CN109425521B - Preparation method of fossil plant dispersed cuticle - Google Patents
Preparation method of fossil plant dispersed cuticle Download PDFInfo
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- CN109425521B CN109425521B CN201710748834.4A CN201710748834A CN109425521B CN 109425521 B CN109425521 B CN 109425521B CN 201710748834 A CN201710748834 A CN 201710748834A CN 109425521 B CN109425521 B CN 109425521B
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Abstract
The invention provides a preparation method of fossil plant dispersed cuticle, which comprises the following steps: pretreatment of the dispersed stratum corneum sample: selecting a cuticle sample and removing water-soluble impurities, carbonate components and siliceous components in the sample to obtain a pre-treatment dispersed cuticle sample; then carrying out oxidation treatment on the dispersed cuticle to obtain an oxidized dispersed cuticle sample; and finally, separating the upper and lower epidermis of the dispersed cuticle to obtain the cuticle. In the preparation process of the fossil plant dispersed cuticle, the rapid preparation of the fossil plant dispersed cuticle is realized by adopting a soaking method, the experimental period is effectively shortened, and the quality of the prepared cuticle is improved.
Description
Technical Field
The invention belongs to the technical field of ancient botany, and relates to a preparation method of a fossil plant dispersed cuticle.
Background
The stratum corneum is present on the outer wall of epidermal cells of plants and is a protective layer of fat. Most of the prior plant fossil cutin layer materials are taken from pressed fossil specimens and are in-situ cutin layers. The fragments of the stratum corneum which have been removed from the plant matrix or plant body and are carried for a long distance before petrochemical processing to break and disperse and preserve in the sedimentary rock are called the dispersed stratum corneum. The discrete stratum corneum characteristics can be used for identifying plant groups or species, and can also be used as an important means in stratigraphic comparison and ancient ecological research. However, the dispersed stratum corneum is often very fragile and difficult to prepare through long geological processes, thereby seriously restricting the development of related researches.
Leaf-mena briefly summarizes the experimental procedures for the treatment of the fossil stratum corneum, which mainly include Schulze solution (Schulze solution) digestion, herd's Method (i.e., sodium hypochlorite solution digestion), and hydrofluoric acid digestion. The suger solution immersion method and the Heder method firstly use strong oxidants such as a suger solution or a sodium hypochlorite solution to oxidize the stratum corneum, and then use alkali liquor (ammonium hydroxide solution, sodium hydroxide solution or potassium hydroxide solution) to separate out the oxidized mesophyll tissue, thereby achieving the purpose of preparing the stratum corneum. The two methods are widely applied to in-situ stratum corneum research. However, the dispersed horny layer is of various types, and different types of horny layers have different reaction sensitivity and reaction time to schulvin solution, sodium hypochlorite solution, ammonium hydroxide solution (ammonia water) and the like, so that the horny layer is easy to shrink, curl or break when encountering the reagents. The hydrofluoric acid leaching method is to dissolve colloidal silica, silicon fluoride and other substances attached to the horny layer after the treatment by the two methods to make the horny layer more pure, but the oxidized horny layer is very fragile and is very easy to damage after the treatment by the hydrofluoric acid solution. In addition, the experimental period of the three methods is long, which also restricts the practical application of the three methods in the research of the dispersed stratum corneum.
Disclosure of Invention
In order to overcome the defects of long experimental period, unsatisfactory experimental effect and the like in the prior art, the invention provides the preparation method of the fossil plant dispersed cuticle, which can quickly obtain a high-quality cuticle sample and provide a material basis for epidermis morphology identification, stratum contrast and ancient ecological research.
The invention is realized by the following method:
a method for preparing a fossil plant dispersed cuticle comprising:
step A: selecting a cuticle sample and removing water-soluble impurities, carbonate components and siliceous components in the sample to obtain a pretreatment dispersion cuticle sample;
and B: b, soaking the pretreated dispersed cuticle sample obtained in the step A in Shu's solution until the leaf membrane is brown and translucent or transparent, stopping the reaction, and washing with water to be neutral to obtain an oxidized dispersed cuticle sample;
and C: soaking the oxidized dispersed cuticle sample in water for a period of time, adding an ammonium hydroxide solution until the residual mesophyll tissues and vascular bundles are changed into brown floccules or filaments, stopping the reaction, washing with water to be neutral, taking out the dispersed cuticle sample, and removing the upper and lower epidermis to obtain the cuticle;
step D: the resulting stratum corneum samples were cleaned of impurities and then soaked in water for subsequent flaking observation.
According to a preferred embodiment of the invention, the schulper solution comprises a saturated potassium chlorate solution and fuming nitric acid, and preferably, the volume ratio of the saturated potassium chlorate solution to the fuming nitric acid is 1: (2-3). The schulper solution used in the present invention ensures that there is no disruption of the stratum corneum during the treatment.
In the present invention, in step a, water-soluble impurities are removed with distilled water; and/or removing the carbonate component with an acid solution, preferably hydrochloric acid; and/or removing the siliceous component with a hydrofluoric acid solution. The purpose of the hydrochloric acid treatment is to eliminate carbonate minerals in the surrounding rock and prevent calcium from reacting in hydrofluoric acid digestion to generate calcium fluoride which is attached to the surface of the stratum corneum so that the prepared stratum corneum is covered by impurities and the structure of the stratum corneum cannot be clearly observed.
According to a preferred embodiment of the present invention, in step a, the dispersed stratum corneum sample pretreatment comprises:
a-1, placing the selected stratum corneum samples in a container with a plurality of sample tanks in sequence, adding distilled water to soak the stratum corneum samples for 40-60min, and then removing the distilled water in the sample tanks;
step A-2, adding a hydrochloric acid solution to soak for 3-4h, and then removing the hydrochloric acid and washing the stratum corneum sample to be neutral;
and step A-3, adding hydrofluoric acid solution to soak for 12-24 hours until the surrounding rock is completely changed into flocculent or powdery, and repeatedly cleaning the flocculent or powdery surrounding rock to be neutral by using distilled water.
In the invention, the distilled water soaking in the step A-1 is preferably carried out at 40-60 ℃, and/or the concentration of hydrochloric acid in the step A-2 is 35-40%, and/or the volume concentration of hydrofluoric acid in the step A-3 is 40-45%, and the hydrofluoric acid soaking is preferably carried out at 40-60 ℃, and preferably at 40 ℃.
According to a preferred embodiment of the present invention, the operation of step A-1 performed at a temperature higher than room temperature is preferably performed by heating in a water bath.
According to a preferred embodiment of the invention, the container is preferably made of polycarbonate material in order to prevent corrosion of the glassware by hydrofluoric acid. The container having a plurality of sample wells used in the present invention can improve efficiency by simultaneously operating with time difference.
In the pretreatment process of the dispersed cuticle, in the step of removing carbonate by using an acid solution, the soaking time of the hydrochloric acid solution is 3-4h, the step is to completely remove some substances which can react with hydrofluoric acid to form insoluble substances on the dispersed cuticle, and the too short reaction time can cause that the carbonate mineral reacts with hydrofluoric acid to form calcium fluoride attached to the surface of the cuticle in the step A-3, so that the prepared cuticle has poor quality and cannot be used for subsequent observation and analysis and the like.
In addition, the stratum corneum is thoroughly removed by treating with 40% -45% hydrofluoric acid solution because the stratum corneum materials are dispersed and a large amount of silicate in the stratum corneum is dispersed.
According to a preferred embodiment of the present invention, in step B, the stratum corneum oxidation treatment time is 3-12 h.
According to a preferred embodiment of the present invention, in step C, the separation of the upper and lower epidermis of the cuticle sample is performed on a glass abutment, which is a flat glass plate having a groove in the center.
According to a preferred embodiment of the invention, the depth of the grooves is 2-5mm and the diameter of the grooves is 1-2 cm.
In the preparation process of the fossil plant dispersed cuticle, the rapid preparation of the fossil plant dispersed cuticle is realized by adopting a soaking method, and the mesophyll tissue is completely carbonized after oxidation treatment and potassium hydroxide treatment of the Shu's solution, so that the cuticle can be easily separated, and the experimental period is effectively shortened. And the cuticle is not damaged in the treatment process, the quality of the prepared cuticle is improved, and the prepared cuticle has no impurities on the surface and no curled edge, and is beneficial to subsequent application.
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FIG. 1 is a schematic view of the structure of the container of the present invention.
Figure 2 is a cross-sectional view of the container of the present invention.
Fig. 3 is a schematic view showing the structure of the container lid of the present invention.
Figure 4 is a cross-sectional view of the container of the present invention.
FIG. 5 is a schematic view of the glass base according to the present invention.
FIG. 6 is a cross-sectional view of the glass base of the present invention.
FIG. 7 is a photograph of the stratum corneum of example 1 dispersed under an optical microscope.
FIG. 8 is a photograph of the stratum corneum of example 1 under a scanning electron microscope.
Fig. 9 is a photograph under an optical microscope of the dispersed horny layer of comparative example 1.
Fig. 10 is a photograph of the dispersed horny layer of comparative example 1 under a scanning electron microscope.
Detailed Description
The present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.
Example 1 the chalky financial component-dispersing stratum corneum in the extended region of Jilin
A method for preparing a fossil plant dispersed cuticle comprising:
step A: pretreatment of a dispersed cuticle sample:
(1) observing under a hand specimen microscope, selecting a carbonaceous mudstone sample with good storage of the dispersed horny layer, taking down the horny layer and the surrounding rock, putting the samples into the container sample tank shown in the figure 1 in sequence, and numbering corresponding container covers;
(2) respectively adding distilled water according to the number until the horny layer and the surrounding rock sample are covered, and heating in water bath at 40 ℃ for 45 minutes;
(3) removing distilled water and surrounding rock debris in the sample tank, adding 35% hydrochloric acid solution, covering a container cover, soaking the sample for 3 hours, and repeatedly cleaning the sample tank with distilled water until the sample tank is neutral;
(4) adding 40% hydrofluoric acid solution, covering the container cover, heating in water bath at 40 deg.C for 18 hr until the surrounding rock is completely changed into flocculent or powder, and cleaning with distilled water repeatedly until the flocculent or powder surrounding rock is neutral;
and B: oxidation treatment of the dispersed horny layer:
(5) 1 part of saturated potassium chlorate solution is added into 2.5 parts of fuming nitric acid to prepare Shu's solution. And after the Shu's solution is cooled to room temperature, adding the Shu's solution into the sample to soak the horny layer, and covering a container cover. Observing in real time, respectively terminating the reaction according to the sequence that the leaf membrane turns brown and is semitransparent or transparent, repeatedly washing the leaf membrane with distilled water to be neutral, and then soaking the leaf membrane with distilled water.
And C: disperse the separation of the upper and lower cuticles:
(6) soaking a sample in distilled water for 1 hour, adding 1 drop (0.02mL) of 5% potassium hydroxide solution by using a dropper at intervals of 2 minutes until residual mesophyll tissues and vascular bundles are changed into brown floccules or filaments, then dropping and scattering, stopping reaction, adding distilled water, and back-washing to be neutral;
(7) the horny layer was carefully transferred to the groove of the glass abutment as shown in fig. 3, a small amount of distilled water was added, the upper and lower epidermis of the horny layer were separated by a dissecting needle under a microscope, and the impurities on the horny layer were removed by a brush, and the upper and lower epidermis and the inside and outside were distinguished and recorded by photographing under a numbered mirror. And repeatedly washing the separated cuticle with distilled water to remove impurities, and soaking in distilled water again to keep the cuticle moist for subsequent tabletting observation.
The optical microscope photograph and the electron microscope photograph of the dispersed horny layer thus obtained are shown in FIGS. 7 and 8. The stratum corneum is intact, no attachments are on the surface, and the characteristics of stratum corneum air pore apparatus, cells, papilla and the like can be clearly obtained.
Comparative example 1 chalky wealth-developing discrete stratum corneum in extended area of Jilin
Comparative example 1 was operated in the same manner as in example 1 except that in comparative example 1, no water bath was used for heating during the distilled water immersion and the hydrofluoric acid immersion in step a, and in comparative example 1, the treatment was performed with hydrofluoric acid and then hydrochloric acid.
The optical microscope photograph and the electron microscope photograph of the dispersed horny layer thus obtained are shown in FIGS. 9 and 10. The stratum corneum is broken, impurities are attached to the surface, and the observation effects of the air pore apparatus, cells and the like are influenced.
Although the invention has been described above with reference to some embodiments, various modifications may be made without departing from the scope of the invention, and the non-exhaustive description of these combinations in this specification is only for the sake of brevity and resource conservation. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (5)
1. A method for preparing a fossil plant dispersed cuticle comprising:
step A: selecting a cuticle sample and removing water-soluble impurities, carbonate components and siliceous components in the sample to obtain a pretreatment dispersion cuticle sample;
and B: b, soaking the pretreated dispersed cuticle sample obtained in the step A in Shu's solution until the leaf membrane is brown and translucent or transparent, stopping the reaction, and washing with water to be neutral to obtain an oxidized dispersed cuticle sample;
and C: soaking the oxidized dispersed cuticle sample in water for a period of time, adding a potassium hydroxide solution, stopping the reaction until the residual mesophyll tissues and vascular bundles become brown floccules or filaments, washing with water to be neutral, taking out the dispersed cuticle sample, and placing the dispersed cuticle sample on a glass base station to remove the upper and lower epidermis to obtain the cuticle;
step D: removing impurities on the obtained horny layer sample, cleaning the horny layer, and then soaking in water for subsequent flaking observation;
the Shu's solution comprises saturated potassium chlorate solution and fuming nitric acid, and the volume ratio of the saturated potassium chlorate solution to the fuming nitric acid is 1 (2-3);
in the step A, the pretreatment of the dispersed horny layer sample comprises the following steps:
a-1, sequentially placing selected cuticle samples in a container with a plurality of sample tanks, adding distilled water to soak the cuticle samples for 40-60min, and then removing the distilled water in the sample tanks;
step A-2, adding a hydrochloric acid solution to soak for 3-4h, and then removing the hydrochloric acid and washing the stratum corneum sample to be neutral;
step A-3, adding hydrofluoric acid solution to soak for 12-24 hours until the surrounding rock is completely changed into flocculent or powdery, and repeatedly cleaning the flocculent or powdery surrounding rock to be neutral by using distilled water;
soaking distilled water in the step A-1 at 40-60 ℃; in the step A-2, the volume concentration of the hydrochloric acid is 35-40%; the volume concentration of the hydrofluoric acid in the step A-3 is 40-45%.
2. The method according to claim 1, wherein the hydrofluoric acid immersion in step a-3 is performed at 40-60 ℃.
3. The method according to claim 1 or 2, wherein in step B the stratum corneum is oxidised for a period of 3-12 h.
4. The method of claim 1 or 2, wherein the glass submount is a flat glass plate with a groove in the center.
5. The method of claim 4, wherein the depth of the grooves is 2-5mm and the diameter of the grooves is 1-2 cm.
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| 化石植物研究方法简介;马清温 等;《生物学通报》;20000120;第35卷(第1期);第23页 * |
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