A method for preparing and transiently genetically transforming pinus massoniana protoplasts based on needles and young stems

Abstract

The application discloses a kind of based on needle and tender stem's Pinus massoniana protoplast preparation and its transient genetic transformation method, belong to plant cell biotechnology field.The application is by optimizing enzyme solution formula, using the enzyme solution containing 1%-3% cellulase and 0.1%-0.5% pectinase is handled Pinus massoniana seedling needle leaf or tender stem explant, successfully obtain high yield, high activity protoplast.Based on this, the application further establishes PEG-mediated transient transformation system: needle leaf protoplast is incubated in 30%PEG, 0.4M mannitol, 100mM CaCl2 Condition is incubated for 15 minutes, and transformation effect is best;And tender stem protoplast is incubated in 15%PEG, 0.6M mannitol, 50mM CaCl2 Condition is incubated for 45 minutes, and reach the highest genetic transformation efficiency.This application provides a kind of efficient, stable and with tissue adaptation's Pinus massoniana gene function research technical scheme.

Description

Technical Field

[0001] This invention relates to the field of plant cell biotechnology, specifically to a method for preparing pine protoplasts based on needles and young stems and their transient genetic transformation. Background Technology

[0002] Masson pine (Pinus massoniana Lamb.) is an important timber and ecological tree species in southern my country, possessing extremely high economic and ecological value. With the development of molecular biology, gene function research and molecular breeding of Masson pine have become key approaches to improving its traits. However, this species is difficult to root under in vitro culture conditions, limiting the progress of its functional gene research. Furthermore, genetic transformation research on Masson pine, and even most coniferous species, is not yet mature, severely restricting the development of functional genomics and the improvement of breeding efficiency.

[0003] Plant protoplasts, or naked plant cells with their cell walls removed, are ideal receptor systems for studying transient gene expression, subcellular protein localization, promoter activity analysis, and protein-protein interactions. Their greatest advantage lies in their ability to rapidly and efficiently validate gene function, making them particularly suitable for woody species like Masson pine, which have complex genetic backgrounds and where establishing stable genetic transformation systems is difficult. Currently, PEG (polyethylene glycol)-mediated transient protoplast transformation has become a widely used standard method in many plants due to its simplicity, low cost, and high efficiency.

[0004] In coniferous species, existing research has attempted to establish protoplast transformation systems. Existing technologies (e.g., Chinese patent application CN109055295A) disclose a method for the isolation and transient transformation of protoplasts from Masson pine needles. This method uses seedling needles as material, employs a specific combination of enzymatic hydrolysates (containing cellulase and pectinase) to separate protoplasts, and achieves transformation of needle protoplasts with an efficiency of approximately 60% by optimizing PEG concentration and plasmid dosage. This technology provides a valuable approach for gene function research in Masson pine.

[0005] However, the existing technology system still has obvious limitations. First, its technical solution relies entirely on needle leaves as the only explant material, without involving other potential sources of high-efficiency materials. It ignores the inherent differences that protoplasts from different tissue sources may have in terms of cell wall composition, physiological state, and membrane stability. This may result in protoplasts from other tissues (such as young stems) failing to achieve optimal transformation efficiency or even failing to transform, thus limiting the versatility and robustness of the technology platform. Summary of the Invention

[0006] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by this application is to provide a method for preparing Masson pine protoplasts based on needles and young stems and their transient genetic transformation.

[0007] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows:

[0008] A highly efficient method for preparing pine protoplasts includes the following steps:

[0009] Tissue from Masson pine seedlings was obtained as explants; the explants were placed in an enzymatic hydrolysate containing cellulase and pectinase and enzymatically hydrolyzed in the dark; after enzymatic hydrolysis, protoplasts were obtained by purification; wherein the enzymatic hydrolysate contained 1%-3% cellulase and 0.1%-0.5% pectinase.

[0010] In some embodiments, the method for cultivating Masson pine seedlings is as follows: sowing Masson pine seeds in nutrient soil, performing photoculture with a photoperiod of 16 h light culture / 8 h dark culture, maintaining a constant temperature of 25℃ and a relative humidity of 60%, and cultivating for 10-15 days.

[0011] In some embodiments, the basic components of the enzymatic hydrolysate are: 0.6 M mannitol, 20 mM MES, 50 mM KCl, 10 mM CaCl2 and 0.1% (w / v) BSA.

[0012] In some embodiments, the explant is a needle, and the enzymatic hydrolysate contains 2%-3% cellulase and 0.125%-0.25% pectinase, with a hydrolysis time of 3-5 hours.

[0013] In some embodiments, the explant is a young stem, and the enzymatic hydrolysate contains 1% cellulase and 0.125%-0.5% pectinase, with a hydrolysis time of 3-4 hours.

[0014] The protoplasts of *Pinus massoniana* prepared by any of the efficient preparation methods described above are *Pinus massoniana* protoplasts.

[0015] A method for PEG-mediated transient genetic transformation of Masson pine protoplasts includes the following steps: mixing a plasmid containing exogenous nucleic acid with the Masson pine protoplasts; adding a PEG solution and incubating at room temperature to complete the transformation; wherein the concentration of the PEG solution is 15%-30%, and it contains 0.4-0.6 M mannitol and 50-100 mM CaCl2.

[0016] In some embodiments, the protoplasts are derived from needles, the polyPEG solution has a concentration of 30%, wherein the mannitol concentration is 0.4 M, the CaCl2 concentration is 100 mM, and the incubation time is 15 minutes.

[0017] In some embodiments, the protoplasts are derived from young stems, the PEG solution has a concentration of 15%, wherein the mannitol concentration is 0.6 M, the CaCl2 concentration is 50 mM, and the incubation time is 45 minutes.

[0018] The application of the protoplasts of young shoots of Pinus massoniana in plant gene function research or genetic transformation.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention represents a breakthrough in overcoming the technological limitation of relying solely on a single source of protoplasts from Masson pine, successfully developing a highly efficient preparation process using young leaves and stems as explants. Compared to traditional needle-based materials, young stems are easier to handle in experimental procedures. Furthermore, by optimizing the enzymatic hydrolysis ratio, this invention achieves new highs in both protoplast yield and viability. This not only provides researchers with crucial freedom in material selection but also significantly expands the recipient cell pool available for gene function research.

[0021] Meanwhile, this application's technical solution utilizes the fundamental differences in physiological characteristics of protoplasts from different tissue sources to establish a new transformation paradigm of "tissue-specific condition adaptation." Experiments have shown that needle and young stem protoplasts have drastically different requirements for PEG concentration, calcium ion environment, and osmotic pressure. Therefore, this invention has developed optimal transformation schemes for both, significantly increasing the transformation efficiency of young stem protoplasts to nearly 60%, thereby improving the reliability and reproducibility of the transformation system.

[0022] In summary, this invention not only provides a new method for preparing high-quality protoplasts, but also offers a technical solution for researchers to flexibly select materials according to experimental needs, thus providing stable and reliable technical support for gene function analysis, molecular breeding, and secondary metabolism research of Masson pine. Attached Figure Description

[0023] Figure 1-1 Figure 1 shows the multi-factor optimization results for protoplast isolation from *Pinus massoniana*; ad: different effects of enzymatic hydrolysis time, seedling stage, and enzymatic hydrolysate on the yield and vigor of needle protoplasts; ef: different effects of enzymatic hydrolysis time and seedling stage on the yield and vigor of young stem protoplasts; (protoplast g) -1 FW mL -1 Vitality (%). Error = ±SE.

[0024] Figure 1-2 Figure showing the multi-factor optimization results for protoplast isolation from *Pinus massoniana*; gh: different effects of enzymatic hydrolysate on the yield and vigor of protoplasts from young stems; il: comprehensive extraction efficiency and yield (protoplasts g) of 16 combined treatments. -1 FW mL -1 ), vitality (%); error = ±SE.

[0025] Figure 2 Procedure for protoplast isolation and viability assessment of *Pinus massoniana*; ad: Phenotypes of seedlings at 10, 15, 20, and 25 days after germination. e, f: Dissected needles and stems of 10-day-old seedlings. g, h: Tissue filaments soaked in enzymatic hydrolysate. i, j: Mixture after enzymatic hydrolysis. k, l: Protoplast suspension filtered from needles and stems. mp: FDA-stained protoplasts 24 hours after isolation (10x magnification). m, o: Bright field microscopy; n, p: Fluorescence (FITC channel). Scale bar: 1 cm (al); 100 µm (m-p);

[0026] Figure 3 Optimization of the transient transformation system for *Pinus massoniana* protoplasts. Optimization factors: (a) PEG concentration, (b) plasmid DNA concentration, (c) protoplast density, (d) incubation time, (e) mannitol concentration, (f) Ca 2+ Concentration. Derived from protoplasts of needles and young stems. Error: mean ± standard deviation;

[0027] Figure 4 Transient expression of GFP in pine protoplasts; (A, B) magnified images of GFP-expressing needle protoplasts at 10x (A) and 63x (B); (C, D) magnified images of GFP-expressing young stem protoplasts at 10x (C) and 63x (D); protoplasts transformed with an empty GFP vector (without GFP insertion) served as a negative control; scale bar: 100 μm (A, C); 20 μm (B, D);

[0028] Figure 5 Subcellular localization of PeWOX5 and PmbHLH44 in Pinus massoniana protoplasts; (A, B) confocal microscopy images, GFP fluorescence localization of transfected protoplasts: empty GFP vector (negative control), PeWOX5-GFP fusion, and PmbHLH44-GFP fusion; scale bar = 20 μm (A, B). Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] Example 1

[0031] 1. Method

[0032] 1.1 Plant materials

[0033] The Masson pine seeds used in this application were collected from the Wuyi State-owned Forest Farm. The seeds were sown in nutrient soil and cultured under the following conditions: 16 h photoperiod / 8 h dark period, constant temperature 25℃, and relative humidity 60%. After 10-25 days of culture, the needles and tender stems of the seedlings were collected for protoplast isolation and purification. Before the experiment, all materials were washed with soapy water to remove any soil residue, and then soaked in sterile water for later use.

[0034] 1.2 Protoplast Extraction

[0035] The enzymatic hydrolysate used in this application consisted of 0.6 M mannitol, 20 mM MES, 50 mM KCl, 10 mM CaCl2, 0.1% BSA, and cellulase, pectinase, and R10 complex enzyme. To systematically evaluate the effects of explant type, seedling development stage, hydrolysis time, and enzyme formulation on protoplast yield and viability, 16 treatment combinations were designed (see Table 1). Needles and young stems were longitudinally cut into strips approximately 1.0 mm wide to enhance enzyme penetration efficiency, and then immersed in the hydrolysate for 3-5 hours at 28°C in the dark. After hydrolysis, the reaction was terminated by adding an equal volume of pre-cooled W5 solution, which consisted of 2 mM MES (pH 5.7), 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl. The enzymatically digested mixture was filtered through a cell sieve, and the filtrate was collected and centrifuged at 85 g for 5 minutes at 4°C. The supernatant was discarded, and the protoplasts were resuspended in 1–2 mL of W5 solution. The protoplast concentration was calculated using a hemocytometer, and the yield was expressed as "protoplasts / g fresh weight". The viability was determined by staining with 0.01% FDA and calculating the proportion of fluorescent cells. All experiments included at least three biological replicates.

[0036] 1.3 Carrier Construction

[0037] To verify the effectiveness of the transient expression system, this application used a p2FGW plasmid with a GFP tag as the basic reporter vector. Two constructs for subcellular localization contained the exogenous gene PeWOX5 and the endogenous gene PmbHLH44 (GenBank: MT989428.1), respectively. The PeWOX5–GFP vector was driven by a cauliflower mosaic virus (CaMV) 35S dual promoter and carried an ampicillin resistance marker (Liu et al., 2018). Specific amplification of PmbHLH44 was performed using F:5'-ATGGGAGCCGAAACGGCA-3' and R:5'-TTAACCAGAAATTGCAGCAACTAATTTTTCT-3'. The amplified products were cloned into the PGC-AMP-GFP-N vector using the Nimble Cloning method, with the GFP tag located at the N-terminus of the insert fragment. All recombinant plasmids were confirmed by Sanger sequencing to ensure the correctness and orientation of the inserted sequences.

[0038] 1.4 PEG-mediated transient conversion

[0039] Both needle and young stem protoplasts were transformed using the p2FGW empty vector carrying the GFP reporter gene for efficiency evaluation. Plasmids were extracted and purified using the TIANGEN TIANprep Mini Plasmid Kit II. During transformation, 5–15 μg of plasmid DNA was added to 100 μL of a 10⁻⁶ m² / L solution. 4 -10 6 A protoplast suspension of 1 protoplasts per mL was added, followed by an equal volume (approximately 110 μL) of 15–45% PEG4000 solution, and gently mixed to ensure homogeneity. The mixture was incubated at 25°C for 15–45 minutes, and then 2.5 volumes of W5 solution were added to terminate the reaction. The mixture was then centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the protoplasts were resuspended in 100 μL of WI solution (containing 4 mM MES, 0.4 M mannitol, and 20 mM KCl) and incubated at room temperature in the dark for 18 hours. GFP fluorescence signals were observed and imaged using an Olympus BX51 fluorescence microscope.

[0040] 2 Results

[0041] 2.1 Optimization of Novel Enzyme Formulation for Protoplast Isolation from Pinus massoniana

[0042] To establish an optimized isolation system for *Pinus massoniana* protoplasts, this application systematically evaluated the tissue type, seedling stage, enzymatic hydrolysis time, and hydrolysate of 16 experimental treatments (Table 1). The protoplast yield and viability under each condition were compared and evaluated, revealing significant differences in the effects of all variables. Figure 1-1 , 1-2 ).

[0043] This application analyzes four different seedling development stages ( Figure 2 The effects on individual development were determined. Ten-day-old seedlings yielded the highest protoplast production in needles, while the yield in young stems did not show a significant difference with seedling stage. The optimal enzymatic digestion time for both tissues was determined to be 3–4 hours, achieving peak yield-vitality balance. Figure 1-1 a,f, Figure 1-2 i). Extended culture time (>4 hours) induced a balance between yield and viability: Although 5 hours of enzymatic hydrolysis increased yield, protoplast viability extracted from needles (protoplast viability (%) = (number of viable protoplasts / total number of protoplasts) × 100%) decreased to 70.78% (treatment 6) and viability extracted from young stems decreased to 67.44% (treatment 13). Needles from 10-day-old seedlings showed superior performance (treatment 1: 34.5 × 10⁻⁶). 6 g -1 FW, with a viability of 87.2%, yield gradually decreases as the seedlings mature. Figure 1-1 b). Conversely, the yield of protoplasts in young stems remains stable at all developmental stages ( Figure 1-2 g), indicating that needles are the best tissue for extracting protoplasts from Masson pine.

[0044] In the optimized system established in this application, more than 10 kJ of 10-day-old needles can be obtained by enzymatic hydrolysis (2% cellulase and 0.125% pectinase) over 3 hours. 7 g -1 The protoplasts from FW tissue exhibited a viability exceeding 90%. Young stems, when enzymatically hydrolyzed for 4 hours with 1% cellulase and 0.125% pectinase, also achieved similar viability. FDA staining showed that the protoplasts from both tissues maintained a viability above 90% within 24 hours of isolation.

[0045] Table 1. Multifactor optimization of protoplast separation efficiency from Pinus massoniana

[0046]

[0047] 2.2 Key factors affecting the protoplast transformation efficiency of Pinus massoniana

[0048] This application modifies six key factors affecting the transient transformation efficiency mediated by PEG, including PEG concentration, plasmid DNA amount, protoplast density, incubation time, mannitol concentration, and Ca2+ concentration. 2+ concentration( Figure 3 (af). Although the PEG-mediated transformation mechanism is universal in plants, the protoplasts of pine needles and young stems show significant differences under optimal conditions.

[0049] PEG concentration has a significant impact on conversion efficiency. Needle protoplasts showed the highest efficiency (66%) at 30% PEG, while young stems achieved the highest efficiency (57%) at 15% PEG. Figure 3 a). Increasing the amount of plasmid can improve transformation efficiency, but in needle protoplasts, the efficiency begins to decline when the amount exceeds 10 μg, while in young stems, the efficiency still shows an increasing trend at 15 μg. Figure 3 c). Protoplast density also has a crucial impact on transformation efficiency; for both explant types, at 1×10⁻⁶, the density at which protoplasts are most effective is significantly higher. 5 The highest efficiency was achieved at a density of [number] cells / mL (needles: 50%; stems: 68%). Higher densities decreased efficiency (P<0.05), possibly due to cell aggregation or resource competition. Figure 3 c). Incubation time exhibited a tissue-specific response: needle protoplasts peaked at 15 minutes (67%), then gradually declined, indicating time sensitivity, while stem protoplasts required 45 minutes to reach maximum efficiency (58%). Figure 3 d).

[0050] The effect of incubation time showed tissue-specific differences: needle protoplasts showed the highest transformation efficiency after 15 minutes of incubation, while young stems required 45 minutes to reach peak efficiency. Mannitol and Ca... 2+ Concentrations exhibited different regulatory effects on stabilizing osmotic pressure and maintaining protoplast membrane stability. Needle protoplasts showed different effects at concentrations of 0.4 M mannitol (57%) and 100 mM Ca2+. 2+ (62%) conditions showed higher efficiency, while young stem protoplasts showed higher efficiency under 0.6 M mannitol (53%) and 50 mM CaO conditions. 2+ (60%) conditions show the best performance. Figure 3 e,f).

[0051] Fluorescence microscopy revealed that the needle and young stem protoplasts transformed under optimized conditions exhibited strong GFP fluorescence signals with extremely low background noise, verifying the robustness and efficiency of the system. Figure 4 ).

[0052] 2.3 Subcellular localization verification of pine protoplasts

[0053] After optimizing protoplast transformation conditions, the subcellular localization of the green fluorescent protein fusion protein of the exogenous gene PeWOX5 and the endogenous gene PmbHLH44 was determined, with transfection using a 35S::GFP empty vector as a control. Confocal microscopy showed that both fusion proteins specifically aggregated in the cell nucleus. Figure 5 ).

[0054] 2.4 Preparation of Masson pine protoplasts and conditions for genetic transformation

[0055] Comparative analysis revealed that pectinase concentration had a more significant impact on protoplast yield and separation efficiency compared to cellulase. This stronger dependence on pectinase has not been widely reported in other species and may reflect the uniqueness of the cell wall composition or intercellular matrix structure of *Pinus massoniana*. This finding has potential reference value for optimizing protoplast separation methods in other coniferous species.

[0056] Although extending the enzymatic hydrolysis time helps to further break down the cell wall and release more protoplasts, viability assays showed that hydrolysis exceeding 5 hours led to a significant decrease in protoplast activity and more pronounced cell damage. This indicates that prolonged enzymatic hydrolysis can have cytotoxic effects, potentially disrupting plasma membrane integrity and reducing protoplast viability. The physiological state of the explant plays a decisive role in the yield and quality of protoplasts. In this example, the highest protoplast yield was observed in 10-day-old Pinus massoniana seedlings, followed by a gradual decline in both yield and viability as the seedlings matured. Under the same enzymatic hydrolysis conditions, the protoplast yield extracted from needles was approximately twice that from young stems. However, young stem tissue is easier to process during enzymatic hydrolysis. Therefore, selecting suitable explants between needles and young stems is of great importance for different experimental purposes.

[0057] Microscopic observations revealed that needle protoplasts were relatively homogeneous in morphology and size, while stem protoplasts exhibited significant morphological heterogeneity. This morphological difference may lead to inconsistent responses to changes in osmotic pressure under uniform transformation conditions, thus reducing overall transformation efficiency.

[0058] While increasing PEG concentration generally promotes DNA uptake, excessively high concentrations can induce cytotoxicity, causing protoplast rupture and reducing transformation efficiency. In Masson pine, needle protoplasts exhibit typical concentration dependence, while young stem protoplasts show a distinctly different response pattern. Furthermore, young stem protoplasts may possess higher physiological heterogeneity and environmental sensitivity, leading to unstable transformation responses. In this application, Ca... 2+ Concentration has little effect on the transformation efficiency of needle protoplasts, but in young stem protoplasts, Ca concentration significantly increases. 2+ Increased concentration significantly reduces conversion efficiency, further confirming its weak membrane stability.

[0059] Furthermore, this application found that protoplasts with lower chloroplast content are more susceptible to genetic transformation; given that protoplasts derived from needles typically contain more chloroplasts, this may pose an additional limitation on PEG-mediated introduction of exogenous genes. Therefore, compared to needle-derived protoplasts, protoplasts derived from young stems exhibit a higher potential advantage in transformation due to their lower chloroplast content. In summary, in applications requiring high morphological consistency and culture stability of protoplasts, needle-derived protoplasts are more suitable; while in applications where the primary goal is to improve the efficiency of PEG-mediated genetic transformation, especially in transient expression or gene function verification, protoplasts derived from young stems are more advantageous.

[0060] The gradient experiments in this application verified that a higher plasmid dosage can improve transformation efficiency, but excessive protoplast density can lead to cell aggregation, thereby reducing transformation efficiency.

[0061] To verify the practicality of this system, needle protoplasts were selected as the transformation material, and subcellular localization analysis was performed using the GFP fusion protein of the exogenous gene PeWOX5 and the endogenous gene PmbHLH44. Confocal microscopy showed that both proteins specifically accumulated in the cell nucleus. These results collectively demonstrate that the protoplast transformation system established in this application is suitable for gene function studies of *Pinus massoniana*.

[0062] In summary, this application optimized the protoplast isolation and PEG-mediated transient transformation of *Pinus massoniana*, establishing an efficient, stable, and reproducible system that significantly improves the efficiency of gene function research in this tree species. This system not only provides a key technological platform for the molecular genetic improvement of *Pinus massoniana*, but also offers important reference for constructing functional gene research systems for other coniferous species that are difficult to genetically transform.

[0063] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A highly efficient method for preparing pine protoplasts, characterized in that, Includes the following steps: Tissue from Masson pine seedlings was obtained as explants; the explants were placed in an enzymatic hydrolysate containing cellulase and pectinase and enzymatically hydrolyzed in the dark; after enzymatic hydrolysis, protoplasts were obtained by purification; wherein the enzymatic hydrolysate contained 1%-3% cellulase and 0.1%-0.5% pectinase.

2. The method according to claim 1, characterized in that, The cultivation method for Masson pine seedlings is as follows: sow Masson pine seeds in nutrient soil, conduct photoculture for 16 hours of light culture and 8 hours of dark culture, maintain a constant temperature of 25℃ and a relative humidity of 60%, and cultivate for 10-15 days.

3. The method according to claim 1, characterized in that, The basic components of the enzymatic hydrolysate are: 0.6 M mannitol, 20 mM MES, 50 mM KCl, 10 mM CaCl2 and 0.1% (w / v) BSA.

4. The method according to claim 1, characterized in that, The explant is a needle, and the enzymatic hydrolysate contains 2%-3% cellulase and 0.125%-0.25% pectinase, with a hydrolysis time of 3-5 hours.

5. The method according to claim 1, characterized in that, The explant is a young stem, and the enzymatic hydrolysate contains 1% cellulase and 0.125%-0.5% pectinase, with a hydrolysis time of 3-4 hours.

6. The *Pinus massoniana* protoplasts prepared by the efficient preparation method according to any one of claims 1-5.

7. A method for PEG-mediated transient genetic transformation of pine protoplasts, characterized in that, Includes the following steps: The plasmid containing exogenous nucleic acid is mixed with the pine protoplasts described in claim 6; Add PEG solution and incubate at room temperature to complete the conversion; the concentration of the PEG solution is 15%-30%, and it contains 0.4-0.6 M mannitol and 50-100 mM CaCl2.

8. The method according to claim 7, characterized in that: The protoplasts were derived from needles, and the polyPEG solution had a concentration of 30%, including 0.4 M mannitol and 100 mM CaCl2, with an incubation time of 15 minutes.

9. The method according to claim 7, characterized in that: The protoplasts were derived from young stems, and the PEG solution had a concentration of 15%, including 0.6 M mannitol and 50 mM CaCl2, with an incubation time of 45 minutes.

10. The application of the pine shoot protoplasts as described in claim 6 in plant gene function research or genetic transformation.

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