CN107964529A - Horehound method for preparing protoplast - Google Patents
Horehound method for preparing protoplast Download PDFInfo
- Publication number
- CN107964529A CN107964529A CN201610908852.XA CN201610908852A CN107964529A CN 107964529 A CN107964529 A CN 107964529A CN 201610908852 A CN201610908852 A CN 201610908852A CN 107964529 A CN107964529 A CN 107964529A
- Authority
- CN
- China
- Prior art keywords
- horehound
- protoplast
- preparing
- fixer
- cleaning
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Abstract
The invention discloses a kind of horehound method for preparing protoplast, follow the steps below：Horehound is chosen, horehound 3~4 times described in tissue-wash solution soaking and washing, soak 30~40s every time, absorb the moisture of horehound blade surface after cleaning with filter paper；The horehound is cut to 3~6mm2, centrifuge tube is put into, the horehound at least 20min is fixed with spire fixer；The spire fixer is absorbed with micropipette rifle, and the tissue-wash solution described in step 1 cleans the horehound 2~3 times；It is less than 10 μm of olm in dark or light intensity‑2s‑1Under conditions of, with when gained horehound at least 0.5 is small in 23~28 DEG C of mixed enzymolysis liquid enzymolysis step 2, obtain horehound protoplast；The horehound method for preparing protoplast of the present invention is easy to operate, time-consuming short, avoids the broken of horehound protoplast during experimental implementation, the shortcomings of physiological status changes.
Description
Technical field
The invention belongs to plant protoplast preparing technical field, relates in particular to a kind of horehound protoplast and prepares
Method.
Background technology
Horehound is Labiatae Marrubium plant, widely distributed, aboundresources.According to pharmacopeia《Plant medicine will in Jiangsu》Record,
" lantern section, is gynecological drug.”《The positive allusion quotation of anaesthetic》Record it and possess the effect of anti-inflammatory diuresis.Modern pharmacology research confirms that it is changing
Kind blood and Lymph microcirculation obstacle, myocardial preservation, anti-inflammatory, anti-oxidant etc. have exact pharmacological action.However, its is thin
Born of the same parents' biological study still lacks so far to be carried out comprehensively.Tradition machinery method prepares horehound protoplast, and there are low output, method are numerous
It is trivial, be only limited to specifically tissue (storage tissue in height vacuolization, larger cell) the shortcomings that.
The content of the invention
In view of the deficiencies of the prior art, it is an object of the invention to provide a kind of preparation method of horehound protoplast,
The preparation method can obtain the ideal conditions and parameter for preparing horehound protoplast.
The purpose of the present invention is what is be achieved by following technical proposals.
A kind of horehound method for preparing protoplast, follows the steps below：
Step 1, the pretreatment of horehound：Selection horehound, horehound 3~4 times described in tissue-wash solution soaking and washing,
30~40s of immersion every time, absorbs the moisture of horehound blade surface with filter paper after cleaning；Wherein, the tissue-wash solution is by going
The compound of ionized water and following concentration forms：
8~9g/l of NaCl
Na2HPO4·7H232~34g/l of O
NaH2PO410~12g/l
Step 2, the horehound is cut to 3~6mm2, centrifuge tube is put into, the horehound is fixed extremely with spire fixer
Few 20min；The spire fixer is absorbed with micropipette rifle, and the tissue-wash solution described in step 1 cleans the horehound 2
~3 times；Wherein, the spire fixer by following volumetric concentrations material composition：
Absolute ethyl alcohol 74~75%
Glacial acetic acid 24~25%
Hydrochloric acid：0.9~1.1%, wherein, the concentration of the hydrochloric acid is 0.9~1.1mol/L；
In the step 2, the 20~45min of horehound is fixed with spire fixer.
Step 3, it is less than 10 μm of olm in dark or light intensity-2s-1Under conditions of, with 23~28 DEG C of mixed enzymolysis liquid enzyme
When gained horehound at least 0.5 is small in solution step 2, horehound protoplast is obtained；Wherein described mixed enzymolysis liquid is by following dense
The material composition of degree：
In the step 3, in enzymolysis process, at interval of 10 minutes mixed enzymolysis rocked immersed with the horehound
Liquid.
In the above-mentioned technical solutions, after the step 3, with 20~25 DEG C of protoplast cleaning solution eccentric cleaning institute
State horehound protoplast 3~4 times, the protoplast cleaning solution is made of the compound of deionized water and following concentration：
In the above-mentioned technical solutions, described in eccentric cleaning during horehound protoplast, 20~25 DEG C of protoplast is washed
The bottom that liquid gently squeezes into centrifuged pellet is washed, be suspended the precipitation.
In the above-mentioned technical solutions, each eccentric cleaning 3~5 minutes.
In the above-mentioned technical solutions, relative centrifugal force is 2000~2500 × g.
Horehound method for preparing protoplast of the invention is easy to operate, takes short (such as two hours), avoids reality
The shortcomings of testing the broken of Summer Solstice grassland life plastid, physiological status change in operating process；On the other hand, Summer Solstice grassland of the invention
Reagent price used in raw plastid preparation method is cheap, easily obtains, and yield is high, easy to the experimental study of researcher.It is logical
Cross to many research such as the composition of horehound physiological status, enzyme liquid and enzymolysis time, there is provided a set of more complete summer
To the technology of preparing of careless protoplast.
Brief description of the drawings
Fig. 1 is the horehound protoplast (μ of scale=25 obtained with the horehound method for preparing protoplast of the present invention
m)；
Fig. 2 is that the horehound protoplast that the present invention obtains is analyzed carefully after DAPI is dyed through panorama multidimensional cytoanalyze
Born of the same parents' quantity result；
Fig. 3 is that the horehound protoplast that the present invention obtains detects after DAPI is dyed through panorama multidimensional cytoanalyze
Picture；
Fig. 4 is displaing micro picture (scale=25 μm) of the horehound protoplast of the invention obtained after DAPI is dyed；
Fig. 5 is the horehound protoplast obtained light field photo (scale overlapping with fluorescence after DAPI is dyed of the invention
=25 μm).
Embodiment
In the embodiment of the present invention, horehound is gathered from Tianjin Xiqing District Tianjin Normal University campus.
Compound medicine used in the embodiment of the present invention is bought from Tianjin Xiqing District Ta Kela experimental articles business department, pure
Degree is 99.5%；Reagent, cellulase and pectase are equally bought from Tianjin Xiqing District Ta Kela experimental articles business department.
Below in conjunction with the accompanying drawings technical scheme is further illustrated with specific embodiment.
A kind of horehound method for preparing protoplast, follows the steps below：
Step 1, the pretreatment of horehound：Selection is sufficiently spread out, the horehound of fresh spire, is soaked with tissue-wash solution clear
Wash horehound 3~4 times, soak 30~40s every time, suitably absorb the moisture of horehound blade surface after cleaning with filter paper；Wherein,
Tissue-wash solution is made of the compound of deionized water and following concentration：
NaCl 8.90g/l
Na2HPO4·7H2O 32.73g/l
NaH2PO4 10.82g/l
Step 2, the blade of three pieces horehound is gripped, is cut the horehound to 5mm with scissors2, 1.5ml centrifuge tubes are put into,
The horehound 20min is fixed with 800 μ l spire fixers；Spire fixer is absorbed with micropipette rifle after 20min, and uses step
Tissue-wash solution described in 1 cleans 2~3 times；Wherein, spire fixer by following volumetric concentrations material composition：
Absolute ethyl alcohol 74.5%
Glacial acetic acid 24.5%
Hydrochloric acid：1%, wherein, the concentration of hydrochloric acid is 1mol/L；
Step 3, it is less than 10 μm of olm in dark or light intensity-2s-1Under the conditions of, with 200 μ l, 26 DEG C of mixed enzymolysis liquid enzyme
When the horehound 0.5 obtained in solution step 2 is small, horehound protoplast is obtained；Wherein, mixed enzymolysis liquid by following concentration thing
Matter forms：
Also, in enzymolysis process, at interval of gently shake within 10 minutes it is several under immersed with horehound mixed enzymolysis liquid.
After step 3, with 600 μ l, 20 DEG C of protoplast cleaning solution eccentric cleaning horehound protoplast 3~4 times
(using first dallying to 20 DEG C before centrifuge cleaning), each eccentric cleaning 3 minutes (relative centrifugal force is 2500 × g).Centrifugation is clear
The method for washing horehound protoplast is：20~25 DEG C of protoplast cleaning solution is gently squeezed into the bottom of centrifuged deposit
Portion, be suspended precipitation.
Wherein, protoplast cleaning solution is made of the compound of deionized water and following concentration：
The dyeing of horehound：During last time cleaning horehound protoplast, supernatant liquor is removed with micropipette rifle, is remained
It is remaining to a drop DAPI coloring agents are added during 60 μ l, under dark condition, dye 30min at 37 DEG C and (gently shaken every 10 minutes several
Under).3min (2500 turns/min) is centrifuged after dyeing at 20 DEG C, is cleaned 3 times with protoplast cleaning solution.
The observation of horehound protoplast：Horehound protoplast is resuspended with 60 μ l protoplasts cleaning solutions, and uses liquid relief
Rifle piping and druming is uniform, and protoplast cleaning solution and sterile water that a drop contains dyeing horehound protoplast are taken in glass slide with dropper
On, coverslip is covered, in just putting the lower 20 times of observations of fluorescence microscope, fluorescence microscope model Leica (Lycra) DM5000, sees
The displaing micro picture examined is as shown in Figure 1.400 mesh cell screen clothes of the remaining protoplast cleaning solution containing horehound protoplast
Filtering, is put into 1.5ml centrifuge tubes, with panorama multidimensional cytoanalyze (Merck Millipore companies, FlowSight by filtrate
Multidimensional panorama flow cytometer) analysis shows that, as shown in figure 3, passage 1 (Ch01) is to be observed under light field as a result, passage 7 (Ch07)
It is the nucleus dyed through DAPI observed when excitation wavelength is 488nm down, with the horehound protoplast of the method preparation
Content is 9642599.51/mL, as shown in Figure 2.Fig. 4 is the horehound protoplast of this method acquisition after DAPI is dyed
Displaing micro picture (scale=25 μm), can clearly show the integrality of protoplasm somatocyte, blue-fluorescence is carried in picture
Round dot is probably protoplast or cell fragment, it is necessary to be further determined that by the way that light field is overlapping with fluorescence；Fig. 5 obtains for this method
Horehound protoplast DAPI dyeing after the light field photo (scale=25 μm) overlapping with fluorescence, what picture was shown can be overlapping
Blue-fluorescence round dot be protoplast, may thereby determine that and the morphological feature of protoplast can be clearly seen, and yield
It is higher.
Exemplary description has been done to the present invention above, it should explanation, in the situation for the core for not departing from the present invention
Under, any simple deformation, modification or other skilled in the art can not spend the equivalent substitution of creative work equal
Fall into protection scope of the present invention.
Claims (8)
1. a kind of horehound method for preparing protoplast, it is characterised in that follow the steps below：
Step 1, the pretreatment of horehound：Selection horehound, horehound 3~4 times described in tissue-wash solution soaking and washing, every time
30~40s is soaked, absorbs the moisture of horehound blade surface after cleaning with filter paper；Wherein, the tissue-wash solution is by deionization
The compound of water and following concentration forms：
8~9g/l of NaCl
Na2HPO4·7H232~34g/l of O
NaH2PO410~12g/l
Step 2, the horehound is cut to 3~6mm2, centrifuge tube is put into, the horehound is fixed at least with spire fixer
20min；Absorb the spire fixer with micropipette rifle, and the tissue-wash solution described in step 1 clean the horehound 2~
3 times；Wherein, the spire fixer by following volumetric concentrations material composition：
Absolute ethyl alcohol 74~75%
Glacial acetic acid 24~25%
Hydrochloric acid：0.9~1.1%, wherein, the concentration of the hydrochloric acid is 0.9~1.1mol/L；
Step 3, it is less than 10 μm of olm in dark or light intensity-2s-1Under conditions of, with 23~28 DEG C of mixed enzymolysis liquid enzymolysis step
When gained horehound at least 0.5 is small in 2, horehound protoplast is obtained；Wherein described mixed enzymolysis liquid by following concentration thing
Matter forms：
2. horehound method for preparing protoplast according to claim 1, it is characterised in that in the step 2, with children
Leaf fixer fixes the 20~45min of horehound.
3. horehound method for preparing protoplast according to claim 1, it is characterised in that in the step 3, in enzyme
In solution preocess, at interval of the 10 minutes mixed enzymolysis liquid rocked immersed with the horehound.
4. horehound method for preparing protoplast according to claim 1, it is characterised in that after the step 3, use
Horehound protoplast 3~4 times described in 20~25 DEG C of protoplast cleaning solution eccentric cleaning, the protoplast cleaning solution by
The compound of deionized water and following concentration forms：
5. horehound method for preparing protoplast according to claim 4, it is characterised in that horehound described in eccentric cleaning
During protoplast, 20~25 DEG C of protoplast cleaning solution is gently squeezed into the bottom of centrifuged pellet, described in suspension
Precipitation.
6. horehound method for preparing protoplast according to claim 5, it is characterised in that each 3~5 points of eccentric cleaning
Clock.
7. horehound method for preparing protoplast according to claim 6, it is characterised in that relative centrifugal force for 2000~
2500×g。
8. horehound protoplast prepared by a kind of horehound method for preparing protoplast as described in claim 1~7 is in list
Application in Cellular gels electrophoretic analysis, fluorescent marker, horehound cell fusion and transgenic technology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610908852.XA CN107964529B (en) | 2016-10-19 | 2016-10-19 | Method for preparing Xiajugao grassland protoplast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610908852.XA CN107964529B (en) | 2016-10-19 | 2016-10-19 | Method for preparing Xiajugao grassland protoplast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107964529A true CN107964529A (en) | 2018-04-27 |
| CN107964529B CN107964529B (en) | 2021-03-02 |
Family
ID=61996793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610908852.XA Expired - Fee Related CN107964529B (en) | 2016-10-19 | 2016-10-19 | Method for preparing Xiajugao grassland protoplast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107964529B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474231A (en) * | 2019-01-23 | 2020-07-31 | 天津师范大学 | Method for detecting duckweed protoplast DNA damage degree by using single cell gel electrophoresis technology |
| CN111484968A (en) * | 2019-01-28 | 2020-08-04 | 天津师范大学 | Preparation method of rehmannia root protoplast |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505004A (en) * | 2011-10-18 | 2012-06-20 | 通化师范学院 | Extraction and fusion method for strawberry protoplast |
| CN105316275A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Extraction method of halogeton mesophyll cell vacuoles |
| CN105670985A (en) * | 2016-04-22 | 2016-06-15 | 中国科学院华南植物园 | Camellia sinensis flower protoplast, preparation method therefor and application thereof |
-
2016
- 2016-10-19 CN CN201610908852.XA patent/CN107964529B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505004A (en) * | 2011-10-18 | 2012-06-20 | 通化师范学院 | Extraction and fusion method for strawberry protoplast |
| CN105316275A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Extraction method of halogeton mesophyll cell vacuoles |
| CN105670985A (en) * | 2016-04-22 | 2016-06-15 | 中国科学院华南植物园 | Camellia sinensis flower protoplast, preparation method therefor and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 牛奎举等: "禾本科牧草及草坪草原生质体培养与融合技术研究进展", 《草地学报》 * |
| 陈泽雄等: "卡特兰叶片原生质体分离条件的研究", 《西南大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474231A (en) * | 2019-01-23 | 2020-07-31 | 天津师范大学 | Method for detecting duckweed protoplast DNA damage degree by using single cell gel electrophoresis technology |
| CN111484968A (en) * | 2019-01-28 | 2020-08-04 | 天津师范大学 | Preparation method of rehmannia root protoplast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107964529B (en) | 2021-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107490511A (en) | A kind of haematoxylin dyeing liquid and HE colouring methods | |
| CN109338493A (en) | A kind of method that rice straw prepares Lyocell fibers for raw material | |
| CN106769315B (en) | A kind of paraffin section production method of hemp callus | |
| CN107964529A (en) | Horehound method for preparing protoplast | |
| CN109136167A (en) | The preparation method of lily mesophyll protoplast | |
| CN103920469A (en) | Preparation method of citric acid silk adsorbent | |
| CN104483178B (en) | The preparation method of epinephelus akaara adult fish chromosome | |
| CN104389032A (en) | Degumming method applicable to green fluorescent protein-containing bave | |
| CN107167350A (en) | A kind of preparation method of the paraffin section of eggplant rhizome portion tissue | |
| CN107923821A (en) | Observe preparation of specimen cell holding substrate holder and the external member including it and the production method for observing sample | |
| CN103808550B (en) | A kind of Myxosporean dyeing method of seal being easy to morphological observation | |
| CN109137133B (en) | Preparation method of loofah sponge cellulose/chitosan composite fiber | |
| CN103278367B (en) | Fast diagnosis method for liberobacter asiaticum | |
| CN112679769A (en) | High-transparency low-haze beta-chitin nanofiber flexible film and preparation method thereof | |
| CN103342826A (en) | Preparation method of chitin nano-fiber/montmorillonite composite membrane material | |
| CN102771394A (en) | Method for cloning and culturing seaweed gametophytes | |
| CN105432598A (en) | Liquid-based cell preservation liquid and preparation method therefor | |
| CN108949665B (en) | Preparation method of lily petal protoplast | |
| CN107964530B (en) | Duckweed protoplast preparation method | |
| CN105754927A (en) | Preparation method of gracilaria salicornia protoplast | |
| CN108593392B (en) | Vaginal secretion staining solution and preparation method thereof | |
| CN109321990A (en) | A kind of preparation method of super-hydrophobic Lyocell fibers | |
| CN103130902B (en) | Method of extracting polysaccharide from pine pollen | |
| CN104568556A (en) | Staining method of ciliates | |
| CN106644644A (en) | Quick and low-toxic mosquito paraffin section manufacturing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210302 Termination date: 20211019 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |