A high-efficiency method for separating protoplasts based on a pear suspension cell line without centrifugal purification

Abstract

The application discloses a high-efficiency protoplast separation method based on a specific state pear suspension cell line and without centrifugal purification. The method is first cut into a physiological window period of an enzymolysis starting material, uses the logarithmic growth period suspension cell of the third day after subculture as the material, uses the transient characteristics of the thinnest cell wall and the loosest cross-linking, cooperates with 1.0% cellulase + 0.1% isolation enzyme, 0.6 mol / L mannitol and the like to optimize an enzymolysis solution, and mildly enzymolyzes for only one hour under the condition of 26 DEG C darkness. Then, the method directly replaces all centrifugal washing steps by using a 40 μm cell screen single-step filtration, and high-purity protoplasts are obtained. The method improves the enzymolysis efficiency by more than 12 times, the protoplast yield is as high as 7.06*10 7 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 6*10 g FW, is improved by one order of magnitude compared to the prior art, the activity is more than 95%, the recovery rate is more than 96%, time is greatly shortened, the yield and the activity are simultaneously broken through, the centrifugal mechanical damage is fundamentally eliminated, and the process is extremely simple.

Description

Technical Field

[0001] This invention belongs to the field of plant protoplast isolation and purification technology, specifically relating to a highly efficient protoplast isolation method based on pear suspension cell lines that does not require centrifugation purification. Background Technology

[0002] Protoplasts are indispensable materials for modern biological technologies such as transient transformation, CRISPR gene editing, and cell fusion. As a globally important fruit tree, the efficient preparation of protoplasts from pears has always faced significant challenges. Existing publicly available techniques, such as the method reported by Zhuang Mengdi et al. (2023), which uses anther callus suspension enzymatic hydrolysis for 12 hours to obtain protoplasts, yield only 3.67 × 10⁻⁶ protoplasts. 6 The concentration is low (e.g., cells / mL), requiring repeated centrifugation and washing for purification. This situation exposes three major technical bottlenecks: First, the enzymatic hydrolysis time is too long. Enzyme treatment lasting up to 12 hours exposes cells to a prolonged environment of hyperosmolar stress and enzyme protein toxicity, leading to plasma membrane lipid peroxidation and membrane receptor protein degradation, which severely impairs protoplast viability and developmental totipotency.

[0003] Second, production is low, remaining at the order of 10. 6 This cannot meet the needs of high-throughput screening and multi-omics research.

[0004] Third, centrifugation purification causes significant damage. The existing standard procedure requires repeating the process of "adding W5 solution → centrifugation → discarding supernatant → resuspending" more than three times. The shear force generated by each centrifugation (120~160 g) can cause 5%~10% of protoplasts to break down, and the cumulative loss after three rounds is as high as 15%~30%, which is the main human factor causing low protoplast yield and insufficient viability.

[0005] The underlying cause of these bottlenecks lies in the failure to recognize that the uniformity of the cell wall structure and the physiological window period of the starting material are fundamental prerequisites for shortening the enzymatic digestion time and simplifying the purification process. If the cell wall thickness of the starting cell population is uneven and the proportion of senescent cells is high, it is necessary to rely on extending the enzymatic digestion time and using high-intensity centrifugation to accommodate these factors, leading to a vicious cycle.

[0006] Therefore, there is an urgent need to develop a systematic separation scheme that can match short-time enzymatic hydrolysis and centrifugation-free purification from the source of materials. Summary of the Invention

[0007] The purpose of this invention is to provide a highly efficient method for separating protoplasts based on suspended cells during a specific window period without the need for centrifugation purification. This method completely overturns the traditional paradigm of "long-term enzymatic digestion + multiple rounds of centrifugation" and achieves a synergistic leap in yield and viability through "golden window cells + short-term targeted enzymatic digestion + one-step filtration".

[0008] This invention is achieved through the following technical solution: A highly efficient method for separating protoplasts based on a pear suspension cell line without centrifugation purification is provided, the method comprising the following steps: (1) In the ultra-clear workbench, take the pear suspension cell line that has been subcultured, filter and collect the cells, mix the suspension with the sterile enzyme digest in a sterile culture dish, and carry out the enzymatic digestion in a shaker. The enzymatic hydrolysate comprises: 1.0% cellulase R-10, 0.1% analyte R-10, 20 mmol / L MES at pH 5.8, 0.3–0.6 mol / L mannitol, 20 mmol / L KCl, 10 mmol / L CaCl2, and 0.1% bovine serum albumin; the suspension cell line is a pear suspension cell line taken on day 3 after subculture. (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is directly filtered through a cell sieve; the filtrate is the protoplast suspension.

[0009] Further, (1) the enzymatic hydrolysate includes 1.0% cellulase R-10, 0.1% dissociative enzyme R-10, 20 mmol / L MES at pH 5.8, 0.6 mol / L mannitol, 20 mmol / L KCl, 10 mmol / L CaCl2 and 0.1% bovine serum albumin.

[0010] Furthermore, the filtration described in (1) is performed using a sterile sieve with a pore size of 200 mesh; Preferably, the sterile sieve is sterilized at 116°C under high temperature and high pressure for 30 minutes.

[0011] Furthermore, the sterile enzymatic hydrolysate described in (1) is obtained by filtering the enzymatic hydrolysate through a 0.22 μm needle-type water filter.

[0012] Furthermore, the enzymatic hydrolysis conditions described in (1) are: enzymatic hydrolysis at 40 rpm under dark conditions at 26°C.

[0013] Furthermore, the enzymatic hydrolysis time described in (1) is 1 hour.

[0014] Furthermore, (2) the cell sieve pore size is 40 μm.

[0015] Furthermore, (2) the cell sieve is a sterile nylon cell sieve.

[0016] The beneficial effects of this invention are as follows: (1) Precise sampling of suspension cells during the "golden window" of enzymatic hydrolysis Based on the established stable suspension cell line, suspension cells were strictly collected on day 3 after subculturing as starting material. At this stage, the cells are in the logarithmic growth phase, with the highest mitotic index. The primary cell walls are dominated by hemicellulose and low-esterified pectin, allowing enzyme molecules to easily penetrate the cell wall network for cleavage. Simultaneously, intracellular metabolites are abundant, and the membrane system remains intact. Cells in the apoptotic phase, 10 days after subculturing, exhibit strong resistance to enzymatic digestion due to thickened cell walls and lignin deposition. During collection, the suspension cell solution was rapidly filtered through a 200-mesh stainless steel sieve to remove as much residual culture medium as possible.

[0017] (2) Precise preparation of high-efficiency and low-loss enzymatic hydrolysate The enzymatic hydrolysate system specifically designed for thin-walled suspension cells has the following exact formulation: Cell wall degrading enzymes: 1.0% (w / v) cellulase R-10 (Yalult, Japan, Cellulase R-10) + 0.1% (w / v) analyte R-10 (Yalult, Japan, Macerozyme R-10). This low concentration of analyte is sufficient to degrade pectin in the intercellular middle lamella. The cellulase targets and breaks down the fibrous network without causing excessive hydrolysis of plasma membrane proteins.

[0018] Osmotic pressure regulator: 0.6 mol / L mannitol. Systematic gradient experiments from 0.3 to 0.6 mol / L confirmed that this concentration precisely matches the intracellular osmotic potential of suspended cells in the logarithmic growth phase, maintaining complete isotonicity of the plasma membrane and preventing protoplast rupture or shrinkage.

[0019] Membrane stabilizing aid: 20 mmol / L MES (pH 5.8) as a buffer system; 20 mmol / L KCl and 10 mmol / L CaCl2 provide K⁺ and Ca. 2+ This enhances transmembrane electrostatic balance and lipid bilayer stability; 0.1% bovine serum albumin (BSA) acts as a protectant to scavenge impurities and free radicals.

[0020] The enzymatic hydrolysate was sterilized by filtration using a 0.22 μm needle-type aqueous filter, with aseptic operation throughout the process.

[0021] (3) Extremely short-time mild enzymatic hydrolysis treatment The cells collected in step (1) were mixed with the enzyme digest from step (2) and transferred to a sterile culture dish. The enzyme digestion was carried out for only 1 hour under the conditions of 26°C, complete darkness, and low-speed shaking at 40 rpm. Low-speed shaking ensured uniform interaction between the enzyme and substrate, and protection from light prevented photo-oxidative damage. The short treatment time maximized the preservation of protoplasmic membrane receptor function and metabolic activity.

[0022] (4) One-step filtration completely eliminates the need for centrifugation purification After enzymatic digestion, the mixture is poured directly onto a sterile nylon cell sieve with a pore size of 40 μm without any centrifugation, relying on gravity for natural filtration. Undigested large cell clusters, tissue fragments, and vascular debris (size > 50 μm) are retained on the sieve surface, while highly viable spherical protoplasts (approximately 20-35 μm in diameter) pass through with the filtrate. The resulting filtrate is a high-purity protoplast suspension that can be directly used for subsequent experiments. This filtration step replaces the traditional three centrifugations, fundamentally eliminating the mechanical breakage caused by the centrifugal gravity field. The total protoplast recovery rate is increased to over 96%, and the purification operation time is drastically reduced from over 30 minutes to approximately 2 minutes.

[0023] (5) Synergistic verification of yield and vitality Using a hemocytometer, this method yields up to 7.06 × 10⁻⁶ protoplasts per gram of fresh weight of suspended cells. 7 The yield is approximately 20 times higher than existing technologies; FDA fluorescence staining analysis showed that the proportion of viable protoplasts exhibiting bright green fluorescence remained consistently above 95%. Under bright-field microscopy, the protoplasts were spherical and intact with clear cytoplasm, demonstrating excellent plasma membrane integrity and metabolic activity. This synergistic effect of "12-fold reduction in time, 20-fold increase in yield, over 95% viability, and purification simplified to a single-step filtration" was previously unforeseen in the field.

[0024] This method increases enzymatic hydrolysis efficiency by more than 12 times, with protoplast yield reaching 7.06 × 10⁻⁶. 7 The yield is increased by an order of magnitude compared to existing technologies, with a viability of over 95% and a recovery rate of >96%. This breakthrough achieves a significant reduction in processing time while simultaneously improving yield and viability. It fundamentally eliminates mechanical damage from centrifugation, and the process is extremely simple. The high-quality protoplasts obtained can be directly applied to research in genetic transformation, gene editing, and single-cell sequencing. Attached Figure Description

[0025] Figure 1 This is a comparison diagram of the effects of suspension systems with different generation days on protoplast yield in embodiments of the present invention, wherein: A shows the yield of protoplasts separated from the suspension system 10 days after generation. B shows the protoplast yield of the suspension system after 3 days of subculturing. This confirms that day 3 is the optimal window period.

[0026] Figure 2 This is a comparison diagram of the effect of different osmotic pressures on protoplast separation in embodiments of the present invention, wherein: A shows the morphology and yield of protoplasts after enzymatic hydrolysis and filtration under 0.3M osmotic pressure; B shows the morphology and yield of protoplasts after enzymatic hydrolysis and filtration under 0.4M osmotic pressure; C represents the protoplast morphology and yield after filtration at 0.5M osmotic pressure; D represents the protoplast morphology and yield after filtration at 0.6M osmotic pressure.

[0027] Figure 3 This is a diagram illustrating the effect of the one-step filtration and purification process of the present invention, wherein: A represents the mixture after enzymatic hydrolysis, while B and C represent the yield and purity of protoplasts after filtration through a 40 μm sieve under 10× and 20× objectives.

[0028] Figure 4 For the FDA staining viability determination of the isolated protoplasts, the following is included: A is a bright-field plot of protoplast yield and viability. B is a fluorescence field image of protoplast yield and viability after FDA staining. The bright field and fluorescence field images show a bright green viability signal of over 95%. Detailed Implementation

[0029] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0030] Example 1: Subculture of suspension systems: Suspension cultures with good condition and high activity were selected for subculture. The culture medium was MS basal medium supplemented with 1.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg / L 6-benzylaminopurine (6-BA), 30 g / L sucrose, and pH 5.8.

[0031] Example 2

[0032] (1) Separation of suspended protoplasts: The suspension cell line was taken on day 3 after subculture (at which time the cells are in the logarithmic growth phase and have the strongest vitality) and filtered through a 200-mesh sterile steel sieve in a clean bench.

[0033] (2) Preparation of enzymatic hydrolysate: 1.0% cellulase R-10 + 0.1% analyte R-10 + 20 mmol / L MES (2-morpholinoethanesulfonic acid, pH 5.8) + 0.6 mol / L mannitol + 20 mmol / L KCl + 10 mmol / L CaCl2 + 0.1% bovine serum albumin (BSA) were diluted with sterile water and filtered and sterilized through a 0.22 μm needle-type water filter.

[0034] The collected cells were mixed with the enzyme digest and transferred to sterile culture dishes (60 mm in diameter). Enzymatic digestion was carried out at 40 rpm for 1 hour under dark conditions at 26°C with shaking.

[0035] After enzymatic hydrolysis, the hydrolysate was directly filtered through a 40 μm sterile nylon cell sieve. Undigested cell clusters and large cell debris were retained on the sieve, while protoplasts passed through with the filtrate. The filtrate was a high-purity protoplast suspension, requiring no further centrifugation or purification. Figure 3 ).

[0036] Example 3: Determination of protoplast yield and viability: I. Setting up existing technology Zhuang Mengdi, Li Tao, Xin Qianyi, et al. (2023). Establishment and application of pear blossom callus induction and suspension culture system. Journal of Fruit Science, 40(03), 577-587. Control group, the specific treatment method is as follows: 1. Callus induction Using anthers of 'Xinli No. 7' cultivar as explants, tissue samples were collected at the compact bud stage (2.7–3.0 mm transverse diameter, 8.0–8.5 mm longitudinal diameter). Callus induction was performed on 1 / 2 MS basal medium supplemented with 1.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.4 mg / L naphthaleneacetic acid (NAA), 30 g / L sucrose, and 7 g / L agar, and cultured in the dark at 24±1℃ for 30 days. After 4–5 subcultures, yellowish-white, granular callus tissue was obtained.

[0037] 2. Establishment of suspension cell lines Relatively healthy callus tissue was selected and inoculated into liquid culture medium consisting of MS basal medium supplemented with 1.5 mg / L 2,4-D, 1.0 mg / L 6-benzylaminopurine (6-BA), and 30 g / L sucrose. Culture conditions were 28℃, 200 r / min shaking, and complete darkness. After 4–5 subcultures in suspension, a suspension cell line was established.

[0038] 3. Protoplast isolation Using suspension cell lines as starting material, protoplasts were isolated by enzymatic digestion. The digestion buffer consisted of 1.0% cellulase R-10 + 0.5% sorbase R-10 + 20 mmol / L MES (pH 5.7) + 0.5 mol / L mannitol + 20 mmol / L KCl + 10 mmol / L CaCl2. Digestion was carried out at 25°C in the dark with shaking at 30 rpm for 12 hours. The digestion product was diluted with an equal volume of W5 solution, centrifuged at 120×g for 4 minutes, and the supernatant was discarded. This centrifugation and washing step was repeated three times to collect and purify the protoplasts.

[0039] 4. Separation Results After 12 hours of enzymatic hydrolysis and three cycles of centrifugation purification, the protoplast yield was 3.67 × 10⁻⁶. 6 Cells / mL, with an activity of 92.00%.

[0040] II. The yield and viability of the protoplasts obtained in Example 2 were compared with those obtained from the control group in the prior art, as follows: Protoplast yield was determined using a hemocytometer. Filtered protoplasts were resuspended in liquid culture medium, gently aspirated with a dropper, and then placed onto the hemocytometer. A coverslip was placed on the spherical surface, and the protoplasts were counted under an inverted microscope. Counts were performed three times, and the average value was taken. The protoplast yield per gram of suspension was calculated based on the amount of enzyme solution added to the suspension.

[0041] Viability was determined using the FDA (fluorescein diacetate) staining method: 1 μL of FDA stock solution (5 mg / mL, dissolved in DMSO) was diluted in 1 mL of liquid culture medium. The diluted FDA staining solution was used to stain protoplasts, which were then incubated at room temperature in the dark for 5 min. Observation was performed under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 515 nm). Protoplasts emitting green fluorescence were considered viable. The proportion of viable protoplasts to the total number of protoplasts was the viability. Results are as follows: Figure 4 As shown.

[0042] Depend on Figure 3 Figure 4 It can be seen that, under the conditions of an enzymatic hydrolysis time of only 1 hour and only one filtration purification, the method of the present invention achieves a protoplast yield of 7.06 × 10⁻⁶. 7 pcs / g FW ( Figure 3 It is approximately 20 times more potent than existing technologies; its viability reaches 95%, a significant improvement over the 92% of existing technologies. FDA staining results ( Figure 4 The images clearly show that the vast majority of protoplasts exhibit bright green fluorescence, confirming their extremely high activity.

[0043] Example 4: Effect of different enzyme hydrolysate concentrations on protoplast isolation This embodiment investigates the effect of different enzyme hydrolysate concentrations on the separation effect of suspended protoplasts.

[0044] Using the established stable suspension cell lines as materials, suspension cells were collected on the 3rd day after subculture and enzymatically digested for 1 hour according to the steps in Example 2 with different enzyme digestion solutions. Protoplasts were then isolated and observed. Each treatment was repeated in 3 replicates.

[0045] The formulation of the enzymatic hydrolysate is shown in Table 1. The enzymatic hydrolysate is diluted with sterile pure water and sterilized by filtration through a 0.22μm needle-type water filter.

[0046] Table 1 1 0.5 0.1 <![CDATA[4.51×10 7 ±0.46b]]> 85.67±1.15c 2 1.0 0.1 <![CDATA[7.06×10 7 ±0.63a]]> 94.90±2.72a 3 1.0 0.2 <![CDATA[7.10×10 7 ±0.84a]]> 88.78±1.07b As shown in Table 1, the protoplast yield and activity reached their highest values ​​when the cellulase concentration was 1.0% and the cleavage enzyme concentration was 0.1%. When the cellulase concentration was reduced, both the protoplast yield and activity decreased. When the cellulase concentration remained constant and the cleavage enzyme concentration was increased, the protoplast yield increased slightly, but the protoplast activity decreased. Therefore, the amount of enzyme added is not necessarily better the higher it is; rather, it is necessary to comprehensively consider both yield and activity and formulate a reasonable solution. The optimal concentration of pear suspension enzymatic hydrolysate is 1.0% cellulase and 0.1% cleavage enzyme.

[0047] Example 5: Effect of different number of generations on protoplast segregation This embodiment examines the effect of different passage numbers of suspended cells on the protoplast separation effect.

[0048] Using the established stable suspension cell line as material, suspension cells were collected on day 3 and day 10 after subculture, respectively. Following the steps in Example 2, the cells were enzymatically digested for 1 hour. The digested mixture was then filtered directly through a 40 μm sterile nylon cell sieve for protoplast separation and observation. Each treatment was performed in triplicate. The results are as follows: Figure 1 As shown.

[0049] Depend on Figure 1 It was found that the suspension cells on day 3 after subculturing entered the logarithmic growth phase, with rapid cell division, thin cell walls, abundant contents, and active metabolism, making it an ideal time for enzymatic hydrolysis. After day 10, they entered the apoptosis phase, cell viability decreased, and the enzymatic hydrolysis effect significantly worsened. This result indicates that accurately selecting cells in the logarithmic growth phase on day 3 after subculturing is one of the key factors in achieving high yield and high viability of protoplasts.

[0050] Example 6: Effect of different osmotic pressures on protoplast separation This example investigates the effect of different mannitol concentrations (osmotic pressure conditions) on protoplast separation.

[0051] Using cells from the stable suspension cell line established in Example 1, dated day 3 after subculturing, mannitol concentration gradients of 0.3, 0.4, 0.5, and 0.6 mol / L were set. Other components of the enzymatic hydrolysate remained unchanged. Following the steps in Example 2, the enzymatic hydrolysate was digested for 1 hour. The resulting mixture was diluted with liquid culture medium and then filtered directly through a 40 μm sterile nylon cell sieve for protoplast morphology and yield observation. The results are as follows... Figure 2 As shown, protoplasts can be isolated at mannitol concentrations ranging from 0.3 to 0.6 M, but the protoplasts exhibit the most regular shape and highest yield at a mannitol concentration of 0.6 mol / L. Figure 2 (D in the text). Therefore, 0.6 mol / L mannitol is the optimal concentration for osmotic pressure stabilizing.

[0052] As can be seen from the above examples, this invention establishes a complete and efficient method for separating pear suspension protoplasts. This invention has the following original contributions: First, based on a stable and highly active suspension material, the enzymatic hydrolysis time is only 1 hour, 12 times shorter than existing technologies; Second, one-step filtration replaces multiple centrifugation purifications, achieving a protoplast recovery rate of over 96%, fundamentally solving the problem of centrifugation loss; Third, it achieves a dual breakthrough in yield and viability, with a yield of 7.06 × 10⁻⁶. 7 With a cell count / g FW and a viability of 95%, this invention achieves a synergistic technological advantage of "shorter time, doubled yield, high viability, and simplified purification." This invention provides an efficient and reliable technical platform for large-scale protoplast manipulation and molecular breeding of pear plants. By closely integrating the quality control of suspension cell lines with the protoplast isolation process, this invention creatively simplifies and efficiencies the separation of pear protoplasts, providing a solid platform for large-scale molecular manipulation of pear plants.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for protoplast isolation based on a pear suspension cell line without centrifugation purification, characterized in that, The method includes the following steps: (1) In an ultra-high-definition workbench, the subcultured pear suspension cell line was taken, the cells were collected by filtration, and the suspension was mixed with sterile enzymatic hydrolysate in a sterile culture dish and subjected to enzymatic hydrolysis by shaking in a shaker; the enzymatic hydrolysate included: 1.0% cellulase R-10, 0.1% analyte R-10, 20 mmol / L MES at pH 5.8, 0.6 mol / L mannitol, 20 mmol / L KCl, 10 mmol / L CaCl2 and 0.1% bovine serum albumin; the suspension cell line was the pear suspension cell line taken on the 3rd day after subculture; the enzymatic hydrolysis time was 1 hour; (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is directly filtered through a cell sieve with a pore size of 40 μm; the filtrate is a pure protoplast suspension.

2. The protoplast separation method according to claim 1, characterized in that, The filtration described in (1) is performed using a sterile sieve with a pore size of 200 mesh.

3. The protoplast separation method according to claim 2, characterized in that, The sterile sieve is sterilized at 116°C under high temperature and pressure for 30 minutes.

4. The protoplast separation method according to claim 1, characterized in that, (1) The sterile enzymatic hydrolysate is obtained by filtering the enzymatic hydrolysate through a 0.22 μm needle-type water filter.

5. The protoplast separation method according to claim 1, characterized in that, (1) The enzymatic hydrolysis conditions described are: enzymatic hydrolysis at 40 rpm under dark conditions at 26°C.

6. The protoplast separation method according to claim 1, characterized in that, (2) The cell sieve mentioned is a sterile nylon cell sieve.

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