CN105695391A - Extraction method of tea tree protoplast - Google Patents
Extraction method of tea tree protoplast Download PDFInfo
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- CN105695391A CN105695391A CN201610175155.8A CN201610175155A CN105695391A CN 105695391 A CN105695391 A CN 105695391A CN 201610175155 A CN201610175155 A CN 201610175155A CN 105695391 A CN105695391 A CN 105695391A
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Abstract
The invention relates to an extraction method of tea tree protoplast, and belongs to the technical field of plant protoplasts. The extraction method utilizes tea tree petals as an extraction material, and comprises the following four steps of flower collection, protoplast enzymolysis, releasing and purification. The extraction method of the tea tree protoplast has the advantages that the obtaining of separating materials is easy, the extraction operation is simple, the number of separated protoplasts is more, the yield is high, the integrity is high, and a foundation is laid for the protoplast transfection and fusion.
Description
Technical field
The invention belongs to Skill of Plant Protoplasts field, be specially the extracting method of a kind of Camellia sinensis protoplast。
Background technology
Camellia sinensis (Camelliasinensis(L.) O.Kuntze) belong to Theaceae Camellia, for perennial woody plant, there is long cultivation history, be important beverage plant。Compared with other plant, containing characteristic secondary metabolites such as extremely abundant catechin, theanine in Camellia sinensis bud-leaf so that Folium Camelliae sinensis has the color quality of uniqueness, also enhance health simultaneously。
Plant protoplast refers to the cell that after eliminating whole cell wall, plasma membrane wraps up, protoplast not only has the character of living cells, and formation hybrid cell can be merged, it can directly absorb the DNA of external source, is by the desirable receptor of genetic transformation and gene functional research。In vitro protoplast has cellular omnipotency, can grow, breaks up, regeneration whole plant under suitable aseptic condition。The research of Camellia sinensis gene function needs to realize the interaction etc. of the Subcellular Localization of Camellia sinensis gene, albumen by means of protoplast, it addition, utilize protoplast fusion to carry out somatic hybridization, it is possible to broken the incompatible dysgenesia of Camellia sinensis sexual hybridization。These are required for obtaining high preparation rate and highly active Camellia sinensis protoplast。At present, from higher plant, separate the protoplast obtained, be herbaceous plant mostly, and xylophyta is difficult to separation and obtains protoplast。Camellia sinensis protoplast is prepared research and is rarely had report, is usually present yield with tea leaf separation protoplast low, and the defect that integrity is also low can not meet the requirement of subsequent experimental。Present invention Camellia sinensis petal is that research material carries out enzymolysis, and release obtains protoplast, obtains the protoplast that quantity is many, purity is high after purification, has established certain technical foundation for researchs such as Camellia sinensis improvement of genes, gene functions。
Summary of the invention
For the above-mentioned problems in the prior art, it is an object of the invention to the technical scheme that design provides the extracting method of a kind of Camellia sinensis protoplast, the method, is completed by flower collection, protoplast enzymolysis, release and four steps of purification as extracting material with Camellia sinensis petal；The separation material of the method can be easily obtained, and extracts simple to operate, separates the protoplast quantity obtained many, and yield is high, and integrity is good, lays a good foundation for protoplast transfection and protoplast fusion。
The extracting method of described a kind of Camellia sinensis protoplast, it is characterised in that comprise the following steps:
1) Flos Camelliae Japonicae choose season in full bloom growing way better, without the Camellia sinensis flower of pest and disease damage；
2) it is placed on the petal front end on clean blank sheet of paper with new blade or shears and after afterbody removal, quickly mid portion is cut into the wide slice of 0.5-1mm, quickly, gently transfers in the culture dish filling enzymolysis solution, and make petal be immersed in completely in enzymolysis solution；
Enzymolysis solution is prepared: 19-21mM2-(N-morpholino) ethyl sulfonic acid, 1.4-1.6 cellulase R10,0.3-0.5 macerozyme R10,0.3-0.5M mannitol, 19-21mM potassium chloride, after adding above reagent, first 50-60 DEG C of heating in water bath 8-15 minute, adds 9-11mM calcium chloride, 4-6mM after being cooled to room temperatureβ-mercaptoethanol, 0.8-0.12 bovine serum albumin, adjust pH to 5.5-5.8,0.45 μm of filtration sterilization, matching while using after constant volume；
3) ware lid is covered, it is placed in vacuum pump under dark condition evacuation 40min-1h, after evacuation, continue to be placed in placement 3.5-4.5h under dark condition, softly rock, if seeing the transparent shape of petal and having wilted, protoplast is described, and release is complete substantially, and culture dish is placed in basis of microscopic observation release conditions；
4) dilute above-mentioned protoplast solution with lavation buffer solution, rock mixing gently；
Lavation buffer solution is prepared: 1.8-2.2mM2-(N-morpholino) ethyl sulfonic acid, 150-158mM sodium chloride, 122-127mM calcium chloride, 4.8-5.2mM potassium chloride, adjusts pH to 5.5-5.8,0.45 μm of filtration sterilization after constant volume；
5) with the nylon wire of sterile water wash 70 μm, with washing buffer liquid wetting nylon wire after drying, filtration step 4) in enzymolysis solution, then wash culture dish 2-3 time with lavation buffer solution, the consumption of lavation buffer solution and enzymolysis solution equal-volume used；
6) Protoplast suspension moving to 50ml centrifuge tube, maximum centrifugal force 100 × g under room temperature, the braking of centrifuge raising speed and reduction of speed is set to 1-2, centrifugal 3.5-4.5min, abandons supernatant, collects protoplast。
The extracting method of described a kind of Camellia sinensis protoplast, it is characterized in that step 2) in: described enzymolysis solution preparation: 20mM2-(N-morpholino) ethyl sulfonic acid, 1.5 cellulase R10,0.4 macerozyme R10,0.4M mannitol, 20mM potassium chloride, first 55 DEG C of heating in water bath 10 minutes after adding above reagent, 10mM calcium chloride, 5mM is added after being cooled to room temperatureβ-mercaptoethanol, 0.1 bovine serum albumin, adjust pH to 5.7,0.45 μm of filtration sterilization after constant volume。
The extracting method of described a kind of Camellia sinensis protoplast, it is characterised in that in step 3): evacuation 45min-55min, continues to be placed in placement 4h under dark condition。
The extracting method of described a kind of Camellia sinensis protoplast, it is characterised in that in step 4): lavation buffer solution is prepared: 2mM2-(N-morpholino) ethyl sulfonic acid, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride, adjusts pH5.7,0.45 μm of filtration sterilization after constant volume。
The extracting method of described a kind of Camellia sinensis protoplast, after it is characterized in that step 6): add the lavation buffer solution of 10mL ice bath in above-mentioned protoplast, ice bath 28-32min, takes a small amount of protoplast during this period and counts with cell counter under the microscope；100 × g is centrifuged 2min, abandons supernatant, and protoplast is transferred to 2mL centrifuge tube, with being used for subsequent experimental after the resuspended protoplast of appropriate permeation protective agent；Permeation protective agent is prepared: comprises 0.38-0.42M mannitol, 14-16mM magnesium chloride in 3.8-4.2mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.5-5.8,0.45 μm of filtration sterilization after constant volume。
The extracting method of described a kind of Camellia sinensis protoplast, it is characterised in that: permeation protective agent is prepared: comprises 0.4M mannitol, 15mM magnesium chloride in 4mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.7,0.45 μm of filtration sterilization after constant volume。
The extracting method of above-mentioned a kind of Camellia sinensis protoplast, with Camellia sinensis petal as extracting material, is completed by flower collection, protoplast enzymolysis, release and four steps of purification；The separation material of the method can be easily obtained, and extracts simple to operate, separates the protoplast quantity obtained many, and yield is high, and integrity is good, lays a good foundation for protoplast transfection and protoplast fusion。
Accompanying drawing explanation
Petal is shredded and is immersed in enzymolysis solution by Fig. 1；
Fig. 2 is the Camellia sinensis petal protoplast figure after enzymolysis of the present invention separation and purification；
Fig. 3 is the Camellia sinensis mature leaf protoplast figure extracted with extracting method of the present invention。
Detailed description of the invention
Below in conjunction with Figure of description and specific embodiment, the invention will be further described。
Embodiment 1
1) Flos Camelliae Japonicae choose season in full bloom 30 growing ways better, without the Camellia sinensis flower of pest and disease damage；
2), after removing with front end by petal of new blade or shears and afterbody, quickly mid portion is cut into the wide slice of 0.5-1mm, quickly, gently transfers in the culture dish filling 15ml enzymolysis solution, and make petal be immersed in completely in enzymolysis solution；
Enzymolysis solution is prepared: 19mM2-(N-morpholino) ethyl sulfonic acid, 1.4 cellulase R10,0.3 macerozyme R10,0.3M mannitol, 19mM potassium chloride, first 50 DEG C of heating in water bath 8 minutes after adding above reagent, 9mM calcium chloride, 4mM is added after being cooled to room temperatureβ-mercaptoethanol, 0.8 bovine serum albumin, adjust pH to 5.5,0.45 μm of filtration sterilization, matching while using after constant volume；
3) ware lid is covered, it is placed in vacuum pump under dark condition evacuation 40min, after evacuation, continue to be placed in placement 3.5h under dark condition, softly rock, if seeing the transparent shape of petal and having wilted, protoplast is described, and release is complete substantially, and culture dish is placed in basis of microscopic observation release conditions；
4) dilute above-mentioned protoplast solution with lavation buffer solution, rock mixing gently；
Lavation buffer solution is prepared: 2mM2-(N-morpholino) ethyl sulfonic acid, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride, adjusts pH to 5.5,0.45 μm of filtration sterilization filtration sterilization after constant volume；
5) clean the nylon wire of 70 μm with water, with washing buffer liquid wetting nylon wire after drying, filtration step 4) in enzymolysis solution, then wash culture dish 2 times with lavation buffer solution, the consumption of lavation buffer solution and enzymolysis solution equal-volume used；
6) Protoplast suspension moving to 50ml centrifuge tube, room temperature 100g is centrifuged 3.5min, and the braking of centrifuge raising speed and reduction of speed is set to 1-2, to prevent protoplast from occurring mechanical damage in centrifugal process, abandons supernatant, collects protoplast；
7) in above-mentioned protoplast, add the lavation buffer solution of 10mL ice bath, ice bath 28min, take a small amount of protoplast during this period and count with cell counter under the microscope；
8) the centrifugal 2min of 100 × g, abandons supernatant, protoplast is transferred to 2mL centrifuge tube, with being used for subsequent experimental after the resuspended protoplast of appropriate permeation protective agent；Permeation protective agent is prepared: comprises 0.4M mannitol, 15mM magnesium chloride in 4mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.5,0.45 μm of filtration sterilization after constant volume。
Embodiment 2
1) Flos Camelliae Japonicae choose season in full bloom 30 growing ways better, without the Camellia sinensis flower of pest and disease damage；
2), after removing with front end by petal of new blade or shears and afterbody, quickly mid portion is cut into the wide slice of 0.5-1mm, quickly, gently transfers in the culture dish filling 15ml enzymolysis solution, and make petal be immersed in completely in enzymolysis solution；
Enzymolysis solution is prepared: 20mM2-(N-morpholino) ethyl sulfonic acid, 1.5 cellulase R10,0.4 macerozyme R10,0.4 mannitol, 20mM potassium chloride, first 55 DEG C of heating in water bath 12 clocks after adding above reagent, 10mM calcium chloride, 5mM is added after being cooled to room temperatureβ-mercaptoethanol, 0.1 bovine serum albumin, adjust pH to 5.7,0.45 μm of filtration sterilization, matching while using after constant volume；
3) cover ware lid, be placed in vacuum pump under dark condition evacuation 50min, after evacuation, continue to be placed in placement 4h under dark condition, softly rock, if seeing the transparent shape of petal and having wilted, protoplast is described, and release is complete substantially, and culture dish is placed in basis of microscopic observation release conditions；
4) dilute above-mentioned protoplast solution with lavation buffer solution, rock mixing gently；
Lavation buffer solution is prepared: 2mM2-(N-morpholino) ethyl sulfonic acid, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride, adjusts pH to 5.7,0.45 μm of filtration sterilization after constant volume；
5) clean the nylon wire of 70 μm with water, with washing buffer liquid wetting nylon wire after drying, filtration step 4) in enzymolysis solution, then wash culture dish 3 times with lavation buffer solution, the consumption of lavation buffer solution and enzymolysis solution equal-volume used；
6) Protoplast suspension moving to 50ml centrifuge tube, room temperature 100xg is centrifuged 4min, and the braking of centrifuge raising speed and reduction of speed is set to 1-2, to prevent protoplast from occurring mechanical damage in centrifugal process, abandons supernatant, collects protoplast；
7) in above-mentioned protoplast, add the lavation buffer solution of 10mL ice bath, ice bath 30min, take a small amount of protoplast during this period and carry out counting (the protoplast that the petal of 30 flowers separates totally 2.56 × 10 with cell counter under the microscope6Individual)；
8) the centrifugal 2min of 100 × g, abandons supernatant, protoplast is transferred to 2mL centrifuge tube, with being used for subsequent experimental after the resuspended protoplast of appropriate permeation protective agent；Permeation protective agent is prepared: comprises 0.4M mannitol, 15mM magnesium chloride in 4mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.7,0.45 μm of filtration sterilization after constant volume。
Embodiment 3
1) Flos Camelliae Japonicae choose season in full bloom 30 growing ways better, without the Camellia sinensis flower of pest and disease damage；
2), after removing with front end by petal of new blade or shears and afterbody, quickly mid portion is cut into the wide slice of 0.5-1mm, quickly, gently transfers in the culture dish filling 15ml enzymolysis solution, and make petal be immersed in completely in enzymolysis solution；
Enzymolysis solution is prepared: 21mM2-(N-morpholino) ethyl sulfonic acid, 1.6 cellulase R10,0.5 macerozyme R10,0.5M mannitol, 21mM potassium chloride, first 60 DEG C of heating in water bath 15 minutes after adding above reagent, 11mM calcium chloride, 6mM is added after being cooled to room temperatureβ-mercaptoethanol, 0.12 bovine serum albumin, adjust pH to 5.8,0.45 μm of filtration sterilization, matching while using after constant volume；
3) cover ware lid, be placed in vacuum pump under dark condition evacuation 1h, after evacuation, continue to be placed in placement 4.5h under dark condition, softly rock, if seeing the transparent shape of petal and having wilted, protoplast is described, and release is complete substantially, and culture dish is placed in basis of microscopic observation release conditions；
4) dilute above-mentioned protoplast solution with lavation buffer solution, rock mixing gently；
Lavation buffer solution is prepared: 2mM2-(N-morpholino) ethyl sulfonic acid, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride, adjusts pH to 5.8,0.45 μm of filtration sterilization after constant volume；
5) clean the nylon wire of 70 μm with water, with washing buffer liquid wetting nylon wire after drying, filtration step 4) in enzymolysis solution, then wash culture dish 3 times with lavation buffer solution, the consumption of lavation buffer solution and enzymolysis solution equal-volume used；
6) Protoplast suspension moving to 50ml centrifuge tube, room temperature 100 × g is centrifuged 4.5min, and the braking of centrifuge raising speed and reduction of speed is set to 1-2, to prevent protoplast from occurring mechanical damage in centrifugal process, abandons supernatant, collects protoplast；
7) in above-mentioned protoplast, add the lavation buffer solution of 10mL ice bath, ice bath 32min, take a small amount of protoplast during this period and count with cell counter under the microscope；
8) the centrifugal 2min of 100 × g, abandons supernatant, protoplast is transferred to 2mL centrifuge tube, with being used for subsequent experimental after the resuspended protoplast of appropriate permeation protective agent；Permeation protective agent is prepared: comprises 0.4M mannitol, 15mM magnesium chloride in 4mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.8,0.45 μm of filtration sterilization after constant volume。
Fig. 2 and Fig. 3 illustrates that tea leaf is higher due to degree of lignification, and wax coat is thicker, the more difficult cracking of palisade tissue, and yield of protoplast is non-normally low, and Camellia sinensis Hua Caineng extracts the protoplast that quantity is many, yield is high, integrity is good。
Claims (6)
1. the extracting method of a Camellia sinensis protoplast, it is characterised in that comprise the following steps:
1) Flos Camelliae Japonicae choose season in full bloom growing way better, without the Camellia sinensis flower of pest and disease damage；
2) it is placed on the petal front end on clean blank sheet of paper with new blade or shears and after afterbody removal, quickly mid portion is cut into the wide slice of 0.5-1mm, quickly, gently transfers in the culture dish filling enzymolysis solution, and make petal be immersed in completely in enzymolysis solution；
Enzymolysis solution is prepared: 19-21mM2-(N-morpholino) ethyl sulfonic acid, 1.4-1.6 cellulase R10,0.3-0.5 macerozyme R10,0.3-0.5M mannitol, 19-21mM potassium chloride, after adding above reagent, first 50-60 DEG C of heating in water bath 8-15 minute, adds 9-11mM calcium chloride, 4-6mM after being cooled to room temperatureβ-mercaptoethanol, 0.8-0.12 bovine serum albumin, adjust pH to 5.5-5.8,0.45 μm of filtration sterilization, matching while using after constant volume；
3) ware lid is covered, it is placed in vacuum pump under dark condition evacuation 40min-1h, after evacuation, continue to be placed in placement 3.5-4.5h under dark condition, softly rock, if seeing the transparent shape of petal and having wilted, protoplast is described, and release is complete substantially, and culture dish is placed in basis of microscopic observation release conditions；
4) dilute above-mentioned protoplast solution with lavation buffer solution, rock mixing gently；
Lavation buffer solution is prepared: 1.8-2.2mM2-(N-morpholino) ethyl sulfonic acid, 150-158mM sodium chloride, 122-127mM calcium chloride, 4.8-5.2mM potassium chloride, adjusts pH to 5.5-5.8,0.45 μm of filtration sterilization after constant volume；
5) with the nylon wire of sterile water wash 70 μm, with washing buffer liquid wetting nylon wire after drying, filtration step 4) in enzymolysis solution, then wash culture dish 2-3 time with lavation buffer solution, the consumption of lavation buffer solution and enzymolysis solution equal-volume used；
6) Protoplast suspension moving to 50ml centrifuge tube, maximum centrifugal force 100 × g under room temperature, the braking of centrifuge raising speed and reduction of speed is set to 1-2, centrifugal 3.5-4.5min, abandons supernatant, collects protoplast。
2. the extracting method of a kind of Camellia sinensis protoplast as claimed in claim 1, it is characterized in that step 2) in: described enzymolysis solution preparation: 20mM2-(N-morpholino) ethyl sulfonic acid, 1.5 cellulase R10,0.4 macerozyme R10,0.4M mannitol, 20mM potassium chloride, first 55 DEG C of heating in water bath 10 minutes after adding above reagent, 10mM calcium chloride, 5mM is added after being cooled to room temperatureβ-mercaptoethanol, 0.1 bovine serum albumin, adjust pH to 5.7,0.45 μm of filtration sterilization after constant volume。
3. the extracting method of a kind of Camellia sinensis protoplast as claimed in claim 1, it is characterised in that in step 3): evacuation 45min-55min, continues to be placed in placement 4h under dark condition。
4. the extracting method of a kind of Camellia sinensis protoplast as claimed in claim 1, it is characterised in that in step 4): lavation buffer solution is prepared: 2mM2-(N-morpholino) ethyl sulfonic acid, 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride, adjusts pH to 5.7,0.45 μm of filtration sterilization after constant volume。
5. the extracting method of a kind of Camellia sinensis protoplast as claimed in claim 1, after it is characterized in that step 6): add the lavation buffer solution of 10mL ice bath in above-mentioned protoplast, ice bath 28-32min, takes a small amount of protoplast during this period and counts with cell counter under the microscope；100 × g is centrifuged 2min, abandons supernatant, and protoplast is transferred to 2mL centrifuge tube, with being used for subsequent experimental after the resuspended protoplast of appropriate permeation protective agent；Permeation protective agent is prepared: comprises 0.38-0.42M mannitol, 14-16mM magnesium chloride in 3.8-4.2mM2-(N-morpholino) ethyl sulfonic acid, adjusts pH to 5.5-5.8,0.45 μm of filtration sterilization after constant volume。
6. the extracting method of a kind of Camellia sinensis protoplast as claimed in claim 5; it is characterized in that: permeation protective agent is prepared: comprise 0.4M mannitol in 4mM2-(N-morpholino) ethyl sulfonic acid; 15mM magnesium chloride, adjusts pH to 5.7,0.45 μm of filtration sterilization after constant volume。
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969718A (en) * | 2016-07-15 | 2016-09-28 | 湖南科技学院 | Preparation and purification methods of sasanqua protoplast |
| CN108085292A (en) * | 2017-12-04 | 2018-05-29 | 湖北省农业科学院果树茶叶研究所 | A kind of isolation and purification method of tea tree mesophyll protoplast |
| CN108949665A (en) * | 2018-07-12 | 2018-12-07 | 北京林业大学 | The preparation method of lily petal protoplast |
| CN109136167A (en) * | 2018-07-12 | 2019-01-04 | 北京林业大学 | The preparation method of lily mesophyll protoplast |
| CN110878281A (en) * | 2019-12-12 | 2020-03-13 | 武汉德翊环境科技有限公司 | Method for extracting submerged plant agave protoplast |
| CN111454875A (en) * | 2020-04-16 | 2020-07-28 | 中国农业科学院蔬菜花卉研究所 | Method for separating colored cell protoplast of hydrangea macrophylla |
| CN112813017A (en) * | 2019-11-18 | 2021-05-18 | 广东省农业科学院环境园艺研究所 | Transient expression system of cymbidium protoplast and construction method and application thereof |
| CN113249296A (en) * | 2021-06-30 | 2021-08-13 | 安徽农业大学 | Method for separating and purifying fresh tissue protoplast of tea tree |
| CN115247145A (en) * | 2022-06-24 | 2022-10-28 | 中南林业科技大学 | Method for separating oil tea petal protoplast and constructing transient transformation system |
| CN116144577A (en) * | 2023-02-24 | 2023-05-23 | 安徽农业大学 | Separation and purification method of plant protoplast |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830896A (en) * | 2015-04-13 | 2015-08-12 | 北京林业大学 | Method for expressing proteins by using plant petal cell protoplast |
| CN105505980A (en) * | 2015-12-19 | 2016-04-20 | 西北农林科技大学 | Method for instantaneously transforming switchgrass protoplast through fluorescence labeling fluorescence maker vector |
-
2016
- 2016-03-25 CN CN201610175155.8A patent/CN105695391A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830896A (en) * | 2015-04-13 | 2015-08-12 | 北京林业大学 | Method for expressing proteins by using plant petal cell protoplast |
| CN105505980A (en) * | 2015-12-19 | 2016-04-20 | 西北农林科技大学 | Method for instantaneously transforming switchgrass protoplast through fluorescence labeling fluorescence maker vector |
Non-Patent Citations (2)
| Title |
|---|
| 中村順行: "チャにおけるプロトプラストの単離", 《茶業研究報告》 * |
| 中村顺行: "茶树原生质体的分离", 《茶叶科学简报》 * |
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| CN105969718B (en) * | 2016-07-15 | 2019-08-23 | 湖南科技学院 | A kind of preparation and purification method of oil tea protoplast |
| CN105969718A (en) * | 2016-07-15 | 2016-09-28 | 湖南科技学院 | Preparation and purification methods of sasanqua protoplast |
| CN108085292A (en) * | 2017-12-04 | 2018-05-29 | 湖北省农业科学院果树茶叶研究所 | A kind of isolation and purification method of tea tree mesophyll protoplast |
| CN109136167B (en) * | 2018-07-12 | 2022-03-08 | 北京林业大学 | Preparation method of lily mesophyll protoplast |
| CN108949665A (en) * | 2018-07-12 | 2018-12-07 | 北京林业大学 | The preparation method of lily petal protoplast |
| CN109136167A (en) * | 2018-07-12 | 2019-01-04 | 北京林业大学 | The preparation method of lily mesophyll protoplast |
| CN108949665B (en) * | 2018-07-12 | 2022-03-04 | 北京林业大学 | Preparation method of lily petal protoplast |
| CN112813017A (en) * | 2019-11-18 | 2021-05-18 | 广东省农业科学院环境园艺研究所 | Transient expression system of cymbidium protoplast and construction method and application thereof |
| CN110878281A (en) * | 2019-12-12 | 2020-03-13 | 武汉德翊环境科技有限公司 | Method for extracting submerged plant agave protoplast |
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| CN113249296A (en) * | 2021-06-30 | 2021-08-13 | 安徽农业大学 | Method for separating and purifying fresh tissue protoplast of tea tree |
| CN113249296B (en) * | 2021-06-30 | 2024-01-30 | 安徽农业大学 | Separation and purification method of fresh tissue protoplast of tea tree |
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