Protoplast
Encyclopedia
- This article relates to protoplasts as a term in biology. For the theological use, see Adam and EveAdam and EveAdam and Eve were, according to the Genesis creation narratives, the first human couple to inhabit Earth, created by YHWH, the God of the ancient Hebrews...
.
Protoplast, from the ancient Greek
Ancient Greek
Ancient Greek is the stage of the Greek language in the periods spanning the times c. 9th–6th centuries BC, , c. 5th–4th centuries BC , and the c. 3rd century BC – 6th century AD of ancient Greece and the ancient world; being predated in the 2nd millennium BC by Mycenaean Greek...
πρῶτον (first) + verb πλάθω or πλάττω (to mould: deriv. plastic), initially referred to the first organized body of a species.
Protoplast has several biological definitions:
- A protoplast is a plantPlantPlants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
, bacterial or fungalFungusA fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria...
cell that had its cell wallCell wallThe cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
completely or partially removed using either mechanical or enzymatic means.- Protoplasts - Have their cell wall entirely removed
- SpheroplastSpheroplastA spheroplast is a cell from which the cell wall has been almost completely removed, as by the action of penicillin. The name stems from the fact that after a microbe's cell wall is digested, membrane tension causes the cell to acquire a characteristic spherical shape...
s - Have their cell wall only partially removed
- More generally protoplast refers to that unit of biology which is composed of a cell's nucleusCell nucleusIn cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
and the surrounding protoplasmProtoplasmProtoplasm is the living contents of a cell that is surrounded by a plasma membrane. It is a general term of the Cytoplasm . Protoplasm is composed of a mixture of small molecules such as ions, amino acids, monosaccharides and water, and macromolecules such as nucleic acids, proteins, lipids and...
ic materials.
Enzymes for the preparation of protoplasts
Cell walls are made of a variety of polysaccharidePolysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...
s. Protoplasts can be made by degrading cell walls with a mixture of the appropriate polysaccharide-degrading enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s:
| Type of cell | Enzyme |
|---|---|
| Plant cell Plant cell Plant cells are eukaryotic cells that differ in several key respects from the cells of other eukaryotic organisms. Their distinctive features include:... s |
Cellulase Cellulase 400px|thumb|right|alt = Colored dice with checkered background|Ribbon representation of the Streptomyces lividans beta-1,4-endoglucanase catalytic domain - an example from the family 12 glycoside hydrolases... , pectinase Pectinase Pectinase is a general term for enzymes, such as pectolyase, pectozyme and polygalacturonase, commonly referred to in brewing as pectic enzymes. These break down pectin, a polysaccharide substrate that is found in the cell walls of plants. One of the most studied and widely used commercial... , xylanase Xylanase Xylanase is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls.... |
| Gram-positive Gram-positive Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink... bacteria |
Lysozyme Lysozyme Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases, enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between... (+EDTA EDTA Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand... ) |
| Fungal Fungus A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria... cells |
Chitinase Chitinase Chitinases are hydrolytic enzymes that break down glycosidic bonds in chitin. As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals , chitinases are generally found in organisms that either need to reshape their own chitin or dissolve and digest the chitin of... |
During and subsequent to digestion of the cell wall, the protoplast becomes very sensitive to osmotic
Osmosis
Osmosis is the movement of solvent molecules through a selectively permeable membrane into a region of higher solute concentration, aiming to equalize the solute concentrations on the two sides...
stress. This means cell wall digestion and protoplast storage must be done in an isotonic solution to prevent rupture of the plasma membrane.
Uses for protoplasts
Protoplasts can be used to study membraneBiological membrane
A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...
biology, including the uptake of macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s and virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es.
Protoplasts are widely used for DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
(for making genetically modified organism
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
s), since the cell wall would otherwise block the passage of DNA into the cell. In the case of plant cells, protoplasts may be regenerated into whole plants first by growing into a group of plant cells that develops into a callus
Callus (cell biology)
Plant callus is a mass of undifferentiated cells derived from plant tissue for use in biological research and biotechnology. In plant biology, callus cells are those cells that cover a plant wound. To induce callus formation, plant tissues are surface sterilized and then plated onto in vitro...
and then by regeneration of shoots (caulogenesis) from the callus using plant tissue culture
Plant tissue culture
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation...
methods. Growth of protoplasts into callus and regeneration of shoots requires the proper balance of plant growth regulators in the tissue culture medium that must be customized for each species of plant.
Unlike protoplasts from vascular plant
Vascular plant
Vascular plants are those plants that have lignified tissues for conducting water, minerals, and photosynthetic products through the plant. Vascular plants include the clubmosses, Equisetum, ferns, gymnosperms and angiosperms...
s, protoplasts from moss
Moss
Mosses are small, soft plants that are typically 1–10 cm tall, though some species are much larger. They commonly grow close together in clumps or mats in damp or shady locations. They do not have flowers or seeds, and their simple leaves cover the thin wiry stems...
es, such as Physcomitrella patens
Physcomitrella patens
Physcomitrella patens is a moss used as a model organism for studies on plant evolution, development and physiology.-Model organism:...
, do not need phytohormones for regeneration, nor do they form a callus during regeneration
Regeneration (biology)
In biology, regeneration is the process of renewal, restoration, and growth that makes genomes, cells, organs, organisms, and ecosystems resilient to natural fluctuations or events that cause disturbance or damage. Every species is capable of regeneration, from bacteria to humans. At its most...
. Instead, they regenerate directly into the filamentous protonema
Protonema
A protonema is a thread-like chain of cells that forms the earliest stage of a bryophyte life cycle...
mimicking a germinating moss spore.
Protoplasts may also be used for plant breeding
Plant breeding
Plant breeding is the art and science of changing the genetics of plants in order to produce desired characteristics. Plant breeding can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics for propagation, to more complex molecular...
, using a technique called protoplast fusion. Protoplasts from different species are induced to fuse by using an electric field
Electric field
In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...
or a solution of polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
. Originally, non Ti-plasmid dependent DNA uptake has been demonstrated 1985 by R. Hain and A.P. Czernilofsky et al., entitled "uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts" using the Ca-phosphate coprecipitation technique (Mol Gen Genet, 199:161-168, 1985). This technique may be used to generate somatic hybrids in tissue culture.