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: 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 seeds or necessary pollinators to produce seeds.
The regeneration of whole plants from plant cells that have been genetically modified.
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 Nepenthes. To clean particular plants of viral and other
infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.
Plant tissue culture relies on the fact that many plant cells have the ability to regenerate
a whole plant. Single cells, plant cells without cell walls, pieces of leaves, stems or 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
aseptic conditions under HEPA filtered air provided by a laminar flow cabinet. Living
plant materials from the environment are naturally contaminated on their surfaces with microorganisms,
so surface sterilization of starting material in chemical solutions (usually alcohol and
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
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 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. Regeneration Pathways
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. Shoot regeneration
efficiency in tissue culture is usually a quantitative trait that often varies between
plant species and within a plant species among subspecies, varieties, cultivars, or ecotypes.
Therefore, tissue culture regeneration can become complicated especially when many regeneration
procedures have to be developed for different genotypes within the same species.
The three common pathways of plant tissue culture regeneration are;
(i) propagation from preexisting meristems (ii) organogenesis
(iii) non-zygotic embryogenesis The propagation of shoots or nodal segments
is usually performed in four stages for mass production of plantlets through in vitro vegetative
multiplication but organogenesis is a common method of micropropagation that involves tissue
regeneration of adventitious organs or axillary buds directly or indirectly from the explants.
Non-zygotic embryogenesis is a noteworthy developmental pathway that is highly comparable
to that of zygotic embryos and it is an important pathway for producing somaclonal variants,
developing artificial seeds, and synthesizing metabolites. Due to the single cell origin
of non-zygotic embryos, they are preferred in several regeneration systems for micropropagation,
ploidy manipulation, gene transfer, and synthetic seed production. Nonetheless, tissue regeneration
via organogenesis has also proved to be advantageous for studying regulatory mechanisms of plant
development. Choice of explant
The tissue obtained from a plant to be cultured is called an explant. Based on work with certain
plants, particularly tobacco, it has often been claimed that a totipotent explant can
be taken from any part of a plant including portions of shoots, leaves, stems, flowers,
roots and single, undifferentiated cells.,. However this is not true for all plants. 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.
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 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. Some cultured tissues are slow in their growth.
For them there would be two options: Optimizing the culture medium; Culturing highly responsive
tissues or varieties. Necrosis can spoil cultured tissues. Generally, plant varieties differ
in susceptibility to tissue culture necrosis. Thus, by culturing highly responsive varieties
it can be managed. Aerial 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 the plant sciences, forestry, and in horticulture.
Applications include: The commercial production of plants used as
potting, landscape, and florist subjects, which uses meristem and shoot culture to produce
large numbers of identical individuals. To conserve rare or endangered plant species.
A plant breeder may use tissue culture to screen cells rather than plants for advantageous
characters, e.g. herbicide resistance/tolerance. Large-scale growth of plant cells in liquid
culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites
and recombinant proteins used as biopharmaceuticals. To cross distantly related species by protoplast
fusion and regeneration of the novel hybrid. To rapidly study the molecular basis for physiological,
biochemical, and reproductive mechanisms in plants, for example in vitro selection for
stress tolerant plants, and in vitro flowering studies.
To cross-pollinate distantly related species and then tissue culture the resulting embryo
which would otherwise normally die. For chromosome doubling and induction of polyploidy,
for example doubled haploids, tetraploids, and other forms of polyploids. This is usually
achieved by application of antimitotic agents such as colchicine or oryzalin.
As a tissue for transformation, followed by either short-term testing of genetic constructs
or regeneration of transgenic plants. Certain techniques such as meristem tip culture
can be used to produce clean plant material from virused stock, such as potatoes and many
species of soft fruit. Production of identical sterile hybrid species
can be obtained. Laboratories
Although some growers and nurseries have their own labs for propagating plants by the technique
of tissue culture, a number of independent laboratories provide custom propagation services.
The Plant Tissue Culture Information Exchange lists many commercial tissue culture labs.
Since plant tissue culture is a very labour-intensive process, this would be an important factor
in determining which plants would be commercially viable to propagate in a laboratory.
See also Hairy root culture
Gottlieb Haberlandt, pioneer of plant tissue culture
Frederick Campion Steward, pioneer and 'champion' of plant tissue culture.
Murashige and Skoog medium, an important plant growth medium
Plant physiology References
George, Edwin F.; Hall, Michael A.; De Klerk, Geert-Jan, ed.. Plant propagation by tissue
culture. Volume 1. The background. Dordrecht: Springer. ISBN 978-1-4020-5004-6.
Yadav, R.; Arora, P.; Kumar, D.; Katyal, D.; Dilbaghi, N.; Chaudhury, A.. "High frequency
direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood".
Plant Biotechnology Reports 3: 175–182. doi:10.1007/s11816-009-0088-5.
Singh, S.K.; Srivastava, S.. Plant Tissue Culture. Campus Book International. ISBN 978-81-8030-123-0.