High-yield and high-activity pear protoplast extraction method based on light shielding and enzymolysis optimization

Abstract

The invention discloses a high-yield and high-activity pear protoplast extraction method based on light shielding and enzymolysis optimization, which comprises the following steps: by taking pear tissue culture seedling leaves growing for 30-40 days or field pear tender leaves as an extraction material, carrying out enzymolysis treatment and protoplast purification by adopting enzymatic hydrolysate to obtain protoplast; before enzymolysis of the pear tissue culture seedlings, the whole pear tissue culture seedlings are placed in a dark environment and subjected to dark pretreatment at 25 + / -2 DEG C for 48-72 hours. According to the method, the yield of the field pear leaf and tissue culture seedling pear leaf protoplast can be increased by more than or equal to 2 times (the highest protoplast yield is greater than or equal to 3.0 * 10 / g FW), the activity is increased to be greater than or equal to 95%, and the technical bottleneck of the existing patent is remarkably broken through. The method has the advantages of high yield, high activity, wide application range and the like, can provide a high-quality protoplast material for pear genetic regeneration, gene editing, single cell transcriptome analysis and metabonomics research, and has wide scientific research and industrial application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant cell engineering, and particularly relates to a high-yield and high-activity extraction method of pear protoplast based on light avoidance and enzymatic optimization. BACKGROUND

[0002] Plant protoplast isolation technology is an important basic tool for cell engineering and gene function research, and its quality directly affects the downstream applications such as protoplast transient transformation efficiency, single cell sequencing data reliability and somatic hybridization. In Rosaceae woody fruit trees, pear is faced with three technical bottlenecks in protoplast isolation due to its unique leaf anatomical structure and secondary metabolic characteristics: ① Physical barrier stubbornness: the dense cutin layer on the surface of pear leaf epidermis (significantly thicker than that of apple, peach and other related species) hinders the penetration of the enzymatic solution; ② Cell wall resistance: the cell wall of mature leaves shows high lignification characteristics, and the density of the cellulose-hemicellulose cross-linked network is much higher than that of herbaceous plants; ③ Metabolic interference effect: the pear leaves are rich in polyphenol oxidase and tannins, which easily cause browning reaction during enzymatic process, resulting in damage to the protoplast membrane system.

[0003] The existing technology mostly uses the enzymatic system of herbaceous plants (such as Arabidopsis and tobacco), which is not fully adapted to the unique cell structure of pear, resulting in difficulty in meeting the subsequent research needs in terms of yield and activity, and seriously restricting the progress of pear protoplast in CRISPR / Cas9 gene editing and single cell map construction and other frontier researches. SUMMARY

[0004] The purpose of the present application is to provide a high-yield and high-activity extraction method of pear protoplast based on light avoidance and enzymatic optimization.

[0005] To achieve the above-mentioned purpose, the following technical solutions are adopted in the present application:

[0006] In a first aspect, the present application claims a method for extracting pear protoplast, which uses the leaves of pear tissue culture seedlings or tender leaves of field pear grown for 30-40 days as extraction materials, and adopts enzymatic solution for enzymatic treatment and protoplast purification to obtain protoplast;

[0007] Before the enzymatic treatment of the pear tissue culture seedlings, the whole plant is placed in a dark environment and subjected to light avoidance pretreatment at 25±2℃ for 48-72 hours;

[0008] The tender leaves of field pear do not need to be subjected to whole plant light avoidance pretreatment, and can be directly collected, put into a sampling bag containing a certain humidity, and taken back to the laboratory for direct enzymatic treatment.

[0009] The formula of the enzymatic solution is: 1.0%-1.5% (w / v, g / 100ml) cellulase, 0.2%-0.6% (w / v, g / 100ml) macerozyme, 0.4-0.6M mannitol, 1%-2% (w / v, g / 100ml) PVP-40, 2-4mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% (w / v, g / 100ml) BSA, 10mM CaCl2, dissolved with deionized water and constant volume; the mass ratio of the cellulase and macerozyme is 1:0.15-0.5.

[0010] Preferably: 1.0%-1.5% cellulase, 0.2%-0.6% macerozyme, 0.4-0.6M mannitol, 1% PVP-40, 2mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% BSA, 10mM CaCl2, dissolved with deionized water and constant volume; the mass ratio of the cellulase and macerozyme is 1:0.2-0.4;

[0011] Most preferably: 1.0% cellulase, 0.4% macerozyme, 0.5M mannitol, 1% PVP-40, 2mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% BSA, 10mM CaCl2, dissolved with deionized water and constant volume.

[0012] Further, the cellulase is Cellulase R-10, and the macerozyme is Macerozyme R-10.

[0013] Further, the process of the enzymatic treatment is: collecting the leaves of pear tissue culture seedlings or field pear young leaves, cutting them into pieces, and then immersing them in the enzymatic solution for 7-11h of enzymatic treatment.

[0014] Further, the leaves of pear tissue culture seedlings are subjected to enzymatic treatment at 28℃, 30 rpm, for 7-10h; and the field pear young leaves are subjected to enzymatic treatment at 28℃, 20 rpm, for 8-11h.

[0015] Further, the leaves of pear tissue culture seedlings or field pear young leaves are cut into 0.5-1mm filaments.

[0016] Further, the process of purifying the protoplasts is:

[0017] (1) The enzymatic solution obtained after the enzymatic treatment is filtered through a 40μm nylon mesh, and the filtrate is collected, centrifuged at 600 rpm, 25℃, a=3, for 10min, and the protoplasts are precipitated, and the supernatant is discarded;

[0018] (2) Slowly add W5 solution along the wall of the tube to resuspend the protoplasts, 600 rpm, 25℃, a=3, centrifuge for 5 min, wash the protoplasts, discard the supernatant;

[0019] (3) Slowly add MMg solution along the wall of the tube to resuspend the protoplasts, 600 rpm, 25℃, a=3, centrifuge for 5 min, facilitate subsequent transformation, discard the supernatant;

[0020] (4) Again, slowly add MMg solution along the wall of the tube to resuspend the protoplasts, count (count and observe fluorescence under Axioscope 5 upright fluorescence microscope).

[0021] In a second aspect, the present application claims to protect an enzymatic solution for extracting pear protoplasts from field leaves and tissue culture seedling leaves, the formula of the enzymatic solution is: 1.0%-1.5% (w / v, g / 100ml) cellulase, 0.2%-0.6% (w / v, g / 100ml) macerase, 0.4-0.6M mannitol, 1%-2% (w / v, g / 100ml) PVP-40, 2-4mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% (w / v, g / 100ml) BSA, 10mM CaCl2, dissolved with deionized water and constant volume; the mass ratio of the cellulase and macerase is 1:0.15-0.5. The preferred formula of the enzymatic solution and the specific cellulase and macerase are as described above.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] Compared with the prior art, the present application can achieve ≥2-fold increase in the yield of protoplasts from field pear leaves and tissue culture seedling pear leaves (the maximum protoplast yield is ≥3.0×10 6 protoplasts per g FW) and the activity is increased to ≥95%, which significantly breaks through the technical bottleneck of the prior art. The method has the advantages of high yield, high activity, wide application range, etc., and can provide high-quality protoplast materials for pear genetic regeneration, gene editing, single-cell transcriptome analysis and metabolomics research, and has wide application potential in scientific research and industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Morphological observation (bright field) and activity detection (fluorescence field) results (activity detected by FDA) of protoplasts extracted from field Korla pear leaves under the formula of pear-specific enzymatic solution. Among them, A is the morphological observation result, and B is the activity detection result.

[0025] Figure 2Morphological observation (bright field) and activity detection (fluorescence field) results of protoplasts extracted from pear seedlings under pear-specific enzyme solution formula (light shielding treatment has been carried out, and activity is detected by FDA).

[0026] Figure 3 Bar chart of the effect of different enzyme concentrations on the yield of leaf protoplasts of Korla pear in the field.

[0027] Figure 4 Bar chart of the effect of different enzyme concentrations on the yield of protoplasts of tissue culture autumn pear seedlings (all combinations are subjected to light shielding treatment).

[0028] Figure 5 Comparison chart of the effect of light shielding treatment on the yield of protoplasts of tissue culture autumn pear seedlings. A is normal light, and B is light shielding pretreatment.

[0029] Figure 6 Comparison chart of the effect of PVP+Vc on the activity of protoplasts of tissue culture autumn pear seedlings. A is the non-addition group, and B is the addition group; both A and B have been subjected to light shielding treatment. DETAILED DESCRIPTION

[0030] The application will be further described below in conjunction with specific examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0031] Example 1: A pear protoplast high-yield high-activity extraction method based on light shielding and enzyme optimization

[0032] I. Solution preparation

[0033] 1. Preparation of mother liquor (stored at -4℃)

[0034] Prepare sterilized ultrapure water, measuring cylinder, 0.45um water filter head, 50ml sterile enzyme-free centrifuge tube.

[0035] 1M MES: weigh 10.66g of MES (213.25g / mol) and add to 50ml sterilized ultrapure water, adjust pH to 5.7 with KOH.

[0036] 1M CaCl2: weigh 5.55g of calcium chloride (110.98 g / mol) and add to 50ml sterilized ultrapure water.

[0037] 1 M MgCl2: Weigh 10.16 g of MgCl2 hexahydrate (203.30 g / mol) into 50 ml of sterile ultrapure water.

[0038] 200 mM KCl: Weigh 0.7455 g of Potassium chbride (74.55 g / mol) into 50 ml of sterile ultrapure water.

[0039] 2. Preparation of enzyme solution:

[0040] Formulation 1: 1.0% (w / v, g / 100 ml) Cellulase R-10, 0.2% (w / v, g / 100 ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100 ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100 ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0041] Formulation 2: 1.0% (w / v, g / 100 ml) Cellulase R-10, 0.4% (w / v, g / 100 ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100 ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100 ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0042] Formulation 3: 1.0% (w / v, g / 100 ml) Cellulase R-10, 0.6% (w / v, g / 100 ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100 ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100 ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0043] Formulation 4: 1.5% (w / v, g / 100 ml) Cellulase R-10, 0.2% (w / v, g / 100 ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100 ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100 ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0044] Formula 5: 1.5% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0045] Formula 6: 1.5% (w / v, g / 100ml) Cellulase R-10, 0.6% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0046] Formula 7: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.2% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0047] Formula 8: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0048] Formulation 9: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.6% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0049] Formulation 10: 1.0% (w / v, g / 100ml) Cellulase R-10, 0.2% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0050] Formulation 11: 1.0% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0051] Formulation 12: 1.0% (w / v, g / 100ml) Cellulase R-10, 0.6% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0052] Formulation 13: 1.5% (w / v, g / 100ml) Cellulase R-10, 0.2% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M Mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM Ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0053] Formulation 14: 1.5% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M Mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM Ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0054] Formulation 15: 1.5% (w / v, g / 100ml) Cellulase R-10, 0.6% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M Mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM Ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0055] Formulation 16: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.2% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M Mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM Ascorbic acid, 20 mM KC1, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and made up to volume.

[0056] Formula 17: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and constant volume.

[0057] Formula 18: 2.0% (w / v, g / 100ml) Cellulase R-10, 0.6% (w / v, g / 100ml) Macerozyme R-10, 0.5% Pectolase Y-23, 0.5 M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and constant volume.

[0058] Formula 19: 1.0% (w / v, g / 100ml) Cellulase R-10, 0.4% (w / v, g / 100ml) Macerozyme R-10, 0.5 M mannitol, 20 mM KCl, 20 mM MES, pH 5.7, 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and constant volume.

[0059] 3. W5 wash solution configuration

[0060] 0.15 M NaCl 、 10 mM CaCl2, 20 mM KCl, 20 mM MES (pH 5.7).

[0061] 4. MMg buffer solution configuration

[0062] 0.5 M D-mannitol, 1 M MgCl2, 20 mM MES (pH 5.7).

[0063] II. Protoplast extraction procedure

[0064] 1. Preparation of extraction material and enzymatic treatment:

[0065] Leaf samples were collected from 30-day-old in vitro seedlings or from field-grown pear seedlings. The field-grown leaves were collected from the top of the seedlings and washed with water. The in vitro seedlings were grown in the dark for 48 hours at 25 ± 2°C.

[0066] About 0.5 g of the leaf was cut with a scalpel (a new blade was used) to remove the tip of the leaf and a 0.5-1 mm filament was quickly cut and placed in a pre-prepared 10 ml enzyme solution (the total amount was adjusted to cover all the leaves). The filament was completely immersed in the enzyme solution using a flat forceps. The Petri dish was wrapped in tin foil to maintain a dark environment and placed in a 28°C shaker for digestion. The field-grown leaves were shaken at 20 rpm for 10 hours and the in vitro seedlings were shaken at 30 rpm for 8.5 hours.

[0067] 2. Protoplast purification

[0068] (1) The enzyme-digested protoplast solution was filtered through a 40 um nylon mesh into a glass dish. The enzyme-digested solution was slowly transferred from the glass dish to a 10 ml round-bottom centrifuge tube using a 2 ml W5 solution to rinse the dish once. The residual leaves were slightly squeezed and the nylon mesh was rinsed once with an additional 1 ml W5 solution. Centrifugation was performed at 600 rpm, 25°C, a = 3 for 10 minutes, and the supernatant was carefully removed.

[0069] (2) The protoplasts were resuspended by slowly adding 1 ml of W5 solution along the wall of the centrifuge tube, gently shaking, and mixing with 2 ml of W5 solution. Centrifugation was performed at 600 rpm, 25°C, a = 3 for 5 minutes, and the supernatant was discarded.

[0070] (3) The protoplasts were resuspended by slowly adding 1 ml of MMg solution along the wall of the centrifuge tube, gently shaking, and mixing with 2 ml of MMg solution. Centrifugation was performed at 600 rpm, 25°C, a = 3 for 5 minutes, and the supernatant was discarded.

[0071] (4) The protoplasts were resuspended with a certain volume of MMg solution, gently shaken, and the tube was rotated horizontally for counting.

[0072] Three, protoplast yield and activity detection

[0073] 1. Cell counting plate counting

[0074] Take 10 uL protoplast suspension drop on 0.1 mm hemocytometer, stand for 1-2 min, observe under optical microscope (10x100), calculate the number of protoplast in 4 corners and the center of the large grid (each large grid has 16 small grids, record the upper line and not the lower line, count the left and not the right), calculate the total number of protoplast according to the formula, repeat 3 times for each sample, finally calculate the yield of protoplast per gram of material (number / g·FW).

[0075] Protoplast number (number / mL) = total number of protoplast in 5 large grids x 5 x 1000 x dilution factor

[0076] Protoplast yield (number / g·FW) = [protoplast number (number / mL) x volume after dilution (mL)] / leaf mass (g)

[0077] 2, FDA staining method to detect protoplast viability

[0078] Take 100 μL protoplast suspension, add 1 μL 10 mg / mL fluorescein diacetate FDA, stand for 1-2 min, take 10 μL and observe with upright fluorescence microscope (Axioscope 5, Germany Carl Zeiss), count the number of green fluorescent protoplast and the total number of protoplast. Protoplast viability is expressed as the percentage of viable protoplast in a field of view to the total number of protoplast in the field of view, randomly select 3 fields for statistics.

[0079] Protoplast viability = (number of green fluorescent protoplast / total number of protoplast) x 100%

[0080] Four, optimization of enzyme solution formula and effect verification

[0081] 1, use the enzyme solution of formula 1-18 to extract pear protoplast (take field Korla pear leaf protoplast and tissue culture autumn pear seedling protoplast as an example, all tissue culture autumn pear seedlings are pretreated in the dark before extraction). The experimental results show that, Figure 3 and 4 , by exploring different enzyme concentrations, under the condition of not adding pectinase, the yield of field Korla pear leaf protoplast and tissue culture autumn pear seedling protoplast extracted by enzyme solution of formula 2 is best ≥3x10 6 number / g·FW, the extraction effect is the best. In addition, the effect of formula 1, formula 5 and formula 6 is second (all ≥2x10 6 number / g·FW), among them, the yield of formula 1 to extract field Korla pear leaf protoplast and formula 5 to extract tissue culture autumn pear seedling protoplast is close to 3x10 60.5M mannitol, 1% (w / v, g / 100ml) PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES (pH 5.7), 1% (w / v, g / 100ml) BSA, 10 mM CaCl2, dissolved with deionized water and constant volume) is the best enzymatic hydrolysis solution formula, and the extraction of protoplasts from the leaves of Korla pear in the field and the tissue culture autumn pear seedlings (which have been subjected to light shielding treatment) is carried out using the enzymatic hydrolysis solution of the best formula (formula 2), and the morphology (bright field) and activity (fluorescence field) of the extracted protoplasts are observed and detected. The detection results are shown in Figure 3 and 4 In the above-mentioned formula, the specific formula number is represented by the concentration of Cellulase R-10 and Macerozyme R-10 in each formula, for example, 1.0% + 0.2% represents formula 1, and the like.

[0082] 2. The extraction of protoplasts from the leaves of Korla pear in the field and the tissue culture autumn pear seedlings (which have been subjected to light shielding treatment) is carried out using the enzymatic hydrolysis solution of the best formula (formula 2), and the morphology (bright field) and activity (fluorescence field) of the extracted protoplasts are observed and detected. The detection results are shown in Figure 1 and Figure 2 The protoplasts extracted from the leaves of Korla pear in the field and the tissue culture autumn pear seedlings using the enzymatic hydrolysis solution of formula 2 all achieve high yield (3 x 10 6 protoplasts extracted from the leaves of Korla pear in the field and the tissue culture autumn pear seedlings using the enzymatic hydrolysis solution of formula 2 all achieve high yield (3 x 10

[0083] 3. The extraction of protoplasts from the tissue culture autumn pear seedlings is carried out using the enzymatic hydrolysis solution of formula 2 and formula 19 (without the addition of PVP and Vc), as shown in Figure 6 The activity of the extracted protoplasts shows that the activity of the protoplasts extracted using the enzymatic hydrolysis solution of formula 19 (without the addition of PVP and Vc) is significantly lower than that of the protoplasts extracted using the enzymatic hydrolysis solution of formula 2 (≥95%).

[0084] 4. Two treatments are carried out on the tissue culture autumn pear seedlings:

[0085] Treatment A: The tissue culture autumn pear seedlings grown for 30 days are used as the extraction material, and the tissue culture autumn pear seedlings grown for 30 days are placed in a dark environment together with the culture container for pretreatment at 25±2°C for 48 hours, and then the extraction of protoplasts is carried out using the enzymatic hydrolysis solution of formula 2 and the protoplast extraction procedure in step two.

[0086] Treatment B: The tissue culture autumn pear seedlings are not subjected to light shielding pretreatment, and the other processes are the same as those of treatment A.

[0087] As shown in Figure 5As shown, even with the optimal enzymatic hydrolysate formulation, the yield of pear tissue culture seedlings without light-protection pretreatment was 8 × 10⁻⁶. 5 The number of cells / g·FW was also significantly lower than that of the light-shielding pretreatment group (3×10). 6 (units / g·FW).

[0088] Existing methods for preparing pear (Pyrus spp.) protoplasts mostly focus on adjusting single parameters, such as changing the enzyme species or enzyme concentration, enzymatic hydrolysis time, and temperature. [1-2] However, pear tissue is rich in phenols and other secondary metabolites, which easily undergo oxidative browning and generate reactive oxygen species during enzymatic hydrolysis. This leads to protein cross-linking and cell membrane damage, reducing protoplast viability and limiting yield and subsequent applications. Therefore, a systematic approach is needed that simultaneously inhibits phenolic oxidation and synergistically optimizes the enzymatic hydrolysis formulation and operating conditions to improve protoplast yield and survival rate while ensuring their division and regeneration capabilities. This would provide a reliable material basis for downstream research such as transient transformation, stable genetic transformation, gene editing, and single-cell sequencing.

[0089] [1] Nanjing Agricultural University. A method for isolating pear stem protoplasts: 202210246470.0[P]. 2023-07-04.

[0090] [2] Gao Chengyu, Wang Yiheng, Jin Jiangzhou, et al. Establishment of a method for preparing pear leaf protoplasts and its transient gene transformation experiment [J]. Journal of Horticulture, 2023, 50(05):1141-1150. DOI:10.16420 / j.issn.0513-353x.2022-0264.

[0091] This invention develops a high-yield, high-activity extraction method for pear protoplasts based on optimized light-shielding and enzymatic hydrolysis, achieving both high yield and high activity. This invention also proposes for the first time a two-stage strategy of "whole-plant light-shielding pretreatment + synergistic optimization of pear-specific compound enzymatic hydrolysate formulation."

[0092] (1) Breakthrough in pretreatment technology: Whole-plant dark pretreatment (pear tissue culture seedlings and culture containers are placed in a dark environment at 25±2℃ for 48-72 hours) induces cell wall relaxation and reduces damage to protoplasts by intracellular phenolic substances, thereby improving protoplast release efficiency. This pretreatment reduces cell wall resistance and thickness without damaging the plant, is simple and easy to operate, and allows leaves to release more and more complete protoplasts in a shorter time using a pear-specific compound enzymatic hydrolysate. This method achieves high yields (≥3×10⁻⁶) of protoplasts extracted from pear tissue culture seedlings and pear field leaves for the first time. 6 The simultaneous achievement of high activity (≥95%) and high activity (cFU / g FW) is a challenge that often exists in existing technologies.

[0093] (2) Innovative enzyme solution formula: a special enzyme solution formula suitable for pear protoplast extraction is developed, a complex enzyme solution containing 1.0%-1.5% cellulase, 0.2%-0.6% isolated enzyme, PVP-40 and ascorbic acid is used, and under the condition of mild shaking, the cell wall is degraded for 7-11 hours, and the balance between high-efficiency degradation and protection of cell activity is achieved.

[0094] The pear protoplast high-efficiency extraction method of the present application realizes the high-yield and high-activity synchronous extraction of pear (Pyrus spp.) leaf protoplasts in the field and in tissue culture by integrating the light-free pretreatment of whole plant tissue culture seedlings and the dynamic optimization of the complex enzyme solution system, and breaks through the limitation that the yield and activity are difficult to be compatible in the prior art.

[0095] The above-mentioned embodiments are only preferred embodiments of the present application, and are used to explain the present application, but not to limit the scope of the present application. For those skilled in the art, other embodiments can be easily obtained by substitution or change based on the technical content disclosed in the present application, and therefore, any changes and improvements made on the principle of the present application shall be included in the scope of the patent application of the present application.

Claims

1. A method for extracting pear protoplasts, characterized in that, Using leaves from pear tissue culture seedlings that have grown for 30-40 days or young pear leaves from the field as extraction materials, protoplasts were obtained by enzymatic hydrolysis and purification using an enzymatic hydrolysate. Before enzymatic hydrolysis, the pear tissue culture seedlings were placed in a dark environment and pretreated at 25±2℃ for 48-72 hours in the dark. The enzymatic hydrolysate is formulated as follows: 1.0%-1.5% cellulase, 0.2%-0.6% analyte, 0.4-0.6M mannitol, 1%-2% PVP-40, 2-4mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% BSA, and 10mM CaCl2. The mass ratio of cellulase to analyte is 1:0.15-0.

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2. The extraction method according to claim 1, characterized in that, The enzymatic hydrolysis process is as follows: collect leaves from pear tissue culture seedlings or young pear leaves from the field, chop them up, soak them in the enzymatic hydrolysis solution, and shake to hydrolyze for 7-11 hours.

3. The extraction method according to claim 2, characterized in that, Pear tissue culture seedling leaves were enzymatically hydrolyzed at 28℃ and 30 rpm for 7-10 h; pear tender leaves from the field were enzymatically hydrolyzed at 28℃ and 20 rpm for 8-11 h.

4. The extraction method according to claim 2, characterized in that, Pear tissue culture seedling leaves or young pear leaves from the field are chopped into 0.5-1 mm fine shreds.

5. The extraction method according to claim 1, characterized in that, The protoplast purification process is as follows: (1) The enzymatic hydrolysis solution obtained after enzymatic hydrolysis was filtered through a 40μm nylon mesh and the filtrate was collected. The solution was centrifuged at 600 rpm, 25℃, a=3 for 10 min to precipitate protoplasts and the supernatant was discarded. (2) Slowly add W5 solution along the tube wall to resuspend the protoplasts, centrifuge at 600 rpm, 25℃, a=3 for 5 min, wash the protoplasts and discard the supernatant; (3) Slowly add MMG solution along the tube wall to resuspend the protoplasts, centrifuge at 600 rpm, 25℃, a=3 for 5 min to facilitate subsequent transformation, and discard the supernatant; (4) Add MMG solution slowly along the tube wall again to resuspend the protoplasts and count them.

6. The extraction method according to claim 1, characterized in that, The formulation of the enzymatic hydrolysate is as follows: 1.0%-1.5% cellulase, 0.2%-0.6% analyte, 0.4-0.6M mannitol, 1% PVP-40, 2mM ascorbic acid, 20mM KCl, 20mM MMES, pH 5.7, 1% BSA, and 10mM CaCl2, wherein the mass ratio of cellulase to analyte is 1:0.2-0.

4. The optimal composition is: 1.0% cellulase, 0.4% analyte, 0.5 M mannitol, 1% PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES, pH 5.7, 1% BSA, and 10 mM CaCl2.

7. The extraction method according to claim 1 or 6, characterized in that, The cellulase is Cellulase R-10, and the analyte is Macerozyme R-10.

8. An enzymatic hydrolysate for extracting pear protoplasts from field leaves and tissue-cultured seedling leaves, characterized in that, The formulation of this enzymatic hydrolysate is as follows: 1.0%-1.5% cellulase, 0.2%-0.6% cleavage enzyme, 0.4-0.6M mannitol, 1%-2% PVP-40, 2-4mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% BSA, and 10mM CaCl2. The mass ratio of cellulase to cleavage enzyme is 1:0.15-0.

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9. The enzymatic hydrolysate according to claim 8, characterized in that, The enzymatic hydrolysate is formulated as follows: 1.0%-1.5% cellulase, 0.2%-0.6% cleavage enzyme, 0.4-0.6M mannitol, 1% PVP-40, 2mM ascorbic acid, 20mM KCl, 20mM MES, pH 5.7, 1% BSA, and 10mM CaCl2, wherein the mass ratio of cellulase to cleavage enzyme is 1:0.2-0.

4. The optimal composition is: 1.0% cellulase, 0.4% analyte, 0.5 M mannitol, 1% PVP-40, 2 mM ascorbic acid, 20 mM KCl, 20 mM MES, pH 5.7, 1% BSA, and 10 mM CaCl2.

10. The enzymatic hydrolysate according to claim 8 or 9, characterized in that, The cellulase is Cellulase R-10, and the analyte is Macerozyme R-10.

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