Agrobacterium rhizogenes-mediated pea hairy root transformation method
Through the Agrobacterium rhizobili-mediated pea bremated root transformation method, the problems of low efficiency and long cycle of pea gene transformation are solved, efficient and rapid transformation is achieved, and a stable set of gene editing and research tools are provided.
Patent Information
- Application Number
- CN202411863980.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-21
AI Technical Summary
In the prior art, peas have low gene transformation efficiency, long transformation cycle, unsuitable trauma methods, and lack an effective Agrobacter rhizobium-mediated hairy root transformation system, which limits the progress of gene editing and gene function research.
Using Agrobacterium rhizobium mediated pea hairoot transformation method, the Agrobacterium bacteria solution was prepared and preserved, and sterile seedlings and explants were prepared, Agrobacterium infection and co-culture were carried out to induce hairy root generation, and the transformation efficiency was verified by GUS staining.
It achieves efficient and rapid pea wool root transformation, improves transformation efficiency and induction rate, reduces transformation cycle, and provides a stable set of gene editing and research tools.
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Figure CN119662705A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of plant biotechnology and genetic engineering, and in particular to a pea hairy root transformation method mediated by Agrobacterium rhizogenes. Background Art
[0002] Agrobacterium rhizogenes is a close relative of Agrobacterium tumefaciens, which has the ability to induce adventitious roots (hairy roots) when plant tissues are damaged. The formation of such adventitious roots depends on the Ri plasmid carried by Agrobacterium, which transfers the target gene into the genome of the host plant through infection, and after integration, transgenic hairy roots can be produced. Based on this principle, the hairy root transformation system mediated by Agrobacterium rhizogenes has been widely used in plant gene function research, metabolic engineering and molecular breeding. Compared with the traditional stable genetic transformation mediated by Agrobacterium tumefaciens, the hairy root transformation system has shown great application potential in many plant species due to its advantages such as short transformation cycle, high transformation efficiency, no need for exogenous hormones and fewer chimeras.
[0003] In the existing technology, the hairy root transformation system mediated by Agrobacterium rhizogenes has been successfully applied to a variety of plants, such as tomatoes, tobacco and carrots. These transformation systems mainly use Agrobacterium rhizogenes to infect plant leaves or stems, and achieve the transformation of target genes through wound induction and Ri plasmid integration. Its core technical steps include:
[0004] (1) Using ultrasound or scalpel wounds to create wounds on plant explants, exposing plant tissues and enhancing the infection efficiency of Agrobacterium;
[0005] (2) Infect injured explants with Agrobacterium rhizogenes bacterial solution, and integrate the target gene in the Ri plasmid into the plant genome through the T-DNA delivery system;
[0006] (3) culturing the infected explants in co-culture medium for several days to allow efficient infection by Agrobacterium;
[0007] (4) Finally, induce hairy root formation and perform gene function verification or downstream analysis on the hairy roots.
[0008] Although these methods have achieved remarkable success in different species, the hairy root transformation system has not been widely used and studied in pea (Pisum sativum). Existing pea transformation systems mainly rely on Agrobacterium tumefaciens-mediated stable transformation, which usually requires a long transformation cycle and has low transformation efficiency, limiting its application in high-throughput gene function research and gene editing technology. In addition, the transient transformation method of leaf injection often leads to unstable expression of exogenous genes due to its easy damage to the leaves, thus affecting the accuracy of the experimental results.
[0009] The main problems and shortcomings of the existing technology include:
[0010] (1) Low transformation efficiency: Genetic transformation of pea is relatively difficult, and the existing Agrobacterium-mediated transformation efficiency is low, especially in high-throughput gene function research and breeding applications.
[0011] (2) Long transformation cycle: Traditional stable transformation methods usually take several months to obtain transgenic plants. The long cycle affects the efficiency of breeding and research progress.
[0012] (3) The wound method is not suitable for peas: Some existing wound treatment methods (such as leaf injection) can easily cause significant damage to crops such as peas, resulting in unstable expression of exogenous genes, especially during transient transformation, where damage affects the accuracy and reliability of exogenous gene expression.
[0013] (4) The hairy root transformation system has not yet been established: Although the hairy root transformation system has been successfully developed in many plant species, there is a lack of an effective Agrobacterium-mediated hairy root transformation system in pea, which limits the rapid development of gene editing and gene function research.
[0014] Therefore, these limitations of the existing technology have brought challenges to the gene function research and molecular breeding of pea. In order to solve these problems, it is necessary to develop an efficient, rapid and stable pea hairy root transformation system to accelerate the process of pea gene function research, gene editing and molecular breeding. Summary of the invention
[0015] In view of this, the present invention provides a pea hairy root transformation method mediated by Agrobacterium rhizogenes.
[0016] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0017] A method for transforming pea hairy roots mediated by Agrobacterium rhizogenes comprises the following steps:
[0018] Step 1: Preparation and storage of Agrobacterium culture solution
[0019] Pipette pC1391-UBI-gusplus plasmid and transform it into Ar.Qual and K599 competent cells respectively, place in ice bath, liquid nitrogen, add preheated TY liquid medium, shake culture, collect bacteria, keep part of the supernatant to suspend the bacteria, and apply it to TY solid medium with Kan and streptomycin resistance;
[0020] Step 2: Preparation of sterile pea seedlings
[0021] Select pea seeds "ZW 6" and spread them in a sterile culture dish, place the culture dish with peas in a well-sealed desiccator, put the NaClO solution in a small beaker, and place the small beaker in the desiccator, add concentrated hydrochloric acid to the beaker with the NaClO solution, quickly cover the lid of the desiccator, seal and disinfect, and then inoculate the pea seeds into the germination medium for culture;
[0022] Step 3: Prepare Agrobacterium culture solution
[0023] Three days before infection, streak the frozen Agrobacterium culture onto TY solid medium containing Kan and Strep resistance and culture for the first bacterial activation;
[0024] The night before infection, use an inoculation loop to dip the activated bacteria into TY liquid medium containing Kan and Strep resistance, culture, and perform secondary activation;
[0025] Step 4: Preparing the explants
[0026] Take out the germinated sterile seedlings, cut the cotyledons in half, and keep part of the hypocotyl;
[0027] Step 5: Infection and co-cultivation of explants
[0028] Take the activated Agrobacterium liquid, adjust it to zero with TY liquid medium containing Kan and Strep resistance, and measure its absorbance OD600 at a wavelength of 600nm;
[0029] Collect the remaining Agrobacterium bacterial liquid, discard the supernatant, resuspend the bacterial cells in the infection liquid medium to make the bacterial liquid OD600 = 0.8, and culture and activate the bacterial cells;
[0030] Place the explant in the resuspended activated bacterial solution, shake it continuously, pour out the bacterial solution, place the explant on a culture dish lined with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-culture medium for culture;
[0031] Step 6: Inducing hairy roots
[0032] The explants were washed with washing solution, placed on a culture dish lined with sterile filter paper, excess liquid was sucked off and blown dry, and then inoculated on a hairy root induction medium and cultured.
[0033] Preferably, the step 1 further comprises: picking 24 single clones, streaking the single clones onto a TY solid culture medium with Kan and Strep resistance, and inverting and culturing at 28°C for 1 day; using an inoculation loop to dip 24 Agrobacterium colonies into a TY liquid culture medium with Kan and Strep resistance, and shaking and culturing at 28°C and 200 rpm / min for 1 day; drawing 500uL of the bacterial solution into a centrifuge tube, adding an equal volume of 60% glycerol, mixing, freezing in liquid nitrogen for 1 minute, and placing the frozen Agrobacterium bacterial solution in a -80°C refrigerator for standby use.
[0034] Preferably, in the step 1, the amount of pC1391-UBI-gusplus plasmid is 10 μL, the ice bath time is 30 min, the liquid nitrogen time is 1 min, 1 mL of TY liquid culture medium preheated at 28°C is added, the shaking rate is 120 rpm / min, the shaking culture time is 3 h, the centrifugation rate is 5,000 rpm, the centrifugation time is 5 min, 100 μL of the supernatant is retained, and the culture conditions are inverted culture at 28°C for 2 days.
[0035] Preferably, in step 2, the germination medium formula is as shown in Table 1:
[0036] Table 1 Germination medium formula
[0037]
[0038] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 °C.
[0039] Preferably, in step 2, the amount of NaClO solution added is 200-400 mL, the volume of the small beaker is 500 mL, the amount of concentrated hydrochloric acid taken is 10-30 mL, and the closed disinfection time is 10-12 h;
[0040] Preferably, the amount of NaClO solution added is 300 mL;
[0041] Preferably, the amount of concentrated hydrochloric acid is 20 ml.
[0042] Preferably, in step three, the culture conditions for the first bacterial activation are: inverted culture at 28°C for 2 days; and the culture conditions for the second bacterial activation are: shaking culture at 28°C and 200 rpm / min overnight.
[0043] Preferably, in step 4, 3-5 mm hypocotyls are retained, 3 replicates are set for each strain, 9 dishes of explants are inoculated in each replicate, and 5 explants are inoculated in each dish.
[0044] Preferably, in step 5, the formula of the infection liquid culture medium is as shown in Table 2:
[0045] Table 2 Infection liquid culture medium formula
[0046]
[0047] The co-culture medium formula is shown in Table 3:
[0048] Table 3 Co-culture medium formula
[0049]
[0050] Preferably, in step 5, 3 to 5 mL of activated Agrobacterium bacterial solution is taken, and the remaining Agrobacterium bacterial solution is centrifuged at 5000 rpm for 10 minutes; the bacteria are activated by culturing at 4°C for more than half an hour, and the explants are placed in the resuspended activated bacterial solution and placed at room temperature for 30 minutes, during which time they are continuously shaken; the culture conditions of the co-cultivation medium are: dark culture at 28°C for 4 days.
[0051] Preferably, the formula of the induction medium in step 6 is as shown in Table 4:
[0052] Table 4 Induction medium formula
[0053]
[0054] The culture conditions are: 28℃ dark culture, culture time is about 3 weeks, subculture every 10 days. BRIEF DESCRIPTION OF THE DRAWINGS
[0055] Figure 1 The present invention is a flowchart of the instantaneous transformation of pea hairy roots based on the Agrobacterium rhizogenes-mediated pea hairy root transformation method;
[0056] Figure 2 The figure is a diagram showing the growth of hairy roots induced by Ar.Qual and K599 strains and the results of GUS staining based on a method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes of the present invention;
[0057] Figure 3 The figure is a graph of the induction rate and positive rate of hairy roots mediated by Ar.Qual and K599 strains in a pea hairy root transformation method mediated by Agrobacterium rhizogenes according to the present invention. DETAILED DESCRIPTION
[0058] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0059] Embodiment 1:
[0060] The present invention discloses a method for transforming pea hairy roots mediated by Agrobacterium rhizogenes, comprising the following steps:
[0061] Step 1: Preparation and storage of Agrobacterium culture solution
[0062] 10 μL of pC1391-UBI-gusplus plasmid was transferred into Ar.Qual and K599 competent cells respectively, and then placed in ice bath for 30 min, liquid nitrogen for 1 min, and 1 mL of TY liquid medium preheated at 28°C was added. The cells were cultured at 120 rpm / min for 3 h, and centrifuged at 5,000 rpm for 5 min to collect the cells. 100 μL of the supernatant was used to suspend the cells, and the cells were spread on TY solid medium with Kan and streptomycin resistance, and cultured at 28°C for 2 days.
[0063] Pick 24 single clones and streak them onto TY solid medium with Kan and Strep resistance, and invert and culture at 28℃ for 1 day; use an inoculation loop to dip 24 Agrobacterium colonies into TY liquid medium with Kan and Strep resistance, and culture at 28℃, 200rpm / min shaking for 1 day; draw 500uL of bacterial solution into a centrifuge tube, add an equal volume of 60% glycerol, mix well, freeze in liquid nitrogen for 1 minute, and put the frozen Agrobacterium bacterial solution in a -80℃ refrigerator for later use;
[0064] Step 2: Preparation of sterile pea seedlings
[0065] Select plump, clean and well-growing pea seeds "ZW 6" and spread them flat in a sterile culture dish. Place the culture dish with peas in a well-sealed desiccator. Place 300 mL of NaClO solution in a 500 mL small beaker, and place the small beaker in the desiccator. Take 20 mL of concentrated hydrochloric acid with a pipette and add it to the beaker with NaClO solution. Quickly cover the lid of the desiccator and seal it for disinfection for 10-12 hours. After disinfection, inoculate the pea seeds into the germination medium and culture it for 5-7 days. The formula of the germination medium is shown in Table 1. The culture conditions are: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 ° C.
[0066] Step 3: Prepare Agrobacterium culture solution
[0067] Three days before infection, streak the frozen Agrobacterium culture onto TY solid medium containing Kan and Strep resistance and incubate at 28°C for 2 days for the first bacterial activation.
[0068] The night before infection, use an inoculation loop to dip the activated bacteria into TY liquid medium containing Kan and Strep resistance, and culture at 28°C, 200 rpm / min, shaking overnight for secondary activation;
[0069] Step 4: Preparing the explants
[0070] Take out the germinated sterile seedlings from the clean bench, cut the cotyledons in half, and leave 3-5 mm hypocotyls; set up 3 replicates for each strain, inoculate 9 dishes of explants in each replicate, and inoculate 5 explants in each culture dish;
[0071] Step 5: Infection and co-cultivation of explants
[0072] Take 3-5 mL of activated Agrobacterium culture medium, adjust to zero with TY liquid medium containing Kan and Strep resistance, and measure its absorbance (OD) at 600 nm. 600 ) The remaining Agrobacterium bacterial solution was centrifuged at 5000 rpm for 10 min, the bacteria were collected, the supernatant was discarded, and the bacteria were resuspended in the infection liquid medium to make the bacterial solution OD 600 =0.8, incubate at 4°C for more than half an hour to activate the bacteria; the formula of the infection liquid culture medium is shown in Table 2;
[0073] Place the explant in the resuspended activated bacterial solution and place it at room temperature for 30 minutes, shaking it continuously during the period, pour out the bacterial solution, place the explant on a culture dish lined with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-culture medium (lay two layers of sterile filter paper on the medium), and culture it in the dark at 28°C for 4 days. The formula of the co-culture medium is shown in Table 3;
[0074] Step 6: Inducing hairy roots
[0075] After the co-cultivation, the explants were washed 4-5 times with washing solution. After washing, the explants were placed on a culture dish lined with sterile filter paper, excess liquid was sucked off and blown dry, and then inoculated on the hairy root induction medium and cultured for about 3 weeks, with subculture every 10 days. The culture conditions were: 28°C dark culture. The formula of the washing induction medium is shown in Table 4.
[0076] Example 2: Observation of hairy roots
[0077] Observe the hairy roots mediated by Ar.Qual and K599 strains, count the number of explants induced to generate hairy roots by these two strains, and take roots for GUS staining to count the positive rates of transformation mediated by these two strains. The steps of GUS staining are as follows:
[0078] (1) Clean the pea hairy roots to be stained and immerse them in a 50 mL centrifuge tube filled with GUS staining solution. Vacuum the centrifuge tube for 20 to 30 minutes with the lid open. During this time, the vacuum box can be stopped to observe whether the GUS staining solution enters the pea hairy roots and whether the pea hairy roots settle to the bottom of the centrifuge tube. Stop the vacuum operation until the GUS staining solution enters the hairy roots and the hairy roots settle to the bottom of the tube.
[0079] (2) Incubate the treated and control groups together at 37°C for 24 h or longer. The staining time can be adjusted according to the degree of staining.
[0080] (3) Pour off the staining solution and add 75% ethanol to decolorize and remove chlorophyll and other pigments. Replace the ethanol solution according to the decolorization situation. It can be replaced every 3 to 4 hours. The entire decolorization time may take 12 to 24 hours or longer until the leaves of the control group are colorless or white.
[0081] (4) Preserve with a solution containing 30% glycerol and 70% ethanol. Take photos under a stereomicroscope to record the staining results.
[0082] Example 3: Experimental results
[0083] like Figure 1 As shown, after the pC1391-UBI-gusplus plasmid was transformed into the rhizogenes Agrobacterium strains Ar.Qual and K599, hairy roots began to form about 10 days later, and more explants were found to be induced to produce hairy roots after 2 weeks.
[0084] like Figure 2 As shown, the upper and lower figures on the left represent the induced hairy roots; the upper and lower figures on the left represent the GUS staining results of the hairy roots; the scale bar is 1 cm;
[0085] After culturing for about 3 weeks, it was observed that the number of hairy roots induced by Ar.Qual was small and the length was short, most of which did not exceed 5 cm; while the number of hairy roots induced by K599 was large, the length could reach more than 10 cm, and there was a tendency to continue to grow and elongate.
[0086] In addition, the study also found that the hairy roots induced by K599 had more obvious staining results, almost the entire root was dyed blue, while the hairy roots induced by Ar.Qual had less obvious staining effect, which indicates that the GUS gene is more expressed in the K599 strain, while the expression level in the Ar.Qual strain is very low or almost not expressed. Therefore, we screened that the K599 rhizogenes Agrobacterium strain is more suitable for mediating the formation of pea hairy roots, with a higher hairy root induction rate (66.67%) and transformation positive rate (62.50%). The results are as follows Figure 2 shown.
[0087] like Figure 3 As shown, the orange column is the hairy root induction rate, and the green column is the transformation positive rate. ** and *** respectively indicate that when P < 0.01 and P < 0.001, they reach the significant and extremely significant levels in the t-test.
[0088] The number of explants that produced hairy roots was counted, and the results showed that 50% of the explants of Ar.Qual induced hairy roots, and 66.67% of the explants of K599 induced hairy roots.
[0089] These hairy roots were taken for GUS staining, and the staining results showed that 28.57% of the hairy roots induced by Ar.Qual were stained blue; while 62.50% of the hairy roots induced by K599 were stained blue.
[0090] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes, characterized in that: The following steps are involved: Step 1: Preparation and storage of Agrobacterium culture solution Pipette pC1391-UBI-gusplus plasmid and transform it into Ar.Qual and K599 competent cells respectively, place in ice bath, liquid nitrogen, add preheated TY liquid medium, shake culture, collect bacteria, keep part of the supernatant to suspend the bacteria, and apply it to TY solid medium with Kan and streptomycin resistance; Step 2: Preparation of sterile pea seedlings Select pea seeds "ZW 6" and spread them in a sterile culture dish. Place the culture dish containing peas in a well-sealed desiccator. Place a NaClO solution in a small beaker, and place the small beaker in the desiccator. Add concentrated hydrochloric acid to the beaker containing the NaClO solution, quickly cover the lid of the desiccator, seal and disinfect, and then inoculate the pea seeds into the germination medium for culture. Step 3: Prepare Agrobacterium culture solution Three days before infection, streak the frozen Agrobacterium culture onto TY solid medium containing Kan and Strep resistance and culture for the first bacterial activation; The night before infection, use an inoculation loop to dip the activated bacteria into TY liquid medium containing Kan and Strep resistance, culture, and perform secondary activation; Step 4: Preparing the explants Take out the germinated sterile seedlings, cut the cotyledons in half, and keep part of the hypocotyl; Step 5: Infection and co-cultivation of explants Take the activated Agrobacterium liquid, adjust it to zero with TY liquid medium containing Kan and Strep resistance, and measure its absorbance OD600 at a wavelength of 600nm; Collect the remaining Agrobacterium bacterial liquid, discard the supernatant, resuspend the bacterial cells in the infection liquid medium to make the bacterial liquid OD600 = 0.8, and culture and activate the bacterial cells; Place the explant in the resuspended activated bacterial solution, shake it continuously, pour out the bacterial solution, place the explant on a culture dish lined with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-culture medium for culture; Step 6: Inducing hairy roots The explants were washed with washing solution, placed on a culture dish lined with sterile filter paper, excess liquid was sucked off and blown dry, and then inoculated on a hairy root induction medium and cultured.
2. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The step 1 also includes picking 24 single clones, streaking the single clones onto a TY solid culture medium with Kan and Strep resistance, and inverting and culturing at 28° C. for 1 day; using an inoculation loop to dip 24 Agrobacterium colonies into a TY liquid culture medium with Kan and Strep resistance, and shaking and culturing at 28° C. and 200 rpm / min for 1 day; drawing 500 uL of bacterial solution into a centrifuge tube, adding an equal volume of 60% glycerol, mixing, freezing in liquid nitrogen for 1 minute, and placing the frozen Agrobacterium bacterial solution in a -80° C. refrigerator for standby use.
3. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 1, the amount of pC1391-UBI-gusplus plasmid is 10 μL, the ice bath time is 30 min, the liquid nitrogen time is 1 min, 1 mL of TY liquid culture medium preheated at 28° C. is added, the shaking rate is 120 rpm / min, the shaking culture time is 3 h, the centrifugation rate is 5,000 rpm, the centrifugation time is 5 min, 100 μL of the supernatant is retained, and the culture conditions are inverted culture at 28° C. for 2 days.
4. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 2, the germination medium formula is shown in Table 1: Table 1 Germination medium formula The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 °C.
5. The method for pea hairy root transformation mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 2, the amount of NaClO solution added is 200-400 mL, the volume of the small beaker is 500 mL, the amount of concentrated hydrochloric acid taken is 10-30 mL, and the closed disinfection time is 10-12 h; Preferably, the amount of NaClO solution added is 300 mL; Preferably, the amount of concentrated hydrochloric acid is 20 ml.
6. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 3, the culture conditions for the first bacterial activation are: inverted culture at 28° C. for 2 days; and the culture conditions for the second bacterial activation are: shaking culture at 28° C. and 200 rpm / min overnight.
7. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 4, 3-5 mm hypocotyls were left, 3 replicates were set for each strain, 9 dishes of explants were inoculated in each replicate, and 5 explants were inoculated in each culture dish.
8. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step 5, the formula of the infection liquid culture medium is shown in Table 2: Table 2 Infection liquid culture medium formula The co-culture medium formula is shown in Table 3: Table 3 Co-culture medium formula 9. The method for transformation of pea hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step 5, 3-5 mL of the activated Agrobacterium bacterial solution is taken, and the remaining Agrobacterium bacterial solution is centrifuged at 5000 rpm for 10 minutes; the bacteria are activated by culturing at 4°C for more than half an hour, and the explants are placed in the resuspended activated bacterial solution and placed at room temperature for 30 minutes with continuous shaking; the culture conditions of the co-cultivation medium are: dark culture at 28°C for 4 days.
10. The method for pea hairy root transformation mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The formula of the induction medium in step 6 is shown in Table 4: Table 4 Induction medium formula The culture conditions are: 28℃ dark culture, culture time is about 3 weeks, subculture every 10 days.