culture is the in vitro aseptic culture of cells, tissues, organs or
whole plants under controlled nutritional and environmental conditions often to
produce the clones of plants.
tissue culture technology is being widely used for large scale plant
from their use as a tool of research, plant tissue culture techniques have in
recent years, become a major industrial importance in the areas of plant
propagation, disease elimination, plant improvement and production of secondary
metabolites. Small pieces of tissues (explants) are used to produce hundreds
and thousands of plants in a continuous process. A single explant is multiplied
into several thousand plants in a relatively short time period and space under
controlled conditions, irrespective of the season and weather on a year-round
basis. Endangered, threatened and rare species are grown and conserved by
micropropagation because of their high coefficient of multiplication and the
small demands on number of initial plants and space.
controlled conditions provide the culture an environment conducive for their
growth and multiplication. These conditions include proper supply of nutrients,
pH medium, adequate temperature and proper gaseous and liquid environment. (Hussain et al., 2012)
culture may be defined as the development of new plants from a part of a plant,
in an artificial nutrient medium under aseptic conditions. A part of a plant
may be a cell (e.g. pollen), tissue (e.g. meristems) or organ (e.g. axillary
bud, apical bud).
basis of cell, tissue or organ culture is the totipotency of plant cells.
Totipotency means that any living somatic cell can produce a plant similar to
the mother plant. Based on this understanding of plant cells, thousands of
plants can be produced through tissue culture under appropriate conditions.
of plant tissue culture takes its roots from the discovery of the cell followed
by propounding of cell theory. In 1838, Schleiden and Schwann proposed that the
cell is the basic structural unit of all living organisms. They visualized that
the cell is capable of autonomy and therefore it should be possible for each
cell to regenerate into a whole plant in a suitable environment. Based on this
premise, in 1902, a German physiologist, Gottlieb Haberlandt for the first time
attempted to culture isolated single
palisade cells from leaves in knop’s salt solution enriched with sucrose. The
cells increased in size, accumulated starch but failed to divide, hence only
remained alive for about one month. Though he was unsuccessful, he laid down
the foundation of tissue culture technology for which he is regarded as the
father of plant tissue culture. Some landmark discoveries in tissue culture
that took place are summarized below:
originated the concept of cell culture, attempted to cultivate isolated plant
cells in vitro on an artificial medium for the first time.
1934 – White generated a continuously
growing culture of meristematic cells of tomato root on a medium containing
salt, yeast extract, sucrose and vitamin B (pyridoxine, thiamine, nicotinic
acid), established the importance of additives.
1939- Goutheret and Nobecourt performed tissue
culture using explants from cambial tissues of carrots and tobacco tumors.
1943- Phillip R White published the first book
on plant tissue culture.
Dr Skoog and his group
identified this as a cytokinin from the effect of degraded DNA preparation on
cell proliferation and named it kinetin. Later another cytokinin, zeatin was
discovered. Many of the early investigators employed either woody or herbaceous
dicot tissues as source of explant.
1950- Miller and Skoog discovered kinetin, a
cytokine that plays an active role in organogenesis.
and Martin generated virus free plants using axillary buds.
successfully cultured monocot tissues with the aid of coconut water, introduced
tissue culture technology to orchid industry
and produced disease-free plants of grape.
and Maheshwari showed that cultured pollen and microsporogenous tissues of
anthers have the potential to produce vast number of haploid embryos.
and Skoog published MS Medium for tissue culture.
1971 International Association for Plant Tissue
Culture (IAPTC) was established and was renamed in 1998 as International Association
for Plant Tissue Culture and Biotechnology (IAPTC).
1989 Genetic transformation of plants became
successful after the discovery of Ti plasmid in crown gall bacterium Agrobacterium tumefaciens. This led to
the development of plants with transformed genes to acquire resistance for
viruses (Hussain et al., 2012)
mass propagation of desirable varieties, to propagate plants that are very
difficult to propagate by cuttings or other traditional methods / and as a
rapid method of propagation
the last 30 years, it has become possible to regenerate plantlets from explants
and/or callus from all types of plants. As a result, laboratory-scale
micropropagation protocols are available for a wide range of species and at
present micropropagation is the widest use of plant tissue-culture technology.
The cost of the labor needed to transfer tissue repeatedly between vessels and
the need for asepsis can account for up to 70% of the production costs of
micropropagation. Problems of vitrification, acclimatization and contamination
can cause great losses in a tissue-culture laboratory. Genetic variations in
cultured lines, such as polyploidy, aneuploidy and mutations, have been
reported in several systems and resulted in the loss of desirable economic
traits in the tissue-cultured products.
are three methods used for micropropagation:, Enhancing axillary-bud breaking. Production
of adventitious buds and Somatic embryogenesis.
latter two methods, organized structures arise directly on the explant or
indirectly from callus.
breaking produces the least number of plantlets, as the number of shoots
produced is controlled by the number of axillary buds cultured, but remains the
most widely used method in commercial micropropagation and produces the most
true to- type plantlets. Adventitious budding has a greater potential for
producing plantlets, as bud primordia may be formed on any part of the
inoculum. Unfortunately, somatic embryogenesis, which has the potential of
producing the largest number of plantlets, can only presently be induced in a
few species (Ajnabi, 2012).
doubled haploids and haploids
plants are of interest to plant breeders because they allow the expression of
simple recessive genetic traits or mutated recessive genes and because doubled
haploids can be used immediately as homozygous breeding lines. The efficiency
in producing homozygous breeding lines via doubled in vitro-produced
haploids represents significant savings in both time and cost compared with
other methods. Three in vitro methods were used to generate haploids
(1) Culture of excised ovaries and ovules;
(2) The bulbosum technique of embryo culture;
(3) Culture of excised anthers and pollen.
171 plant species are used to produce haploid plants by pollen, microspore and
anther culture. These include cereals (barley, maize, rice, rye, triticale and
wheat), forage crops (alfalfa and clover), fruits (grape and strawberry),
medicinal plants (Digitalis and Hyoscyamus), ornamentals (Gerbera and sunflower), oil seeds
(canola and rape), trees (apple, litchi, poplar and rubber), plantation crops
(cotton, sugar cane and tobacco), and vegetable crops (asparagus, brussels
sprouts, cabbage, carrot, pepper, potato, sugar beet, sweet potato, tomato and
wing bean) Purohit, 2012).
produce new plant varieties by wide hybridization
requirement for crop improvement is the introduction of new genetic material into
the cultivated lines of interest. It could be via single genes, multiple genes,
genetic engineering, conventional hybridization or tissue-culture techniques.
During fertilization in angiosperms, pollen grains reach the stigma of the host
plant, germinate and produce a pollen tube. The pollen tube penetrates through
the stigma and style and reaches the ovule. The discharge of sperm within the
female gametophyte triggers syngamy and the two sperm nuclei fuse with their
respective partners. The egg nucleus and fusion nucleus then form a developing
embryo and the nutritional endosperm, respectively. This process can be blocked
at any number of stages, resulting in a functional barrier to hybridization and
the blockage of gene transfer between the two plants.
barriers to hybridization (barriers prior to fertilization), such as the
failure of pollen to germinate or poor pollen-tube growth, may be overcome
using in vitro fertilization. Post-zygotic barriers (barriers after
fertilization), such as lack of endosperm development, may be overcome by
embryo, ovule or pod culture. Protoplast fusion was successful in producing the
desired hybrids where fertilization could not be induced by in vitro
treatments. In vitro fertilization (IVF) was used to facilitate both
interspecific and intergeneric crosses, to overcome physiological-based
self-incompatibility and to produce hybrids. A wide range of plant species were
recovered through IVF via pollination of pistils and self and cross-pollination
of ovules. This range includes agricultural crops, such as tobacco, clover,
com, rice, cole, canola, poppy and cotton. The use of delayed pollination,
distant hybridization, pollination with abortive or irradiated pollen, and
physical and chemical treatment of the host ovary were used to induce haploidy
1-fused cell 2-Dividing hybrid cell, 3-cell
cluster of hybrid 4-shoot formation from hybrid callus 5-regenerated somatic
(source: Terada et al 1987)
For virus eradication, genetic manipulation, somatic hybridization and other
procedures that benefit propagation, plant improvement, and basic research.
plants, especially vegetatively propagated varieties, are generally infected
with pathogens. Strawberry plants are susceptible to over 60 viruses and
mycoplasmas and this often necessitates the yearly replacement of mother plants.
In many cases, although the presence of viruses or other pathogens may not be
obvious, yield or quality may be substantially reduced as a result of the
infection. In China, for example, virus-free potatoes, produced by culture in
vitro, gave higher yields than the normal field plants, with increases up to
150%. As only about 10% of viruses are transmitted through seeds, careful
propagation from seed can eliminate most viruses from plant material.
Fortunately, the distribution of viruses in a plant is not uniform and the
apical meristems either have a very low incidence of virus or are virus-free.
The excision and culture of apical meristems, coupled with thermo- or
chemo-therapy, have been successfully employed to produce virus-free and
generally pathogen-free material for micropropagation (Ajnabi, 2012).
For Germplasm conservation – In-vitro conservation of rare and endangered
is an alternative to seed banks and specially to field collections of clonally
propagated crops. It is preserved by in vitro storage under slow-growth
conditions (at low temperature and/or with growth-retarding compounds in the
medium), cryopreserved or preserved as a desiccated synthetic seed. The
technologies are all directed towards reducing or stopping growth and metabolic
activity. Techniques have been developed for a wide range of plants. The most
serious limitations are lack of a common method suitable for all species and
genotypes, the high costs and the possibility of somaclonal variation and
non-intentional cell-type selection in the stored material (e.g. aneuploidy due
to cell division at low temperatures or non-optimal conditions giving one cell
type a selective growth advantage).
tissue-culture technology is playing an increasingly important role in basic
and applied studies, including crop improvement. In modern agriculture, only
about 150 plant species are extensively cultivated. Many of these are reaching
the limits of their improvement by traditional methods. The application of
tissue-culture technology, as a central tool or as an adjunct to other methods,
including recombinant DNA techniques, is at the vanguard in plant modification
and improvement for agriculture, horticulture and forestry;
new hybrids e.g. through protoplast fusion and similar techniques
incompatibility as hybridization through in vitro fertilization
new varieties though mutation induction
transgenic plants (Ajnabi, 2012)
To produce artificial seeds through somatic embryogenesis
synthetic or artificial seed is defined as a somatic embryo encapsulated inside
a coating and is analogous to a zygotic seed. There are several different types
of synthetic seeds: somatic embryos encapsulated in a water gel; dried and
coated somatic embryos, dried and uncoated somatic embryos, somatic embryos
suspended in a fluid carrier; and shoot buds encapsulated in a water gel. Other
applications include the maintenance of male sterile lines, the maintenance of
parental lines for hybrid crop production, and the preservation and
multiplication of elite genotypes of woody plants that have long juvenile
developmental phases. However, before the widespread application of this
technology, somaclonal variation will have to be minimized, large-scale
production of high quality embryos must be perfected in the species of interest
and the protocols will have to be made cost-effective compared with existing
seed or micropropagation technologies (Ajnabi, 2012).
conditions do plant cells need to multiply in vitro?
early tissue culture experiments failed. The primary reason for this was
because they were not maintained in sterile conditions. Isolated fragments of a
plant are extremely disadvantageous compared to pathogenic competitors that are
complete, unhindered and flourishing in a culture environment. Bacteria, fungi
and other organisms which can be resisted to some degree by a whole plant can
easily compete an isolated fragment of tissue from the plant in the relatively
nutrient-rich environment of a culture flask. Therefore, it is necessary to
remove competitor organisms from the culture and isolate it in aseptic
conditions. This is usually done by surface sterilization of the explant with
chemicals such as bleach, at a suitable concentration and for a duration that
will kill or remove pathogens without injuring the plant cells beyond recovery.
The medium and culture flasks used must also be sterile (Nair, 2010).
proper plant hormones, and removal of waste products
When a small portion of a plant is isolated, it is no
longer able to receive nutrients or hormones from the plant, hence these must
be provided to in vitro (Nair, 2010). The composition of the nutrient
medium is for the most part similar, although the exact components and
quantities will vary for different species and purpose of culture. Types and
amounts of hormones
vary greatly. In addition, the culture must be provided
with the ability to continue its growth and to excrete the waste products of
cell metabolism. This is accomplished by culturing on or in a defined culture
medium, which is periodically replenished (Nair, 2010).
A Controlled Environment
Tissue cultures, sustained by the nutrient medium and
confined in a protective vessel, equire a stable and suitable environment.
Thus, light and temperature must be more carefully regulated than in the case
for a whole plant (Nair, 2010). The optimum temperature for growth of culture
is 25±2°C. Some cultures can be kept in complete darkness; however, most
culture rooms need to be illuminated at 1000 lux (134.5 µmol/m2/s)
with some upto 5000 to 10,000 lux (672-1345 µmol/m2/s).