CN113049338B - Method for obtaining complete plant vein - Google Patents
Method for obtaining complete plant vein Download PDFInfo
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- CN113049338B CN113049338B CN202110327920.4A CN202110327920A CN113049338B CN 113049338 B CN113049338 B CN 113049338B CN 202110327920 A CN202110327920 A CN 202110327920A CN 113049338 B CN113049338 B CN 113049338B
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- 210000003462 vein Anatomy 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 46
- 239000000243 solution Substances 0.000 claims abstract description 56
- 239000003761 preservation solution Substances 0.000 claims abstract description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 31
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000011259 mixed solution Substances 0.000 claims abstract description 11
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 7
- 238000004043 dyeing Methods 0.000 claims abstract description 7
- QUBBAXISAHIDNM-UHFFFAOYSA-N ethyldimethylbenzene Natural products CCC1=CC=CC(C)=C1C QUBBAXISAHIDNM-UHFFFAOYSA-N 0.000 claims abstract description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 57
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- 241000196324 Embryophyta Species 0.000 claims description 39
- 239000012153 distilled water Substances 0.000 claims description 31
- 238000005204 segregation Methods 0.000 claims description 18
- 238000004140 cleaning Methods 0.000 claims description 13
- 238000002791 soaking Methods 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 239000003086 colorant Substances 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 6
- 230000001680 brushing effect Effects 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 4
- 238000003672 processing method Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 241000169680 Aphloia theiformis Species 0.000 description 3
- 241001013934 Erigeron breviscapus Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 240000001549 Ipomoea eriocarpa Species 0.000 description 2
- 235000005146 Ipomoea eriocarpa Nutrition 0.000 description 2
- 241000830535 Ligustrum lucidum Species 0.000 description 2
- 240000007695 Nandina domestica Species 0.000 description 2
- 241000219492 Quercus Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000037039 plant physiology Effects 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000002341 toxic gas Substances 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000008096 xylene Substances 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
- 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
-
- 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
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a method for obtaining a complete plant vein, which comprises the following steps: preparing a fixed preservation solution A; isolating the leaves; removing the fur of the blade; transferring the leaves into a sodium hypochlorite solution; firstly, placing the blade into a fixed preservation solution A with the mass concentration of 31-35% for treatment for 10-30min, then placing the blade into the fixed preservation solution A with the mass concentration of 64-70% for treatment for 10-30min, and finally placing the blade into the fixed preservation solution A for treatment for 10-14 h; dyeing; placing into a mixed solution of absolute ethyl alcohol and dimethylbenzene, transferring the leaves into dimethylbenzene, naturally airing the leaves until the leaves are transparent and veins are completely exposed, and slicing and preserving the leaves; the tail end veins and free ducts of the blade obtained by the invention can be completely reserved, and the blade is suitable for blades with various textures and fur types, not only can fresh blades be processed, but also can cured blade specimen blades be processed, and the veins obtained after the processing can be made into slices for permanent preservation.
Description
Technical Field
The invention belongs to the technical field of research on plant physiology and ecology, and particularly relates to a method for obtaining a complete plant vein.
Background
The characteristics of plant veins are stable, and the plant veins are often used as important classification characters in the field of taxonomy. Meanwhile, the plant veins have the physiological and ecological functions of transporting moisture, nutrient, mineral substances, supporting and protecting the leaves and the like. As important functional properties, the leaf vein network characteristics composed of leaf veins of each level can reflect ecological adaptation ability and ecological adaptation countermeasures of plants. The terminal veins and free ducts are key structural characters for determining the water supply and demand balance of the leaves, and have important influence on physiological functions such as plant growth, water transportation, gas exchange, photosynthesis and the like. In recent years, the structures and functions of the leaf vein network (especially the characteristics of the terminal leaf vein network) and the ecological significance of the leaf vein network have become research hot spots of plant physiology and ecology.
A prerequisite for successful development of this study is the acquisition of a clear and complete vein network, but it is also difficult to acquire an ideal vein network due to factors such as leaf texture, epidermal hair cover, etc. At present, people often adopt different blade treatment methods according to the characteristics of the texture, the fur quilt and the like of the blades, and the blade treatment methods can be roughly classified into the following categories: (1) Separating method, boiling in water to soften or fermenting in water, separating chemical liquid such as sodium hydroxide solution, and brushing off skin and mesophyll with brush; (2) The chemical transparent method comprises soaking chemical liquid such as potassium hydroxide solution, separating or fixing FAA fixing solution, bleaching with hydrogen peroxide, and dehydrating with gradient alcohol solution; (3) Mixing method, soaking in chemical liquid, separating or fixing with FAA fixing solution, and brushing skin, fur and mesophyll in mixed solution of xylene and acetone. The above methods have certain defects, and the first type of method can cause damage to the terminal veins, especially the blind veins and free ducts of the blade; the second method cannot effectively remove the surface fur, has complicated gradient dehydration treatment procedures, and is difficult to treat a large amount of blade samples; the third type of method removes the skin and hair from the toxic agent, and the toxic gas volatilized during the operation may cause harm to the human body. In addition, the current method has the following two problems: firstly, the method is suitable for processing thicker and harder leaves or cured leaf specimen leaves. The blade which is not suitable for the thin and soft blade and has developed surface fur is damaged in the process of isolation and dehairing, and the complete blade vein is difficult to obtain; secondly, the above methods are often designed separately for plant leaves with different texture and epidermis characteristics, while ecological studies often involve many plant species and require a large number of samples of the population, dynamic samples at different developmental stages. Different treatment methods are adopted for different blade types, so that the method is very inconvenient. Meanwhile, the reliability of the research result is greatly reduced due to the non-uniformity of the method.
Disclosure of Invention
In order to solve the problems, the invention provides a method for obtaining the complete plant veins, wherein the veins at the tail ends of the leaves and free ducts can be completely reserved, the method is suitable for the leaves with various textures and fur types, not only can fresh leaves be processed, but also leaves of cured leaves can be processed, and the veins obtained after the processing can be made into slices for permanent preservation.
The invention adopts the technical scheme for solving the technical problems that: a method of obtaining a whole plant vein comprising the steps of:
step one: taking anhydrous ethanol and glacial acetic acid according to the volume ratio of 96:4-94:6 to prepare a fixed preservation solution A;
step two: cleaning the collected plant leaves with clear water, soaking the plant leaves in boiled water for 0-10 min, then placing the plant leaves in sodium hydroxide solution with the mass concentration of 4-6% for segregation for 3-7 days, and transferring the plant leaves into distilled water for cleaning after the mesophyll of the plant leaves is separated out in a large amount;
step three: removing the fur of the blade, and then putting the blade into distilled water for cleaning;
step four: carefully transferring the leaves washed by the distilled water in the third step into a sodium hypochlorite solution with the mass concentration of 2-4%, soaking for 25-35 min, and transferring into the distilled water for washing after the leaves are completely whitened;
step five: preparing a fixed preservation solution A with the mass concentration of 31-35% and a fixed preservation solution A with the mass concentration of 64-70% from the fixed preservation solution A and distilled water, firstly placing the blade subjected to the fourth treatment into the fixed preservation solution A with the mass concentration of 31-35% for treatment for 10-30min, then placing the blade into the fixed preservation solution A with the mass concentration of 64-70% for treatment for 10-30min, and finally placing the blade into the fixed preservation solution A for treatment for 10-14 h;
step six: putting the blade treated in the fifth step into a safranin alcohol coloring agent with the mass concentration of 0.5-1.5% for dyeing for 25-35 min;
step seven: placing the dyed leaves in the step six into a mixed solution of absolute ethyl alcohol and dimethylbenzene for 5-10 min, transferring the leaves into dimethylbenzene for 5-15 min, and slicing after the leaves are transparent and veins are completely exposed;
step eight: and naturally airing the slices for 6-8 days, and placing the slices into a slice box for preservation.
Further, the leaf in the second step may be a fresh leaf or a cured leaf specimen leaf.
Further, the processing method of the different types of blades in the second step comprises the following steps: the method comprises the steps of firstly soaking thicker and harder blades in boiled water for 1-2 min, and then placing the blades in sodium hydroxide solution with the mass concentration of 4-6% for segregation; soaking the leaf of the cured leaf specimen in boiled water for 5-10 min, and then placing the leaf into a sodium hydroxide solution with the mass concentration of 4-6% for segregation; the herbaceous plant leaves with thin and soft texture are directly placed into sodium hydroxide solution with the mass concentration of 4-6% for segregation.
Further, the method for removing the fur of the blade in the third step comprises the following steps: transferring the leaves washed by distilled water into absolute ethyl alcohol, flattening the leaves by using glass sheets if the leaves are uneven, and brushing the leaves gently towards one direction by using a hairbrush in the absolute ethyl alcohol after 8-12 min.
Further, in the mixed solution in the step seven, the volume fraction of the absolute ethyl alcohol is 40-60%, and the volume fraction of the dimethylbenzene is 40-60%.
The beneficial effects of the invention are mainly represented in the following aspects: the invention provides a method for obtaining complete plant veins, which is simple and easy to implement, and the obtained veins are clear and complete in network, and comprise terminal blind veins and free ducts. The method is suitable for blades with various textures and fur types, can be used for fresh blades and also can be used for cured leaf specimen blades, and the processed vein network can be made into slices for permanent storage.
Detailed Description
The present invention will be described in detail with reference to examples, which give detailed embodiments and specific operation procedures on the premise of the technical solution of the present invention, but the scope of protection of the present invention is not limited to the following examples.
Examples
A method of obtaining a whole plant vein comprising the steps of:
step one: taking anhydrous ethanol and glacial acetic acid according to the volume ratio of 96:4-94:6 to prepare a fixed preservation solution A;
step two: the leaf disclosed by the invention is suitable for leaves with various textures and fur types, can be used for fresh leaves and also can be used for leaves of cured leaves, the collected plant leaves are washed by clean water, the leaves with thicker textures such as Nandina domestica, oak, glossy privet and the like are soaked in boiled water for 1-2 min, and then are put into sodium hydroxide solution with the mass concentration of 5% for segregation; soaking the leaf of the cured leaf specimen in boiled water for 5-10 min, and then placing the leaf into a sodium hydroxide solution with the mass concentration of 5% for segregation; the herb leaves with thin and soft texture such as plant leaves of small flower mountain peach grass, round leaf morning glory, and erigeron breviscapus are directly placed into 5% sodium hydroxide solution for segregation. The segregation time is 3-7 days, the thicker the texture of the leaf is, the longer the leaf needs to be treated, the more turbid the solution is observed every day, the new sodium hydroxide solution is replaced in time, and after a great amount of mesophyll of the leaf is separated out, the leaf is transferred into distilled water for cleaning;
step three: the leaves with more hair, such as plant leaves of the small flower, the mountain peach, the erigeron breviscapus and the like, are transferred into absolute ethyl alcohol, if the leaves are uneven, the leaves are flattened by glass sheets, and after 10 minutes, the leaves are gently brushed in one direction by a hairbrush in the absolute ethyl alcohol, so that the hair can be removed well, and then the leaves are put into distilled water for cleaning;
step four: carefully transferring the leaves washed by the distilled water in the third step into a sodium hypochlorite solution with the mass concentration of 3%, soaking for 30min, and transferring into the distilled water for washing after the leaves are completely whitened;
step five: preparing a fixed preservation solution A with the mass concentration of 33% and a fixed preservation solution A with the mass concentration of 67% from the fixed preservation solution A and distilled water, firstly placing the blade treated in the step four into the fixed preservation solution A with the mass concentration of 33% for treatment for 10-30min, then placing the blade into the fixed preservation solution A with the mass concentration of 67% for treatment for 10-30min, and finally placing the blade into the fixed preservation solution A for treatment for 12h; if the next step is not performed, the blade can be stored in the solution A in a sealing way for more than 4 weeks;
step six: putting the leaves treated in the fifth step into a safranin alcohol coloring agent with the mass concentration of 1% for dyeing for 30min;
step seven: putting the dyed leaves in the step six into a mixed solution of absolute ethyl alcohol and dimethylbenzene for 5-10 min, wherein the volume part of the absolute ethyl alcohol in the mixed solution is 50%, the volume part of dimethylbenzene is 50%, then transferring the leaves into dimethylbenzene for 5-15 min, and slicing after the leaves are transparent and veins are completely exposed;
step eight: the small leaves can be directly sealed by neutral resin, the larger leaves can be cut into a plurality of parts according to the sequence of up, down and up, the small leaves are made into slices, the slices are naturally dried for 7 days, and the slices are put into a slice box for preservation.
Embodiment case 1:
step one, preparing a fixed preservation solution: preparing a fixed preservation solution A with the volume fraction of 95:5 by using absolute ethyl alcohol and glacial acetic acid;
and secondly, cleaning the collected plant leaves with clear water, soaking the plant leaves with thicker and harder textures such as Nandina domestica, oak, glossy privet and the like in boiled water for 1-2 min, then placing the plant leaves into 5% sodium hydroxide solution for segregation, wherein the segregation treatment time is 3-7 d, the thicker and harder the texture of the plant leaves is, the longer the time required to be treated is, and the time for daily observation is longer, and when the solution becomes turbid, the new sodium hydroxide solution is replaced in time. After a great amount of mesophyll of the leaf blade is separated out, the leaf blade is transferred into distilled water for cleaning;
carefully transferring the leaves washed by distilled water into a 3% sodium hypochlorite solution for about 30min, and transferring to distilled water for washing after the leaves are completely whitened;
and fourthly, preparing the fixed preservation solution A and distilled water into a solution A with the concentration of 33% and a solution A with the concentration of 67%, firstly placing the blade into the solution A with the concentration of 33% for treatment for 10-30min, then placing the blade into the solution A with the concentration of 67% for treatment for 10-30min, and finally placing the blade into the solution A for treatment for 12h. If the next step is not performed, the blade can be stored in the solution A in a sealing way for more than 4 weeks;
step five, putting the last treated leaf into a 1% safranin alcohol coloring agent for dyeing for about 30min;
step six, putting the dyed leaves into a mixed solution of 5:5 parts by volume of absolute ethyl alcohol and dimethylbenzene for 5-10 min, transferring the leaves into dimethylbenzene for 5-15 min, and slicing the leaves after the leaves are transparent and veins are completely exposed;
and seventh, the small leaves can be directly sealed by neutral resin, the larger leaves can be cut into a plurality of parts according to the sequence of up, down, and made into slices, naturally dried for a week, and placed into a slice box for permanent storage.
Embodiment case 2:
step one, preparing a fixed preservation solution: preparing a fixed preservation solution A from anhydrous ethanol and glacial acetic acid according to a volume ratio of 95:5;
and step two, cleaning the collected plant leaves with clear water. The leaf of the wax leaf specimen is soaked in boiled water for 5-10 min, then is put into 5% sodium hydroxide solution for segregation, the segregation treatment time is 3-7 d, the thicker the texture of the leaf is, the longer the leaf is required to be treated, the observation is carried out every day, and when the solution becomes turbid, the new sodium hydroxide solution is replaced in time. After a great amount of mesophyll of the leaf blade is separated out, the leaf blade is transferred into distilled water for cleaning;
and thirdly, carefully transferring the leaves washed by the distilled water into a 3% sodium hypochlorite solution for about 30min, and transferring the leaves to distilled water for washing after the leaves are completely whitened:
step four, preparing a fixed preservation solution A and distilled water into a solution A with the concentration of 33% and a solution A with the concentration of 67%, firstly placing the blade into the solution A with the concentration of 33% for treatment for 10-30min, then placing the blade into the solution A with the concentration of 67% for treatment for 10-30min, and finally placing the blade into the solution A for treatment for 12h, wherein if the next step is not performed, the blade can be stored in the solution A in a sealing way and can be stored for more than 4 weeks;
step five, putting the leaves treated in the step four into a safranin alcohol coloring agent with concentration of 1% for dyeing for 30min;
step six, putting the dyed leaves into a mixed solution of 5:5 parts by volume of absolute ethyl alcohol and dimethylbenzene for 5-10 min, transferring the leaves into dimethylbenzene for 5-15 min, and slicing the leaves after the leaves are transparent and veins are completely exposed;
and seventh, the small leaves can be directly sealed by neutral resin, the larger leaves can be cut into a plurality of parts according to the sequence of up, down, and made into slices, naturally dried for a week, and placed into a slice box for permanent storage.
Embodiment 3:
step one, preparing a fixed preservation solution: preparing a fixed preservation solution A from absolute ethyl alcohol and glacial acetic acid according to a volume ratio of 95:5;
step two, the herb leaves with thin and soft texture, such as plant leaves of small flower mountain peach grass, round leaf morning glory, erigeron breviscapus and the like, are directly put into 5% sodium hydroxide solution for segregation after being washed by clean water, the segregation treatment time is 3-7 d, the thicker and harder the leaf texture is, the longer the time needed to be treated is, and the new sodium hydroxide solution is timely changed when the solution becomes turbid after daily observation. After a great amount of mesophyll of the leaf blade is separated out, the leaf blade is transferred into distilled water for cleaning;
transferring the leaves washed by distilled water into absolute ethyl alcohol, flattening the leaves by using glass sheets if the leaves are uneven, brushing the leaves in the absolute ethyl alcohol slightly towards one direction by using a hairbrush after 10 min, and washing the leaves in distilled water;
carefully transferring the blade washed by distilled water into 3% sodium hypochlorite solution for about 30min until the blade becomes white completely, and transferring to distilled water for washing;
and fifthly, preparing a fixed preservation solution A and distilled water into a solution A with the concentration of 33% and a solution A with the concentration of 67%, firstly placing the blade into the solution A with the concentration of 33% for treatment for 10-30min, then placing the blade into the solution A with the concentration of 67% for treatment for 10-30min, and finally placing the blade into the solution A for treatment for 12h. If the next step is not performed, the blade can be stored in the solution A in a sealing way for more than 4 weeks;
step six, putting the leaves treated in the step five into a safranin alcohol coloring agent with concentration of 1% for dyeing for about 30min;
step seven, putting the dyed leaves into a mixed solution of 5:5 parts by volume of absolute ethyl alcohol and dimethylbenzene for 5-10 min, transferring the leaves into dimethylbenzene for 5-15 min, and slicing the leaves after the leaves are transparent and veins are completely exposed;
and step eight, the small leaves can be directly sealed by neutral resin, the larger leaves can be cut into a plurality of parts according to the sequence of up, down, and made into slices, naturally dried for a week, and placed into a slice box for permanent storage.
Firstly, putting the blade into a solution A with the concentration of 33% for treatment for 10-30min, then putting the blade into a solution A with the concentration of 67% for treatment for 10-30min, and finally putting the blade into the solution A for treatment for 12h; the purpose is to further isolate (mesophyll isolate), and can make the veins elastic, so that the whole leaves (otherwise easy to break) can be conveniently taken out, and the veins are easier to dye.
The invention provides a method for obtaining complete plant veins, which is simple and easy to implement, and the obtained veins are clear and complete in network, and comprise terminal blind veins and free ducts. The method is suitable for blades with various textures and fur types, can be used for fresh blades and also can be used for cured leaf specimen blades, and the processed vein network can be made into slices for permanent storage.
The present invention is not limited to the technical scheme and embodiments, but is described in detail with reference to the preferred examples, and those skilled in the art will understand that: modifications may be made to the specific embodiments of the present invention or equivalents may be substituted for part of the technical features thereof; without departing from the spirit of the invention, it is intended to cover the scope of the invention as claimed. It should also be noted that relational terms are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Claims (5)
1. A method for obtaining a whole plant vein, characterized by: the method comprises the following steps:
step one: taking anhydrous ethanol and glacial acetic acid according to the volume ratio of 96:4-94:6 to prepare a fixed preservation solution A;
step two: cleaning the collected plant leaves with clear water, soaking the plant leaves in boiled water for 0-10 min, then placing the plant leaves in sodium hydroxide solution with the mass concentration of 4-6% for segregation for 3-7 days, and transferring the plant leaves into distilled water for cleaning after the mesophyll of the plant leaves is separated out in a large amount;
step three: removing the fur of the blade, and then putting the blade into distilled water for cleaning;
step four: carefully transferring the leaves washed by the distilled water in the third step into a sodium hypochlorite solution with the mass concentration of 2-4%, soaking for 25-35 min, and transferring into the distilled water for washing after the leaves are completely whitened;
step five: preparing a fixed preservation solution A with the mass concentration of 31-35% and a fixed preservation solution A with the mass concentration of 64-70% from the fixed preservation solution A and distilled water, firstly placing the blade subjected to the fourth treatment into the fixed preservation solution A with the mass concentration of 31-35% for treatment for 10-30min, then placing the blade into the fixed preservation solution A with the mass concentration of 64-70% for treatment for 10-30min, and finally placing the blade into the fixed preservation solution A for treatment for 10-14 h;
step six: putting the blade treated in the fifth step into a safranin alcohol coloring agent with the mass concentration of 0.5-1.5% for dyeing for 25-35 min;
step seven: placing the dyed leaves in the step six into a mixed solution of absolute ethyl alcohol and dimethylbenzene for 5-10 min, transferring the leaves into dimethylbenzene for 5-15 min, and slicing after the leaves are transparent and veins are completely exposed;
step eight: and naturally airing the slices for 6-8 days, and placing the slices into a slice box for preservation.
2. A method of obtaining whole plant veins according to claim 1 wherein: the leaf in the second step can be a fresh leaf or a cured leaf specimen leaf.
3. A method of obtaining whole plant veins according to claim 1 wherein: the processing method of the different types of blades in the second step comprises the following steps: the method comprises the steps of firstly soaking thicker and harder blades in boiled water for 1-2 min, and then placing the blades in sodium hydroxide solution with the mass concentration of 4-6% for segregation; soaking the leaf of the cured leaf specimen in boiled water for 5-10 min, and then placing the leaf into a sodium hydroxide solution with the mass concentration of 4-6% for segregation; the herbaceous plant leaves with thin and soft texture are directly placed into sodium hydroxide solution with the mass concentration of 4-6% for segregation.
4. A method of obtaining whole plant veins according to claim 1 wherein: the method for removing the fur of the blade in the third step comprises the following steps: transferring the leaves washed by distilled water into absolute ethyl alcohol, flattening the leaves by using glass sheets if the leaves are uneven, and brushing the leaves gently towards one direction by using a hairbrush in the absolute ethyl alcohol after 8-12 min.
5. A method of obtaining whole plant veins according to claim 1 wherein: in the mixed solution in the seventh step, the volume fraction of the absolute ethyl alcohol is 40-60%, and the volume fraction of the dimethylbenzene is 40-60%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110327920.4A CN113049338B (en) | 2021-03-26 | 2021-03-26 | Method for obtaining complete plant vein |
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