Tissue Culture

W ha t is T is sue Cultur e? Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth media, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is used for plants. Tissue culture is when a piece of plant is put into the tiniest of greenhouses...a test tube....and a miracle takes place. If the test tube is free of micro-organisms and the media in the test tube has the correct balance of chemicals and growth hormones, that little piece of plant will reproduce replicas or clones of the single parent plant. That little piece of plant material can replicate in incredible numbers so theoretically, one small piece can produce an infinite number of clones. W hy is tissue

cultur e used?

Tissue culture is used for many reasons but the 3 major reasons DNA Gardens are concerned with are 1) Large quantities of plant production for the sake of volume, 2) to speed up the production of new varieties into the market place. Large amounts of plants can be produced in a very short time compared to conventional propagation methods, 3) to establish and maintain virus free stock as in the case of the seed potato industry. Working in a tissue culture lab is like working in a hospital operating room. Sanitation is imperative. If the plant cultures become infected with fungus or bacteria, the contaminants soon over take and kill the cultures. Other applications include protoplast fusion and the induction and selection of mutants and the biosynthesis of plant metabolites.

A. Animal T issue s The development of a fertilized egg into a newborn child requires an average of 41 rounds of mitosis. During this period, the cells produced by mitosis enter different pathways of differentiation; some becoming blood cells, some muscle cells, and so on. There are more than 100 visibly-distinguishable kinds of differentiated cells in the vertebrate animal. These are organized into tissues; the tissues into organs. Groups of organs make up the various systems - digestive, excretory, etc. - of the body.

TYPE S OF ANIMAL

T ISSU ES

1. Epithelial – covering 2. Connective – support 3. Muscular – movement 4. Nervous – control

EPITHELIAL TISSUES

General Characteristics 1. Cellularity – cells are closely-packed with only tiny amount of extra cellular material in between 2. Specialized contacts – adjacent cells are bound together at many points by lateral contacts (e.g., gap junctions, tight junctions, etc.) 3. Polarity – have an apical surface (free surface exposed to exterior/cavity) and an attached basal surface • Apical surface usually has microvillus or cilia • Basal lamina is noncellular and adhesive (glycoproteins) 4. Supported by connective tissue – just deep to basal lamina is reticular lamina (layer of extra cellular material containing collagen fibers); these two lamina form the basement membrane which reinforces epithelial sheet and defines its boundary 5. Avascular but innervated – nourished by substances diffusing from blood vessels in underlying connective tissue 6. Regeneration – high regenerative capacity as long as they are nourished • General functions: 1. Covering and lining 2. Glandular or secretive Types: -based on arrangement 1. Simple – composed of single cell layer; located where thin barrier is needed for absorption or filtration 2. Stratified – composed of 2 or more cell layers stacked on each other; located where protection is important (areas of high abrasion) - based on shape in apical layer 1. Squamous- flattened and scale like; nucleus is disc-shaped 2. Cuboidal – boxlike; as tall as they are wide; nucleus is spherical 3. Columnar – tall and column-shaped; nucleus is elongated from top to bottom and located near base 4. Pseudo stratified – vary in height but all rest on basement membrane; only tallest reach free surface; cell nuclei lie at different levels giving false impression that layers are present

• Specific types of epithelial tissue: 1. Simple squamous – cells flattened laterally; sparse cytoplasm; thin layered; found where filtration or exchange by diffusion is important; can have secretory function

Figure 7B: Schematic representation of simple squamous epithelium. 2. Simple cuboidal – single layer of cells as tall as wide; found where secretion and absorption is important

3. Simple columnar – single layer of tall cells with oval nuclei; may bear cilia to propel mucus; may contain mucus secreting unicellular glands (goblet cells); found where secretion (mucus, enzymes, etc.) and absorption is important.

Figure 4: Schematic representation of simple columnar epithelium. 4. Pseudo stratified – single layer of cells of differing heights; may contain goblet cells and Bear cilia; found where secretion and absorption is important.

Figure 9A: Schematic representation of pseudostratified epithelium. 5. stratified squamous – several layers thick; free surface cells are squamous while deeper layers are cuboidal or columnar; can be keratinized (epidermis) or no keratinized (linings); function in protection from wear and tear underlying tissues.

Figure 8A: Schematic representation of stratified squamous epithelium.

6. Glandular – 1+ cell that make and secrete a product; classified on basis of site of product release and # of cells (unicellular or multicellular) • Endocrine – ductless glands that produce hormones; products secreted by exocytosis directly into extra cellular space; diverse • Exocrine – secrete products onto body surfaces or into body cavities; products secreted by exocytosis or via epithelial-lined duct that transports secretion to surface

CONNECTIVE TISSUE

• General Characteristics: 1. Common origin – all arise from embryonic tissue (mesenchyme) 2. Degree of vascularity – variable; can be avascular, poorly vascularized or richly vascularized have 3 main elements – ground substance, fibers, and cells • Ground substance = interstitial fluid, cell adhesion proteins, proteoglycans; holds large amounts of fluid and functions as molecular sieve. • Fibers = collagen, elastic, reticular fibers • Cells = fibroblasts (connective tissue proper), chondroblasts (cartilage), osetoblasts (bone), accessory cells (WBCs, mast cells, and macrophages), blood cells 4. Extra cellular matrix – nonliving; separates living cells from non-living; makes tissue more capable of bearing weight and withstanding tension (= ground substance and fibers)

• General functions: 1. Binding and support 2. Protection 3. Insulation 4. Transportation of substances • Specific types of connective tissue: 1. Areolar tissue (loose) – functions include supporting and binding other tissues, holding body fluids, defending against infection, and storing nutrients and water (holds as much water as in bloodstream) • Ground substance is semi fluid or gelatinous • Fibers = all 3 types but loosely arranged • Cells = fibroblasts, macrophages, fat cells, mast cells, WBCs 2. Adipose tissue – functions include providing reserve food fuel, insulation, support and protection of organs; richly vascularized b/c of high metabolic activity • Ground substance is semi fluid or gelatinous, but scanty so cells are packed closely • Fibers = all 3 types but sparsely arranged • Cells = adiposities, fat cells predominate and are packed closely together giving a chicken-wire appearance to tissue; oil droplets occupy most of the volume; 3. Reticular – forms internal framework that can support many free blood cells in lymph nodes, spleen, and bone marrow • Ground substance is loose • Fibers = only reticular fibers • Cells = fibroblasts (reticular cells) are scattered on fiber network 4. Dense fibrous – fibers are primary component of tissue A. dense regular connective tissue – forms tendons (attach muscle to bone), aponeuroses (attach muscle to other muscle or bone), ligaments (bind bones) • Ground substance is scant • Fibers = collagen fibers mainly, some elastic fibers; all run in same direction and in direction of pull • Cells = fibroblasts in rows along fibers B. dense irregular connective tissue – found in areas where tension is exerted in many directions at once, such as skin (dermis), joint capsules, fibrous coverings of testes, kidneys, bones, cartilages, muscles, and nerves • Ground substance is scant • Fibers = thick bundles of collagen fibers, irregularly arranged

• Cells = fibroblasts in rows along fibers 5. Cartilage – avascular and aneural • Ground substance has large amounts of glycosaminoglycans, making it more stiff and lots of tissue fluid (up to 80% water) • Fibers = collagen and elastic fibers • Cells = chondrocytes and chondroblasts (immature cells) A. hyaline cartilage (gristle) – lots of collagen fibers and few chondrocytes; provides firm support and some pliability B. fibro cartilage – rows of chondrocytes alternate with rows of thick collagen fibers; compressible to resist tension C. elastic cartilage – more elastic fibers to that it has both strength and stretchability. 6. Bone – functions in support and protection, provides levers for muscles to act on, stores calcium and other minerals and fats; marrow is site of RBC formation • Matrix is similar to cartilage but more rigid b/c of presence of inorganic calcium salts; salts are deposited on and between fibers • Cells = osteoblasts that produce the organic matrix 7. Blood – functions as transport vehicle for cardiovascular system (carries nutrients, wastes, gases, etc. throughout body) • Matrix is blood plasma (nonliving fluid) + soluble protein molecules that can form fibers (visible only during clotting) • Cells = RBCs

MUSCLE TISSUE

• General characteristics: - Highly cellular - Well-vascularized - possess myofilaments (composed of actin and myosin molecules) that bring about movement • General functions: According to types: 1. Skeletal (voluntary muscle) – packaged by connective tissue into sheet-like organs; have long, cylindrical, multinucleate cells; have striations reflective of alignment of Myofilaments 2. Cardiac (involuntary muscle) – found only in wall of heart; cells are uninucleate and Branching and fit together at junctions called intercalated discs; have striations 3. Smooth (involuntary muscle) – spindle-shaped cells with centrally located nucleus; no obvious striations; found in walls of hollow organs; functions to squeeze substances through organs NERVOUS TISSUE

• General characteristics - makes up brain, spinal cord, and nerves • General functions: 1. Transmit electrical signals from sensory receptors to effectors (muscles and glands) • Cell types: • Neurons – highly specialized nerve cells that generate and conduct nerve impulses; are branching cells

• support cells (neuroglial cells or glial cells) – non-conducting cells that support, insulate and protect neurons. Wound Healing - repair of tissues • Regeneration - destroyed tissues replaced with tissues of same kind • Fibrosis - destroyed tissues replaced by fibrous connective tissue BODY MEMBRANES – comprised of both epithelial and connective tissue A. Cutaneous (skin) – consists of keratinized stratified squamous epithelium attached to a thick layer of dense irregular connective tissue (dermis); is a dry membrane B. Mucous – consists of either stratified squamous or simple columnar epithelium attached to a layer of loose connective tissue called lamina propria, which may in turn sit on a layer of smooth muscle; is a wet membrane (secretes mucus) that lines all body cavities that open to the exterior C. Serous – consists of simple squamous epithelium resting on areolar connective tissue; is a wet membrane (secretes serous fluid) that lines all body cavities (peritoneum, pleural, Pericardial) that are closed to the exterior; has 2 layers • Parietal – pertains to the walls of the cavity (outermost layer) • Visceral – pertains to outer wall covering of internal organ or inner part of structure. B . P lant Tissue s Plants are composed of three major organ groups: roots, stems and leaves. As we know from other areas of biology, these organs are comprised of tissues working together for a common goal (function). In turn, tissues are made of a number of cells which are made of elements and atoms on the most fundamental level. In this section, we will look at the various types of plant tissue and their place and purpose within a plant. It is important to realize that there may be slight variations and modifications to the basic tissue types in special plants. Plant tissues are characterized and classified according to their structure and function. The organs that they form will be organized into patterns within a plant which will aid in further classifying the plant. A good example of this is the three basic tissue patterns found in roots and stems which serve to delineate between woody dicot, herbaceous dicot and monocot plants. We will look at these classifications later on in the tutorial.

Types o f Plant Tissues Examples of tissue in other multicellular organisms are vascular tissue in plants, such as xylem and phloem. Plant tissues are categorized broadly into three tissue systems: the epidermis, the ground tissue, and the vascular tissue. Together they are often referred to as biomass. • • •

Epidermis - Cells forming the outer surface of the leaves and of the young plant body. Vascular tissue - The primary components of vascular tissue are the xylem and phloem. These transport fluid and nutrients internally. Ground tissue - Ground tissue is less differentiated than other tissues. Ground tissue manufactures nutrients by photosynthesis and stores reserve nutrients.

Plant tissues can also be divided differently into two types: 1. MERISTEMATIC TISSUES 2. PERMANENT TISSUES

Meristimatic tissues The growth of plant occurs only in certain specific regions. At these regions, the meristimatic tissues are present. New cells produced by meristem are initially those of meristem itself, but as they grow and mature, their characteristics slowly change and they become differentiated as components of other tissues. Depending on the region of occurrence of meristimatic tissues they are classified as: a) Apical Meristem - It is present at the growing tips of stems and roots and increases the length of the stem and root. The girth of the stem or root increases due to lateral meristem (cambium).This meristem is responsible for the linear growth of an organ. b) Lateral Meristem - This meristem consist of cells which mainly divide in one plane and cause the organ to increase in diameter and girth. Lateral Meristem usually occurs beneath the bark of the tree in the form of Cork Cambium and in vascular bundles of dicots in the form of vascular cambium. The activity of this cambium results in the formation of secondary growth. c) Intercalary Meristem - This meristem is located in between permanent tissues. It is usually present at the base of node, inter node and on leaf base. They are responsible for growth in length of the plant.

Characteristics of meristematic tissues The cells of meristematic tissues are similar in structure and have thin and elastic primary cell wall made up of cellulose. They are compactly arranged without inter molecular spaces between them. Each cell contains a dense cytoplasm and a prominent nucleus. Dense protoplasm of meristematic cells contains very few vacuoles. Normally the meristimatic cells are oval, polygonal or rectangular in shape.

Permanent tissue The meristematic tissues that take up a specific role and lose the ability to divide. This process of taking up a permanent shape, size and a function is called cellular differentiation. Cells of meristematic tissue differentiate to form different types of permanent tissue. There are 2 type of permanent tissues:

Simple permanent tissues These tissues are called simple because they are composed of similar types of cells which have common origin and function. They are further classified into: 1. Parenchyma 2. Collenchyma 3. Sclerenchyma

Parenchyma Parenchyma is Greek word where "parn" means besides and "enchien" means to pour. Parenchyma is the most specialized primitive tissue. It mainly consist of thin-walled cells which have intermolecular spaces between them. The cell wall is made up of cellulose. Each parenchymatous cell is iso-diametric, spherical, or oval in shape. It is widely distributed in various plant organs like root, stem, leaf, flowers and fruits. They mainly occur in cortex epidermis, pith and mesophyll of leaves.

A line drawing of a parenchyma

Collenchyma Collenchyma is Greek word where "Collen" means gum and "enchyma" means infusion. It is a living tissue of primary body like Parenchyma. Cells are thin-walled but possess thickening of cellulose and pectin substances at the corners where number of cells join together. This tissue gives a tensile strength to the plant and the cells are compactly arranged and do not have intermolecular spaces. It occurs chiefly in hypodermis of stems and leaves. It is absent in monocots and in roots. Collenchymatous tissue acts as a supporting tissue in stems of young plants. It provides mechanical support, elasticity, and tensile strength to the plant body. It helps in manufacturing sugar and storing it as starch. It is present in margin of leaves and resist tearing effect of the wind.

Sclerenchyma

Sclerenchyma is Greek word where "Sclrenes" means hard and "enchyma" means infusion. This tissue consists of thick-walled, dead cells. These cells have hard and extremely thick secondary walls due to uniform distribution of lignin. Lignin deposition is so thick that the cell walls become strong, rigid and impermeable to water. Sclerenchymatous cells are closely packed without intra-cellular spaces between them. Thus, they appear as hexagonal net in transverse section. The cells are cemented with the help of lamella. The middle lamella is a wall that lies between adjacent cells. Sclerenchymatous cells mainly occur in hypodermis, pericycle, secondary xylem and phloem. They also occur in endocorp of almond and coconut. It is made of pectin, lignin, protein. The cells of sclerenchyamtous cells can be classified as:

1. Fibres- Fibres are long, elongated sclerenchyamtous cells with pointed ends. 2. Sclerides- Sclerenchymatous cells which are short and possess extremely thick, lamellated, lignified walls with long singular piths. 3. They are called sclerides. The main function of Sclerenchymatous tissues is to give support to the plant.

A line drawing of a sclerenchyma cell.

Complex permanent tissue A complex permanent tissue may be classified as a group of more than one type of tissue having a common origin and working together as a unit to perform a function. These tissues are concerned with transportation of water, mineral, nutrients and organic substances. The important complex tissues in vascular plants are xylem, phloem.

Xylem Xylem is a chief, conducting tissue of vascular plants. It is responsible for conduction of water and inorganic solutes. 1. Tracheids- Trachids are elongated, tube-like dead cells with elongated end-walls. End walls remain intact and possess piths. In transverse section, They usually occur as polygonal cells and lignified walls. 2. Vessels - Vessels are placed one upon another. Their end walls are perforated. They form long tubes or channels for conduction of water and minerals. 3. Xylem Parenchyma - Xylem Parenchymatous cells are living cells present in xylem. They help in lateral conduction of organic solutes and storage reserves. 4. Xylem Fibres - Xylem Fibres are lignified fibres present in xylem which provide mechanical strength to the plant body. Xylem is a major conducting tissue of vascular plants. It serves in upward movement of water and minerals from root to different parts of plant.

Phloem Phloem is a chief conducting tissue of vascular plants. It is regarded as a living tissue responsible for translocation of organic solutes. 1. Sieve tube - Sieve tubes are long tubular structures composed of elongated sieve tube elements placed one above other forming a continuous tube. 2. Companion cell - Companion cells are living cells always associated with sieve tubes. Sieve tube elements and companion cells arrive from the same, initial cell and therefore forms a single functional unit. Each companion cell shows presence of fine piths with all the living components of the cell. 3. Phloem Parenchyma - These cells are living parenchymatous cells that are present in phloem. These cells help in storage of food. 4. Phloem Fibres - Phloem fibres are formed by dead, sclerenchymatous fibres. The main function of phloem is translocation of organic solutes from the leaves to the storage organ and later from the storage organ to the growing part. Sieve tube allow free diffusion of soluble, organic substances across sieve plates due to the presence of large number of sieve pores.