A red pine cell fusion method based on combination of chemical induction and magnetic bead method

Abstract

The present application belongs to the field of plant biotechnology, and particularly relates to a Korean pine cell fusion method based on chemical induction combined with a magnetic bead method. The method comprises the following steps: S1, preparing first protoplasts and second protoplasts; S2, preparing passivated first protoplasts and passivated second protoplasts; S3, mixing the passivated first protoplasts with the passivated second protoplasts in equal volumes after the passivated first protoplasts are cultured with a sodium silicate aqueous solution to obtain mixed protoplasts; S4, performing electrofusion on the mixed protoplasts in a cell fusion instrument to obtain fusion products; and S5, screening hybrid cells from the fusion products by using a magnetic bead method. The present application adopts a double screening mechanism of chemical passivation and magnetic bead screening, inactivates parent cells by treating with iodoacetamide and rose Bengal B, and then specifically enriches hybrid cells by using the magnetic bead method, so that the acquisition rate and screening accuracy of hybrid cells are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and in particular relates to a method for fusion of red pine cells based on a combination of chemical induction and magnetic bead method. Background Technology

[0002] As an important ecological and economic tree species, the genetic improvement of Korean pine is of great significance for the sustainable development of forestry. Traditional hybridization breeding is time-consuming, inefficient, and limited by reproductive isolation. Somatic cell hybridization technology can overcome the incompatibility of sexual hybridization and realize the exchange of genetic material between distant relatives, which is an important approach to the genetic improvement of Korean pine.

[0003] Somatic cell hybridization (protoplast fusion) technology can overcome the incompatibility of distant hybridization, enabling the combination of genetic material from different species, genera, and even families, providing a new approach for forest genetic improvement. This technology involves fusing protoplasts from two parents under artificial induction conditions to form hybrid cells, which are then cultured and regenerated into complete plants, significantly shortening the breeding cycle and creating new germplasm resources. Existing somatic cell hybridization methods mainly employ chemical induction (such as polyethylene glycol induction) or physical induction (such as electrofusion). Chemical induction is simple to operate but has a low fusion rate and is somewhat toxic to cells; electrofusion offers controllable parameters and good reproducibility, but when used alone, hybrid cell screening is difficult, making it hard to effectively distinguish fused hybrid cells from unfused parental cells.

[0004] In summary, there are currently no mature and stable somatic cell hybridization methods reported for the specific tree species, Korean pine. The preparation of Korean pine protoplasts is difficult, their cell wall regeneration ability is weak, and the screening of hybrid cells after fusion is a major technical bottleneck. Therefore, developing an efficient and stable somatic cell hybridization method for Korean pine has significant theoretical and practical value. Summary of the Invention

[0005] The main objective of this invention is to provide a method for fusion of red pine cells based on a combination of chemical induction and magnetic bead method, so as to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: According to a first aspect of the present invention, a method for fusion of red pine cells based on a combination of chemical induction and magnetic bead method is provided, comprising the following steps: S1. Protoplast preparation: First protoplasts were prepared from the first Korean pine parent, and second protoplasts were prepared from the second Korean pine parent; S2. Chemical passivation: The first protoplast is treated with iodoacetamide solution, and the second protoplast is treated with rose red B solution. Then they are washed and cultured to a preset density to obtain the passivated first protoplast and the passivated second protoplast. S3, Silicate induction: The passivated first protoplast is cultured in an aqueous sodium silicate solution and then mixed with the passivated second protoplast in an equal volume to obtain a mixed protoplast; S4. Electrofusion: The mixed protoplasts are placed in a cell fusion instrument for electrofusion to obtain fusion products; S5. Magnetic bead screening: Hybrid cells are screened from the fusion product using the magnetic bead method.

[0007] Furthermore, the protoplast preparation in step S1 includes: taking young tissues from the first and second Korean pine parents, cutting them into small pieces, placing them in an enzyme solution containing cellulase, pectinase and dissociation enzyme for enzymatic hydrolysis, then filtering, centrifuging and purifying, and collecting the precipitate to obtain the protoplasts.

[0008] Furthermore, the enzymatic hydrolysis conditions are: enzymatic hydrolysis with shaking at 25-30℃ for 3-5 hours; the centrifugal purification includes first centrifuging at 450-550 r / min to collect the precipitate, then washing the precipitate with CPW solution, and repeating the centrifugation and washing 2-3 times.

[0009] Further, in step S2, the concentration of the iodoacetamide solution is 5-7 mmol / L, the concentration of the rose red B solution is 45-55 μg / mL, the treatment time is 15-25 minutes, and the treatment temperature is room temperature; the washing is performed by centrifugation at a speed of 750-850 r / min for 4-6 minutes each time, with two washes; the culture involves placing the washed first and second protoplasts in the dark and culturing them to a density of 4 × 10⁻⁶. 5 -6×10 5 per mL.

[0010] Furthermore, in step S3, the concentration of the sodium silicate aqueous solution is 0.5-0.8 mg / L, the incubation time is 10-15 minutes, and the incubation temperature is 20-30℃.

[0011] Furthermore, the parameters for the electrofusion described in step S4 are: AC field strength 150-250V / cm, AC frequency 450-650kHz, AC duration 100-300s, DC pulse field strength 1.5-2.5kV / cm, pulse width 30-50μs, and pulse count 1-3 times.

[0012] Furthermore, in step S4, before electrofusion, the mixed protoplasts are pre-equilibrated in the fusion solution for 2-5 minutes.

[0013] Furthermore, the magnetic bead method in step S5 includes: incubating the fusion product with magnetic beads conjugated with antibodies targeting markers on the surface of Korean pine hybrid cells, and then collecting the hybrid cells by magnetic separation.

[0014] Furthermore, the antibody is an antibody targeting a marker on the surface of Korean pine hybrid cells.

[0015] Furthermore, the first and second Korean pine parents are Korean pine plants of different varieties.

[0016] Compared with the prior art, the advantages of the present invention include: This invention provides a method for Korean pine cell fusion based on a combination of chemical induction and magnetic beading. It employs a dual screening mechanism of chemical passivation and magnetic bead selection. Parental cells are first inactivated by treatment with iodoacetamide and rose red B, and then hybrid cells are specifically enriched using magnetic beads, significantly improving the hybrid cell yield and screening accuracy. This invention introduces a silicate induction step, altering the surface charge state of the protoplast membrane, promoting protoplast adhesion, and improving electrofusion efficiency. The method is controllable and reproducible, and holds significant application potential for Korean pine germplasm resource innovation and superior variety breeding. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is an electron microscope image of cell fusion in a typical embodiment of the present invention; Figure 2 This is one of the photographs of plant seedlings formed after cell fusion in a typical embodiment of the present invention; Figure 3 This is the second photograph of a plant seedling formed after cell fusion in a typical embodiment of the present invention; Figure 4 This is the third photograph of a plant seedling formed after cell fusion in a typical embodiment of the present invention; Figure 5 This is the fourth photograph of a plant seedling formed after cell fusion in a typical embodiment of the present invention; Figure 6 This is the fifth photograph of a plant seedling formed after cell fusion in a typical embodiment of the present invention. Detailed Implementation

[0018] In view of the shortcomings of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. The following will further explain and illustrate this technical solution, its implementation process, and its principles.

[0019] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0020] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials used in the following embodiments are commercially available.

[0021] This invention provides a method for fusion of red pine cells based on a combination of chemical induction and magnetic bead method, comprising the following steps: S1. Protoplast preparation includes: taking young tissues from the first and second Korean pine parents, cutting them into small pieces, and placing them separately in enzyme solutions containing cellulase, pectinase, and ionizing enzyme for enzymatic hydrolysis. Then, filtering, centrifuging for purification, and collecting the precipitate yields the first and second protoplasts. The enzymatic hydrolysis conditions are: hydrolysis with shaking at 25-30℃ for 3-5 hours. The centrifugal purification includes first centrifuging at 450-550 r / min to collect the precipitate, then washing the precipitate with CPW solution, repeating the centrifugation and washing 2-3 times. This low-speed centrifugation condition effectively precipitates intact protoplasts while avoiding cell damage. S2. Chemical passivation: The first protoplast is treated with iodoacetamide solution, and the second protoplast is treated with rose red B solution. Then, they are washed and cultured to a preset density to obtain passivated first and second protoplasts. The concentration of the iodoacetamide solution is 5-7 mmol / L, the concentration of the rose red B solution is 45-55 μg / mL, the treatment time is 15-25 minutes, and the treatment temperature is room temperature (20-30℃). The washing is performed by centrifugation at 750-850 r / min for 4-6 minutes each time, with two washes. The culture involves placing the washed first and second protoplasts in the dark and culturing them to a density of 4 × 10⁻⁶. 5 -6×10 5 Cells / mL; Iodoacetamide irreversibly inhibits cell metabolism, and rose red B causes photosensitivity inactivation of cells. Neither of the parental protoplasts treated with either treatment can divide and regenerate independently. However, the hybrid cells formed after fusion can regain vitality due to metabolic complementarity, thus achieving preliminary screening of hybrid cells. The concentration range and treatment time have been optimized to ensure passivation without causing excessive cell damage that could affect subsequent fusion. S3, Silicate Induction: The passivated first protoplast is cultured in a sodium silicate aqueous solution, and then mixed with an equal volume of the passivated second protoplast to obtain a mixed protoplast. The concentration of the sodium silicate aqueous solution in step S3 is 0.5-0.8 mg / L (based on silicon), the culture time is 10-15 minutes, and the culture temperature is 20-30℃. Silicates can alter the surface charge state of the protoplast membrane, increase membrane fluidity, and promote adhesion and fusion between protoplasts, thereby improving electrofusion efficiency. S4. Electrofusion: The mixed protoplasts are pre-equilibrated in the fusion solution for 2-5 minutes to allow them to fully adapt to the fusion solution environment and improve fusion stability. Then, the mixed protoplasts are placed in a cell fusion instrument for electrofusion to obtain the fusion product. The parameters for electrofusion are: AC field strength 150-250 V / cm, AC frequency 450-650 kHz, AC duration 100-300 s, DC pulse field strength 1.5-2.5 kV / cm, pulse width 30-50 μs, and pulse count 1-3. This parameter range is suitable for the electrofusion of Korean pine protoplasts and can achieve good protoplast queuing and fusion effects. S5. Magnetic Bead Screening: Hybrid cells are screened from the fusion product using a magnetic bead method. The magnetic bead method involves incubating the fusion product with magnetic beads conjugated with antibodies targeting surface markers of Korean pine hybrid cells, followed by magnetic separation to collect the hybrid cells. The antibodies are specifically designed to recognize differences in surface antigens between Korean pine hybrid cells and parental cells, thereby achieving efficient sorting of hybrid cells.

[0022] Preferably, the first and second Korean pine parents are Korean pine plants of different varieties. Hybridization between different varieties can yield richer genetic variation, which is beneficial for the combination of superior traits and the utilization of heterosis.

[0023] This invention employs a combination of chemical passivation and magnetic bead screening: first, the two parent protoplasts are treated with iodoacetamide and rose red B respectively, causing the parent cells to lose their individual regeneration ability, and only the hybrid cells formed by fusion can complement each other and restore vitality; then, silicate induction and electrofusion are combined to improve fusion efficiency; finally, the magnetic bead method is used to specifically screen hybrid cells, thereby establishing a complete somatic cell hybridization technology system for red pine.

[0024] To better understand the technical solution of the present invention, the following detailed discussion is provided in conjunction with specific embodiments.

[0025] Example 1 This embodiment provides a method for fusion of red pine cells based on a combination of chemical induction and magnetic bead method, including the following steps: (1) Preparation of protoplasts Young needles of the current year from Korean pine variety A (high fruit yield) and variety B (strong adaptability and fast growth) were selected as parent materials. 1.0g of each parent's young needles were taken, washed with sterile water, and then cut into small pieces 0.5-1mm wide. The cut tissue pieces were placed into sterile Erlenmeyer flasks containing 10mL of enzyme solution. The enzyme solution composition was: 1.5% cellulase R-10, 0.5% pectinase Y-23, 0.3% cleavage enzyme R-10, and 0.6mol / L mannitol as an osmotic pressure stabilizer, pH 5.8.

[0026] Seal the Erlenmeyer flask with sealing film and place it in a shaker at 28℃ and 40 rpm for 4 hours for enzymatic hydrolysis. After hydrolysis, filter the hydrolysate through 80-mesh and 200-mesh stainless steel sieves to remove undigested tissue residue and collect the filtrate. Transfer the filtrate to a centrifuge tube and centrifuge at 500 rpm for 5 minutes, discard the supernatant, and collect the precipitate. Add 5 mL of CPW solution (containing 0.6 mol / L mannitol) to the precipitate, gently resuspend by pipetting, and centrifuge again at 500 rpm for 5 minutes, discarding the supernatant. Repeat the washing process twice to obtain the purified protoplast precipitate. Resuspend the protoplast in CPW solution and adjust the density for later use.

[0027] (2) Chemical passivation The prepared protoplasts of Korean pine variety A were suspended in 6 mmol / L iodoacetamide solution and treated at room temperature (25℃) for 20 minutes. Protoplasts of Korean pine variety B were suspended in 50 μg / mL rose cinnabar B solution and treated at room temperature for 20 minutes. After treatment, both types of protoplasts were centrifuged at 800 r / min for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in CPW solution and washed again by centrifugation at 800 r / min for 5 minutes, repeating the washing twice. The washed protoplasts were resuspended in CPW solution and cultured in the dark, with the density adjusted to 5 × 10⁶. 5 Protoplasts of variety A and variety B were obtained by passing on protoplasts at a density of 1 / mL.

[0028] (3) Silicate-induced Take the passivated protoplast suspension of variety A, add sodium silicate aqueous solution to make the final silica concentration 0.6 mg / L, and incubate at room temperature for 12 minutes. After incubation, mix equal volumes of silicate-treated protoplasts of variety A and untreated protoplasts of variety B to obtain a mixed protoplast suspension.

[0029] (4) Magnetic assisted queuing and electrical fusion Centrifuge the mixed protoplast suspension, discard the supernatant, and resuspend the precipitate in fusion buffer (containing 0.6 mol / L mannitol and 0.5 mmol / L CaCl2), adjusting the density to 2 × 10⁻⁶. 5Protoplast suspension was added to the fusion chamber and allowed to stand for 3 minutes to allow the protoplasts to pre-equilibrate. Before applying an alternating electric field, a weak magnetic field (approximately 0.2T) was applied to the fusion chamber for 5 minutes to induce the protoplasts carrying magnetic beads to move in a specific direction and come into contact with unlabeled protoplasts.

[0030] After magnetic field treatment, the proportion of heterologous pairings of AB cells in the formed cell clusters was 35.5% ± 2.8%, which was significantly higher than that of heterologous contact pairs formed by random collisions.

[0031] Set the cell fusion instrument parameters as follows: AC field strength 200V / cm, AC frequency 500kHz, AC duration 200s, DC pulse field strength 2.0kV / cm, pulse width 40μs, pulse count 2. Start the fusion program for electrofusion. After fusion, let stand for 10 minutes, then gently transfer the fusion product into a centrifuge tube for later use.

[0032] (5) Magneto-optical sorting Magnetic beads conjugated with antibodies against the surface antigen of Korean pine protoplasts were prepared and used to label protoplasts of variety B. Simultaneously, protoplasts of variety A were labeled during the preparation process by transfecting them with the green fluorescent protein (GFP) gene. The fusion products were subjected to magneto-optical sorting within 2 hours post-fusion: first, cells positive for magnetic bead labeling were enriched using a magnetic separation column, and the eluent was collected; then, the eluent was subjected to flow cytometry to collect cells that were both positive for GFP and magnetic bead labeling.

[0033] From the initial 1×10 6 Starting with a single protoplast, magneto-optical sorting was used to obtain 2.1 × 10⁻⁶ protoplasts in a single operation. 2 Double-positive target cells were identified. The sorted cell suspension was analyzed by flow cytometry; the purity of the double-positive cells was greater than 65%.

[0034] The sorted double-positive cells were seeded in regeneration medium (containing 2.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.2 mg / L NAA, 500 mg / L glutamine, and 100 mg / L inositol) and cultured. After 7 days of culture, more than 70% of the cell clusters showed signs of division, increased cell cluster volume, and thickened cytoplasm, initiating the regeneration process.

[0035] Fifty double-positive cells were randomly selected from the sorted cells for single-cell genomic PCR identification to detect parent-specific molecular markers. The results showed that 26 cells carried both parent-specific markers, and the true hybrid identification rate was 52%.

[0036] like Figure 1 As shown, Figure 1 This is an image of cell fusion under an electron microscope; Figure 2-6 It is a photo of a plant seedling formed after cell fusion.

[0037] Example 2 This embodiment is basically the same as that of embodiment 1, except that in step (2), the concentration of iodoacetamide is adjusted to 5 mmol / L, the concentration of rose red B is adjusted to 45 μg / mL, and the treatment time is 15 minutes; in step (3), the concentration of sodium silicate is 0.5 mg / L, and the culture time is 10 minutes; in step (4), the magnetic field treatment time is shortened to 3 minutes, and the electrofusion parameters are adjusted as follows: AC field strength 150 V / cm, AC frequency 450 kHz, AC duration 300 s, DC pulse field strength 1.5 kV / cm, pulse width 30 μs, and pulse count 3 times.

[0038] The results showed that, after magnetic field treatment, the proportion of heterologous pairing (AB) under microscopic counting was 28.3% ± 2.1%; 1.8 × 10⁸ double-positive cells were obtained by magneto-optical sorting. 2 The purity of the cells was 58% as verified by flow cytometry; single-cell PCR identification showed a true hybrid identification rate of 45%; after 7 days of culture in regeneration medium, approximately 62% of the cell clusters showed division signals.

[0039] Example 3 This embodiment is basically the same as that of embodiment 1, except that in step (2), the concentration of iodoacetamide is adjusted to 7 mmol / L, the concentration of rose red B is adjusted to 55 μg / mL, and the treatment time is 25 minutes; in step (3), the concentration of sodium silicate is 0.8 mg / L, and the culture time is 15 minutes; in step (4), the magnetic field treatment time is extended to 7 minutes, and the electrofusion parameters are adjusted as follows: AC field strength 250 V / cm, AC frequency 650 kHz, AC duration 100 s, DC pulse field strength 2.5 kV / cm, pulse width 50 μs, and pulse count 1.

[0040] The results showed that the passivation effect was more thorough, but the protoplast viability decreased slightly. After magnetic field treatment, the AB heterologous pairing ratio was 32.1% ± 2.5%; magneto-optical sorting yielded 1.9 × 10⁻⁶ double-positive cells. 2 The cell count was 62% pure; single-cell PCR identification showed a true hybrid identification rate of 49%; after 7 days of culture in regeneration medium, approximately 58% of the cell clusters showed division signals.

[0041] Comparative Example 1 (Traditional PEG Method) This comparative example uses the traditional polyethylene glycol (PEG) induced fusion method, without chemical passivation, silicate induction, or magnetic bead screening.

[0042] Protoplasts of Korean pine varieties A and B were taken and mixed in a 1:1 ratio, with the total density adjusted to 5 × 10⁻⁶. 5Cells / mL. Add PEG fusion buffer (containing 40% PEG 6000, 0.1 mol / L CaCl2, 0.3 mol / L mannitol, pH 7.0) and let stand at room temperature for 15 minutes. Then slowly add diluent to elute PEG, centrifuge to collect cells, and resuspend in culture medium for culture.

[0043] Microscopic counting showed that closely contacting AB protoplast pairs accounted for only 1.3% ± 0.2% of all contact pairs, indicating extremely low heteronuclear body formation efficiency.

[0044] After fusion, the cells were cultured for 4 weeks, and cell clusters suspected of exhibiting amphipathic characteristics were selected using a manual microscope. The initial cell count was 1×10⁻⁶. 6 Starting with a single protoplast, only 16 suspected hybrid cell clusters were selected. Molecular identification was performed on the 16 selected cell clusters, of which only 3 were confirmed to be true hybrids containing the genomes of both parents, with a true hybrid identification rate of approximately 18.7%. Of the 3 true hybrid callus tissues, only 1 was able to continuously proliferate and differentiate into embryoids, with a regeneration initiation success rate of 13.3%.

[0045] Comparative Example 2 This comparative example is basically the same as Example 1, except that in step (4), magnetic field-assisted queuing is not performed, and electric fusion is performed directly.

[0046] The results showed that the proportion of heterologous pairing of AB cells counted under a microscope was 5.8% ± 0.7%, and the fusion rate was significantly lower than that in Example 1. Subsequent magneto-optical sorting yielded 6.5 × 10⁻⁶ double-positive cells. 1 The purity of the sorted samples was 41%, which was lower than that of Example 1.

[0047] Table 1

[0048] The above examples and comparative examples demonstrate that the Korean pine cell fusion method based on chemical induction and magnetic bead method provided by the present invention inactivates parental cells through chemical passivation, improves heterologous pairing efficiency by combining silicate induction and magnetic field-assisted queuing, and then efficiently enriches hybrid cells using magneto-optical sorting technology. This significantly improves the heterokaryotic formation efficiency, hybrid cell screening purity, and true hybrid identification rate, ultimately obtaining hybrid cells with high regeneration power, providing a reliable technical means for the genetic improvement of Korean pine.

[0049] The above descriptions are merely some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for fusing Korean pine cells based on a combination of chemical induction and magnetic bead method, characterized in that, Includes the following steps: S1. Protoplast preparation: First protoplasts were prepared from the first Korean pine parent, and second protoplasts were prepared from the second Korean pine parent; S2. Chemical passivation: The first protoplast is treated with iodoacetamide solution, and the second protoplast is treated with rose red B solution. Then they are washed and cultured to a preset density to obtain the passivated first protoplast and the passivated second protoplast. S3, Silicate induction: The passivated first protoplast is cultured in an aqueous sodium silicate solution and then mixed with the passivated second protoplast in an equal volume to obtain a mixed protoplast; S4. Electrofusion: The mixed protoplasts are placed in a cell fusion instrument for electrofusion to obtain fusion products; S5. Magnetic bead screening: Hybrid cells are screened from the fusion product using the magnetic bead method.

2. The method according to claim 1, characterized in that, The protoplast preparation in step S1 includes: taking young tissues from the first and second Korean pine parents, cutting them into small pieces, placing them in an enzyme solution containing cellulase, pectinase and dissociation enzyme for enzymatic hydrolysis, then filtering, centrifuging and purifying, and collecting the precipitate to obtain the protoplasts.

3. The method according to claim 2, characterized in that, The enzymatic hydrolysis conditions are: enzymatic hydrolysis with shaking at 25-30℃ for 3-5 hours; the centrifugal purification includes first centrifuging at 450-550 r / min to collect the precipitate, then washing the precipitate with CPW solution, and repeating the centrifugation and washing 2-3 times.

4. The method according to claim 1, characterized in that, In step S2, the concentration of the iodoacetamide solution is 5-7 mmol / L, the concentration of the rose red B solution is 45-55 μg / mL, the treatment time is 15-25 minutes, and the treatment temperature is room temperature. The washing is performed by centrifugation at 750-850 r / min for 4-6 minutes each time, with two washes. The culture involves culturing the washed first and second protoplasts separately in the dark until a density of 4 × 10⁻⁶ is reached. 5 -6×10 5 per mL.

5. The method according to claim 1, characterized in that, The concentration of the sodium silicate aqueous solution in step S3 is 0.5-0.8 mg / L, the incubation time is 10-15 minutes, and the incubation temperature is 20-30℃.

6. The method according to claim 1, characterized in that, The parameters for electrofusion in step S4 are: AC field strength 150-250V / cm, AC frequency 450-650kHz, AC duration 100-300s, DC pulse field strength 1.5-2.5kV / cm, pulse width 30-50μs, and pulse count 1-3 times.

7. The method according to claim 1, characterized in that, In step S4, before electrofusion, the mixed protoplasts are pre-equilibrated in the fusion solution for 2-5 minutes.

8. The method according to claim 1, characterized in that, The magnetic bead method described in step S5 includes: incubating the fusion product with magnetic beads conjugated with antibodies targeting markers on the surface of Korean pine hybrid cells, and then collecting the hybrid cells by magnetic separation.

9. The method according to claim 8, characterized in that, The antibody is an antibody that targets a marker on the surface of Korean pine hybrid cells.

10. The method according to claim 1, characterized in that, The first and second Korean pine parents are Korean pine plants of different varieties.

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