CN106480085A - A kind of efficient moment gene expression method of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor - Google Patents
A kind of efficient moment gene expression method of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor Download PDFInfo
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Abstract
The invention discloses a kind of efficient moment gene expression method of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor, including：The preparation of hybridized Chinese tuliptree mesophyll protoplast, the preparation of plasmid, conversion and observation.The present invention with the protoplast of hybridized Chinese tuliptree mesophyll cell as genetic transformation receptor system, the method mediated by PEG, using green fluorescent protein GFP gene as reporter gene, detect the Transient Expression of GFP gene efficient by fluorescence microscope.The present invention is drawn materials conveniently using hybridized Chinese tuliptree blade as the source of protoplast, energetic, and the Transient Expression system of foundation can provide a quickly and easily operating platform for the research of Liriodendron and Magnoliacea plant gene function.
Description
Technical field
The present invention relates to hybridized Chinese tuliptree genetic transforming method, and in particular to a kind of efficient moment heredity of hybridized Chinese tuliptree
Method for transformation.
Background technology
Liriodendron belongs to original angiosperm in ancient Magnoliacea plant from system generation.To Magnoliaceae
Plant gene and its research of function, will have important impact to illustrating angiospermous system.And currently with normal
The model plant arabidopsis seen, the transformation system of tobacco conduct a research and may deposit to the gene function of Liriodendron and signal transduction
The problems such as the false positive that Heterologous System brings, the transformation system of Liriodendron is hence set up for Liriodendron and Magnoliacea plant
The research of gene function suffers from significance.Transformation system mainly includes the transformation system of stable transfection and Transient Expression
Gene system, is affected by the long feature of xylophyta growth cycle, and Transient Expression gene system is plant gene function high flux
Research provides a quickly and easily operating platform.At present, Transient transformation system mainly have Agrobacterium-mediated genetic transformation,
The protoplast transformation of particle bombardment, PEG mediation or electroporation mediation, the protoplast Transient transformation of wherein PEG mediation
It is most widely used Transient Expression system.PEG has cell adhesion and upsets the effect of cell membrane phospholipid bilayer, and it can
Make DNA molecular bridge be formed with film, promote mutual contact and adhesion；It can also cause film surface charge disorderly, interference cell
Identification, the fusion for being conducive to cell intermembranous and foreign DNA enter protoplast.PEG is used at present in the species such as arabidopsis, willow
The mesophyll protoplast transient transfection systems of mediation have been widely used in the signal transmission of research gene expression, albumen
Interaction between interaction, Subcellular Localization and protein-dna and albumen between etc..But the plasm of PEG mediation
The transformation frequency of body conversion is influenced by many factors, the concentration including foreign gene and conformation, the molecular weight of PEG and concentration,
The ratio of plasmid/protoplast, transfection time etc..Only set up efficiently in plants such as arabidopsis, paddy rice, cotton, willows at present
PEG mediation Transient Expression system, affected by species difference, Magnoliacea plant substantial amounts of wax, mesh as blade contains
Front not yet have the method for its mesophyll protoplast of quick separating and effective Transient Expression system in the short time.
Content of the invention
Goal of the invention：For the deficiencies in the prior art, it is an object of the invention to provide one kind is with mesophyll plasm
Body is the efficient moment gene expression method of hybridized Chinese tuliptree of acceptor, it is established that the hybridized Chinese tuliptree mesophyll of PEG mediation is primary
Plastid Transient transformation system, be Liriodendron and Magnoliacea plant gene function research provide one quickly and easily operate flat
Platform.
Technical scheme：In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows：
A kind of efficient moment gene expression method of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor, comprises the following steps：
1）Using QIAGEN Plasmid Midi Kits extracting and purifying plasmid, after being adjusted to the concentration of 2 μ g/ μ L, -20
DEG C save backup；
2）Isolating and purifying for hybridized Chinese tuliptree mesophyll protoplast, obtains concentration for 105The protoplast of/mL；
3）Plasmid is taken in 2 mL centrifuge tubes, is subsequently adding protoplast, with hand gently by DNA and protoplast solution
Mix；40% PEG-4000 solution is slowly added to, soft mixing, place 15 min；W5 solution is added, soft mixing；1100
Rpm abandons supernatant after being centrifuged 2 min, adds WI solution gently to suspend protoplast, and is transferred on Tissue Culture Plate；
4）Tissue Culture Plate is placed on after 23 DEG C of dark 18 h of quiescent culture, is placed in fluorescence microscopy Microscopic observation.
Step 1）In, detailed process is as follows：
1）Well dispersed single bacterium colony in picking flat board, the LB culture medium of 2 mL being inoculated in containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 8 h of shaken cultivation；
2）The bacterium solution that shakes is taken, by 1:500 ratio is added to the LB culture medium of 50 mL containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 12～16 h of shaken cultivation；
3）15 minutes collects thalline precipitations, complete reject supernatant is centrifuged at 4 DEG C with 6000 × g；
4）With the resuspended thalline of 4 mL Buffer P1；
5）4 mL Buffer P2 are added, is turned upside down 4-6 time and mixes completely, room temperature standing 5 min of cracking；
6）The Buffer P3 of 4 mL precoolings is added, overturns 4-6 mixing immediately, ice bath staticly settles 15min；
7）30 min are centrifuged at 4 DEG C with >=20000 × g, by the supernatant containing plasmid be carefully transferred to one new clean
Centrifuge tube in；And 15 min are centrifuged at 4 DEG C again with >=20000 × g；
8）4 mL Buffer QBT are added on 100 centrifugal column of QIAGEN-tip, by Action of Gravity Field treat its flow completely through from
Stem；
9）By 7）In the supernatant containing plasmid be immediately transferred on 100 centrifugal column of QIAGEN-tip that balanced, rely on gravity
Effect plasmid is attached on the resin of centrifugal column；
10）Add 2 × 10 mL Buffer QC cleaning centrifugal column；
11）Add 5 mL Buffer QF that the plasmid combined on the resin of centrifugal column is eluted in a clean centrifuge tube；
12）To in plasmid eluent, add 3.5 mL room temperatures to place isopropanol precipitating DNA, after mixing completely, at 4 DEG C
30 min are centrifuged with >=15000 × g and precipitate DNA, careful reject supernatant；
13）70% ethanol that is placed with 2 mL room temperatures washs DNA precipitation, is centrifuged 10 min with >=15000 × g, carefully abandons
Except supernatant should not disturb the precipitation of centrifugation bottom of the tube；
14）After 5～10 min of drying at room temperature；The ddH for adding 30 uL to sterilize2O, dissolves plasmid；
15）After the concentration and purity of agarose gel electrophoresis and ultramicrospectrophotometer detection plasmid, 2 μ are adjusted to
After g/ μ L, -20 DEG C save backup.
Step 2）In, the culture body embryo seedling leaf that health stretches in blake bottle is taken, and the thin of 0.5～1mm is cut into blade
Silk, is then put in ready enzymolysis liquid rapidly, is placed under 28 DEG C of dark conditions, and 45 rpm slight oscillatory, 3 hr carries out enzyme
Solution；After the completion of enzymolysis, appropriate W5 solution is added, is jiggled, is filtered to remove using 300 mesh sieve and does not digest sufficient blade；
Filtrate is centrifuged 2 min through 1100 rpm, abandons supernatant, is washed after protoplast 2 times with the W5 solution of 2 mL ice precoolings, adds 2 mL
The resuspended protoplast of the W5 solution of ice precooling, stands 30 min, 1100 rpm on ice and is centrifuged 2 min, abandon supernatant, add MMg
Solution suspension protoplast, and protoplast concentration is adjusted to 105/mL.
The preparation of enzymolysis liquid：Weigh 0.15 g, Macerozyme R10 of Cellulase R-10,0.1 g and
0.01 g of Pectolase Y-23 is in 6.25 mL, 0.8 M mannitol, 2.5 mL, 80 mM KCl, 1 mL, 200 mM MES
（pH5.7）Solution in, stir evenly, put 4 DEG C of refrigerator overnight and fully dissolve；Add 0.1 mL, 1 M CaCl2, 0.01 g BSA,
It is settled to 10 mL；Above-mentioned solution is filtered with the sterilising filter of 0.45 m, standby.
Step 3）In, 20 μ g plasmids conversion 1 × 104Individual cell.
Beneficial effect：Compared with prior art, source of the present invention using hybridized Chinese tuliptree blade as protoplast, passes through
PEG is mediated, using GFP gene as reporter gene, it is established that hybridized Chinese tuliptree mesophyll protoplast efficient moment table
System is reached, is that the research of Liriodendron and Magnoliacea plant gene function provides a quickly and easily operating platform.
Description of the drawings：
Fig. 1 is the displaing micro photo figure of hybridized Chinese tuliptree mesophyll protoplast；
Fig. 2 is the displaing micro photo figure of PEG mediation hybridized Chinese tuliptree mesophyll protoplast conversion；Left figure is the light field visual field,
Middle figure is the GFP visual field, after right figure is superposition.
Specific embodiment
With reference to instantiation, the present invention is further illustrated.
The formula of the reagent that following examples are used is：
PEG solution（40%, V/V）（10mL）：4g PEG4000,3mL H2O, 2.5mL 0.8M mannitol, 1mL 1M CaCl2.
W5 solution：154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES（pH5.7）, 5mM glucose.
MMg solution：0.4M mannitol, 15mM MgCl2, 4mM MES（pH5.7）.
WI solution：4mM MES（pH5.7）, 0.5M mannitol, 20mM KCl.
Prepared by 1 plasmid of embodiment
Using QIAGEN Plasmid Midi Kits extracting and purifying plasmid（PJIT166-GFP）, quickly and easily can obtain
The higher plasmid of purity, comprises the following steps that：
1）Well dispersed single bacterium colony in picking flat board, the LB culture medium of 2 mL being inoculated in containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 8 h of shaken cultivation；
2）The bacterium solution that shakes is taken, by 1:500 ratio is added to the LB culture medium of 50 mL containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 12～16 h of shaken cultivation；
3）15 minutes collects thalline precipitations, complete reject supernatant is centrifuged at 4 DEG C with 6000 × g；
4）With the resuspended thalline of 4 mL Buffer P1；
5）4 mL Buffer P2 are added, is turned upside down 4-6 time and mixes completely, room temperature standing 5 min of cracking；
6）The Buffer P3 of 4 mL precoolings is added, overturns 4-6 mixing immediately, ice bath staticly settles 15min；
7）30 min are centrifuged at 4 DEG C with >=20000 × g, by the supernatant containing plasmid be carefully transferred to one new clean
Centrifuge tube in；And 15 min are centrifuged at 4 DEG C again with >=20000 × g；
8）4 mL Buffer QBT are added on 100 centrifugal column of QIAGEN-tip, by Action of Gravity Field treat its flow completely through from
Stem；
9）By 7）In the supernatant containing plasmid be immediately transferred on 100 centrifugal column of QIAGEN-tip that balanced, rely on gravity
Effect plasmid is attached on the resin of centrifugal column；
10）Add 2 × 10 mL Buffer QC cleaning centrifugal column；
11）Add 5 mL Buffer QF that the plasmid combined on the resin of centrifugal column is eluted in a clean centrifuge tube；
12）To in plasmid eluent, add 3.5 mL room temperatures to place isopropanol precipitating DNA, after mixing completely, at 4 DEG C
30 min are centrifuged with >=15000 × g and precipitate DNA, careful reject supernatant；
13）70% ethanol that is placed with 2 mL room temperatures washs DNA precipitation, is centrifuged 10 min with >=15000 × g, carefully abandons
Except supernatant should not disturb the precipitation of centrifugation bottom of the tube；
14）After 5～10 min of drying at room temperature；The ddH for adding 30 uL to sterilize2O, dissolves plasmid；
15）After the concentration and purity of agarose gel electrophoresis and ultramicrospectrophotometer detection plasmid, 2 μ are adjusted to
After g/ μ L, -20 DEG C save backup.
The the isolating and purifying of 2 hybridized Chinese tuliptree mesophyll protoplast of embodiment
The quality of protoplast directly affects transformation efficiency, and when the species of the growth conditions of vegetable material, enzyme and purity, enzymolysis
Between and the factors such as temperature can all affect efficiency and the quality of the protoplast being separated to.Mesophyll protoplast more great Cheng
That spent remains many physiological responses of plant and the activity of cell, has therefore widely been elected to be plant protoplast
Material source.The mesophyll protoplast of current many plants such as arabidopsis, tobacco, willow, paddy rice, corn, cotton is
Through efficiently being separated at short notice, and the blade of Liriodendron plant, similar to Ginkgoaceae and wide yulan, epicuticle
Blade wax all containing homologous series, therefore, its epidermis it is difficult with and tear as arabidopsis, tobacco, cereal and poinsettia etc.
Method remove.The enzyme liquid conjugate fiber element enzyme R-10 and macerozyme R10 being usually used can not efficiently separate miscellaneous at short notice
Hand over Liriodendron mesophyll protoplast.
This example based on special enzymolysis liquid, is established effectively and prepares hybridization using hybridized Chinese tuliptree blade as material
The method of Liriodendron mesophyll protoplast.Comprise the following steps that：
1）The healthy and strong body embryo seedling in blake bottle of culture is taken as experiment material, will more be stretched with scissors and growth conditions are good
Good 2-5 piece blade is sheared off（Often with a piece of cut a piece of）, be placed on clean blank sheet of paper, 0.5～1mm is cut into single-edge blade
Band, cut after pick up one end with tweezers rapidly, be put in ready enzymolysis liquid（Note：When being put into, first by one side immersion enzyme
Solution liquid, takes out and immerses another side again, and purpose makes blade bar section completely dip in enzymolysis liquid, digests more abundant）.It is subsequently placed in
Under 28 °C of dark conditions, 3 hr of 45rpm slight oscillatory is digested.
2）After the completion of enzymolysis, several seconds promotion digestion is jiggled, appropriate W5 solution is added, is jiggled, using 300 mesh sieves
Son is filtered to remove and does not digest sufficient blade.
3）Filtrate is centrifuged 2min through 1100rpm, suctions out supernatant with pipette tips, it is seen that protoplast pellet is in bottom.
4）The resuspended protoplast of W5 solution of 2 mL ice precoolings is added in the precipitation of gained, and 1100rpm is centrifuged 2min, uses
Pipette tips suction out supernatant, and jiggling makes protoplast suspend.
5）Repeat once.
6）The resuspended protoplast of the W5 solution of addition 2mL ice precooling, stands 30min on ice again.1000rpm is centrifuged
2min, suctions out supernatant with pipette tips, and jiggling makes protoplast suspend.
7）Add volume required MMg solution（Each reaction about needs 100 μ L）Suspension protoplast.Take a drop re-suspension liquid to enter
Row microscopy, observes quality and the concentration of protoplast, and adjusts protoplast concentration to 105/mL.Hybridized Chinese tuliptree mesophyll cell
The microphoto of protoplast, as shown in Figure 1.
The preparation of special enzymolysis liquid：Weigh 0.15 g, Macerozyme R10 of Cellulase R-10,0.1 g and
0.01 g of Pectolase Y-23 is in 6.25 mL, 0.8 M mannitol, 2.5 mL, 80 mM KCl, 1 mL, 200 mM MES
（pH5.7）Solution in, stir evenly, put 4 DEG C of refrigerator overnight and fully dissolve；Add 0.1 mL, 1 M CaCl2, 0.01 g BSA,
It is settled to 10 mL；Above-mentioned solution is filtered with the sterilising filter of 0.45 m, standby.
Embodiment 3 is converted
So far, established in a lot of plants such as arabidopsis, tobacco, willow, diversiform-leaved poplar, grape, corn efficiently primary
Plastid Transient Expression system.But, the parameter of the Transient transformation system of the PEG mediation of the protoplast in different materials source is present
Certain difference.In the diversiform-leaved poplar Suspension Cells protoplast Transient transformation system of the PEG mediation that sets up recently, optimum ginseng
Number is：In the transformation system of 240 μ L, 1 × 105Protoplast and 20 μ g plasmids under the mediation of 20% PEG-4000, quiet
Put 20 min of transfection, it is possible to obtain the conversion ratio more than 50%；And the optimized parameter of grape Suspension Cells protoplast is：?
In the transformation system of 220 μ L, 4 × 105Protoplast be added in 20 μ g plasmids, under the mediation of 25% PEG-4000, standing
Transfect 30 min, it is possible to obtain the conversion ratio more than 60%.Therefore the protoplast moment of the PEG mediation in different materials source turns
Change parameter differences are very big, from the point of view of the Transient transformation system of the protoplast of the PEG mediation having built up at present, affect its conversion
The factor of rate mainly has DNA concentration and quantity, the ratio of plasmid/protoplast, PEG molecular weight and concentration, transfection time etc..
The method of the hybridized Chinese tuliptree mesophyll protoplast Transient transformation that this example is mediated with PEG, concrete steps are such as
Under：
1）For single reaction, add DNA totally 10 μ L in 2mL centrifuge tube.
2）Each reaction draws the above-mentioned protoplast for preparing of 100 μ L in the centrifuge tube for adding DNA, and each is anti-
Should be sure to before absorption resuspended once, otherwise protoplast can sink to bottom.
3）With hand DNA and protoplast solution being mixed gently.
4）The PEG solution that equal-volume is 110 μ L is added, PEG can sink to solution bottom, often add a reaction and use hand immediately
Mixing solution gently.
5）Standing transfection 15min.
6）It is 440 L W5 solution to add 4 times of volumes in each reaction, and mixing of gently turning upside down terminates transfection reaction.
7）Under room temperature, 1100 rpm are centrifuged 2 min, suction out supernatant with wide head pipette tips, add 1 mL WI solution gently to suspend
Protoplast, and be transferred in 6 porocyte culture plates.
8）About 18hr is cultivated under the conditions of 23 DEG C of low-light.
Observe after culture：Tissue Culture Plate is placed on after 23 DEG C of dark 18 h of quiescent culture, is placed under fluorescence microscope and sees
Examine.As a result as shown in Fig. 2 under light field, it can be observed that complete protoplast, under GFP, it can be seen that green fluorescence divides
Cloth in whole protoplast, in terms of the ratio that the protoplast of fluoresced green under the visual field accounts for observed protoplast
Calculate, the conversion ratio of protoplast has reached 60%.
Test confirms, with 20 μ g plasmids conversion 1 × 104Individual cell, it is possible to obtain higher conversion ratio, reaches 60%.
Claims (5)
1. the efficient moment gene expression method of a kind of hybridized Chinese tuliptree with mesophyll protoplast as acceptor, it is characterised in that
Comprise the following steps：
1）Using QIAGEN Plasmid Midi Kits extracting and purifying plasmid, after being adjusted to the concentration of 2 μ g/ μ L, -20
DEG C save backup；
2）Isolating and purifying for hybridized Chinese tuliptree mesophyll protoplast, obtains concentration for 105The protoplast of/mL；
3）Plasmid is taken in 2 mL centrifuge tubes, is subsequently adding protoplast, with hand gently by DNA and protoplast solution
Mix；40% PEG-4000 solution is slowly added to, soft mixing, place 15 min；W5 solution is added, soft mixing；1100
Rpm abandons supernatant after being centrifuged 2 min, adds WI solution gently to suspend protoplast, and is transferred on Tissue Culture Plate；
4）Tissue Culture Plate is placed on after 23 DEG C of dark 18 h of quiescent culture, is placed in fluorescence microscopy Microscopic observation.
2. the efficient moment gene expression side of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor according to claim 1
Method, it is characterised in that step 1）In, detailed process is as follows：
1）Well dispersed single bacterium colony in picking flat board, the LB culture medium of 2 mL being inoculated in containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 8 h of shaken cultivation；
2）The bacterium solution that shakes is taken, by 1:500 ratio is added to the LB culture medium of 50 mL containing 100 mg/L ampicillins
In, 37 DEG C, 300 rpm, 12～16 h of shaken cultivation；
3）15 minutes collects thalline precipitations, complete reject supernatant is centrifuged at 4 DEG C with 6000 × g；
4）With the resuspended thalline of 4 mL Buffer P1；
5）4 mL Buffer P2 are added, is turned upside down 4-6 time and mixes completely, room temperature standing 5 min of cracking；
6）The Buffer P3 of 4 mL precoolings is added, overturns 4-6 mixing immediately, ice bath staticly settles 15min；
7）30 min are centrifuged at 4 DEG C with >=20000 × g, by the supernatant containing plasmid be carefully transferred to one new clean
Centrifuge tube in；And 15 min are centrifuged at 4 DEG C again with >=20000 × g；
8）4 mL Buffer QBT are added on 100 centrifugal column of QIAGEN-tip, by Action of Gravity Field treat its flow completely through from
Stem；
9）By 7）In the supernatant containing plasmid be immediately transferred on 100 centrifugal column of QIAGEN-tip that balanced, rely on gravity
Effect plasmid is attached on the resin of centrifugal column；
10）Add 2 × 10 mL Buffer QC cleaning centrifugal column；
11）Add 5 mL Buffer QF that the plasmid combined on the resin of centrifugal column is eluted in a clean centrifuge tube；
12）To in plasmid eluent, add 3.5 mL room temperatures to place isopropanol precipitating DNA, after mixing completely, at 4 DEG C
30 min are centrifuged with >=15000 × g and precipitate DNA, careful reject supernatant；
13）70% ethanol that is placed with 2 mL room temperatures washs DNA precipitation, is centrifuged 10 min with >=15000 × g, carefully abandons
Except supernatant should not disturb the precipitation of centrifugation bottom of the tube；
14）After 5～10 min of drying at room temperature；The ddH for adding 30 uL to sterilize2O, dissolves plasmid；
15）After the concentration and purity of agarose gel electrophoresis and ultramicrospectrophotometer detection plasmid, 2 μ are adjusted to
After g/ μ L, -20 DEG C save backup.
3. the efficient moment gene expression side of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor according to claim 2
Method, it is characterised in that step 2）In, the culture body embryo seedling leaf that health stretches in blake bottle is taken, 0.5 is cut into blade～
The filament of 1mm, is then put in ready enzymolysis liquid rapidly, is placed under 28 DEG C of dark conditions, 45 rpm slight oscillatory, 3 hr
Digested；After the completion of enzymolysis, appropriate W5 solution is added, is jiggled, be filtered to remove using 300 mesh sieve and do not digest sufficiently
Blade；Filtrate is centrifuged 2 min through 1100 rpm, abandons supernatant, is washed after protoplast 2 times with the W5 solution of 2 mL ice precoolings, plus
Enter the resuspended protoplast of W5 solution of 2 mL ice precoolings, stand 30 min, 1100 rpm on ice and 2 min are centrifuged, supernatant is abandoned, plus
Enter MMg solution suspension protoplast, and protoplast concentration is adjusted to 105/mL.
4. the efficient moment gene expression side of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor according to claim 2
Method, it is characterised in that the preparation of enzymolysis liquid：Weigh 0.15 g, Macerozyme R10 of Cellulase R-10,0.1 g and
0.01 g of Pectolase Y-23 is in 6.25 mL, 0.8 M mannitol, 2.5 mL, 80 mM KCl, 1 mL, 200 mM MES
Solution in, stir evenly, put 4 DEG C of refrigerator overnight and fully dissolve；Add 0.1 mL, 1 M CaCl2, 0.01 g BSA, be settled to
10 mL；Above-mentioned solution is filtered with the sterilising filter of 0.45 m, standby.
5. the efficient moment gene expression side of the hybridized Chinese tuliptree with mesophyll protoplast as acceptor according to claim 2
Method, it is characterised in that step 3）In, 20 μ g plasmids conversion 1 × 104Individual cell.
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| CN107267549A (en) * | 2017-07-06 | 2017-10-20 | 江苏省中国科学院植物研究所 | A kind of method of the middle mesophyll protoplast of mountain China fir kind 406 separation, purifying and Efficient Conversion |
| CN108546670A (en) * | 2018-04-19 | 2018-09-18 | 贵州大学 | A kind of method for transformation of the preparation method of Sorghum Protoplast and the protoplast of preparation |
| CN110499278A (en) * | 2019-09-25 | 2019-11-26 | 南京林业大学 | A kind of ginkgo leaf protoplast separation purification and the method for instantaneous conversion |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107267549A (en) * | 2017-07-06 | 2017-10-20 | 江苏省中国科学院植物研究所 | A kind of method of the middle mesophyll protoplast of mountain China fir kind 406 separation, purifying and Efficient Conversion |
| CN107267549B (en) * | 2017-07-06 | 2021-01-05 | 江苏省中国科学院植物研究所 | Method for separating, purifying and efficiently converting mesophyll protoplast of taxus chinensis variety 406 |
| CN108546670A (en) * | 2018-04-19 | 2018-09-18 | 贵州大学 | A kind of method for transformation of the preparation method of Sorghum Protoplast and the protoplast of preparation |
| CN110499278A (en) * | 2019-09-25 | 2019-11-26 | 南京林业大学 | A kind of ginkgo leaf protoplast separation purification and the method for instantaneous conversion |
| 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 |
| CN115386530A (en) * | 2022-09-02 | 2022-11-25 | 西南大学 | Rapid preparation and transformation method of plant leaf protoplast, kit and application |
| CN117363558A (en) * | 2023-12-07 | 2024-01-09 | 三亚中国农业科学院国家南繁研究院 | Preparation method of protoplast, transformation method of protoplast and application of protoplast |
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