Introduction to Cell & Tissue Culture

By Dr. Amit Kumar Dutta, Ph.D

Plant Tissue Culture ?

“… the aseptic culture of plant protoplasts, cells, tissues or organs under conditions which lead to cell multiplication or regeneration of organs or whole plants “

Tissue can be regenerated from explants such as cotyledons, hypocotyls, leaf, ovary, protoplast, petiole, root, anthers, etc.

Plant Tissue

Culture

Tissue

Definition

The culture of plant seeds, organs,

tissues, cells, or protoplasts on

nutrient media under sterile conditions.

What for ?

Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture).

1838-39 Cellular theory (Cell is autonom and totipotent)

Schleiden-Schwann

1902 First Attempt of Plant Tissue Culture

Harberlandt

1939 Continuously growing Callus Culture

White

1946 Whole Plant developed from Shoot Tip

Ball

1950 Organs regenerated on Callus Ball

1954 Plant from Single cell Muir

1960 Protoplast Isolation Cocking

Look Back Before…………..

A more recent advance is the use of plant and animal tissue culture along with genetic modification using viral and bacterial vectors and gene guns to create genetically engineered Organisms.

Look Back Before…………..

1962 MS Media Murashige - Skoog

1964 Clonal Propagation of Orchids

Morel

1964 Haploids from Pollen Guha

1970 Fusion of Protoplasts Power

1971 Plants from Protoplasts Takebe

1981 Somaclonal Variation Larkin

Basic Requirements

Tissue culture has several critical requirements:

Aseptic (Sterile) Conditions,

Microorganisms grow much more quickly than plant and animal tissue

and can over run a culture.

A Suitable Growth Medium

Containing energy sources and

inorganic salts to supply cell growth needs. This can be liquid

or semisolid.

Appropriate Tissue

Some tissues culture better than others

Other Requirements…..

Growth Regulators

In Plants, both Auxins & Cytokinins.

Frequent Subculturing

To ensure adequate nutrition and to avoid the build up of

waste metabolites.

Early Cell Culture……Haberlandt .. early 1900’s… proposed concept of totipotency… cells cultured under right conditions

Callus cultured from tree cambium(Gautheret, Nobecourt, Whire in the 1930s.… cells kept alive but did not develop

The first commercial use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920’s.

Early Cell Culture ……

In the 1950’s and 60’s there was a great deal of research, but it was only after the development of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really ‘took off’ commercially.

Early Cell Culture……

Culture Medium Constituents

Inorganic salt formulationsSource of carbohydrate

VitaminsPlant Hormones - Auxins,

Cytokinins, GA’sSolidifying agents

Water

• Growth Media– Minerals, Growth factors, Carbon source, Hormones• Environmental Factors– Light, Temperature, Photoperiod, Sterility, Media• Explant Source– Usually, the younger, less differentiated the explant, the better for tissue culture

Factors Affecting Plant Tissue Culture

• Genetics–Different species show differences in amenability to tissue culture.–In many cases, different genotypes within a species will have variable responses to tissue culture.--Response to somatic embryogenesis has been transferred between melon cultivars through hybridization.

Factors Affecting Plant Tissue Culture

• A single explant can be multiplied into several thousand plants in less than a year – this allows fast commercial propagation of new Cultivars.

• 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.

Why do Plant Tissue Culture?

• In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants.

• Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture.

• Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner).

Why do Plant Tissue Culture?

Tissue Culture Applications

- Micropropagation- Germplasm preservation- Somaclonal variation & mutation selection- Embryo Culture

Haploid & Dihaploid ProductionIn vitro hybridization – Protoplast Fusion

- Industrial Products from Cell Cultures

Basis for Plant Tissue Culture• Two Hormones Affect Plant Differentiation:

– Auxin: Stimulates Root Development– Cytokinin: Stimulates Shoot Development

• Generally, the ratio of these two hormones can determine plant development:– Auxin ↓Cytokinin = Root Development– Cytokinin ↓Auxin = Shoot Development– Auxin = Cytokinin = Callus Development

Three Fundamental Abilities of PlantsTotipotency

the potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated.It implies that all the information necessary for growth and reproduction of the organism is contained in the cell

DedifferentiationCapacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganise into new organ

Competencythe endogenous potential of a given cells or tissue to develop in a particular way

Types of In Vitro Culture Culture of intact plants (seed and seedling

culture) Embryo culture (immature embryo culture)Organ culture

1. shoot tip culture2. root culture3. leaf culture4. anther culture

Callus cultureCell suspension cultureProtoplast culture

Tissue Culture ApplicationsMicropropagationGermplasm preservationSomaclonal variationdihaploid productionProtoplast fusionSecondary metabolites productionGenetic engineering

Seeds of P. hexandrum

Germinated seedling

Callus culture

Suspension culture

Batch cultivation

Batch cultivation with fluorescence probe

Continuous cultivation with cell retention

Setric impeller

Various steps involved in cell culture