CN106546461A - A kind of Citrus phloem sap preparation method - Google Patents
A kind of Citrus phloem sap preparation method Download PDFInfo
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- CN106546461A CN106546461A CN201610976797.8A CN201610976797A CN106546461A CN 106546461 A CN106546461 A CN 106546461A CN 201610976797 A CN201610976797 A CN 201610976797A CN 106546461 A CN106546461 A CN 106546461A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- Sampling And Sample Adjustment (AREA)
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Abstract
The invention discloses a kind of Citrus phloem sap preparation method：From tree body clip branch, hermetic bag labelling is preserved and to be put into ultra cold storage freezer frozen；Branch blade is cut off when extracting juice, will be rinsed well outside branch with sterile deionized water and the water on surface is wiped away, branch bark is longitudinally scratched with blade, then bark is peeled away with woody part, retain the bark part with phloem tissue；Bark is cut into into segment and loads liner separating pipe, then liner separating pipe is placed in into external support collecting pipe, then separated with refrigerated centrifuger；After the completion of centrifugation, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem sap.This method sampling is simple, and ultralow temperature freeze thawing had both kept the biological activity of sample, is easy to branch phloem and xylem to be easily isolated, improves the extraction ratio of phloem sap.
Description
Technical field
The present invention relates to plant physiology, pathology, technological field of biochemistry, more particularly to a kind of Citrus phloem sap system
Preparation Method.
Background technology
Citruss fruit tree phloem plays a significant role in nutrition transport, information transmission and defense reaction, phloem juice
Containing saccharide, protein, aminoacid, inorganic salt, various RNAs and hormone etc. in liquid, for research plant physiology and biochemistry, nutrition
There is important researching value with the aspect such as pathology.Traditional phloem sap acquisition methods are mainly the aphid kiss skill of handling needles and bleeding
Method.
Aphid kisses the skill of handling needles：Using insecticide mouthpart --- the method that kiss pin collects phloem sap, sucking insecticide mouthpart can
Help its kiss to be needled into phloem sieve tube molecule to secrete pectase, after the kiss of insecticide is needled into sieve tube element, use CO2By its
Anesthesia, cuts off parent and leaves kiss pin.Due to the presence of screen casing normal pressure, phloem sap can continuously from kiss pin stream
Go out, the collection phloem sap method is used for the succulence class plant such as vegetable and field crop, is rarely used in fruit tree kind plant, receive
Collection amount is few, and operation difficulty is high, and repeatability is bad.
Bleeding method：The stem of plant is cut with blade, the juice for being flowed out from the stem of root side naturally is collected, what the method was collected
Not only there is phloem sap, while substantial amounts of xylem sap and Cell sap are mixed with, and many plants pass through the bleeding method very
It is difficult to obtain nature effluent.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes a kind of Citrus phloem sap preparation method, has
Body step is as follows：
(1) from tree body clip branch, per plant takes 3-5 branch, and hermetic bag labelling is preserved；
(2) the sample branch that step (1) is obtained is put into into ultra cold storage freezer rapidly frozen；
(3) preparation of liner separating pipe；
(4) preparation of external support collecting pipe；
(5) the sample branch of step (2) is taken out from ultra cold storage freezer, branch blade, Ran Houyong is removed with clean shears
Sterile deionized water will be rinsed well outside branch, is wiped away the water on surface with aseptic filter paper, and this process should be complete within 3-5 minutes
Into；
(6) branch bark is longitudinally scratched with aseptic sharp blade, as deep as xylem, then bark is shelled with woody part
Leave, remove xylem, retain the bark part with phloem tissue；
(7) bark is cut into into segment with the shears or blade of sterilizing, is prepared by a branch of loading step (3) of 5-10 segment
Good liner separating pipe, then liner separating pipe is placed in be formed in the external support collecting pipe that step (4) is prepared is collected by centrifugation
Pipe, is then placed within pre-cooling on ice；
(8) the effective refrigerated centrifuger that is collected by centrifugation that step (7) is prepared is separated；
(9) after the completion of being centrifuged, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem juice
Liquid；
(10) supernatant in collecting pipe is supported with the careful suck up outside of sterile pipette tip, another sterilizing is transferred to clean
In net frozen centrifuge tube, and save backup.
Preferably, the long 10-20cm of branch, diameter 0.5cm in the step (1).
Preferably, the temperature of the ultra cold storage freezer in the step (2) is -70 DEG C and the frozen time is 2-6 hours.
Preferably, the segment length being cut in the step (7) is 0.6-1.0cm.
Preferably, in the step (8) refrigerated centrifuger working condition：4 DEG C, 12000rpm × 15min.
Preferably, saving backup in the step (10) is located in the ultra cold storage freezer that temperature is -70 DEG C.
Compared with the prior art, the present invention has the beneficial effect that：
1st, the material involved by this technology method is the convenient versatile material for easily obtaining of general biology laboratory, it is only necessary to
Do simple transformation or process；This method is not only applicable to the Citrus nursery stock of greenhouse or solarium's culture, is also applied for field cultivation
Various adult mandarin tree.
2nd, sampling request is simple, and branch sample had both kept the biological activity of sample by ultralow temperature freeze thawing, was easy to again
Branch phloem and xylem are easily isolated, it is possible to which a large amount of phloem saps are collected in extraction, will not be oozed by xylem
Go out the impact of liquid.
3rd, using prepare phloem sap be collected by centrifugation device and high-speed low temperature centrifugation (4 DEG C, 12000rpm ×
15min), it is possible to achieve the isolated phloem sap of one-step method simultaneously goes the removal of impurity, overall process to be kept at low temperature completely
Sample biological activity.
4th, Sample Preparation Procedure standardization, simple to operate, Subjective Factors are few, the high-volume short time can process sample
Product, facilitate scientific research.
Description of the drawings
Structural representations of the Fig. 1 for liner separating pipe；
Structural representations of the Fig. 2 for external support collecting pipe；
Fig. 3 is the structural representation that pipe is collected by centrifugation；
Schematic diagrams of the Fig. 4 for sample branch；
Fig. 5 liners separating pipe fills thing schematic diagram；
Fig. 6 is the state diagram that juice is collected by centrifugation；
In figure：1- liner separating pipes, 2- weepage holes, 3- external support collecting pipes, 4- be collected by centrifugation pipe, 5- sample branches,
6- xylem, 7- phloems.
Specific embodiment
The present invention is further explained with reference to specific embodiment.
Embodiment 1
The present invention proposes a kind of Citrus phloem sap preparation method, comprises the following steps that：
Experimental subject：The Citrus nursery stock (2 years age of trees) of hot-house culture
(1) from the long 10cm of tree body clip, the branch of diameter 0.5cm, per plant takes 3 branches, and hermetic bag labelling is preserved；
(2) step (1) acquisition sample branch is put into into -70 DEG C of ultra cold storage freezers rapidly frozen 2 hours；
(3) preparation of liner separating pipe：Prepare the clean 0.6ml centrifuge tubes of sterilizing, centrifugation bottom of the tube needs to use nothing in advance
Bacterium large size syringe needle stabs the circular weepage holes of an a diameter of 1mm, cuts off lid；
(4) preparation of external support collecting pipe：Prepare the clean 1.5ml centrifuge tubes of sterilizing, make the tube wall of 1.5ml centrifuge tubes
Edge support just as the liner separating pipe mouth of pipe, and can be enclosed within outside liner separating pipe；
(5) the sample branch of step (2) is taken out from ultra cold storage freezer, branch blade, Ran Houyong is removed with clean shears
Sterile deionized water will be rinsed well outside branch, is wiped away the water on surface with aseptic filter paper, and this process should be complete in 3 minutes
Into；
(6) branch bark is longitudinally scratched with aseptic sharp blade, as deep as xylem, then bark is shelled with woody part
Leave, remove xylem, retain the bark part with phloem tissue；
(7) bark is cut into the segment of 0.6cm with the shears or blade of sterilizing, with 5 segments as a branch of loading step (3)
The liner separating pipe for preparing, will be equipped with sample liner separating pipe be placed in external support collecting pipe formed pipe is collected by centrifugation,
Then pre-cooling on ice is placed in pipe is collected by centrifugation；
(8) the effective refrigerated centrifuger that is collected by centrifugation that step (7) is prepared is separated, centrifugal work condition：4 DEG C,
12000rpm×15min；
(9) after the completion of being centrifuged, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem juice
Liquid；
(10) supernatant in collecting pipe is supported with the careful suck up outside of sterile pipette tip, another sterilizing is transferred to clean
In net frozen centrifuge tube, and it is stored in standby in -70 DEG C of ultra cold storage freezer.
Embodiment 2
The present invention proposes a kind of Citrus phloem sap preparation method, comprises the following steps that：
Experimental subject：Adult mandarin tree (10 years age of trees)
(1) from the long 20cm of tree body clip, the branch of diameter 0.5cm, per plant takes 5 branches, and hermetic bag labelling is preserved；
(2) step (1) acquisition sample branch is put into into -70 DEG C of ultra cold storage freezers rapidly frozen 6 hours；
(3) preparation of liner separating pipe：Prepare the clean 0.6ml centrifuge tubes of sterilizing, centrifugation bottom of the tube needs to use nothing in advance
Bacterium large size syringe needle stabs the circular weepage holes of an a diameter of 1mm, cuts off lid；
(4) preparation of external support collecting pipe：Prepare the clean 1.5ml centrifuge tubes of sterilizing, make the tube wall of 1.5ml centrifuge tubes
Edge support just as the liner separating pipe mouth of pipe, and can be enclosed within outside liner separating pipe；
(5) the sample branch of step (2) is taken out from ultra cold storage freezer, branch blade, Ran Houyong is removed with clean shears
Sterile deionized water will be rinsed well outside branch, is wiped away the water on surface with aseptic filter paper, and this process should be complete in 5 minutes
Into；
(6) branch bark is longitudinally scratched with aseptic sharp blade, as deep as xylem, then bark is shelled with woody part
Leave, remove xylem, retain the bark part with phloem tissue；
(7) bark is cut into the segment of 1.0cm with the shears or blade of sterilizing, with 10 segments as a branch of loading step
(3) the liner separating pipe for preparing, will be equipped with sample liner separating pipe be placed in external support collecting pipe formed be collected by centrifugation
Pipe, is then placed in pre-cooling on ice pipe is collected by centrifugation；
(8) the effective refrigerated centrifuger that is collected by centrifugation that step (7) is prepared is separated, centrifugal work condition：4 DEG C,
12000rpm×15min；
(9) after the completion of being centrifuged, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem juice
Liquid；
(10) supernatant in collecting pipe is supported with the careful suck up outside of sterile pipette tip, another sterilizing is transferred to clean
In net frozen centrifuge tube, and it is stored in standby in -70 DEG C of ultra cold storage freezer.
Embodiment 3
The present invention proposes a kind of Citrus phloem sap preparation method, comprises the following steps that：
Experimental subject：The Citrus nursery stock (4 years age of trees) of solarium's culture
(1) from the long 15cm of tree body clip, the branch of diameter 0.5cm, per plant takes 4 branches, and hermetic bag labelling is preserved；
(2) step (1) acquisition sample branch is put into into -70 DEG C of ultra cold storage freezers rapidly frozen 4 hours；
(3) preparation of liner separating pipe：Prepare the clean 0.6ml centrifuge tubes of sterilizing, centrifugation bottom of the tube needs to use nothing in advance
Bacterium large size syringe needle stabs the circular weepage holes of an a diameter of 1mm, cuts off lid；
(4) preparation of external support collecting pipe：Prepare the clean 1.5ml centrifuge tubes of sterilizing, make the tube wall of 1.5ml centrifuge tubes
Edge support just as the liner separating pipe mouth of pipe, and can be enclosed within outside liner separating pipe；
(5) the sample branch of step (2) is taken out from ultra cold storage freezer, branch blade, Ran Houyong is removed with clean shears
Sterile deionized water will be rinsed well outside branch, is wiped away the water on surface with aseptic filter paper, and this process should be completed in 4 minutes；
(6) branch bark is longitudinally scratched with aseptic sharp blade, as deep as xylem, then bark is shelled with woody part
Leave, remove xylem, retain the bark part with phloem tissue；
(7) bark is cut into the segment of 0.8cm with the shears or blade of sterilizing, with 7 segments as a branch of loading step (3)
The liner separating pipe for preparing, will be equipped with sample liner separating pipe be placed in external support collecting pipe formed pipe is collected by centrifugation,
Then pre-cooling on ice is placed in pipe is collected by centrifugation；
(8) the effective refrigerated centrifuger that is collected by centrifugation that step (7) is prepared is separated, centrifugal work condition：4 DEG C,
12000rpm×15min；
(9) after the completion of being centrifuged, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem sap；
(10) supernatant in collecting pipe is supported with the careful suck up outside of sterile pipette tip, another sterilizing is transferred to clean
In net frozen centrifuge tube, and it is stored in standby in -70 DEG C of ultra cold storage freezer.
Citrus nursery stock (4 years age of trees) with solarium's culture kisses the skill of handling needles using the inventive method, aphid respectively as experimental subject
Citrus phloem sap is obtained with bleeding method, Comparative result is as follows：
1 Comparative result table of table
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (6)
1. a kind of Citrus phloem sap preparation method, it is characterised in that comprise the following steps that：
(1) from tree body clip branch, per plant takes 3-5 branch, and hermetic bag labelling is preserved；
(2) the sample branch that step (1) is obtained is put into into ultra cold storage freezer rapidly frozen；
(3) preparation of liner separating pipe；
(4) preparation of external support collecting pipe；
(5) the sample branch of step (2) is taken out from ultra cold storage freezer, branch blade is removed with clean shears, then with sterilizing
Deionized water will be rinsed well outside branch, is wiped away the water on surface with aseptic filter paper, and this process should be completed within 3-5 minutes；
(6) branch bark is longitudinally scratched with aseptic sharp blade, as deep as xylem, then bark is peeled off with woody part
Open, remove xylem, retain the bark part with phloem tissue；
(7) bark is cut into into segment with the shears or blade of sterilizing, is prepared with a branch of loading step (3) of 5-10 segment
Liner separating pipe, then by liner separating pipe be placed in the external support collecting pipe that step (4) is prepared formed pipe is collected by centrifugation, so
After be put on ice for pre-cooling；
(8) the effective refrigerated centrifuger that is collected by centrifugation that step (7) is prepared is separated；
(9) after the completion of being centrifuged, liner separating pipe is discarded, the supernatant in external support collecting pipe is Citrus phloem sap；
(10) supernatant in collecting pipe is supported with the careful suck up outside of sterile pipette tip, the clean jelly of another sterilizing is transferred to
Deposit in centrifuge tube, and save backup.
2. a kind of Citrus phloem sap preparation method according to claim 1, it is characterised in that in the step (1)
The long 10-20cm of branch, diameter 0.5cm.
3. a kind of Citrus phloem sap preparation method according to claim 1, it is characterised in that in the step (2)
Ultra cold storage freezer temperature be -70 DEG C and frozen time be 2-6 hours.
4. a kind of Citrus phloem sap preparation method according to claim 1, it is characterised in that in the step (7)
The segment length being cut into is 0.6-1.0cm.
5. a kind of Citrus phloem sap preparation method according to claim 1, it is characterised in that in the step (8)
The working condition of refrigerated centrifuger：4 DEG C, 12000rpm × 15min.
6. a kind of Citrus phloem sap preparation method according to claim 1, it is characterised in that in the step (10)
Save backup and be located in the ultra cold storage freezer that temperature is -70 DEG C.
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| CN201610976797.8A CN106546461B (en) | 2016-11-07 | 2016-11-07 | A kind of citrus phloem sap preparation method |
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| CN106546461B CN106546461B (en) | 2019-03-22 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109459293A (en) * | 2019-01-16 | 2019-03-12 | 山东农业大学 | A kind of corn stem bleeding liquor collecting device |
| CN114018677A (en) * | 2021-11-01 | 2022-02-08 | 天津大学 | Method for extracting potassium ions in liquid flow of woody part |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109459293A (en) * | 2019-01-16 | 2019-03-12 | 山东农业大学 | A kind of corn stem bleeding liquor collecting device |
| CN109459293B (en) * | 2019-01-16 | 2023-09-26 | 山东农业大学 | Corn stalk hurts liquid collection device that flows |
| CN114018677A (en) * | 2021-11-01 | 2022-02-08 | 天津大学 | Method for extracting potassium ions in liquid flow of woody part |
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| Publication number | Publication date |
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| CN106546461B (en) | 2019-03-22 |
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