Plant tissue culture (PTC)

Definition- Plant tissue culture (PTC) refers to the cultivation of undifferentiated plant
cells, tissues or organs on synthetic media under aseptic environment and suitable
controlled physical conditions.

It is an important tool for both basic research as well as commercial

applications.
Plant tissue culture is based upon the unique characteristic of a plant cell i.e.,
totipotency.
Totipotency is the ability of a vegetative cell to divide and differentiate into any
type of specialised cell or to regenerate into a whole plant.

Historical Perspective In 19th century, German scientists Theodor Schwann and

Matthias Schleiden, drew attention to the fact that a cell is the basic unit of life and
has the capacity to divide and grow.

During 1890’s, Gottlieb Haberlandt (German Botanist), pioneered the field of PTC with
his idea to achieve continuous cell divisions in plant cells on nutrient media. He
attempted to culture fully differentiated plant cells on simple nutrient media. He laid
down several principles of plant tissue culture in 1902. Therefore he is regarded as the
‘Father of Plant Tissue Culture’

Major landmark discoveries in plant tissue culture and its applications

1902 Gottlieb Haberlandt proposed that plant cells can be cultured on artificial media
and developed the concept of in vitro cell culture.

1904 Hannig initiated work on excised embryo culture and later cultured embryos from
several cruciferous species.

1922 Kotte and Robbins suggested root and stem tips as possible explants to initiate
in vitro tissue culture.

1926 Went discovered the first plant growth hormone i.e., Indole Acetic Acid (IAA).

1934 The role of vitamin B as growth supplement in plant tissue culture was reported
by White. He could successfully establish continuous growing cultures from tomato
root tips.

1937 White formulated the first synthetic plant tissue culture medium (WM).

1941 Van Overbeek introduced coconut water as a media component and

demonstrated its beneficial effects on in vitro tissue culture.

1946 Ball raised whole plants from shoot tips of Lupinus.

1954 Muir successfully induced cell division in mechanically isolated single cells.
1955 Skoog and Miller reported the discovery of Kinetin, which is a type of cytokinin
and promotes cell division.

1957 Skoog and Miller described chemical control hypothesis of root and shoot
differentiation by manipulating the ratio of concentrations of auxin and kinetin.

1962 Murashige and Skoog formulated MS medium with higher salt concentrations.

1964 Guha and Maheshwari produced the first androgenic haploid Datura plant by
anther culture.

1971 Protoplasts were subcultured in vitro, and plants were regenerated from their
culture.

1972 Protoplast from two different species of Nicotiana were isolated, fused together
and somatic hybrids were generated successfully.

1976 Gynogenic haploid plants were successfully cultured from unfertilised ovaries of
barley by San Noeum.

1978 Melchers and colleagues produced ‘Pomato’, which was a hybrid of potato and
tomato, and was produced through somatic hybridisation.

Plant Cell and Tissue Culture Techniques

• Virtually any part of the plant like leaf, apical meristem, embryo, cotyledon,
hypocotyl, etc., can be used as a starting material called explant.
• These explants are transferred on to the nutrient media and whole plants can
be regenerated through in vitro culture.
• It has been observed in various research experiments that different plant
organs of different plant species respond in different ways as per their
nutritional requirement and physical conditions under in vitro culture conditions.
• However, the response of different plant organs varies for in vitro culture. For
example, immature embryos are more responsive than apical meristem, which
are generally more responsive than leaf explants on a particular tissue culture
media and culture conditions.
• Plant regeneration in cultures can mostly be achieved by two morphogenetic
pathways—organogenesis and somatic embryogenesis.
• Inducing the formation of various vegetative organs from cells or tissues in
plant tissue culture is called organogenesis.
• First, the specialised cells of explants start dividing under certain specific
conditions and form a mass of undifferentiated cells. This process is called
dedifferentiation.
• This is followed by the formation of organ primordia, like shoot or root, and is
called redifferentiation.
 • Relative concentration of growth hormones (especially auxins and cytokinins)
play an important role in organogenesis.
• The process of formation of an embryo from somatic cells is called somatic
embryogenesis.
• Resulting embryos are called somatic embryos. Somatic embryogenesis
follows embryogenic pathways of zygotic embryogenesis.
• Somatic embryos are very similar to zygotic embryos.

Steps of plant tissue culture

Following is the step-by-step procedure that explains the in vitro plant tissue cultures
using tomato cotyledons as explants.

(i) Selection of a suitable nutrient media and its sterilisation by autoclaving or

passing through micropore filters to avoid microbial contamination.
(ii) Selection of a starting desired material for tissue culture i.e., explants. Any
plant tissue, organ or part, which is used in plant tissue culture to regenerate
mass of dedifferentiated cells, tissues, organ or whole plant, is called
explant. Most commonly used explants are root or shoot apical meristems,
leaves, cotyledons, hypocotyls and immature embryos. Fig. 7.1 (a) shows
the aseptically grown tomato seedlings, which can be used for preparing
explants.
(iii) Surface sterilisation of the explant is done using suitable disinfectants
followed by washing with sterile distilled water. Sodium hypochlorite is the
most commonly used disinfectant for sterilisation of explants. Explants
harvested from seedlings grown in sterile conditions [Fig. 7.1 (a)] need not
to be sterilised again.
(iv) Inoculation of explants takes place onto the nutrient media (Fig. 7.1 (b))
(v) Growing the cultures in the plant tissue culture room under suitable physical
conditions like light, temperature and humidity [Fig. 7.1 (c)], small calli are
regenerated on cotyledon explant on suitable tissue culture media.
(vi) Transfer of growing cultures on to suitable media to regenerate shoots, and
their elongation.[Fig. 7.1 (d)], (vii) Excision of regenerated shoots and
transfer onto the rooting medium [Fig. 7.1 (e)].
(vii) Transfer of plantlets to sterilised soil in pots for hardening in green house or
growth room [Fig. 7.1(f)] followed by their transfer to the field conditions.

Nutrient Media
A variety of nutrients and suitable environmental conditions are required for
optimal growth and development of an explant.
Depending upon the type of plant species, like monocot or dicot;
domesticated or wild, etc., composition of the culture media varies.
Even different tissues from the same plant may have different nutritional
requirements for optimal growth. Therefore, success of in vitro culture of
plants mainly depends upon the selection of the right composition of culture
medium.
 Culture media used for in vitro plant cultures broadly contain the following
components:
1. Inorganic components
2. Organic supplements
3. Carbon source
4. Plant growth hormones
5. Gelling agents
6. Antibiotics

Macronutrients
Many inorganic components are required in large amounts (milli molar
concentrations) and are categorised as macronutrients.
These include carbon (C), calcium (Ca), hydrogen (H), potassium (K),
magnesium (Mg), nitrogen (N), oxygen (O), phosphorus (P) and sulphur (S).
Micronutrients
Several other essential inorganic components are required in small amounts
(micro molar concentrations) and are categorised as micronutrients.
These include boron (B), cobalt (Co), copper (Cu), iron (Fe), manganese
(Mn), molybdenum (Mo) and zinc (Zn).
Amino acids and vitamins are common organic supplements that are used
in culture media. Amino acids serve as nitrogen source and commonly used
amino acids are arginine, asparagine, glycine or proline.
Vitamins added in the culture medium are thiamine (Vitamin B1), nicotinic
acid (Vitamin B3) and myoinositol.
Carbon source-For in vitro cultures, sucrose is considered as the best
carbon source and is used at concentration of about 2–5 percent. Other
sources for carbon are glucose, fructose and mannose.
Plant growth hormones play a vital role in plant’s growth and development,
and are critical components of media. These are required in minute
quantities in the nutrient media. There are five main categories of growth
hormones i.e.,
(i) Auxin,
(ii) Cytokinin
(iii) Gibberellin
(iv) Abscisic acid
(v) Ethylene
However, auxins and cytokinin are most commonly used in plant tissue culture.
Ratio of their concentrations determines the type of organs produced from the
cultured cell or tissues.
For example, higher concentration of cytokinin results in shoot regeneration in
general. Further, different plant tissues need different amount of hormones for
their growth and therefore, depending upon the type of explant, their
concentration may vary in the nutrient media.
Gelling agent is required for solid culture media. Agar is the most commonly
used gelling agent and is ideal for routine applications.

pH adjustment- Further, pH of the nutrient media is usually adjusted to about

5.8 to 6.0. Increase in the pH increases the hardness of the medium and
decrease in pH leads to poor solidification of the media.

Antibiotics can be used to suppress the bacteria and antifungal agents for
mould infections in cultures. The recipe of plant tissue culture media is usually
directed by plant species; however (MS) / media is the most commonly used
media composition in plant tissue culture.
Culture Types
Plant Tissue Culture may be categorised as organ culture, callus culture, cell
suspension culture, protoplast culture, etc.

Cultivation of plant organs like roots, anther, ovary, embryo, endosperm, seeds, etc.,
under laboratory conditions is called organ culture. Depending upon the type of organ,
the culture is called root culture, anther culture, embryo culture and so on.

Cell culture:
• The culture of isolated individual cells.
• Carried out in dispersion medium.

Callus culture:
• The growth of callus from explant by providing appropriate conditions.
• Darkness and solid medium gelled by agar stimulate callus formation.

Protoplast culture:
• The protoplast culture is aimed to develop genetically transformed plant.
• The cell in which the cell wall has been removed.
• In this, isolated protoplasts are cultured on a suitable medium under the aseptic
condition.

Organ culture:
• Isolated plant organs (Shoot, root, leaf, and flower) are used as explant.
• The organ culture may be organized or unorganized.
• New growth (differentiated structures) continues given that the organ retains its:
• Physiological features,
• Provide information on patterns of growth, as well as development.

Seed culture:
• For this method, explants are obtained from an in-vitro derived plant.
• Introduced into an artificial environment.
• Where they get to proliferate.
• Seed culture is primarily used for plants such as orchids

Embryo Culture:
• For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is
sterilized.
• Culturing them in nutrient media, help in developing viable seedlings.
• Embryo culture is the isolation and growth of mature or immature embryo in-vitro.

Meristem culture:
• A meristem is a localized group of cells, which are actively dividing and give rise to
permanent tissue.
• The apical meristem of shoots is cultured.
• To get the disease-free plants.
Anther Culture:
• Anthers of some plants are cultured on a suitable medium to produce haploid plants,
it is called anther culture.
• Separated anthers from flowers.
• Cultured on a suitable medium.
• This technique was first used in India to produce haploids of Datura.
Applications of Plant tissue culture
Clonal Propagation:
• Useful tool to get a large population of plant species having desirable traits.
• This technique is very much used in horticulture and silviculture—orchids and many
fruit plants.
• Large scale production of plants of same genetic stock.

Secondary Metabolites:
• Secondary metabolites produced by plant cell cultures are rather small in amount.
• By clonal selection the particular high yielding clone of cells can be isolated.
• Production of many useful compounds can be done by plant cell culture.

Genetic Variability:
• The chromosomal instability in the cultured cells play an important role in
polyploidization of cells and genetically variable plants can be raised.
• Such kind of variations may show some useful characters such as:
• Different kinds of morphological variations in leaf and flowering.

Somatic Embryogenesis and Synthetic Seed:

• Direct or indirect somatic embryogenesis may be achieved from:
(a) Pro-embryonic cell of the direct explant
(b) The embryoids developed within the callus tissue.

The application of this technique is the mass production of adventitious embryos

which ultimately develop into complete plantlet.

Haploid Plants:
• Haploid plants can be obtained through anther or pollen culture (androgenesis).
• These haploids produced homozygous diploid or polyploid lines.
• Colchicine treatment within a very short period.

Somatic Hybrids:
• Somatic hybridization, using isolated somatic protoplasts is a new tool to make the
wide
hybridization successful.
• Obtain somatic hybrid plants between sexually compatible and incompatible plants.
• Production of cybrid is also of immense importance in the plant breeding program.

Transgenic Plants:
• The genetically modified (GM) plants, in which a functional foreign gene has been
incorporated by biotechnological method.

• A number of transgenic plants have been produced carrying genes for different traits.

• The direct DNA uptake through protoplast is the most ideal method.

Germplasm Conservation:
• Many of the important crop species produce recalcitrant seeds.
• Plant tissue culture may be applied for this purpose.
• Mainly the plant species which are endangered, needed to be conserved by ex-situ
method of
germplasm conservation.