Agrobacterium tumefaciens-mediated instant transformation method of polygonatum cyrtonema and application of agrobacterium tumefaciens-mediated instant transformation method

Abstract

The invention provides a polygonatum cyrtonema instantaneous transformation method based on agrobacterium tumefaciens mediation and application of the polygonatum cyrtonema instantaneous transformation method, and belongs to the technical field of genetic engineering. The instantaneous transformation method comprises the following steps: taking sterile rhizomes of polygonatum cyrtonema as explants, placing the explants in an agrobacterium infection bacterium solution, and carrying out vacuumizing infection; and then transferring into a co-culture medium for co-culture to obtain the instant transformation tuber of polygonatum cyrtonema with the key gene. The method can improve the genetic transformation efficiency of polygonatum cyrtonema, can be used for verifying the key gene function of polygonatum cyrtonema, and provides technical support for molecular assisted breeding of polygonatum cyrtonema.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an Agrobacterium-mediated transient transformation method of Polygonatum cyrtonema and an application thereof in functional verification of Polygonatum cyrtonema genes. Background Art

[0002] Polygonatum cyrtonema Hua (scientific name: Polygonatum cyrtonema Hua) is a perennial herb belonging to the genus Polygonatum of the family Liliaceae, Liliales. It combines medicinal, edible and ornamental uses and has high economic value. According to the "Flora of China", there are more than 40 species of Polygonatum, of which 31 are distributed in China.

[0003] Conventional breeding suffers from long cycles, low efficiency, and the limitations of hybrid compatibility. Transgenic technology, however, can overcome species limitations and accelerate breeding progress. In recent years, with the rapid development of various molecular biology techniques, stable transgenic technologies and gene silencing systems applicable to various crops have been successfully established, creating opportunities for the advancement of crop molecular biology research. Currently, genetic transformation systems for Polygonatum cyrtonema using Agrobacterium infection have been reported. For example, Jin Qing and Li Xiaochun induced hairy roots in Polygonatum cyrtonema and obtained transgenic Polygonatum cyrtonema through root-mediated genetic transformation. However, a more efficient, mature, and reliable genetic transformation system remains to be explored. Therefore, establishing a high-frequency regeneration system and a stable and reliable genetic transformation system for Polygonatum cyrtonema will lay the theoretical and applied foundation for further gene function research and cultivar innovation in Polygonatum cyrtonema. Agrobacterium-mediated transient transformation is an efficient method for studying gene function. As a supplement to stable transformation systems, it provides a powerful tool for rapid gene function studies and opens the door to homologous expression and reverse genetics studies in plants that lack stable genetic transformation systems.

[0004] The reported genetic transformation efficiency of Polygonatum multiflorum is 88.8%. However, the initial genetic transformation and positive strain screening process is time-consuming and time-consuming. Transient transformation directly from sterile tissue culture rhizomes has not been reported. Therefore, establishing an efficient and stable transient transformation technique for Polygonatum multiflorum using sterile rhizomes to verify gene function is of great significance. Summary of the Invention

[0005] In order to provide a high-frequency regeneration system and a stable and reliable genetic transformation system for Polygonatum cyrtonema, the present invention takes Polygonatum cyrtonema rhizomes as research objects and establishes a Polygonatum cyrtonema transient transformation system through Agrobacterium-mediated transient transformation technology.

[0006] To achieve the above objectives, the inventors provide the following technical solutions:

[0007] The invention discloses a transient transformation method of Polygonatum cyrtonema based on Agrobacterium-mediated transformation. The transient transformation method comprises the following steps: transforming a target gene into Agrobacterium, resuspending the target gene in MS liquid culture medium, and obtaining an infection solution; placing sterile rhizomes of Polygonatum cyrtonema as explants in the infection solution and performing vacuum infection; and then transferring the rhizomes to a co-culture culture medium for co-cultivation; and obtaining rhizomes transiently transformed with the target gene of Polygonatum cyrtonema after the co-culture is completed.

[0008] The method for obtaining the explant is as follows: the polygonatum multiflorum bulb is cut along the crease between the rhizome and the bulb and transferred to the tissue culture medium, cultured for 30 to 60 days to obtain the tissue culture seedling, the leaves and brown parts are removed, the rhizome tissue is washed with sterile water for 2 to 4 times, each washing time is less than 1 minute; and cut into 1 to 2 cm pieces. 3 After the cells are formed into blocks, they are transferred to the co-culture medium and pre-cultured at 28±2℃ and light intensity of 1800-2200lx for 1-2 days.

[0009] The MS liquid culture medium comprises 4-6% sucrose (mass percentage), 4-5g / L MS powder, 4-6mg / L AgNO3, and 90-110μmol / L acetosyringone (AS), preferably 5% sucrose, 4.47g / L MS powder, 5mg / L AgNO3, and 100μmol / L AS.

[0010] The co-cultivation medium comprises 4-6% sucrose (by mass), 4-5g / L MS powder, 4-6mg / L AgNO3, 90-110μmol / L acetosyringone, 4-6g / L agar, and the pH is adjusted to 5.8-6.2. Preferably, the co-cultivation medium comprises 5% sucrose, 4.47g / L MS powder, 5mg / L AgNO3, 100μmol / L AS, 5g / L agar, and the pH is adjusted to 5.8-6.2.

[0011] The tissue culture medium comprises 4-5 g / L MS powder, 20-40 g / L sucrose, 4-6 g / L agar, 0.8-1.2 mg / L 6-benzyladenine, and the pH is adjusted to 5.8-6.2. Preferably, the medium comprises 4.43 g / L MS powder, 30 g / L sucrose, 4.8 g / L agar, 1.0 mg / L 6-benzyladenine, and the pH is adjusted to 5.8-6.2.

[0012] The bacterial concentration of the infection solution is OD 600 =0.4~1.5. Preferably OD 600 =1.2.

[0013] The infection time is 5 to 20 minutes, preferably 15 minutes.

[0014] The co-cultivation method comprises the following steps: firstly placing the cells in darkness at 28±2°C for co-cultivation for 20-28 hours; and then transferring the cells to a temperature of 28±2°C and a light intensity of 1800-2200 lx for co-cultivation for 3-4 days.

[0015] Furthermore, the target gene is the PcWRKY33 gene.

[0016] Furthermore, the Agrobacterium is transformed by constructing the PcWRKY33 gene into the BGV007 plant expression vector.

[0017] The method for transforming Agrobacterium with the target gene comprises placing the competent Agrobacterium on ice until it is partially melted, inserting it into ice when it is in an ice-water mixed state, adding plasmid DNA containing the target gene, mixing, and sequentially standing on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 28°C water bath for 5 minutes, and in an ice bath for 5 minutes; adding LB liquid culture medium without antibiotics, and shaking culturing at 28°C for 2-3 hours; centrifuging at 6000 rpm for 1 minute to collect the bacteria; and smearing the resuspended bacterial block on an LB solid culture medium plate containing kanamycin and rifampicin antibiotics, and culturing the plate upside down in a 28°C incubator for 3 days.

[0018] Furthermore, the described method of transient transformation of Polygonatum cyrtonema mediated by Agrobacterium is applied in the functional verification of Polygonatum cyrtonema genes.

[0019] Furthermore, the application of the Agrobacterium-mediated transient transformation method of Polygonatum cyrtonema in molecular breeding of Polygonatum cyrtonema.

[0020] Furthermore, the described transient transformation method of Polygonatum cyrtonema mediated by Agrobacterium is applied in pathogen-resistant transgenic Polygonatum cyrtonema.

[0021] Furthermore, the pathogenic bacteria include Fusarium oxysporum.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention does not require inducing callus tissue or inducing the production of hairy roots. The sterile rhizomes of Polygonatum cyrtonema are used as explants. The positive transformed tubers of the target gene can be obtained efficiently and quickly by simply infecting the sterile tubers of Polygonatum cyrtonema with Agrobacterium, thus saving the time for tissue culture and positive tuber screening.

[0024] (2) The transient transformation system of Polygonatum cyrtonema of the present invention has a rapid infection process, a short culture period after transformation, and a high transformation efficiency, providing technical support for the molecular-assisted breeding channel for large-scale and efficient gene function verification and variety design.

[0025] (3) The technology of the present invention will not interfere with the stability of the genome in Polygonatum cyrtonema, and the expression of genes will not be affected by position effects; this technology does not require the regeneration of transformed cells for analysis of gene function.

[0026] (4) The present invention is a transient gene transformation system that can significantly accelerate the time consumed for transformation, greatly shorten the research process, and allow genes to be differentially expressed in Polygonatum cyrtonema after transient transformation.

[0027] (5) The present invention verifies the anti-pathogen function of the PcWRKY33 gene based on the transient transformation method. The discovery of this disease-resistance-related gene provides a reference for deepening the understanding of plant immune mechanisms and promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the tissue culture seedling of Polygonatum cyrtonema described in the specific implementation method.

[0029] Figure 2 This is a map of the BGV007 expression vector described in the specific embodiment.

[0030] Figure 3 This is the identification result of a single colony transformed with Agrobacterium as described in the specific embodiment.

[0031] Figure 4 Co-cultivation of transiently transformed tubers as described in a specific embodiment.

[0032] Figure 5 This is the PCR identification result of the transiently transformed tuber described in the specific embodiment.

[0033] Figure 6 OD described in the specific embodiment 600 The DNA electrophoresis diagrams of samples from the 0.4 and 1.5 treatment groups.

[0034] Figure 7 This is the target gene sequence alignment result described in the specific implementation method.

[0035] Figure 8 These are the symptoms of disease on transformed tubers after infection by the pathogen described in the specific implementation manner. DETAILED DESCRIPTION

[0036] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0037] Unless otherwise specified, the technical means and terms used in the following examples are understood according to the conventional usage of ordinary technicians in the relevant fields. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0038] Example 1

[0039] 1. Experimental Materials

[0040] 1.1 Materials

[0041] MS (Murashige & Skoog Basal Medium with Vitamins) culture medium and other materials were purchased from PhytoTechnology Laboratories, Agar agar powder was purchased from Yisheng Biological Company, and sucrose (analytical grade) was purchased from Xilong Science Co., Ltd.

[0042] 1.2 Explant collection and processing

[0043] The Polygonatum multiflorum bulbs were cut along the crease between the rhizome and bulb and transferred to the proliferation medium.

[0044] 2. Test methods

[0045] 2.1 Preparation of tissue culture medium

[0046] To prepare 1 L of MS solid medium, accurately weigh 4.43 g of MS reagent, dissolve the MS reagent and sucrose in distilled water, and dilute to 1000 mL. Adjust the pH to 5.8-6.2. Then, add agar, 6-benzyladenine (6-BA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and thiadiazole urea (TDZ). Specific amounts are shown in Table 1. Autoclave at 121°C for 20 min.

[0047] Table 1 Hormone formulas for different experimental groups

[0048]

[0049] 2.2 Different culture test groups

[0050] In a cleanroom, Polygonatum cyrtonema bulbs were transferred to tissue culture medium containing different experimental hormone formulations to induce adventitious buds. The growth of the Polygonatum cyrtonema rhizomes in tissue culture was observed after 30-40 days of culture.

[0051] 3. Experimental Results

[0052] 3.1 Effects of different hormones on adventitious bud induction in Polygonatum cyrtonema rhizomes

[0053] According to the results in Table 2, Group 1 produced the most leaves and the highest average induction rate (89.47%) from adventitious buds. Group 4 showed significant variation in the number of leaves induced per bottle, with the lowest average induction rate (50.00%). Similarly, Group 1 had the highest browning rate (56.88%). Groups 2 and 4 had similar browning rates, with no significant difference between these two groups and Group 1. Furthermore, although Group 3's adventitious buds did not produce leaves, they did exhibit browning of the rhizomes, with a browning rate significantly different from that of Group 1.

[0054] Table 2 Growth statistics of different experimental groups

[0055]

[0056] Note: Different letters in the same column indicate significant differences (p < 0.05).

[0057] Induction rate (%) = number of explants with induced buds / total number of inoculated cells * 100. Browning rate (%) = number of explants with browned buds / total number of inoculated cells * 100.

[0058] 3.2 Growth of Polygonatum cyrtonema rhizome tissue culture

[0059] After 30 days of culture, the results showed that among the four culture media, except for Group 3, which failed to induce adventitious buds, the other three groups were able to induce adventitious buds. Among them, Group 1 induced the largest number of leaves, followed by Group 2, and the least in Group 4. After 40 days of culture, there were differences in the elongation of leaves in each group. The leaves in Group 1 grew the best; the leaves in Groups 2 and 4 had significant growth effects, as shown in Figure 2. Figure 1 .

[0060] Therefore, the first group of MS solid medium containing only 1.0 mg / L 6-BA hormone was used as tissue culture medium, and the tissue culture seedlings of Polygonatum cyrtonema were the best, which could be used as explants for transient transformation.

[0061] Example 2

[0062] 1 Experimental Materials

[0063] (1) Vector and strain

[0064] Plant overexpression vector BGV007, Agrobacterium GV3101 competent cells. Infection was performed with Agrobacterium carrying the BGV007 (GV3103) plant expression vector, which contained the kanamycin (Kan) selectable marker gene, and stored at the Medicinal Plant Center of the Crop Research Institute of the Fujian Academy of Agricultural Sciences.

[0065] (2) Test reagents

[0066] Acetosyringone (AS) was purchased from Langbolide Biotechnology Co., Ltd. 192 mg of AS was weighed and dissolved in a small amount of NaOH, the volume was adjusted to 10 ml, and the AS was filtered and stored at -20°.

[0067] LB liquid medium: yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0068] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0069] MS liquid culture medium: 5% sucrose, MS powder 4.47 g / L, 5 mg / LAgNO3, 100 uM AS.

[0070] Co-culture medium: 5% sucrose, MS powder 4.47 g / L, 5 mg / LAgNO3, 100 uMAS, agar 5 g / L, pH adjusted to 5.8-6.2.

[0071] 50 mg / L Kanamycin (Kan): Dissolve in double-distilled water and sterilize by filtration at 0.22 μM. Working solution: 50 μg / ml.

[0072] 50 mg / L Rifampicin (Rif): Dissolve in DMSO and sterilize by filtration at 0.22 μM. Working solution: 20 μg / ml.

[0073] 2 Experimental methods

[0074] 2.1 Obtaining Polygonatum cyrtonema Explants

[0075] In a clean bench, remove the leaves and browned parts of the multiflora Polygonatum cyrtonema tissue culture seedlings, and use sterile water to wash the rhizome tissues 2 to 4 times, with each washing time less than 1 minute. Cut into 1cm 3 After the cells have formed into blocks, they are transferred to tissue culture medium and pre-cultured at 28±2℃ and 2000lx light intensity for 1 day. The pre-culture process is completed to improve the survival rate of the materials and ensure that the materials adapt to the new culture medium.

[0076] 2.2 Transformation of the target gene PcWRKY33

[0077] WRKYs are one of the largest families of transcription factors in plants. They are typical multifunctional regulatory factors involved in various signaling pathways and are associated with plant stress resistance. WRKYs not only play a crucial role in regulating responses to abiotic stresses, but also participate in a variety of biotic stress responses. Based on our full-length transcriptome data from Polygonatum cyrtonema and Polygonatum cyrtonema, we identified the WRKY gene family in Polygonatum cyrtonema and identified and validated the gene PcWRKY33 (transcript1513424) that may be involved in resistance to Fusarium oxysporum in Polygonatum cyrtonema. This study provides a reference for breeding disease-resistant Polygonatum cyrtonema varieties.

[0078] Take the competent Agrobacterium stored at -80℃ and place it on ice until it partially melts. When it is in an ice-water mixing state, insert it into ice. Add 1μg (volume not more than 10μl) PcWRKY33-BGV007 plasmid for every 100μl competent cell, pull the bottom of the tube by hand to mix, and let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 28℃ water bath for 5 minutes, and ice bath for 5 minutes. Add 700μl LB liquid culture medium without antibiotics and culture at 28℃ with shaking for 2-3 hours. Centrifuge at 6000rpm for 1min to collect the bacteria, take about 100μl of supernatant and gently blow to resuspend the bacteria, spread it on the LB solid culture medium plate containing Kan and Rif antibiotics, and place it upside down in a 28℃ incubator for 3 days.

[0079] 2.3 Bacterial proliferation culture and verification

[0080] After 72 hours, single colonies were picked and cultured overnight in LB liquid medium containing Kan and Rif antibiotics. The next day, an appropriate amount of fresh bacterial culture was aspirated for PCR verification. Colony PCR verification primers are shown in Table 3. Once the bacterial culture was verified, an appropriate amount of fresh bacterial culture was taken and expanded in LB liquid medium containing Kan and Rif antibiotics.

[0081] Table 3 Colony PCR verification primers and their sequences

[0082]

[0083] 2.4 Instantaneous conversion method

[0084] OD 600 The bacterial solution was centrifuged at 5000 rpm for 10 min to collect the cells, and the concentration of the bacterial solution was adjusted to OD 600= 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5, and allowed to stand at room temperature for two hours to obtain an infection solution. The pre-cultured Polygonatum cyrtonema explants were transferred to the infection solution and infected in a vacuum machine for 5, 10, 15, 20, and 30 minutes, respectively. After infection, the bacterial solution on the surface of the explants was blotted dry with sterile filter paper and then transferred to MS solid medium for co-cultivation. The culture dish was sealed and placed in the dark at 28±2°C for 24 hours. Then, the culture was transferred to 28±2°C and co-cultivated at 2000 lx light intensity for 3 days. After co-cultivation, transiently transformed rhizomes of Polygonatum cyrtonema with PcWRKY33 were obtained.

[0085] 2.5 Verification of amplified bands based on the Agrobacterium-mediated transient transformation system of Polygonatum cyrtonema

[0086] After sampling the transformed Polygonatum cyrtonema rhizomes, DNA was extracted for PCR verification. After electrophoresis, the amplification was observed and the number of bands in each group was counted to analyze the transient transformation efficacy of the Polygonatum cyrtonema samples under different treatments.

[0087] 3Comparative study results of different combinations of factors in the transient transformation method of Polygonatum cyrtonema

[0088] 3.1 Effects of different concentrations on browning rate and contamination rate

[0089] For different concentrations (OD 600 Statistical analysis of browning and contamination rates was conducted for treatments with OD values of 1.2 and 0.0%, respectively. Table 4 shows that concentration changes significantly affect both rates. Specifically, as concentration increases, the browning and contamination rates exhibit a fluctuating trend. At an OD value of 1.2, the average browning rate was the lowest, at only 0.0%, and the contamination rate was 25.0%. 600 When OD is 0.4, although the browning rate is low at 27.1%, the contamination rate reaches 77.1%, indicating that the contamination problem is more serious. 600 The treatment group with a value of 1.2 had the best overall performance, with the lowest browning rate and relatively low contamination rate.

[0090] 3.2 Effects of different infection times on browning rate and contamination rate

[0091] The effect of infection time on browning and contamination rates was also significant. The 5-minute infection group had an average browning rate of 15.9% and a contamination rate of 47.2%, both of which were relatively low. The 15-minute infection group showed a significant increase in browning to 40.0%, while the contamination rate only slightly increased to 48.3%. The 20-minute infection group showed a slight decrease in browning to 36.7%, but a significant increase in contamination to 75.6%. The 30-minute infection group showed a significant decrease in browning to 9.7%, but a significant increase in contamination to 58.3%. Considering the balance between browning and contamination rates, the 15-minute infection group achieved the best performance.

[0092] Based on the above results, combined with different OD 600 Comprehensive analysis of infection time showed that 600 When the OD value is 1.2 and the infection time is 15 minutes, the best balance between browning rate (0.0%) and contamination rate (25.0%) can be achieved, which has better practical application value. Under this condition, the browning phenomenon is significantly inhibited, and the contamination rate is controlled within a reasonable range, which provides an important reference for subsequent process optimization. The results show that the appropriate treatment conditions are OD 600 =1.2, infection time is 15 minutes.

[0093] Table 4 Pollution status of different treatments

[0094]

[0095]

[0096] Note: Browning rate (%) = number of explants with browned buds / total number of inoculated samples * 100. Contamination rate (%) = number of explants with contaminated buds / total number of inoculated samples * 100.

[0097] 4. Verification of infection effect

[0098] 4.1 Extraction of genomic DNA from Polygonatum cyrtonema tubers using CTAB

[0099] After the co-culture period, OD was extracted using the CTAB method. 600 = 1.2 of the polygonatum tuber DNA, PCR identification was performed to identify whether the PcWRKY33-BGV007 recombinant plasmid was successfully transformed into the polygonatum tuber. At the same time, the OD 600 = 0.4 and 1.5 treated samples with different infection times, whether they also successfully completed transient transformation.

[0100] 4.2 Amplification of target genes and agarose gel electrophoresis

[0101] Dilute the DNA stock solution to 200 ng / μL and perform PCR amplification. Observe the amplified target fragment band by electrophoresis and send the PCR product for sequencing. The primers used for PCR amplification are shown in Table 5. The PCR reaction system and reaction procedure are shown in Tables 6 and 7.

[0102] Table 5 PCR verification primers and their sequences

[0103]

[0104] Table 6 PCR reaction system

[0105]

[0106] Table 7 PCR amplification program

[0107]

[0108] 4.3 Results of genomic DNA identification of Polygonatum multiflorum tubers

[0109] After PCR, the results of agarose gel electrophoresis showed that 600 =1.2 infected Polygonatum cyrtonema tuber DNA showed an obvious amplification band around 1000bp, which was consistent with the position of the target band, indicating that the transient transformation was successful ( Figure 3 The results showed that the recombinant plasmid PcWRKY33-BGV007 had been transformed into the rhizomes of Polygonatum cyrtonema.

[0110] At the same time, by extracting OD 600 PCR supplementary verification was performed on the DNA of the samples from the 0.4 and 1.5 treatment groups. It was found that each treatment group had a clear and obvious amplification band around 1000 bp, which was consistent with the target fragment ( Figure 6 ), indicating that the conversion rate of this experiment reached 100%.

[0111] 4.4 DNAMAN sequence alignment analysis

[0112] The PCR products were sent to Sangon Biotech for DNA sequencing. The sequencing results were compared and analyzed using DNA MAN software to verify sequence consistency.

[0113] The sequence comparison results of genes ( Figure 7 ) showed that the PCR amplified sequence of the transiently transformed rhizomes of Polygonatum cyrtonema successfully aligned with the PcWRKY33-BGV007 sequence. PCR product sequencing results further confirmed that the PcWRKY33-BGV007 recombinant plasmid had been successfully transformed into the rhizomes of Polygonatum cyrtonema.

[0114] Example 3

[0115] Transiently transformed Polygonatum cyrtonema tubers obtained by co-cultivation using the method of Example 2 were subjected to a pathogen infection test and the disease status of the rhizomes was observed and recorded. The strain used in the test was Fusarium oxysporum isolated from a Polygonatum cyrtonema root rot sample, strain number 33245.

[0116] 1. Pathogen infection and observation methods

[0117] Preparation of bacterial cakes: Culture the GFP-tagged Fusarium oxysporum strain on a PDA plate in a 28°C incubator for 3-5 days. Use a hole punch or pipette to cut the bacteria from the plate into cakes less than 1 cm in diameter. The prepared cakes are ready for inoculation.

[0118] Experimental Treatment: Select PcWRKY33-BGV007 transformed rhizomes of uniform size and growth. After disinfection by soaking twice in 75% ethanol, rinse three times with sterile water, and blot dry the rhizomes with sterile filter paper. A control group (rhizomes treated with clean water) and a treatment group (rhizomes transformed transiently) were set up. Prepared bacterial cakes were picked with a sterile needle and placed on the rhizomes. Six transiently transformed rhizomes were inoculated into each group and incubated in an incubator at 26 ± 2°C.

[0119] Infection Observation: Observe the rhizomes infected with Fusarium oxysporum regularly after inoculation, including 3 and 5 days after inoculation. Observe and record the area of the rhizomes infected with the pathogen and take photos.

[0120] 2 Observation results

[0121] according to Figure 8 The results showed that 3 days after inoculation, the control group had obvious lesions and some tubers had superficial rot, while the PcWRKY33 transformed materials had smaller surface lesions and no rot. 5 days after inoculation, the degree of rot in the control group was more severe than that in the PcWRKY33 transformed materials.

[0122] In summary, the transient genetic transformation system for Polygonatum cyrtonema established in this invention enables efficient and rapid production of rhizomes overexpressing key genes in Polygonatum cyrtonema, with a conversion rate of 100%. Transformation of Polygonatum cyrtonema tubers with the PcWRKY33 gene via Agrobacterium-mediated transient transformation of Polygonatum cyrtonema yielded PcWRKY33-overexpressing tubers with significantly enhanced resistance to infection by Fusarium oxysporum. This transient transformation method, constructed in this invention, can be used to cultivate pathogen-resistant transgenic Polygonatum cyrtonema and lays the foundation for functional verification of key genes in Polygonatum cyrtonema.

[0123] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the present invention's specification and drawings, and direct or indirect application of the above technical solutions to other related technical fields, are all included in the scope of patent protection of the present invention.

Claims

1. A method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation, characterized in that: The transient transformation method comprises the following steps: transforming the target gene into Agrobacterium, resuspending the culture in MS liquid medium, and obtaining an infection solution; placing the sterile rhizome of Polygonatum cyrtonema as an explant in the infection solution and performing vacuum infection; and then transferring the culture solution to a co-culture medium for co-cultivation. After the co-cultivation, rhizomes transiently transformed with the target gene of Polygonatum cyrtonema were obtained.

2. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1, characterized in that: The method for obtaining the explant is as follows: the polygonatum multiflorum bulb is cut along the crease between the rhizome bulb and the bulb and transferred to the tissue culture medium, cultured for 30 to 60 days to obtain the tissue culture seedling, the leaves and brown parts are removed, the rhizome tissue is washed with sterile water for 2 to 4 times, each washing time is less than 1 minute; cut into 1 cm 3 After the cells have formed into clumps, they are transferred to a co-culture medium and pre-cultured.

3. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1, characterized in that: The components of the MS liquid culture medium include 4-6% sucrose, 4-5g / L MS powder, 4-6mg / L AgNO3 and 90-110μmol / L acetosyringone.

4. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1 or 2, characterized in that: The co-cultivation culture medium comprises 4-6% sucrose, 4-5g / L MS powder, 4-6mg / L AgNO3, 90-110μmol / L acetosyringone, 4-6g / L agar, and the pH is adjusted to 5.8-6.

2.

5. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 2, characterized in that: The tissue culture medium comprises 4-5 g / L of MS powder, 20-40 g / L of sucrose, 4-6 g / L of agar, and 0.8-1.2 mg / L of 6-benzyladenine, and the pH is adjusted to 5.8-6.

2.

6. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1, characterized in that: The bacterial concentration of the infection solution is OD 600 =0.4~1.5, and the infection time is 5~20min.

7. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1, characterized in that: The co-cultivation method comprises the following steps: firstly placing the cells in darkness at 28±2°C for co-cultivation for 20-28 hours; and then transferring the cells to a temperature of 28±2°C and a light intensity of 1800-2200 lx for co-cultivation for 3-4 days.

8. The method for transient transformation of Polygonatum cyrtonema based on Agrobacterium-mediated transformation according to claim 1, characterized in that: The target gene is the PcWRKY33 gene.

9. Use of the Agrobacterium-mediated transient transformation method of Polygonatum cyrtonema as claimed in any one of claims 1 to 7 in the functional verification of Polygonatum cyrtonema genes.

10. Use of the Agrobacterium-mediated transient transformation method of Polygonatum cyrtonema as claimed in any one of claims 1 to 8 in pathogen-resistant transgenic Polygonatum cyrtonema.

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