CN101556215A - Sampling method for non-damage fixed position observation of plant stomata in field experiment - Google Patents
Sampling method for non-damage fixed position observation of plant stomata in field experiment Download PDFInfo
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- CN101556215A CN101556215A CNA2009100224726A CN200910022472A CN101556215A CN 101556215 A CN101556215 A CN 101556215A CN A2009100224726 A CNA2009100224726 A CN A2009100224726A CN 200910022472 A CN200910022472 A CN 200910022472A CN 101556215 A CN101556215 A CN 101556215A
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Abstract
The invention discloses a sampling method for non-damage fixed position observation of plant stomata in a field experiment, which comprises the following steps of: according to experimental design, selecting functional leaves, avoiding overripe leaves, over tender leaves, leaves damaged by diseases and insects and malformed leaves, and cleaning object leaves; evenly coating a thin layer of nail polish at a position to be observed, and natural drying or blowing to be nearly dry by using a washing ear ball; flatly and tightly pasting a transparent adhesive tape on a clean glass slide, using a dissecting needle or a clean blade for slowly guiding to prevent air bubbles, and tightly pasting the adhesive tape with the width being at least 2mm around the nail polish on the glass slide; and numbering, clean packaging, and taking back to the laboratory for microscopic observation and measurement. The method has the advantages of being capable of bulk sampling in field living leaves, having no damage to the leaves, fixed position recording stomatal conditions, observing daily change of the stomata of the leaves under different processing conditions, and the like, and can be used for the research and practice in the fields such as bioecology, plant physiology, plant germplasm resource, plant thremmatology, cultivation science, etc.
Description
Technical field
The present invention relates to the plant physioecology studying technological domain, particularly under the field test condition of living body, not damaged, location sampling in a large number, with the technical method of research plant stomata feature and dynamic change thereof.
Background technology
Blade is the major organs that plant carries out photosynthesis and transpiration, the needed CO of leaf photosynthesis
2From atmosphere, advance people's blade interior by pore.Meanwhile, the moisture in the plant is lost to the atmosphere from pore by the transpiration of blade, and therefore, the physiological function of the form of leaf epidermis pore, quantity and distribution and blade is closely related.In addition, also closely related with the growing environment of plant.
In the evaluation of germ plasm resource, pore can be used as the record project of observing as one of important botany proterties.Pore is the vitals that remain under natural selection and artificial selection, has bigger stability, thereby it can be used for studying sibship, evolutionary process, the ecotype, chromosome characteristic and the geographic origin of plant.The variation that belongs between plant not of the same race general stomatal frequency and pore length together is less relatively, and the plant that does not belong to together then changes bigger.Stomatal frequency, Stomacal guard cell number of chloroplast are relevant with ploidy.Li Runtang etc. think that the pore form can be used as research fruit tree genetic breeding, the important microscopic feature of difference kind, type.This shows that the research of pore observational technique is the important content (intercalation loyal industry, 2007) in the botany research.
(2006) such as Wang Xiao ravines have studied the stomata characteristics of test-tube plantlet with the adhesive tape method, can transplant with domestication for the morphological feature of illustrating test-tube plantlet foundation is provided.
Shao Ke etc. (2008) have carried out the Primary Study of cotyledon pore method selection beet new varieties, result of study shows, beet cotyledon stomatal frequency is the characteristic of its kind, high glycoform beet cotyledon stomatal frequency is obviously greater than high yield type kind, closely related with sugar content, can be used as the index of selecting the high glycoform kind.Chosen period is in 4 true leaves latter stage in seedling stage, and this system of selection has live body, easy and characteristic of accurate comparatively, to improving efficiency of selection, quickening the seed selection process and have important theory and practice significance.
At present, the method for observation of plant blade pore has multiple, as water load method, blotting, electron microscope method etc.
Water load method is promptly got plant leaf blade, tears to get its lower epidermis and make Temporary slide, examines under a microscope.This method often can not reflect the real conditions of pore, mostly stomatal closure under the situation.Thanking to national wealth etc. (2007) adopts this method research to think that the freshwater mussel orchid is the good material of observing pore and probing into vegetable cell suction and dehydration.
Blotting, promptly smear ox-hide glue, guncotton gum-solution method or cellulose acetate glue on the surface of leaves of plants, film just congeals into after colloid is air-dry, this film just is printed on the boundary's mark edge tracking and the air hole structure of each cell of epidermal tissue, can represent the situation of pore at that time, as aperture, but operation also is difficult for the film-making success than trouble.Huang Liping etc. (2008) have studied the stomata characteristics of 36 jujube cultivars and the relation of dehiscent fruit with collodion process; (2008) such as the victorys of horse have studied peach blade pore shape and big or small gas cell distribution with collodion process, prove that stomata characteristics is relevant with the peach evolution.
The acid dissociation method: 10% salpeter solution and 10% chromic acid solution mix in 1: 1 ratio, are made into the blade dissociation solution.Blade is got 1cm
2About size.Soak 16-20h with the blade dissociation solution, lower epidermis is taken out with tweezers in the back of dissociating, and embathes with clear water, observes under optical microscope, measures visual field diameter (D) with micrometer, investigates the stoma number in every visual field.
Sticking following the example of: with the sticking blade lower epidermis observation of plant pore of getting of common plastics adhesive tape than blotting with tear the method for taking off epidermis observation pore and have advantage simple to operate, that speed is fast, authenticity is strong, especially be suitable for the research (Chen Baihong etc., 2004) of the pore change dynamics and the morphological indexes of young tender materials such as test-tube plantlet.This method can prevent to cause because of guard cell's dehydration the variation of pore opening degree, truly the observation of plant different phase and period pore change dynamics.Use the adhesive tape method separately, because vane thickness is inhomogeneous, blade has concavo-convex or because influence such as vein, can not guarantee that whole visual field evenly copies the blade lower surface, the sampling area has only 70～80% of leaf area, influence statistical accuracy as a result such as stomatal frequency, many or have obducent leaf effect also not so good to epidermal hair.
Fixation: cut the about 60cm of area in the middle part of each is handled institute's leaf of surveying
2, fixing with FAA (50% ethanol, 5% glacial acetic acid, 10% formaldehyde, 35% distilled water), take back laboratory observation.Select nearly vein place, leaf middle part, scrape mesophyll with scalper from the leaf front and stay the epidermis film-making.With OLYMPUS (AHBS) digital micro-analysis sem observation, take pictures, measure (Gao Zhiying etc., 2008) with Computerized image processing system.
Chen Wenfu etc. (2000) have invented that rice leaf stoma proterties research new method---method for making is scraped in desiliconization.This method is finally made the envelope that can forever preserve and is hidden sheet by fixing, desiliconization, striking, dyeing and transparent to material.Advantages such as the method for making device therefor is scraped in desiliconization and reagent is few, easy to operation, have clear picture, observed result is accurate; But the pore that weak point is a blade to be carried out under isolated condition, seen is in closed condition, so can not be used for stomatal movement situation or aperture Study of variation law, becomes the fatal shortcoming of this method.
Pore with fluorescence microscope leaves of plants epidermis, need not do any processing, can the supracutaneous stomatal apparatus of Direct observation leaves of plants, and can observe the situations of change such as form, density and stomatal movement degree of pore easily, for a kind of new pore observational technique (Cui Hongan has been found in the research of plant microtechnic, 2007), but need equipment disposition higher, blade need exsomatize.
Envelope great waves (2008) are improved the chromic acid segregation process, have set up to be fit to monocotyledonous pore sampling observational technique.
Research such as neat red rock (2009) water deficit is to tomato leaf stomatal properties and chloroplast ultrastructure influence, and method is to cut 1～2mm between blade middle part vein and blade edge
2Square fritter, with fixing before 2.5% glutaraldehyde, fixing behind 1% osmic acid.Serial routinely ethanol acetone dehydration, the isoamyl acetate displacement is at liquid CO
2It is bonding to carry out sample after the middle drying, it is carried out metal spraying handles, under S-450 type scanning electron microscope, observe and respectively handle blade stoma number and pore degree of opening, measure the transverse diameter of stomatal apparatus and the length in vertical footpath, statistics stomatal movement situation, measure pore length and wide and calculating stomatal frequency, choose the several representative visual field and take a picture.
More than the method for several observation pores certain advantage and operability are all arranged, some method is simple, cost is little, easy operating but observed pore often is closed condition; Although some degree of accuracy height but complicated operation or need exact instrument.These methods all have certain limitation, as are subjected to the influence of quality, leaf epidermis hair or the dermal appendage and the production effect of blade, the more important thing is researchs such as the open-air sampling of stomatal properties, behavioral characteristics are restricted.
Summary of the invention
At above-mentioned problems of the prior art and defective, the object of the invention is to provide a kind of sampling method of field test not damaged position observation of plant pore.This method has and can take a sample in a large number at field live body blade, to the blade not damaged, but position the record pore situation, the diurnal variation of different disposal condition lower blade pore etc. can be observed, the research and the practice in fields such as bioecology, plant physiology, plant germplasm resource, thremmatology, cultivation can be used for.
To achieve these goals, the technical solution adopted in the present invention is a kind of sampling method of field test not damaged position observation of plant pore, it is characterized in that, comprises the following steps:
1) according to test design, the selection function blade was avoided old, tender excessively, disease and pest and lopsided blade, and the object blade is cleaned up;
2) intending evenly very thin coating one deck nail polish of look-out station, about 5～10mm * 5～10mm, air dry or blow near with ear washing bulb and to do, the blade of hairiness is thrown off after earlier detected part being coated with one deck nail polish drying, is coated with nail polish more again and is used for sampling;
3) be affixed on the nail oil film with adhesive tape, the centre is aimed at;
4) slowly take off, the nail oil film is separated with blade with dissecting needle is auxiliary together with the nail oil film;
5) it is sturdy on clean slide to take off the adhesive tape and the nail oil film composite membrane plane of getting, slowly guide to prevent bubble with dissecting needle or clean blade, must have 1～2mm adhesive tape and sturdy on microslide around the nail oil film at least, the nail polish membrane closure on microslide;
6) handle numbering according to test, simultaneously with number record at experimental record on this;
7) clean packaging is used for microscopic examination and measurement after taking back the laboratory.
The general thought of the present invention's sampling method is to take all factors into consideration from aspect such as harmless, continuous, a large amount of, accurate, combine the advantage of nail polish blotting and adhesive tape blotting, improve the tabletting technology scheme, formed the sampling method of the field test not damaged position observation of plant pore of this practicality.
Sampling method of the present invention is applicable to agricultural, forestry industry, the research and the practice in fields such as plant ecology, plant physiology, plant germplasm resource, thremmatology, cultivation.
Compared with prior art, of the present invention having the following advantages:
1) can be used for living body sampling, photosynthetic blade is not caused damage;
2) pore that can be used for fixing the position is observed sampling, the variation of research same position of blade or a plurality of positions of same blade pore;
3) the diurnal variation research that can be used for stomata characteristics is taken a sample;
4) the sampling film-making can be preserved and can not taken place air porous expanded or contraction the long term;
5) can be used for the relatively large film-making in field, taking back arranges the time behind the laboratory observes etc.;
6) also be applicable to blade table chrotoplast size and the observation of arranging, and the blade different parts (as around blade center, vein, the vein, leaf margin etc.) research such as different parts epidermal cell form.
Description of drawings
Fig. 1 receives lattice grape pore observation figure for the embodiment of the invention 1 wood.
Fig. 2 is that the embodiment of the invention 2 red earth grape pores are observed figure.
Fig. 3 is that figure is observed in the variation in the morning of the red grape pore in the embodiment of the invention 3 Wensus.
Fig. 4 is that figure is observed in the variation at noon of the red grape pore in the embodiment of the invention 3 Wensus.
Fig. 5 is that figure is observed in the variation in afternoon of the red grape pore in the embodiment of the invention 3 Wensus.
Embodiment
Below in conjunction with accompanying drawing and example the present invention is described in further detail.
Embodiment 1 wood is received lattice grape pore and is observed
In August, 2008, draw Da Han village, township grape gallery in Aksu Prefecture Wenxiu County, Xinjiang holder, in the research that lattice grape photosynthesis characteristics influences was received in soil and foliage dressing to wood, entering the coffin-die was received lattice grape leave pore and is taken a sample to research.Method is to handle according to different soils fertilizer treatment and foliage application, choose the normal photosynthetic functional leaf of sunny slope (being generally up the 3rd or the 4th leaf of branch base portion), because experimental field in the roadside, village, dust is very big, to the blade surface dust by blowing means cleanings such as wiping, brush.At position near blade main lobe arteries and veins, careful coating nail oil film, area is less than 1cm
2Blow with mouth and to quicken dryly, take off with the transparent plastic adhesive tape general of the 15mm * 15mm nail oil film of drying and to take off, paste rapidly on the microslide, write numbering with marking pen, comprise and handling name, time etc. that every processing repeated sampling packages more than 3, take back microexamination behind the laboratory, the results are shown in Figure 1, about figure (a, b figure) be respectively Beijing time 12:00, the wood during 17:00 is received the stomata characteristics of lattice grape.
Embodiment 2 red earth grape pores are observed
In August, 2008,, in the research to red earth grape photosynthesis characteristics influence, take a sample to research with regard to red earth grape leave pore at soil and foliage dressing on farm, Xinjiang Aksu Prefecture red flag slope.Method is to handle according to different soils fertilizer treatment and foliage application, choose the normal photosynthetic functional leaf of sunny slope (the branch base portion is the 3rd or the 4th leaf up), earlier to the blade table top blast, wipe clean, at position near blade main lobe arteries and veins, careful coating nail oil film, area 5mm * 5mm, dry to quicken drying with ear washing bulb, (15mm * 15mm) takes off the nail oil film of drying and takes off with the transparent plastic adhesive tape, paste rapidly on the microslide, each microslide is all write numbering, comprises handling name, time etc., every processing repeated sampling is more than 3, package, take back microexamination behind the laboratory, the results are shown in Figure 2, about figure be respectively Beijing time 12:00, the stomata characteristics of the red earth grape during 17:00.
The red grape pore diurnal variation of embodiment 3 Wensus is observed
In August, 2008, in Aksu Prefecture Wenxiu County, Xinjiang, during the red grape variety in Wensu is preferably studied, take a sample to research with regard to the red grape leave pore in Wensu, particularly intend fruit morphology is showed visibly different nutrition Variation Lines, by characteristics such as diurnal variation such as analysis pore opening degrees, in the hope of preferably providing foundation for kind.Method the results are shown in Figure 3～5 with example 2, and figure is respectively Beijing time 10:00,12:00 about Fig. 3; Fig. 4 is respectively 14:00,16:00; Fig. 5 is respectively 18:00, the stomata characteristics of the red grape in Wensu during 20:00.
Claims (1)
1. the sampling method of a field test not damaged position observation of plant pore is characterized in that, comprises the following steps:
1) according to test design, the selection function blade was avoided old, tender excessively, disease and pest and lopsided blade, and the object blade is cleaned up;
2) intending evenly very thin coating one deck nail polish of look-out station, about 5～10mm * 5～10mm, air dry or blow near with ear washing bulb and to do, the blade of hairiness is thrown off after can detected part being coated with one deck nail polish drying earlier, is coated with nail polish more again and is used for sampling;
3) be affixed on the nail oil film with adhesive tape, the centre is aimed at;
4) slowly take off, the nail oil film is separated with blade with dissecting needle is auxiliary together with the nail oil film;
5) it is sturdy on clean slide to take off the adhesive tape and the nail oil film composite membrane plane of getting, slowly guide to prevent bubble with dissecting needle or clean blade, must have 1～2mm adhesive tape and sturdy on microslide around the nail oil film at least, the nail polish membrane closure on microslide;
6) handle numbering according to test, simultaneously with number record at experimental record on this;
7) clean packaging is taken back and is used for microscopically behind the laboratory and observes and measure.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2009100224726A CN101556215A (en) | 2009-05-13 | 2009-05-13 | Sampling method for non-damage fixed position observation of plant stomata in field experiment |
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| CNA2009100224726A CN101556215A (en) | 2009-05-13 | 2009-05-13 | Sampling method for non-damage fixed position observation of plant stomata in field experiment |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101769836B (en) * | 2010-01-25 | 2011-11-09 | 河南科技大学 | Pine needle section observation method |
| CN105067620A (en) * | 2015-07-30 | 2015-11-18 | 广西南亚热带农业科学研究所 | Hevea brasiliensis leaf pore observation method |
| CN105547740A (en) * | 2015-12-10 | 2016-05-04 | 大连大学 | Method for acquiring epidermis of plant leaf |
| CN105628704A (en) * | 2015-11-30 | 2016-06-01 | 江苏大学 | Quick measurement method based on light interference technology and used for blade surface microstructure |
| CN106770943A (en) * | 2016-12-12 | 2017-05-31 | 甘肃农业大学 | Potato stomatal conductance analyzer and its assay method |
| CN107179268A (en) * | 2017-05-26 | 2017-09-19 | 安徽科技学院 | A kind of estimating and measuring method of Leaf-Blade of Poaceae epidermis stomatal frequency |
| CN108469434A (en) * | 2018-04-11 | 2018-08-31 | 山东农业大学 | A kind of monitoring fruit tree whether the device and method of water shortage |
| CN109060480A (en) * | 2018-09-11 | 2018-12-21 | 南京林业大学 | Silicon preprocess method is removed before a kind of paraffin section for the leaf of bamboo |
| CN109060801A (en) * | 2018-06-22 | 2018-12-21 | 德清绿色阳光农业生态有限公司 | A kind of seedling detection device of forestry |
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2009
- 2009-05-13 CN CNA2009100224726A patent/CN101556215A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101769836B (en) * | 2010-01-25 | 2011-11-09 | 河南科技大学 | Pine needle section observation method |
| CN105067620A (en) * | 2015-07-30 | 2015-11-18 | 广西南亚热带农业科学研究所 | Hevea brasiliensis leaf pore observation method |
| CN105628704A (en) * | 2015-11-30 | 2016-06-01 | 江苏大学 | Quick measurement method based on light interference technology and used for blade surface microstructure |
| CN105547740A (en) * | 2015-12-10 | 2016-05-04 | 大连大学 | Method for acquiring epidermis of plant leaf |
| CN106770943B (en) * | 2016-12-12 | 2023-02-03 | 甘肃农业大学 | Potato porosity conductivity tester and testing method thereof |
| CN106770943A (en) * | 2016-12-12 | 2017-05-31 | 甘肃农业大学 | Potato stomatal conductance analyzer and its assay method |
| CN107179268A (en) * | 2017-05-26 | 2017-09-19 | 安徽科技学院 | A kind of estimating and measuring method of Leaf-Blade of Poaceae epidermis stomatal frequency |
| CN108469434A (en) * | 2018-04-11 | 2018-08-31 | 山东农业大学 | A kind of monitoring fruit tree whether the device and method of water shortage |
| CN108469434B (en) * | 2018-04-11 | 2022-01-25 | 山东农业大学 | Device and method for monitoring whether fruit trees lack water |
| CN109060801A (en) * | 2018-06-22 | 2018-12-21 | 德清绿色阳光农业生态有限公司 | A kind of seedling detection device of forestry |
| CN109060480A (en) * | 2018-09-11 | 2018-12-21 | 南京林业大学 | Silicon preprocess method is removed before a kind of paraffin section for the leaf of bamboo |
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN109556933B (en) * | 2018-12-12 | 2021-08-17 | 宁波中盛产品检测有限公司 | Method for quickly preparing perineal pattern of root-knot nematode |
| CN110231443A (en) * | 2019-07-30 | 2019-09-13 | 吉林大学 | A kind of plant disease diagnostic device |
| CN110231443B (en) * | 2019-07-30 | 2024-05-31 | 吉林大学 | Plant disease diagnosis device |
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