In this article we will discuss about:- 1. Study of Typical Plant Cells 2. Streaming Movement of Protoplasm 3. Study of Living Cell Inclusions 4. Study of Non-Living Cell Inclusions 5. Study of Schizogenous and Lysigenous Cavities 6. Study of Plasmodesmata 7. Study of the Chemical Nature of the Cell Wall 8. Study of Cell Types and Cell Wall Thickening from Macerated Tissues.
Study of Typical Plant Cells (Fig. 3.1):
Material:
Fleshy scale leaf of onion.
Remove the dry scale leaves from a piece of onion bulb and take a colourless fleshy scale leaf. Peel off its epidermis, stain a part of this epidermis in 1% aqueous iodine solution and mount in 20 to 50% glycerine. Observe under both low and high power of microscope.
Note the almost rectangular cells with nucleus (deep stain), cytoplasm (light stain) and vacuoles (very light stain), and thin cell wall with middle lamella.
Streaming Movement of Protoplasm (Cyclosis):
(a) Rotation (Fig. 3.2):
Material:
Vallisneria spiralis leaf (fresh).
Take a tangential section of the leaf. Mount in water. Observe under high power and note the rotation of the chloroplasts around the central vacuole.
(b) Circulation (Fig. 3.3):
Material:
Staminal hairs of Rhoeo discolor (R. spathecea).
Pick up some hairs from the filament of a stamen. Mount in water and observe under high power. Note the circulation of granular cytoplasm around the vacuoles.
Study of Living Cell Inclusions:
(a) Chloroplasts (Fig. 3.2):
Material — Leaf of Vallisneria or any green leaf.
Cut a transverse or longitudinal section through mesophyll tissue of the leaf. Mount in 50% glycerine and observe under high power. Note the elliptical or oval green plastids in cytoplasm.
(b) Chromoplasts (Fig. 3.4):
Material:
Fruit of Lycopersicon esculentum (tomato), root of Daucus carota (Carrot).
Scoop out a little soft fleshy pulp from a ripe tomato, mount in water or 50% glycerine and observe under high power. Note the rod-like acicular chromoplasts in cytoplasm.
Cut a T.S. of carrot root, mount in 50% glycerine and observe under high power. Note the rod like chromoplasts in cytoplasm.
Study of Non-Living Cell Inclusions (Ergastic Maters):
(a) Starch Grains (Fig. 3.5):
Material:
Pea seed, banana fruit, sweet potato tuber, maize grain, gram seed and potato tuber. Cut through the above-mentioned plant organs to expose the inner tissues. Scrape the surface of the exposed tissue with a razor or scalpel. Mount the scraped tissue on a slide in 1% aqueous iodine solution, or in 0.3 g iodine, 1.5 g potassium iodide and 100 ml distilled water solution.
Observe under high power. The innumerable starch grains appear blue in colour. The blue colour disappears on heating but reappears on cooling. Note the nature of the grains in each material. Every grain has a shiny refractive point called hilum.
Starchy materials are deposited in layers around the hilum. These are lines of stratification. When the hilum lies at the centre of the grain, it is a concentric grain. If the hilum lies at one end of the grain, it is an excentric grain.
A grain with one hilum is a simple grain. Two or more grains may be adpressed together forming a compound grain. When a compound grain develops some common line stratification, it is called a semi-compound or half-compound. Identify the different types of grains are concentric and simple or compound. In banana, the grains are concentric and simple or compound and almost round.
In sweet potato, they are concentric, simple or semi-compound and irregular in outline. In maize grain, the starch grains are concentric, simple or compound and often angular in outline. In gram, they are concentric, simple or compound and elliptical or round in outline. The starch grains of potato are excentric, simple compound or semi-compound and various in shape, often oval with the hilum end being pointed.
(b) Inulin Crystals (Fig. 3.6):
Material:
Root tuber of Dahlia.
Inulin is a polysaccharide carbohydrate which forms a powdery compound and occurs in the cell sap in colloidal condition in plants like Dahlia. Put small pieces of fresh Dahlia root tubers in 70% alcohol for 2 to 4 days in order to allow inulin to form crystals. Cut sections from the fixed tubers and observe under high power. Beautiful fan-shaped crystals are noted in the cells.
Add a drop of chloral hydrate solution (5 parts chloral hydrate and 2 parts water), and note the concentric layers of the crystals. Add a drop of 15% solution of thymol in alcohol and a drop of concentrated sulphuric acid. The crystals turn carmine-red immediately and quickly dissolve in solution. With acid phloroglucin inulin turns yellowish-brown.
(c) Aleurone Grains (Fig. 3.7):
Material:
Wheat grains, maize grains and castor seeds. Cut sections of the grains and endosperms of castor seeds. In wheat and maize and, in fact, in all grains, the layer of cells just below the grain coat is full of small round aleurone grains.
In castor endosperm the aleurone grains are much larger, having a protein matrix in which lies 1 or 2 large crystalline bodies called crystalloid, and a small round body which is known as globoid. The former is nitrogenous and the latter a double phosphate of calcium and magnesium. Actually, all aleurone grains are made up of protein crystals and globules in a protein matrix.
Tests:
1. Saturate absolute alcohol with picric acid and nigrosin dye. Place the sections on a slide, add a few drops of this reagent and observe under microscope. When the ground substance of the grains turns blue, stop the reaction by adding absolute alcohol.
The crystalloids turn yellowish green and the globoids remain colourless. The sections can be cleared with clove oil and mounted in Canada balsam to make permanent slides.
2. Use eosin in place of nigrosin. The matrix turns dark red, the crystalloids yellow and the globoids become slightly tinted red.
3. Xanthoprotein test — Treat the sections in water with strong nitric acid. A yellow colour is developed. Now add a few drops of strong ammonium hydroxide. The colour changes to orange.
4. Biuret test — Treat sections with 1 ml of 20% sodium hydroxide and 1 drop of 1% copper sulphate solution. The grains turn violet.
(d) Mineral Crystals:
(i) Calcium oxalate crystals (Fig. 3.8):
Material:
Petioles of arum, papaw and water-hyacinth, leaf of Pistia and dry scale leaf of onion. Cut sections of the above plant organs. In case of onion, take a small piece from a dry scale leaf. Mount in water or 20% to 50% glycerine and observe under microscope. In the dry scale leaf of onion, solitary calcium oxalate crystals — which are rod-like, prismatic etc. — are present in the cells?
In the petioles of arum and water-hyacinth, bunches of needle-like crystals — which look like brooms — are present in special cells called idioblasts. These are called raphides. In Pistia and papaw the crystals are aggregated into roundish stellate bodies within the idioblasts. These are called sphaeraphides or druses. Pistia has both raphides and sphaeraphides.
(ii) Calcium carbonate crystals (Fig. 3.9):
Material:
Leaves of banyan and India rubber plant (Moraceae), (Momordica) Cucurbitaceae (Ruellia) Acanthaceae. Cut transverse sections of the leaves, mount in water or 20% to 50% glycerine and observe under microscope. The leaves have multiple epidermis being made up of 2 to 4 layers of cells.
Some cells of the innermost layer are much larger and calcium carbonate crystals are aggregated on a peg-like projection of the cell wall. The crystal aggregate looks like a bunch of grapes. These are called cystolith and the cell a lithocyst.
Study of Schizogenous and Lysigenous Cavities (Fig. 3.10):
Material:
Stems of sunflower, pine and maize, fruits of lemon and orange (Citrus fruit).
Cut transverse sections of the stems and vertical sections of skins of the fruits. Mount in water or 20% to 50% glycerine. Observe under microscope.
Stems of sunflower and pine show resin ducts in the cortical regions which are schizogenous in origin. Schizogenous cavities arise by the separation of adjacent cell walls along the areas of contact followed by contraction of separated parts (schizos = split). The resin ducts are circular in T.S. and are lined by small thin-walled cells called epithelial cells.
Skins of Citrus fruits show innumerable ovoid cavities containing essential oil. These are lysigenous cavities (lysis = decomposition). These arise by the decomposition or destruction of a group of cells. Stems of maize show large protoxylem lacunae or cavities where the dis-organising protoxylem elements can be actually seen in the vascular bundles. These are schizo-lysigenous cavities.
Study of Plasmodesmata (Fig. 3.11):
Material:
Seeds of date palm, potato tuber.
Plasmodesmata (sing, plasmodesma) are the extremely delicate and fine strands of cytoplasm which connect the protoplasts of adjacent cells. It is difficult to demonstrate their existence because of the extremely delicate nature.
The following solutions should be prepared beforehand:
1. Killing solution:
0.75 g potassium iodide, 1.5 g iodine, 100 ml distilled water.
2. Mordanting solution:
1.25 g potassium iodide, 1 g iodine, 100 ml 5% aqueous sulphuric acid.
3. Staining solution:
(a) 5% aqueous sulphuric acid, (b) 0.5 g crystal violet, 100 ml distilled water.
4. Mounting solution:
30 ml glycerine, 60 ml distilled water, 2 g zinc chloride, 0.2 g iodine, potassium iodide — a trace.
The killing and mordanting solutions are warmed until the iodine dissolves to the point of saturation. The mounting solution is also slightly warmed and a small crystal of potassium iodide is added, which dissolves some of the excess iodine. All iodine-containing solutions are stored in brown bottles in darkness.
Make sections of date palm endosperm and the central part of potato tuber tissues and place them in the killing solution for 5 minutes and then swell them for another 5 minutes in 10% sulphuric acid. Transfer the sections to the mordanting solution for 5 minutes and then wash them in 5% sulphuric acid till the iodine starts to fade.
To prepare the staining solution, take 1 ml of sulphuric acid in a watch glass and add crystal violet solution drop by drop until a deep green colour is obtained. This staining solution is unstable and should be used at once. Transfer sections to the stain and allow to remain until they become darkened.
Mount the sections in the mounting solution, observe under microscope and locate the plasmodesmata. The sections-last for several weeks. In date palm endosperm the excessive thickness of the cell wall is due to the deposition of hemi-cellulose.
Study of the Chemical Nature of the Cell Wall:
(a) Cellulose:
It is the most abundant of the cell wall materials and is a polysaccharide carbohydrate.
Make some transverse sections of any suitable plant organ — such as sunflower or maize stem — and perform the following tests:
(i) Treat some sections with chlor-zinc-iodine solution. Cellulose walls turn blue to violet.
(ii) Treat some sections with iodine solution (0.3 g iodine, 1.5 g potassium iodide and 100 ml distilled water) and 50% sulphuric acid. Cellulose walls turn blue.
(b) Lignin:
It is an organic compound of high carbon content — distinct from carbohydrates — and is present in the walls of cells constituting the mechanical tissues such as sclerenchyma, xylem vessels, tracheids, etc.
Make some sections of any suitable plant organ — such as sunflower or maize stem — and perform the following tests:
(i) Treat some sections with chlor-zinc-iodine solution. Lignified cell walls turn yellow.
(ii) With phloroglucin and conc. HCl, lignin turns red.
(iii) With aniline sulphate, lignin turns bright yellow.
(c) Cutin:
It is a fatty substance almost impermeable to water. It is present on the outer walls of epidermal cells of serial plant organs, where it forms a waterproof layer called cuticle.
Take sections of sunflower or maize stem or mango leaf and perform the following tests:
(i) Treat some sections with Sudan IV solution. Cutinized cell walls turn orange to pink- ish-red.
(ii) If treated with iodine solution and 50% sulphuric acid, it turns yellowish-brown,
(iii) When treated with KOH solution, it turns yellow.
(d) Suberin:
It is a fatty substance like cutin but differs from cutin in having phellic acid. It also is impermeable to water.
Make some sections of bottle cork or shola (Aescynomene aspera) or potato tuber skin and perform the following tests:
(i) Suberin turns brown with KOH solution,
(ii) When treated with chlor-zinc-iodine solution, suberized cell walls turn yellowish-brown.
(e) Mucilage:
It is a derivative of carbohydrate and is present in many aquatic plants. It is also present in the husks of ‘isabgul’ — Plantago ovata.
Take some husks of isabgul or some sections of an aquatic plant — such as Nymphaea sp. — and perform the following tests:
(i) Mucilage turns violet when treated with iodine solution and 50% sulphuric acid,
(ii) If treated with ruthenium red, it turns pink.
(f) Silica:
Silica particles often remain impregnated on the cell walls of grasses (Poaceae), sedges (Cyperaceae), Equisetum, etc. Take some sections of a grass leaf or stem and mount them in saturated carbolic acid solution. (Do not touch the solution with your hand). Silica particles turn pink.
Study of Cell Types and Cell Wall Thickening from Macerated Tissues:
Maceration involves the dissolution of the middle lamellae — is the cementing layer in-between the adjacent cells — and, consequently, the cells become completely free from one another.
Single isolated cells can thus be obtained by maceration and their shape, size as well as the patterns of secondary cell wall thickening can be studied.
Although Jeffrey’s method of maceration can be applied to all kinds of tissues, it is a long-drawn process and, hence, not suitable for usual class-work. Maceration with KOH for soft stem, with HNO3 and chromic acid for semi-woody tissues, such as Cestrum stem, and with HNO3 and KCIO3 for woody tissues, such as pine and mango wood, are well-suited for usual class-work.
As KOH is a mild macerating agent, it leaves almost all the cells intact but loosened, including the thin-walled parenchyma and collenchyma cells.
HNO3 and KClO3, on other hand, is the strongest macerating element and so it practically dissolves all thin-walled elements, such as parenchyma, collenchyma, phloem etc., leaving only the thick-walled and lignified elements such as fibres, tracheids and vessel segments intact. This, therefore, is the ideal macerating agent for studying the elements which constitute wood along with their wall-thickening patterns.
Macerate some representative types of plant organs and study the cell types along-with wall-thickening patterns after staining or without staining:
(i) With KOH solution — Cucurbita stem.
(ii) With nitric acid and chromic acid — Cestrum stem, fern rhizome (Pteris or Dryopteris or Polypodium).
(iii) With nitric acid and potassium chlorate — Gymnosperm and angiosperm wood (Pine and Mango stem).