P L A N T T I S S U E C U LT U R E

CONTENT
• INTRODUCTION
• HISTORY
• BASIC TERMS IN TISSUE CULTURE
• DIFFERENT STEPS IN TISSUE CULTURE
• TYPES OF PLANT TISSUE CULTURE
• FACTORS EFFECTING PLANT TISSUE CULTURE
• APPLICATIONS
• USES
• ADVANTAGES
• REFERENCES.
INTRODUCTION
• Plant tissue culture is an in vitro culture or growth of cells,
tissue or organs of plant in a sterile condition and well
formulated media to produce an entire plant.
• The 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.
HISTORY OF PLANT TISSUE CULTURE.
• 1902 - Haberlandt proposed concept of in vitro cell culture
• 1922 - Kolte and Robbins successfully cultured root and stem tips
respectively
• 1926 - Went discovered first plant growth hormone –Indole acetic
acid
• 1939 - Gautheret, White and Nobecourt established endless
proliferation of callus cultures
• 1957 - Skoog and Miller gave concept of hormonal control (auxin:
cytokinin) of organ formation
• 1960 - Cocking was first to isolate protoplast by enzymatic
degradation of cell wall.

• 1962 - Murashige and Skoog developed MS medium with higher salt

concentration.

• 1964 - Guha and Maheshwari produced first haploid plants from

pollen grains of Datura (Anther culture).

• 1970 - Power et al. successfully achieved protoplast fusion.

• 1971 - Takebe et al.regenerated first plants from protoplasts.

• 1981- Larkin and Scowcroft introduced the term somaclonal

variation.
BASIC TERMS USED IN TISSUE CULTURE.

• Explant
• An excised piece of differentiated tissue or organ is regarded as an
explant. The explant may be taken from any part of plant body
e.g.,leaf, stem, root.

• Callus
• The unorganized and undifferentiated mass of plant cells is referred
to as callus. Generally ,when plant cells are cultured in a suitable
medium, they divide to form callus.
CONTINUE-
De-differentiation
• The phenomenon of mature cells reverting to meristematic state to
produce callus is dedifferentiation.
Re-differentiation
• The ability of the callus cells to differentiate into a plant organ or a
whole plant is regarded as re-differentiation.
Totipotency
• The ability of an individual cell to develop into a whole plant is referred
to as totipotency.
STEPS INVOLVED IN PLANT TISSUE CULTURE.

• Preparation of suitable nutrient medium:

• MS medium, White’s medium, B5 medium are few culture media
that can be obtained from the market for the lab preparation of
nutrient medium needed for tissue culture.
• The culture medium consists of :
• Inorganic nutrients:
• 2 types of inorganic nutrients are used in the culture medium.
• 6 macronutrients like nitrogen, phosphorus, sulphur, calcium,
potassium and magnesium and 6 micronutrients like iron,
molybdenum, copper, zinc, boron and manganese are also added in
the medium.
.

Organic nutrients:
•Carbon and nitrogen containing compounds are used.
•Sucrose, fructose and other carbohydrates are the carbon source.
•Vitamins and amino acids are the nitrogen source.
•Plant growth hormones : Auxins, Gibberellins, Cytokinins,
Abscisic acid, Ethylene etc. are some examples of plant hormones.
Auxins, cytokinins and Gibberellins are the most commonly used growth
hormones in culture medium prepared for tissue culture.

•Agar : It solidifies the medium and acts as a substratum. It doesn’t have

any nutritive importance. It is obtained from sea weeds and is used to
provide solid surface for growth.
•All these important nutrients are incorporated in the culture medium
and transferred into suitable containers and then sterilized.
2.Selection of explant:
•Explants are any excised part of the plant to be used in tissue culture
like axillary buds, leaf and stem segments, root tip, shoot tip, anther,
ovary and endosperm.
•Always young and healthy parts of plants are selected as explants.

3.Sterilization of the explant:

•Explants are sterilized by using different types of disinfectants like
sodium hypochlorite (2%), silver nitrate (1%) calcium hypochlorite (9-
10%), bromine water (1-2%), mercuric chloride (0.1-1%), hydrogen
peroxide etc. and different types of antibiotics.

4.Inoculation of explant:
•The sterile explant is inoculated on the surface of the solidified
nutrient medium under aseptic (free from contamination) condition.
•Laminar air flow can be used for this inoculation process.
5.Incubation:
•Incubation is done at 25+/- 2 0C, with a relative humidity of 50-60%
with 16 hours of photoperiod.
•After defined period of incubation, an unorganized and
undifferentiated (no root and shoot) mass of cells called callus is
obtained from each explant. In some cases, root and shoot directly
develop from the explant after incubation.

6.Transfer of callus (subculture) and regeneration:

•The callus obtained from incubation of explant is transferred into
another medium and reincubated.
•Plantlet regenerates from the callus with the induction of roots
and shoot directly from the callus.
7.Hardening or acclimatization:

•Hardening is the gradual exposure of the plantlets for acclimatization

or adjustment to the environmental conditions.

8.Transfer of plantlets to the field:

•After the hardening process, plantlets are transferred to either the

green house or the field conditions outside.
•These plantlets will grow into independent and healthy plants.
FACTORS AFFECTING TISSUE
CULTURE
• Growth media
• Minerals, carbon source, vitamins , hormones.
• Environmental factors
• Light, temperature, photoperiod, humidity.
• Explant source
• Usually younger ,less differentiated, explant is used for tissue culture.
TYPES OF TISSUE CULTURE.
• Micropropagation
Micropropagation is the rapid vegetative propagation of plants
under in-vitro condition.
• Germplasm Conservation
The conservation of germplasm involves the preservation of the
genetic diversity of a particular plant or genetic stock.
• Somaclonal Variation
The genetic variation found in the in-vitro cultured cells are
collectively referred to as somaclonal variation.
CONTINUE
• Haploid & dihaploid production
Plants which possess only a single set of chromosomes in their
sporophyte are known as haploid plants.
• In vitro hybridization – protoplast fusion
It is the technique which allows the manipulation of cellular genomes by
protoplast fusion.
• Embryo rescue
It is an in vitro technique used to development of an immature embryo
into a viable plant.
• Synthetic seed production.
Seeds produce by encapsulation of somatic embryo.
 USES OF PLANT TISSUE CULTURE.
Plant tissue culture now has direct commercial applications as well as value
in basic research into cell biology, genetics and biochemistry. The
techniques include culture of cells, anthers, ovules and embryos on
experimental to industrial scales, protoplast isolation and fusion, cell
selection and meristem and bud culture. Applications include:
• micropropagation using meristem and shoot culture to produce large
numbers of identical individuals.
• screening programmes of cells, rather than plants for advantageous
characters.
• large-scale growth of plant cells in liquid culture as a source of secondary
product.
CONTINUE-
• crossing distantly related species by protoplast fusion and regeneration
of the novel hybrid.
• production of dihaploid plants from haploid cultures to achieve
homozygous lines more rapidly in breeding programmes.
• as a tissue for transformation, followed by either short-term testing of
genetic constructs or regeneration of transgenic plants.
• removal of viruses by propagation from meristematic tissues.
• To conserve endangered plant species to avoid extinction.
• To produce disease free plants due to its production in sterile
environment.
ADVANTAGES OF PLANT TISSUE CULTURE
• fast commercial propagation of new cultivars. Prevention of starvation in
third world countries with higher productivity if its genome is changed.
• Taking an explant does not usually destroy the mother plant, so rare and
endangered plants can be cloned safely.
• Once established, a plant tissue culture line can give a continuous supply
of young plants throughout the year.
• The production of clones of plants that produce particularly good flowers,
fruits, or have other desirable traits.
• The regeneration of whole plants from plant cells that have been
genetically modified.
CONTINUE-

• Tissue culture has been exploited to create genetic variability from

which crop plants can be improved.
• To improve the state of health of the planted material and to increase
the number of desirable germplasms available to the plant breeder .
• The culture of single cells and meristems can be effectively used to
eradicate pathogens from planting material and thereby dramatically
improve the yield of established cultivars.
• Little space needed for multiplying large number of plants .
REFERENCES
• Singh,B.D ,Kalyani Publishers,Third edition,Chapter-8 ,Plant tissue
culture, Page no-282-286.
• By Altaf Hussain, Iqbal Ahmed Qarshi, Plant Tissue Culture [Online],
Available on https://www.intechopen.com/books/recent-advances-
in-plant-in-vitro-culture/plant-tissue-culture-current-status-and-
opportunit culture,
• By Aditi Raj, Application of Plant Tissue Culture, [online ] Available
on www.biologydiscussion.com.
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