CN105969718A - Preparation and purification methods of sasanqua protoplast - Google Patents
Preparation and purification methods of sasanqua protoplast Download PDFInfo
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Abstract
The invention discloses preparation and purification methods of sasanqua protoplast, belonging to the technical field of plant protoplast. Compared with an enzyme method for preparing protoplast, the preparation and purification methods of the sasanqua protoplast, disclosed by the invention, have the advantages that the prepared protoplast can not be injured by chemical reagents, such as enzymes, the required lime is short and the cost is low. The preparation and purification methods of the sasanqua protoplast, disclosed by the invention, can supply a lot of materials for protoplast downstream experiments of culture, fusion and gene conversion of the sasanqua protoplast.
Description
Technical field
The present invention relates to Skill of Plant Protoplasts field, particularly relate to a kind of oil tea protoplast
Preparation and purification method.
Background technology
Oil tea belongs to evergreen dungarunga.Because of its seed can extract oil (Oleum Camelliae) edible, therefore named.Tea
The clear taste of oil colours is fragrant, and nutritious, storage tolerance, is high-quality edible oil；Also can as lubricating oil,
Antirust oil is for industry.Tea cake is pesticide, is again fertilizer, can improve farmland water-holding capacity and
Preventing and treating rice pest.Peel is the raw material obtaining through refining tannic extract.
Oil tea happiness is warm, is afraid of cold, it is desirable to average temperature of the whole year 16～18 DEG C, florescence temperature on average
It it is 12～13 DEG C.Unexpected low temperature or late frost can cause fallen flowers, shedding.Requirement has more sufficient
Sunlight, the longest branch and leaf, result is few, and oil content is low.Requirement moisture is sufficient, annual precipitation
General more than 1000 millimeters, but florescence continuous rainfall, impact pollination.Require gentle in the gradient,
Planting in the weak place of corrosion function, requires soil the strictest, and general suitable soil layer is deep
Acid soil, and it is unsuitable for the place that stone is many and soil property is hard.
Protoplast (protoplast): slough the cell of cell wall protoplast, is a biological work
The concept of Cheng Xue.Protoplast one word derives from protoplasm (protoplasm).Protoplasm refers to group
Become the general name of the living substance of cell, be the concept of material.Protoplast is composition cell
One morphosis unit.In cytology's history, " cell " (cell) word is to be carried by Hooke
Go out, mean " cell ".It was found that protoplasm later, to the understanding of cell and Hooke period
Different, within 1880, Hanstein proposes " protoplast " word.Appearance due to cell one word
Relatively protoplast early, therefore is used till today.
Plant protoplast has important function in scientific research, at research cell wall-deficient mutant machine
Reason, cell to functions of hormones mechanism, the cultivation of somatic cell hybrid, proceed to exogenous gene cultivate
The aspects such as transfer-gen plant have application.The method being typically prepared protoplast has Mechanical Method and enzyme
Method.Enzyme process is due to simple to operate, it is thus achieved that cell number many, be widely used at present, but enzyme
Cell can be produced harmful effect Deng chemical reagent, affect the downstream experiment of protoplast.Machinery
Method does not the most come into one's own due to the quantity preparing protoplast.Mechanical Method is not by reagent such as enzymes
Impact, the protoplast form of preparation is complete and active, and required time is short, and expense is low,
It it is a kind of the most promising method.At present, the preparation research of oil tea protoplast is reported very
Few, with Mechanical Method prepare oil tea protoplast research it is not yet reported that.
Summary of the invention
The invention provides a kind of preparation and purification method of oil tea protoplast.
The present invention adopts the following technical scheme that
Specifically comprising the following steps that of the preparation and purification method of the oil tea protoplast of the present invention
A preparation method:
A1 cuts oil tea annotinous branch from outdoor, takes back water planting 1-2 days；
A2 takes 6 climax leaves, cuts edge and the blade master pulse of blade with blade, and every at blade
Cut some gaps every the position of 0.5-1cm, but do not cut off blade；
Apertured for band leaf block is immersed and carries out plasmolysis 0.5-1h in sucrose solution by A3, and period is used
Tweezers are by floating leaf block press-in sucrose solution；
A4 takes out leaf block, is cut into elongate strip with sharp blade and at 45 ° leaf block of leaf block, the thinnest
The best；
Spire bar is transferred to fill in the container of 3ml leaf bar cleaning mixture by A5, places 30s-1min；6
Sheet leaf can divide 3-4 to criticize cutting to join in cleaning mixture, so that material is the most crowded be
Limit；
Each washed leaf bar is taken out by A6 tweezers, puts into 2ml 13% mannitol CPW solution
In, place 5-15min；3-4 criticizes the protoplast of spire bar and is collected in successively in same pipe solution；
A7 tweezers remove leaf bar, obtain oil tea protoplast solution.
2 protoplast purification:
B1 adds sucrose solution, protoplast solution and sucrose in the bottom of oil tea protoplast solution
The volume ratio of solution is 1: 1, rinses protoplast 1-5min；
B2 transfer upper strata protoplast solution, in centrifuge tube, is centrifuged, removes supernatant；
B3 precipitation equivalent sucrose solution suspends, then is centrifuged, and removes supernatant；
B4 precipitation equivalent sucrose solution suspends, then is centrifuged, and leaves supernatant, removes precipitation；
B5 supernatant, through recentrifuge, removes supernatant, precipitates by a small amount of quality-percent by volume
Concentration is that 18% sucrose solution suspends and transfers in new centrifuge tube；
Protoplast suspension is added on sucrose solution rinsing 1-5min by B6, takes out upper liquid and is
The protoplast solution of purification.
In step A3 and B1, the quality-concentration of volume percent of sucrose solution is 35%.
In step A5, the composition of described leaf bar cleaning mixture is containing 0.03%CaCl2、
0.1%NaCl and 0.075%MgCl235% sucrose solution, wherein, the concentration of various compositions
Being quality-concentration expressed in percentage by volume, unit is g/ml.
In step A6, the composition of 13% described mannitol CPW solution is: 10A0mg/L
Potassium nitrate, 27.2mg/L carbonic acid potassium dihydrogen, 1480.0mg/L calcium chloride dihydrate, 264.0mg/L
Magnesium sulfate heptahydrate, 0.16mg/L potassium iodide, 0.025mg/L copper sulphate pentahydrate, 13%W/V
Mannitol.
In step B2, centrifugal speed is 1500rpm, and the time is 8min.
In step B3, centrifugal speed is 1000rpm, and the time is 8min
In step B4, centrifugal speed is 100rpm, and the time is 1-2min.
In step B2 and B3, the quality-concentration of volume percent of described sucrose solution is
18%.
In step B5, centrifugal speed is 500rpm, and the time is 12min.
In step B6, the quality-concentration of volume percent of described sucrose solution is 25%.
The positive effect of the present invention is as follows:
The preparation and purification method of the oil tea protoplast of the present invention and enzyme process prepare protoplast
Comparing, protoplast prepared by the present invention will not be injured by chemical reagent such as enzymes, required time
Short, expense is low.The preparation and purification method of the oil tea protoplast of the present invention can be that oil tea is former
The protoplast downstream experiments such as the raw cultivation of plastid, fusion and gene transformation provide lot of materials.
Accompanying drawing explanation
Fig. 1 is the microscope figure of protoplast prepared by Mechanical Method.
1. protoplast, 2. impurity.
Take annual oil tea branch and bring indoor water planting 1-2 days into, it is possible to promote in blade cell
Starch Conversion becomes soluble sugar, increases the cell resistance to adverse circumstance, and beneficially blade cell exists
Quality-percent by volume is to carry out holding cell under conditions of plasmolysis in 35% sucrose solution
Activity.Putting in cleaning mixture after plasmolytic maturated oil Folium Camelliae sinensis sheet cutting
30s-1min, it is possible to remove the materials such as substantial amounts of pectin in cell, polysaccharide, protein and tannin
With blade fragment；Cleaning mixture contains the Na of higher concentration simultaneously+Fruit in cell wall can be reduced
The material such as glue and polysaccharide and the interaction of protoplast, Ca2+And Mg2+Protoplasm can be kept
The stability of film and the material aggregations such as pectin can be made, make spire bar shifts from cleaning mixture
The when of carrying out plasmolysis demobilization in 13% mannitol CPW solution, from the otch of blade
It is clean much that protoplast is flowed out at place smoothly.Still further aspect, the spire bar after plasmolysis
Transfer in batch 13% a small amount of mannitol CPW solution (2ml) is divided by matter wall
The protoplast of 6 Camellia Leaves (about 1.2g) can be collected from demobilization method, so may be used
To reduce the outflow of the material such as pectin and polysaccharide in spire bar, but do not affect the outflow of cell.Institute
With, in the protoplast solution prepared in this way, cell quantity is many and form clearly (is schemed
1), impurity is relatively fewer, beneficially the purification of protoplast.
Fig. 2 be the embodiment of the present invention 3 prepare, the microscope figure of the protoplast of purification.
1. protoplast, 2. impurity
The protoplast solution (2ml) collected with 13% mannitol CPW solution, volume is relatively
Big and also to there is impurity many.In purge process, this solution centrifugal through 3 reductions of speed and
The sucrose solution of 2 reduction concentration is floating eliminates substantial amounts of impurity, it is thus achieved that purer is primary
Plastid.The volume of protoplast solution is reduced to 200 μ L by 2ml before purification so that
The density of the protoplast in solution greatly increases (Fig. 2), and density is 3.1 × 106/ ml,
Impurity is few.The form of protoplast after purification is complete, and cell membrane edge is clean.With this
The quantity of protoplast prepared by method can reach 5.1 × 105Individual/g FW, therefore. with this
Method is prepared protoplast and is had using value.
Fig. 3 is the microscope figure of the protoplast through trypan blu e dyeing.
1. protoplast
With trypan blu e dyeing protoplast method: taking containing quality-percentage by volume is 18%
The protoplast solution of sucrose 1, drops on microscope slide.Molten with the trypan blu e that concentration is 0.4%
Liquid dyes, and makes the final concentration of dyeing reach 0.04%.Dyeing 3-5min.Time can not be long,
Otherwise living cells also can be colored.Be placed on basis of microscopic observation, colors blue for dead cell,
Do not catch color for living cells.Fig. 3 shows, protoplast does not become blue, illustrates to use
Protoplast prepared by this method is great-hearted.
Fig. 4 is the microscope figure of the protoplast merged.
1. the protoplast that 2 cells are merging
High Ca is combined with PEG2+The induced fusion method of-high pH carrys out the protoplasm of fusion oil Folium Camelliae sinensis sheet
Body.Step is: 1. take protoplast solution with glue head dropper, drips 2 and drops in one piece of clean load
Stand 8-10min on slide, make protoplast form a thin layer on microscope slide.2. by 2
PEG fusion drop, on protoplast, stands 10-15min, makes to stick between protoplast
Together.3. 2 high Ca are dripped every 5min2+-high pH solution, on protoplast, carries out 3 times totally 6
Drip.4. covered, sucks unnecessary solution with absorbent paper, is placed in basis of microscopic observation thin
Born of the same parents merge situation (Fig. 4).It is observed that the percent of 2 cell fusions is 11%, explanation
The protoplast prepared in this way is activated.Protoplast through trypan blu e dyeing and
Cell fusion experiment, result all shows that the protoplast prepared in this way is activated,
Therefore, the protoplast prepared by this Mechanical Method may be used for cell and cultivates, merge and turn base
Because waiting downstream experiment of protoplast.
Detailed description of the invention
The following examples are that the present invention is described in further detail.
Embodiment 1
Specifically comprising the following steps that of the preparation and purification method of the oil tea protoplast of the present invention
1 preparation method:
A1 cuts oil tea annotinous branch from outdoor, takes back water planting 1-2 days；
A2 takes 6 climax leaves, cuts edge and the blade master pulse of blade with blade, and every at blade
Cut some gaps every the position of 0.5cm, but do not cut off blade；
Apertured for band leaf block is immersed in sucrose solution and carries out plasmolysis 0.5h, period tweezer by A3
Son is by floating leaf block press-in sucrose solution；
A4 takes out leaf block, is cut into elongate strip with sharp blade and at 45 ° leaf block of leaf block, the thinnest
The best；
Spire bar is transferred to fill in the container of leaf bar cleaning mixture by A5, places about 30s；6
Leaf can divide 3-4 to criticize to join in cleaning mixture, the most crowded as limit with material；
Each washed leaf bar is taken out by A6 tweezers, puts in 13% mannitol CPW solution,
Place 5min；3-4 criticizes the protoplast of spire bar and is collected in successively in same pipe solution；
A7 tweezers remove leaf bar, obtain oil tea protoplast solution；
2 protoplast purification:
B1 adds sucrose solution, protoplast solution and sucrose in the bottom of oil tea protoplast solution
The volume ratio of solution is 1: 1, rinses protoplast 1min；
B2 transfer upper strata protoplast solution, in centrifuge tube, is centrifuged, removes supernatant；
B3 precipitation equivalent sucrose solution suspends, then is centrifuged, and removes supernatant；
B4 precipitation equivalent sucrose solution suspends, then is centrifuged, and leaves supernatant, removes precipitation；
B5 supernatant, through recentrifuge, removes supernatant, precipitates by a small amount of quality-percent by volume
Concentration is 18% sucrose solution suspension protoplast and transfers in centrifuge tube；
Protoplast suspension is added on sucrose solution rinsing 1-5min by B6, takes out upper liquid and is
The protoplast solution of purification.
In step A3 and B1, the quality-concentration of volume percent of sucrose solution is 35%.
In step A5, the composition of described leaf bar cleaning mixture is containing 0.03%CaCl2、
0.1%NaCl and 0.075%MgCl235% sucrose solution, wherein, the concentration of various compositions
Being quality-concentration of volume percent, unit is g/ml.
In step A6, the composition of 13% described mannitol CPW solution is: 10A0mg/L
Potassium nitrate, 27.2mg/L carbonic acid potassium dihydrogen, 1480.0mg/L calcium chloride dihydrate, 264.0mg/L
Magnesium sulfate heptahydrate, 0.16mg/L potassium iodide, 0.025mg/L copper sulphate pentahydrate, 13%W/V
Mannitol.
In step B2, centrifugal speed is 1500rpm, and the time is 8min.
In step B3, centrifugal speed is 1000rpm, and the time is 8min.
In step B4, centrifugal speed is 100rpm, and the time is 1-2min.
In step B2 and B3, the quality-concentration of volume percent of described sucrose solution is
18%.
In step B5, centrifugal speed is 500rpm, and the time is 12min.
In step B6, the quality-concentration of volume percent of described sucrose solution is 25%.
Embodiment 2
Specifically comprising the following steps that of the preparation and purification method of the oil tea protoplast of the present invention
1 preparation method:
A1 cuts oil tea annotinous branch from outdoor, takes back water planting 1-2 days；
A2 takes 6 leaves, cuts edge and the blade master pulse of blade with blade, and by blade every 1cm
Position cut some gaps, but do not cut off blade；
Apertured for band leaf block is immersed in sucrose solution and carries out plasmolysis 1h, period tweezers by A3
By in floating leaf block press-in sucrose solution；
A4 takes out leaf block, becomes 45o that leaf block is cut into elongate strip with sharp blade with leaf block；
Spire bar is transferred to fill in the container of leaf bar cleaning mixture by A5, places 1min；6 leaves can
Criticizing with a point 3-4 joins in cleaning mixture, the most crowded as limit with material；
Each washed leaf bar is taken out by A6 tweezers, puts in 13% mannitol CPW solution,
Place 15min；3-4 criticizes the protoplast of spire bar and is collected in successively in same pipe solution；
A7 tweezers remove leaf bar, obtain oil tea protoplast solution；
2 protoplast purification:
B1 adds sucrose solution, protoplast solution and sucrose in the bottom of oil tea protoplast solution
The volume ratio of solution is 1: 1, rinses protoplast 5min；
B2 transfer upper strata protoplast solution, in centrifuge tube, is centrifuged, removes supernatant；
B3 precipitation equivalent sucrose solution suspends, then is centrifuged, and removes supernatant；
B4 precipitation equivalent sucrose solution suspends, then is centrifuged, and leaves supernatant, removes precipitation；
B5 supernatant, through recentrifuge, removes supernatant, precipitates by a small amount of quality-percent by volume
Concentration is the sucrose solution suspension protoplast of 18% and transfers in centrifuge tube；
Protoplast suspension is added on sucrose solution rinsing 5min by B6, takes out upper liquid and is pure
The protoplast solution changed.
In step A3 and B1, the quality-concentration of volume percent of sucrose solution is 35%.
In step A5, the composition of described leaf bar cleaning mixture is containing 0.03%CaCl2、
0.1%NaCl and 0.075%MgCl235% sucrose solution, wherein, the concentration of various compositions
Being quality-concentration of volume percent, unit is g/ml.
In step A6, the composition of 13% described mannitol CPW solution is: 10A0mg/L
Potassium nitrate, 27.2mg/L carbonic acid potassium dihydrogen, 1480.0mg/L calcium chloride dihydrate, 264.0mg/L
Magnesium sulfate heptahydrate, 0.16mg/L potassium iodide, 0.025mg/L copper sulphate pentahydrate, 13%W/V
Mannitol.
In step B2, centrifugal speed is 1500rpm, and the time is 8min.
In step B3, centrifugal speed is 1000rpm, and the time is 8min.
In step B4, centrifugal speed is 100rpm, and the time is 1-2min.
In step B2 and B3, the quality-concentration of volume percent of described sucrose solution is
18%.
In step B5, centrifugal speed is 500rpm, and the time is 12min.
In step B6, the quality-concentration of volume percent of described sucrose solution is 25%.
Embodiment 3
Specifically comprising the following steps that of the preparation and purification method of the oil tea protoplast of the present invention
Preparation method；
A cuts oil tea annotinous branch from outdoor, takes back laboratory water planting 1-2 days,
B takes 6 leaves, cuts edge and the blade master pulse of blade with blade, and by blade every
Some gaps are cut in the position of 0.5-1CM, but do not cut off blade.
3. apertured for band leaf block is immersed and 35% sucrose solution carries out plasmolysis about 0.5-1h,
Floating leaf block is pressed in sucrose solution by period with tweezers.
4. take out leaf block to be placed on microscope slide, be cut into at 45 ° with leaf block leaf block of sharp blade
The strip that 0.5mm is wide, the thinnest more good.
5. transfer to spire bar to fill in the test tube of 3ml leaf bar cleaning mixture, place 30s-1min left
Right.6 leaves can divide 3-4 to criticize to join in cleaning mixture, so that material is the most crowded be
Limit.
6. being taken out by each washed leaf bar with tweezers, the 13% mannitol CPW putting into 2ml is molten
In liquid, place about 10min.3-4 criticizes the protoplast of spire bar and is collected in same pipe successively
In solution；
7. remove leaf bar with tweezers, obtain oil tea protoplast solution.
Protoplast purification process:
A liquid-transfering gun puts into 2ml 35% sucrose solution bottom the test tube of oil tea protoplast solution,
Rinsing protoplast about 3min.
B transfer upper strata protoplast solution is in centrifuge tube, and 1500rpm is centrifuged 8min, removes supernatant
Liquid.
3. precipitation 2ml 18% sucrose solution suspends, and 1000rpm is centrifuged 8min, removes supernatant.
4. precipitation 2ml 18% sucrose solution suspends, and 100rpm is centrifuged 1-2min, leaves supernatant,
Remove precipitation.
5. supernatant is centrifuged 12min through 500rpm, removes supernatant, precipitate with 200 μ L mass-
Concentration of volume percent is 18% sucrose solution suspension protoplast and transfers to the centrifugal of A5ml
Guan Zhong.
6. it is to float on 25% sucrose solution that Protoplast suspension is added in quality-concentration of volume percent
Wash about 3min, take out supernatant and be the protoplast solution of purification.
Although an embodiment of the present invention has been shown and described, for the ordinary skill of this area
For personnel, it is possible to understand that without departing from the principles and spirit of the present invention can be to this
A little embodiments carry out multiple change, revise, replace and modification, and the scope of the present invention is by appended power
Profit requires and equivalent limits.
Claims (9)
1. the preparation and purification method of an oil tea protoplast, it is characterised in that: described method
Specifically comprise the following steps that
A preparation method:
A1 cuts oil tea annotinous branch from outdoor, takes back water planting 1-2 days；
A2 takes 6 climax leaves, cuts edge and the blade master pulse of blade with blade, and every at blade
Cut some gaps every the position of 0.5-1cm, but do not cut off blade；
Apertured for band leaf block is immersed and carries out plasmolysis 0.5-1h in sucrose solution by A3, and period is used
Tweezers are by floating leaf block press-in sucrose solution；
A4 takes out leaf block, is cut into elongate strip with blade and at 45 ° leaf block of leaf block, the thinnest more good；
Spire bar is transferred to fill in the container of leaf bar cleaning mixture by A5, places 30s-1min；6
Leaf can divide 3-4 to criticize cutting to join in cleaning mixture, so that material is the most crowded in cleaning mixture be
Limit；
Each washed leaf bar is taken out by A6 tweezers, puts in 13% mannitol CPW solution,
Place 5-15min；3-4 criticizes the protoplast of spire bar and is collected in successively in same pipe solution；
A7 tweezers remove leaf bar, obtain oil tea protoplast solution；
B protoplast purification:
B1 adds sucrose solution, protoplast solution and sucrose at the bottom of oil tea protoplast solution
The volume ratio of solution is 1: 1, rinses protoplast 1-5min；
B2 transfer upper strata protoplast solution, in centrifuge tube, is centrifuged, removes supernatant；
B3 precipitation equivalent sucrose solution suspends, then is centrifuged, and removes supernatant；
B4 precipitation equivalent sucrose solution suspends, then is centrifuged, and leaves supernatant, removes precipitation；
B5 supernatant, through recentrifuge, removes supernatant, precipitates by 200 μ L mass-percent by volume
Concentration is 18% sucrose solution suspension protoplast and transfers in centrifuge tube；
Protoplast suspension is added on sucrose solution rinsing 1-5min by B6, takes out upper liquid and is
The protoplast solution of purification.
2. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step A3 and B1, the quality-concentration of volume percent of sucrose solution is 35%.
3. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step A5, the composition of described leaf bar cleaning mixture is containing 0.03%CaCl2、
0.1%NaCl and 0.075%MgCl235% sucrose solution, wherein, the concentration of various compositions
Being quality-concentration of volume percent, unit is g/ml.
4. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step A6, the composition of 13% described mannitol CPW solution is: 101.0mg/L
Potassium nitrate, 27.2mg/L carbonic acid potassium dihydrogen, 1480.0mg/L calcium chloride dihydrate, 264.0mg/L
Magnesium sulfate heptahydrate, 0.16mg/L potassium iodide, 0.025mg/L copper sulphate pentahydrate, 13%W/V
Mannitol.
5. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step B2, centrifugal speed is 1500rpm, and the time is 8min；In step B3,
Centrifugal speed is 1000rpm, and the time is 8min.
6. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step B4, centrifugal speed is 100rpm, and the time is 1-2min.
7. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step B2 and B3, the quality-concentration of volume percent of described sucrose solution is 18%.
8. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step B5, centrifugal speed is 500rpm, and the time is 12min.
9. the preparation and purification method of oil tea protoplast as claimed in claim 1, its feature exists
In: in step B6, the quality-concentration of volume percent of described sucrose solution is 25%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN111705033A (en) * | 2020-07-08 | 2020-09-25 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
| CN113549591A (en) * | 2021-08-10 | 2021-10-26 | 中南林业科技大学 | Method for separating and purifying tea-oil camellia mesophyll protoplast |
| 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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| CN105695391A (en) * | 2016-03-25 | 2016-06-22 | 中国农业科学院茶叶研究所 | Extraction method of tea tree protoplast |
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| CN113549591A (en) * | 2021-08-10 | 2021-10-26 | 中南林业科技大学 | Method for separating and purifying tea-oil camellia mesophyll 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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