A method for preparing and transforming different tissue protoplasts of soybean

Abstract

The application discloses a preparation and transformation method suitable for different tissue protoplasts of soybean, and belongs to the technical field of biology. The method comprises the following steps: taking leaf, stem or root tissue of 10-12 day old soil culture yellowing soybean seedlings, chopping, and then carrying out light-free enzymolysis in a mixed enzyme solution; collecting precipitated cells after filtration and centrifugation; resuspending the precipitated cells to obtain a protoplast suspension; uniformly mixing the protoplast suspension with a plasmid, and adding 50% PEG transformation liquid to carry out transformation for 15 min under room temperature. The method optimizes an enzymolysis system and key parameters of a PEG transformation liquid, and obtains a protoplast preparation and transformation method suitable for multiple tissues and multiple genotypes of soybean, so that the problems of material limitation, low enzymolysis efficiency and poor transformation efficiency in the preparation and transformation process of soybean protoplasts in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for the preparation and transformation of protoplasts from different soybean tissues. Background Technology

[0002] Protoplasts, plant cells with their cell walls removed, are crucial tools for studying gene expression, protein localization, gene editing, and cell fusion. Currently, plant protoplast preparation primarily employs enzymatic hydrolysis. The key technology lies in utilizing enzymatic hydrolysates such as cellulase and pectinase to obtain high-yield, high-activity protoplasts. PEG-mediated transient transformation has become the mainstream method for protoplast transformation due to its ease of operation, low cost, and lack of genotype limitations. As a globally important dual-purpose crop for both oil and forage, soybean requires efficient protoplast preparation and transformation methods for significant research on soybean gene function.

[0003] Invention patent CN201510597136.X (publication date 2019-03-29) discloses a method for separating protoplasts from soybean leaves for subcellular localization. Using fully expanded young leaves of 10-30 day old Xiangdou No. 3 soybean seedlings as material, the leaves are cut into 0.5-1 mm strips and placed in a CPW + 13% mannitol solution for plasmolysis in the dark for 2 hours. The strips are then transferred to an enzymatic hydrolysate containing CPW + 0.1% MES + 0.5% Onozuka R-10 cellulase + 0.8% Hemicellulase + 0.8% Macerozyme R-10 dissociative enzyme + 0.4% Pectolyase Y-23 + 10% mannitol (pH 6.0). The hydrolysate is carried out in the dark at 27℃ and 45 rpm for 6 hours. After filtration through a 75 μm nylon mesh membrane and washing with W5 solution, a yield of 1.75 × 10⁻⁶ protoplasts is obtained. 7 Protoplasts with a count / g·FW and a survival rate of 82.86% were mainly used for subcellular localization studies, but no transformation efficiency data were provided.

[0004] Invention patent CN201910480976.6 (publication date 2019-09-17) uses soybean embryogenic callus or leaves as materials, employing an enzymatic hydrolysate of 2% cellulase R-10 + 1% cleavage enzyme R-10 + 0.1% pectinase Y-23 + MES (pH 5.8), at a ratio of 0.2 g callus or leaf / 5 mL enzyme solution, enzymatically hydrolyzing at 25-26℃, 50 rpm in the dark for 5 h. The solution is then purified by filtration through a 200-mesh cell sieve and centrifugation with W5-23% solution, adjusting the protoplast density to 2.5 × 10⁻⁶. 6After achieving a protoplast density of 3.6 × 10⁶ cells / mL, the protoplasts were transformed with 30% PEG4000 at room temperature for 10 minutes, resuspended in WI solution, and cultured in the dark for 24 hours. This method significantly improved protoplast yield and transformation efficiency compared to the traditional cotyledon method, achieving a yield of 3.6 × 10⁶ cells / mL. 6 The conversion rate reached 20%, but the drawback was that the induction of embryogenic callus depended on the young soybean embryo, the induction time was long and required aseptic operation.

[0005] Invention patent CN202510958372.3 (publication date 2025-10-17) optimizes the preparation and transformation process of soybean stem protoplasts. It uses the first internode stem segment of Williams 82 sterile soybean seedlings (taken when the first pair of trifoliate leaves have just emerged) or stem segments of 10-day-old soil-grown seedlings. An enzymatic hydrolysate containing 1.75% cellulase R-10 + 0.75% analyte R-10 + 10 mM MES + 0.6M mannitol + 0.1% BSA + 1 mM CaCl2 + 5 mM β-mercaptoethanol (pH 5.7) is used. The hydrolysate is carried out at 28℃ and 80 rpm in the dark for 4 hours (no vacuum filtration required). After filtration through a 40 μm cell sieve and washing with W5 buffer, the protoplast yield reaches 2.1 × 10⁻⁶. 6 Transformation was achieved by incubating at room temperature for 15-20 minutes with 20% PEG4000 (containing 0.6 M mannitol and 1 mM CaCl2). Protein expression could be detected after incubation at 28°C in the dark or with weak light for 12-16 hours, but the conversion rate was not determined.

[0006] Although there are technologies related to the preparation and transformation of soybean protoplasts, there are obvious defects: (1) The materials and genotypes are highly limited. For example, patent CN201510597136.X is only applicable to leaves of Xiangdou No. 3, CN202510958372.3 uses Williams 82 stem segments, while CN201910480976.6 requires the induction of embryogenic callus, depends on immature embryos, has a short sampling window, and a long subculture time. (2) The protoplast yield is low. For example, the protoplast yield of patent CN201510597136.X is 1.75×10 7 The protoplast yield was 3.6 × 10⁶ cells / g·FW, but there was a discrepancy between its yield and viability, with a survival rate of only 82.86%; CN201910480976.6 had a protoplast yield of 3.6 × 10⁶ cells / g·FW. 6 The protoplast yield of CN202510958372.3 was 2.1 × 10⁻⁶ cells / mL. 6(3) Insufficient conversion efficiency, such as patents CN201510597136.X and CN201910480976.6 which do not specify the conversion rate value, and the conversion rate of CN201910480976.6 is about 20%. Therefore, it is very necessary to provide a method for the preparation of protoplasts with multiple tissues and multiple genotypes in soybeans and a high conversion rate. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and transforming protoplasts from different soybean tissues. Specifically, this invention optimizes key parameters of the enzymatic hydrolysis system and PEG conversion solution to obtain a method for preparing and transforming protoplasts from multiple tissues and genotypes of soybean, solving the problems of material limitations, low enzymatic hydrolysis efficiency, and poor transformation efficiency in existing technologies.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a method for preparing protoplasts suitable for different soybean tissues, comprising the following steps:

[0010] Leaves, stems, or roots of etiolated soybean seedlings are taken, chopped, and enzymatically hydrolyzed in a mixed enzymatic hydrolysate in the dark. After filtration and centrifugation, the precipitated cells are collected and resuspended to obtain a protoplast suspension. The mixed enzymatic hydrolysate consists of 2% cellulase, 0.1% pectinase, 1% cleavage enzyme, 0.6M mannitol, 10mM MES, 0.1% BSA, and 3.4mM CaCl2, with ddH2O as the solvent.

[0011] Preferably, the mass-to-volume ratio of the tissue to the mixed enzymatic hydrolysate is (0.1-0.4) g : 5 mL, and the enzymatic hydrolysis conditions are: first, enzymatic hydrolysis at room temperature and 60 r / min for 3-4 hours, and then enzymatic hydrolysis at 90 r / min for 5 min.

[0012] Preferably, the filtration and centrifugation conditions are as follows: after filtering the enzyme hydrolysate through a 70µm filter, the filtrate is centrifuged at 150g for 2 minutes, and the supernatant is discarded to obtain the precipitated cells.

[0013] Preferably, the resuspension is performed by: resuspending the precipitated cells in W5 solution, centrifuging at 150g for 2 minutes, discarding the supernatant, and adding MMG solution to the precipitate to resuspend the cells.

[0014] The components of the W5 solution are: MES 2mM, CaCl2 125mM, NaCl 154mM and KCl 5mM, with ddH2O as the solvent;

[0015] The MMG solution consists of: 0.4 M mannitol, 15 mM MgCl2 and 4 mM MES, with ddH2O as the solvent.

[0016] Preferably, the etiolated soybean seedlings are etiolated soybean seedlings grown in soil for 10-12 days; the varieties of the etiolated soybean seedlings include Zhonghuang 301, Heihe 43, and Chengdou 17, but are not limited to the above-mentioned soybean varieties.

[0017] This invention also provides a method for transforming soybean protoplasts, comprising the following steps:

[0018] A protoplast suspension was obtained using the aforementioned preparation method. The protoplast suspension was then mixed uniformly with plasmids, and a PEG conversion solution was added and converted at room temperature for 15 min. The PEG conversion solution consisted of 0.2 M mannitol, 100 mM CaCl2, and 50% w / v PEG4000, with ddH2O as the solvent.

[0019] Preferably, the density of the protoplast suspension is 2 × 10⁻⁶. 6 The volume ratio of the protoplast suspension, the plasmid, and the PEG conversion solution is 10:1:11.

[0020] Preferably, after adding the PEG conversion solution, the following steps are also included: terminating the conversion with 500 μL W5 solution, centrifuging, discarding the supernatant, adding 1 mL W5 solution to resuspend the cells, centrifuging again, discarding the supernatant, adding 500 μL W5 solution to resuspend the cells, and transferring to a culture plate for overnight culture for 12-18 hours.

[0021] The present invention discloses the following technical effects:

[0022] (1) This invention breaks through the limitations of materials and genotypes and develops a universal method applicable to multiple organ tissues (roots, stems and leaves) of soybean and multiple cultivars (such as Zhonghuang 301, Heihe 43 and Chengdou 17).

[0023] (2) This invention improves protoplast yield and survival rate by optimizing the enzymatic hydrolysis system.

[0024] (3) This invention clarifies the key parameters of PEG-mediated transformation (such as PEG concentration, incubation time, Ca...). 2+ (Concentration), which improves conversion efficiency. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Soybean protoplasts prepared by enzymatic hydrolysis;

[0027] Figure 2 Soybean protoplasts transformed with GFP plasmid;

[0028] Figure 3 Soybean protoplasts co-expressing GFP and mCherry. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The reagents involved in the following examples:

[0035] (1) Mixed enzymatic hydrolysate: 2% cellulase, 0.1% pectinase, 1% ionase, 0.6M mannitol, 10mM MES, dissolved in sterile water, pH adjusted to 5.7, water bath at 65℃ for 10min, filtered through a 0.22µm membrane, and after cooling to room temperature, BSA was added to a final concentration of 0.1%, and then CaCl2 was added to a final concentration of 3.4mM.

[0036] (2) W5 solution: MES 2mM, CaCl2 125mM, NaCl 154mM, KCl 5mM, dissolved in ddH2O, pH adjusted to 5.7.

[0037] (3) MMG solution: Mannitol 0.4M, MgCl2 15mM, MES 4mM, dissolved in ddH2O, and pH adjusted to 5.7.

[0038] (4) PEG conversion solution: 0.2M mannitol, 100mM CaCl2, 50% (w / v) PEG4000, dissolved in ddH2O, and in a 65℃ water bath for 10 min until completely dissolved.

[0039] Example 1: Preparation and transformation of soybean protoplasts

[0040] 1. Preparation of soybean protoplasts

[0041] (1) Take leaves, stems or roots of soybean (Zhonghuang 301) etiolated seedlings that have been grown in soil for about 10 days, cut the tissue into 0.5mm strips or thin slices with a blade, and take about 0.3g of each sample.

[0042] (2) Add the chopped sample to 5 mL of mixed enzymatic hydrolysate and enzymatically hydrolyze for about 4 hours at room temperature and 60 r / min, and then enzymatically hydrolyze for 5 min at 90 r / min. The enzymatic hydrolysis process should be carried out in the dark.

[0043] (3) Filter the enzyme hydrolysate using a 70µm filter, transfer all the filtrate into a centrifuge tube, and centrifuge at 150g for 2 minutes;

[0044] (4) Discard the supernatant, add 5 mL of W5 solution to resuspend the cells, and centrifuge at 150 g for 2 minutes;

[0045] (5) Discard the supernatant, resuspend the cells in about 500 µL of MMG solution, and examine the number and morphology of protoplasts under a microscope (see Figure 1 ).

[0046] 2. Transformation of soybean protoplasts

[0047] (1) Adjust the density of the leaf protoplast suspension to 2×10 6 Take 100µL of MMG cell suspension per mL and transfer it to a 2mL round-bottom EP tube.

[0048] (2) Add 10 µL of plasmid to the protoplast cell suspension and mix well. The plasmid concentration is 1 µg / µL. The plasmid is a plasmid with 35S-driven green fluorescent protein GFP, abbreviated as pCAMBIA-GFP; or a plasmid used to study the subcellular localization of proteins, the empty vector is a plasmid with both 35S-driven green fluorescent protein GFP and CmYLCV-driven red fluorescent protein mCherry, abbreviated as pCAMBIA-GFP-mCherry;

[0049] (3) Add 110 µL of PEG conversion buffer to the cell suspension and mix gently. Incubate at room temperature for 15 min;

[0050] (4) Add 500µL of W5 solution to terminate the conversion, and centrifuge at 150g for 2 minutes.

[0051] (5) Remove the supernatant, add 1 mL of W5 solution to resuspend the cells, and centrifuge at 150 g for 2 minutes;

[0052] (6) Remove the supernatant, resuspend the cells in 500 µL of W5 solution, transfer to a 24-well plate and incubate overnight for 12-18 hours. Observe the transformation efficiency under a microscope the next day (see...). Figure 2 ) or protein subcellular localization (see Figure 3 ).

[0053] Example 2

[0054] The difference from Example 1 is that the leaves are replaced with stems; all other steps are the same.

[0055] Example 3

[0056] The difference from Example 1 is that the leaves are replaced with roots; all other steps are the same.

[0057] Example 4

[0058] The difference from Example 1 is that the soybean variety is replaced with Heihe 43; all other methods and steps are the same.

[0059] Example 5

[0060] The difference from Example 4 is that the leaves are replaced with stems; all other steps are the same.

[0061] Example 6

[0062] The difference from Example 4 is that the leaves are replaced with roots; all other steps are the same.

[0063] Example 7

[0064] The difference from Example 1 is that the soybean variety is replaced with Chengdou 17; all other methods and steps are the same.

[0065] Example 8

[0066] The difference from Example 7 is that the leaves are replaced with stems; all other steps are the same.

[0067] Example 9

[0068] The difference from Example 7 is that the leaves are replaced with roots; all other steps are the same.

[0069] Comparative Example 1

[0070] The difference from Example 1 is that no 0.1% pectinase was added to the mixed enzymatic hydrolysate, and the concentration of PEG4000 in the PEG conversion solution was 30%; all other methods and steps were the same.

[0071] Comparative Example 2

[0072] The difference from Example 1 is that no 0.1% pectinase was added to the mixed enzymatic hydrolysate, and the concentration of PEG4000 in the PEG conversion solution was 20%; all other methods and steps were the same.

[0073] The results of the number, morphology, and conversion rate of protoplasts prepared in the above embodiments and comparative examples are shown in Table 1 below. The results show that the method of the present invention can significantly improve the conversion rate, up to 31%.

[0074] Table 1

[0075]

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing protoplasts from different tissues of soybean, characterized by, The method comprises the following steps: The leaf, stem or root tissue of the yellow soybean seedling is cut into pieces and then subjected to enzymolysis in a mixed enzyme solution in the dark. The precipitated cells are collected after filtration and centrifugation, and the precipitated cells are resuspended to obtain a protoplast suspension; wherein the components of the mixed enzyme solution are 2% cellulase, 0.1% pectinase, 1% macerozyme, 0.6M mannitol, 10mM MES, 0.1% BSA and 3.4mM CaCl2; The enzymolysis conditions are: first, enzymolysis at room temperature at 60r / min for 3-4 hours, and then enzymolysis at 90r / min for 5 minutes; The mass-volume ratio of the tissue to the mixed enzyme solution is (0.1-0.4) g:5mL; The filtration and centrifugation conditions are: the enzyme solution is filtered with a 70µm filter screen, and then the filtrate is centrifuged at 150g for 2 minutes, and the supernatant is discarded to obtain the precipitated cells.

2. The production method according to claim 1, wherein The resuspension is: the precipitated cells are resuspended with a W5 solution, and then centrifuged at 150g for 2 minutes, and the supernatant is discarded. The MMG solution is added to the precipitate to resuspend the cells. The components of the W5 solution are: 2mM MES, 125mM CaCl2, 154mM NaCl and 5mM KCl. The components of the MMG solution are: 0.4M mannitol, 15mM MgCl2 and 4mM MES.

3. The production method according to claim 1, wherein The yellow soybean seedling is a soil-cultivated yellow soybean seedling for 10-12 days. The varieties of the yellow soybean seedling include Zhonghuang 301, Heihe 43 and Chengdou 17.

4. A method for transforming a soybean protoplast, characterized by, The method comprises the following steps: The protoplast suspension is obtained by the preparation method of claim 1, and then the protoplast suspension is uniformly mixed with a plasmid and 500μL of a PEG transformation solution is added for transformation at room temperature for 15 minutes; wherein the components of the PEG transformation solution are: 0.2M mannitol, 100mM CaCl2 and 50% w / v PEG4000; The density of the protoplast suspension is 2 x 10 6 The volume ratio of the protoplast suspension, the plasmid and the PEG transformation solution is 10:1:

11.

5. The conversion process of claim 4 wherein, After the PEG transformation solution is added, the following steps are further included: 500μL of W5 solution is added to terminate the transformation, centrifuged, and the supernatant is discarded. 1mL of W5 solution is added to resuspend the cells, and then centrifuged, and the supernatant is discarded. 500μL of W5 solution is added to resuspend the cells, and then the culture plate is transferred for overnight culture for 12-18 hours.

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