Efficient construction method of cedarwood genetic transformation system taking seeds as materials

Abstract

The invention discloses an efficient construction method of a cunninghamia lanceolata genetic transformation system with seeds as materials, and belongs to the technical field of genetic transformation. The efficient construction method of the cunninghamia lanceolata genetic transformation system taking the seeds as the materials mainly comprises the following steps: disinfecting and peeling the cunninghamia lanceolata seeds, sequentially performing germination culture, adding the cunninghamia lanceolata seeds into an agrobacterium suspension, performing vacuum-pumping infection treatment, performing co-culture, performing degerming culture to form seedlings, taking stem segments of the seedlings as explants, and culturing the explants to obtain the cunninghamia lanceolata genetic transformation system. And inoculating to an adventitious bud induction culture medium added with screening antibiotics, and carrying out screening induction culture until the heredity-transformed regenerated adventitious buds of the cunninghamia lanceolata are obtained. The technical system can be widely applied to the field of genetic improvement of cunninghamia lanceolata, and a reliable technical platform is provided for efficient transformation of disease-resistant genes, stress-resistant genes and wood quality related genes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic transformation, and more particularly relates to a high-efficiency construction method of a genetic transformation system of Chinese fir using seeds as materials. BACKGROUND

[0003] Coniferous trees have long growth cycles, tall tree bodies, high heterozygosity and high genetic load due to long-term outcrossing, which makes traditional breeding methods face challenges such as long cycle and low efficiency, and it is difficult to meet the demand for genetic improvement of fast-growing tree species. As a frontier field of forest biotechnology, forest genetic engineering not only provides a new way for germplasm innovation, but also is an important means for gene function research. Combining genetic engineering technology with conventional breeding can significantly shorten the breeding cycle and accelerate the genetic improvement process. Therefore, using genetic engineering methods for molecular design breeding and directional improvement of key traits of Chinese fir has become a research hotspot.

[0004] The applicant provided a set of Agrobacterium transformation system of Chinese fir based on suspension cells (publication number: CN103088058A) in the early stage. By Agrobacterium infection of Chinese fir embryogenic callus cells, Chinese fir cotyledon embryos were induced to obtain, and the efficient acquisition of transgenic plants was realized, solving the problems of low transformation efficiency and difficult regeneration in Chinese fir genetic engineering. The method can be used for introducing disease-resistant, stress-resistant and wood formation-related genes into Chinese fir to cultivate fast-growing, high-quality and stress-resistant Chinese fir new varieties. For the regeneration system and Chinese fir lines sensitive to tissue culture, a new genetic transformation system needs to be established to fully meet the application of Chinese fir in disease resistance, stress resistance, wood property improvement and other aspects of gene function verification and molecular breeding. SUMMARY

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide a high-efficiency construction method of a genetic transformation system of Chinese fir using seeds as materials, which is used for constructing excellent transgenic lines of Chinese fir.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] A high-efficiency construction method of a genetic transformation system of Chinese fir using seeds as materials, comprising:

[0008] After the Chinese fir seeds are sterilized and peeled, the seeds are subjected to germination culture, vacuum infection treatment by adding Agrobacterium suspension, co-culture, and degerming culture to form seedlings. Then, stems of the seedlings are taken as explants, inoculated on an adventitious bud induction medium added with a screening antibiotic for screening and induction culture, until regenerated adventitious buds of Chinese fir that have completed genetic transformation are obtained.

[0009] In some embodiments, the method for efficiently constructing a genetic transformation system of Chinese fir using seeds as material comprises the following steps:

[0010] S1: Put the disinfected Chinese fir seeds without seed coat into a germination medium for 2-4 days to obtain easily infected Chinese fir germination seeds;

[0011] S2: Put the easily infected Chinese fir germination seeds into a bacterial suspension for vacuum infection, and then co-culture to obtain infected Chinese fir germination seeds;

[0012] S3: After washing the infected Chinese fir germination seeds with sterile water containing antibiotics, transfer them to a seed germination medium containing antibiotics for decontamination culture to obtain Chinese fir seedlings;

[0013] S4: Take the Chinese fir stem segments obtained by removing the leaves of the Chinese fir seedlings as explants, inoculate them into an adventitious bud induction medium containing a selection antibiotic for selection and induction culture until genetically transformed Chinese fir regenerated adventitious buds are obtained.

[0014] In some embodiments, in step S1, the germination medium is specifically 1 / 2DCR medium with 10 mg / L VC and 30 g / L sucrose, and the water crystal agar is 2.8 g / L; the light and temperature conditions for germination are constant temperature dark culture at 23°C.

[0015] In some embodiments, in step S2, the Agrobacterium is specifically EHA105 with a target gene and a resistance selection gene.

[0016] In some embodiments, the target gene is a 35S:iGUS gene, and the resistance selection gene is an NPT-Ⅱ gene.

[0017] In some embodiments, the preparation method of the Agrobacterium suspension is as follows: inoculate a single colony of EHA105 into LB liquid medium, shake culture until the OD 600 is 0.2-0.3, centrifuge and collect the bacterial cells, and then resuspend the bacterial cells with a seed germination liquid medium, which is 1 / 2DCR medium with 10 mg / L VC and 30 g / L sucrose.

[0018] 7In some embodiments, the negative pressure for vacuum infection in step S2 is 0.06-0.08 MPa for 13-17 min.

[0019] In some embodiments, the co-culture method is as follows: after vacuum infection of the easily infected Chinese fir germination seeds in the bacterial suspension, inoculate them into the germination medium, and co-culture in a constant temperature incubator at 23°C for 48 h.

[0020] In some embodiments, the seed germination medium with antibiotics is a seed germination medium with 400 mg / L of cephalosporin; the step of degerming culture is: dark culture for 10 days in a 23℃ constant temperature incubator; and then placed in a 23℃ culture for 20 days with light for 16 hours per day;

[0021] In some embodiments, the adventitious bud induction medium containing screening antibiotics is an adventitious bud induction medium with 200 mg / L of cephalosporin and 6 mg / L of kanamycin, and the adventitious bud induction medium is a DCR basic medium with 10 mg / L of VC, 1.0 mg / L of 6-BA, 0.001 mg / L of TDZ, 0.1 mg / L of NAA, 30 g / L of sucrose and 7 g / L of agar.

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

[0023] The genetic transformation method of the application uses mature seeds of Chinese fir as the recipient material, uses iGUS gene as the reporter gene, and realizes plant regeneration through adventitious bud regeneration by the method of Agrobacterium-mediated genetic transformation, thereby establishing an efficient genetic transformation system of Chinese fir. In the technical system, the recipient material is easy to obtain, is not limited by genotype, has a short transformation period, has a high transformation efficiency, and can quickly obtain excellent transgenic lines of Chinese fir.

[0024] The application uses Chinese fir seeds as the recipient for genetic transformation, which has the following advantages: first, the material is easy to obtain, the seeds are easy to obtain, store and transport across regions, and the reserved seeds can be called, which is not limited by seasons; second, the genotype limitation is broken, and the universality is strong, especially for Chinese fir lines that are difficult to establish a regeneration system or sensitive to tissue culture; third, the genetic stability is high, the somatic cell variation can be reduced, the de-differentiation process is not experienced, and chromosome aberration or epigenetic disorder commonly seen in the callus stage is avoided. Therefore, the method provided by the application can be widely applied to the field of genetic improvement of Chinese fir, and provides a reliable technical platform for efficient transformation of disease-resistant genes, stress-resistant genes and wood quality-related genes. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Pre-culture photo of mature seeds of Chinese fir;

[0026] Figure 2 GUS transient expression detection diagram of Chinese fir zygotic embryos;

[0027] Figure 3 Chinese fir regenerated adventitious bud;

[0028] Figure 4 GUS expression detection diagram of Chinese fir regenerated adventitious bud. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described below with specific examples. In the following examples, the technical means used are all conventional means well known to those skilled in the art, unless otherwise specified.

[0030] In the following examples, the medium formula used is as follows, and the conventional medium is not particularly listed.

[0031] In the following examples, the composition and standard formula of the DCR (Gupta and Durzan medium) basic medium used are as follows: 340 mg / L KNO3, 556 mg / L Ca(NO3)2·4H2O, 400 mg / L NH4NO3, 170 mg / L KH2PO4, 85 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 37.3 mg / L Na2-EDTA·H2O, 27.9 mg / L FeSO4·7H2O, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.83 mg / L KI, 0.025 mg / L CoCl2, 200 mg / L MyO-Inositol, 2.0 mg / L Glycine, 1.0 mg / L Thiamine·HCl, 0.5 mg / L Nictinic·acid, 0.5 mg / L Pyridoxine·HCl. 4, 85mg / L CaCl2·2H2O,370mg / L MgSO4·7H2O,37.3mg / LNa2-EDTA·H2O,27.9mg / L FeSO4·7H2O,6.2mg / L H3BO3,22.3mg / LMnSO4·4H2O,8.6mg / L ZnSO4·7H2O,0.25mg / L Na2MoO4·2H2O,0.25mg / LCuS04·5H2O,0.83mg / L KI,0.025mg / L CoC12,200mg / L MyO-Inositol,2.0mg / LGlycine,1.0mg / L Thiamine·HC1,0.5mg / L Nictinic·acid,0.5mg / L Pyridoxine·HCl。

[0032] LB medium formula: 5 g / L Yeast extract, 10 g / L Tryptone, 5 g / L NaCl.

[0033] Example 1: Establishment of transformation receptor

[0034] (1) Take mature and full-fledged Chinese fir seeds and place them in 100 mL triangular bottles, add an appropriate amount of detergent, wrap the bottle opening with gauze, rinse with running water for 30 min, quickly soak in 75% alcohol for 30 s, sterilize in 30% sodium hypochlorite for 10 min, and then wash with water for 3-5 times before being divided and placed in new triangular bottles.

[0035] (2) Remove the seed coat with a scalpel and forceps, inoculate into Chinese fir seed germination medium, and cultivate in a 23°C constant temperature incubator in the dark. The Chinese fir seed germination medium is 1 / 2 DCR medium, with the addition of VC 10 mg / L, sucrose 30 g / L, and agar 2.8 g / L.

[0036] (3) After 3 days of pre-culture, cedar seeds can be used for Agrobacterium infection. A photo of cedar seeds with the seed coat removed on the third day of pre-culture is shown below. Figure 1 As shown.

[0037] Plant transgenic recipient systems refer to plant materials or cell systems that can efficiently and stably regenerate clones through tissue culture or other non-tissue culture methods, accept the integration of exogenous genes, and are sensitive to screening antibiotics. They are a key foundation for achieving stable expression and inheritance of exogenous genes, possessing the following characteristics: recipient materials must have the ability to differentiate into complete plants efficiently and with good reproducibility; the integration of exogenous genes must not affect the original genetic background of the recipient and must be stably inherited to offspring; and they must be sensitive to antibiotics, facilitating the screening of transformed cells using selection marker genes (such as antibiotic resistance genes). Depending on the genetic transformation method and purpose, a wide variety of recipient materials can be selected, including seeds, leaves, petioles, cotyledons, cotyledonary petioles, hypocotyls, stem segments, immature embryos, callus tissue, embryogenic suspension cells, and protoplasts. Based on preliminary exploratory experiments, this embodiment selected sterilized and dehulled Chinese fir seeds that had germinated for three days as transformation recipients. If the seeds failed to germinate or germinated for too long, subsequent infection steps could not achieve the desired effect. Furthermore, the choice of germination culture medium is also a crucial factor. During seed germination, increased metabolic activity produces a large amount of reactive oxygen species (ROS), which can damage cells. Using a 1 / 2 DCR medium supplemented with vitamin C can protect seeds from ROS damage during germination, reduce oxidative stress on seed cells, thereby improving seed viability and facilitating infection. However, continued addition of plant hormones such as ABA and IAA inhibited germination and had the opposite effect.

[0038] Example 2 Preparation of Agrobacterium tumefaciens bacterial solution

[0039] (1) The EHA105 bacterial culture containing the 35S:iGUS gene (for transformation detection) and NPT-II gene (for screening) stored at -70℃ was thawed on ice. In a clean bench, a small amount of bacterial culture was taken with an inoculation loop and inoculated onto LB solid medium containing kanamycin (50 mg / L). The culture was then placed in a constant temperature incubator at 28℃ for 72 h.

[0040] (2) After a single colony grows, pick up a single colony with a pipette tip and inoculate it into a centrifuge tube containing 750 μL of LB liquid medium with kanamycin (50 mg / L), and culture at 28°C and 220 r / min with shaking.

[0041] (3) After about 12 hours, take 100 μL of bacterial culture and inoculate it into a centrifuge tube containing 2 mL of LB liquid medium with kanamycin (50 mg / L). Incubate at 28°C and 220 r / min with shaking.

[0042] (4) After about 20h, 2mL bacterial solution was taken and inoculated into a 50mL flask containing 50mL LB liquid medium with appropriate amount of antibiotic (kanamycin 50mg / L), and cultured at 28℃ with 280r / min shaking until OD6 00 was 0.6-0.8.

[0043] (5) When the Agrobacterium was cultured to OD 600 was 0.6-0.8, 25mL bacterial solution was taken in a 50mL centrifuge tube, and centrifuged at 5000r / min at 4℃ for 10min, the supernatant was removed, and the bacterial body was collected.

[0044] (6) The bacterial body was resuspended with Chinese fir seed germination liquid medium, and the concentration of the bacterial solution was diluted to OD 600 was about 0.2, and was ready for use.

[0045] Preparation of Agrobacterium infection solution with high purity, vigorous growth and strong activity is the key to genetic transformation. When the OD 600 of the bacterial solution reached 0.6-0.8, it corresponded to the middle logarithmic growth phase of the strain, at which stage the Agrobacterium was in the vigorous division period, the Ti plasmid was actively replicated, the expression amount of Vir gene was high, which was conducive to T-DNA cutting and transfer, and was conducive to genetic transformation.

[0046] Infection and co-culture of Example 3

[0047] (1) The bacterial solution was shaken and 15mL diluted bacterial solution was measured with a measuring cylinder and poured into a 100mL flask. Chinese fir seeds (Example 1) cultured for 3 days were added, the flask was placed in a vacuum pump, and maintained at a negative pressure of 0.06-0.08MPa for 13-17min, then placed in a constant temperature shaker at 23℃ and 85r / min for continuous shaking culture for 5min; then the seeds were inoculated in germination medium and co-cultured in a constant temperature incubator at 23℃ for 48h.

[0048] Infection refers to the process of contacting Agrobacterium or other carrier systems with plant cells and introducing exogenous genes (T-DNA) into the host genome. The infection effect directly affects the delivery efficiency of exogenous genes. Excessive infection can cause significant cytotoxicity, resulting in a decrease in the survival rate of the recipient material; while insufficient infection can result in failure of exogenous gene delivery. The Agrobacterium solution cannot penetrate into the tissue without using vacuum infection for the genetic transformation of Chinese fir seeds, resulting in failed transformation. The metabolic activity of ungerminated seeds is low, and the cell wall is compact, making it difficult for Agrobacterium to adhere; the cell wall of seeds with too long germination time thickens, and lignin is deposited, making it difficult for Agrobacterium to invade; for these two types of recipient materials, even if vacuum infection is used, genetic transformation cannot be successfully performed. The applicant found that after 3 days of culture, the radicle of Chinese fir seeds broke through the seed coat, the meristem was exposed, and the cell division was vigorous, and under a negative pressure of 0.06-0.08 MPa for 13-17 minutes, neither the cells nor the tissues of Chinese fir seeds were damaged, allowing them to continue to grow and germinate, nor did it effectively promote the infection of Agrobacterium into the cells and tissues. Under conditions other than this, either the infection effect is not achieved, or the Chinese fir seeds are prone to death.

[0049] Co-culture refers to the stage of co-culturing Agrobacterium with plant cells under suitable conditions to allow T-DNA to be stably integrated into the plant genome. During co-culture, the Vir genes of Agrobacterium are activated, driving the cutting, transport and integration of T-DNA into plant cell DNA.

[0050] Example 4: Antibiotic-free culture

[0051] (1) Place the seeds after co-culture for 48 hours in a triangular flask and wash them several times with sterile water containing cefotaxime (400 mg / L) until the supernatant in the triangular flask is clear. This process is called washing.

[0052] (2) Transfer the washed Chinese fir seeds to a seed germination medium containing cefotaxime (400 mg / L), with 10 seeds per dish, and place them in a 23°C constant temperature incubator for dark culture for 3 days.

[0053] (3) Take part of the Chinese fir seeds, remove the endosperm, and perform GUS staining on the embryo to observe its transient expression. The GUS transient expression detection chart of Chinese fir zygotic embryos is as follows Figure 2 .

[0054] (4) Place the remaining seeds in a 23°C, 16h / d light incubator for 20 days, and the Chinese fir seeds grow into seedlings.

[0055] After the co-cultivation stage with Agrobacterium, a large number of symbiotic Agrobacterium cells will be enriched on the surface and shallow tissue of the seedling tissue of Chinese fir germinated seeds. In order to ensure that the stem segments of the transformed seedlings can grow normally as explants in the subsequent tissue culture steps, it is necessary to effectively kill and inhibit the residual Agrobacterium through a strict decontamination treatment program. This key step not only eliminates the toxic effects of Agrobacterium overgrowth on seedlings, but also creates favorable conditions for subsequent callus induction and plant regeneration. In this implementation case, the optimal concentration of cefotaxime for killing and inhibiting the growth of Agrobacterium is 400 mg / L.

[0056] Implementation Case 5 Screening Culture

[0057] (1) After the Chinese fir seeds germinate into seedlings, remove the leaves, and inoculate the Chinese fir stem segments as explants into the adventitious bud induction medium containing 200 mg / L cefotaxime and 4 mg / L kanamycin for screening induction culture.

[0058] (2) After one month of screening induction culture, the regenerated buds have been induced, and the Chinese fir regenerated adventitious bud diagram is as follows Figure 3 .

[0059] In the process of plant genetic transformation, the selective addition of antibiotics plays a key role in the screening of transformed cells. In this implementation case, NPT-II (neomycin phosphotransferase) is used as a selection marker gene, which confers kanamycin resistance to transformed cells, so kanamycin is chosen as a selection agent to establish a selection pressure system.

[0060] According to the different periods of selection pressure addition, it can be divided into three types, namely, early selection, delayed selection and late selection. Because in the medium containing selection pressure, untransformed cells cannot survive, and transformed cells with weak growth potential also have difficulty forming effective transformants, in order to improve the transformation efficiency, this implementation case uses late selection.

[0061] This application uses mature Chinese fir seeds as materials to prepare genetic transformation receptors, and obtains genetically transformed Chinese fir regenerated adventitious buds, with a total process time of about 60 days, which is 25 days shorter than using callus as material, and the conditions are simpler, especially can be applied to Chinese fir strains which are difficult to tissue culture, so it has strong universality.

[0062] Implementation Case 6 GUS Cell Detection

[0063] Take the transformed seeds of Example 3 and the regenerated adventitious buds in the initial stage of Example 5 as GUS staining materials. Soak the prepared materials in the staining solution at 37°C for 2 hours to overnight. The GUS staining formula is:

[0064] (1) 50 mmol / L sodium phosphate buffer solution (pH 7.0): A solution: weigh NaH2PO4·2H2O 3.12 g and dissolve in sterile water, and make up to 100 mL. B solution: weigh Na2HPO4·12H2O 7.17 g and dissolve in sterile water, and make up to 100 mL. Mix 39 mL of A solution with 61 mL of B solution;

[0065] (2) Staining solution: 7.1 mL of 50 mmol / L sodium phosphate buffer solution, 0.2 mL of 10 mmol / L Na2EDTA, 0.2 mL of 5 mmol / L K4[Fe(CN)6], 0.2 mL of K3[Fe(CN)6], 0.05 mL of 0.05% (v / v) Triton-100, 2 mL of 20% methanol, 0.25 mL of 0.5 mg / mL X-Gluc. Prepare 10 mL of GUS staining solution for standby.

[0066] GUS gene as a reporter gene system, the use of histochemical staining method to detect the transient expression of GUS gene, long-term expression. As shown in Figure 2 、 Figure 4 , observed with the naked eye or under a microscope, the blue in the material is the GUS expression site, it can be seen that the foreign gene has been efficiently transformed.

[0067] GUS gene (β-glucuronidase gene) is a reporter gene derived from microorganisms such as Escherichia coli, and its encoded product β-glucuronidase (β-glucuronidase, GUS) can specifically catalyze the hydrolysis reaction of various β-glucuronidase substances. Because most higher plant cells lack endogenous GUS enzyme activity, this gene system has become one of the most widely used reporter genes in plant genetic transformation research.

[0068] In this embodiment, we use histochemical staining method to detect the expression of GUS gene. This method uses 5-bromo-4-chloro-3-indole-β-D-glucuronide (X-Gluc) as a specific substrate, and realizes in situ detection of GUS activity through enzymatic color reaction. Its technical principle is that when the transformed material containing GUS gene is incubated with the substrate solution, the expressed GUS enzyme can catalyze the hydrolysis of X-Gluc to produce insoluble blue indole derivative precipitate. This color reaction can be directly observed at the tissue, cell or protoplast level without the need for enzyme extraction and purification steps, and has the advantages of simple operation and direct results.

[0069] The foregoing shows and describes the primary features of the present application and the advantages of the present application. It is understood that the present application is not limited to the above-described embodiments and that various changes and modifications can be made to the present application without departing from the spirit and scope of the present application, and such changes and modifications are intended to fall within the scope of the present application.

Claims

1. A method for efficiently constructing a genetic transformation system for Chinese fir using seeds as material, characterized in that, include: After disinfecting and peeling the seeds, the cedar seeds were subjected to germination culture, vacuum infection treatment in Agrobacterium suspension, co-culture, and sterilization culture to form seedlings. The stem segments of the seedlings were then used as explants and inoculated on adventitious bud induction medium containing screening antibiotics for screening and induction culture until genetically transformed cedar regenerated adventitious buds were obtained.

2. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 1, characterized in that, Includes the following steps: S1: Place the disinfected and seed-coat-removed Chinese fir seeds into a germination medium and germinate for 2-4 days to obtain easily infected Chinese fir germination seeds; S2: Easily infected germinating seeds of Chinese fir were placed in Agrobacterium suspension and vacuum-infected, and then co-cultured to obtain infected germinating seeds of Chinese fir. S3: After washing the infected germinating seeds with sterile water containing antibiotics, transfer them to a seed germination medium containing antibiotics for destere culture to obtain cedar seedlings. S4: The stem segments of Chinese fir obtained by removing the leaves from Chinese fir seedlings were used as explants and inoculated onto adventitious bud induction medium containing screening antibiotics for screening and induction culture until genetically transformed Chinese fir regenerated adventitious buds were obtained.

3. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, In step S1, the germination medium is specifically 1 / 2 DCR medium supplemented with VC 10 mg / L, sucrose 30 g / L, and crystal agar 2.8 g / L; the germination light and temperature conditions are: constant temperature dark culture at 23℃.

4. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, In step S2: the Agrobacterium is specifically EHA105, which carries the target gene and the resistance selection gene.

5. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 4, characterized in that, The target gene is the 35S:iGUS gene, and the resistance selection gene is the NPT-II gene.

6. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 4, characterized in that, The method for preparing the Agrobacterium suspension is as follows: a single colony of EHA105 is inoculated into LB liquid medium and cultured with shaking until OD... 600 The concentration was 0.2-0.

3. After centrifugation and collection of bacterial cells, the bacterial cells were resuspended in seed germination liquid medium, which was 1 / 2 DCR medium supplemented with 10 mg / L VC and 30 g / L sucrose.

7. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, In step S2, the negative pressure for vacuum impregnation is 0.06-0.08 MPa, and the duration is 13-17 min.

8. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, The co-culture method is as follows: the easily infected germinating seeds of Chinese fir are placed in Agrobacterium suspension and vacuum-infected, and then inoculated into germination medium and co-cultured in a constant temperature incubator at 23°C for 48 hours.

9. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, The seed germination medium containing antibiotics is a seed germination medium containing 400 mg / L cephalosporin; the sterilization culture steps are as follows: dark culture in a constant temperature incubator at 23℃ for 10 days; then culture at 23℃ with a light time of 16 h / d for 20 days.

10. The method for efficiently constructing a Cunninghamia lanceolata genetic transformation system using seeds as material according to claim 2, characterized in that, The adventitious bud induction medium containing the screening antibiotics was prepared by screening culture on an adventitious bud induction medium supplemented with 200 mg / L cephalosporin and 6 mg / L kanamycin. The adventitious bud induction medium was DCR basal medium supplemented with 10 mg / L VC, 1.0 mg / L 6-BA, 0.001 mg / L TDZ, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar.

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