10.1 Plant microscopy and histology

Plant microscopy and histology are essential tools for understanding plant structure and function. These techniques allow scientists to examine the intricate details of plant cells, tissues, and organs at various magnifications, revealing their complex organization and relationships.

From light microscopy to electron microscopy, researchers use a variety of methods to study plant anatomy. These techniques, combined with specialized tissue preparation and staining, provide insights into plant cell ultrastructure, tissue systems, and growth processes, enhancing our knowledge of plant biology.

Microscopy in plant biology

• Microscopy is a crucial tool in plant biology that allows researchers to study the intricate structures and processes within plants at various magnifications
• Different microscopy techniques, such as light and electron microscopy, provide complementary information about plant cells, tissues, and organs
• Advances in microscopy have greatly expanded our understanding of plant anatomy, physiology, and development

Light microscopy techniques

Bright field microscopy

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• Most basic and widely used form of light microscopy
• Specimens appear dark against a bright background
• Useful for observing general plant cell and tissue morphology (leaf cross-sections)
• Limited resolution and contrast compared to other techniques

Dark field microscopy

• Illuminates specimens from the side, causing them to appear bright against a dark background
• Enhances contrast for unstained or low-contrast samples (plant fibers)
• Useful for visualizing small, transparent structures

Phase contrast microscopy

• Converts phase shifts in light passing through a specimen into brightness differences
• Improves contrast without staining, particularly for live, unstained cells (algal cells)
• Reveals internal cell structures and organelles

Fluorescence microscopy

• Uses fluorescent dyes or proteins to label specific structures or molecules within cells
• Allows for selective visualization of targeted components (chloroplasts, cell walls)
• Enables the study of dynamic processes and interactions within living cells

Confocal laser scanning microscopy

• Optical sectioning technique that eliminates out-of-focus light, providing high-resolution 3D images
• Allows for the reconstruction of detailed 3D structures (plant vascular systems)
• Commonly used with fluorescent labeling for precise localization of cellular components

Electron microscopy techniques

Scanning electron microscopy (SEM)

• Uses a focused beam of electrons to scan the surface of a specimen, generating high-resolution 3D images
• Reveals surface topography and morphology of plant structures (leaf stomata, pollen grains)
• Requires special sample preparation, including fixation, dehydration, and coating

Transmission electron microscopy (TEM)

• Passes a beam of electrons through ultra-thin sections of a specimen, providing high-resolution 2D images
• Reveals internal ultrastructure of plant cells and organelles (thylakoid membranes, cell wall layers)
• Requires extensive sample preparation, including fixation, embedding, and sectioning

Plant tissue preparation

Fixation and embedding

• Fixation preserves plant tissues by cross-linking proteins and stabilizing cellular structures
• Common fixatives include formaldehyde, glutaraldehyde, and osmium tetroxide
• Embedding involves infiltrating fixed tissues with a supportive medium (paraffin wax, resin) for sectioning

Sectioning techniques

• Sectioning allows for the preparation of thin, uniform slices of plant tissue for microscopic examination
• Paraffin-embedded tissues are typically sectioned using a microtome
• Resin-embedded tissues are sectioned using an ultramicrotome for electron microscopy

Staining methods

• Staining enhances contrast and selectively highlights specific structures or components within plant cells and tissues
• Common stains include safranin, fast green, and toluidine blue for light microscopy
• Electron-dense stains, such as uranyl acetate and lead citrate, are used for electron microscopy

Plant cell ultrastructure

Cell wall composition and layers

• Plant cell walls are composed primarily of cellulose, hemicellulose, and pectin
• Primary cell walls are thin and flexible, allowing for cell growth and expansion
• Secondary cell walls are thicker and more rigid, providing structural support and specialized functions (xylem vessels)

Plasma membrane structure

• The plasma membrane is a selectively permeable barrier that controls the movement of substances in and out of the cell
• Composed of a phospholipid bilayer with embedded proteins
• Plays a crucial role in cell signaling, transport, and cell-to-cell communication

Cytoplasmic organelles

• Chloroplasts are the site of photosynthesis, containing chlorophyll and thylakoid membranes
• Mitochondria are responsible for cellular respiration and energy production
• Endoplasmic reticulum and Golgi apparatus are involved in protein synthesis, modification, and transport

Nucleus and chromosomes

• The nucleus contains the cell's genetic material (DNA) organized into chromosomes
• Nuclear envelope, with its pores, regulates the movement of molecules between the nucleus and cytoplasm
• Nucleolus is the site of ribosomal RNA synthesis and ribosome assembly

Plant tissue systems

Dermal tissue system

• Consists of the epidermis and periderm, which cover and protect the plant body
• Epidermis is a single layer of cells that secretes a waxy cuticle to prevent water loss
• Periderm replaces the epidermis in woody plants, providing protection and gas exchange

Ground tissue system

• Comprises the majority of the plant body, including parenchyma, collenchyma, and sclerenchyma cells
• Parenchyma cells are living, thin-walled cells involved in various metabolic functions and storage
• Collenchyma and sclerenchyma provide mechanical support

Vascular tissue system

• Consists of xylem and phloem tissues, which transport water, nutrients, and photosynthates throughout the plant
• Xylem conducts water and dissolved minerals from roots to leaves
• Phloem transports sugars and other organic compounds from leaves to other parts of the plant

Meristematic tissues

Apical meristems

• Located at the tips of roots and shoots, responsible for primary growth and elongation
• Consist of rapidly dividing, undifferentiated cells that give rise to primary tissues
• Examples include the root apical meristem and shoot apical meristem

Lateral meristems

• Found along the sides of stems and roots, responsible for secondary growth and thickening
• Vascular cambium produces secondary xylem and phloem
• Cork cambium (phellogen) produces the periderm

Intercalary meristems

• Located at the base of internodes in monocot stems (grasses)
• Allow for rapid elongation of internodes and regrowth after damage
• Contribute to the growth of leaves and other organs in some plants

Permanent tissues

Parenchyma

• Most abundant and versatile plant cell type, found in various tissues and organs
• Living cells with thin cell walls, involved in photosynthesis, storage, and secretion
• Contain large vacuoles for storage of water, nutrients, and waste products

Collenchyma

• Living cells with unevenly thickened primary cell walls, providing structural support
• Commonly found in young, growing parts of the plant (petioles, stems)
• Elongated cells arranged in strands or sheets

Sclerenchyma

• Dead cells with heavily thickened, lignified secondary cell walls, providing mechanical support
• Two main types: fibers and sclereids
• Fibers are long, slender cells found in bundles (phloem fibers, xylem fibers)
• Sclereids are shorter, irregular-shaped cells found in various tissues (nut shells, seed coats)

Epidermis and periderm

Cuticle and wax layers

• Cuticle is a waxy, water-resistant layer secreted by epidermal cells
• Helps prevent water loss, protects against pathogens, and reduces UV damage
• Composition and thickness vary among plant species and organs

Stomata and guard cells

• Stomata are pores in the epidermis that allow for gas exchange and transpiration
• Guard cells are specialized epidermal cells that regulate the opening and closing of stomata
• Stomatal movements are controlled by changes in turgor pressure within guard cells

Trichomes and emergences

• Trichomes are hair-like outgrowths of the epidermis, serving various functions (protection, water absorption, secretion)
• Glandular trichomes secrete essential oils, resins, or digestive enzymes (sundew plants)
• Emergences are multicellular outgrowths that involve both epidermal and subepidermal tissues (thorns, prickles)

Cork and lenticels

• Cork (phellem) is a layer of dead, suberized cells produced by the cork cambium
• Provides protection, insulation, and reduces water loss in woody plants
• Lenticels are raised pores in the periderm that allow for gas exchange

Primary vs secondary growth

• Primary growth originates from apical meristems and results in the elongation of roots and shoots
• Involves the formation of primary tissues (epidermis, ground tissue, vascular tissue)
• Secondary growth originates from lateral meristems and results in the thickening of stems and roots
• Involves the formation of secondary tissues (secondary xylem, secondary phloem, periderm)

Xylem tissue

Tracheids and vessel elements

• Tracheids are elongated, tapered cells with lignified secondary walls and pits for water transport
• Vessel elements are shorter, wider cells arranged end-to-end to form continuous tubes (vessels)
• Vessel elements have perforated end walls, allowing for more efficient water transport

Primary vs secondary xylem

• Primary xylem develops from the procambium during primary growth
• Consists of protoxylem (first-formed) and metaxylem (later-formed)
• Secondary xylem develops from the vascular cambium during secondary growth
• Includes axial and radial systems (fibers, tracheids, vessel elements, parenchyma, rays)

Xylem development and differentiation

• Xylem cells undergo a series of changes during differentiation, including cell elongation, secondary wall thickening, and programmed cell death
• Lignification of secondary walls provides mechanical support and waterproofing
• Bordered pits in cell walls allow for lateral water transport between xylem elements

Phloem tissue

Sieve tube elements and companion cells

• Sieve tube elements are living, elongated cells that form continuous tubes for translocation of sugars and other organic compounds
• Companion cells are specialized parenchyma cells closely associated with sieve tube elements
• Companion cells provide metabolic support and help load and unload substances from sieve tube elements

Primary vs secondary phloem

• Primary phloem develops from the procambium during primary growth
• Consists of protophloem (first-formed) and metaphloem (later-formed)
• Secondary phloem develops from the vascular cambium during secondary growth
• Includes axial and radial systems (sieve tube elements, companion cells, parenchyma, fibers, rays)

Phloem development and differentiation

• Phloem cells differentiate from meristematic cells in a similar manner to xylem cells
• Sieve tube elements lose their nucleus and most organelles during differentiation
• Sieve plates with pores develop at the end walls of sieve tube elements, allowing for continuous flow of phloem sap
• P-proteins and callose accumulate in sieve plates, helping to regulate flow and seal off damaged sieve tube elements