Construction method of agrobacterium tumefaciens-mediated masson pine genetic transformation system

Abstract

The invention discloses a construction method of an agrobacterium tumefaciens mediated masson pine genetic transformation system, and belongs to the technical field of genetic engineering. According to the construction method of the agrobacterium tumefaciens-mediated masson pine genetic transformation system, immature masson pine cones are collected, embryogenic calluses are induced and cultured, the embryogenic calluses of the masson pine in a PEM III stage are used as a material, suspension pre-culture is performed to obtain an acceptor material, and the acceptor material is subjected to genetic transformation to obtain the agrobacterium tumefaciens-mediated masson pine genetic transformation system. The method comprises the following steps: firstly, carrying out tissue culture on a transgenic somatic embryo, infecting by adopting an agrobacterium infection solution with an OD600 value of 0.2-0.6, carrying out co-culture, recovery culture and screening culture, carrying out histochemical staining by adopting GUS, carrying out molecular identification to obtain a transgenic embryogenic callus, and carrying out maturation treatment to finally obtain the transgenic somatic embryo. The masson pine genetic transformation system established by the invention is stable in transformation performance and high in transformation positive rate, and when an acceptor material is masson pine embryonic callus with the genotype of 1-3-5, the transformation positive rate is as high as 100%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and more specifically, relates to a method for constructing a Masson pine genetic transformation system mediated by Agrobacterium tumefaciens. Background Art

[0002] Masson pine (Pinus massoniana Lamb.) is a tree of the genus Pinus in the Pinaceae family. It is drought-resistant and barren. It is widely distributed in many provinces in southern my country. It is the main tree species for afforestation on barren hills and has important ecological value. At the same time, it has a wide range of uses and extremely high economic value. It is widely used in construction engineering, fuel, medicine and industrial raw materials.

[0003] However, due to the long growth cycle and high genetic load of Masson pine, traditional breeding methods are difficult to meet the needs of rapid genetic improvement. Facing this challenge, genetic engineering technology provides a new solution. Genetic transformation technology, especially Agrobacterium-mediated method, has become the main method for genetic improvement of coniferous species due to its high efficiency and repeatability.

[0004] The efficiency of Agrobacterium-mediated genetic transformation is mainly affected by key factors such as the receptor material used, the type and concentration of Agrobacterium strains, infection time and co-cultivation time. In terms of the selection of receptor materials, embryonic callus is one of the ideal materials for studying plant genetic transformation. It has strong exogenous integration ability, is extremely sensitive to antibiotics but has good tolerance to Agrobacterium, and has efficient and stable regeneration ability. At the same time, the genotype of callus also has a great influence on transformation efficiency. Given the importance of pine species, its genetic transformation research has attracted much attention and has made great progress in recent years, but it is still relatively slow compared with other species. So far, the number of successful transgenic species among the 119 pine species is still very limited. At present, some researchers have used embryonic callus as the receptor material, and successfully cultivated transgenic plants of radiata pine (P. radiata), loblolly pine (P. taeda), coastal pine (P. pinaster), and splay pine (P. patula) by selecting different Agrobacterium strains (LBA4404, EHA105 or GV3101, etc.) and appropriate infection concentrations, and successfully constructed transient gene expression systems of North American pine (P. strobus) (Doi.org / 10.48130 / forres-0024-0007) and Chinese pine (Pinus tabuliformis) (Doi: 10.1186 / s13007-020-00594-5). Therefore, for the genetic transformation of specific pine species, it is particularly important to select appropriate receptor materials, determine suitable strains for transformation, and appropriate infection concentrations. As an important native tree species in my country, the research progress of genetic transformation of Masson pine is relatively slow. Although some researchers have successfully cultivated transgenic plants using mature zygotic embryos of Masson pine as receptor materials (Doi:10.1007 / s11240-018-1388-7), the stability and efficiency of its genetic transformation system still need to be improved. At present, there is a lack of reports on the genetic transformation system using Masson pine embryonic callus as receptor material. Therefore, the establishment of an efficient and stable genetic transformation system of Masson pine is crucial to accelerate the mining and research of Masson pine functional genes and shorten the genetic improvement process of Masson pine. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for constructing a Masson pine genetic transformation system mediated by Agrobacterium tumefaciens, which has the advantages of stable genetic transformation performance and high positive rate.

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

[0007] A method for constructing a genetic transformation system of Masson pine mediated by Agrobacterium tumefaciens, collecting immature cones of Masson pine and inducing embryonic callus, using the embryonic callus of Masson pine at the PEMIII stage as material, performing suspension pre-culture to obtain receptor material, and using OD 600 The cells were infected with Agrobacterium infection solution with a value of 0.2-0.6. After the infection, co-culture, recovery culture, screening culture and maturation culture were performed, and GUS was used for histochemical staining and molecular identification to finally obtain transgenic embryonic callus.

[0008] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens, wherein the Agrobacterium is selected from Agrobacterium tumefaciens AGL1, LBA4404, EHA105, and GV3101.

[0009] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens is characterized in that the genotype of the Masson pine embryonic callus is 1-3-5.

[0010] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens is that the genotype of the Masson pine embryonic callus is 1-3-5, and the Agrobacterium is Agrobacterium tumefaciens AGL1.

[0011] In the method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens, the genotype of the Masson pine embryonic callus is 1-3-5, and the Agrobacterium is LBA4404 or EHA105 or GV3101.

[0012] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens, the genotype of the Masson pine embryonic callus is 1-3-5, the Agrobacterium is LBA4404 or EHA105 or AGL1, the infection concentration OD 600 The value is 0.6.

[0013] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens, the genotype of the Masson pine embryonic callus is 1-3-5, the Agrobacterium is GV3101, the infection concentration OD 600 The value is 0.2-0.6.

[0014] The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens comprises the following specific steps:

[0015] 1) sterilizing immature female gametophytes of Masson pine with genotype 1-3-5 and inoculating them into induction medium for induction culture to obtain embryonic callus;

[0016] The formula of the induction medium is: LP basic medium + 2mg / L 2,4-D + 1mg / L 6-BA + 0.25mg / LVC + 30g / L maltose + 1g / L inositol + 0.5g / L hydrolyzed casein + 0.45g / L glutamine, pH 5.8;

[0017] 2) inoculating the stably proliferating embryonic callus into a proliferation medium for proliferation culture;

[0018] The formula of the proliferation medium is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / LVC + 30 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar, pH 5.8;

[0019] 3) transferring the Masson pine embryonic callus at the PEMIII stage into a liquid suspension culture medium for suspension pre-culture;

[0020] The liquid suspension culture medium formula is: LP basic medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 100 μM acetosyringone, pH 5.8;

[0021] 4) Transform the plasmid containing the target gene into AGL1 Agrobacterium and prepare the infection solution;

[0022] 5) Use OD 600 The infection solution with a value of 0.6 infects the callus tissue after pre-culture;

[0023] 6) After infection, the callus tissue is transferred to the co-cultivation medium for co-cultivation;

[0024] The co-culture medium formula is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 100 μM acetosyringone, pH 5.8;

[0025] 7) Degerming the co-cultured callus tissue and transferring it to a recovery medium for recovery culture;

[0026] The recovery medium formula is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / LVC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime, pH 5.8;

[0027] 8) transferring the callus tissue after recovery culture to a screening medium for screening culture, and finally obtaining positive embryonic callus tissue after genetic transformation;

[0028] The screening culture medium formula is: LP basic culture medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / LVC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime + 25 mg / L hygromycin, pH 5.8.

[0029] 9) Perform histochemical staining and molecular identification on the positive transformed callus tissue after screening and culture, and finally obtain transgenic embryonic callus tissue.

[0030] 10) transferring the transgenic embryonic callus to a maturation medium for maturation treatment to obtain transgenic somatic embryos;

[0031] The formula of the mature culture medium is: LP basic culture medium + 5 mg / L ABA + 0.5 mg / L GA3 + 0.25 mg / L VC + 130 g / L PEG8000 + 1.5 g / L activated carbon + 20 g / L maltose + 1 g / L glutamine + 1 g / L inositol + 3.5 g / L plant gel, pH 5.8.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens of the present application comprises the following steps: sterilizing immature female gametophytes of Masson pine and inoculating them into an induction medium for induction culture to obtain embryonic callus; inoculating the obtained embryonic callus into a proliferation medium for proliferation culture; transferring the well-growing callus at the PEMIII stage into a liquid suspension culture medium for suspension pre-culture; transforming Agrobacterium with a plasmid containing a target gene to prepare an infection solution; using the infection solution to infect the pre-cultured callus; after the infection is completed, transferring the callus to a co-culture medium for co-culture; degerming the co-cultured callus and transferring it to a recovery medium for recovery culture; transferring the recovery cultured callus to a screening medium for screening culture, and finally obtaining genetically transformed positive embryonic callus.

[0034] 2) This application uses immature female gametophytes of Masson pine as explants to induce embryonic callus, obtains embryonic callus that can stably proliferate, and is used as experimental material for the subsequent construction of a Masson pine genetic transformation system. After 6 days of co-cultivation after infection, the callus can be successfully degermed and grow normally.

[0035] 3) The examples of the present application demonstrate that positive transformation callus tissues were successfully obtained by infection and transformation using Agrobacterium tumefaciens strains AGL1, GV3101, LBA4404, and EHA105, and the positive rate was not less than 93% and as high as 100%, indicating that the system is stable and efficient, providing a basis for subsequent research on gene function and quality improvement of Masson pine.

[0036] 4) The examples of the present application demonstrate that after maturation culture of transgenic embryonic callus, the maturation-treated transgenic callus differentiates into somatic embryos. After 50 days of maturation treatment, the somatic embryos are blue in color after GUS staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a real picture of the embryonic callus of Masson pine; the left picture is the embryonic callus just induced, and the right picture is the picture of the embryonic callus of Masson pine that has been stably proliferated;

[0038] Figure 2 This is a microscopic picture of the embryonic callus of Masson pine at the stable proliferation PEMIII-SE stage;

[0039] Figure 3 This is a plate coating image after the vector pCAMBIA1301 was transformed into Agrobacterium tumefaciens AGL1, GV3101, LBA4404, and EHA105;

[0040] Figure 4It is a gel electrophoresis verification diagram after the vector pCAMBIA1301 was transformed into Agrobacterium tumefaciens AGL1, GV3101, LBA4404, and EHA105; in the figure, lane M is a standard sample (2000bp); A0, G0, L0, and E0 correspond to the empty competent cells of AGL1, GV3101, LBA4404, and EHA105, and A1-A3, G1-G3, L1-L3, and E1-E3 correspond to the competent cells of AGL1, GV3101, LBA4404, and EHA105 after the plasmid was introduced;

[0041] Figure 5 This is a picture of pre-culture of embryonic callus before infection;

[0042] Figure 6 The co-culture picture (left) and the recovery culture picture (right) after the embryonic callus tissue was infected with Agrobacterium tumefaciens AGL1 bacterial solution 1-3-5;

[0043] Figure 7 This is the result of two screenings of positive embryonic callus; from left to right are AGL1, EHA105, GV3101, LBA4404;

[0044] Figure 8 This is the result of GUS staining and molecular detection of positive embryonic callus; the molecular detection of AGL1 provides some results. The leftmost lane in the electrophoresis is Maker, the second lane is CK, and the other lanes are sample lanes;

[0045] Fig. 9 The figure is the transgenic somatic embryo after infection with Agrobacterium AGL1 solution after maturation (left) and GUS staining (right);

[0046] Fig.10 This is a diagram showing the screening results of positive embryogenic callus in Example 5 (GUS staining provides partial results, the leftmost lane in the electrophoresis diagram is Maker, the second lane is CK, and the other lanes are sample lanes). DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0048] The immature cones of Pinus massoniana used in this application were collected from Guangyun Forest Farm, Pingle County, Guilin City, Guangxi Zhuang Autonomous Region. The immature cones were transported in cold storage after collection and placed in a 4°C refrigerator upon receipt for 7 days before use. The above-mentioned immature cones of Masson pine were used as explants for disinfection and callus induction. The process was as follows: wipe and disinfect the immature cones of Masson pine, the surface of the pruning shears and tweezers with alcohol cotton, cut the cones along the direction of the seed scales, pick up the seeds with tweezers, wrap them with sterile gauze in a clean bench, tie them with sterile cotton ropes and disinfect them with 75% ethanol for 3 minutes, rinse them with sterile water 3 times, use sterile tweezers to peel off the seed shell, seed coat and caruncle of the immature seeds in turn, take out the intact female gametophyte, place it horizontally on the induction medium (LP basic medium + 2mg / L 2,4-D + 1mg / L 6-BA + 0.25mg / L VC + 30g / L maltose + 1g / L inositol + 0.5g / L hydrolyzed casein + 0.45g / L glutamine, pH 5.8, the same below), and culture them in the dark at 25±1℃ for 60d. Figure 1 As shown, embryonic callus was successfully induced, and the embryonic callus was smooth, white and translucent (left picture); the induced embryonic callus was able to stably proliferate on the proliferation medium (right picture).

[0049] The embryonic callus tissues from different immature cones of Masson pine were named as: genotype 1-3-5, genotype 20-1-4, and genotype 20-1-7. The applicant has conducted multiple studies on this genotype biological material and has published it publicly (Doi: 10.3389 / fpls.2023.1130471). This type of biological material is already known to the public, and the public can obtain this type of biological material from the applicant.

[0050] Example 1: Callus proliferation culture

[0051] The induced embryonic callus was inoculated into proliferation medium (LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 30 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar, pH 5.8, the same below) and cultured for proliferation until the callus no longer browned. The cells were subcultured every 14 days and some callus cells were taken for microscopic examination to determine their embryonic nature.

[0052] According to the double staining method of Gupta and Holmstrom, a small amount of suspended cells was placed on a slide, first stained with 2% acetic acid magenta for 1 minute, then stained with 0.5% Evans blue for 30 seconds, covered with a coverslip and gently rinsed with sterile water 5-6 times, and after the staining solution was removed, the cells were placed under a Zeiss stereo microscope (SteREO Discovery.V20) for observation. According to the classification criteria of Filonova et al., whether the cell line is embryonic and the developmental stage it is in was determined.

[0053] The results are as follows Figure 2 As shown, the embryonic callus is at the PEMIII stage, with obvious bundle-like head and tail structures, indicating good embryonic quality.

[0054] Example 2 Preparation of infection solution

[0055] 1. Plasmid pCAMBIA1301 (containing GUS reporter gene and hygromycin (Hyg) resistance gene, commercially available) was used to transform Agrobacterium tumefaciens AGL1, LBA4404, EHA105, GV3101 (all commercially available)

[0056] Take 60 μL of Agrobacterium competent cells and melt them on ice, add 5 μL of plasmid, mix gently with a pipette, place on ice for 30 minutes, freeze in liquid nitrogen for 5 minutes, heat shock at 37°C for 5 minutes, and quickly ice bath for 5 minutes again; add 500 μL LB (without antibiotics), culture in a shaker in the dark for 2 hours, 200 rpm, 28°C; centrifuge for 3 minutes, 5000 rpm, remove part of the supernatant, and mix the remaining bacterial solution; take 80 μL of bacterial solution and evenly spread it on an LB plate (containing Rif and Kan), and culture in the dark at 28°C for 2 days; pick a single colony into 500 μL LB liquid culture medium (containing 50 mg / L Kan and Rif), 200 rpm, 28°C, culture for 8-10 hours, take all the bacterial solution and transfer it to 10 mL of new LB liquid culture medium and culture overnight, take the overnight cultured bacterial solution for positive clone detection, and use the GUS gene (1081 bp) in the pCAMBIA1301 vector as the detection gene. The detection primer sequences are:

[0057] F: 5'-GTCGCGCAAGACTGTAACCA-3',

[0058] R: 5'-CGGCGAAATTCCATACCTG-3'.

[0059] The PCR reaction system was: 2 μL bacterial solution, 10 μL Taq DNA polymerase mix, 1 μL each of upstream and downstream primers, and 6 μL RNase-free H2O.

[0060] The PCR reaction conditions were: 95°C for 30 s; 95°C for 30 s, 58°C for 30 s, 72°C for 40 s, 30 cycles; 72°C for 7 min.

[0061] After the reaction was completed, the PCR product was detected by electrophoresis on 1.2% agarose gel and observed by gel imaging instrument (UVP GelDoc-It). After the detection, the remaining bacterial solution was stored at -80°C for subsequent infection tests.

[0062] The results are as follows Figure 3 and Figure 4 As shown, positive clones were successfully grown on the resistance plates ( Figure 3 ); Three positive clones were selected from each strain for detection. Molecular detection showed that the pCAMBIA1301 vector had been successfully introduced into four types of Agrobacterium tumefaciens, with water as the blank control and Agrobacterium competent cells as the negative control ( Figure 4 ).

[0063] 2. Prepare infection solution

[0064] The GUS-introduced Agrobacterium bacterial suspension (stored at -80°C) was streaked on LB plates containing 50 mg / L Kan and Rif for activation and cultured at 28°C for 2 days. A single colony was picked and inoculated into 500 μL LB liquid medium (containing 50 mg / L Kan and Rif), cultured at 200 rpm and 28°C for 8-10 hours, and the entire bacterial suspension was transferred to 20 mL of new LB liquid medium for overnight culture.

[0065] Take the overnight culture solution and centrifuge it at 25℃ and 5000rpm for 5min, then discard the supernatant and collect the bacteria; then resuspend the bacteria in liquid suspension culture medium (LP liquid culture medium) as the infection medium, repeat twice, and the OD of the infection medium is 600 The value is 0.2-0.6.

[0066] Example 3 Construction of genetic transformation system

[0067] 1. Suspension pre-culture of embryonic callus

[0068] Weigh 4 g of embryonic callus tissue of genotype 1-3-5 that has developed to the PEMIII stage and has grown well for 14 days of proliferation culture, and inoculate it into a 250 mL conical flask containing 120 mL of liquid suspension culture medium (LP basic medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine, pH 5.8, after high temperature sterilization and cooling, add 250 μL of acetosyringone (final concentration 100 μM) to every 500 mL of suspension), break it up and place it in a shaker (dark, 25°C, 90 rpm) for pre-culture for 7 days. The results are as follows Figure 5 As shown, embryogenic callus grew well in suspension culture.

[0069] 2. Infection and Co-cultivation

[0070] Collect the pre-cultured callus into a 250mL triangular flask, put it in the infection solution (covering the callus) and infect for 15 minutes. During this process, shake gently to disperse the callus evenly in the infection solution and contact the infection solution with the largest area. After the infection is completed, discard the supernatant, use a 60-mesh cell sieve to sieve out the infected callus, spread the callus on a sterile culture dish with more than 7 layers of filter paper, take another piece of sterile filter paper and cover the callus. Use tweezers to gently press the callus to absorb the surface bacterial solution, discard the filter paper on it, and repeat the above operation to dry the callus until there is no moisture on the surface. Use tweezers to transfer the fully dried callus to the co-culture medium: LP basic medium + 1mg / L 2,4-D + 0.5mg / L 6-BA + 0.25mg / L VC + 15g / L maltose + 1g / L inositol + 0.5g / L hydrolyzed casein + 0.45g / L glutamine + 6.24g / L agar + 100μM acetosyringone, pH 5.8, 25℃ dark culture for 6 days. After 6 days of co-culture, if Figure 6 (Left) As shown, Agrobacterium successfully attached to the surface of embryonic callus and grew together with the embryonic callus.

[0071] 3. Degerming and recovery culture

[0072] After the co-cultivation, the callus tissue was collected into a triangular flask using a 60-mesh sterile cell sieve, washed 10 times with sterile distilled water, and washed 11 times with a solution containing Cef (final concentration 300 mg / L) and Tim (final concentration 300 mg / L); the callus tissue was sucked up with a 5 mL pipette and sprayed evenly on the filter paper. After the filter paper was dried, it was transferred to the recovery medium (LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / LVC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime, pH 5.8), and cultured at 26°C for 7 days. After 7 days of recovery culture, if Figure 6 (Right) Callus was successfully degermed and continued to grow on recovery medium.

[0073] In this embodiment, during the degerming and recovery culture, the callus tissue is evenly sprayed on the filter paper, so that the callus tissue can better contact the culture medium, which is conducive to its growth. By using this method, all callus cells are evenly distributed on the filter paper, which can not only reduce the probability of continued proliferation of Agrobacterium, but also allow the callus tissue to more fully contact the recovery culture medium to promote its recovery.

[0074] 4. Screening and cultivation

[0075] The filter paper on the recovery medium together with the callus was transferred to the screening medium (LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime + 25 mg / L hygromycin, pH 5.8) and cultured in the dark at 25°C for 20 days. Figure 7 As shown in the figure, the first screened callus tissue with normal growth was initially judged to be the successfully transformed callus tissue. Under the same culture conditions, the first screened callus tissue was clamped into blocks with tweezers and placed in the screening medium for re-screening (if the callus tissue was too small, it was transferred to the screening medium together with the filter paper, such as Fig. 9 ), obtain the embryonic callus after genetic transformation, and record the number of embryonic callus (number of pieces, with a diameter of not less than 0.5 cm, the same below).

[0076] 5. Verification of transgenic callus

[0077] The positive callus tissues that can still grow normally after two Hyg screenings were subjected to GUS histochemical staining and molecular verification. The specific methods are as follows:

[0078] Hyg-resistant tissue was immersed in 1.5 mL of GUS staining solution (SL7160, Beijing Coolable Technology Co., Ltd.) and placed in a 35°C incubator for 1 day. Non-transformed tissue was used as negative control. The GUS staining of resistant tissue was observed and counted. Tissues that showed blue were transgenic tissues, and colorless, transparent or slightly yellow resistant tissues were false positive tissues.

[0079] Callus tissue that can still grow normally after two Hyg screenings was used as material, and total RNA was extracted using an RNA extraction kit (ER101, Beijing Quanshijin Biotechnology). The qualified RNA was reverse transcribed into cDNA using a reverse transcription kit (KR116-02, Beijing Tiangen Biotechnology). RT-PCR detection was performed using cDNA as a template. Actin was used as an internal reference gene, and the primer sequences were 5′-TGCCTGCCATGTATGTTGCA-3′ and 5′-GTCAGATCACGCCCAGCAAG-3′. The GUS gene in the plasmid was used as a detection gene, and the primer sequences were 5′-GTCGCGCAAGACTGTAACCA-3′ and 5′-CGGCGAAATTCCATACCTG-3′. The PCR reaction system was 2 μL cDNA, 10 μL Taq DNA polymerase mix, 1 μL of each primer, and 6 μL RNase-free H2O. Reaction conditions: 95℃30s; 95℃30s, 58℃30s, 72℃40s, 33 cycles; 72℃7min. After the reaction, the PCR product was detected by electrophoresis on 1.2% agarose gel. The result was positive if the fragment size was correct and the band was single.

[0080] The callus tissues that were positive for GUS staining and molecular verification were considered to be successfully genetically transformed, and the number of transgenic callus tissues (blocks) was recorded.

[0081] The above method was used, with the embryonic callus of genotype 1-3-5 at the PEMIII stage prepared in Example 1 as the receptor material, and the infection solution (OD 600 The number of positive callus tissues obtained was counted, and the results are shown in Table 1. Figure 7 , Figure 8 shown.

[0082] Table 1 Comparison of different combinations on genetic transformation rate and positive transgenic rate of Masson pine

[0083] Infection method 1 2 3 4 Infectious strain AGL1 LBA4404 EHA105 GV3101 Number of successful screening and cultivation (blocks) 75 13 2 5 Conversion efficiency (block / g) 7.5 6.5 0.2 1.5 GUS stained transgenic number (blocks) 70 13 2 5 Number of transgenic samples detected by molecular method (blocks) 70 13 2 5 Positive transgenic rate (%) 93.3 100 100 100

[0084] Table 1 Figure 7 , Figure 8The results showed that the infection method constructed in this example can successfully obtain transformed positive callus tissue, and the highest positive transgenic rate is 100%.

[0085] 6. Maturity training

[0086] Take 1g of transgenic embryonic callus and inoculate it into a conical flask containing 30mL of suspension culture medium. Use sterile tweezers to gently break it up so that it is in full contact with the suspension medium. Then place the conical flask in a 25℃ shaker and culture it at 90rpm for 7 days. After 7 days, take 5mL of the suspended cell liquid with the supernatant removed and spray it evenly on a sterile filter paper, which is then placed horizontally in a maturation medium and cultured in the dark for 75 days.

[0087] The results are as follows Fig. 9 As shown, the transgenic callus after maturation treatment can differentiate into somatic embryos, and GUS staining is shown in blue.

[0088] Example 4

[0089] Agrobacterium tumefaciens strains AGL1, GV3101, LBA4404, and EHA105 were used, and the infection solution OD 600 The value was 0.6, and the infected genotypes were 1-3-5, 20-3-9, and 20-4-4 embryonic callus at the PEM I stage. All other steps were the same as in Example 3. The statistical results are shown in Table 2.

[0090] Table 2 Comparison of genetic transformation rate and positive transgenic rate of Masson pine with different receptor materials and strain types

[0091]

[0092]

[0093] The results in Table 2 showed that positive transformation could not be successfully obtained when embryogenic callus at PEM I stage was used as the recipient material for infection.

[0094] Example 5

[0095] The Agrobacterium tumefaciens strain used in this example is GV3101, and the infection fluid OD 600 The value is 0.2, the embryonic callus genotype used is 1-3-5, and all other steps are the same as in Example 3.

[0096] The results are as follows Fig.10 As shown, after the secondary screening, a total of 9 hygromycin-resistant callus tissues were obtained. The results of histochemical staining and molecular detection using GUS showed that 8 of the 9 hygromycin-resistant callus tissues obtained after the secondary screening were successfully transformed positive callus tissues, and the positive transgenic rate was 88.9%.

[0097] In summary, the present application induces callus with good embryogenicity, which can be used as a receptor material for genetic transformation of Masson pine. Taking callus with genotype 1-3-5 as an example, infection experiments were carried out using four Agrobacterium tumefaciens strains AGL1, GV3101, LBA4404, and EHA105, and successfully transformed callus was obtained, among which the AGL1 strain had the highest efficiency. The construction method of the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens provided in the present application is illustrated, and the genetic transformation performance is stable and efficient.

Claims

1. A method for constructing a genetic transformation system of Masson pine mediated by Agrobacterium tumefaciens, characterized in that: Immature cones of Masson pine were collected and induced to culture embryonic callus. Embryonic callus of Masson pine at PEMIII stage was used as the material for suspension pre-culture to obtain receptor material. 600 The cells were infected with Agrobacterium infection solution with a value of 0.2-0.

6. After the infection, co-culture, recovery culture, screening culture and maturation culture were performed, and GUS was used for histochemical staining and molecular identification to finally obtain transgenic embryonic callus.

2. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The Agrobacterium is selected from Agrobacterium tumefaciens AGL1, LBA4404, EHA105, and GV3101.

3. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The genotype of the Masson pine embryonic callus is 1-3-5.

4. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The genotype of the Masson pine embryonic callus is 1-3-5, and the Agrobacterium is Agrobacterium tumefaciens AGL1.

5. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The genotype of the Masson pine embryonic callus is 1-3-5, and the Agrobacterium is LBA4404, EHA105 or GV3101.

6. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The genotype of the Masson pine embryonic callus is 1-3-5, the Agrobacterium is LBA4404 or EHA105 or AGL1, and the infection concentration OD 600 The value is 0.

6.

7. The method for constructing the Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The genotype of the Masson pine embryonic callus was 1-3-5, the Agrobacterium was GV3101, and the infection concentration OD 600 The value is 0.2-0.

6.

8. The method for constructing a Masson pine genetic transformation system mediated by Agrobacterium tumefaciens according to any one of claims 1 to 7, characterized in that: The specific steps include: 1) sterilizing immature female gametophytes of Masson pine with genotype 1-3-5 and inoculating them into induction medium for induction culture to obtain embryonic callus; The formula of the induction medium is: LP basic medium + 2 mg / L 2,4-D + 1 mg / L 6-BA + 0.25 mg / L VC + 30 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine, pH 5.8; 2) inoculating the induced embryonic callus into a proliferation medium for proliferation culture; The formula of the proliferation medium is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 30 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar, pH 5.8; 3) transferring the Masson pine embryonic callus at the PEMIII stage into a liquid suspension culture medium for suspension pre-culture; The liquid suspension culture medium formula is: LP basic medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 100 μM acetosyringone, pH 5.8; 4) Transform the plasmid containing the target gene into AGL1 Agrobacterium and prepare the infection solution; 5) Use OD 600 The infection solution with a value of 0.6 infects the pre-cultured callus tissue; 6) After infection, the callus tissue is transferred to the co-cultivation medium for co-cultivation; The co-culture medium formula is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 100 μM acetosyringone, pH 5.8; 7) Degerming the co-cultured callus tissue and transferring it to a recovery medium for recovery culture; The recovery medium formula is: LP basic medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime, pH 5.8; 8) transferring the callus tissue after recovery culture to a screening medium for screening culture, and finally obtaining positive embryonic callus tissue after genetic transformation; The screening culture medium formula is: LP basic culture medium + 1 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.25 mg / L VC + 15 g / L maltose + 1 g / L inositol + 0.5 g / L hydrolyzed casein + 0.45 g / L glutamine + 6.24 g / L agar + 300 mg / L timentin + 300 mg / L cefotaxime + 25 mg / L hygromycin, pH 5.

8. 9) Perform histochemical staining and molecular identification on the positive transformed callus tissue after screening and culture, and finally obtain transgenic embryonic callus tissue. 10) transferring the transgenic embryonic callus to a maturation medium for maturation treatment to obtain transgenic somatic embryos; The formula of the mature culture medium is: LP basic culture medium + 5 mg / L ABA + 0.5 mg / L GA3 + 0.25 mg / L VC + 130 g / L PEG8000 + 1.5 g / L activated carbon + 20 g / L maltose + 1 g / L glutamine + 1 g / L inositol + 3.5 g / L plant gel, pH 5.8.

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