CN114136741B - Fluorescent staining tabletting method for observing morphology of plant leaf epidermis hair, quilt and non-gland hair - Google Patents
Fluorescent staining tabletting method for observing morphology of plant leaf epidermis hair, quilt and non-gland hair Download PDFInfo
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- CN114136741B CN114136741B CN202111487907.1A CN202111487907A CN114136741B CN 114136741 B CN114136741 B CN 114136741B CN 202111487907 A CN202111487907 A CN 202111487907A CN 114136741 B CN114136741 B CN 114136741B
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- 210000004209 hair Anatomy 0.000 title claims abstract description 99
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 22
- 210000002615 epidermis Anatomy 0.000 title claims description 24
- 210000004907 gland Anatomy 0.000 title description 13
- 230000000762 glandular Effects 0.000 claims abstract description 84
- 241000196324 Embryophyta Species 0.000 claims abstract description 56
- JPYHHZQJCSQRJY-UHFFFAOYSA-N Phloroglucinol Natural products CCC=CCC=CCC=CCC=CCCCCC(=O)C1=C(O)C=C(O)C=C1O JPYHHZQJCSQRJY-UHFFFAOYSA-N 0.000 claims abstract description 32
- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229960001553 phloroglucinol Drugs 0.000 claims abstract description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000012153 distilled water Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 240000006891 Artemisia vulgaris Species 0.000 claims abstract description 12
- 235000003261 Artemisia vulgaris Nutrition 0.000 claims abstract description 12
- 229960000583 acetic acid Drugs 0.000 claims abstract description 11
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 11
- 239000011259 mixed solution Substances 0.000 claims abstract description 10
- 239000011521 glass Substances 0.000 claims abstract description 9
- 238000004043 dyeing Methods 0.000 claims abstract description 8
- 238000004140 cleaning Methods 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 8
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 8
- 235000003092 Artemisia dracunculus Nutrition 0.000 claims description 5
- 240000001851 Artemisia dracunculus Species 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 10
- 230000005284 excitation Effects 0.000 abstract description 2
- 210000002268 wool Anatomy 0.000 description 7
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000035699 permeability Effects 0.000 description 2
- 241000842962 Apoda limacodes Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000000339 bright-field microscopy Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The invention relates to a fluorescent staining sheet making method for observing the forms of plant leaf surface fur and glandular fur, which can effectively solve the problem of observing the plant surface fur and glandular fur by using a fluorescent microscope, wherein the leaves of fresh healthy plants are taken, washed by distilled water in an ultrasonic mode, small pieces are cut, immersed in a mixed solution of alcohol and glacial acetic acid for treatment, rinsed by distilled water, then kept stand in a phloroglucinol solution, the leaves are taken out, one surface with the surface fur is placed upwards on a glass slide, a drop of diluted glycerol is dripped above the leaves, and the blue light or green light is used for excitation under the fluorescent microscope respectively, so that bright yellow fluorescence is observed for the glandular fur and the non-glandular hair, and the fluorescent staining sheet for observing the forms of the plant leaf surface fur and glandular fur and the non-glandular fur is obtained. The method is scientific and reasonable, simple in operation, low in cost, small in workload, high in success rate, clear in observation and good in effect, and is effectively used for innovation of fluorescent dyeing tabletting methods for microscopic observation of the surface coat and the glandular hair and the non-glandular hair of the plant leaves and the surface coat of the mugwort.
Description
Technical Field
The invention relates to the technical field of plant structures, in particular to a fluorescent staining sheet-making method for observing the morphology of the hair of the epidermis of a plant leaf by carrying out fluorescent staining treatment on the surface of the plant and further observing the morphological characteristics of the hair of the epidermis of the plant leaf by a fluorescent microscope.
Background
The epidermis hair cover of a plant is a specialized structure on the surface of the plant, and is generally distributed on leaves, stems or flower cover sheets of the plant, and has various forms, and can be divided into glandular hair and non-glandular hair according to whether the plant has secretion capacity or not; according to the cell composition, single-cell hair and multicellular hair are divided into plant hair, single-column hair and multicellular hair; the shape can be classified into head shape, star shape, hook shape, scale shape, etc. The observation and determination of the morphology, distribution and type of plant coat plays an important guiding role in classification and identification of plants. In general, observing and determining the morphology and type of plant epidermis coat is usually accomplished using bright field microscopy and scanning electron microscopy. When the leaf epidermis is observed by a bright field microscope, the epidermis is usually required to be torn off and dissociated by adopting a leaf epidermis flaking method, but paper leaves, particularly leaves of dense gland wool and (or) non-gland wool, are easy to tear, and plant leaf epidermis required for an ideal experiment is difficult to obtain, even if the leaf epidermis can be obtained, dyeing is often required, and the dyeing inevitably stains epidermal cells, air holes, gland wool and non-gland wool, so that the identification and the quantity statistics of the gland wool and the non-gland wool are not facilitated. In addition, when the epidermis of the close-packed glandular hair and/or non-glandular hair is observed by a bright field microscope and a scanning electron microscope, for example, mugwort, tarragon and the like have weak penetration, and thus cannot be efficiently observed by microscopy.
At present, a fluorescence microscope generally adopts an epi-illumination light as a light source to irradiate a plant leaf sample to be detected so as to emit fluorescence, and then the shape and the position of an object are observed under the microscope. For the epidermal hair of the plant with dense gland hair and/or non-gland hair, the morphological characteristics and distribution of the epidermal hair of the plant can be clearly known by utilizing the strong penetrability of a fluorescence microscope, the difference of colors after the autofluorescence of different chemical components and fluorescent dye dyeing and the background treatment, so that the morphological observation of the epidermal hair of the plant leaf with dense gland hair and/or non-gland hair can be better solved theoretically, and the quality and the medicinal value of the plant are ensured. However, there has been no disclosure of how to prepare a leaf for fluorescence microscopic observation of the glandular wool of the gland Mao Huofei.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a fluorescent staining sheet-making method for observing the glandular hair and non-glandular hair forms of the epidermis hair of a plant, which can effectively solve the problem of observing the epidermis hair of the plant by using a fluorescent microscope.
The technical scheme of the invention is that the fluorescent staining film-making method for observing the forms of the plant leaf epidermis hair, the coat hair and the non-coat hair comprises the following steps:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 3-8 min, wherein the ultrasonic frequency is less than or equal to 30KHz;
the plant is mugwort or tarragon;
(2) Immersing: cutting small blocks with the size of 8-10 mm from the blade after ultrasonic cleaning, immersing in 3-5 mL of mixed solution of alcohol and glacial acetic acid for 1-3 h, wherein the volume ratio of the alcohol to the glacial acetic acid is 2:1;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing the blade with distilled water for 1-3 min;
(4) Standing: placing the rinsed leaves into 3-5 mL of phloroglucinol solution, and standing for 10-30 min;
the phloroglucinol solution is prepared by adding phloroglucinol into 95% alcohol serving as a solvent to prepare a phloroglucinol solution with the mass content of 5% -10%;
(5) And (3) observation: the leaves are taken out from the phloroglucinol solution, one surface with the surface fur is placed on a glass slide upwards, a drop of diluted glycerol (about 0.02 mL) with the mass concentration of 20% -30% is dripped above the leaves, the leaves are excited by blue light or green light respectively under fluorescence microscopy, and bright yellow fluorescence is observed from glandular hair and non-glandular hair, thus obtaining the fluorescent staining sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves.
The method is scientific and reasonable, simple in operation, low in cost, small in workload, high in success rate, clear in observation and good in effect, can satisfactorily solve the technical problem of leaf epidermis hair observation, has low technical requirements on operators, can achieve an ideal effect, and is effectively used for innovation of fluorescent staining flaking methods for mugwort and tarragon, but is not limited to mugwort and tarragon, and microscopic observation of the leaf epidermis hair, the glandular hair and the non-glandular hair of plants.
Drawings
Fig. 1 is a scanning electron microscope effect diagram of upper surface glandular hair (oval) and non-glandular hair (T-shaped non-glandular hair) of mugwort leaf.
Fig. 2 is a graph showing the effect of the upper surface glandular hair (oval) and the non-glandular hair (T-shaped non-glandular hair) of mugwort leaf under the electron microscope.
FIG. 3 is a graph showing the effect of the lower surface of mugwort leaf and the non-glandular hair (T-shaped non-glandular hair) and glandular hair (oval shape, most of which are covered by non-glandular hair) by the scanning electron microscope of the present invention.
Fig. 4 is a graph showing the effect of the lower surface glandular hair (oval spot) and the non-glandular hair (T-shaped non-glandular hair) of mugwort leaf under the low power lens of the present invention.
Fig. 5 is a graph showing the effect of the lower surface glandular hair (oval spot) and the non-glandular hair (T-shaped non-glandular hair) of mugwort leaf under the high power microscope.
Detailed Description
The following describes in detail the embodiments of the present invention with reference to specific cases and examples.
The invention, in its practice, can be illustrated by the following examples.
Example 1
The invention discloses a fluorescent staining flaking method for observing the morphology of plant leaf epidermis hair, including the following steps:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 5.5min at an ultrasonic frequency of 25KHz;
(2) Immersing: cutting 9mm small blocks from the blade after ultrasonic cleaning, and immersing in 4mL of mixed solution of alcohol and glacial acetic acid for 2 hours;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 2min;
(4) Standing: placing the rinsed leaves into 4mL of phloroglucinol solution, and standing for 20min, wherein the mass content of the phloroglucinol solution is 7.5%;
(5) And (3) observation: the leaves are taken out from the phloroglucinol solution, one surface with the surface fur is placed on a glass slide upwards, a drop of 25% mass concentration diluted glycerol (about 0.02 mL) is dripped above the leaves, the leaves are excited by blue light or green light respectively under fluorescence microscopy, and bright yellow fluorescence is observed from glandular hair and non-glandular hair, thus obtaining the fluorescent staining sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves.
Example 2
The invention discloses a fluorescent staining flaking method for observing the morphology of plant leaf epidermis hair, including the following steps:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 4min at an ultrasonic frequency of 28KHz;
(2) Immersing: cutting small blocks of 8mm from the blade after ultrasonic cleaning, and immersing in 3mL of mixed solution of alcohol and glacial acetic acid for 1.2h;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 1min;
(4) Standing: placing the rinsed leaves into 3mL of phloroglucinol solution, and standing for 30min, wherein the mass content of the phloroglucinol solution is 6%;
(5) And (3) observation: the leaves are taken out from the phloroglucinol solution, one surface with the surface fur is placed on a glass slide upwards, a drop of diluted glycerol (about 0.02 mL) with the mass concentration of 22% is dripped above the leaves, and the leaves are excited by blue light or green light respectively under fluorescence microscopy, so that bright yellow fluorescence is observed from glandular hair and non-glandular hair, and the fluorescent staining sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves is obtained.
Example 3
The invention discloses a fluorescent staining flaking method for observing the morphology of plant leaf epidermis hair, including the following steps:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 8min at an ultrasonic frequency of 20KHz;
(2) Immersing: cutting small pieces of 10mm from the blade after ultrasonic cleaning, and immersing in 5mL of mixed solution of alcohol and glacial acetic acid for 2.8h;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 3min;
(4) Standing: placing the rinsed leaves into 5mL of phloroglucinol solution, and standing for 10min, wherein the mass content of the phloroglucinol solution is 9%;
(5) And (3) observation: the leaves are taken out from the phloroglucinol solution, one surface with the surface fur is placed on a glass slide upwards, a drop of 28% mass concentration diluted glycerol (about 0.02 mL) is dripped above the leaves, and the leaves are excited by blue light or green light respectively under fluorescence microscopy, so that bright yellow fluorescence is observed from glandular hair and non-glandular hair, and the fluorescent staining sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves is obtained.
From the above, the method is scientific and reasonable, easy to operate and good in observation effect, the quality and the medicinal value of the mugwort can be effectively ensured, and the result shows that after repeated trial and experiment (taking example 1 as an example), the method is simple and convenient to operate: pigment in chloroplast can be destroyed after the treatment of the alcohol mixed solution, and interference on fluorescent color development is avoided, so that the observation background is changed, and meanwhile, non-glandular hairs on epidermis are softened, so that the permeability of glandular Mao Jiaozhi layer is increased; in addition, the 95% alcohol is used as a solvent to ensure that the phloroglucinol has high solubility and high concentration, and the permeability is enhanced, so that the phloroglucinol is beneficial to the combination of the phloroglucinol and the epidermal hair; and then emits bright yellow fluorescence under the excitation light of a fluorescence microscope. The morphology and distribution of glandular and non-glandular hairs can be clearly seen, and the glandular and non-glandular hairs are quite clear even under a low power microscope, so that the statistics of the number and the density of glandular and non-glandular hairs is very favorable.
The same experiment was performed on the example 1 and other examples, and the same and similar results were obtained, and the method of the present invention was also used for producing sheets of plant festoon sheets or a section of stalk sheets, and the technical effects were very good, and are not listed here.
Therefore, the invention provides a fluorescent staining flaking method for observing the morphology of plant epidermis hair, solves the technical problem that a bright field optical microscope and a scanning electron microscope cannot be used for observing the morphology of leaf epidermis hair, has low technical requirements on operators, is simple to operate, has a one-time flaking success rate of about 99%, and can satisfactorily complete the statistics of the number and density of glandular hair and non-glandular hair and the analysis and research of the surface structure of a leaf blade. The invention is a great innovation in the technology of observing the morphological structure of the plant leaf surface fur and the non-glandular fur, and has wide application prospect.
Claims (4)
1. A method for fluorescent staining and tabletting for observing the morphology of the glandular hair and the non-glandular hair of the epidermis of a plant leaf, comprising the steps of:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 3-8 min, wherein the ultrasonic frequency is less than or equal to 30KHz;
the plant is mugwort or tarragon;
(2) Immersing: cutting small blocks with the size of 8-10 mm from the blade after ultrasonic cleaning, immersing in 3-5 mL of mixed solution of alcohol and glacial acetic acid for 1-3 h, wherein the volume ratio of the alcohol to the glacial acetic acid is 2:1;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing the blade with distilled water for 1-3 min;
(4) Standing: placing the rinsed leaves into 3-5 mL of phloroglucinol solution, and standing for 10-30 min;
the phloroglucinol solution is prepared by adding phloroglucinol into 95% alcohol serving as a solvent to prepare a phloroglucinol solution with the mass content of 5% -10%;
(5) And (3) observation: taking out the leaves from the phloroglucinol solution by using forceps, placing the surface with the surface fur on a glass slide, dripping a drop of diluted glycerol with the mass concentration of 20-30% above the leaves, respectively exciting the leaves by blue light or green light under a fluorescence microscope, and observing that the glandular hair and the non-glandular hair emit bright yellow fluorescence, thus obtaining the fluorescent dyeing sheet for observing the glandular hair and the non-glandular hair forms of the surface fur of the plant leaves.
2. A method of fluorescent dye flaking for observing plant leaf surface coat glandular and non-glandular morphology according to claim 1, comprising the steps of:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 5.5min at an ultrasonic frequency of 25KHz;
(2) Immersing: cutting 9mm small blocks from the blade after ultrasonic cleaning, and immersing in 4mL of mixed solution of alcohol and glacial acetic acid for 2 hours;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 2min;
(4) Standing: placing the rinsed leaves into 4mL of phloroglucinol solution, and standing for 20min, wherein the mass content of the phloroglucinol solution is 7.5%;
(5) And (3) observation: and taking out the leaves from the phloroglucinol solution, placing the surface with the surface fur on a glass slide, dripping a drop of diluted glycerol with the mass concentration of 25% above the leaves, respectively exciting the leaves by blue light or green light under a fluorescence microscope, and observing bright yellow fluorescence of glandular hair and non-glandular hair, thus obtaining the fluorescent dyeing sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves.
3. A method of fluorescent dye flaking for observing plant leaf surface coat glandular and non-glandular morphology according to claim 1, comprising the steps of:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 4min at an ultrasonic frequency of 28KHz;
(2) Immersing: cutting small blocks of 8mm from the blade after ultrasonic cleaning, and immersing in 3mL of mixed solution of alcohol and glacial acetic acid for 1.2h;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 1min;
(4) Standing: placing the rinsed leaves into 3mL of phloroglucinol solution, and standing for 30min, wherein the mass content of the phloroglucinol solution is 6%;
(5) And (3) observation: taking out the leaves from the phloroglucinol solution, placing the surface with the surface fur on a glass slide, dripping a drop of diluted glycerol with the mass concentration of 22% above the leaves, respectively exciting the leaves with blue light or green light under a fluorescence microscope, and observing bright yellow fluorescence of glandular hair and non-glandular hair, thus obtaining the fluorescent dyeing sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves.
4. A method of fluorescent dye flaking for observing plant leaf surface coat glandular and non-glandular morphology according to claim 1, comprising the steps of:
(1) Selecting the blade: taking fresh healthy plant leaves, and ultrasonically cleaning the leaves in distilled water for 8min at an ultrasonic frequency of 20KHz;
(2) Immersing: cutting small pieces of 10mm from the blade after ultrasonic cleaning, and immersing in 5mL of mixed solution of alcohol and glacial acetic acid for 2.8h;
(3) Rinsing: taking out the blade immersed in the step (2), and rinsing with distilled water for 3min;
(4) Standing: placing the rinsed leaves into 5mL of phloroglucinol solution, and standing for 10min, wherein the mass content of the phloroglucinol solution is 9%;
(5) And (3) observation: taking out the leaves from the phloroglucinol solution, placing the surface with the surface fur on a glass slide, dripping a drop of 28% mass concentration diluted glycerol above the leaves, respectively exciting the leaves with blue light or green light under a fluorescence microscope, and observing bright yellow fluorescence of glandular hair and non-glandular hair to obtain the fluorescent dyeing sheet for observing the forms of glandular hair and non-glandular hair of the surface fur of the plant leaves.
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