A preparation method for simultaneously observing and counting glandular hairs, non-glandular hairs and stomata under mugwort leaves

Abstract

The invention relates to a preparation method for simultaneously observing and counting glandular hairs, non-glandular hairs and stomata of mugwort leaves, and can effectively solve the problem that the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface of mugwort leaves cannot be simultaneously observed. The technical solution is as follows: firstly, fresh mugwort leaves are taken, small pieces of leaves are cut from leaves with a knife, the leaves are placed in methanol for immersion and fixation, the leaves are rinsed with distilled water and then transferred to a sodium hypochlorite solution for bleaching until colorless, the leaves are rinsed with distilled water and then transferred to a tissue transparent agent for transparency, the materials are taken out and dyed with a safranin solution, the slides are sealed with water, and then observed with an optical microscope, the non-glandular hair handles and glandular hairs are dyed red, the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface can be clearly observed and the counting is completed. The method of the invention has the characteristics of simple operation, low cost, small workload, high success rate and clear observation, and has low technical requirements for operators, and is an innovation in the preparation method.

Description

Technical Field

[0001] The invention relates to the field of plant microscopic identification, in particular to a preparation method for simultaneously observing and counting glandular hairs, non-glandular hairs and stomata of mugwort leaves. Background Art

[0002] Ai Artemisia argyi ) belongs to the genus Artemisia (Compositae) Artemisia ) plant, mugwort leaf is the dried leaf of mugwort, which is a commonly used Chinese medicine with the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain. Mugwort is widely distributed in my country, and is almost everywhere in the country except in extremely arid and high-cold areas. The "mugwort wool" obtained by crushing mugwort leaves is used to make moxibustion sticks, and moxibustion wool is the main material for moxibustion. The quality of mugwort is closely related to the origin and variety. The quality of mugwort leaves and its wool yield and the quality of moxibustion wool vary greatly due to the different latitudes, light and varieties of different origins. The quality of mugwort leaves is related to the content of chemical components such as volatile oils and flavonoids. The glandular hairs on the surface of mugwort leaves are the main source of volatile oils and flavonoids in mugwort leaves, and the non-glandular hairs on the lower surface of mugwort leaves are the main components of moxibustion wool. Therefore, the number of glandular hairs and non-glandular hairs on mugwort leaves is directly related to the quality evaluation of mugwort leaves and mugwort wool. Research on the number and distribution characteristics of glandular hairs and non-glandular hairs on mugwort leaves can provide experimental data and theoretical guidance for the high-quality production and quality control of mugwort leaves. The size and density of stomata are affected by sunlight, temperature, water and other factors, which are of certain significance for the identification of mugwort varieties and physiological and ecological research. Therefore, mugwort researchers need to observe and count the distribution of glandular hairs, non-glandular hairs and stomata of mugwort.

[0003] Artemisia argyi is a papery leaf. The glandular and non-glandular hairs on the upper surface are easy to observe and count. The hairs on the lower surface are thick and difficult to observe the epidermis by dissociating the epidermis. The leaves are bleached with sodium hypochlorite and then transparently prepared with chloral hydrate under a bright field microscope. It is also impossible to observe and count the glandular hairs, non-glandular hairs and stomata on the lower surface at the same time. The scanning electron microscope can only see the surface of the sample, so it is impossible to observe and count the glandular hairs, non-glandular hairs and stomata on the lower surface at the same time by observing the thick hairs. Therefore, so far, domestic and foreign researchers have found it difficult to observe and count the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface at the same time, whether using optical microscopes or scanning electron microscopes. Since the T-shaped non-glandular hairs on the lower surface of the wormwood leaf are densely distributed and their extremely long apical cells are intertwined, it seems that effective observation and counting can only be completed by observing and counting the stalks; the glandular hairs on the lower surface are covered by the apical cells of the densely distributed T-shaped non-glandular hairs intertwined. In addition, due to the background produced by the autofluorescence of chlorophyll, it is also difficult to observe the glandular hairs under a fluorescence microscope, resulting in the inability to simultaneously observe the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface of the wormwood leaf and complete the counting. Therefore, new special film-making technology is needed to solve the above problems. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a preparation method for simultaneously observing and counting the glandular hairs, non-glandular hairs and stomata of mugwort leaves, which can effectively solve the problem of not being able to simultaneously observe the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface of mugwort leaves.

[0005] The technical solution provided by the present invention is that fresh moxa leaves are firstly taken, small pieces of leaves are cut from the leaves with a knife, the leaves are placed in methanol for immersion and fixation for 6-12 hours, the leaves are rinsed with distilled water and then transferred to a sodium hypochlorite solution for bleaching until colorless, the leaves are rinsed with distilled water and then transferred to a tissue transparent agent for transparentization for 5-10 minutes, the materials are then taken out and dyed with a 0.01-0.1% safranin solution for 0.5-5 minutes, the pieces are sealed with water, and then observed with an optical microscope, the non-glandular hair handles and glandular hairs are dyed red, the distribution of glandular hairs, non-glandular hairs and stomata on the lower surface can be clearly observed, and the counting can be completed.

[0006] The method of the present invention has the characteristics of simple operation, low cost, small workload, high success rate and clear observation. It can satisfactorily solve the technical difficulties in observing and counting the glandular hairs, non-glandular hairs and stomata of mugwort leaves, and has low technical requirements for operators. It is an innovation in the production method. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 It is a schematic diagram of the observation of glandular hairs and non-glandular hairs on the lower surface of Artemisia argyi according to the present invention.

[0008] Figure 2 It is a schematic diagram of observing the non-glandular hairs of the lower surface stomata according to the present invention.

[0009] Figure 3 This is a schematic diagram of observing the upper surface of the present invention. DETAILED DESCRIPTION

[0010] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0011] Example 1

[0012] The present invention, when implemented, specifically comprises the following steps:

[0013] 1) Take fresh mugwort leaves and rinse them with distilled water for 1-3 minutes to remove dust on the surface;

[0014] 2) Dry the surface moisture of the rinsed leaves, cut small pieces of 8-10 mm in length and width with a knife, and try not to leave large leaf veins, and soak them in methanol for 6-12 hours;

[0015] 3) Remove the leaves from methanol and transfer them to n-hexane for 1-3 hours;

[0016] 4) Remove the leaves from the n-hexane and rinse them with distilled water for 1-3 minutes; then transfer them to 5-8% sodium hypochlorite solution and bleach them until they are colorless;

[0017] 5) Remove the leaves from the sodium hypochlorite solution, rinse with distilled water, and transfer to a tissue clearing agent for 5-10 minutes;

[0018] 6) Take out the material and dye it with 0.01~0.1% safranin aqueous solution for 0.5~5 minutes;

[0019] 7) After removing the leaves from the safranin solution, rinse with water for 5-10 seconds, seal with 10-20% dilute glycerol to make a temporary glass slide, flatten it, and observe it under an optical microscope;

[0020] 8) After adjusting the focus and brightness under a bright field microscope, you can observe that the handles of the T-shaped non-glandular hairs and the heads of the glandular hairs are dyed red by safranin, so you can clearly see the distribution of glandular hairs and non-glandular hairs and count them easily;

[0021] 9) If you observe with a fluorescence microscope, after observing clearly under bright field, use blue light to excite, you can not only see the distribution of glandular hairs and non-glandular hairs, but also the distribution of stomata and complete the counting.

[0022] Example 2

[0023] The present invention, when implemented, specifically comprises the following steps:

[0024] 1) Take fresh mugwort leaves and rinse them with distilled water for 2 minutes to remove dust on the surface;

[0025] 2) Dry the surface moisture of the rinsed leaves, cut small pieces of 9 mm in length and width with a knife, and try not to leave large leaf veins, and soak them in methanol for 9 hours;

[0026] 3) Remove the leaves from methanol and transfer them to n-hexane for 2 hours;

[0027] 4) Remove the leaves from the hexane and rinse them with distilled water for 2 minutes; then transfer them to 6% sodium hypochlorite solution to bleach them until they are colorless;

[0028] 5) Remove the leaves from the sodium hypochlorite solution, rinse with distilled water, and transfer to RapiClear 1.52 tissue clearing agent for 7 minutes;

[0029] 6) Take out the material and dye it with 0.05% safranin aqueous solution for 3 minutes;

[0030] 7) After removing the leaves from the safranin aqueous solution, rinse with water for 7 seconds, seal with 15% dilute glycerol to make a temporary glass slide, flatten it, and observe it under an optical microscope;

[0031] 8) After adjusting the focus and brightness under a bright field microscope, you can observe that the handles of the T-shaped non-glandular hairs and the heads of the glandular hairs are dyed red by safranin, so you can clearly see the distribution of glandular hairs and non-glandular hairs and count them easily;

[0032] 9) If you observe with a fluorescence microscope, after observing clearly under bright field, use blue light to excite, you can not only see the distribution of glandular hairs and non-glandular hairs, but also the distribution of stomata and complete the counting.

[0033] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

[0034] The preparation method of the present invention has been repeatedly tried and tested for many times. The results show that methanol fixation can not only maintain the morphology of the leaf epidermis, but also destroy the pigment in the chloroplast to avoid interference with fluorescence color development, thereby changing the observation background. N-hexane can dissolve the wax layer on the surface of non-glandular hairs and glandular hairs to avoid interference with their autofluorescence when observed under a fluorescence microscope. Sodium hypochlorite bleaching completely avoids the influence of pigments on light transmission during bright field observation. RapiClear 1.52 tissue clearing agent replaces the commonly used chloral hydrate to avoid self-fluorescence affecting the observation of the target. The observation results are as follows: Figure 1-3 As shown, it is proved that the present invention can see the distribution of glandular hairs and non-glandular hairs under bright field of view ( Figure 1 ), using blue light excitation, we can see not only the distribution of glandular hairs and non-glandular hairs, but also the distribution of stomata, so that we can observe glandular hairs, non-glandular hairs and stomata at the same time and complete the counting ( Figure 2 ), after turning the leaf over, the glandular hairs, non-glandular hairs (stalk) and epidermal cell shapes on the upper surface can be observed ( Figure 3 ).

[0035] This preparation technology is simple to operate. The distribution of glandular and non-glandular hairs can be clearly seen when observed only with a bright-field microscope, which is very helpful for counting the number of glandular and non-glandular hairs. If stomata need to be observed at the same time, the stomata distribution can be observed and counted under a fluorescence microscope. This method can be used to see the distribution of glandular and non-glandular hairs (hair bases) on the upper surface and the shape of epidermal cells with the same material.

[0036] In summary, the present invention has found a staining preparation method for observing the morphology of plant epidermal hairs, which can satisfactorily solve a technical problem that has long plagued researchers in observing the distribution and counting of mugwort epidermal hairs, and the stomatal distribution can be observed and counted at the same time under a fluorescence microscope with the same material as needed, and the instrument requirements are simple and the technical requirements for operators are not high. It has the characteristics of simple operation, low cost, small workload, high success rate, clear observation and good effect, and satisfactorily solves the existing technical problems. It is an innovation in the preparation method for simultaneously observing and counting mugwort glandular hairs, non-glandular hairs and stomata, and has good economic and social benefits.

Claims

1. A method for simultaneously observing and counting glandular hairs, non-glandular hairs and stomata of mugwort leaves, characterized in that: The following steps are involved: 1) Take fresh mugwort leaves and rinse them with distilled water for 1 to 3 minutes to remove dust on the surface; 2) Dry the surface moisture of the rinsed leaves, cut small pieces of leaves with a length and width of 8 to 10 mm with a knife, and soak them in methanol for 6 to 12 hours; 3) Remove the leaves from methanol and transfer them to n-hexane for 1 to 3 hours; 4) Remove the leaves from the n-hexane and rinse them with distilled water for 1-3 minutes; then transfer them to 5-8% sodium hypochlorite solution and bleach them until they are colorless; 5) Remove the leaves from the sodium hypochlorite solution, rinse with distilled water, and transfer to RapiClear 1.52 tissue clearing agent for 5 to 10 minutes; 6) The material is then taken out and dyed with a 0.01-0.1% safranin aqueous solution for 0.5-5 minutes; 7) After removing the leaves from the safranin aqueous solution, rinse with water for 5-10 seconds, seal with 10-20% dilute glycerol to make a temporary glass slide, flatten it, and observe it under an optical microscope; 8) After adjusting the focus and brightness under a bright field microscope, you can observe that the handles of the T-shaped non-glandular hairs and the heads of the glandular hairs are dyed red by safranin, so you can clearly see the distribution of glandular hairs and non-glandular hairs and count them conveniently; 9) If you observe with a fluorescence microscope, after observing clearly under bright field, use blue light to excite, you can not only see the distribution of glandular hairs and non-glandular hairs, but also the distribution of stomata and complete the counting.

2. The method for simultaneously observing and counting glandular hairs, non-glandular hairs and stomata of mugwort leaves according to claim 1, characterized in that: The following steps are involved: 1) Take fresh mugwort leaves and rinse them with distilled water for 2 minutes to remove dust on the surface; 2) Dry the surface moisture of the rinsed leaves, cut small pieces of 9 mm in length and width from the leaves with a knife, and soak them in methanol for 9 hours; 3) Remove the leaves from methanol and transfer them to n-hexane for 2 h; 4) Remove the leaves from the n-hexane and rinse them with distilled water for 2 minutes; then transfer them to a 6% sodium hypochlorite solution to bleach them until they are colorless; 5) Remove the leaves from the sodium hypochlorite solution, rinse with distilled water, and transfer to RapiClear 1.52 tissue clearing agent for 7 minutes; 6) The material was then taken out and dyed with 0.05% safranin aqueous solution for 3 minutes; 7) After removing the leaves from the safranin aqueous solution, rinse with water for 7 seconds, seal with 15% dilute glycerol to make a temporary glass slide, flatten it, and observe it under an optical microscope; 8) After adjusting the focus and brightness under a bright field microscope, you can observe that the handles of the T-shaped non-glandular hairs and the heads of the glandular hairs are dyed red by safranin, so you can clearly see the distribution of glandular hairs and non-glandular hairs and count them conveniently; 9) If you observe with a fluorescence microscope, after observing clearly under bright field, use blue light to excite, you can not only see the distribution of glandular hairs and non-glandular hairs, but also the distribution of stomata and complete the counting.

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