High-Efficiency Pinus Protoplast Isolation and Purification Technology for Gene Function Analysis in Gymnosperms

Abstract

The present invention relates to a method for isolating and purifying protoplasts derived from pine trees. To optimize the conditions for isolating protoplasts from pine trees, optimization analyses were performed on tissue pretreatment conditions, enzymatic hydrolysis conditions, washing buffer conditions, and the purification process. Additionally, genetic transformation and transcriptional activity were analyzed for the isolated protoplasts, confirming that protoplasts can be isolated from pine trees and that optimal conditions for isolation and the isolated protoplasts can be used for transient transformation were also confirmed.

Description

Technology Field

[0001] The present invention relates to a method for isolating and purifying a protoplast derived from pine trees. Background Technology

[0002] Protoplasts are plant cells from which the cell wall has been removed. Possessing totipotency and high flexibility, they serve as useful tools for genetics and molecular research. The removal of the cell wall enables the effective delivery of foreign molecules such as DNA, RNA, and proteins, thereby facilitating both transient expression and stable transformation. Transient expression induces rapid and ephemeral gene expression over short periods (several hours to days), whereas stable transformation refers to the integration of foreign genes into the plant genome for long-term maintenance and inheritance.

[0003] Protoplast-based transformation methods generally utilize polyethylene glycol (PEG)-mediated transformation, electroporation, and microinjection, and are widely used in molecular breeding, genome editing (e.g., CRISPR-Cas9, TALEN), gene silencing (e.g., dsRNA, miRNA, siRNA), transcriptional studies, transcriptome analysis (e.g., single-cell RNA-seq), cell fusion, and plant regeneration through tissue culture. Recently, with the advancement of automation technology, high-efficiency protoplast isolation and transformation using robotic systems have become possible, further improving the efficiency and reproducibility of experiments.

[0004] However, while protoplast isolation and transformation techniques have advanced significantly in angiosperms, the development of efficient experimental protocols remains a major challenge in gymnosperms. To date, protoplasts of gymnosperms such as Pinus and Picea have been isolated from immature seedlings, somatic embryos, cell suspensions, and developing xylem tissues. However, phenolic compounds and resins released from thick epidermal layers and damaged tissues inhibit enzyme activity, leading to prolonged digestion times and reduced yield and viability of protoplasts.

[0005] In the past, studies using gymnosperm protoplasts primarily focused on transient gene expression using electroporation, but specific data on transformation efficiency were rarely reported. On the other hand, Arabidopsis thaliana, an angiosperm ( Arabidopsis thaliana ), cigarette( Nicotiana tabacum ), and the major crop, rice ( Oryza sativa ), corner( Zea mays A high-yield protoplast isolation protocol using mesophyll tissues has been established. However, due to characteristics such as gymnosperms' unique tissue structure and seasonal dormancy, obtaining suitable plant material is limited, and thus an efficient protoplast isolation method is urgently needed.

[0006] Accordingly, the inventors of the present invention are gymnosperms, specifically pine trees ( Pinus densifloraWe developed a protocol to rapidly and easily isolate protoplasts with high yield and high viability from red pine. In particular, we secured protoplasts with excellent viability and high transformation efficiency through optimization based on enzyme composition, buffer components, and tissue type (mesophyll tissue and xylem tissue (including developing xylem and whole stem)). In addition, we performed a transient transcriptional activation assay using the isolated protoplasts and confirmed that this protocol can be utilized in functional genomics studies. Prior art literature

[0007] Methods in Enzymology Volume 118, 1986, Pages 549-578 The problem to be solved

[0008] The objective of the present invention is to provide a method for isolating protoplasts derived from the mesophyll tissue of a pine tree.

[0009] Another objective of the present invention is to provide a method for isolating protoplasts derived from the stem tissue of a pine tree.

[0010] Another objective of the present invention is to provide a protoplasm separated through the above method.

[0011] Another objective of the present invention is to provide a method for temporary transformation. means of solving the problem

[0012] To achieve the above objective, the present invention provides a method for isolating protoplasts derived from the mesophyll tissue of a pine tree, comprising: 1) cutting the mesophyll tissue of a pine tree; 2) treating the cut tissue with an enzyme; and 3) washing the enzyme-treated tissue with a washing buffer.

[0013] In addition, the present invention provides a method for isolating protoplasts derived from pine tree stem tissue, comprising: 1) cutting the stem tissue of a pine tree; 2) washing the cut tissue; 3) enzymatically treating the washed tissue; and 4) performing sucrose gradient centrifugation.

[0014] In addition, the present invention provides a protoplast separated through the above method.

[0015] In addition, the present invention provides a transformation method comprising: 1) a step of isolating a protoplast according to the above method; and 2) a step of introducing a foreign gene into the protoplast. Effects of the invention

[0016] The present invention relates to a method for isolating and purifying protoplasts derived from pine trees. To optimize the conditions for isolating protoplasts from pine trees, optimization analyses were performed on tissue pretreatment conditions, enzymatic hydrolysis conditions, washing buffer conditions, and the purification process. Additionally, genetic transformation and transcriptional activity were analyzed for the isolated protoplasts, confirming that protoplasts can be isolated from pine trees. Furthermore, by confirming the optimal conditions for isolation and the ability to use the isolated protoplasts for transformation, the invention can be usefully applied in related fields. Brief explanation of the drawing

[0017] Fig. 1 is a two-year-old red pine ( P. densiflora This is a diagram showing the effect of the needle pretreatment method on the protoplast yield in seedlings. Figure 2 is a figure showing various needle sources for protoplast separation. Figure 3 shows the results of the optimization analysis of protoplast separation from the mesophyll tissue of a pine tree. A: Protoplast yield in various mesophyll sources B: Protoplast yield according to enzyme and hydrolysis time C: Protoplast survival rate according to washing buffer solution D: Optical and FDA staining images of the isolated protoplast Figure 4 is a figure showing the effect of washing buffer on the integrity and morphology of protoplasts. Figure 5 shows the results of the optimized analysis of protoplast separation from the stem tissue of a pine tree. A: Protoplast yield and viability according to hydrolysis time B: Optical microscope and FDA stained images of protoplasts separated by 5 hours of hydrolysis Fig. 6 is red pine ( P. densiflora This is a diagram showing the process of protoplast isolation and the results of survival rate analysis from the developing xylem of a stem. A: Red pine ( P. densiflora ) Process of protoplast separation in the developing xylem of the stem B: Optical and FDA staining images of the isolated protoplast Fig. 7 is a young red pine ( P. densiflora This is a diagram showing the dendritic tubes identified in the anatomical analysis of the stem. Figure 8 shows the protoplast purity and survival rate according to the sucrose density gradient. A: Protoplast purification process based on sucrose density gradient B: Optical microscope, FDA, and PI stained images according to sucrose density gradient Figure 9 shows the results of GFP transformation and transient transcriptional activation analysis in red pine and Arabidopsis protoplasts. A and B: Gene introduction efficiency via GFP expression in Arabidopsis leaf protoplasts (a) and pine cotyledon protoplasts (b) C: Activation of Pro_AtCesA4::GUS reporter following treatment with Arabidopsis MYB46 transcription factor (35S::AtMYB46) in Arabidopsis and Japanese Red Pine protoplasts D: Activation of Pro_PdeCesA7::GUS reporter following treatment with PdeMYB46 transcription factor (35S::PdeMYB46) in Arabidopsis and Pseudomonas red protoplasts Specific details for implementing the invention

[0018] The terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0019] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0021] The present invention provides a method for isolating protoplasts derived from the mesophyll tissue of a pine tree, comprising: 1) cutting the mesophyll tissue of a pine tree; 2) treating the cut tissue with an enzyme; and 3) washing the enzyme-treated tissue with a washing buffer.

[0022] In one embodiment of the present invention, the pine tree is a red pine ( Pinus densiflora It may be characterized by being ), but is not limited thereto.

[0023] In one embodiment of the present invention, step 1) may be characterized by cutting the mesophyll tissue longitudinally, but is not limited thereto.

[0024] In one embodiment of the present invention, the enzyme may comprise 2 to 6% (w / v) hemicellulase, 3 to 9% (w / v) cellulase, and 0.5 to 0.9% (w / v) pectinase, and preferably may comprise 4% (w / v) hemicellulase, 6% (w / v) cellulase, and 0.7% (w / v) pectinase, but is not limited thereto.

[0025] In one embodiment of the present invention, step 2) may involve treating the cut tissue with an enzyme to hydrolyze it for 4 to 6 hours, preferably for 5 hours, but is not limited thereto.

[0026] In one embodiment of the present invention, the washing buffer may consist of 154 mM NaCl, 125 mM CaCl, 5 mM KCl, 2 mM MES, and 5 mM glucose, but is not limited thereto.

[0028] In addition, the present invention provides a method for isolating protoplasts derived from pine tree stem tissue, comprising: 1) cutting the stem tissue of a pine tree; 2) washing the cut tissue; 3) enzymatically treating the washed tissue; and 4) performing sucrose gradient centrifugation.

[0029] In one embodiment of the present invention, the invention may relate to a composition for isolating protoplasts of pine trees comprising 2 to 6% (w / v) hemicellulase, 3 to 9% (w / v) cellulase, and 0.5 to 0.9% (w / v) pectinase as active ingredients.

[0030] In one embodiment of the present invention, step 3) may involve treating the cut tissue with an enzyme and hydrolyzing it for 4 to 6 hours, but is not limited thereto.

[0031] In one embodiment of the present invention, step 4) may involve separating protoplasts using a sucrose gradient in the concentration range of 13 to 17% (w / v), but is not limited thereto.

[0033] In addition, the present invention provides a protoplast separated through the above method.

[0035] In addition, the present invention provides a transformation method comprising: 1) a step of isolating a protoplast according to the above method; and 2) a step of introducing a foreign gene into the protoplast.

[0036] The transformant of the present invention can be constructed by introducing a vector into a host cell in a manner in which a promoter can act.

[0037] In the present invention, "transformation" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. Transformation includes any method of introducing nucleic acid molecules into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0038] Since the expression levels and modifications of proteins vary depending on the host cells transformed by the expression vector, the host cell most suitable for the purpose should be selected and used. Host cells that can be used in this invention may include insect cell lines, yeast, fungi, bacteria, or algae.

[0040] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0042] <Example 1> Analysis Method

[0043] 1-1. Preparation of Plant Materials and Setting of Growth Conditions

[0044] Red pine ( Pinus densiflora ) was used as a raw material for the separation of protoplasts of the present invention. Red pine seeds were obtained from Aram Seed Company (Seoul, South Korea), subjected to low-temperature treatment (vernalization) for one week at 4°C, and hydroponically germinated ( in vitro Germination was performed. For seed germination, seeds were sterilized and cultured in half-strength MS medium containing 1% (w / v) sucrose (Murashige and Skoog, Duchefa, Haarlem, The Netherlands). The culture conditions were a photoperiod of 14 hours (light intensity 150 μmol m⁻²). -2 s -1 ), and the temperature was maintained at 23 ± 2°C. In addition, some seeds were placed in pots filled with soil and grown in a growing room, and the soil used was a landscaping potting mix (Taeheung F&G, Gyeonggi-do, South Korea) composed of a mixture of coco peat (40%), peat moss (35%), vermiculite (24.5%), and humic acid (0.5%).

[0046] 1-2. Separation of Protoplasts

[0047] Protoplast isolation was performed by modifying the existing protocol applied to Arabidopsis thaliana. Protoplasts were isolated from cotyledons (1-month cultured seedlings, potted seedlings), young leaves (2-month-old seedlings, 2-year-old potted plants), mature trees (Kyung Hee University, Suwon, South Korea), and young stems of mature trees. Protoplasts were isolated from each tissue according to the following methods.

[0048] For conifer tissue, 250 mg of leaves were lengthwise cut or finely chopped and immersed in 2.5 mL of enzyme solution. For stem tissue, bark-removed stem sections (approx. 1 cm in diameter) were cultured in a 50 mL centrifuge tube with enzyme solution. For whole stems, young stems with a diameter of approximately 3 mm and a weight of 3.5 g were cut into transverse sections (approx. 0.05 mm in thickness) in distilled water, washed three times, and cultured in a Petri dish containing 20 mL of enzyme solution.

[0049] The enzyme solution for cell wall degradation contained 0.4 M mannitol, 20 mM KCl, and 20 mM MES, and was heated at 65 °C for 5 minutes and then cooled to room temperature. Subsequently, 10 mL of CaCl2, 0.1% (w / v), FBS (A7906-100G, Sigma, St. Louis, MO, USA), and the enzyme were added, followed by filtration through a 0.45 μm injection filter. Cellulase R-10 (Yakult, Honsha, Japan), pectinase (P2401-1KU, Sigma, St. Louis, MO, USA), and hemicellulase (H2125-150KU, Sigma, St. Louis, MO, USA) were used as enzymes. Tissue samples were immersed in the enzyme solution and subjected to vacuum infiltration for 30 minutes, after which they were incubated at room temperature with gentle shaking at 15 rpm. In the above process, the yield and viability of protoplasts were evaluated, and after staining with Wiesner reagent (phloroglucinol-HCl) and toluidine blue O (TBO), they were analyzed using a microscope (KCS3-SS, Optinity, Korea Lab Tech, Seoul, South Korea).

[0051] 1-3. Protoplast Purification

[0052] To purify protoplasts from conifer samples, the enzyme solution containing the protoplasts was filtered through a 150 μm nylon mesh (Miracloth, 475855-1RCN, Sigma, St. Louis, MO, USA) and then diluted with an equal volume of wash buffer. The mixture was left on ice for 30 minutes to precipitate the protoplasts. W5 (154 mM NaCl, 125 mM CaCl, 25 mM KCl, 2 mM MES), W1 (80 mM KCl, 0.4 M sucrose), W2 (half-strength MS medium, 4 mM CaCl, 20.7 M glucose), and W5-modified (W5 with 5 mM glucose added) were used as wash buffers.

[0053] For stem samples, the enzyme solution was filtered through a mesh and then carefully layered onto a sucrose solution (13%, 15%, or 17%) contained in a 15 mL conical tube. The tube was then centrifuged at 120 Φ g for 15 minutes at room temperature to obtain an intermediate layer containing condensed purified protoplasts.

[0054] Protoplasts were observed using a microscope, and the number of cells was counted using a hemacytometer (Hausser Scientific, Horsham, PA, USA). Cell viability was evaluated by staining living cells with fluorescein diacetate (FDA; Sigma) and dead cells with propidium iodide (PI; Sigma).

[0056] 1-4. Analysis of Protoplast Transduction and Transient Activation

[0057] Transient transient transduction of protoplasts was performed using a polyethylene glycol (PEG)-mediated method. Transduction efficiency was calculated via fluorescence analysis using GFP. Protoplasts were 3 × 10⁻¹⁰ in MMG solution (0.4 M mannitol, 15 mM MgCl, 4 mM MES, pH 5.7).5 The solution was diluted to a concentration of cells / mL. Subsequently, a mixture of 10 μg of 35S::GFP / pK2GW7 plasmid, 100 μL of MMG solution containing the protoplast, and 110 μL of PEG-calcium 40% solution was incubated at room temperature for 5 minutes. Afterward, the protoplast was washed with 400 μL of W5-modified solution, diluted with 500 μL of WI solution, and incubated overnight. Fluorescence signals were observed at wavelengths of 450–490 nm (blue light) using a microscope.

[0058] In addition, for the transient activation assay (TAA), 4 μg of reporter plasmid (Pro_PdeCesA7::GUS / pTrOX or Pro_AtCesA4::GUS / pTrOX), 5 μg of factor plasmid (35S::PdeMYB46 / pTrOX or 35S::AtMYB46 / pTrOX), and 1 μg of NAN plasmid as an internal control were used. After incubation overnight, GUS and NAN enzyme activities were measured using MUG (4-Methylumbelliferyl β-D-Glucuronide) and MUN (2'-(4-Methylumbelliferyl)-α-DN-acetylneuraminic acid) as substrates. Activity values ​​were quantified based on the MU (4-Methylumbelliferone) standard curve and using a Hoefer TK 100 fluorometer (excitation: 355 nm, emission: 460 nm). The analysis was repeated three times, and the results were expressed as the activity ratio of GUS / NAN.

[0060] <Example 2> Analysis Results

[0061] 2-1. Optimization of Protoplast Separation Materials

[0062] To verify an efficient method for isolating and purifying protoplasts from Japanese red pine, the yield and viability of the protoplasts were optimized by setting various conditions according to Example 1-1. First, young needles from biennial potted plants were used (Fig. 1a), and the needles were cut or split lengthwise and cultured in an enzyme solution containing 3% (w / v) cellulase and 0.14% (w / v) pectinase. The culture results are shown in Fig. 1. As shown in Fig. 1, the yield was approximately 20 times higher when the needles were split lengthwise compared to when they were cut.

[0063] For further optimization, various coniferous materials were used (Fig. 2), and the yield was verified. The protoplast yield is shown in Fig. 3a. Looking at Fig. 3a, the highest yield (5.0 × 10⁻⁶) was observed when the cotyledons of 1-month-old potted seedlings were split lengthwise (B). 6 It showed ( / g FW), whereas the cotyledons (A) of seedlings cultured in vitro were 1.2×10 6 It showed a low yield of / g FW. In the case of 2-month-old and 2-year-old conifers (C and D), the yield was found to decrease, and mature conifers grown outdoors (E) were found not to decompose. Through the above results, it was confirmed that 1-month-old pot-grown cotyledons (B) are the most suitable material for protoplast isolation.

[0065] 2-2. Optimization of Enzyme Hydrolysis Conditions

[0066] To optimize the enzymatic hydrolysis treatment conditions, separation efficiency was determined according to six enzyme combinations (E1-E6) and hydrolysis time in accordance with Example 2-2. E1 (3% cellulase, 0.14% pectinase) was used as a control, and E2 (2% hemicellulase, 3% cellulase, 0.14% pectinase), E3 (2% hemicellulase, 3% cellulase, 0.5% pectinase), E4 (2% hemicellulase, 3% cellulase, 0.7% pectinase), E5 (3% hemicellulase, 4.5% cellulase, 0.7% pectinase), and E6 (4% hemicellulase, 6% cellulase, 0.7% pectinase) were used. The analysis results are shown in Figure 3b. Looking at Figure 3b, the highest yield of approximately 12 x 10⁶ / g FW was observed when hydrolysis was performed for 4 to 5 hours with the E5 enzyme. However, E1 and E6 showed low yields, and even E6, which used the highest enzyme concentration, showed a lower yield compared to E5. Through the above results, it was confirmed that the concentrations of hemicellulase and sufficient pectinase play an important role in cell wall degradation, and that the highest yield was observed when the hydrolytic enzyme E5 was reacted for 5 hours.

[0068] 2-3. Optimization of Washing Buffer

[0069] After protoplast isolation, the protoplasts tended to aggregate in standard W5 wash buffer, which significantly reduced protoplast viability (Fig. 4). To address this phenomenon, various wash buffer conditions were investigated. Cell viability was assessed in W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, mM MES), W1 (80 mM KCl, 0.4 M sucrose), W2 (half-strength MS medium, 4 mM CaCl2, 0.7 M glucose), and W5-m (W5 with 5 mM glucose added) wash buffers. Fig. 3c shows the appearance of protoplasts in the wash buffers. As shown in Fig. 3c, the protoplasts did not maintain their shape and aggregated in the standard W5 and W2 wash buffers, while some distorted protoplasts were observed in the W1 wash buffer. However, in the W5-m wash buffer, the protoplasts were found to maintain a round shape. Furthermore, the cell viability of protoplasts significantly increased in the W5-m wash buffer compared to W5, W1, and W2, exhibiting a cell viability of 86.2%. Additionally, the morphology of protoplasts in the W5-m wash buffer was confirmed through optical and FDA staining image analysis. The analysis results are shown in Figure 3d. Looking at Figure 3d, it can be seen that the protoplasts maintain a round shape in the optical image, and the viability of the protoplasts can be confirmed through the fluorescence signal in the FDA staining image. Through these results, it was confirmed that the W5-m wash buffer, modified from W5, can increase the morphology and viability of protoplasts.

[0071] 2-4. Separation of protoplasts from woody tissue

[0072] According to Examples 1-2, protoplasts were isolated from developing xylem (DX), a woody tissue, and the whole stem using the same process as the protoplast isolation process described above. Protoplast isolation was possible from the developing xylem (DX), with a cell viability of 85.3% and a yield of 8.9 x 10⁻⁶ 5 It was found to be / g FW. In addition, the cell size ranged from 5 to 60 μm (Fig. 6).

[0073] In addition, young stems of mature Japanese red pine were used to isolate protoplasts from the entire stem. Since a large amount of resin was present due to the resin ducts in the entire stem (Fig. 7), stem sections were cut in distilled water to reduce resin flow and repeatedly washed with distilled water. Subsequently, hydrolysis analysis over time was performed to optimize protoplast release from the entire stem tissue. The results of the hydrolysis analysis are shown in Fig. 5. As shown in Fig. 5, protoplasts were first released after 2 hours of hydrolysis, and the yield increased rapidly after 5 hours of hydrolysis (7.4 x 10⁻¹⁰). 4 / g FW, viability 68.4%). However, it was found that cell viability decreased due to an increase in apoptotic cells after 7 hours, and the number of single cells decreased due to an increase in cell aggregation after 9 hours. Based on the above results, it was confirmed that the optimal hydrolysis time is 5 hours.

[0075] 2-5. Purification of stem-derived protoplasts using a sucrose density gradient

[0076] Protoplasts derived from the stems of Japanese red pine presented difficulties during the purification process due to cell sizes of various sizes (5–60 μm) and high residue content (Fig. 5b). Therefore, to resolve these issues, a sucrose gradient purification method was used. The same sucrose was used at concentrations of 13%, 15%, and 17%, and among them, protoplasts and residues could be clearly separated in the 17% sucrose gradient (Fig. 8). After purification, the yield and viability of the stem-derived protoplasts were higher than before purification (7.4 × 10⁻¹⁰). 4 The yield relative to / g FW, 68.4%) is 7.5 X 10 4 / g FW, the survival rate was found to have increased to 81.9%. Through the above results, it was confirmed that the purification of protoplasts can be optimized using the sucrose gradient method.

[0078] 2-6. Analysis of Gene Transformation and Transcriptional Activity Using Protoplasts Derived from Red Pine

[0079] Protoplasts purified according to Examples 1-4 above were transformed using a polyethylene glycol (PEG)-mediated method. The GFP transformation rate of protoplasts derived from red pine cotyledons was 94.1% and the cell viability was 82.9%, which is similar to the transformation rate of 83.3% and the cell viability of 83.7% of Arabidopsis mesophyll protoplasts performed under the same conditions (Figs. 9a and b). Through these results, it was confirmed that PEG-mediated transformation can also be used as a method for gene transformation in pine trees.

[0080] In addition, to confirm the functionality of the protoplasts, a transient activation assay (TAA) was performed using gene-activating regulatory elements derived from Arabidopsis thaliana and Pinus densiflora. The Arabidopsis thaliana MYB46 gene (35S::AtMYB46) strongly activated the ProAtCesA4::GUS reporter within Arabidopsis thaliana protoplasts (Fig. 9c), and activated the same reporter in Pinus densiflora protoplasts, but with a relatively lower level of activation. The Pinus densiflora MYB46 gene (35S::PdeMYB46) effectively activated the ProPdeCesA7::GUS reporter within Pinus densiflora protoplasts (Fig. 9d) and also showed activation in Arabidopsis thaliana protoplasts, confirming that the transcription factor of Pinus densiflora can be compatible with the transcription factor of angiosperms under specific conditions.

[0082] Through the above examples, optimization analyses were performed on tissue pretreatment conditions, enzymatic hydrolysis conditions, washing buffer conditions, and purification processes for the isolation of pine-derived protoplasts. Additionally, genetic transformation and transcriptional activity were analyzed for the isolated protoplasts, confirming that protoplasts can be isolated from pine and verifying the optimal conditions for isolation and the ability to use the isolated protoplasts for transformation.

Claims

Claim 1 A method for isolating protoplasts derived from pine needle tissue, comprising: 1) a step of cutting the needle tissue of a pine tree; 2) a step of enzymatically treating the cut tissue; and 3) a step of washing the enzymatically treated tissue with a washing buffer. Claim 2 In paragraph 1, the above pine tree is a red pine ( Pinus densiflora A method for isolating protoplasts derived from pine needle tissue, characterized by being ). Claim 3 A method for isolating protoplasts derived from pine needle tissue, wherein, in claim 1, step 1) involves longitudinally cutting the needle tissue. Claim 4 A method for isolating protoplasts derived from pine needle tissue, wherein, in claim 1, the enzyme comprises 2 to 6% (w / v) hemicellulase, 3 to 9% (w / v) cellulase, and 0.5 to 0.9% (w / v) pectinase. Claim 5 A method for isolating protoplasts derived from pine needle tissue, wherein, in claim 1, step 2) involves treating the cut tissue with an enzyme and hydrolyzing it for 4 to 6 hours. Claim 6 A method for isolating protoplasts derived from pine mesophyll tissue according to claim 1, wherein the washing buffer consists of 154 mM NaCl, 125 mM CaCl, 25 mM KCl, 2 mM MES, and 5 mM glucose. Claim 7 A method for isolating protoplasts derived from pine stem tissue, comprising: 1) a step of cutting the pine stem tissue; 2) a step of washing the cut tissue; 3) a step of enzymatically treating the washed tissue; and 4) a step of performing sucrose gradient centrifugation. Claim 8 A method for isolating protoplasts derived from pine stem tissue, wherein, in claim 7, the enzyme comprises 2 to 6% (w / v) hemicellulase, 3 to 9% (w / v) cellulase, and 0.5 to 0.9% (w / v) pectinase. Claim 9 A method for isolating protoplasts derived from pine stem tissue, wherein, in claim 7, step 3) involves treating the cut tissue with an enzyme and hydrolyzing it for 4 to 6 hours. Claim 10 A method for isolating protoplasts derived from pine stem tissue, wherein, in claim 7, step 4) above is to isolate protoplasts using a sucrose gradient in the concentration range of 13 to 17% (w / v). Claim 11 Protoplast separated by the method of paragraph 1 or 7. Claim 12 A transformation method comprising: 1) a step of isolating a protoplast according to the method of claim 1 or 7; and 2) a step of temporarily introducing an exogenous gene into the protoplast.

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