 0.1M MES lysate (pH 5.7) were used to dissolve the final bacterial solution to the infect concentration.

[0055] Leaf infection

[0056] Injection infection

[0057] Wipe camphor leaves twice with sterile water. Gently scrape away the cuticle on the back of the leaf with a syringe needle to create obvious scratches, without damaging the underlying epidermis. Draw up the inoculum using a needle-free syringe and inject it into the scratched area, about 2 cm deep. 2 Approximately 150-200 μL of infection solution is needed to inoculate the leaf area. Successful penetration is indicated when the injection site darkens in color. Figure 1 The entire leaf was wrapped in aluminum foil to keep it in the dark, and then the camphor plant was placed in an incubator to continue culturing so that Agrobacterium could successfully infect the leaf cells.

[0058] Immersion and contamination

[0059] After washing camphor leaves with sterile water, gently scrape the underside of the leaf about one-third of the way from the base and about 0.4 cm from the main vein on both sides with a blade to remove the cuticle, without damaging the lower epidermis. Immerse the area with the cuticle removed in 10-20 mL of infection solution for 5-8 minutes to encourage Agrobacterium to penetrate the leaf. Successful penetration is indicated when the leaf color darkens. Figure 1 After soaking, wash the leaves with sterile water and blot off the surface water with filter paper. Then wrap the entire leaf with aluminum foil to keep it in the dark. Place the camphor plant in an incubator to continue culturing so that Agrobacterium can successfully infect the leaf cells.

[0060] in, Figure 1 A: Untreated; B: Scratches; C: Injection contamination; D: Immersion contamination.

[0061] Determining the optimal co-culture time, infection concentration, and cell state

[0062] In Agrobacterium-mediated transient transformation, the co-culture time between Agrobacterium and plant material, the concentration of the infection medium, and the cell state are important factors affecting transient transformation. To determine the optimal co-culture time, the OD after amplification was measured. 600 The bacterial culture was centrifuged at 0.7 μL, and the OD value was measured. 600 Camphor tree leaves were infected with a 0.6% solution and incubated in an incubator for 12, 24, 48, 60, and 72 hours. The Ca2+ levels were measured. 2+ Fluorescence intensity was used to determine the optimal co-culture time.

[0063] To determine the optimal concentration of the infection solution, the OD after amplification was... 600 The bacterial culture was centrifuged to a concentration of 0.6, and then the OD of the infection solution was determined using the lysate. 600 The concentrations were adjusted to 0.5, 0.6, 0.7, 0.8, and 0.9 respectively, and then used to infect camphor leaves. The Ca2+ levels after 48 hours of co-cultivation were measured. 2+ Fluorescence intensity is used to determine the optimal concentration of the inoculum.

[0064] Agrobacterium viability varies significantly at different growth stages. To clarify the impact of Agrobacterium viability on infection efficacy, the OD400 values ​​after amplification were analyzed. 600 Bacterial solutions with concentrations of 0.3, 0.5, 0.7, 0.9, and 1.1 were centrifuged and adjusted to OD values ​​using lysate. 600 The infection solution was 0.7%. After infecting camphor leaves, they were placed in an incubator and co-cultured for 48 hours. The Ca2+ concentration was measured. 2+ Fluorescence intensity is used to determine the optimal cell state.

[0065] High temperature on Ca 2+ Detection of the expression effect of fluorescent indicator protein genes

[0066] High temperature can induce calcium in plant cell cytoplasm 2+ Increased concentration of calcium in plants induces a response to heat stress through signal transduction. Therefore, we subjected camphor trees to heat stress after rapid transformation and measured the calcium concentration. 2+ After the fluorescent indicator protein gene is expressed, it interacts with cytoplasmic Ca2+. 2+ The effectiveness of this instantaneous rotation technique can be assessed by combining the observed fluorescence changes.

[0067] For OD 600 The bacterial suspension was centrifuged to 0.5 μL, and then adjusted to OD using lysate. 600The concentration of the inoculum was 0.7 to prepare the infection solution. Three to four leaves of each plant were infected and cultured in an incubator (28℃) for 48 hours. The temperature was then rapidly increased to 40℃ and maintained for 30 minutes as a high-temperature treatment, while the temperature was treated at 28℃ for 30 minutes as a control. The fluorescence intensity of the two was then measured.

[0068] Fluorescence observation and fluorescence intensity measurement

[0069] Contains Ca 2+ Agrobacterium, containing the fluorescent indicator protein gene (GCaMP3), can express Ca after successfully infecting leaf cells. 2+ Fluorescent indicator protein, which interacts with cytoplasmic calcium 2+ The infected material exhibits green fluorescence upon binding. Therefore, changes in fluorescence intensity can be used to detect the infection effect. Infected areas were cut and temporary slides were prepared. Excitation with blue light (488 nm) was performed, and the green fluorescence (emission wavelength 507 nm) was observed and photographed using a fluorescence microscope or laser confocal microscope. ImageJ software was used to analyze the fluorescence intensity of the camphor leaves, and one-way ANOVA was performed and plotted using OriginPro 8.5.

[0070] Experimental results

[0071] Effect of infection method on fluorescence intensity

[0072] Using OD 600 After 48 hours of injection and immersion in a 0.9% solution to infect camphor leaves, obvious fluorescence was observed in both cases. Figure 2 ), and there was no significant difference in fluorescence intensity among them. Figure 3 This indicates that both infection methods are applicable to the instantaneous transformation of camphor trees. Figure 2 Note: A: Injection infection; B: Immersion infection. Figure 3 Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0073] Effect of co-culture time on fluorescence intensity

[0074] Before infection, camphor leaves did not exhibit green fluorescence; after 12 hours of infection, green fluorescence began to appear, gradually increasing in intensity, reaching its peak at 48 hours, but showing no significant difference compared to 24 hours. Subsequently, the fluorescence intensity gradually weakened, and by 72 hours, the fluorescence had completely disappeared. Figure 4 , Figure 5 Therefore, 24-48 hours is the optimal co-culture time, during which the fluorescence intensity is strongest and most suitable for the determination and analysis of relevant indicators after transient transformation. Figure 4 Note: A: Not infected; B: Infected for 12 hours; C: Infected for 24 hours; D: Infected for 48 hours; E: Infected for 60 hours; F: Infected for 72 hours. Figure 5Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0075] Effect of dye concentration on fluorescence intensity

[0076] Using OD 600 After 48 hours of infection with inoculants of 0.5, 0.6, 0.7, 0.8, and 0.9 g, the fluorescence intensity of camphor leaves first increased and then decreased with the concentration of the inoculant. The fluorescence intensity at different inoculant concentrations (OD) was observed. 600 The fluorescence intensity reaches its maximum value at a value of 0.7, but it is related to the OD. 600 There was no significant difference when the value was 0.8, which indicates that OD 600 The optimal concentration of the inoculum for the instantaneous transformation of camphor trees is 0.7-0.8. Figure 6 ). Figure 6 Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0077] Effect of cell state on fluorescence intensity

[0078] At bacterial concentration OD 600 At concentrations of 0.5 and 0.7, the fluorescence intensity after infecting camphor leaves was the highest, with no significant difference between the two concentrations. However, the fluorescence intensity at other concentrations decreased significantly (P<0.05), and at OD... 600 The values ​​were lowest at 0.3 and 1.1, indicating that Agrobacterium growth to OD values ​​was at which point the value was lowest. 600 The activity and infection effect are strongest at a concentration of 0.5-0.7. Figure 7 ). Figure 7 Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0079] High temperature affects the Ca2+ in camphor leaves 2+ Effect of fluorescence intensity

[0080] High temperature stress can induce cytoplasmic calcium 2+ Concentration increases. For instantaneous Ca... 2+ Camphor tree leaves treated with the fluorescent binding protein gene were subjected to 40°C high-temperature stress for 30 minutes, resulting in increased Ca2+ levels. 2+ The fluorescence intensity was significantly enhanced (P<0.05). Figure 8 ), an increase of 20% compared to the control ( Figure 9 This demonstrates that this instantaneous conversion technology has excellent application results. Figure 8 Note: A: Control, treated at room temperature (28℃); B: Treated at high temperature (40℃). Figure 9 Note: Different lowercase letters indicate significant differences at the P<0.05 level.

[0081] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0082] 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 instantaneous conversion of camphor trees, characterized in that: The instantaneous conversion method for camphor trees includes: To cultivate camphor trees, take camphor leaves and scrape off the cuticle. After the camphor leaves were cleaned, they were infected with a dyeing solution and then co-cultured. Methods for preparing the infiltration solution include: Will contain Ca 2+ Agrobacterium GV3101 with the fluorescent indicator protein gene was rejuvenated by streaking on LB plates containing kanamycin, rifampin, and streptomycin; Single colonies were picked and activated in LB liquid medium containing kanamycin, rifampin and streptomycin until the bacterial culture became turbid; Aspirate bacterial culture into culture medium to amplify to OD. 600 The value is 0.3-1.1; After centrifugation, the bacterial cells were collected and dissolved to the infective concentration using a lysate containing sucrose, Silwet-77, and MES. Using 5g·L -1 Sucrose, 0.04% Silwet-77, and 0.1M MES lysate were used to dissolve the bacteria to the infective concentration, and the pH of the MES lysate was 5.

7. After the cuticle was scraped off, the camphor leaves were washed and then infected with the above-mentioned inoculum and co-cultured.

2. The method for instantaneous conversion of camphor trees according to claim 1, characterized in that: The leaves are infected by either injection or immersion.

3. The instantaneous conversion method for camphor trees according to claim 1, characterized in that: Aspirate bacterial culture into culture medium to amplify to OD. 600 It is 0.5-0.

7.

4. The instantaneous conversion method for camphor trees according to claim 1, characterized in that: Concentration of the infiltration solution (OD) 600 It is 0.7-0.

8.

5. The instantaneous conversion method for camphor trees according to claim 1, characterized in that: The co-cultivation time is 24-48 hours.

6. The method for instantaneous conversion of camphor trees according to claim 1, characterized in that: The LB plate contains 50 mg·L⁻¹ -1 Kanamycin, 25 mg / L -1 Rifampin and 25 mg / L -1 Streptomycin.

7. The instantaneous conversion method for camphor trees according to claim 1, characterized in that: The centrifugation is performed at 4000-6000 rpm for 10-15 minutes.

8. The method for instantaneous conversion of camphor trees according to claim 1, characterized in that: Single colonies were activated in LB liquid medium for 18 hours until the bacterial culture became turbid.

Images