CN114993793B - Application of sudan red in plant glandular hair specific dyeing - Google Patents
Application of sudan red in plant glandular hair specific dyeing Download PDFInfo
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- CN114993793B CN114993793B CN202210681649.9A CN202210681649A CN114993793B CN 114993793 B CN114993793 B CN 114993793B CN 202210681649 A CN202210681649 A CN 202210681649A CN 114993793 B CN114993793 B CN 114993793B
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- 230000000762 glandular Effects 0.000 title claims abstract description 44
- 238000004043 dyeing Methods 0.000 title claims abstract description 34
- 239000001044 red dye Substances 0.000 claims abstract description 18
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- 239000000843 powder Substances 0.000 claims description 22
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Images
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- 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
- G01N2001/302—Stain compositions
Abstract
The invention discloses an application of sudan red in specific dyeing of plant glandular hairs, which belongs to the technical field of plant tissue observation, and is characterized in that plant leaves with good growth are placed in a fixing solution, so that on one hand, morphological characteristics of plant epidermal hairs are maintained, on the other hand, chlorophyll in the leaves is removed, influence of autofluorescence of the chlorophyll on the observation of the plant epidermal features is eliminated, lipid can be specifically indicated by using sudan red dye, the lipid is dyed into orange to dark red, and glandular hairs of synthetic and stored essential oil are specifically dyed, so that glandular hairs and non-glandular hairs are effectively distinguished, and the quantity and distribution of the glandular hairs and the non-glandular hairs are counted. The application of sudan red in plant glandular hair specific dyeing provided by the invention has the advantages of simple operation method, strong practicality, low cost, low expensive equipment dependence, small workload and high success rate, and can effectively solve the problem that the plant leaves are indistinct in various epidermis hair types.
Description
Technical Field
The invention relates to the technical field of plant tissue observation, in particular to application of sudan red in plant glandular hair specific dyeing.
Background
Almost all plant surfaces are covered with hair-like structures, the upper part being called the epidermis and the lower part being called the root hair. The epidermal hair is developed from plant epidermal cells, is widely distributed in terrestrial plants, and is a specific structure growing in plant epidermal tissues. Depending on the characteristics and functions, the epidermal hair can be divided into branched or unbranched, single-or multicellular, and glandular Mao Huofei glandular hair. Non-glandular hairs are the first protective barrier of plants against natural hazards such as animal feeding, ultraviolet (UV) radiation, etc., whereas glandular hairs are important sites for the synthesis, storage and secretion of natural products. The activity of medicinal plants can be attributed to the natural products synthesized by the medicinal plants, and the epidermis hair is one of important sites for the synthesis and storage of natural products of plants, so that the type and quantity of epidermis hair are important factors affecting the quality of medicinal materials.
In order to improve the quality of medicinal materials, researchers have intensively studied the epidermis Mao Jin of the plant and observe the morphology and type of the leaf epidermis of the plant by means of a series of electronic devices, which are usually implemented by an optical microscope, a fluorescence microscope, a scanning electron microscope, etc., and the discrimination effect is sequentially improved and the instrument price is increased. The plant leaves of patchouli, peppermint and the like are attached with dense epidermis hairs of different types, and a large number of non-glandular hairs generate serious interference on observing the morphology, the type, the distribution and the like of glandular hairs; in addition, the epidermis Mao Touming, which has a certain height, has difficulty focusing on the same horizontal plane to distinguish the structure and type of epidermis. In general, a laboratory adopts a flaking method such as a peeling method, a transparent flaking method, a tissue slicing method and the like, but the operation difficulty of the above-mentioned means is higher, the process is complex, and the destruction of plant tissues is easy to cause, thereby influencing the morphology and the distribution state of plant glandular hairs. Thus, there is a need to provide a technique for specific staining of plant glandular hairs for effectively distinguishing the type of plant epidermal hair and its distribution.
Disclosure of Invention
The invention provides application of sudan red in specific dyeing of plant glandular hairs, effectively solves the technical problems that the existing tabletting process is complex and the morphology and the distribution state of the plant glandular hairs are easy to damage, and simultaneously overcomes the technical defect that the existing observation means highly depend on expensive instruments.
The invention provides an application of sudan red in plant glandular hair specific dyeing, which is characterized by comprising the following steps:
s1, collecting plant tissues: selecting leaves with good plant growth state, and taking part of the leaves for standby;
s2, fixing: placing part of the blades in the S1 into FAA fixing liquid, vacuumizing, refrigerating and fixing after the blades sink into the bottom of the fixing liquid, and rinsing to obtain the fixed blades;
s3, dyeing: placing the fixed blade in the S2 in Sudan red dye, vacuumizing, dyeing at constant temperature after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a dyed blade;
s4, tabletting and observing: the stained leaves of S3 were pelleted and then observed under an optical fiber and photographed.
Preferably, in S3, the sudan red dye is sudan red 7B dye solution, sudan red i dye solution, sudan red iii dye solution, or sudan red iv dye solution.
Preferably, the preparation method of the sudan red dye comprises the steps of dissolving 10-50 mg of sudan red powder in polyethylene glycol, and adding 70-100% of glycerin after the powder is completely dissolved.
Preferably, the feed liquid ratio of the sudan red powder to the polyethylene glycol is 10mg: 2-10 mL.
Preferably, the feed liquid ratio of the sudan red powder to the glycerol is 10mg: 5-10 mL.
Preferably, in S3, the vacuumizing time is 3-15 min, and the vacuum pressure is 10-20 hPa.
Preferably, in S3, the constant temperature dyeing temperature is 28-37 ℃ and the time is 16-48 h.
Preferably, in S1, the partial blade is a circular blade with a diameter of 0.2-1 cm taken at both sides thereof avoiding the main vein.
Preferably, in S2, the vacuumizing time is 2-10 min, and the vacuum pressure is 10-20 hPa.
Preferably, in S2, the refrigerating fixed temperature is 4 ℃ and the time is 24-72 hours.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, the plant leaves with good growth are placed in the fixing liquid, so that on one hand, the morphological characteristics of the plant epidermal hair are maintained, on the other hand, chlorophyll in the leaves is removed, the influence of autofluorescence of the chlorophyll on observing the plant epidermal characteristics is eliminated, the sudan red dye is adopted to specifically indicate grease substances, the grease is dyed into orange to dark red, and the glandular hair synthesized and stored with essential oil is specifically dyed, so that glandular hair and non-glandular hair are effectively distinguished, and the quantity and distribution of glandular hair and non-glandular hair are counted.
(2) The application of sudan red in plant glandular hair specific dyeing provided by the invention has the advantages of simple operation method, strong practicality, low cost, low expensive equipment dependence, small workload and high success rate, and can effectively solve the problem that the plant leaves are indistinct in various epidermis hair types.
Drawings
FIG. 1 is an optical microscope image of patchouli leaf surface coat of example 1; representing the glandular hairs, [ V ] representing the glandular hairs;
FIG. 2 is a graph of patchouli leaf surface fur under a high power microscope in example 1; figure a is the shield hair, figure B is the head hair, and figure C is the non-hair;
FIG. 3 is an optical microscope image of patchouli leaves of comparative example 1;
FIG. 4 is an optical microscope image of patchouli leaf of comparative example 2.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention. The following test methods and detection methods, if not specified, are conventional methods; the reagents and starting materials, unless otherwise specified, are commercially available.
The invention uses patchouli as raw material to carry out dyeing experiment.
The FAA fixing solution is prepared from 3.7mL of formaldehyde, 5mL of glacial acetic acid, 60mL of absolute ethyl alcohol, 5mL of glycerol and deionized water, and the volume is fixed to 100 mL.
The polyethylene glycol adopts polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or polyethylene glycol 600.
The preparation method comprises the following steps: transfer the stained leaves to the slide, fix the stained leaves with 20% -50% glycerol.
Example 1
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.2cm from two sides of the main veins by using a puncher for standby;
s2, fixing: placing the round blade in the FAA fixing solution, vacuumizing for 2min under the vacuum pressure of 10hPa, refrigerating and fixing for 24h at 4 ℃ after the blade is immersed into the bottom of the fixing solution, and rinsing to obtain the fixed blade;
s3, dyeing: placing the fixed blade in S2 in Sudan red 7B staining solution, vacuumizing for 3min under the vacuum pressure of 10hPa, staining at constant temperature of 28 ℃ for 16h after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain the stained blade;
the preparation method of the sudan red 7B staining solution comprises the steps of dissolving 10mg of sudan red 7B powder in 5mL of polyethylene glycol 300, and adding 2mL of 70% glycerol by volume after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 20% glycerol, they were pelleted, observed under an optical fiber, and photographed.
Example 2
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 1cm from two sides by using a puncher for standby;
s2, fixing: placing the round blade in the fixing liquid, vacuumizing for 10min under the vacuum pressure of 20hPa, refrigerating and fixing for 72h at 4 ℃ after the blade is immersed into the bottom of the fixing liquid, and rinsing to obtain the fixed blade;
s3, dyeing: placing the fixed blade in S2 in Sudan red I staining solution, vacuumizing for 15min under the vacuum pressure of 20hPa, staining at a constant temperature of 37 ℃ for 48h after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a stained blade;
the preparation method of the sudan red I staining solution comprises the steps of dissolving 50mg of sudan red I powder in 10mL of polyethylene glycol 200, and adding 10mL of 100% glycerol after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 50% glycerol, they were pelleted, observed under an optical fiber, and photographed.
Example 3
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.6cm from two sides of the main veins by using a puncher for standby;
s2, fixing: placing the round blade in the fixing liquid, vacuumizing for 6min under the vacuum pressure of 15hPa, refrigerating and fixing for 48h at 4 ℃ after the blade is immersed into the bottom of the fixing liquid, and rinsing to obtain the fixed blade;
s3, dyeing: placing the fixed blade in S2 in Sudan red III staining solution, vacuumizing for 9min under the vacuum pressure of 15hPa, staining the blade at a constant temperature of 32 ℃ for 32h after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a stained blade;
the preparation method of the sudan III staining solution comprises the steps of dissolving 30mg of sudan III powder in 8mL of polyethylene glycol 400, and adding 6mL of 85% glycerol after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 35% glycerol, they were pelleted, observed under an optical fiber, and photographed.
Example 4
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.4cm from two sides of the main veins by using a puncher for standby;
s2, fixing: placing the round blade in the fixing liquid, vacuumizing for 4min under the vacuum pressure of 12hPa, refrigerating and fixing for 36h at 4 ℃ after the blade is immersed into the bottom of the fixing liquid, and rinsing to obtain the fixed blade;
s3, dyeing: placing the fixed blade in the S2 in Sudan red IV staining solution, vacuumizing for 6min under the vacuum pressure of 12hPa, staining for 24h at the constant temperature of 30 ℃ after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain the stained blade;
the preparation method of the sudan IV staining solution comprises the steps of dissolving 20mg of sudan IV powder in 6mL of polyethylene glycol 600, and adding 4mL of glycerin with the volume percentage of 78% after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 28% glycerol, they were pelleted, observed under an optical fiber, and photographed.
Example 5
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.8cm from two sides of the main veins by using a puncher for standby;
s2, fixing: placing the round blade in the fixing liquid, vacuumizing for 8min under the vacuum pressure of 18hPa, refrigerating and fixing for 60h at 4 ℃ after the blade is immersed into the bottom of the fixing liquid, and rinsing to obtain the fixed blade;
s3, dyeing: placing the fixed blade in S2 in Sudan red 7B staining solution, vacuumizing for 12min under the vacuum pressure of 18hPa, staining the blade at a constant temperature of 35 ℃ for 40h after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a stained blade;
the preparation method of the sudan red 7B staining solution comprises the steps of dissolving 40mg of sudan red 7B powder in 9mL of polyethylene glycol 300, and adding 8mL of 93% glycerol by volume after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 42% glycerol, they were pelleted, observed under an optical fiber, and photographed.
To further illustrate the effect of the present invention, the present invention provides a comparative example as follows:
comparative example 1
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.6cm from two sides of the main veins by using a puncher for standby;
s2, fixing without fixing liquid;
s3, dyeing: placing the round blade in S1 in Sudan red 7B staining solution, vacuumizing under 15hPa for 9min, after the blade is immersed into the bottom of Sudan red dye, staining at constant temperature of 32deg.C for 32h, rinsing to obtain dyed blade, decolorizing the dyed blade with ethanol to obtain decolorized blade
The preparation method of the sudan red 7B staining solution comprises the steps of dissolving 30mg of sudan red 7B powder in 8mL of polyethylene glycol 300, and adding 6mL of 85% glycerol after the powder is completely dissolved.
S4, tabletting and observing: the decolorized leaves of S3 were transferred to a glass slide, and the stained leaves were fixed with 35% glycerol, and then observed under an optical fiber and photographed.
Comparative example 2
The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting blades with good growth state of herba Agastaches, avoiding main veins, and taking round blades with diameter of 0.6cm from two sides of the main veins by using a puncher for standby;
s2, fixing without fixing liquid;
s3, dyeing: placing the fixed blade in S2 in Sudan red 7B staining solution, vacuumizing for 9min under the vacuum pressure of 15hPa, staining the blade at a constant temperature of 32 ℃ for 32h after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a stained blade;
the preparation method of the sudan red 7B staining solution comprises the steps of dissolving 30mg of sudan red 7B powder in 8mL of polyethylene glycol 300, and adding 6mL of 85% glycerol after the powder is completely dissolved.
S4, tabletting and observing: the stained leaves of S3 were transferred to a glass slide, and after the stained leaves were fixed with 35% glycerol, they were pelleted, observed under an optical fiber, and photographed.
As can be seen from fig. 1 and 2, the sudan red dye can be used for specifically dyeing plant glandular hairs, and can be used for better distinguishing the scutellar glandular hairs, the cephalic glandular hairs and the non-glandular hairs of plant leaves, so that the problem that the plant leaves are indistinct in various epidermis hair types is effectively solved. In comparative example 1, patchouli leaves were not fixed with a fixing solution, were directly dyed with a sudan red dyeing solution, and were decolorized with alcohol; comparative example 2 the patchouli leaves were not fixed with the fixing solution, were directly dyed with sudan red dyeing solution and were not decolorized with alcohol, as can be seen from fig. 3 and 4, both of which were not able to distinguish the plant glandular hair species at all under an optical microscope.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (4)
1. The application of sudan red in the specific dyeing of plant glandular hairs is characterized by comprising the following steps:
s1, collecting plant tissues: selecting leaves with good plant growth state, and taking part of the leaves for standby; the partial blades are round blades with the diameter of 0.2-1 cm at two sides of the main veins;
s2, fixing: placing part of the blades in the S1 into FAA fixing liquid, vacuumizing, refrigerating and fixing after the blades sink into the bottom of the fixing liquid, and rinsing to obtain the fixed blades; the vacuumizing time is 2-10 min, and the vacuum pressure is 10-20 hPa; the fixed temperature of the refrigeration is 4 ℃ and the time is 24-72 hours;
s3, dyeing: placing the fixed blade in the S2 in Sudan red dye, vacuumizing, dyeing at constant temperature after the blade is immersed into the bottom of the Sudan red dye, and rinsing to obtain a dyed blade; the preparation method of the sudan red dye comprises the steps of dissolving 10-50 mg of sudan red powder in polyethylene glycol, and adding 70-100% of glycerin after the powder is completely dissolved; the vacuumizing time is 3-15 min, and the vacuum pressure is 10-20 hPa; the constant-temperature dyeing temperature is 28-37 ℃ and the constant-temperature dyeing time is 16-48 h;
s4, tabletting and observing: the stained leaves of S3 were pelleted and then observed under an optical microscope and photographed.
2. The use of sudan red according to claim 1 for specific staining of plant glandular hairs, wherein in S3 the sudan red dye is sudan red 7B dye solution, sudan red i dye solution, sudan red iii dye solution or sudan red iv dye solution.
3. The use of sudan red according to claim 1 for specific staining of plant glandular hairs, wherein the sudan red powder to polyethylene glycol feed liquid ratio is 10mg: 5-10 mL.
4. Use of sudan red according to claim 1 for specific staining of plant glandular hairs, characterized in that the sudan red powder to glycerol feed liquid ratio is 10mg: 2-10 mL.
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|---|---|---|---|---|
| CN101975780B (en) * | 2010-04-28 | 2012-01-11 | 湖南农业大学 | Observation and density measurement method for glandular hairs on Artemisia annua leaf surfaces |
| CN103743611A (en) * | 2014-01-16 | 2014-04-23 | 西南大学 | Staining solution and method for glandular hair and glandular hair secreta of tobacco |
| CN106802252A (en) * | 2016-12-16 | 2017-06-06 | 江苏省中国科学院植物研究所 | A kind of method of counting of mint peltate glandular hairs |
| CN110174298B (en) * | 2019-04-30 | 2021-12-28 | 长江大学 | Dyeing method for observing microstructure inside plant |
| CN114608910A (en) * | 2022-03-09 | 2022-06-10 | 河南中医药大学 | Method for preparing slices by simultaneously observing and counting glandular hairs, non-glandular hairs and pores under folium artemisiae argyi |
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