Plant Anatomy: Practical Course - Panyuta O.O. 2019

Introduction

Plant anatomy is a branch of botany that explores the Structure of Plant Cells, Tissues, and Organs in close connection with their Functions and development.

Understanding the architecture of the plant Organism opens up new horizons for researchers in plant growing, breeding, genetics, biotechnology, and ecology. It helps enhance crop productivity, improve resistance to extreme environmental factors and pathogens, ensure successful plant Introduction, provide a scientific foundation for phytobiotechnology, facilitate phytomonitoring studies, and deepen our grasp of the patterns governing Plant GROWTH AND DEVELOPMENT.

Laboratory classes in plant anatomy introduce students to the structural diversity of cells, tissues, and organs.

This study guide covers four main topics: "The Cell," "Tissues," "Structure of Vegetative Organs," and "Seed Structure." Each topic features a theoretical Overview, practical laboratory Assignments complete with detailed descriptions of specimens, illustrative drawings, and Review Questions. The Appendix at the end of the manual outlines various stains and Reagents used for dyeing, fixing, and preparing plant specimens, as well as for Microchemical Reactions aimed at identifying Organic compounds, among other techniques.

A thorough grasp of the life processes and Structural Organization of plants is essential for any modern plant biologist conducting successful research. The wide array of prepared slides featured in this manual makes it a compelling resource for a broad audience, including undergraduate students, graduate researchers, and instructors in biological sciences.

STRUCTURE OF THE Light Microscope. Preparing Temporary Mounts for Microscopic Examination

The light microscope is an optical instrument that produces a magnified, inverted image of a study object, allowing observers to examine minute structural details whose dimensions lie beyond the resolving power of the human eye.

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Fig. 1. Light microscope:

1 - eyepiece, 2 - body tube, 3 - tube holder, 4 - coarse adjustment knob, 5 - fine adjustment knob, 6 - base-stand, 7 - mirror, 8 - condenser, iris Diaphragm and light filter, 9 - mechanical stage, 10 - objective lens

The microscope consists of two main systems: optical and mechanical. The Optical System includes the objective lenses, eyepieces, and illumination device (Fig. 1).

The objective lens is one of the most crucial Components of the microscope. It forms a magnified, real, yet inverted image of the specimen and reveals the fine details of its structure.

An objective lens consists of a metal cylinder housing a variable number of lenses. The bottom lens facing the specimen is called the frontal (front) lens. The upper part of the objective features a screw

thread used to screw it into the revolving nosepiece socket. The magnification of the objective is indicated directly on it. The MBR-1 microscope is equipped with three objectives: x8, x40, and x90, whereas the Biolam microscope features five: x10, x20, x40, x60, and x90. For educational purposes, x8 or x10 and x40 objectives are most commonly used.

The quality of an objective is defined by its resolving power. Naturally, the greater the resolution, the clearer the smaller elements of the observed specimen appear. The resolving power is 1.68 µm for the x8 objective, 0.52 µm for the x40 objective, and 0.27 µm for the x90 objective. The resolution value is marked on each objective. Resolution is directly influenced by the diameter of the front lens: the smaller the diameter, the higher the resolution. Objectives must be handled with utmost care and precision. This is particularly true for high-magnification objectives, where the working distance—that is, the clearance between the coverslip and the front lens—is measured in fractions of a millimeter. The working distance is 13.8 mm for the x8 objective, 0.6 mm for the x40 objective, and 0.12 mm for the x90 objective. Low-power objectives provide the maximum working distance and the largest field of view. Image quality, especially when using high-power objectives, also depends on the thickness of the Glass slides and coverslips. The standard thickness is 1.2 mm for microscope slides and 0.17 mm for coverslips.

The eyepiece (ocular), much like a magnifying glass, produces an upright, virtual, magnified image of the specimen created by the objective lens. It does not reveal any new structural details, which is why its magnification is termed virtual. The eyepiece has a simpler construction than the objective, consisting of two or three lenses mounted inside a metal cylinder. A fixed diaphragm is positioned between the lenses to define the BOUNDARIES OF THE field of view. The lower lens focuses the intermediate image formed by the objective within the plane of the diaphragm, while the upper lens serves directly for observation. Eyepiece magnifications are designated by numbers such as x7, x10, and x15. To determine the total magnification of the microscope, multiply the magnification of the objective by the magnification of the eyepiece (for example, 90x10).

The illumination device comprises a mirror and a condenser equipped with an iris diaphragm, both located beneath the stage. This system is designed to illuminate the specimen with a concentrated beam of light.

The mirror directs a beam of light through the condenser and the stage aperture onto the specimen. It has two surfaces: flat and concave. The concave surface is used in conditions of diffused ambient light. The mirror is mounted on the stand in a way that allows it to rotate in two mutually perpendicular planes.

The condenser consists of two or three lenses housed in a metal cylinder. Raising or lowering the condenser via a dedicated adjustment screw concentrates or diffuses the light passing from the mirror onto the specimen.

The iris diaphragm is situated between the mirror and the condenser. It serves to adjust the diameter of the light beam directed by the mirror through the condenser to match the diameter of the objective's front lens. It consists of thin metal leaves that can be closed to completely block the lower lens of the condenser or opened to widen the light beam.

The mechanical system of the microscope includes the base, the adjustment box housing the fine-focus mechanism, the tube holder, the coarse adjustment knob, the condenser bracket, the condenser adjustment screw, the revolving nosepiece, and the mechanical stage.

The fine-adjustment gear box, built on THE PRINCIPLE OF interacting gears, is rigidly attached to the base.

The fine adjustment knob is used for minute (micrometer-scale) vertical shifts of the tube holder and, consequently, the objective lens. One full Rotation of the fine adjustment knob moves the tube holder by 100 µm, while turning it by a single graduation raises or lowers it by 2 µm. To prevent damage to the fine-focus mechanism, it should never be turned in one direction by more than half a revolution.

The body tube is a cylinder into which the eyepiece is inserted from above. The tube is movably connected to the HEAD of the tube holder and secured in position by a locking screw. Loosening the locking screw allows the tube to be removed.

The revolving nosepiece is designed for quick switching between objective lenses. A centered position of the objective is maintained by a click-stop mechanism located inside the nosepiece.

The body tube carrier serves to mount the body tube and the revolving nosepiece. In modern microscopes with an inclined body tube, the carrier is movably connected to the fine adjustment box by means of a rack and pinion mechanism driven by the coarse adjustment knob.

The coarse adjustment knob is used for substantial vertical Displacement of the body tube carrier—and consequently the objective lens—to achieve initial focus under low magnification.

The stage is designed to hold the specimen slide in place. At the center of the stage, There is a circular aperture through which the front lens of the condenser passes. The microscope features a circular stage equipped with a movable mechanical stage disk. This disk can be rotated around its axis and translated in two mutually perpendicular directions using two control knobs located on the right and left sides of the stage. Such movements allow for precise centering of specific areas of the specimen, which is crucial when working with high-magnification objectives. A stop screw allows the disk to be locked in a desired position. The stage is also fitted with two spring clips to securely hold the slide.

Preparation of temporary biological slides. When preparing temporary slides, the specimen is placed on a microscope slide in a drop of Water, glycerin, reagent, or stain, and then covered with a coverslip. Such preparations typically last no longer than a month. Specimens that can be preserved for extended periods are referred to as permanent slides. Certain plant organs, such as spores or the leaves of specific species, can be examined directly under a biological microscope as whole mounts without prior sectioning. However, the number of specimens that can be studied in this manner is quite small. More often, it is necessary to prepa-

re thin sections of the organs under investigation. These sections are made from either fresh plant material or specimens fixed in alcohol or formalin. Temporary slides are prepared following this sequence of steps:

1) wash and thoroughly dry the microscope slide and coverslip. To avoid breaking the extremely fragile coverslip, rinse it in water, place it within the fold of a towel between the thumb and forefinger of your right hand, and gently wipe it with circular finger movements;

2) place a drop of liquid (water, glycerin, a reagent, or a stain) onto the microscope slide;

3) cut a thin section of the organ under study using a razor blade or scalpel;

4) select the thinnest section and place it into the drop of liquid on the slide;

5) lower the coverslip over the section so that no air bubbles are trapped underneath. To do this, hold the coverslip by its edges between two fingers, bring the lower edge into contact with the drop of liquid, and gently lower it;

6) if there is an excess of liquid and it spills out from under the coverslip, blot it away using a piece of filter paper. Conversely, if air pockets remain beneath the coverslip, add more liquid by placing a drop right at the edge of the coverslip.