Plant tissue culture
Encyclopedia
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. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:
Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplast
s), pieces of leaves, or (less commonly) roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.
conditions under filtered air. Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (explants) in chemical solutions (usually Sodium or calcium hypochlorite or mercuric chloride) is required. Mercuric chloride is seldom used as a plant sterilant today,unless other sterilizing agents are found to be ineffective, as it is dangerous to use, and is difficult to dispose of. Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media
are prepared from liquid media with the addition of a gelling agent, usually purified agar.
The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin
will often result in a proliferation of roots, while an excess of cytokinin
may yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells, or callus
, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard.
As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.
The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins.
The pathways through which whole plants are regenerated from cells and tissues or explants such as meristems broadly fall into three types:
The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.
Tissue cultured plants are clones, if the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem, conversely any positive traits would remain within the line also.
- The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.
- To quickly produce mature plants.
- The production of multiples of plants in the absence of seedSeedA seed is a small embryonic plant enclosed in a covering called the seed coat, usually with some stored food. It is the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant...
s or necessary pollinatorsPollinationPollination is the process by which pollen is transferred in plants, thereby enabling fertilisation and sexual reproduction. Pollen grains transport the male gametes to where the female gamete are contained within the carpel; in gymnosperms the pollen is directly applied to the ovule itself...
to produce seedSeedA seed is a small embryonic plant enclosed in a covering called the seed coat, usually with some stored food. It is the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant...
s. - The regeneration of whole plants from plant cells that have been genetically modifiedGenetic engineeringGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
. - The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.
- The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthesNepenthesThe Nepenthes , popularly known as tropical pitcher plants or monkey cups, are a genus of carnivorous plants in the monotypic family Nepenthaceae. The genus comprises roughly 130 species, numerous natural and many cultivated hybrids...
. - To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticultureHorticultureHorticulture is the industry and science of plant cultivation including the process of preparing soil for the planting of seeds, tubers, or cuttings. Horticulturists work and conduct research in the disciplines of plant propagation and cultivation, crop production, plant breeding and genetic...
and agriculture.
Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplast
Protoplast
Protoplast, from the ancient Greek πρῶτον + verb πλάθω or πλάττω , initially referred to the first organized body of a species.Protoplast has several biological definitions:...
s), pieces of leaves, or (less commonly) roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.
Techniques
Modern plant tissue culture is performed under asepticAseptic technique
Aseptic technique refers to a procedure that is performed under sterile conditions. This includes medical and laboratory techniques, such as with microbiological cultures. It includes techniques like flame sterilization...
conditions under filtered air. Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (explants) in chemical solutions (usually Sodium or calcium hypochlorite or mercuric chloride) is required. Mercuric chloride is seldom used as a plant sterilant today,unless other sterilizing agents are found to be ineffective, as it is dangerous to use, and is difficult to dispose of. Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
are prepared from liquid media with the addition of a gelling agent, usually purified agar.
The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin
Auxin
Auxins are a class of plant hormones with some morphogen-like characteristics. Auxins have a cardinal role in coordination of many growth and behavioral processes in the plant's life cycle and are essential for plant body development. Auxins and their role in plant growth were first described by...
will often result in a proliferation of roots, while an excess of cytokinin
Cytokinin
Cytokinins are a class of plant growth substances that promote cell division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth, and leaf senescence...
may yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells, or 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...
, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard.
As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.
Choice of explant
The tissue obtained from the plant to culture is called an explant. Based on work with certain model systems, particularly tobacco, it has often been claimed that a totipotent explant can be grown from any part of the plant. However, this concept has been vitiated in practice. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture.The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins.
The pathways through which whole plants are regenerated from cells and tissues or explants such as meristems broadly fall into three types:
- The method in which explants that include a meristem (viz. the shoot tips or nodes) are grown on appropriate media supplemented with plant growth regulators to induce proliferation of multiple shoots, followed by rooting of the excised shoots to regenerate whole plants,
- The method in which totipotency of cells is realized in the form of de novo organogenesis, either directly in the form of induction of shoot meristems on the explants or indirectly via a callus ( unorganised mass of cells resulting from proliferation of cells of the explant) and plants are regenerated through induction of roots on the resultant shoots,
- Somatic embryogenesis, in which asexual adventive embryos( comparable to zygotic embryos in their structure and development) are induced directly on explants or indirectly through a callus phase.
The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.
Tissue cultured plants are clones, if the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem, conversely any positive traits would remain within the line also.
Applications
Plant tissue culture is used widely in plant science; it also has a number of commercial applications. Applications include:- Micropropagation is widely used in forestryForestryForestry is the interdisciplinary profession embracing the science, art, and craft of creating, managing, using, and conserving forests and associated resources in a sustainable manner to meet desired goals, needs, and values for human benefit. Forestry is practiced in plantations and natural stands...
and in floriculture. Micropropagation can also be used to conserveConservation biologyConservation biology is the scientific study of the nature and status of Earth's biodiversity with the aim of protecting species, their habitats, and ecosystems from excessive rates of extinction...
rare or endangeredEndangered speciesAn endangered species is a population of organisms which is at risk of becoming extinct because it is either few in numbers, or threatened by changing environmental or predation parameters...
plant species. - A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicideHerbicideHerbicides, also commonly known as weedkillers, are pesticides used to kill unwanted plants. Selective herbicides kill specific targets while leaving the desired crop relatively unharmed. Some of these act by interfering with the growth of the weed and are often synthetic "imitations" of plant...
resistance/tolerance. - Large-scale growth of plant cells in liquid culture in bioreactorBioreactorA bioreactor may refer to any manufactured or engineered device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This...
s for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticalBiopharmaceuticalBiopharmaceuticals are medical drugs produced using biotechnology. They include proteins , nucleic acids and living microorganisms like virus and bacteria where the virulence of viruses and bacteria is reduced by the process of attenuation, they can be used for therapeutic or in vivo diagnostic...
s. - To cross distantly related speciesSpeciesIn biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
by protoplast fusionSomatic fusionSomatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between the different varieties of...
and regeneration of the novel hybrid. - To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (EmbryoEmbryoAn embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
Rescue). - For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicineColchicineColchicine is a medication used for gout. It is a toxic natural product and secondary metabolite, originally extracted from plants of the genus Colchicum...
which causes doubling of the chromosomeChromosomeA chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
number. - As a tissue for transformation, followed by either short-term testing of geneticGenetic engineeringGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
constructs or regeneration of transgenicTransgenesisthumb|300px|right|A diagram comparing the genetic changes achieved through conventional plant breeding, transgenesis and cisgenesisTransgenesis is the process of introducing an exogenous gene – called a transgene – into a living organism so that the organism will exhibit a new property and transmit...
plants. - Certain techniques such as meristemMeristemA meristem is the tissue in most plants consisting of undifferentiated cells , found in zones of the plant where growth can take place....
tip culture can be used to produce clean plant material from virusVirusA 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...
ed stock, such as potatoPotatoThe potato is a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family . The word potato may refer to the plant itself as well as the edible tuber. In the region of the Andes, there are some other closely related cultivated potato species...
es and many species of soft fruit. - micropropagation using meristem and shoot culture to produce large numbers of identical individuals.
- production of identical sterile hybrid species can be obtained