CDB simple jasmine flower genetic transformation method

Abstract

The invention belongs to the technical field of gene inheritance, and particularly relates to a CDB simple arabian jasmine flower genetic transformation method which comprises the following specific steps: selecting different physiological parts of arabian jasmine flowers as candidate explants, performing scratch pretreatment, setting time gradient for pre-culture, and screening the optimal duration; the method comprises the following steps: screening three agrobacterium adaptive strains, namely AR1193, GV3101 and EHA105, and constructing a vector containing an RUBY visual reporter gene; according to the application, the RUBY reporter gene is adopted, the positive transformant can be directly recognized through red color development under natural light, DNA extraction and PCR detection do not need to be carried out in the early stage, the screening process is simplified, the screening period is shortened, the transformation efficiency can be improved only by lightly scratching an explant with a sterile blade, and complex tissue culture for inducing callus is not needed; the soaking method is adopted for infection, ultrasonic-assisted delivery is matched, operation is convenient and fast, professional high-end equipment is not needed, clear gradients are set for key conditions, optimized parameters can be directly reused, the test exploration cost is reduced, and the method is suitable for being popularized in different laboratories.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic technology, in particular to a CDB simple jasmine genetic transformation method. BACKGROUND

[0002] CDB refers to a cut-dip-budding (Cut-dip-budding) delivery system, which is a plant genetic transformation technology mainly used to realize genetic modification and gene editing of plants. The CDB delivery system uses Agrobacterium rhizogenes to inoculate explants, and realizes gene transformation by means of plant root sprout characteristics without tissue culture and sterile conditions, and is simple to operate. The specific process is to cut the junction of the plant rhizome under non-sterile conditions, take the upper part to be infected with Agrobacterium rhizogenes, and then cultured on vermiculite medium, take the positive new root, cut into segments and culture, and obtain regenerated transformed plants. The current transformation method still has the following technical problems: The current transformation method is difficult to operate, so that the technical threshold in the process is high, and the transformation system is not accurate enough, which greatly affects the efficiency and stability.

[0003] Therefore, the present application provides a CDB simple jasmine genetic transformation method to solve the above problems. SUMMARY

[0004] The present application aims to provide a CDB simple jasmine genetic transformation method to solve the problems in the background art.

[0005] To achieve the above purpose, the present application provides the following technical scheme: a CDB simple jasmine genetic transformation method, the specific step process of which is as follows: Step 1, explant selection and treatment: select different physiological parts of jasmine as candidate explants, pretreat by scratching, set time gradient pre-culture, and screen the best time length; Step 2, selection of Agrobacterium strains and vectors: screen three Agrobacterium strains AR1193, GV3101 and EHA105, construct a vector containing RUBY visual reporter gene, and verify the correctness by PCR; Step 3, optimization of Agrobacterium infection conditions: set OD600 bacterial liquid concentration, infection time gradient, and co-culture temperature combination, and screen the optimal parameters; Step 4, optimization of additive concentration: test the influence of 100-300 μmol / L acetyl-syringone (AS) and 0.02%-0.1% Tween-20 on the transformation efficiency; Step five, establishment of Agrobacterium rhizogenes-mediated transformation system: positive transformants were screened by RUBY gene coloration, and the accuracy was verified by PCR. Three repeated tests (CV≤10%) were performed to ensure the stability of the system. Step six, application and optimization of CDB delivery system: ultrasonic power and time gradient were set for stem tip explants, and the co-culture time was optimized in a dark environment at 25°C. Step seven, verification and functional study of the transformation system: DNA was extracted by CTAB method, and the positive rate was verified by double PCR. Step eight, gene function verification: CRISPR / Cas9 vector was introduced into jasmine flowers, and GC-MS was used to detect the differences in flower fragrance components between edited plants and wild type plants to verify gene function.

[0006] Preferably, the specific steps of step one are as follows: S1, selection of potential explants: select different physiological parts of jasmine flowers as candidate explants, set 3 biological repeats for each explant, and select 50 samples for each repeat. The different physiological parts of jasmine flowers include stem segments, leaves, axillary buds, and tender shoots. S2, explant pretreatment: compare the transformation efficiency of the uncut group, and the specific operation of the cut group is as follows: lightly scratch the surface with a sterile blade to cause slight damage, with a depth of 0.5-1 mm, and scratch 3-5 lines per square centimeter. S3, pre-culture parameter test: set a pre-culture time gradient, place the treated explants in a sterile humid environment for culture, and select the optimal pre-culture time; the pre-culture time gradient is set to 0d, 2d, 4d, and 6d, the humidity of the sterile humid environment is 90%, and the temperature is 25°C.

[0007] Preferably, the specific steps of step two are as follows: S1, strain selection: select Agrobacterium rhizogenes AR1193, GV3101, and EHA105 strains, prepare bacterial solution (OD600=0.6) for each strain, infect the same explants, and count the hairy root induction rate and positive rate after 7 days to select suitable strains. S2, vector construction: select a vector carrying the RUBY visual reporter gene, insert the target gene into the T-DNA region of the vector by enzyme digestion and ligation, transform it into the selected Agrobacterium strain, and verify the correct construction of the vector by PCR.

[0008] Preferably, the specific steps of step three are as follows: S1, bacterial solution concentration gradient: set the OD600 value gradient, process 50 explants for each gradient, count the positive transformation efficiency after infection, and determine the optimal concentration. The OD600 value gradient is set to 0.4, 0.6, 0.8, and 1.0. S2, dipping time test: under the optimal bacteria solution concentration, set the infection time gradient, use the soaking method to dip, compare the transformation effect of different time; the infection time gradient is set to 10 min, 15 min, 20 min, 25 min and 30 min respectively; S3, co-culture parameters: the co-culture temperature is set to 22℃, 25℃, 28℃, the co-culture time is set to 2d, 3d, 4d, the positive rate is observed by RUBY gene coloration after each group treatment, and the optimal temperature combination is selected.

[0009] Preferably, the specific steps of step four are as follows: S1, acetyl-syringone (AS) concentration gradient: add AS in the dipping solution and co-culture environment, the concentration is set to 100 μmol / L, 200 μmol / L, 300 μmol / L, and the group without AS is used as a control to count the transformation efficiency improvement range; S2, surfactant test: add 0.02%, 0.05%, 0.1% Tween-20 to the dipping solution, and compare the influence of the surfactant on the adhesion of Agrobacterium and the transformation efficiency.

[0010] Preferably, the specific steps of step five are as follows: S1, visual selection system operation positive identification: the dipped explants are placed under natural light for observation, the positive transformants are identified by RUBY gene coloration, and the coloration time and coloration intensity are recorded to replace the early screening of traditional molecular detection; S2, screening efficiency verification: the coloration positive explants are subjected to PCR detection, the coloration positive-molecular positive coincidence rate is calculated to ensure the screening accuracy; S3, system stability test repeated transformation test: using the determined explants and conditions, 3 independent transformation tests are continuously carried out, 100 explants are treated each time, the coefficient of variation (CV) of the transformation efficiency is calculated, and CV≤10% is regarded as system stability.

[0011] Preferably, the specific steps of step six are as follows: S1, basic parameter test ultrasonic treatment optimization: set the ultrasonic power and time gradient, perform ultrasonic assisted Agrobacterium infection on the explants of jasmine flower shoot tips, count the transient transformation rate, the ultrasonic power gradient is set to 200W, 300W and 400W respectively, and the time gradient is set to 30s, 60s and 90s respectively; S2, co-culture conditions: after ultrasonic treatment, the explants are co-cultured with Agrobacterium, the time gradient is set, the temperature is kept at 25℃, and the environment is dark, the transformation efficiency change is detected, and the time gradient is set to 1d, 2d and 3d.

[0012] Preferably, the specific steps of step seven are as follows: S1, molecular identification operation DNA extraction: CTAB method is used to extract the genomic DNA of positive transformants, 3 times for each sample, and the purity of the DNA is ensured; S2, double PCR verification: specific primers of target genes and RUBY genes are designed, double PCR amplification is carried out, positive bands are detected by electrophoresis, and the positive rate is calculated.

[0013] Preferably, the specific steps of step eight are as follows: S1, gene editing operation: a CRISPR / Cas9 vector containing a target gene is constructed, and the edited plant is obtained by introducing the vector into jasmine through an optimized transformation system; S2, trait detection: phenotype analysis is performed on the edited plant, such as detecting the content of flower fragrance components by gas chromatography-mass spectrometry (GC-MS), comparing the differences with wild type, and verifying the function of the gene.

[0014] Compared with the prior art, the beneficial effects of the present application are: 1) The present application uses RUBY reporter gene, and the positive transformant can be directly identified by red color under natural light without early DNA extraction and PCR detection, which simplifies the screening process, shortens the screening period, improves the transformation efficiency of explants by only needing to be lightly cut with a sterile blade, and does not need complex tissue culture to induce callus; the infection adopts soaking method combined with ultrasonic assisted delivery, which is convenient to operate and does not need professional high-end equipment, and the key conditions are set with clear gradient, the optimized parameters can be directly reused, the experimental groping cost is reduced, and it is suitable for promotion in different laboratories; 2) The present application optimizes the whole process gradient from explants, strains, infection conditions, additives to delivery system, specifically improves the transformation efficiency of jasmine, avoids blind test, verifies through 3 independent repeated tests, the coefficient of variation (CV) of transformation efficiency is less than or equal to 10%, and 3 biological repeats are set, the data reliability is high, solves the pain point of poor stability of traditional genetic transformation method, visual coloration and double PCR verification linkage, coloration positive-molecular positive coincidence rate is guaranteed, which simplifies early screening, and eliminates false positives through targeted primer PCR, and ensures the purity of transformants. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The step flowchart of the present application. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application. Embodiment

[0017] Please refer to Figure 1 The present application provides a technical solution: A CDB simple jasmine genetic transformation method, the specific steps of the CDB simple jasmine genetic transformation method are as follows: Step one, explant selection and treatment: select different physiological parts of jasmine as candidate explants, after scratch pretreatment, set time gradient pre-culture, and screen the best time length; Step two, selection of agrobacterium strains and vectors: screen three agrobacterium strains AR1193, GV3101 and EHA105, construct a vector containing RUBY visual reporter gene, and verify the correctness by PCR; Step three, optimization of agrobacterium infection conditions: set OD600 bacterial liquid concentration, infection time gradient, and co-culture temperature combination, and screen the optimal parameters; Step four, additive concentration optimization: test the influence of 100-300 μmol / L acetyl syringone (AS) and 0.02%-0.1% Tween-20 on the transformation efficiency; Step five, establishment of Agrobacterium rhizogenes mediated transformation system: screen positive transformants by RUBY gene coloration, verify the accuracy by PCR, and ensure the stability of the system through 3 repeated experiments (CV≤10%); Step six, application and optimization of CDB delivery system: set ultrasonic power and time gradient for stem tip explants, and optimize co-culture time in 25℃ dark environment; Step seven, verification and function research of the transformation system: extract DNA by CTAB method, and verify the positive rate by double PCR; Step eight, gene function verification: construct CRISPR / Cas9 vector to introduce into jasmine, detect the differences in flower fragrance components between edited plants and wild type by GC-MS, and verify the gene function.

[0018] The specific steps of step one are as follows: S1, screen potential explants: select different physiological parts of jasmine as candidate explants, set 3 biological repeats for each explant, and select 50 samples for each repeat, and the different physiological parts of jasmine include stem segments, leaves, axillary buds and tender shoots; S2, explant pretreatment: compare the transformation efficiency difference of the uncut group, and the specific operation of the scratch treatment of the candidate explants is as follows: lightly scratch the surface with a sterile blade to slightly damage, the depth is 0.5-1mm, and scratch 3-5 lines per square centimeter; S3, pre-culture parameter test: set the pre-culture time gradient, place the treated explants in a sterile humid environment for culture, and select the best pre-culture time; the pre-culture time gradient is set to 0d, 2d, 4d, and 6d, the humidity of the sterile humid environment is 90%, and the temperature is 25°C.

[0019] In step one, by multi-site screening and 3 biological repeats (50 samples each time), the explant adaptation range is expanded, the test data reliability is improved, the scratch pretreatment increases the contact area of Agrobacterium with the explant, the efficiency is improved compared with the unscratched group, the gradient pre-culture and standardized environment are used to select the best physiological state of the explant, and the subsequent infection failure rate is reduced; The specific steps of step two are as follows: S1, strain screening: select Agrobacterium rhizogenes AR1193, GV3101 and EHA105 strains, prepare bacterial liquid (OD600=0.6) respectively, and infect the same explants, count the hairy root induction rate and positive rate after 7 days, and select the suitable strain; S2, vector construction: select a vector carrying RUBY visual reporter gene, insert the target gene into the T-DNA region of the vector by enzyme digestion and ligation, transform it into the selected Agrobacterium strain, and verify by PCR to ensure that the vector construction is correct.

[0020] 3 kinds of Agrobacterium are screened for specific selection, matching the genetic characteristics of jasmine flowers, and improving the strain-explant adaptation degree; the vector carrying the RUBY visual reporter gene does not need early molecular detection, and directly identifies positive transformants through color development, simplifying the screening process; enzyme digestion and ligation construction and PCR verification ensure that the target gene is correctly inserted into the T-DNA region, reducing the risk of invalid transformation.

[0021] The specific steps of step three are as follows: S1, bacterial liquid concentration gradient: set the OD600 value gradient, process 50 explants per gradient, count the positive transformation efficiency after infection, and determine the best concentration, the OD600 value gradient is set to 0.4, 0.6, 0.8 and 1.0; S2, immersion time test: under the best bacterial liquid concentration, set the infection time gradient, use the immersion method for immersion, and compare the transformation effects of different times; the infection time gradient is set to 10min, 15min, 20min, 25min and 30min; S3, co-culture parameters: the co-culture temperature is set to 22°C, 25°C and 28°C, the co-culture time is set to 2d, 3d and 4d, the positive rate is observed by RUBY gene color development after each group is treated, and the optimal temperature combination is selected.

[0022] Bacterial liquid concentration (OD600=0.4 / 0.6 / 0.8 / 1.0) and infection time (10-30min) gradient optimization, balance Agrobacterium activity and explant tolerance; co-culture temperature combination (22 / 25 / 28℃, 2-4d) and RUBY color screening, find the optimal infection environment, reduce explant browning death.

[0023] The specific steps of step four are as follows: S1, acetyl eugenitol (AS) concentration gradient: add AS in the immersion liquid and co-culture environment, the concentration is set to 100 μmol / L, 200 μmol / L, 300 μmol / L, and the no-AS group is used as a control, and the transformation efficiency improvement amplitude is counted. S2, surfactant test: add 0.02%, 0.05%, 0.1% Tween-20 to the immersion liquid, and compare the influence of the surfactant on the adhesion of Agrobacterium and the transformation efficiency.

[0024] Acetyl eugenitol promotes Agrobacterium T-DNA transfer, and the concentration, synergistic correlation are determined; Tween-20 improves the adhesion ability of Agrobacterium, and the optimal dosage is determined through comparison tests to avoid excessive damage to the explants caused by additives.

[0025] The specific steps of step five are as follows: S1, visual screening system operation positive identification: after immersion and infection, the explants are placed under natural light for observation, and the positive transformants are identified by RUBY gene coloration (red spot area), the coloration time and coloration intensity are recorded, and the early screening of traditional molecular detection is replaced; S2, screening efficiency verification: PCR detection (primers are RUBY gene specific primers) is performed on the coloration positive explants, the coloration positive-molecular positive coincidence rate is calculated, and the screening accuracy is ensured; S3, system stability test repeated transformation test: using the determined explants and conditions, 3 independent transformation tests are continuously carried out, 100 explants are treated each time, the variation coefficient (CV) of the transformation efficiency is counted, and CV≤10% is regarded as system stability.

[0026] Visual screening replaces traditional molecular detection, shortens the screening cycle, and reduces the technical threshold; coloration positive and PCR verification linkage ensure the accuracy of positive identification and reduce false positive interference; 3 independent repeated tests and CV≤10% standard ensure the stability of the system, which is convenient for reuse in different laboratories.

[0027] The specific steps of step six are as follows: S1, basic parameter test ultrasonic treatment optimization: set ultrasonic power and time gradient, ultrasonic assisted Agrobacterium infection of jasmine stem tip explants, count the transient transformation rate, the gradient of ultrasonic power is set to 200W, 300W and 400W, the time gradient is set to 30s, 60s and 90s; S2, co-culture conditions: after ultrasonic treatment, the explants are co-cultured with Agrobacterium, the time gradient is set, the temperature is kept at 25℃, the dark environment, the change of transformation efficiency is detected, the time gradient is set to 1d, 2d and 3d.

[0028] Ultrasonic parameter gradient assisted infection, enhance the penetration of Agrobacterium to the stem tip, improve the transient transformation rate; 25℃ dark environment and co-culture gradient, adapt to the recovery needs of explants after ultrasonic treatment, further improve the transformation success rate.

[0029] The specific steps of the step seven are as follows: S1, molecular identification operation DNA extraction: CTAB method is used to extract the genomic DNA of positive transformants, 3 times for each sample, to ensure the purity of DNA (OD260 / 280=1.8-2.0); S2, double PCR verification: design specific primers for target genes and RUBY genes, perform double PCR amplification (annealing temperature 55℃, 35 cycles), electrophoresis detection of positive band, calculate the positive rate.

[0030] CTAB method is used to extract DNA, which ensures the quality of nucleic acid and provides a reliable template for subsequent identification; double PCR and standardized amplification conditions can accurately eliminate false positives and determine the final transformation efficiency; The specific steps of the step eight are as follows: S1, gene editing operation: construct CRISPR / Cas9 vector containing target gene, introduce into jasmine flower through optimized transformation system, and obtain edited plants; S2, trait detection: phenotype analysis of edited plants, such as GC-MS detection of flower fragrance components, comparison of differences with wild type, verification of gene function.

[0031] Compatible with CRISPR / Cas9 gene editing, quickly obtain edited plants through optimized system, meet the demand of genetic improvement; GC-MS detection of flower fragrance components and other phenotypic differences, direct verification of target gene function, provide direct basis for jasmine flower quality improvement.

[0032] The foregoing illustrates and describes embodiments of the present application. However, it is to be understood that the application is not limited to the above-described embodiments, and that various changes and modifications can be suggested to one skilled in the art. It is intended that the present application encompass any and all such changes and modifications without departing from the spirit or scope of the underlying principles of the application. In addition, it should be understood that the figures are not necessarily drawn to scale and that, where appropriate, reference numerals have been maintained from one figure to another and from one embodiment to another in order to illustrate similar constructions.

[0033] While the application has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, since the scope of the application includes not only the embodiments presently described and illustrated, but also other embodiments which are within its spirit and scope. For a clearer understanding of the present application, other and further objects thereof, together with various features of the application, reference is made to the following claims.

Claims

1. A simplified CDB method for genetic transformation of jasmine, characterized in that, The specific steps of this CDB simplified jasmine genetic transformation method are as follows: Step 1: Explant selection and treatment: Different physiological parts of jasmine flowers were selected as candidate explants. After pretreatment by scratching, they were pre-cultured in time gradients to screen for the optimal duration. Step 2: Selection of Agrobacterium strains and vectors: Three Agrobacterium strains, AR1193, GV3101, and EHA105, were screened to select suitable strains. A vector containing the RUBY visual reporter gene was constructed, and its correctness was verified by PCR. Step 3: Optimization of Agrobacterium infection conditions: Set the OD600 bacterial concentration, infection time gradient, and co-culture temperature and time combination to screen the optimal parameters; Step 4: Optimization of additive concentration: Test the effect of 100-300 μmol / L acetylsuccinone (AS) and 0.02%-0.1% Tween-20 on conversion efficiency; Step 5: Establishment of Agrobacterium rhizogenes-mediated transformation system: Positive transformants were screened by RUBY gene colorimetric detection, and the accuracy was verified by PCR. The system was stabilized by three replicate experiments (CV≤10%). Step 6: Application and optimization of the CDB delivery system: Ultrasonic power and time gradient were set for shoot tip explants, and the co-culture time was optimized in a dark environment at 25℃. Step 7: Validation and functional study of the transformation system: DNA was extracted using the CTAB method, and the positive rate was verified by double PCR; Step 8: Gene function verification: A CRISPR / Cas9 vector was constructed and introduced into jasmine flowers. GC-MS was used to detect the differences in fragrance components between the edited plants and the wild type to verify gene function.

2. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step one are as follows: S1. Screening potential explants: Different physiological parts of jasmine flowers were selected as candidate explants. Each explant was set up with 3 biological replicates, and 50 samples were processed in each replicate. Different physiological parts of jasmine flowers were selected, including stem segments, leaves, axillary buds, and tender shoots. S2. Explant pretreatment: The candidate explants were scratched to compare the transformation efficiency of the unscratched group. The specific procedure for scratching the candidate explants is as follows: Use a sterile blade to lightly scratch the surface to cause minor damage, with a depth of 0.5-1 mm, and make 3-5 scratches per square centimeter. S3. Pre-culture parameter test: Set the pre-culture time gradient, place the treated explants in a sterile and humid environment for culture, and screen the optimal pre-culture time; set the pre-culture time gradients as 0d, 2d, 4d, and 6d, with a humidity of 90% and a temperature of 25℃ in the sterile and humid environment.

3. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step two are as follows: S1. Strains screening: Agrobacterium rhizogenes strains AR1193, GV3101 and EHA105 were selected, and bacterial solutions (OD600=0.6) were prepared for each strain. The same explant was then infected, and the hairy root induction rate and positive rate were counted after 7 days to screen suitable strains. S2. Vector construction: A vector carrying the RUBY visual reporter gene was selected. The target gene was inserted into the T-DNA region of the vector by enzyme digestion and ligation. The vector was then transformed into the selected Agrobacterium strain. PCR verification was performed to ensure that the vector construction was correct.

4. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step three are as follows: S1. Bacterial concentration gradient: Set up an OD600 value gradient, with 50 explants treated per gradient. Calculate the positive transformation efficiency after infection to determine the optimal concentration. The OD600 value gradients were set to 0.4, 0.6, 0.8 and 1.0, respectively. S2. Infection time test: Under the optimal bacterial concentration, an infection time gradient was set, and the immersion method was used for infection to compare the transformation effect at different times; the infection time gradients were set to 10 min, 15 min, 20 min, 25 min and 30 min respectively; S3. Co-culture parameters: Co-culture temperatures were set to 22℃, 25℃, and 28℃, and co-culture times were set to 2d, 3d, and 4d. After each treatment, the positive rate was observed by RUBY gene colorimetry to screen the optimal temperature and time combination.

5. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step four are as follows: S1, Acetyleugenol (AS) concentration gradient: AS was added to the inoculum and co-culture environment at concentrations of 100 μmol / L, 200 μmol / L, and 300 μmol / L, with the group without AS as the control, and the improvement in transformation efficiency was statistically analyzed. S2. Surfactant test: Add 0.02%, 0.05%, and 0.1% of Tween-20 to the dyeing solution and compare its effects on Agrobacterium attachment and conversion efficiency.

6. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step five are as follows: S1. Visualized screening system for positive identification: After infection, the explants are placed under natural light for observation. Positive transformants are identified by RUBY gene color development. The color development time and intensity are recorded, replacing the early screening of traditional molecular detection. S2. Screening efficiency verification: Perform PCR detection on chromogenic positive explants, calculate the concordance rate between chromogenic positive and molecular positive to ensure screening accuracy; S3. System stability test: Repeated transformation experiment: Using the determined explants and conditions, conduct 3 consecutive independent transformation experiments, treating 100 explants each time, and calculate the coefficient of variation (CV) of transformation efficiency. The system is considered stable if CV ≤ 10%.

7. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step six are as follows: S1. Basic parameter testing and ultrasonic processing optimization: Set ultrasonic power and time gradient, perform ultrasonic-assisted Agrobacterium infection on jasmine stem tip explants, and count the instantaneous conversion rate. The ultrasonic power gradient was set to 200W, 300W and 400W respectively, and the time gradient was set to 30s, 60s and 90s respectively. S2. Co-culture conditions: After sonication, the explants were co-cultured with Agrobacterium, with a time gradient set, the temperature maintained at 25℃, and the environment in darkness. The transformation efficiency was detected, with the time gradient set to 1d, 2d, and 3d.

8. The CDB simplified jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step seven are as follows: S1. Molecular identification procedure: DNA extraction: Genomic DNA of positive transformants was extracted using the CTAB method, with each sample extracted 3 times to ensure DNA purity; S2. Double PCR verification: Design specific primers for the target gene and the RUBY gene, perform double PCR amplification, detect positive bands by electrophoresis, and calculate the positive rate.

9. The simplified CDB jasmine genetic transformation method according to claim 1, characterized in that: The specific steps of step eight are as follows: S1. Gene editing operation: Construct a CRISPR / Cas9 vector containing the target gene, introduce it into jasmine through an optimized transformation system, and obtain edited plants; S2. Phenotypic analysis: Phenotypic analysis of edited plants, such as using gas chromatography-mass spectrometry (GC-MS) to detect the content of floral fragrance components, compare the differences with wild type, and verify gene function.

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