CN104718900B - A kind of micro-damage phloem collects the method for cotton bleeding sap - Google Patents
A kind of micro-damage phloem collects the method for cotton bleeding sap Download PDFInfo
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- CN104718900B CN104718900B CN201510053386.7A CN201510053386A CN104718900B CN 104718900 B CN104718900 B CN 104718900B CN 201510053386 A CN201510053386 A CN 201510053386A CN 104718900 B CN104718900 B CN 104718900B
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Abstract
The invention discloses a kind of method that micro-damage phloem collects cotton bleeding sap.It adopts stem base diameter to be not less than the cotton plant of 0.5cm, first by the incision that supporter is fixedly drafted, strike off the epidermis of 1/4 girth, cortex, phloem and formation layer, excision xylem and marrow, bleeding sap is absorbed with absorbent cotton after cleaning up otch, for subsequent use with concentrate eluant after deionized water wash-out.Bleeding sap collection method of the present invention, by retaining most of phloem, maintain the contact of overground part to root system, the bleeding sap collected more can reflect growth and development of plants and metabolic condition truly, there is significant application value, can be used for the research that the dicotyledonous crops such as cotton carry out overground part and root system interaction, as the research of overground part and root system interaction under drought stress, salt stress.
Description
Technical field
The present invention relates to a kind of method of collecting cotton bleeding sap, destroy epidermis, cortex, phloem and formation layer and take out a bit of stem stalk xylem and marrow specifically by fraction, micro-damage collects the concrete grammar of cotton bleeding sap.
Background technology
Cotton stem ecto-entad is primarily of epidermis, cortex, phloem, formation layer, xylem and marrow composition (see accompanying drawing 1).Epidermis and cortex shield, and the Main Function of phloem from top to bottom transports organic matter, and xylem is mainly responsible for the moisture of root absorption and the ion be dissolved in inside water upwards to transport, and utilizes for other organ-tissues.Recent study finds, phloem and xylem also have the effect of trafficking signal material, are responsible for the information interchange between overground part branches and leaves and underground part root system.
Bleeding sap is the juice of the conducting tissue (xylem) of plants wound, flows out by the effect of root pressure makes it move to acrial part in the catheter; Meanwhile, the outflows such as the cell sap of plant and tissue are inevitably also had.Plant Root Bleeding amount is the important indicator of root physiological activity power, can reflected well plant absorption moisture, nutrient situation, and improving activity of root system and root system metabolism situation.Meanwhile, research in recent years also proves to contain plant hormone (basic element of cell division, abscisic acid etc.) in bleeding sap, is preced with as “ Gen – " make signaling molecule mutually, growing and playing an important role in various abiotic stress response.Therefore, Plant Root Bleeding collection and research very important.
The method of collecting plant bleeding sap is a lot of and constantly perfect, but what adopt so far is all directly cut off plant stem base portion to collect.This method is large for plant injury, and after cut-out stem, overground part and root system is made to lose contact, also acrial part has been cut off to the nutrient transport of root system and regulating action, can not reflect that plant is in the truth of growing or under environment stress, will have a greatly reduced quality according to the accuracy that bleeding sap conducts a research completely.Therefore, set up one and can collect bleeding sap, the bleeding sap collection method of overground part to root system nutrient transport and regulating action can be maintained again very necessary.
Summary of the invention
The object of the invention is the deficiency that will solve conventional acquisition bleeding sap method, provide a kind of micro-damage phloem to collect the method for cotton bleeding sap.The method adopts diameter of stem to be not less than the cotton plant of 0.5cm, destroy the epidermis of its 1/4 girth, cortex, phloem and formation layer, excise inner xylem and marrow, retain most of phloem with on the basis maintaining overground part and contact root system, collection Xylem sap.
The technical solution adopted in the present invention is: a kind of micro-damage phloem collects the method for cotton bleeding sap, it is characterized in that, adopts diameter of stem to be not less than the cotton plant of 0.5cm, first by the incision that supporter is fixedly drafted; Then destroy the epidermis of cotton plant 1/4 girth, cortex, phloem and formation layer, excise inner xylem and marrow, after cleaning up otch, absorbent cotton is put into otch, collect bleeding sap.
Specifically comprise the following steps:
(1) cutting of cotton plant stem
Choose the above true leaf of 7-8 sheet, stem base diameter is not less than the cotton plant of 0.5cm; Parallelly with stem in stem base portion (under cotyledonary node 2cm place) side bind a support bar (steel bar, iron wire etc.), fracture to support after cotton plant prevents cutting; At stem base portion opposite side, excise 1/4 girth with sharp cutter, be highly the epidermis of 0.3cm, cortex, phloem and formation layer, expose xylem; At the two ends blade exposing xylem, xylem is cut off and marrow, carefully take out xylem and the marrow of cut-out with dissecting needle and blade, note not injuring the tissues such as remaining phloem, keep lower tangent plane smooth (see accompanying drawing 2);
(2) collection of bleeding sap
Absorbent cotton watery hydrochloric acid (0.1mol/L), distilled water, washed with de-ionized water, dry, and loads in clean Eppendorf pipe stand-by;
First use deionized water rinsing otch inner 3-5 time, to remove the overflow of damaged cell, after cleaning, blot otch inside and surface moisture with the above-mentioned absorbent cotton handled well; Then the cotton 0.1g of new extracting degreasing puts into otch, then rayon balls and plant is bandaged with sealed membrane; After acquisition time, carefully cut sealed membrane with blade, open, take out absorbent cotton with tweezers, weigh, calculate the weight of collected bleeding sap, with 2-3ml deionized water wash-out, then with Vacuum Concentration instrument, eluent is concentrated to 0.5-1ml for subsequent use.
Above-mentioned bleeding sap acquisition time can experimentally require to be adjusted, and generally collects 2 ~ 12 hours.
The general long 3cm of above-mentioned support bar, diameter are 0.3cm.
Bleeding sap collection method of the present invention is compared with conventional method, has the following advantages: the accuracy the method increasing result.Remain most of phloem, the bleeding sap of collecting can reflect the upgrowth situation of cotton more accurately, particularly be applicable to research overground part signal to the impact of root system, especially larger on root system impact Stress treatment, as arid, Salt Strees Condition etc., Root exude is more weak, and bleeding sap collects relative difficulty, and long-time collection after bleeding sap root system loses nutrient and semiochemicals input changes greatly, and can not simulate the physiological situation of cotton exactly.This method remains most of phloem, is conducive to the regulation and control maintaining root system nutrient supply and semiochemicals, simulates the real conditions of cotton more accurately; The present invention is not only applicable to cotton, is also applicable to other most of dicotyledons.
Accompanying drawing explanation
Accompanying drawing 1 is the schematic cross-section of cotton stem;
Accompanying drawing 2 is schematic diagrames that micro-damage phloem collects cotton bleeding sap method; Wherein left figure is the schematic diagram cut cotton plant stem; Right figure is the schematic cross-section of incision; As can be seen from left figure: parallelly with stem in stem side bind a support bar; The cutting of 1/4 girth is carried out at stem base portion opposite side; As can be seen from right figure: method of the present invention has excised the whole xylem of incision and marrow, the epidermis of 1/4, cortex, phloem and formation layer are excised;
In figure, 1, epidermis; 2, cortex; 3, phloem; 4, layer is formed; 5, xylem; 6, marrow.
Embodiment
Embodiment 1:
Get matrix in greenhouse and cultivate the strain of 8 true leaf cottons 6, diameter of stem is the cotton plant of 0.6cm.With 50mmol/L sodium chloride solution process 1 week, then at the urea of blade spraying 1%, every strain 10ml, the every day of each spray sooner or later once.Process after 3 days, wherein 3 strain conventional methods take out bleeding sap, cane is cut off at cotyledonary node place, remove head, with deionized water rinsing otch 3 times, by ready absorbent cotton (watery hydrochloric acid, distilled water, washed with de-ionized water after cleaning, dry, load in clean 50mlEppendorf pipe stand-by) blot otch inside and surface moisture, getting above-mentioned absorbent cotton 0.1g is placed on otch, finally use 5cm × 5cm to sample plastic sack to package, absorbent cotton and otch are fully contacted but not too tightly, with rubber band or cotton rope, plastic sack is tied up on the cotton stalk in bottom.All the other 3 strain cottons method of micro-damage phloem collects cotton bleeding sap, concrete operations are as follows: in basal part of stem side, sealed membrane parallel with stem binds a long 3cm, the iron wire of diameter 0.3cm, fixing point is selected at the two ends of iron wire, fractures after preventing cutting with fixing cotton plant.At basal part of stem opposite side 1cm place, excise 1/4 girth with sharp cutter, the height epidermis of 0.3cm, cortex, phloem and formation layer, expose xylem.At the two ends blade exposing xylem, xylem and marrow are cut off, otch can not be excessively dark, in order to avoid destroy the phloem retained, carefully takes out xylem and the marrow of cut-out with dissecting needle and blade, the remaining xylem of further destruction also takes out, and notes keeping lower tangent plane smooth.First use deionized water rinsing otch inner 3 times, to remove the overflow of damaged cell, otch inside and surface moisture is blotted with ready absorbent cotton after cleaning, get above-mentioned absorbent cotton 0.1g and put into otch, finally with sealed membrane, rayon balls and plant are bandaged, do not wrap up too tight, so that cotton balls absorbs bleeding sap.12 h before harvest bleeding saps, carefully cut sealed membrane with blade, open, take out absorbent cotton with tweezers, weigh, and calculate the weight of collected bleeding sap, and with 3ml deionized water wash-out, constant volume is to 25ml.The same constant volume of bleeding sap that conventional method is taken out, to 25ml, measures wherein sodium, potassium content with Metalization of skin, result following (table 1):
Na in the bleeding liquid measure that table 1 distinct methods obtains and bleeding sap
+, K
+content
* numerical value significant difference (P<0.05) between same column different letter in showing
Result shows, and the amount that new method (fraction destroys phloem and collects cotton bleeding sap method) obtains bleeding sap slightly declines, but does not reach significance level, Na in bleeding sap
+content is remarkable decline compared with conventional method.K in bleeding sap
+changes of contents is not obvious.Therefore, compared with routine side's method, new method bleeding sap amount to obtain is a little more than conventional method, but the heavy Na of bleeding sap
+content significantly declines.Illustrate in micro-damage method that the most of phloem retained exists certain regulating action to root system, and conventional method cannot detect this regulating and controlling effect.
Embodiment 2:
Get 6 strain Greenhouse Water Cultures, what two graftings were formed has a head, and (see patent of invention ZL201010190429.3) 6 true leaf cotton plants of the grafting root-planting of two roots, diameter of stem is the cotton plant of 0.5cm.After recovering 2 weeks, add the sodium chloride solution of 100mmol/L at the side root system of the cotton of all 6 strain graftings, opposite side adds normal nutrition liquid.Process the bleeding sap of getting the root system grown in normal nutrition liquid side after 6 hours, 3 strain conventional methods, cane is cut off at base portion 1cm place, remove head, with deionized water rinsing otch 3 times, with ready absorbent cotton (watery hydrochloric acid after cleaning, distilled water, washed with de-ionized water, dry, load in clean 50mlEppendorf pipe stand-by) blot otch inside and surface moisture, getting above-mentioned absorbent cotton 0.1g is placed on otch, finally use 5cm × 5cm to sample plastic sack to package, absorbent cotton and otch are fully contacted but too not tight, with rubber band or cotton rope, plastic sack is tied up on the cotton stalk in bottom.3 strain new methods (fraction destroys phloem and collects cotton bleeding sap method) get bleeding sap, get its bleeding sap method as follows: sealed membrane parallel with stem binds a long 3cm in basal part of stem side, the iron wire of diameter 0.3cm, fixing point is selected at the two ends of iron wire.At basal part of stem opposite side 1cm place, excise 1/4 girth with sharp cutter, the height epidermis of 0.3cm, cortex, phloem and formation layer, expose xylem.At the two ends blade exposing xylem, xylem is cut off, otch can not be excessively dark, in order to avoid destroy the phloem retained, carefully takes out xylem and the marrow of cut-out with dissecting needle and blade, the remaining xylem of further destruction and marrow also take out, and note keeping lower tangent plane smooth.First use deionized water rinsing otch inner 3 times, to remove the overflow of damaged cell, otch inside and surface moisture is blotted with ready absorbent cotton after cleaning, get above-mentioned absorbent cotton 0.1g and put into otch, finally with sealed membrane, rayon balls and plant are bandaged, do not wrap up too tight, so that cotton balls absorbs bleeding sap.12 h before harvest bleeding saps, carefully cut sealed membrane with blade, open, take out absorbent cotton with tweezers, weigh, and calculate the weight of collected bleeding sap, and with 3ml deionized water wash-out, constant volume is to 25ml.The same constant volume of bleeding sap that conventional method is taken out, to 25ml, measures wherein sodium, potassium content with Metalization of skin, result following (table 2):
Na in the bleeding liquid measure that table 2 distinct methods obtains and bleeding sap
+, K
+content
* numerical value significant difference (P<0.05) between same column different letter in showing
Result shows, and new method (fraction destroys phloem and collects cotton bleeding sap method) obtains the amount of bleeding sap, Na in bleeding sap
+content and K
+content is all significantly higher than conventional method.Therefore, in grafting root-planting system, there is certain regulating and controlling effect to normal nutrient solution side root system in the most of phloem retained in new method, and conventional method cannot detect the existence of this regulating action.
Claims (4)
1. micro-damage phloem collects a method for cotton bleeding sap, and its step is,
(1) cutting of cotton plant stem
Choose more than 7 true leaves, stem base diameter is not less than the cotton plant of 0.5cm; Parallelly with stem in stem base portion side bind a support bar; At stem base portion opposite side, excise 1/4 girth with sharp cutter, be highly the epidermis of 0.3cm, cortex, phloem and formation layer, expose xylem; At the two ends of exposing xylem, whole xylem and marrow are cut off, take out the xylem and marrow that cut off;
(2) collection of bleeding sap
First use deionized water rinsing otch inner 3-5 time, after cleaning, absorbent cotton blots otch inside and surface moisture; Then new extracting degreasing cotton puts into otch, then absorbent cotton and cotton plant is bandaged with sealed membrane; After acquisition time, cut sealed membrane, open, take out absorbent cotton, weigh, calculate the weight of collected bleeding sap, use deionized water wash-out, then eluent is concentrated for subsequent use with Vacuum Concentration instrument.
2. a kind of micro-damage phloem as claimed in claim 1 collects the method for cotton bleeding sap, it is characterized in that, watery hydrochloric acid, distilled water, the washed with de-ionized water of the absorbent cotton priority 0.1mol/L that described step (2) uses, dry, and loads in clean Eppendorf pipe stand-by.
3. a kind of micro-damage phloem as claimed in claim 1 collects the method for cotton bleeding sap, and it is characterized in that, described bleeding sap acquisition time is 2 ~ 12 hours.
4. a kind of micro-damage phloem as claimed in claim 1 collects the method for cotton bleeding sap, and it is characterized in that, the long 3cm of described support bar, diameter are 0.3cm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106546461B (en) * | 2016-11-07 | 2019-03-22 | 赣南师范大学 | A kind of citrus phloem sap preparation method |
| CN107396766B (en) * | 2017-09-07 | 2022-05-03 | 湖南省水稻研究所 | Plant wound fluid collecting and recovering device |
| CN109738589B (en) * | 2019-02-13 | 2021-12-17 | 南京禾稼春生物科技有限公司 | Method for rapidly extracting xylem sap of plant |
| CN112268731B (en) * | 2020-09-25 | 2023-02-17 | 江苏大学 | Low-damage plant xylem juice sampling method |
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| SU1404026A1 (en) * | 1986-02-21 | 1988-06-23 | Центральный Научно-Исследовательский Институт Лесной Генетики И Селекции | Method of producing phloem juice of wood plants |
| CN1115595A (en) * | 1994-07-23 | 1996-01-31 | 黄才芬 | Method of obtaining fresh bamboo liquid |
| CN103063655A (en) * | 2012-12-28 | 2013-04-24 | 中国科学院新疆生态与地理研究所 | Method for detecting mineral nutrient cycling from cotton seedling stage to cotton flowering and boll-forming stage by applying pressure |
| CN103616370A (en) * | 2013-12-14 | 2014-03-05 | 山东省农业科学院玉米研究所 | Method for detecting phosphorus recirculation in growing period of corns |
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Patent Citations (4)
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| SU1404026A1 (en) * | 1986-02-21 | 1988-06-23 | Центральный Научно-Исследовательский Институт Лесной Генетики И Селекции | Method of producing phloem juice of wood plants |
| CN1115595A (en) * | 1994-07-23 | 1996-01-31 | 黄才芬 | Method of obtaining fresh bamboo liquid |
| CN103063655A (en) * | 2012-12-28 | 2013-04-24 | 中国科学院新疆生态与地理研究所 | Method for detecting mineral nutrient cycling from cotton seedling stage to cotton flowering and boll-forming stage by applying pressure |
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