CN109370976A - Chinese small iris mesophyll protoplast and preparation method thereof - Google Patents
Chinese small iris mesophyll protoplast and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of Chinese small iris mesophyll protoplasts and preparation method thereof, it is handled by the pre-treatment and plasmolysis of Chinese small iris material, enzymatic hydrolysis Chinese small iris mesophyll tissue is combined using cellulase, macerozyme and pectase, effectively realize the preparation of Chinese small iris protoplast, it overcomes Chinese small iris protoplast and is difficult to free technical bottleneck, effectively establish a kind of high efficiency preparation method of Chinese small iris mesophyll protoplast.The present invention is to realize Chinese small iris fast genetic transformation, gene transient expression research etc. provides a kind of efficient operation sample platform, provides technical support to establish Chinese small iris cell engineering system progress Chinese small iris Protoplast cuhnre, protoplast fusion, protobiont genetic transformation, gene transient expression research etc..
Description
Technical field
The invention belongs to cell biology and field of biotechnology, and in particular to Chinese small iris mesophyll protoplast and its preparation side
Method.
Background technique
Plant protoplast has unique because it does not have the characteristics of cell wall relative to tissue, organ or individual level
A little: protobiont can get rid of cytoskeleton constraint and influence each other with intercellular first, can individually study a cell
Function and totipotency；Secondly, protoplast is good " cell " group of a homogeneity, a large amount of one can be obtained in a short time
The research object of cause property.Meanwhile protoplast can be used for the cytology and molecular biology research of different levels, protoplast one
A ideal unicellular system can study cell wall-deficient mutant, cell division and differentiation, intake organelle, virus infection machine
The fundamental researches such as reason, membrane permeability and ion transport, it can also be used to cell culture, protoplast fusion, protoplast heredity
The initiative of the new material of conversion etc..
In recent years, Skill of Plant Protoplasts is rapidly developed, and is mainly manifested in and is obtained again by protoplast
Raw plant and the floristics for establishing genetic conversion system are continuously increased, including cereal crops, vegetables, flowers, medicinal plant, fruit
Tree etc..Chinese small iris (Iris lactea var.chinensis), alias Ma Lian, kalimeris are that Iridaceae Jris perennial root is close
Clump herbaceous plant, is most study in irides, a kind of iris class ornamental plant being most widely used, and be me
Institute of state is exclusive, has many advantages and important application value.Flower, seed, the root of Chinese small iris can be used as medicine.Chinese small iris not only has weight
The medical value wanted, and due to its well developed root system, the green phase is long, and pattern is gorgeous, and the florescence is longer, seldom needs people after the planting of greenery patches
Work management has the characteristics such as drought resisting, cold-resistant, impoverishment tolerant, saline-alkali tolerant, be used as water shortage urban afforestation in recent years, slope protection fixes the sand,
Alkaline land improving, afforestation and the quality material of revegetation.Chinese small iris is as a kind of raw plant of typical ornamental types salt simultaneously
Object, well developed root system select to absorb and salinity are transported in selection by root cells, not only refuse to absorb excessive harmful salt ion, but also
Limitation transports it into the significant points such as blade.It can replace Taro Aso and produce paper, rope, leaf is basketry as fibre plant
The raw material of product, root can also make brush, unique ecological and biological characteristic and utility value, determine that it is green with city
Change and gardens construction to ecological harmonious environment friendly transformation, as city street green, public lawn plant effect and
Prospect is immeasurable.Therefore, in order to preferably develop and utilize Chinese small iris ecological functions and genetic resources, by the way that tradition is educated
Kind means, which combine improvement with modern molecular biology and cultivate new degeneration-resistant strong Chinese small iris germ plasm resource, is made more gardens
Skill character is fully utilized, especially technique for gene engineering, and completely new thinking is provided for its character improvement, is enabled people to
Directional operation some or certain objective traits are possibly realized.
As the Chinese small iris of fibre plant, organizationally efficient regeneration itself is exactly a restricted problem, and because it is numerous with seedling
It grows, progeny population gene heterozygosity is high, thus causes to establish the efficient genetic conversion system of Chinese small iris to be always that Chinese small iris biology is ground
The technical bottleneck studied carefully.Due to free barrier of the protoplast without plant cell wall, plasma membrane can directly absorb external source through processing
Substance (such as organelle, virus, DNA) frequently as the ideal receptor system of genetic transformation, thus is attempted to pass through protobiont approach
Obtaining Chinese small iris genetic conversion system is ideal selection, and Chinese small iris can be overcome to be difficult to the technical bottleneck of genetic transformation, fast run-up
Immediately Lin's protobiont genetic transfoumation, realizes the function of instantaneous verifying target gene, but establishes the premise of this technical system
Condition is to obtain a large amount of high quality Chinese small iris protoplasts.But it is not yet had been reported that about the research of Chinese small iris protoplast electrofusion at present.
Summary of the invention
The technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide a kind of efficient Chinese small iris plasm
Preparation establishes to break through the unicellular technical bottleneck for being difficult to separate and be difficult to carry out Chinese small iris genetic transformation of Chinese small iris
A kind of effective technical system for obtaining Chinese small iris protobiont, mentions for Chinese small iris protoplast genetic transformation, Protoplast cuhnre, fusion etc.
For technical support.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is as follows:
A kind of preparation method of Chinese small iris mesophyll protoplast, includes the following steps:
(1) the Chinese small iris blade of suitable seedling age is chosen as processing material, is sterilized on superclean bench after rinsing well, and
It is completely stand-by with aseptic water washing；
(2) the Chinese small iris blade that step (1) is cleaned is cut into spire silk；
(3) the spire silk that step (2) obtains is put into sterile plasmolysis liquid, is protected from light processing；
(4) the spire silk by step (3) by plasmolysis processing is put into sterile enzymolysis liquid, and sealing is protected from light enzymatic hydrolysis, when
It is placed on constant-temperature table and shakes when enzymolysis liquid greening, protoplast liberation is promoted to come out；
(5) in the cut tobacco solution of step (4) enzymolysis processing, the cell isotonic solution of equivalent is added according to 1:1 volume, shakes up
It is filtered afterwards using cell sieve, removes non-disintegrated tissue；It collects filtrate and is centrifuged, abandon supernatant collection protoplast pellet,
Then cell isotonic solution suspension protoplast is used, by micro- sem observation protoplast form, is collected completely green without broken and leaf
The protoplast that body is evenly distributed.
Preferably, in step (1), selected Chinese small iris blade seedling age is 30~60d.
The mode of the disinfection is that 70vt% dehydrated alcohol sterilizes 1min, then 5wt% sodium hypochlorite re-disinfection 10-
The influence that 15min, elimination pathogen and environmental microorganism separate protobiont.
Preferably, in step (2), the width of the spire silk is 1~2mm.
In step (3), the plasmolysis liquid composition are as follows: cell-protoplast cleaning solution+130g/L sorbierite+0.6g/
L morpholino b acid adjusts pH=5.6~6.0；The spire silk and plasmolysis liquid proportional are 1g:30ml；Be protected from light processing 2~
3h carries out plasmolysis, and cytoskeleton exposure is made to be easy to digest.
In step (4), the enzymolysis liquid composition are as follows: cell-protoplast cleaning solution+90g/L sorbierite+60g/L fiber
Plain enzyme R-10+4g/L macerozyme R-10+4g/L pectase Y-23+0.6g/L morpholino b acid adjusts pH=5.6~6.0.
Preferably, the spire silk and enzymatic hydrolysis liquid proportional be 1g:20ml, 22~28 DEG C of hydrolysis temperature, enzymolysis time 8~
12h；The temperature of the constant-temperature table is 22~28 DEG C, revolving speed 50rpm, and the concussion time is 30~60min.By enzymatic hydrolysis, disappear
Except cell wall fetters, it is easy to protobiont separate out.
In step (5), the cell isotonic solution composition are as follows: cell-protoplast cleaning solution+90g/L sorbierite+0.6g/L
Morpholino b acid adjusts pH=5.6~6.0.It is handled using isotonic solution and maintains the isotonic state of cell, prevent protobiont broken.
Preferably, the cell sieve is 300 mesh；Centrifugal rotational speed is 800rpm, is centrifuged 5min at 4 DEG C, in centrifugal process, is added
Speed is adjusted to 0 grade with deceleration.Damage of the external environment to protobiont is reduced using low-speed centrifugal, guarantees that it maintains cell viability.
The Chinese small iris mesophyll protoplast that above-mentioned preparation method is prepared is also within protection scope of the present invention.
The utility model has the advantages that
The present invention uses cellulose by chopping processing and plasmolysis processing to Chinese small iris spire piece in isotonic solution
To digest Chinese small iris mesophyll cell wall, the height for effectively realizing Chinese small iris mesophyll protoplast is gone for an outing for enzyme, macerozyme and pectase combination
From, overcome the high iris class ornamental plant protoplast of content of cellulose and be difficult to free technical bottleneck, establish it is a kind of efficiently
The method for preparing Chinese small iris mesophyll protoplast.Relevant cell engineering technology operation can be carried out on this basis, establish Chinese small iris original
The technical systems such as raw plastid culture, protobiont fusion, the instantaneous table of target gene and protoplast genetic transformation, it is raw to Chinese small iris cell
Object and molecular biology research, the initiative of Chinese small iris new germ plasm and rearing new variety are of great significance.
Detailed description of the invention
The present invention is done with reference to the accompanying drawings and detailed description and is further illustrated, of the invention is above-mentioned
And/or otherwise advantage will become apparent.
Fig. 1 is the preparation process figure of Chinese small iris protoplast of the present invention；A.45d seedling age Chinese small iris Reducing sugar；B.45d seedling
The Chinese small iris protoplast (Bar=10 μm) that age Chinese small iris vanes 3h is protected from light processing and 12h enzymolysis processing obtains.
Specific embodiment
According to following embodiments, the present invention may be better understood.
In following embodiment, used reagent composition is as follows:
Cell-protoplast cleaning solution (CPW) solution formula: KH2PO427.2mg/L；KNO3101.0mg/L；
CaCl2·2H2O 1480.0mg/L；MgSO4·7H2O 246.0mg/L；CuSO4·5H2O 0.025mg/L, adds ddH2O to 1L,
Adjusting pH is 5.6~6.0, and with the filter filtration sterilization of 0.22 μ l, 4 DEG C of filtrate are saved backup.
Plasmolysis formula of liquid: CPW solution+130g/L sorbierite+0.6g/L morpholino b acid (MES), pH be 5.6~
6.0；
The preparation of plasmolysis liquid: weighing 2.6g sorbierite and 0.012g morpholino b acid (MES) to be dissolved in 20ml CPW molten
Plasmolysis treatment fluid is prepared in liquid, pH is 5.6~6.0, and with the filter filtration sterilization of 0.22 μ l, 4 DEG C of filtrate are saved backup.
Isotonic formula of liquid: CPW solution+90g/L sorbierite+0.6g/L morpholino b acid (MES), pH 5.6~6.0；
The preparation of isotonic solution: weighing 4.5g sorbierite and 0.03g MES is dissolved in 50ml CPW solution and prepares isotonic solution, pH
It is 5.6~6.0, with the filter filtration sterilization of 0.22 μ l, 4 DEG C of filtrate are saved backup.
The preparation of enzymolysis liquid: based on CPW solution, cellulase R-10 (Cellulase is added wherein respectively
R10), macerozyme R-10 (Mecerozyme R10), pectase Y-23 (Pectolyase Y-23), sorbierite (Sorbitol)
It with morpholino b acid (MES), then mixes, tune pH is 5.6~6.0,55 DEG C of heating water bath 10min；It is used after being cooled to room temperature
The filter filtration sterilization of 0.22 μ l, 4 DEG C of filtrate save backup.Concentration of each component in enzymolysis liquid is as follows: sorbierite 90g/
L, cellulase R-10 are 60g/L, and macerozyme R-10 is 4g/L, and pectase Y-23 is 4g/L, MES 0.6g/L.Wherein, used
Cellulase, macerozyme and pectase are all from the original product of Japanese Yakult company production.
Embodiment 1
(1) choose robust growth seedling age be 30,45, the Chinese small iris spire of 60d as processing material, with putting after rinsing with ruinning water
Enter superclean bench, 70vt% dehydrated alcohol sterilizes 1min, 5wt% hypochlorite disinfectant 10-15min, and aseptic water washing is clean
For use；
(2) material for obtaining step (1) is placed on the filter paper of sterile drying in superclean bench, is cut with sterile
Blade is cut into the spire silk of 1~2mm wide along vein direction by knife or scalpel；
(3) in superclean bench, the spire silk cut is put into 1g:30ml ratio containing sterile plasmolysis liquid
In beaker, beaker then is wrapped up with sterile aluminium-foil paper, is carried out in the dark plasmolysis processing 2h；
(4) take the above-mentioned spire silk handled by plasmolysis respectively, be put into prepare in advance equipped with 20ml enzymolysis liquid
It in beaker, is then allowed to stand in 22~28 DEG C of environment, is protected from light enzymatic hydrolysis 8h, 22~28 DEG C of constant-temperature tables are placed in when enzymolysis liquid greening
On, revolving speed 50rpm, the concussion time is 30~60min, and protoplast liberation is promoted to come out；
(5) by the above-mentioned spire silk solution by enzymatic hydrolysis, isometric isotonic solution is added, is filtered, is gone with 300 mesh cell sieves
Except non-disintegrated tissue, then by 4 DEG C of filtrate, 800rpm, accelerates (Accel) and deceleration (Decel) to be adjusted to 0 grade, is centrifuged 5min,
Supernatant is removed, protoplast pellet is resuspended with isometric isotonic solution.A small amount of protoplast re-suspension liquid is taken, it is former with micro- sem observation
Raw plastide morphology, and (being shown in Table 1) is counted with blood counting chamber, collect the plasm being completely evenly distributed without broken and chloroplaset
Body, 4 DEG C save backup.
The yield of the different seedling age Chinese small iris spire separated protoplasts of table 1
| Seedling age | The plasmolysis time | Enzymolysis time | Protoplast yield (× 106A/g) |
| 30d | 2h | 8h | 5.82 |
| 45d | 2h | 8h | 6.50 |
| 60d | 2h | 8h | 5.32 |
Embodiment 2
(1) choose robust growth seedling age be 30,45, the Chinese small iris spire of 60d as processing material, with putting after rinsing with ruinning water
Enter superclean bench, 70vt% dehydrated alcohol sterilizes 1min, 5wt% hypochlorite disinfectant 10-15min, and aseptic water washing is clean
For use；
(2) material for obtaining step (1) is placed on the filter paper of sterile drying in superclean bench, is cut with sterile
Blade is cut into the spire silk of 1~2mm wide along vein direction by knife or scalpel；
(3) in superclean bench, the spire silk cut is put into 1g:30ml ratio containing sterile plasmolysis liquid
In beaker, beaker then is wrapped up with sterile aluminium-foil paper, is carried out in the dark plasmolysis processing 2h；
(4) take the above-mentioned spire silk handled by plasmolysis respectively, be put into prepare in advance equipped with 20ml enzymolysis liquid
It in beaker, is then allowed to stand in 22~28 DEG C of environment, is protected from light enzymatic hydrolysis 12h, 22~28 DEG C of constant temperature are placed in when enzymolysis liquid greening and are shaken
On bed, revolving speed 50rpm, the concussion time is 30~60min, and protoplast liberation is promoted to come out；
(5) by the above-mentioned spire silk solution by enzymatic hydrolysis, isometric isotonic solution is added, is filtered, is gone with 300 mesh cell sieves
Except non-disintegrated tissue, then by 4 DEG C of filtrate, 800rpm, accelerates (Accel) and deceleration (Decel) to be adjusted to 0 grade, is centrifuged 5min,
Supernatant is removed, protoplast pellet is resuspended with isometric isotonic solution.A small amount of protoplast re-suspension liquid is taken, it is former with micro- sem observation
Raw plastide morphology, and (being shown in Table 2) is counted with blood counting chamber, collect the plasm being completely evenly distributed without broken and chloroplaset
Body, 4 DEG C save backup.
The yield of the different seedling age Chinese small iris spire separated protoplasts of table 2
| Seedling age | The plasmolysis time | Enzymolysis time | Protoplast yield (× 106A/g) |
| 30d | 2h | 12h | 7.10 |
| 45d | 2h | 12h | 8.31 |
| 60d | 2h | 12h | 6.97 |
Embodiment 3
(1) choose robust growth seedling age be 30,45, the Chinese small iris spire of 60d as processing material, with putting after rinsing with ruinning water
Enter superclean bench, 70vt% dehydrated alcohol sterilizes 1min, 5wt% hypochlorite disinfectant 10-15min, and aseptic water washing is clean
For use；
(2) material for obtaining step (1) is placed on the filter paper of sterile drying in superclean bench, is cut with sterile
Blade is cut into the spire silk of 1~2mm wide along vein direction by knife or scalpel；
(3) in superclean bench, the spire silk cut is put into 1g:30ml ratio containing sterile plasmolysis liquid
In beaker, beaker then is wrapped up with sterile aluminium-foil paper, is carried out in the dark plasmolysis processing 3h；
(4) take the above-mentioned spire silk handled by plasmolysis respectively, be put into prepare in advance equipped with 20ml enzymolysis liquid
It in beaker, is then allowed to stand in 22~28 DEG C of environment, is protected from light enzymatic hydrolysis 8h, 22~28 DEG C of constant-temperature tables are placed in when enzymolysis liquid greening
On, revolving speed 50rpm, the concussion time is 30~60min, and protoplast liberation is promoted to come out；
(5) by the above-mentioned spire silk solution by enzymatic hydrolysis, isometric isotonic solution is added, is filtered, is gone with 300 mesh cell sieves
Except non-disintegrated tissue, then by 4 DEG C of filtrate, 800rpm, accelerates (Accel) and deceleration (Decel) to be adjusted to 0 grade, is centrifuged 5min,
Supernatant is removed, protoplast pellet is resuspended with isometric isotonic solution.A small amount of protoplast re-suspension liquid is taken, it is former with micro- sem observation
Raw plastide morphology, and (being shown in Table 3) is counted with blood counting chamber, collect the plasm being completely evenly distributed without broken and chloroplaset
Body, 4 DEG C save backup.
The yield of the different seedling age Chinese small iris spire separated protoplasts of table 3
| Seedling age | The plasmolysis time | Enzymolysis time | Protoplast yield (× 106A/g) |
| 30d | 3h | 8h | 5.81 |
| 45d | 3h | 8h | 7.03 |
| 60d | 3h | 8h | 5.69 |
Embodiment 4
(1) choose robust growth seedling age be 30,45, the Chinese small iris spire of 60d as processing material, with putting after rinsing with ruinning water
Enter superclean bench, 70vt% dehydrated alcohol sterilizes 1min, 5wt% hypochlorite disinfectant 10-15min, and aseptic water washing is clean
For use；
(2) material for obtaining step (1) is placed on the filter paper of sterile drying in superclean bench, is cut with sterile
Blade is cut into the spire silk of 1~2mm wide along vein direction by knife or scalpel；
(3) in superclean bench, the spire silk cut is put into 1g:30ml ratio containing sterile plasmolysis liquid
In beaker, beaker then is wrapped up with sterile aluminium-foil paper, is carried out in the dark plasmolysis processing 3h；
(4) take the above-mentioned spire silk handled by plasmolysis respectively, be put into prepare in advance equipped with 20ml enzymolysis liquid
It in beaker, is then allowed to stand in 22~28 DEG C of environment, is protected from light enzymatic hydrolysis 12h, 22~28 DEG C of constant temperature are placed in when enzymolysis liquid greening and are shaken
On bed, revolving speed 50rpm, the concussion time is 30~60min, and protoplast liberation is promoted to come out；
(5) by the above-mentioned spire silk solution by enzymatic hydrolysis, isometric isotonic solution is added, is filtered, is gone with 300 mesh cell sieves
Except non-disintegrated tissue, then by 4 DEG C of filtrate, 800rpm, accelerates (Accel) and deceleration (Decel) to be adjusted to 0 grade, is centrifuged 5min,
Supernatant is removed, protoplast pellet is resuspended with isometric isotonic solution.A small amount of protoplast re-suspension liquid is taken, it is former with micro- sem observation
Raw plastide morphology, and (being shown in Table 4) is counted with blood counting chamber, collect the plasm being completely evenly distributed without broken and chloroplaset
Body, 4 DEG C save backup.
The yield of the different seedling age Chinese small iris spire separated protoplasts of table 4
| Seedling age | The plasmolysis time | Enzymolysis time | Protoplast yield (× 106A/g) |
| 30d | 3h | 12h | 7.82 |
| 45d | 3h | 12h | 8.33 |
| 60d | 3h | 12h | 7.32 |
By testing implementation data above, show that the Chinese small iris seedling leaves of 45d seedling age by chopping, are protected from light plasmolysis
After digesting 12h, it is best to obtain protoplast yield by 3h.Implement according to preferred embodiment, it is seen that the Chinese small iris mesophyll protoplast of acquisition
In suspension, protoplast form is full, and chloroplaset is evenly distributed, and protoplast fragment is few (see Fig. 1), illustrates under this condition
Can obtain has the Chinese small iris of higher vigor primary, for the Chinese small iris Protoplast cuhnre, protoplast fusion, primary based on this technology
Body genetic transformation, gene transient expression research etc. provide technical support.
The present invention provides the thinkings and method of a kind of Chinese small iris mesophyll protoplast and preparation method thereof, implement the skill
There are many method and approach of art scheme, the above is only a preferred embodiment of the present invention, it is noted that this technology is led
For the those of ordinary skill in domain, various improvements and modifications may be made without departing from the principle of the present invention, these
Improvements and modifications also should be regarded as protection scope of the present invention.The available prior art of each component part being not known in the present embodiment
It is realized.
Claims (10)
1. a kind of preparation method of Chinese small iris mesophyll protoplast, which comprises the steps of:
(1) the Chinese small iris blade of suitable seedling age is chosen as processing material, is sterilized on superclean bench after rinsing well, and use nothing
Bacterium water is rinsed well for use；
(2) the Chinese small iris blade that step (1) is cleaned is cut into spire silk；
(3) the spire silk that step (2) obtains is put into sterile plasmolysis liquid, is protected from light processing；
(4) the spire silk by step (3) by plasmolysis processing is put into sterile enzymolysis liquid, and sealing is protected from light enzymatic hydrolysis, works as enzymatic hydrolysis
It is placed on constant-temperature table and shakes when liquid greening, protoplast liberation is promoted to come out；
(5) in the cut tobacco solution of step (4) enzymolysis processing, the cell isotonic solution of equivalent is added according to 1:1 volume, is adopted after shaking up
It is filtered with cell sieve, removes non-disintegrated tissue；It collects filtrate and is centrifuged, abandon supernatant collection protoplast pellet, then
It is collected completely by micro- sem observation protoplast form without broken and chloroplaset point with cell isotonic solution suspension protoplast
The uniform protoplast of cloth.
2. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that selected in step (1)
The Chinese small iris blade seedling age taken is 30~60d.
3. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (1)
The mode of disinfection is that 70vt% dehydrated alcohol sterilizes 1min, then 5wt% sodium hypochlorite re-disinfection 10-15min.
4. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (2)
The width of spire silk is 1~2mm.
5. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (3)
Plasmolysis liquid composition are as follows: cell-protoplast cleaning solution+130g/L sorbierite+0.6g/L morpholino b acid adjusts pH=
5.6~6.0；The spire silk and plasmolysis liquid proportional are 1g:30ml；It is protected from light 2~3h of processing.
6. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (4)
Enzymolysis liquid composition are as follows: cell-protoplast cleaning solution+90g/L sorbierite+60g/L cellulase R-10+4g/L macerozyme R-10
+ 4g/L pectase Y-23+0.6g/L morpholino b acid adjusts pH=5.6~6.0.
7. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (4)
Spire silk and enzymatic hydrolysis liquid proportional be 1g:20ml, 22~28 DEG C of hydrolysis temperature, 8~12h of enzymolysis time；The temperature of the constant-temperature table
Degree is 22~28 DEG C, revolving speed 50rpm, and the concussion time is 30~60min.
8. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (5)
Cell isotonic solution composition are as follows: cell-protoplast cleaning solution+90g/L sorbierite+0.6g/L morpholino b acid adjusts pH=5.6
~6.0.
9. the preparation method of Chinese small iris mesophyll protoplast according to claim 1, which is characterized in that described in step (5)
Cell sieve is 300 mesh；Centrifugal rotational speed is 800rpm, is centrifuged 5min at 4 DEG C, in centrifugal process, accelerates to be adjusted to 0 grade with slowing down.
10. the Chinese small iris mesophyll protoplast that any one preparation method is prepared in claim 1~9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358722A (en) * | 2019-08-16 | 2019-10-22 | 中南林业科技大学 | Preparation method of litsea cubeba mesophyll cell protoplast |
| CN110577925A (en) * | 2019-10-16 | 2019-12-17 | 中国农业科学院生物技术研究所 | Composition and method for preparing rice root protoplast |
| CN112063576A (en) * | 2020-09-23 | 2020-12-11 | 山东师范大学 | Method for rapidly extracting epidermal cell protoplast by taking tender and complete plant leaves as material |
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| CN112063576A (en) * | 2020-09-23 | 2020-12-11 | 山东师范大学 | Method for rapidly extracting epidermal cell protoplast by taking tender and complete plant leaves as material |
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