CN114894827B - Method for rapidly treating wax coating of leaf surface of wax leaf specimen - Google Patents
Method for rapidly treating wax coating of leaf surface of wax leaf specimen Download PDFInfo
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- CN114894827B CN114894827B CN202210432085.5A CN202210432085A CN114894827B CN 114894827 B CN114894827 B CN 114894827B CN 202210432085 A CN202210432085 A CN 202210432085A CN 114894827 B CN114894827 B CN 114894827B
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000011248 coating agent Substances 0.000 title abstract description 13
- 238000000576 coating method Methods 0.000 title abstract description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 210000002615 epidermis Anatomy 0.000 claims abstract description 18
- 238000005520 cutting process Methods 0.000 claims abstract description 11
- 238000010438 heat treatment Methods 0.000 claims abstract description 10
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- 238000002791 soaking Methods 0.000 claims abstract description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 7
- 210000003491 skin Anatomy 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims 1
- 210000001595 mastoid Anatomy 0.000 abstract description 43
- 235000017166 Bambusa arundinacea Nutrition 0.000 abstract description 19
- 235000017491 Bambusa tulda Nutrition 0.000 abstract description 19
- 241001330002 Bambuseae Species 0.000 abstract description 19
- 235000015334 Phyllostachys viridis Nutrition 0.000 abstract description 19
- 239000011425 bamboo Substances 0.000 abstract description 19
- 241000196324 Embryophyta Species 0.000 abstract description 17
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 210000000056 organ Anatomy 0.000 abstract description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 abstract description 3
- 238000011282 treatment Methods 0.000 description 43
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 38
- 238000001000 micrograph Methods 0.000 description 24
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 20
- 239000008096 xylene Substances 0.000 description 18
- 241000267040 Sasa borealis Species 0.000 description 8
- 241000544461 Sasa senanensis Species 0.000 description 8
- 239000011148 porous material Substances 0.000 description 8
- 241000209504 Poaceae Species 0.000 description 7
- 240000005499 Sasa Species 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 238000001493 electron microscopy Methods 0.000 description 4
- 239000000356 contaminant Substances 0.000 description 3
- 241001330024 Bambusoideae Species 0.000 description 2
- 239000011247 coating layer Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000004544 sputter deposition Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000233614 Phytophthora Species 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000004018 waxing Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for rapidly treating a wax coating of a leaf surface of a wax leaf specimen. Cutting a cured leaf specimen, placing the cured leaf specimen in chloroform for soaking, then replacing new chloroform, heating at constant temperature, then replacing the cured leaf specimen with an ethanol aqueous solution, and performing ultrasonic treatment, thus repeating the steps for a plurality of times to obtain a pretreated scanning electron microscope sample. Because the flowering period of the bamboo subfamily plants is long, most of the collected plants can be observed in the field, and therefore, the classification characters of the bamboo subfamily plants are often mainly nutrition organs. The characteristics of stomata, mastoid, hair quilt and the like in the epidermis of the bamboo leaves have important classification value. The invention can clean the leaf epidermis of the cured leaf specimen, and has great value for classifying plants of the bamboo subfamily.
Description
Technical field:
the invention belongs to the field of electron microscopy, and particularly relates to a method for rapidly processing a wax cover of a leaf surface of a wax leaf specimen.
The background technology is as follows:
some features of plant leaves are controlled by genes, and leaf epidermis micro-morphology features have important taxonomic value in plant taxonomy. Some plant leaf wax is thick and covers the pores completely, and the wax has the main functions of protecting leaf epidermal cells and preventing foreign insects and bacteria from erosion. The observation of micro-morphology structures such as air hole types, mastoid processes, fur quilts and the like is influenced to a certain extent, and glue is often used for modern binding specimens, so that more impurities are contained in leaf epidermis and the cleaning is difficult. The characteristics of the type and size of the air holes, the type and size of the hair of the epidermis, the morphology of the epidermis cells and the like can not be seen under a scanning electron microscope. At present, a rapid method for removing wax from the epidermis of a leaf specimen is not available.
The invention comprises the following steps:
the invention aims to provide a method for rapidly processing a wax coating of a leaf epidermis of a wax leaf specimen.
The invention relates to a method for rapidly processing a wax coating of a leaf surface of a wax leaf specimen, which comprises the following steps:
cutting a cured leaf specimen, placing the cured leaf specimen in chloroform for soaking, then replacing new chloroform, heating at constant temperature, then replacing the cured leaf specimen with an ethanol aqueous solution, and performing ultrasonic treatment, thus repeating the steps for a plurality of times to obtain a pretreated scanning electron microscope sample.
Preferably, the constant temperature heating is heating at 50 ℃ for 8 hours. Further heating the mixture for 8 hours at the temperature of 50 ℃ in a constant-temperature metal bath.
Preferably, the soaking time of the material is 12 hours after the material is soaked in chloroform.
Preferably, the ethanol aqueous solution is 70% ethanol aqueous solution by volume fraction, ultrasonic treatment is carried out for 3min, and the ethanol aqueous solution by volume fraction 70% is repeatedly replaced for three times, and each ultrasonic treatment is carried out for 3min.
Preferably, the cutting of the cured leaf sample is cutting the cured leaf sample into rectangles of 0.8cm multiplied by 0.6 cm.
Because the flowering period of the bamboo subfamily plants is long, most of the collected plants can be observed in the field, and therefore, the classification characters of the bamboo subfamily plants are often mainly nutrition organs. The characteristics of stomata, mastoid, hair quilt and the like in the epidermis of the bamboo leaves have important classification value. The invention can clean wax of leaf epidermis of the cured leaf specimen, and has great value for classifying plants of the bamboo subfamily.
Description of the drawings:
FIG. 1 is a scanning electron microscope image of a Hua Chizhu Sinosasa longiligulata prior art method;
FIG. 2 is a scanning electron microscope image of Hua Chizhu Sinosasa longiligulata, a method of the present invention;
FIG. 3 is a scanning electron microscope image of a multi Mao Huachi bamboo Sinosasa polytricha, prior art method;
FIG. 4 is a scanning electron microscope image of a multi Mao Huachi bamboo Sinosasa polytricha, method of the present invention;
FIG. 5 is a scanning electron microscope image of Sasa senanensis, a prior art method;
FIG. 6 is a scanning electron microscope image of Sasa senanensis, a method of the present invention;
FIG. 7 is a scanning electron microscope image of Sasa borealis, a prior art method;
FIG. 8 is a scanning electron microscope image of Sasa borealis, a method of the invention;
FIG. 9 is a scanning electron microscope image of Hua Chizhu Sinosasa longiligulata, untreated direct mount;
FIG. 10 is a scanning electron microscope image of Hua Chizhu Sinosasa longiligulata, acetone treatment;
FIG. 11 is a scanning electron microscope image of Hua Chizhu Sinosasa longiligulata, xylene treatment;
FIG. 12 is a scanning electron microscope image of Hua Chizhu Sinosasa longiligulata, xylene+acetone (1:1 v/v) treatment;
FIG. 13 is a scanning electron microscope image of a multi Mao Huachi bamboo Sinosasa polytricha, untreated direct mount;
FIG. 14 is a scanning electron microscope image of bamboo Sinosasa polytricha, duobu Mao Huachi, acetone treatment;
FIG. 15 is a scanning electron microscope image of Duobu Mao Huachi bamboo Sinosasa polytricha, xylene treatment;
FIG. 16 is a scanning electron microscope image of a multi Mao Huachi bamboo Sinosasa polytricha, xylene+acetone (1:1 v/v) treatment;
FIG. 17 is a scanning electron microscope image of Sasa senanensis, untreated direct mount;
FIG. 18 is a scanning electron microscope image of Sasa senanensis, acetone treatment;
FIG. 19 is a scanning electron microscope image of Sasa senanensis, xylene treatment;
FIG. 20 is a scanning electron microscope image of Sasa senanensis, xylene+acetone (1:1 v/v) treatment;
FIG. 21 is a scanning electron microscope image of Sasa borealis, without processing the direct mount;
FIG. 22 is a scanning electron microscope image of Sasa borealis, acetone treatment;
FIG. 23 is a scanning electron microscope image of Sasa borealis, xylene treatment;
FIG. 24 is a scanning electron microscope image of Sasa borealis, xylene+acetone (1:1 v/v) treatment.
Detailed Description
The following examples are illustrative of the invention and should not be construed as limiting the invention.
Example 1:
in the invention, the wax leaf of the wax leaf specimen is selected to cover all air holes as a test material. The study subjects were 2 Sasa species and 2 Sasa species of the genus Sasa of the subfamily Bambusae of the family Gramineae, the specific materials and sources are shown in Table 1, and the evidence specimens and the collection places are marked after the collection numbers. The species in the table are mainly arranged according to the systems of Chinese Phytophthora and Flora of China.
Because the flowering period of the bamboo subfamily plants is long, most of the collected plants can be observed in the field, and therefore, the classification characters of the bamboo subfamily plants are often mainly nutrition organs. The characteristics of stomata, mastoid, hair quilt and the like in the epidermis of the bamboo leaves have important classification value. .
Table 1 Experimental materials and credential specimens
Note that: IBSC is an abbreviation for the national academy of sciences, south China, and the plant garden specimen museum.
The experimental method comprises the following steps:
cutting leaf of cured leaf sample into rectangle of 0.8cm×0.6cm, soaking in test tube filled with chloroform for 12 hr, replacing chloroform, heating with constant temperature metal bath JS-400A at 50deg.C for 8 hr, replacing with 70% ethanol, ultrasonic (WIGGENS UAIOMFD 59HZ 250W) for 3min, and repeatedly replacing 70% ethanol for three times each ultrasonic for 3min. And (3) carrying out serial dehydration 80%, 90% (10 min/time) and 100% (10 min/time and 5 times in total) according to a conventional scanning electron microscope method, drying critical points, adhering a table, plating platinum on an ion sputtering instrument, and observing and photographing under a JSM-6360LV vacuum scanning electron microscope.
Control:
scanning electron microscopy (scanning electron microscope, SEM) of the prior art observes the blade processing method and procedure:
selecting mature leaves, cutting a rectangle with the length of 0.5cm multiplied by 1cm at the middle part close to a midrib, uniformly dividing the rectangle into two squares with the length of 0.5cm multiplied by 0.5cm, selecting one square, placing the upper surface of the square face upwards, cutting the upper right corner of the square face so that the square face is easily distinguished from the lower surface of the square face, and placing the square face into a centrifuge tube; dropwise adding a proper amount of chloroform into the centrifuge tube, cutting a small piece of filter paper, plugging into the centrifuge tube, enabling the leaves to be completely immersed into the chloroform, and standing for 12 hours to remove wax on the surfaces of the leaves; clamping the treated leaves, washing off chloroform with distilled water, placing the leaves on filter paper for airing, and adhering the leaves on a sample table in a positive-negative way; and (3) placing the electrode in an ion sputtering instrument for conducting treatment, and then placing the electrode under a JSM-6360LV vacuum scanning electron microscope for observation and photographing. Long cells, short cells, stomata and appendages of the upper and lower epidermis of the leaf are mainly observed, the number of long cells and stomata columns is mainly counted, and the length and width of the stomata are measured (at least 20 stomata are measured for each species, and the average value is calculated). The voucher specimens were stored in the national academy of sciences, south China vegetable garden public laboratory.
The method is compared with the following steps: after soaking in chloroform for 12 hours, the method of the invention heats the solution for 8 hours by using a constant-temperature metal bath at 50 ℃, and then obtains the result of completely removing the wax after ultrasonic cleaning by 70% ethanol. The wax is not completely removed by soaking for 12 hours in the prior art.
The results of the picture comparison are as follows:
1. hua Chizhu Sinosasa longiligulata: the leaf surface of the genus Hua Chi of the subfamily Bambusae of Gramineae has stomata, mastoid (spherical mastoid, rod-like mastoid) and micromouch. As shown in fig. 1, the prior art treatment method scans the leaf skin photograph of an electron microscope: the treatment is not clean. As shown in fig. 2, the leaf epidermis scanning electron microscope photograph of the treatment method of the present invention: all the treatment is clean.
2. Many Mao Huachi bamboo Sinosasa polytricha: the leaf surface of the genus Hua Chi of the subfamily Bambusae of Gramineae has stomata, mastoid (spherical mastoid, rod-like mastoid) and micromouch. As shown in fig. 3, a scanning electron microscope leaf skin photograph of the prior art treatment method: the treatment is not clean. As shown in fig. 4, the leaf epidermis scanning electron microscope photograph of the treatment method of the present invention: all the treatment is clean.
3.Sasa senanensis (k.yonekura 92403, japan): the leaf surface of the plant of Sasa genus of Bambusoideae of Gramineae family has stomata, mastoid (spherical mastoid, rod-like mastoid), micropouch, and thorn hair. As shown in fig. 5, a scanning electron microscope leaf skin photograph of the prior art treatment method: the treatment is not clean. As shown in fig. 6, the leaf epidermis scanning electron microscope photograph of the treatment method of the present invention: all the treatment is clean.
4.Sasa borealis (k. Yonekura 8528, japan): the leaf surface of the plant of Sasa genus of Bambusoideae of Gramineae family has stomata, mastoid (spherical mastoid, rod-like mastoid), micropouch, and thorn hair. As shown in fig. 7, a scanning electron microscope leaf skin photograph of the prior art treatment method: the treatment is not clean. As shown in fig. 8, the leaf epidermis scanning electron microscope photograph of the treatment method of the present invention: all the treatment is clean.
From the above, the method for rapidly removing the wax coating on the leaf surface of the wax leaf specimen can clearly observe the surface micro-morphology structures such as mastoid (spherical mastoid, rod-shaped mastoid), air holes, micro-hair and the like.
To confirm that the method of the invention works better, a series of treatments with different organic chemical reagents were completed. Untreated direct sticking stations were control groups, while acetone, xylene, acetone+xylene (1:1 v/v) were used as controls instead of chloroform according to the invention, with the other being identical to the method according to the invention. Compared with the method, the density of the chloroform is higher than that of the acetone and the dimethylbenzene, the chloroform is cleanly removed, the effect is best, and a high-quality photo is provided for a taxonom.
The method comprises the following steps:
1. hua Chizhu Sinosasa longiligulata: the untreated direct sticking table is of the genus Hua Chi of the subfamily Bambusae of the family Gramineae. As a result of observation under an electron microscope, the wax coating was unable to observe leaf pores, mastoid processes (globular mastoid processes, rod-shaped mastoid processes) and micro-hair morphology (fig. 9). Acetone treatment: as a result of observation under an electron microscope, the wax coating layer was reduced in some places, and it was observed that the leaf pores and the circumferentially distributed mastoid (globular mastoid, rod-like mastoid) did not remove all the wax coating (FIG. 10). Xylene treatment: the results were observed under an electron microscope and the wax coating was not completely removed (fig. 11). Xylene+acetone (1:1) treatment: as a result of observation under an electron microscope, the wax-coated layer was sparse in some places and was not peeled off (FIG. 12).
2. Many Mao Huachi bamboo Sinosasa polytricha: untreated direct sticking table. As a result of observation under an electron microscope, the wax coating failed to observe the leaf pore distribution, mastoid (globular mastoid, rod-like mastoid) and the micro-hair morphology (fig. 13). Acetone treatment: some wax had been detached as observed under electron microscopy. Contaminants were also present as above untreated cleanup (fig. 14). Xylene treatment: the results were observed under an electron microscope, with untreated clean wax and contaminants also present above (fig. 15). Xylene+acetone (1:1) treatment: as a result of observation under an electron microscope, the leaf epidermis wax was sparsely visible (fig. 16).
3.Sasa senanensis (k.yonekura 92403, japan): as a result of observation under an electron microscope with a direct adhesion table without leaf treatment, clear leaf pore distribution, mastoid (spherical mastoid, rod-like mastoid) and micro-hair morphology were not observed with wax coating (FIG. 17). Acetone treatment: the wax coverage and contamination was also severe as observed under electron microscopy (fig. 18). Xylene treatment: as a result of observation under an electron microscope, the wax coating layer had been removed in some places. The contamination was not treated cleanly (fig. 19). Xylene+acetone (1:1) treatment: as a result of observation under an electron microscope, mastoid (globular mastoid, rod-like mastoid), microvilli and epidermic waxing were not completely treated with the contaminants (fig. 20).
Sasa borealis (k. Yonekura 8528, japan): the genus Sasa of the subfamily Bambusae of the family Gramineae, the untreated direct sticking table. As a result of observation under an electron microscope, the wax coating was not able to observe the distribution of leaf pores and the morphology and structure of mastoid (globular mastoid, rod-like mastoid) clearly (fig. 21). Acetone treatment: some wax had been shed as observed under electron microscopy (fig. 22). Xylene treatment: as a result of observation under an electron microscope, leaf pore distribution, mastoid (globular mastoid, rod-like mastoid), micropilose morphology and also wax coating were observed (FIG. 23). Xylene+acetone (1:1) treatment: as a result of observation under an electron microscope, a small amount of wax was also coated on leaf pores and mastoid (globular mastoid, rod-like mastoid) morphology and structure (FIG. 24).
Claims (2)
1. A method for rapidly processing a leaf epidermis wax quilt of a cured leaf specimen, which is characterized by comprising the following steps:
cutting a cured leaf specimen, placing the cured leaf specimen in chloroform for soaking, then replacing new chloroform, heating at constant temperature, then replacing the cured leaf specimen with an ethanol aqueous solution, and performing ultrasonic treatment to obtain a pretreated scanning electron microscope sample;
the constant temperature heating is heating at 50 ℃ for 8 hours;
the constant temperature heating is to heat the metal bath for 8 hours at 50 ℃;
the preparation method comprises the steps of soaking in chloroform for 12 hours;
the ethanol aqueous solution is 70% ethanol aqueous solution by volume fraction, ultrasonic treatment is carried out for 3min, and the ethanol aqueous solution by volume fraction 70% is repeatedly replaced for three times, and each ultrasonic treatment is carried out for 3min.
2. The method for rapidly processing a wax cover of a leaf skin of a cured leaf specimen according to claim 1, wherein the cutting of the cured leaf specimen is cutting the cured leaf specimen into a rectangle of 0.8cm x 0.6 cm.
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| CN105651573A (en) * | 2016-01-05 | 2016-06-08 | 中国科学院华南植物园 | Plant leaf epidermal hair removing method |
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| CN110644240A (en) * | 2019-07-26 | 2020-01-03 | 南通大学 | Preparation method of multifunctional durable and self-repairing super-hydrophobic fabric surface |
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| CN105651573A (en) * | 2016-01-05 | 2016-06-08 | 中国科学院华南植物园 | Plant leaf epidermal hair removing method |
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