Tagetes erecta genetic transformation method

Abstract

The invention discloses a marigold genetic transformation method which comprises the following steps: constructing an overexpression vector and transforming and identifying agrobacterium competent cells, in the transformation of the agrobacterium competent cells, mixing an agrobacterium transformation solution of the constructed expression vector with seeds, putting the mixture into a gas storage tank, covering a cover, and storing the mixture in the gas storage tank; and carrying out vacuumizing infection treatment by using an oil-free air compressor. According to the marigold genetic transformation method, the gas storage tank is adopted, and the innovation lies in that continuous and stable gas supply can be provided for various culture environments, especially high-sensitivity experiments such as marigold genetic transformation, with unprecedented precision; compared with a traditional method, the marigold genetic transformation process adopting the innovative air storage tank system can obviously improve the obtaining rate of transgenic seedlings, optimizes the hereditary characteristics and growth quality of the marigold, and opens up a new path for research and application in the field of agricultural biotechnology.

Description

Technical Field

[0001] The invention relates to the field of molecular biology of garden plants, in particular to a marigold genetic transformation method. Background Art

[0002] Marigold (Tagetes erecta L.) is an annual herbaceous plant of the genus Tagetes in the Asteraceae family. It is native to Mexico and is widely planted throughout China. It is often used in flower beds, flower borders, and cut flower production. It is also a functional flower for extracting lutein. Because of its bright color, long flowering period, and strong adaptability, it occupies an important position in the field of ornamental gardening. The flower has the effects of clearing away heat and detoxifying, resolving phlegm and relieving cough, and the root detoxifies and reduces swelling. In recent years, with the increasing diversification of market demand for flowers, the improvement and innovation of marigold varieties has become a research hotspot. Traditional marigold breeding mainly relies on natural variation and artificial hybridization, but these methods have limitations such as long breeding cycles and limited genetic variation. In the prior art, the success rate of genetic transformation of marigold is very low, and it is urgent to develop a method that can improve the transformation rate. Summary of the invention

[0003] The technical problem to be solved by the invention is to provide a marigold genetic transformation method which can improve the transformation rate and is easy to operate.

[0004] The marigold genetic transformation method of the invention comprises the construction of an overexpression vector and the transformation and identification of Agrobacterium competent cells. In the transformation of the Agrobacterium competent cells, the Agrobacterium transformation liquid of the constructed expression vector is mixed with seeds, put into an air storage tank, covered with a lid, and vacuumed and infected by using an oil-free air compressor.

[0005] In the marigold genetic transformation method of the present invention, the pressure of the gas storage tank is 0.6 MPa, the treatment time is set to 12 hours, the temperature is room temperature, and the pressurized treatment is performed in a dark gas storage tank.

[0006] The marigold genetic transformation method of the present invention comprises the following steps: after the vacuum treatment is completed, the valve is opened to slowly release the gas, and after the gas is exhausted, the heavy suspension in the culture bottle is replaced with clean water and washed twice; the infected seeds are placed in a constant temperature incubator, the temperature is set to 22° C., and the light cycle is 16 hours of light / 8 hours of darkness; after culturing in the incubator for 2 days, the seeds are taken out and dried, and then planted in the soil.

[0007] The marigold genetic transformation method of the present invention, wherein the method for constructing the overexpression vector specifically comprises the following steps:

[0008] (1) Double restriction enzyme digestion of pMDC85 vector;

[0009] (2) Amplification of the TeWRKY1 target fragment;

[0010] (3) Construction of expression vectors using seamless cloning technology;

[0011] (4) Transformation;

[0012] (5) Screening of positive clones and verification of bacterial culture by PCR.

[0013] The genetic transformation method of marigold of the present invention, wherein the transformation and identification method of Agrobacterium competent cells comprises the steps of transforming Agrobacterium competent cells with overexpression vectors and transforming marigold 'No. 65' mediated by Agrobacterium.

[0014] In the marigold genetic transformation method of the present invention, the method of transforming Agrobacterium competent cells with the overexpression vector comprises transforming Agrobacterium EHA105 competent cells by heat shock transformation method and identifying the recombinant Agrobacterium.

[0015] The method for genetic transformation of marigold of the present invention, wherein the method for Agrobacterium-mediated transformation of marigold 'No. 65' specifically comprises the following steps:

[0016] (1) Collect mature and plump seeds of Marigold 'No. 65', ensuring the quality and quantity of the seeds, 60-70 seeds are required; these seeds should have a healthy color and be free from diseases and insect pests;

[0017] (2) The Agrobacterium culture solution of the constructed pMDC85-TeWRKY1 expression vector was shaken overnight (about 10 hours, 500 μL YEB in a 2 mL centrifuge tube). After the culture solution was verified by PCR, it was shaken overnight (about 10 hours, 100 mL YEB in a 500 mL conical flask, OD value around 0.8).

[0018] (3) Collect the Agrobacterium cells by centrifuging the overnight shaken culture at 5000 rpm for 10 min at room temperature. Remove the supernatant after centrifugation and suspend the cells in the transformation solution to a constant OD 600 = 0.6-0.8, let stand at 28°C for 3 hours, mix with the collected seeds after standing, and put into a clean tissue culture bottle with a cotton wool bottom and let stand for 1 hour;

[0019] (4) Place the culture bottle containing the mixed solution and seeds into a gas storage tank and cover it with a lid. The design parameters of the gas storage tank are: pressure 1.6MPa, working pressure 1MPa, design temperature 100°C, volume 0.036m 3 , Volume 65kg;

[0020] Use an oil-free air compressor to perform vacuum infection treatment. The pressure of the gas tank during the treatment process is 0.6MPa, the treatment time is set to 12h, the temperature is room temperature 25℃, and the pressure treatment is performed in a dark gas tank to ensure that the Agrobacterium transformation liquid can fully penetrate and infect the seeds. After the vacuum treatment is completed, open the valve to slowly release the gas. After the gas is exhausted, replace the heavy suspension in the culture bottle with clean water and wash twice;

[0021] (5) Place the infected seeds in a constant temperature incubator with a set temperature of 22°C and a photoperiod of 16 h light / 8 h dark;

[0022] (6) After culturing in the incubator for 2 days, remove the seeds, dry them, and then plant them in the soil. After 2-3 weeks, observe the growth of the plants and take leaves for testing.

[0023] The marigold genetic transformation method of the present invention is different from the prior art in that:

[0024] The invention discloses a method for genetic transformation of marigolds using a gas storage tank. The gas storage tank is a device that integrates gas storage and regulation functions. Its innovation lies in that it can provide a continuous and stable gas supply with unprecedented accuracy for various culture environments, especially for highly sensitive experiments such as genetic transformation of marigolds. The core of this technology is to utilize the gas storage tank of the invention, and its system can store a large amount of gas resources and a pressure maintenance mechanism. According to the specific needs in the genetic transformation process of marigolds, the gas storage tank can be maintained within a constant and suitable pressure range. The gas storage tank is specially designed according to specific parameters and meets national product standards.

[0025] The carrier used in the present invention is pMDC85 and carries the GFP (green fluorescent protein) gene. Introducing GFP in the pMDC85 carrier can realize real-time, non-invasive monitoring of the target gene or protein in the cell, greatly improving the experimental efficiency and accuracy. GFP can be stably inherited to the offspring cells, which makes the pMDC85 carrier with GFP become an ideal genetic marker tool. Through genetic engineering technology, GFP can be fused with the target gene, thereby continuously expressing in the offspring cells, realizing the tracking and monitoring of the target gene.

[0026] In order to further improve the efficiency and quality of genetic transformation, the use of oil-free vacuum pump technology to accurately extract the gas in the gas tank to a specific negative pressure state (such as 0.6MPa) in an efficient and pollution-free manner is crucial to promote the transformation liquid to effectively infect seed cells. The negative pressure environment not only helps the transformation liquid to penetrate deeper into the seeds, but also promotes the permeability of the cell membrane to a certain extent, making it easier for exogenous genes to be accepted by cells and integrated into their genomes. Therefore, compared with traditional methods, the marigold genetic transformation process using this innovative gas tank system can significantly increase the acquisition rate of transgenic seedlings, while optimizing their genetic characteristics and growth quality, opening up a new path for research and application in the field of agricultural biotechnology.

[0027] The marigold genetic transformation method of the present invention is further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the GFP band and positive seedlings in the identification of transgenic positive seedlings of marigold 'No. 65' in the method of the present invention;

[0029] Figure 2 It is a PCR detection diagram of a comparative test in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] 1 Test materials

[0031] 1.1 Plant materials

[0032] The plant material used for transgenic was Marigold 'No. 65' which was stored in the automated greenhouse of Beijing Academy of Agriculture and Forestry Sciences, with normal water and fertilizer management, and full seeds were collected.

[0033] 1.2 Strains and vectors

[0034] Bacterial strain: Agrobacterium tumefaciens EHA105 heat shock competent cells were purchased from Beijing Biomed Gene Technology Co., Ltd.

[0035] Vector: The plant overexpression vector pMDC85 plasmid is preserved in our laboratory.

[0036] 1.3 Equipment and Instruments

[0037] Gas storage tank (Yingtong Pressure Vessel Co., Ltd., GB / T150, product code is 22Y11299), design pressure 1.6MPa, working pressure 1MPa, design temperature 100℃, volume 0.036m 3 , Volume 65kg;

[0038] Oil-free air compressor (Shanghai Luodi Air Compressor Co., Ltd., 2-550-24L), PCR instrument (Bio-rad, T100), microcentrifuge (Thermo Scientific Heraeus, Pico 17), -80℃ ultra-low temperature refrigerator (SANYO, MDF-U53V), -20℃ refrigerator (Haier, DW-25L262), 4℃ refrigerator (Haier, BCD-225TMPM), vertical pressure steam sterilizer (Jiangyin Binjiang Medical Equipment Co., Ltd., LS-B75-II), ultra-micro spectrophotometer (Food ALYTPhotometer), laser confocal scanning system (Nikon, ECLIPSE Ti), electric shock instrument (Bio-rad, Gene PulserXcell TM ), UV gel imaging system (Tanon, 1600), gel electrophoresis instrument (Beijing Liuyi Instrument, JY-SPCT), clean bench (Suzhou Purification, SW-CJ-1FD), biological microscope (BU200i), water bath (Shanghai Yiheng, DK-8D), electronic balance (JA2003), etc.

[0039] 1.4 Preparation of test reagents and culture medium

[0040] 1.4.1 Main reagents

[0041] Seamless Cloning Kit ( HD Cloning Kit User Manual, Takara), the target fragment was recovered and purified using the recovery kit ( SV Grl and PCR Clean-Up System, Promega), PCR rapid amplification kit (2×Taq Plus Master Mix, Novozymes). DL2000 DNA Marker was purchased from Takara, restriction enzymes Pac I and Asc I were purchased from New England Biotechnology, Kanamycin (Kan), ampicillin (Amp), and rifampicin (Rif) were purchased from Solarbio, MES Buffer and acetosyringone (AS) were purchased from Beijing Coolbo Co., Ltd., and 75% alcohol was purchased from Lierkang.

[0042] 1.4.2 Main culture medium

[0043] ① Configuration of YEB culture medium (taking 1L as an example):

[0044]

[0045] Adjust the pH to 5.8-6.0 and sterilize in a vertical pressure steam sterilizer at 117°C for 17 minutes.

[0046] ②LB solid culture medium (taking 1L as an example):

[0047]

[0048]

[0049] Adjust the pH to 5.8-6.0 and sterilize in a vertical pressure steam sterilizer at 117°C for 17 minutes.

[0050] 1.4.3 Conversion solution configuration

[0051]

[0052] 1.5 PCR primers required for the experiment

[0053] Table 1 Primer sequences

[0054]

[0055] 2 Test methods

[0056] 2.1 Construction of overexpression vector

[0057] 2.1.1 Double restriction digestion of pMDC85 vector

[0058] According to the characteristics of the multiple cloning site of the pMDC85 expression vector and the sequence characteristics of the TeWRKY1 gene, the upstream and downstream primers of the Pac I and Asc I recognition site sequences were added: C85-TeWRKY1-F and C85-TeWRKY1-R (Table 1), respectively. The TeWRKY1 plasmid product with correct sequencing was diluted 10 times as a template, and the reaction system and procedure were used to amplify the ORF fragment of TeWRKY1 with restriction sites.

[0059] The pMDC85 empty expression vector with correct sequencing was double-digested with restriction endonucleases Pac I and Asc I. The enzyme digestion system is as follows:

[0060]

[0061]

[0062] The enzyme was digested at 37°C for 6-8h, and the result was detected by 1% agarose gel electrophoresis. The expected bands were excised and recovered.

[0063] 2.1.2 Amplification of TeWRKY1 target fragment

[0064] (1) TeWRKY1 target fragment amplification system:

[0065]

[0066] Program system:

[0067]

[0068] 2.1.3 Construction of expression vector using seamless cloning technology

[0069] (1) Seamless cloning technology reaction system:

[0070]

[0071] The solution was mixed with a pipette, incubated in a metal bath at 50°C for 15 min, and then placed on ice.

[0072] 2.1.4 Conversion

[0073] The ligation product was transformed into E. coli DH5α competent cells by heat shock method, and the transformed bacterial solution was shaken at 37℃ and 180rpm for 1h. After the bacterial solution was flicked to mix, 300μL of the bacterial solution was evenly spread on the LB plate medium containing Kan resistance using a spreader, and inverted cultured at 37℃ for 12-16h.

[0074] 2.1.5 Positive clone screening and bacterial liquid PCR verification:

[0075] (1) The next day, take out the plate from the previous step, pick up 8 monoclonal colonies on the plate with a 2.5μL pipette tip in a clean bench, put them into a 1.5mL centrifuge tube containing 500μL LB+Kan, and shake at 37℃ for 3-5h. Then use 1μL of the mixed bacterial solution as a template for Pac I and Asc I double enzyme digestion detection.

[0076] The reaction system is: bacterial solution 1μL; 10×Cutsmart Buffer 5μL; Pac I 1μL; Asc I 1μL; ddH 2 O was added to 50 μL. The reaction procedure was: enzyme digestion in a metal bath at 37°C for 3 h.

[0077] (2) 1% agarose gel electrophoresis was used to detect the presence of the target band. 200 μL of the bacterial solution showing the target band was aspirated in a clean bench and placed in a 1.5 mL centrifuge tube and sent to Shanghai Biotechnology for sequencing.

[0078] (3) After obtaining the sequencing results, use software to compare and select samples with correct sequences for Agrobacterium transformation.

[0079] 2.2 Transformation and identification of Agrobacterium competent cells

[0080] 2.2.1 Transformation of Agrobacterium competent cells with overexpression vector

[0081] Heat shock transformation method was used to transform Agrobacterium EHA105 competent cells:

[0082] (1) Thaw the EHA105 Agrobacterium competent cells stored at -80°C in an ice water bath.

[0083] (2) Add 5 μL of plasmid DNA to the competent cells in a clean bench, mix gently, and place in an ice water bath for 5 min.

[0084] (3) Place the centrifuge tube in liquid nitrogen for 5 minutes.

[0085] (4) Then quickly place the centrifuge tube in a 37°C water bath and keep it for 5 minutes.

[0086] (5) Place the centrifuge tube back into the ice water bath for 5 minutes.

[0087] (6) Add 500 μL of antibiotic-free YEB liquid culture medium to the clean bench, shake and culture at 28°C for 2-3 h, collect the bacteria by centrifugation at 6000 rpm for 5 min, retain about 100 μL of supernatant, gently blow to resuspend the bacteria, take an appropriate amount of bacterial liquid, spread it on the YEB plate with the corresponding antibiotics (50 mg / L Kan and 50 mg / L Rif), and culture it upside down at 28°C on a shaker for 48-72 h.

[0088] 2.2.2 Identification of recombinant Agrobacterium

[0089] Pick a single colony into 5 mL YEB liquid medium containing 50 mg / L Kan and 50 mg / L Rif, and culture at 28°C, 150 rpm shaking for 36-48 h. Take the bacterial solution as a template and water as a negative control. Use primers C85-TeWRKY1-F / C85-TeWRKY33-F (Table 1) to perform bacterial solution PCR to check positive clones. The reaction system is as follows:

[0090]

[0091] After mixing, place in PCR instrument according to the following reaction conditions:

[0092]

[0093] The denaturation to extension step was repeated for 35 cycles. The PCR product was detected by 1% agarose gel electrophoresis, and 25% sterile glycerol was added to the Agrobacterium strain that amplified the expected band and stored at -80°C for later use.

[0094] 2.3 Agrobacterium-mediated transformation of Marigold 'No. 65'

[0095] (1) Collect mature and plump seeds of Marigold 'No. 65' to ensure the quality and quantity of the seeds, about 60-70 seeds are needed. These seeds should have a healthy color and be free from pests and diseases to ensure the accuracy and reliability of subsequent experiments.

[0096] (2) The Agrobacterium culture solution of the constructed pMDC85-TeWRKY1 expression vector was shaken overnight (about 10 hours, 500 μL YEB in a 2 mL centrifuge tube). After the culture solution was verified by PCR, it was shaken overnight (about 10 hours, 100 mL YEB in a 500 mL conical flask, OD value around 0.8).

[0097] (3) Collect the Agrobacterium cells by centrifuging the overnight shaken culture at 5000 rpm for 10 min at room temperature. Remove the supernatant after centrifugation and suspend the cells in the transformation solution to a constant OD 600 =0.6-0.8, let it stand at 28℃ for 3h, then mix it with the collected seeds and put it into a clean tissue culture bottle with a cotton wool bottom and let it stand for 1h.

[0098] (4) Place the culture bottle containing the mixed solution and seeds into a gas storage tank (design pressure 1.6MPa, working pressure 1MPa, design temperature 100℃, volume 0.036m 3 , volume 65kg), cover the lid, use an oil-free air compressor to vacuum infection treatment, pressurize the gas tank at 0.6MPa, set the treatment time to 12h, and the temperature is room temperature 28°C. Perform pressure treatment in a dark gas tank to ensure that the Agrobacterium transformation liquid can fully penetrate and infect the seeds. After the vacuum treatment is completed, open the valve and slowly release the gas. After the gas is exhausted, replace the resuspended liquid in the culture bottle with clean water and wash twice.

[0099] (5) Place the infected seeds in a constant temperature incubator with a set temperature of 22°C and a photoperiod of 16 h light / 8 h dark.

[0100] (6) After 2 days of cultivation in the incubator, remove the seeds, dry them, and then plant them in the soil. After 2-3 weeks, observe the growth of the plants and take leaves for testing.

[0101] 2.4 Identification of transgenic positive seedlings of Marigold 'No. 65'.

[0102] In order to identify positive seedlings carrying the 35S::TeWRKY1::GFP transgenic structure, it is necessary to design specific primers for the GFP gene and amplify the target fragment through PCR amplification technology. First, based on the known sequence of the GFP (green fluorescent protein) gene, primer design software is used to design a primer pair with high specificity and sensitivity. Next, a standard plant DNA extraction method is used to extract pure DNA from the leaves of the seedlings through grinding, lysis, purification and other steps.

[0103] After obtaining the DNA sample, the GFP-specific primers designed previously will be used to amplify the target fragment through PCR technology. Finally, the PCR amplification product will be detected and analyzed by gel electrophoresis. If a fragment that matches the size of the GFP gene is successfully amplified from the DNA sample of the suspected transgenic seedling, then it can be preliminarily determined that the seedling is a positive transgenic strain. It should be noted that in order to ensure the accuracy and reliability of the identification results, it is necessary to repeat the experiment many times and perform bioengineering sequencing on the amplified bands. Figure 1 As shown, 4 positive seedlings were obtained (20, 24, 28, and 29 were positive seedlings); M was DL2000, 1-30 were T0 generation strains, 31 (WT) was a negative control (wild type), and 32 was a positive control (Agrobacterium bacterial solution).

[0104] 3. Comparative test:

[0105] First, select 80-100 seeds respectively and place them in the corresponding transformation solution and clean water. Subsequently, dark treatment is carried out at room temperature of 28°C for 12 hours. Next, take out the seeds from the treatment solution, replace it with clean water, and then place them in a constant temperature incubator (temperature is 22°C, light cycle is 16h light / 8h dark). After two days of cultivation, take out the seeds, dry them, and then plant them in the soil. After the seeds germinate and grow, pick the leaves to extract DNA. The subsequent experimental steps are consistent with the detection method mentioned in the present invention to detect positive seedlings. The results are as follows Figure 2 As shown, no positive seedlings were obtained through the detection comparison test, and the final conversion rate was 0, while the present invention obtained 4 positive seedlings with a conversion rate of 4%-5%.

[0106] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for genetic transformation of marigold, characterized in that: The invention comprises the construction of overexpression vector and transformation and identification of Agrobacterium competent cells. In the transformation of the Agrobacterium competent cells, the Agrobacterium transformation liquid of the constructed expression vector is mixed with seeds, put into a gas storage tank, covered with a lid, and vacuumed and infected by an oil-free air compressor.

2. The method for genetic transformation of marigold according to claim 1, characterized in that: The pressure of the gas storage tank is 0.6 MPa, the treatment time is set to 12 h, the temperature is room temperature, and the pressurized treatment is performed in a dark gas storage tank.

3. The method for genetic transformation of marigold according to claim 2, characterized in that: After the vacuum treatment is completed, open the valve and slowly release the gas. After the gas is exhausted, replace the heavy suspension in the culture bottle with clean water and wash twice; place the infected seeds in a constant temperature incubator, set the temperature to 22°C, and the light cycle to 16h light / 8h dark; after culturing in the incubator for 2 days, take out the seeds and dry them, and then plant them in the soil.

4. The method for genetic transformation of marigold according to claim 1, characterized in that: The method for constructing the overexpression vector specifically comprises the following steps: (1) Double restriction enzyme digestion of pMDC85 vector; (2) Amplification of the TeWRKY1 target fragment; (3) Construction of expression vectors using seamless cloning technology; (4) Transformation; (5) Screening of positive clones and verification of bacterial culture by PCR.

5. The method for genetic transformation of marigold according to claim 4, characterized in that: The transformation and identification method of Agrobacterium competent cells comprises the steps of transforming Agrobacterium competent cells with overexpression vectors and transforming marigold 'No. 65' mediated by Agrobacterium.

6. The method for genetic transformation of marigold according to claim 5, characterized in that: The method for transforming Agrobacterium competent cells with an overexpression vector comprises transforming Agrobacterium EHA105 competent cells with a heat shock transformation method and identifying the recombinant Agrobacterium.

7. The method for genetic transformation of marigold according to claim 6, characterized in that: The method for Agrobacterium-mediated transformation of Marigold 'No. 65' specifically comprises the following steps: (1) Collect mature and plump seeds of Marigold 'No. 65', ensuring the quality and quantity of the seeds, 60-70 seeds are required; these seeds should have a healthy color and be free from diseases and insect pests; (2) The Agrobacterium culture solution of the constructed pMDC85-TeWRKY1 expression vector was shaken overnight for 10 hours, and then 500 μL YEB was placed in a 2 mL centrifuge tube. After the culture solution was verified by PCR, it was shaken overnight for 10 hours, and then 100 mL YEB was placed in a 500 mL conical flask, and the OD value was 0.8; (3) Collect the Agrobacterium culture solution that has been shaken overnight by centrifugation at 5000 rpm for 10 min at room temperature to collect the cells. After centrifugation, remove the supernatant and suspend the cells in the transformation solution to a constant OD 600 = 0.6-0.8, let stand at 28°C for 3 hours, mix with the collected seeds after standing, and put into a clean tissue culture bottle with a cotton wool bottom and let stand for 1 hour; (4) Place the culture bottle containing the mixed solution and seeds into a gas storage tank and cover it with a lid. The design parameters of the gas storage tank are: pressure 1.6MPa, working pressure 1MPa, design temperature 100°C, volume 0.036m 3 , Volume 65kg; Use an oil-free air compressor to perform vacuum infection treatment. The pressure of the gas tank during the treatment process is 0.6MPa, the treatment time is set to 12h, the temperature is room temperature 28°C, and the pressure treatment is performed in a dark gas tank to ensure that the Agrobacterium transformation liquid can fully penetrate and infect the seeds. After the vacuum treatment is completed, open the valve to slowly release the gas. After the gas is exhausted, replace the heavy suspension in the culture bottle with clean water and wash twice; (5) Place the infected seeds in a constant temperature incubator with a set temperature of 22°C and a photoperiod of 16 h light / 8 h dark; (6) After culturing in the incubator for 2 days, take out the seeds, dry them, and then plant them in the soil. After 2-3 weeks, observe the growth of the plants and take leaves for testing.

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