Construction method of cunninghamia lanceolata genetic transformation system

Abstract

The invention discloses a construction method of a cunninghamia lanceolata genetic transformation system, and belongs to the technical field of genetic transformation. The invention discloses a construction method of a cunninghamia lanceolata genetic transformation system, which comprises the following steps: preparing an EHA105 agrobacterium infection solution with OD600 of 1.2 by taking a 11C genotype cunninghamia lanceolata tissue culture seedling as a transformation receptor, carrying out-60kPa vacuumizing treatment for 25 minutes, and co-culturing in a dark environment at 23 DEG C for 48 hours after infection to complete transformation. According to the method, an efficient instantaneous genetic transformation system which is simple and convenient to operate is successfully established, the problem that traditional breeding genetic gain of the cunninghamia lanceolata is limited is solved, key parameters such as bacterial liquid concentration, infection time and co-culture conditions are optimized through an orthogonal test by utilizing an agrobacterium tumefaciens-mediated instantaneous transformation technology, and the transformation efficiency is remarkably improved. GUS enzyme activity determination and dyeing verification show that the system is high in transformation efficiency and good in repeatability, and an efficient technical platform is provided for Chinese fir gene function research and genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic transformation technology, and more specifically, relates to a method for constructing a genetic transformation system for Chinese fir. Background Technology

[0002] Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is a large evergreen tree belonging to the family Taxodiaceae and the genus Cunninghamia. It is tall with a straight trunk and is an important fast-growing timber species in my country. Chinese fir is characterized by its rapid growth, high quality, and high yield, and is widely planted in subtropical and tropical regions of my country, significantly increasing the stock volume of afforestation species and making a significant contribution to forest resource growth and ecological balance maintenance. For a long time, traditional breeding of Chinese fir has achieved success in areas such as source selection, hybridization breeding, and the establishment of high-generation seed orchards, but it is gradually facing challenges such as soil degradation and limited genetic gain.

[0003] Unlike stable transformation, Agrobacterium-mediated transient genetic transformation, while unable to integrate exogenous genes into chromosomes, offers advantages such as short cycle time, high efficiency, and less restriction on the size of gene fragments that T-DNA can carry, making it an ideal choice for efficient gene expression pattern research. This technology is particularly suitable for species for which stable transformation systems have not yet been established, providing a foundation for research on stable genetic transformation. For example, researchers used vacuum staining to treat needles and stem segments of western white pine (Pinus monticola) and observed transient GUS expression activity in cross-sections of the recipient tissue, thus establishing a transient genetic transformation system. Similar transient genetic transformation systems have also been established in callus, cotyledons, and needles of Chinese pine (Pinus tabuliformis) using the same method.

[0004] Reporter genes (such as GUS, GFP, RFP, etc.) encode proteins or enzyme products that are easy to detect. By co-transforming with the target gene, the expression and regulation of the target gene can be effectively tracked. Among them, the GUS (β-glucuronidase) gene is widely used in research to establish genetic transformation systems due to its stable product and flexible detection methods. Histochemical staining uses 5-bromo-4-chloro-3-indole-β-glucuronide (X-Gluc) as a substrate, generating a blue product under enzymatic digestion, allowing direct observation of the transformation site. To ensure the accuracy of experimental results and perform quantitative analysis, fluorescence spectrophotometry can be used. This method uses 4-methyl-2-oxo-2H-1-benzopyran-7-ylβ-D-glucopyranosiduronic acid (4-MUG) as the reaction substrate. By detecting the fluorescence intensity of the enzymatic hydrolysis product 4-methylumbelliferone (4-MU), GUS activity is quantitatively analyzed, providing a precise assessment of transformation efficiency and gene expression levels. Genetic improvement based on modern technologies such as genetic engineering has become a new direction for overcoming breeding bottlenecks. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for constructing a genetic transformation system of Chinese fir, which is used to construct superior transgenic Chinese fir lines.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for constructing a genetic transformation system for Chinese fir, comprising:

[0008] 1) Preparation of infection solution: After culturing the Agrobacterium strain, adjust the OD of the infection solution. 600 The value is 0.2-1.2;

[0009] 2) Genetic transformation: Using 11C genotype Chinese fir tissue culture seedlings as transformation recipients, infection was performed using an infection solution; after infection, the seedlings were rinsed with washing solution and placed in a co-culture medium for dark culture to obtain genetically transformed Chinese fir tissue culture seedlings.

[0010] Furthermore, the Agrobacterium strain is one or more of EHA105, GV3101, LBA4404, Ar1193, C58C1, K599, or K599 (pSoup-p19).

[0011] Furthermore, the Agrobacterium strain is EHA105.

[0012] Furthermore, the infection step involves cutting a 1cm section of newly proliferated seedlings from 11C genotype Chinese fir tissue culture seedlings as the transformation recipient, and immersing them in OD... 600 Infect the material with a concentration of 0.2-1.2, shake at 90 rpm, remove the material, discard the conversion solution, quickly rinse the material with washing solution, blot dry on filter paper, and transfer it to a co-culture medium for dark incubation.

[0013] Furthermore, the OD of the infiltration solution 600 It is 1.2.

[0014] Furthermore, the infection time is 10 min to 6 h or vacuum treatment for less than 30 min.

[0015] Furthermore, the vacuum treatment is -60 kPa.

[0016] Furthermore, the infection time is 25 minutes of vacuum treatment.

[0017] Furthermore, the co-culture conditions are: 4℃ dark culture for 48h, or 23℃ dark culture for 48h, or 28℃ dark culture for 48h, or 4℃ dark culture for 12h followed by 23℃ dark culture for 36h, or 4℃ dark culture for 12h followed by 28℃ dark culture for 36h.

[0018] Furthermore, the co-culture conditions are 23°C dark culture for 48 hours.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) This invention optimizes the Agrobacterium strain (e.g., EHA105) and the concentration of the infection solution (OD). 600 Key parameters such as GUS enzyme activity assay and staining verification were used to determine the transformation efficiency, with an average enzyme activity of 166 pmol 4-MU / μg·min and a GUS expression level of 97-fold.

[0021] 2) This invention employs Agrobacterium-mediated transient genetic transformation technology, which has the advantages of short cycle and high efficiency. It has few limitations on the size of the T-DNA-carrying gene fragment, making it suitable for gene expression pattern research and laying the foundation for stable genetic transformation. This system has good reproducibility and is easy to operate, providing an efficient technical platform for the study of Cunninghamia lanceolata gene function and the construction of superior transgenic lines, which is of great significance for forest resource growth and ecological balance maintenance. Attached Figure Description

[0022] Figure 1 Figure showing the determination and comparison of GUS enzyme activity in Cunninghamia lanceolata tissue culture seedlings of different treatment groups in the orthogonal experiment;

[0023] Figure 2 GUS staining images of Cunninghamia lanceolata tissue culture seedlings in each treatment group of the orthogonal experiment (ay corresponds to orthogonal experiment combination number 1-25, scale bar is 2mm);

[0024] Figure 3 The transformation results are shown in the diagrams after different initial concentrations of infection and dilution concentrations of infection at different time ratios (af represents the GUS staining results of treatment groups ①-⑥, with a scale bar of 2 mm; g represents the GUS enzyme activity assay and comparison; h represents the qRT-PCR results of GUS expression level).

[0025] Figure 4 The transformation results after treatment with different Agrobacterium strains are shown in the figure (a is EHA105; b is GV3101; c is LBA4404; d is Ar1193; e is C58C1; f is K599; g is K599 (pSoup-p19); h is the GUS enzyme activity assay and comparison; i is the GUS expression level qRT-PCR result).

[0026] Figure 5 The graphs show the transformation results after different vacuum treatments (a = 0; b = 5; c = 10; d = 15; e = 25; f = 30; g = GUS enzyme activity assay and comparison; h = GUS expression level qRT-PCR results).

[0027] Figure 6 The graphs show the transformation results after different co-culture temperature treatments (a is 4; b is 4+23; c is 4+28; d is 23; e is 28; f is the GUS enzyme activity assay and comparison; g is the GUS expression level qRT-PCR result).

[0028] Figure 7 GUS staining results of the transformation of the "Yang 061" clone material (a is the GUS staining result of the WT transformed plant; be is the GUS staining result of the transformed bud, scale bar is 2mm). Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0030] In the following examples, 1 cm of newly proliferated seedlings of the Chinese fir 11C genotype with good growth on proliferation medium (DCR + 0.6 mg / L 6-BA + 0.3 mg / L IBA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8) were selected as transformation recipients.

[0031] Example 1

[0032] 1. Preparation of inoculum

[0033] Healthy *Cunninghamia lanceolata* 11C tissue culture seedlings were thoroughly ground in liquid nitrogen. Total RNA was extracted using the Nanjing Novizan FastPure Total RNA Isolation Kit, and cDNA was obtained using the Nanjing Novizan HiScriptⅢ 1st Strand cDNA Synthesis Kit reverse transcription reagent (specific experimental procedures were performed according to the manufacturer's instructions). The *ClCesA3* gene (its nucleotide sequence is shown in SEQ ID NO.1) was cloned based on the cDNA template, ligated into the pClone007 vector, and transformed into DH5α competent cells. The target gene was obtained through colony PCR screening and sequencing verification. Based on multi-parameter analysis (GC content, genomic location, sgRNA secondary structure and specificity, etc.), the optimal target site was screened to construct a CRISPR / Cas9 knockout vector. The verified vector was transformed into *Agrobacterium* EHA105 competent cells, and positive clones were screened for subsequent genetic transformation experiments.

[0034] Take 20 μL of positive bacterial suspension and inoculate it into 2 mL of liquid culture medium. Incubate at 28℃ and 220 rpm with shaking for 12 h (vigorous shaking). Take 2 mL of the vigorously shaken bacterial suspension and transfer it to 50 mL of liquid culture medium containing Kana. Expand the culture to OD. 600 =0.6-0.8 (logarithmic growth phase). Take an appropriate amount of bacterial culture, centrifuge at 6000 rpm for 8 min, and collect the bacterial cells. Resuspend the bacterial cells in transformation buffer (MES 2mM, CaCl2 40mM, AS 120μM, mannitol 270mM, sucrose 2%, Tween 0.02%, 5-AzaC 200μM, DTT 200mg / L, 6-BA 2mg / L, NAA 0.5mg / L) to adjust the OD... 600 Once the desired concentration is achieved, the inoculum is prepared.

[0035] 2. Genetic transformation of Chinese fir

[0036] L25(5) 6An orthogonal experiment was conducted, setting five levels (five factors each) for bacterial concentration, infection time, co-culture time, transformation solution osmotic pressure (sucrose concentration), and acetylsylgenin (AS) concentration (Table 1). Transformation recipients were excised and immersed in the infection solution, with shaking at 90 rpm for infection. After the set infection time, the recipients were removed. When half of the infection time had elapsed, 0.5 times the original volume of transformation solution (MES 2 mM, CaCl2 40 mM, AS 120 μM, mannitol 270 mM, sucrose 2%, Tween 0.02%, 5-AzaC 200 μM, DTT 200 mg / L, 6-BA 2 mg / L, NAA 0.5 mg / L) was added to complete the remaining infection time. Discard the infection solution and quickly rinse the material with washing buffer (CaCl2 240mM, AS 120μM, sucrose 3%, 6-BA 2mg / L, NAA 0.5mg / L, DTT 200mg / L). Blot dry thoroughly on filter paper and transfer to co-culture medium for dark incubation. After co-culturing at 23℃ for 0-24 hours, samples are taken for various measurements. Each treatment infects 9 tissue culture seedlings (6 for GUS staining, 3 for GUS enzyme activity measurement), with 3 replicates. Range analysis is used to determine the influencing factors, and a highly efficient transient genetic transformation method for *Cunninghamia lanceolata* tissue culture seedlings is preliminarily screened.

[0037] Table 1. Preliminary optimization of the orthogonal experiment table.

[0038]

[0039]

[0040] 3. GUS staining and enzyme activity assay

[0041] 1) GUS staining

[0042] After co-culturing, the *Cunninghamia lanceolata* tissue culture seedlings were removed and placed in 10 mL centrifuge tubes, with different treatments placed separately. 90% acetone solution was added to the centrifuge tubes to cover the plant material, and the tubes were fixed on ice for 20 min. The acetone solution was discarded, and the plant material was rinsed with buffer solution, repeated three times. The buffer solution was discarded, and GUS staining solution was added to cover the plant material. The material was then vacuum-permeable for 20–30 min, removed, and placed in a 37°C oven for staining for 12 h. The staining solution was discarded, and the plant material was rinsed three times with 75% anhydrous ethanol solution, followed by soaking in a gradient of 50%–90% ethanol solutions for 5 min each. Finally, the plant material was soaked in anhydrous ethanol for 1 h to decolorize. To avoid incomplete decolorization with anhydrous ethanol and material brittleness, after discarding the anhydrous ethanol, a clearing solution was added to immerse the material. The staining results were observed and photographed after the tissue became completely clear.

[0043] 2) GUS enzyme activity assay

[0044] ① Protein extraction: Add 3 times the volume of GUS enzyme extraction solution according to the sample weight, grind thoroughly on ice, centrifuge at 13000 rpm and 4℃ for 10 min, and collect the supernatant for later use.

[0045] ② Prepare a protein standard curve: Use 10 μg / mL BSA solution, dilute it serially (Table 2), measure the absorbance of each concentration, and establish a standard curve.

[0046] Table 2. Preparation of Protein Standard Curve

[0047] BSA (μL) 0 1 2 4 6 8 10 <![CDATA[ddH2O(μL)]]> 1000 999 998 996 994 992 990 Final concentration (ng / μL) 0 10 20 40 60 80 100

[0048] ③ Take 5 μL of the supernatant obtained from protein extraction, add water to make up to 4 mL; add 1 mL of Coomassie Brilliant Blue G250, vortex to mix, and react in the dark for 5-10 min; take 200 μL of the reaction solution into a 96-well plate and measure the absorbance at 595 nm; calculate the protein concentration according to the standard curve.

[0049] ④ Prepare a 4-MU standard curve: Dilute 2 μM 4-MU stock solution in a serial manner (Table 3), measure the fluorescence value at each concentration, and establish a standard curve.

[0050] Table 3. Construction of 4-MU Standard Curve

[0051]

[0052]

[0053] ⑤ Take 50 μL of the protein extraction supernatant, add 450 μL of preheated 2 mM MUG reaction solution (37℃), mix immediately, and add 50 μL to 950 μL of stop solution (0.2 M Na2CO3) as a T0 control. Take 50 μL samples at 5 min, 10 min, 20 min, and 30 min to terminate the reaction; transfer 200 μL of all assay solutions to a 96-well microplate and measure the fluorescence value of the reaction solution under 365 nm excitation light and 455 nm emission light conditions; calculate the concentration of 4-MU produced by each sample at each time point according to the formula obtained from the 4-MU standard curve. GUS enzyme activity (unit: nmol / μg·min) is defined as the amount of 4-MU produced per unit protein of the sample per minute. The formula for calculating GUS enzyme activity is as follows:

[0054]

[0055] The results are as follows Figure 1As shown, differences exist among different treatments. Combinations with higher mean enzyme activity (e.g., experiments 16 and 23) yielded mean GUS enzyme activities ≥100 pmol 4-MU / μg·min, while combinations with lower activity (e.g., experiments 13 and 25) yielded mean activities ≤30 pmol 4-MU / μg·min. The small coefficients of variation within groups indicate good experimental repeatability, making the experiments suitable for range calculation and analysis in subsequent orthogonal experiments.

[0056] The results are as follows Figure 2 As shown, there were significant differences in expression among the treatment groups, consistent with the trend of enzyme activity assay results. The combination with higher enzyme activity showed significantly better staining effect than other treatment groups. In treatment groups 8, 10, 12, and 23, the vascular tissue and leaf veins of the plant were clearly visible as blue, while in treatment groups 13 and 25, almost no blue was visible.

[0057] Using the GUS enzyme activity data obtained from a total of 25 treatments in an orthogonal experimental table as an indicator, the optimal treatment was calculated and analyzed using the range method.

[0058] The results are shown in Table 4. The order of the major effects of the five factors on GUS enzyme activity was: co-culture time (C, R = 52.09) > bacterial concentration (A, R = 31.18) > infection time (B, R = 30.176) > acetylsuccinone (AS) concentration (E, R = 23.824) > osmotic pressure (D, R = 8.736). The optimal combination was A5B4C4D2E4 (treatment 26), with the specific parameters being: bacterial concentration OD... 600 =1.2 (A5), osmotic pressure of 30 g / L sucrose (D2), 200 μM AS (E4) added to the conversion solution, infection for 3 h (diluted to OD at 1.5 h). 600 =0.8, B4), co-cultured for 48h (C4).

[0059] Table 4. Calculation results of the orthogonal experiment range method

[0060]

[0061]

[0062]

[0063] Note: The five levels corresponding to the factors in the table are simplified from 1 to 5. For example, in factor A (bacterial concentration), columns 1 to 5 represent OD. 600 =0.2~1.2.

[0064] Example 2: Conversion results of infection time ratios at different initial concentrations and dilution concentrations

[0065] Based on the experimental steps of Example 1, the optimal concentration of the inoculum was determined to be OD.600 =1.2, the dilution method is still to add 0.5 times the volume of the original inoculum solution (i.e., the inoculum concentration OD) midway through the process. 600 =1.2 OD after dilution 600 =0.8); Set two total durations, 2 and 3 hours, and compare three time ratios (1:0, 1:1, 1:1.5) for initial concentration infection and diluted concentration infection at each duration (taking 2 hours as an example, a time ratio of 1:0 represents the initial concentration infection at OD). 600 =1.2 infection time 2 hours, 1:1 represents OD 600 =1.2 hours of infection, OD 600 =0.8 infection time 1 hour, 1:1.5 represents OD 600 =1.2 infection time 0.8h, OD 600 The effect of 0.8 μg / L sucrose (infection time 1.2 h) on transformation results was investigated, with a total of 9 treatments. The co-culture time was set at 48 h, the osmotic pressure at 30 g / L sucrose, and the AS concentration in the transformation solution at 200 μM. Each experiment involved infecting 15 tissue culture seedlings (6 for GUS staining, 6 for GUS enzyme activity measurement, and 3 for GUS quantitative PCR measurement), with 3 replicates. The GUS staining and GUS enzyme activity procedures were as described in Example 1. Quantitative PCR detection: Transformed and co-cultured *Cunninghamia lanceolata* tissue culture seedlings were used as materials. Total RNA was extracted using the Nanjing Novizan FastPure Total RNA Isolation Kit, and cDNA was obtained using the Nanjing Novizan HiScriptⅢ 1st Strand cDNA Synthesis Kit reverse transcription reagent (specific experimental procedures were performed according to the manufacturer's instructions). Specific primers were designed for the target sequence, and quantitative PCR experiments were performed using the Nanjing Novizan AceQ qPCR SYBR GreenMaster Mix reagent.

[0066] Table 5 Optimized Processing Combinations

[0067]

[0068]

[0069] The results are as follows Figure 3 As shown, the entire process is in OD format. 600 The optimal treatment concentration was 1.2, with an infection time of 2 hours, resulting in an average GUS enzyme activity of 160.5 pmol 4-MU / μg·min; GUS expression was also significantly increased (10⁹-fold); and clear blue staining was visible throughout the entire plant by GUS staining. Figure 3 (a) In addition, after 3 hours of infection, the bacterial concentration was diluted to OD at 1.5 hours. 600 The treatment with a value of 0.8 is a suboptimal solution and can be considered as a method for young materials such as infective embryos and cotyledons.

[0070] Example 3: Transformation results of different Agrobacterium strains

[0071] Based on the experimental procedures of Example 1, the pCAMBIA1301 vector was used to transform three strains of Agrobacterium tumefaciens (EHA105, GV3101, LBA4404) and four strains of Agrobacterium rhizogenes (Ar1193, C58C1, K599, K599 (pSoup-p19)) to prepare infection solutions, and transformation was performed separately. The transformation results of the seven strains were compared. The co-culture time was set at 48 h, the osmotic pressure was set at 30 g / L, and the AS concentration in the transformation solution was set at 200 μM. Each experiment infected 15 tissue culture seedlings (6 strains were stained with GUS, 6 strains were used to measure GUS enzyme activity, and 3 strains were used for GUS fluorescence quantitative PCR detection), with 3 replicates. The GUS staining and GUS enzyme activity procedures were the same as in Example 1. The fluorescence quantitative PCR detection procedures were the same as in Example 2.

[0072] The results are as follows Figure 4 As shown, among the Agrobacterium tumefaciens strains, the transformation efficiency mediated by strain EHA105 (GUS enzyme activity 514 pmol 4-MU / μg·min, GUS expression level 213-fold) was significantly better than that of strains GV3101 and LBA4404 (p<0.05); among the Agrobacterium rhizogenes strains, strains Ar1193 and C58C1 showed good transformation results (GUS enzyme activity >200 pmol 4-MU / μg·min, GUS expression level >70-fold), significantly better than the two K599 strains (p<0.05). GUS histochemical staining results showed that the GUS staining effect of plants transformed by strain EHA105 was generally better than that of other strains (…). Figure 4 (a).

[0073] Example 4: Conversion Results of Different Vacuuming Treatments

[0074] Based on the experimental procedures of Example 1, vacuum treatment (-60 kPa) was performed for 0, 10, 15, 25, and 30 minutes during the infection process. Samples were taken after co-culture for various measurements. The co-culture time was set to 48 hours, the osmotic pressure to 30 g / L, and the AS concentration in the transformation solution to 200 μM. Each experiment involved 15 tissue culture seedlings (6 for GUS staining, 6 for GUS enzyme activity measurement, and 3 for GUS quantitative PCR detection), with 3 replicates. The GUS staining and GUS enzyme activity measurement procedures were as described in Example 1. The quantitative PCR detection procedures were as described in Example 2.

[0075] The results are as follows Figure 5As shown, vacuum treatment for 25 minutes during infection yielded the best results, with significantly higher mean GUS enzyme activity (112 pmol 4-MU / μg·min) and GUS expression level (76-fold) compared to other treatments (p<0.05). GUS histochemical staining revealed a distinct blue color throughout the entire plant. Figure 5 f).

[0076] Example 5: Transformation results under different co-culture temperature treatments

[0077] Based on the experimental procedures of Example 1, five treatments were set up: dark incubation at 4, 23, and 28°C for 48 hours; and dark incubation at 4°C for 12 hours followed by co-incubation at 23 and 28°C for 36 hours. Various measurements were then performed. The osmotic pressure was set at 30 g / L, and the AS concentration in the transformation solution was set at 200 μM. Each experiment involved infecting 15 tissue culture seedlings (6 for GUS staining, 6 for GUS enzyme activity measurement, and 3 for GUS quantitative PCR detection), with three replicates. The GUS staining and GUS enzyme activity measurement procedures were as described in Example 1. The quantitative PCR detection procedures were as described in Example 2.

[0078] The results are as follows Figure 6 As shown, continuous co-culturing at 23℃ for 48 h yielded the best results. The mean GUS enzyme activity (166 pmol 4-MU / μg·min) and GUS expression level (97-fold) in this treatment were significantly higher than those in other treatments (p<0.05). GUS histochemical staining showed that blue was clearly visible in the shoot tip, vascular tissue, and leaves. Figure 6 d).

[0079] Example 6: Conversion Results of Other Materials

[0080] This embodiment uses the "Yang 061" clone for transformation, and employs strain EHA105; the infection solution OD... 600 =0.9; osmotic pressure set at 20 g / L, AS concentration in conversion solution set at 120 μM; infection time 3 h, OD 600 =0.9 infection 1.5h + OD 600 =0.6 infection for 1.5h; no vacuum treatment, co-culture conditions were 23℃ dark culture for 48h.

[0081] The results are as follows Figure 7 As shown, the overall staining effect is poor, with staining only appearing on a small number of leaf veins at the stem tip, failing to achieve the effect of the entire plant turning blue.

[0082] The foregoing has shown and described the main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for constructing a genetic transformation system for Chinese fir, characterized in that, include: 1) Preparation of infection solution: After culturing the Agrobacterium strain, adjust the OD of the infection solution. 600 The value is 0.2-1.2; 2) Genetic transformation: Using 11C genotype Chinese fir tissue culture seedlings as transformation recipients, infection was performed using an infection solution; after infection, the seedlings were rinsed with washing solution and placed in a co-culture medium for dark culture to obtain genetically transformed Chinese fir tissue culture seedlings.

2. The construction method according to claim 1, characterized in that, The Agrobacterium strain is one or more of EHA105, GV3101, LBA4404, Ar1193, C58C1, K599, or K599 (pSoup-p19).

3. The construction method according to claim 2, characterized in that, The Agrobacterium strain is EHA105.

4. The construction method according to claim 1, characterized in that, The infection step involves cutting a 1cm section of newly proliferated seedlings from 11C genotype Chinese fir tissue culture seedlings as the transformation recipient, and immersing them in OD... 600 Infect the material with a concentration of 0.2-1.2, shake at 90 rpm, remove the material, discard the conversion solution, quickly rinse the material with washing solution, blot dry on filter paper, and transfer it to a co-culture medium for dark incubation.

5. The construction method according to claim 4, characterized in that, The OD of the infecting solution 600 It is 1.

2.

6. The construction method according to claim 4, characterized in that, The inoculation time is 10 min to 6 h or vacuum treatment for less than 30 min.

7. The construction method according to claim 6, characterized in that, The vacuum treatment is -60 kPa.

8. The construction method according to claim 6, characterized in that, The infection time is 25 minutes of vacuum treatment.

9. The construction method according to claim 4, characterized in that, The co-culture conditions are: 4℃ dark culture for 48h, or 23℃ dark culture for 48h, or 28℃ dark culture for 48h, or 4℃ dark culture for 12h followed by 23℃ dark culture for 36h, or 4℃ dark culture for 12h followed by 28℃ dark culture for 36h.

10. The construction method according to claim 9, characterized in that, The co-culture conditions were: dark incubation at 23°C for 48 hours.

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