CN110669718A - Method for separating and purifying root, stem and leaf protoplasm body of larch and performing instantaneous high-efficiency conversion - Google Patents
Method for separating and purifying root, stem and leaf protoplasm body of larch and performing instantaneous high-efficiency conversion Download PDFInfo
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Abstract
The invention discloses a method for separating, purifying and instantly and efficiently converting the protoplasm of roots, stems and leaves of larch, which comprises the following steps: 1) selecting the seedling of the larch with 1-3 round leaves, and culturing for 24h in a dark place; cleaning with 75% alcohol for 1 time, cleaning with deionized water for 3 times, and separating the leaves, stem segments and roots of the seedling with a blade; 2) enzymolyzing the root, stem and leaf of the larch into protoplast with cellulase and pectinase, and separating, purifying and pretreating the obtained protoplast; 3) different tissue protoplasts are taken as receptors, the transient expression vector containing the exogenous gene is transferred into the protoplasts by utilizing a PEG mediated transformation method, and the exogenous gene is expressed in the protoplasts after culture. The method is simple and easy to operate, can obtain a large amount of complete and pure root, stem and leaf protoplasts of the larch, has the transformation efficiency of over 50 percent, and provides technical basis for the transient expression detection of the larch gene, the protoplast fusion and the like.
Description
Technical Field
The invention belongs to the technical field of cell biology and biology, and particularly relates to a method for separating, purifying and instantly and efficiently converting a protoplasm of roots, stems and leaves of larch.
Background
Plant cells form protoplasts, including cell membranes, cytoplasm and nucleus, after removal of the cell wall. Briefly, plant protoplasts are "naked cells" surrounded by a plasma membrane. With the continuous development of scientific technology, protoplasts have gradually become an effective tool for researchers to study gene and protein biological functions and analyze plant signal transduction pathways. Protoplast isolation is the first step of protoplast culture, and directly affects whether the protoplast culture is successful or not. The isolation of protoplasts by enzymatic hydrolysis is currently the most simple and efficient method recognized. Cellulase is used to digest the cell wall, pectinase is used to soften the cell wall, the mesoglea is degraded, and the single cells are released. Meanwhile, in addition to the kind and combination of the enzyme, it is also important for the selection of materials, the osmotic pressure of the enzyme solution, the stability of the plasma membrane, the time and temperature of the enzymolysis, the method of separation and purification, and the like.
After pure plant protoplast is obtained, the genetic transformation of the protoplast is carried out in the next step, the protoplast is taken as a receptor, and exogenous genes are introduced into the plant protoplast by utilizing a certain technical means, so that the exogenous genes are stably expressed. Plant protoplast genetic transformation is divided into stable transfection and transient expression, the transient expression is common, and the method has the advantages that the gene activity can be easily detected in a short time after the introduction of the exogenous gene, and the technology is widely applied to promoter activity analysis, gene subcellular localization, protein localization and the like. There are several methods for transient expression of protoplasts, such as: PEG mediated transformation method, electroporation hole transformation method, liposome mediated transformation method and Agrobacterium co-culture transformation method, among which PEG mediated transformation method is most commonly used.
Larch (Taxodium distichum (L.) Rich) is an ancient wiggery plant, has the advantages of fast growth, beautiful tree shape, good material quality, strong adaptability and less plant diseases and insect pests, and is an excellent tree species for using materials, protecting and viewing. Native north america, now widely introduced around the world. At present, the larval is listed as an important afforestation and greening tree species in China, and large-area larval artificial forests are available in Su, Zhe, Wan, Hu, Xiang, Jian, Min, Chuan, Henan, Lu, Shaan and other provinces. In recent decades, forestry and researchers have developed the hybridization and breeding of larch species, morphological, physiological and molecular biological studies, and have achieved certain results. However, as most of needle-leaved trees have immature genetic transformation systems, further mechanism research is limited, most of the current researches on protoplasts are concentrated in mesophyll cells of model plants, namely arabidopsis thaliana and some broad-leaved plants, the needle-leaved plants are only reported in a few of coniferous plants, and related researches on the protoplasts of the larch are blank at home and abroad, so that the larch protoplasts are utilized to provide a convenient and powerful tool for analyzing molecular mechanisms of the larch trees, and technical bases are provided for transient expression detection of the larch genes, protoplast fusion and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for separating and purifying the protoplasm of the roots, stems and leaves of the larch. Can obtain a large amount of complete and pure procaryotic bodies of the roots, stems and leaves of the larch. The invention also aims to provide a method for the instantaneous and efficient transformation of the root, stem and leaf protoplasm of the larch. The method is simple and easy to operate, and the plasmid conversion rate reaches more than 50%.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
a method for separating, purifying and instantaneously and efficiently converting the protoplasm of the root, stem and leaf of larch comprises the following steps:
1) selecting the seedling of the larch with 1-3 round leaves, and culturing for 24h in a dark place; cleaning with 75% alcohol for 1 time, cleaning with deionized water for 3 times, and separating the leaves, stem segments and roots of the seedling with a blade;
2) enzymolyzing the root, stem and leaf of the larch into protoplast by using a tissue enzyme solution containing 1-8% vol of cellulase and 1-5% vol of pectinase, and separating, purifying and pretreating the obtained protoplast;
3) different tissue protoplasts are taken as receptors, the transient expression vector containing the exogenous gene is transferred into the protoplasts by utilizing a PEG mediated transformation method, and the exogenous gene is expressed in the protoplasts after culture.
In the step 2), the specific operation is as follows:
1) preparation of different tissue enzyme solutions: 2mL of 200mM MES solution, 15mL of 0.8M mannitol, 2mL of 0.2M KCl, 2mL of ddH2Mixing O, carrying out water bath at 70 ℃ for 10min, averagely dividing the mixed solution into 3 parts, and adding enzymes required by root, stem and leaf enzymolysis respectively, wherein the cellulase required by the root enzymolysis is 2% vol, and the pectinase is 1% vol; cellulase required by stem enzymolysis is 6% vol, and pectinase is 4% vol; cellulase required by leaf enzymolysis is 4% vol, and pectinase is 2% vol; water bath at 55 deg.C for 20min, and filtering with 0.22 μm filter membrane;
2) cutting the leaves, stem segments and roots into filaments with the width of 0.5-1 mm; placing the cut tissue into enzyme solution in a stretched manner, placing in dark, vacuumizing for 15min to make the enzyme solution penetrate into the cell gap of the material, and performing enzymolysis for 5 hours at 28 ℃ in a dark place;
3) respectively adding pre-cooled 10mL of W5 solution into the reaction solution obtained in the step 2), rinsing and diluting the protoplast, and filtering by using a 35-75 mu m nylon net or a 60-100 mesh metal sieve to remove impurities which are not subjected to enzymolysis; adding the filtrate into a 50mL round-bottom centrifuge tube, centrifuging at 4 ℃ and 900rpm for 5min, and precipitating protoplasm; after removing the supernatant, 5mL of W5 solution was addedResuspending and rinsing the protoplast precipitated at the bottom; the protoplasts were allowed to stand on ice for 30min, after removal of the supernatant, the MMG solution was used to resuspend the protoplasts to a final concentration of 6X 105one/mL.
In the step 3), the specific operation is as follows:
adding 10 μ L of target plasmid vector containing exogenous gene with concentration not less than 1 μ g/μ L into EP tube, and adding 100 μ L of 6 × 105The protoplast of the root, stem and leaf of the separated and purified larval fir is added with 220 mu L of PEG-Ca with the mass volume of 30-50 percent2+The solution is flicked and mixed evenly; in the dark, the transformation mixture was induced for 15 minutes in an ice bath; then adding 800 mu L of W5 solution, mixing uniformly, and stopping the conversion reaction; centrifuging at 600rpm for 5min, and sucking off the supernatant; add 200. mu.L of WI solution to resuspend the protoplasts; culturing at 25 deg.C in dark for 18-24 hr.
The formula of the W5 solution is as follows: 2mM MES, 154mM NaCl, 125mM CaCl2，5mM KCl。
The formula of the MMG solution is as follows: 4mM MES, 0.4M mannitol, 15mM MgCl2，100mM CaCl2。
PEG-Ca2+The formula of the solution is as follows: PEG 4000, 0.2M mannitol, 100mM CaCl2。
Further, 30% by mass volume of PEG-Ca was added to the root protoplasts2+Solution, stem protoplast with 50% by mass of PEG-Ca2+Adding 50% PEG-Ca by mass volume to the solution and the leaf protoplasm2+And (3) solution.
The formula of the WI solution is as follows: 4mM MES, 0.5M mannitol, 20mM KCl, pH 5.7.
Furthermore, the target plasmid vector containing the exogenous gene is p2FGW7.0, contains a green fluorescent protein GFP label, is started by a 35S promoter, and is fused with the ThRAP2.1 gene of the larch-the taxus chinensis and the p2FGW7.0 by using Gateway and TA cloning technology to obtain the target plasmid vector.
Has the advantages that: the invention can simply and quickly obtain a large amount of complete and pure protoplasts of the larval cypress with different tissues and organs, and the protoplasts of different tissues can reach higher plasmid conversion rate, even the protoplasts of roots with lower conversion rate can reach 51 percent. Provides convenient and effective technical basis for the subcellular localization of the protein and the expression regulation of the gene in the research of the larch species.
Drawings
FIG. 1 is a diagram of a seedling of Lueizia juvenia, a material for preparing a protoplast of Lueizia juvenia;
FIG. 2 is a graph of protoplast activity after FDA staining;
FIG. 3 is a diagram of the elements of the plant transient expression vector contained therein;
FIG. 4 is a graph showing the transformation and localization of the protoplasts of larval fir; in the figure, A: root protoplasts; b: a stem protoplast; c: mesophyll protoplasts; d: and (4) plasmid transformation.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are not intended to limit the present invention.
Example 1 selection of Material of Lumeria decipiens for the preparation of protoplasts
1) Collecting seeds of the larch in the classification garden of the Nanjing Zhongshan botanical garden in autumn and winter, and naturally airing the seeds in a dry and ventilated place;
2) washing seeds with tap water, soaking the seeds in deionized water for 3 weeks, and changing water every 3 days;
3) putting the cleaned seeds in a specific substrate (V (peat soil): V (perlite): 1), covering fine soil with the thickness of 1-2 cm on the surfaces of the seeds, watering thoroughly, covering with straws, and preserving heat, moisture and promoting seedling emergence;
4) removing grass when the seedling of the larval fir emerges, placing the seedling in an illumination constant temperature incubator at 25 + -2 deg.C under strong illumination of 50 μ Em-2s-1The illumination time is 16 h/d. Watering once every 2 days until the seedlings grow a round of complete leaves, generally not more than three rounds of leaves, and the method can be used for preparing protoplasts;
5) the material from which protoplasts are isolated will generally be young tissue with low keratinization and fibrosis. The method selects the seedling of the larval fir growing 1-3 rounds of leaves (about 30 days are generally needed from germination to 3 rounds of complete leaves growing), takes the root, stem and leaf materials (as shown in figure 1), thoroughly cleans the seedling before separating the protoplast, cleans the seedling with 75% alcohol for 1 time, cleans the seedling with deionized water for 3 times, quickly separates the leaves, stem segments and roots of the larval fir with a blade, and reduces water loss in a culture dish.
EXAMPLE 2 isolation and purification of protoplasts from different tissues
1) Optimal enzyme liquid system for separating protoplast of different tissues
The invention optimizes the enzymolyzing system of the larch protoplast based on the optimization research of the separation condition of the arabidopsis thaliana mesophyll protoplast published by Liao Jiaming, etc. 2010, cellulase sets a gradient from 1-8% vol every 1%, pectinase sets a gradient from 1-5% vol every 1%, three different tissues of root, stem and leaf are counted to 120 groups of enzyme liquid proportion, the enzymolysis condition is observed and counted after 5 hours of enzymolysis, the counting is repeated three times each time, the results are shown in tables 1-3: when the cellulase is 2% vol, the pectinase is 1% vol, the root enzymolysis effect is the best, and the protoplast yield is 4.32 multiplied by 106/gFW; when the cellulase is 6% vol, the pectinase is 4% vol, the stem enzymolysis effect is the best, and the protoplast yield is 5.89 multiplied by 106/gFW; when the cellulase is 4% vol and the pectinase is 2% vol, the leaf enzymolysis effect is the best, and the protoplast yield is 7.67X 106/gFW。
TABLE 1 different enzyme combinations on root protoplast yield (× 10) of seedlings of Luquoia6/gFW) influence
TABLE 2 different enzyme combinations on yield of protoplasts from the stems of seedlings of Aleurites Lucifica (x 10)6/gFW) influence
TABLE 3 yield of protoplasts from the leaf of larval seedlings by different enzyme combinations (× 10)6/gFW) influence
Preparation of different tissue enzyme solutions: 2mL of 200mM MES solution, 15mL of 0.8M mannitol, 2mL of 0.2M KCl, 2mL of ddH2Mixing O, performing water bath at 70 ℃ for 10min, averagely dividing the mixed solution into 3 parts, and respectively adding enzymes required by root, stem and leaf enzymolysis, wherein the enzymes required by the root enzymolysis are 150 mu L cellulase and 75 mu L pectinase; the enzyme required by stem enzymolysis is 500 mu L cellulase and 300 mu L pectinase; the enzyme required by leaf enzymolysis is 300 mu L cellulase and 150 mu L pectinase; water bath at 55 deg.C for 20min, and filtering with 0.22 μm filter membrane.
2) Gently cutting the leaves, stem segments and roots into filaments of 0.5-1mm width by using an operating blade along the longitudinal direction; placing the cut tissue in enzyme solution in a stretched manner, placing in dark, vacuumizing for 15min to make the enzyme solution penetrate into the cell gap of the material, and then carrying out enzymolysis at constant temperature of 28 ℃ for 5 hours in a dark place, wherein the enzymolysis solution becomes turbid after clarification;
3) gently add 4 ℃ pre-cooled 10mL W5 solutions (2mM MES, 154mM NaCl, 125mM CaCl) along the sides of the culture vessels, respectively25mM KCl), the protoplast is rinsed and diluted, and then a 35-75 mu m nylon net or a 60-100 mesh metal screen is used for filtering to remove the impurities which are not enzymolyzed;
4) further adding the filtrate into a 50mL round-bottom centrifuge tube, centrifuging at 4 ℃ and 900rpm for 5min, and precipitating protoplasts;
5) placing the centrifugal tube on ice, carefully removing the supernatant, and then re-suspending and rinsing the protoplast precipitated at the bottom by using 5mL of W5 solution;
6) observing and counting by using a hemocytometer under a common optical microscope, ensuring that the following transformation can be carried out only when the number of the protoplasts in a large square lattice in the center of the hemocytometer reaches more than 200, further detecting the activity of the protoplasts, and observing by using 0.02 percent Fluorescein Diacetic Acid (FDA) under a fluorescence microscope;
7) the protoplasts were again allowed to stand on ice for 30min, and after removing the supernatant, the appropriate amount of MMG solution (4mM MES, 0.4M Glycine)Lutol, 15mM MgCl2，100mM CaCl2) Resuspend protoplasts to a final concentration of 6X 105and/mL, used for later plasmid transformation.
The number and activity detection method of the protoplast comprises the following steps:
the number of the yield of the taxus chinensis protoplasts is counted by a 0.1mm haemocytometer, and 1 drop of the protoplast suspension after separation and purification is taken out and put on a glass slide for microscopic examination under a 10-time objective lens. Protoplast density (one/mL) in large square (0.1 mm)3I.e., 0.1. mu.L) of protoplasts × 104Protoplast yield (per g) ═ protoplast density (per mL) × total protoplast suspension volume (mL)]Fresh weight of material used for protoplast preparation (gFW).
Protoplast activity was measured using 0.02% Fluorescein Diacetate (FDA). 100 mu L FDA solution with the concentration of 0.02% is taken, added into the protoplast suspension on the glass slide, slowly and uniformly mixed, and stood for 5-10min at room temperature. The fluorescence excitation light wavelength was 450-490nm and the emission wavelength was 250nm, examined under a fluorescence microscope (OLYMPUS BX51, Japan). It was observed that the highly viable protoplasts had yellow-green fluorescence (as shown in FIG. 2). The number of active protoplasts and the total number of protoplasts in each field were counted. Protoplast survival (%) was calculated. Protoplast viability is the number of yellow-green fluorescent protoplasts/total number of observed protoplasts × 100%.
Example 3 transformation of protoplasts of different tissues by PEG-mediated method
Expression of plasmids after transformation into protoplasts
The vector used for transformation in this example is p2FGW7.0, which is a commercial vector, and the vector map is shown in FIG. 2, the vector contains a constitutive strong expression promoter P35S, can be efficiently expressed in protoplasts, contains a green fluorescent protein GFP tag, and can observe a green fluorescent signal under the irradiation of an ultraviolet lamp. At the early stage, the target plasmid vector is obtained by fusing ThRAP2.1 (GenBank: KY463469) gene of the tree of the genus Aleurites and p2FGW7.0 by using Gateway and TA cloning technology.
1) Pipetting 10 mu L of target plasmid vector (more than 10 mu g) containing the exogenous gene and adding the target plasmid vector into a 2mL round-bottom EP tube;
2) sucking 100 μ L of separated and purified protoplast of root, stem and leaf of the larch, and mixing the plasmid and the protoplast with the bottom of a finger flick centrifugal tube;
3) 30 to 50 percent of PEG-Ca is prepared2+Solution [ PEG 4000(M/v), 0.2M mannitol, 100mM CaCl2]Draw 220. mu.L of PEG-Ca2+Solution (root-30% PEG-Ca)2+Stem-50% PEG-Ca2+Leaf-50% PEG-Ca2 +) Flicking the bottom of the centrifugal tube by fingers again to mix uniformly;
4) in the dark, the transformation mixture was induced for 15 minutes with ice-bath;
5) at room temperature, absorbing 800 mu L of W5 solution to dilute the conversion mixed solution, slightly reversing and uniformly mixing, and stopping the conversion reaction;
6) at room temperature, a desk centrifuge is used for 600rpm, the vertical centrifugation is carried out for 5min, the acceleration is set to be 4, and then a liquid transfer gun is used for gently removing the supernatant;
7) gently resuspend protoplasts using 200. mu.L of WI solution (4mM MES, 0.5M mannitol, 20mM KCl (pH 5.7);
8) culturing in dark at 25 deg.C in a constant temperature incubator, inducing protoplast expression and transformation for 18-24 hr, and observing GFP label expression under Olimpus fluorescence microscope (OLYMPUS BX51, Japan), wherein the fluorescence excitation wavelength is 450-490nm, and the emission wavelength is 250 nm. When the green fluorescence is excited, the fluorescence generation of the protoplast nucleus, cytoplasm and cell membrane can be observed.
9) And detecting the transformation degree by using an enzyme-labeling instrument, and counting the transformation efficiency, wherein the transformation efficiency of the root protoplast is 51%, the transformation efficiency of the stem protoplast is 62%, and the transformation efficiency of the leaf protoplast is 77%.
Claims (9)
1. A method for separating, purifying and instantaneously and efficiently converting the protoplasm of the root, the stem and the leaf of a larch is characterized by comprising the following steps:
1) selecting the seedling of the larch with 1-3 round leaves, and culturing for 24h in a dark place; cleaning with 75% alcohol for 1 time, cleaning with deionized water for 3 times, and separating the leaves, stem segments and roots of the seedling with a blade;
2) enzymolyzing the root, stem and leaf of the larch into protoplast by using a tissue enzyme solution containing 1-8% vol of cellulase and 1-5% vol of pectinase, and separating, purifying and pretreating the obtained protoplast;
3) different tissue protoplasts are taken as receptors, the transient expression vector containing the exogenous gene is transferred into the protoplasts by utilizing a PEG mediated transformation method, and the exogenous gene is expressed in the protoplasts after culture.
2. The method for separating, purifying and transiently efficiently transforming the protoplasm of the roots, stems and leaves of the larch as claimed in claim 1, wherein in the step 2), the specific operations are as follows:
1) preparation of different tissue enzyme solutions: 2mL of 200mM MES solution, 15mL of 0.8M mannitol, 2mL of 0.2M KCl, 2mL of ddH2Mixing O, carrying out water bath at 70 ℃ for 10min, averagely dividing the mixed solution into 3 parts, and respectively adding enzymes required by root, stem and leaf enzymolysis, wherein the cellulase required by the root enzymolysis is 2% vol, and the pectinase is 1% vol; cellulase required by stem enzymolysis is 6% vol, and pectinase is 4% vol; cellulase required by leaf enzymolysis is 4% vol, and pectinase is 2% vol; water bath at 55 deg.C for 20min, and filtering with 0.22 μm filter membrane;
2) cutting the leaves, stem segments and roots into filaments with the width of 0.5-1 mm; placing the cut tissue into enzyme solution, placing in dark, vacuumizing for 15min to make the enzyme solution penetrate into the material cell gap, and performing enzymolysis for 5 hr at 28 deg.C in dark;
3) respectively adding pre-cooled 10mL of W5 solution into the reaction solution obtained in the step 2), rinsing and diluting the protoplast, and filtering by using a 35-75 mu m nylon net or a 60-100 mesh metal sieve to remove impurities which are not subjected to enzymolysis; adding the filtrate into a 50mL round-bottom centrifuge tube, centrifuging at 4 ℃ and 900rpm for 5min, and precipitating protoplasts; after removing the supernatant, the protoplasts precipitated at the bottom were resuspended and rinsed with 5mL of W5 solution; the protoplasts were allowed to stand on ice for 30min, after removal of the supernatant, the MMG solution was used to resuspend the protoplasts to a final concentration of 6×105one/mL.
3. The method for separating, purifying and transiently efficiently transforming the protoplasm of the roots, stems and leaves of the larch as claimed in claim 1, wherein in the step 3), the specific operations are as follows:
adding 10 μ L of the target plasmid vector containing the foreign gene at a concentration of not less than 1 μ g/. mu.L into an EP tube, and adding 100 μ L of 6X 105The protoplast of the root, stem and leaf of the separated and purified larval fir is added with 220 mu L of PEG-Ca with the mass volume of 30-50 percent2+The solution is flicked and mixed evenly; in the dark, the transformation mixture was induced for 15 minutes with ice-bath; then adding 800 mu L of W5 solution, mixing uniformly, and stopping the conversion reaction; centrifuging at 600rpm for 5min, and sucking off the supernatant; add 200. mu.L of WI solution to resuspend the protoplasts; culturing at 25 deg.C in dark for 18-24 hr.
4. The method for separating, purifying and transiently transforming the protoplasm of the root, stem and leaf of the larch as claimed in claim 2, wherein the formula of the W5 solution in the step 3) is as follows: 2mM MES, 154mM NaCl, 125mM CaCl2，5mM KCl。
5. The method for separating, purifying and transiently efficiently transforming the protoplasm of the roots, stems and leaves of the larch as claimed in claim 2, wherein the formula of the MMG solution in the step 3) is as follows: 4mM MES, 0.4M mannitol, 15mM MgCl2，100mMCaCl2。
6. The method for separating, purifying and transiently transforming the protoplasm of the root, stem and leaf of the larch as claimed in claim 3, wherein the PEG-Ca is selected from the group consisting of2+The formula of the solution is as follows: PEG 4000, 0.2M mannitol, 100mM CaCl2。
7. The method for separating, purifying and transiently transforming the protoplasm of the root, stem and leaf of the larch as claimed in claim 3, wherein 30% by mass volume of the protoplasm of the root is added to the protoplasm of the rootPEG-Ca2+Solution, stem protoplast with 50% by mass of PEG-Ca2+Adding 50% PEG-Ca by mass volume to the solution and the leaf protoplasm2+And (3) solution.
8. The method for separating, purifying and transiently converting the protoplasm of the root, stem and leaf of the larch as claimed in claim 3, wherein the WI solution is formulated as follows: 4mM MES, 0.5M mannitol, 20mM KCl, pH 5.7.
9. The method for separating, purifying and transiently transforming the protolast of the root, stem and leaf of the larch as claimed in claim 3, wherein the target plasmid vector containing the exogenous gene is p2FGW7.0, contains a green fluorescent protein GFP tag, is started by a 35S promoter, and is obtained by fusing the ThRAP2.1 gene and the p2FGW7.0 gene of the larch tree of the genus larch by using the Gateway and TA cloning technology.
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| CN116803257A (en) * | 2023-08-09 | 2023-09-26 | 中国农业科学院植物保护研究所 | Rapid enrichment and separation method for plant parasitic nematodes in plant tissues |
| CN117143797A (en) * | 2023-09-14 | 2023-12-01 | 九江学院 | Separation method and instantaneous transformation method of camellia oleifera protoplast |
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| CN117143797A (en) * | 2023-09-14 | 2023-12-01 | 九江学院 | Separation method and instantaneous transformation method of camellia oleifera protoplast |
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