CN107219103B - Preparation method of perennial ryegrass epidermal cell slice - Google Patents

Abstract

The invention relates to experimental utensils, and specifically discloses a method for preparing perennial ryegrass epidermal cell slices. The method comprises: (1) selecting leaves of perennial ryegrass, putting them into FPA fixative for at least 24 hours, taking them out for freehand slicing, Obtain the perennial ryegrass leaves to be observed; (2) wash the immobilized liquid remaining on the surface of the leaves, scrape off the epidermis and part of the mesophyll on one side of the leaves, and put them into a mixed enzyme solution with a concentration of 0.9-1.1%, under the condition of 26-28 ℃ After dissociation for 1 to 3 hours, wash and remove the mixed enzyme solution; (3) scrape off the remaining tissue until the leaf epidermis is transparent, that is, slices of perennial ryegrass epidermal cells; wherein, the mixed enzyme solution contains an equal mass ratio of Pectinases, cellulases and hemicellulases. By the method of the invention, a complete and clear view of the epidermal cell slices of the leaves of perennial ryegrass can be obtained, and clear stomata, epidermal cell structures, epidermal hairs and other accessory organs can be observed under a microscope.

Description

Preparation method of perennial ryegrass epidermal cell slices

technical field

The present invention relates to the field of experimental utensils, in particular, to a microscopically observed cell section.

Background technique

Perennial ryegrass is an important cool-season turfgrass species native to Europe, temperate Asia and North Africa. Ryegrass is suitable for cool conditions without severe winters and harsh summers. As one of the important grass species for turf construction, perennial ryegrass is quick to grow into lawn, resistant to low mowing, and can form dense turf. It is often used as the pioneer grass of cold-season mixed-seeding turf to build football field turf, and can also be used as supplementary seeding. and overcast material. At the same time, the characteristics of bright leaves and dark green color make it widely used in landscape lawn, sports field lawn and golf course fairway lawn. Due to the wide application of perennial ryegrass and the correlation between leaf morphology and physiological characteristics, researchers need to observe the leaf epidermal cells.

The observation of leaf epidermal cell characteristics includes cell number, size, shape, cell classification (such as: long cells, short cells), the number, size, shape of epidermal hairs, and the observation of the number, size, and shape of stomata. Different varieties of perennial ryegrass leaf epidermal cells have some differences in morphology, and this difference may cause differences in physiological resistance among different varieties of perennial ryegrass. Therefore, it is of great significance to observe and study the epidermal cells of perennial ryegrass.

A large number of researchers have made freehand slices by directly scraping the surface of turfgrass leaves to observe the epidermal cells of turfgrass leaves. However, this method is not suitable for perennial ryegrass. The leaves of perennial ryegrass contain more water and the epidermal cells are closely arranged. It is impossible to obtain complete epidermal cells by directly scraping the epidermis. completeness.

The surface of perennial ryegrass leaves contains waxy, epidermal hairs and other accessory organs, which protect the leaf epidermis, which makes it difficult to make leaf epidermis slices. Both the scotch tape method and the nail polish imprinting method use the adhesiveness of the scotch tape and the nail polish to the leaf surface to tear the leaf epidermal cells, and make a mount for microscopic observation. However, these two methods have obvious shortcomings. First, the epidermis of perennial ryegrass leaves is more waxy, which makes the adhesiveness of scotch tape and nail polish to the surface of the leaves not strong, and it is impossible to obtain the complete epidermal cells of perennial ryegrass; After some epidermal cells are removed, a large amount of chlorophyll will be torn off together, which will hinder the visual field during the observation process and affect the observation clarity of epidermal cells.

The method for isolating leaf epidermal cells with hypochlorous acid is to isolate the leaf epidermal cells and mesophyll by the isolation method, and after tearing the epidermal cells, carry out staining, and make them into slices for observation. The disadvantage of this method is that the use of sodium hypochlorite will cause irreversible damage to the epidermal cells of perennial ryegrass leaves. contradictory results.

Therefore, there is an urgent need to develop a method for freehand sectioning of leaf epidermal cells suitable for perennial ryegrass, which can quickly obtain complete and high-definition freehand sectioning of epidermal cells on the basis of minimal damage to the epidermal cells.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a preparation method of slices of epidermal cells of perennial ryegrass.

In order to realize the purpose of the present invention, the technical scheme of the present invention is as follows:

The present invention first provides a method for preparing perennial ryegrass epidermal cell slices, comprising the following steps:

(1) Select perennial ryegrass leaves, put them into FPA fixative for at least 24 hours, take them out for free-hand slicing, and obtain perennial ryegrass leaves to be observed,

(2) Rinse the residual fixative solution on the leaf surface, scrape off the epidermis and part of the mesophyll on one side of the leaf, put it into a mixed enzyme solution with a concentration of 0.9-1.1%, dissociate at 26-28 °C for 1-3 hours, and wash remove the mixed enzyme solution;

Specifically, if you want to observe the upper epidermis of the leaves, scrape off the lower epidermis and part of the mesophyll; if you want to observe the lower epidermis of the leaves, scrape off the upper epidermis and part of the mesophyll;

(3) scrape off the remaining tissue until the leaf epidermis is transparent, that is, the epidermal cell slices of perennial ryegrass;

Wherein, the mixed enzyme solution contains pectinase, cellulase and hemicellulase in equal mass ratios. That is, pectinase, cellulase and hemicellulase are composed of mixed enzymes in a mass ratio of 1:1:1.

Preferably, the concentration of the mixed enzyme in the mixed enzyme solution is 1%, and the solvent is a PBS buffer with pH 5.7. The concentration of 1% is understood as containing 1 mg of mixed enzyme per 100 mL of mixed enzyme solution, and the aforementioned mixed enzyme solution with a concentration of 0.9 to 1.1% is understood to contain 0.9 to 1.1 mg of mixed enzyme per 100 mL of mixed enzyme solution. Since the present invention finds that the optimal mixed enzyme concentration is 1% through experiments, for those skilled in the art, a little adjustment (concentration of 0.9-1.1%) on this basis can also realize the technology of the present invention. Effect.

The FPA fixative, PBS buffer, pectinase, cellulase and hemicellulase are reagents and enzymes routinely used in the art, all of which are commercially available commodities, which are not otherwise limited in the present invention.

Preferably, in the step (2), the dissociation is carried out in a water bath environment of 26-28° C. The water bath environment will provide a better constant temperature effect, which is conducive to the dissociation.

More preferably, the best dissociation effect can be obtained by dissociating in a 27°C water bath for 2 hours.

Further, in the step (1), the FPA fixative solution is put into the fixation solution for 24 to 48 hours, and the fixation effect is better.

The perennial ryegrass epidermal cell slice prepared according to the above method can observe clear stomata, epidermal cell structure, epidermal hair and other accessory organs under a microscope.

Based on this, the perennial ryegrass epidermal cell slice prepared by the above method also falls within the protection scope of the present invention.

The beneficial effects of the present invention are:

The invention provides a preparation method of perennial ryegrass epidermal cell slices, through which a complete and clear view of perennial ryegrass leaf epidermal cell slices can be obtained, and clear stomata, epidermal cell structures, epidermal hairs and the like can be observed under a microscope accessory organs.

Description of drawings

FIG. 1 is a microscopic observation diagram of the section obtained in Example 1 in Experimental Example 1 of the present invention.

FIG. 2 is a microscopic observation diagram of the section obtained by the test group 2 in the comparative example 1 of the present invention.

FIG. 3 is a microscopic observation diagram of the section obtained in Test Group 3 in Comparative Example 1 of the present invention.

FIG. 4 is a microscopic observation diagram of the section obtained in test group 4 in Comparative Example 1 of the present invention.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

In this example, the preparation of epidermal cell slices of perennial ryegrass is taken as an example to illustrate the preparation method of perennial ryegrass epidermal cell slices according to the present invention.

The specific operations are as follows:

1. Prepare mixed enzyme solution:

Mix pectinase, cellulase and hemicellulase in a ratio of 1:1:1 and dissolve them in PBS buffer with pH 5.7 to prepare a mixed enzyme solution with a concentration of 1% (per 300 mL of mixed enzyme solution). , containing 1 mg pectinase, 1 mg cellulase and 1 mg hemicellulase).

2. Preparation of epidermal cell slices on perennial ryegrass:

The leaves of perennial ryegrass were put into the FPA fixative solution, and after 24 hours of fixation, they were taken out and sliced by hand. Rinse several times with distilled water to remove fixative from the leaf surface. After gently scraping off part of the leaf epidermis and part of the mesophyll on the back of the leaf with a blade,

Put it into the mixed enzyme solution (1/3 pectinase + 1/3 cellulase + 1/3 hemicellulase) obtained in step 1, dissociate in a 27°C water bath for 2 hours and then take it out. Immerse the dissociated leaf tissue segments in a petri dish filled with distilled water for washing, and remove the mixed enzyme solution. Lay the leaf tissue segment on a glass slide, gently scrape off the remaining mesophyll and other tissues on the back of the leaf, and repeat this process until the leaf epidermis is transparent, that is, the epidermal cell slices on the perennial ryegrass.

Experimental example 1

The epidermal cell sections of perennial ryegrass prepared in Example 1 were observed under a 10×20 magnification Olympus microscope, and the results are shown in FIG. 1 . It can be seen from Figure 1 that the epidermal hairs, leaf stomata, leaf epidermal cells and motile cells are clearly visible.

Comparative Example 1

This comparative example is used to investigate the effects of different enzyme solutions of the same concentration on the preparation of slices under the same conditions of enzymatic hydrolysis time.

Test group 1: same as Example 1;

Test group 2: The difference from Example 1 is that the 1% mixed enzyme solution consists of 1/2 pectinase and 1/2 cellulase;

Test group 3: The difference from Example 1 is that the 1% mixed enzyme solution consists of 1/3 pectinase and 2/3 cellulase;

Test group 4: The difference from Example 1 is that the 1% mixed enzyme solution consists of 2/3 pectinase and 1/3 cellulase.

The sections prepared in test groups 1 to 4 were observed under a microscope, and the results were shown in Figures 1 to 4 respectively. It can be seen from the figures that when the mixed enzyme solution was composed of cellulase, pectinase and hemicellulase, the The structures of epidermal hairs, leaf stomata, leaf epidermal cells, and motile cells are clearer than those obtained by other treatments, and have the best observation effect.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (3)

1. A preparation method of perennial ryegrass epidermal cell slices is characterized by comprising the following steps:

(1) selecting perennial ryegrass leaves, putting the perennial ryegrass leaves into FPA stationary liquid for fixing for at least 24 hours, taking out the perennial ryegrass leaves, and slicing the perennial ryegrass leaves by hands to obtain perennial ryegrass leaves to be observed;

(2) washing the residual fixing solution on the surface of the leaf, scraping off the epidermis and part of the mesophyll on one side of the leaf, putting into the mixed enzyme solution with the concentration of 1%, dissociating for 2 hours in a water bath environment at the temperature of 27 ℃, and cleaning to remove the mixed enzyme solution;

(3) scraping residual tissues until the epidermis of the leaves is transparent, and obtaining perennial ryegrass epidermal cell slices;

wherein the mixed enzyme solution contains pectinase, cellulase and hemicellulase in equal mass ratio; the solvent of the mixed enzyme solution is PBS buffer solution with pH5.7.

2. The method according to claim 1, wherein in the step (1), the FPA fixative is added for 24 to 48 hours.

3. A perennial ryegrass epidermal cell slice prepared by the method of claim 1 or 2.

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