Lecture 8

Plant tissue culture and applications

What is Plant Tissue Culture?

Plant Tissue Culture - The aseptic growth or maintenance of plant cells, organs and
tissues in vitro. The goal is to genetically modify a plant cell or tissue in vitro, such as
transformation with the gene gun or Agrobacterium and then regenerate an entire plant
with the new gene trait.

Totipotency - The ability to regenerate the entire organism from a single somatic cell, i.e.,
trigger the use of the genetic information present to direct the entire regenerative and
developmental programs needed to create the whole organism from a single cell.

Callus - A proliferating mass of undifferentiated plant cells; this type of cells can be
produced by plants as a result of a wound. Undifferentiated - Cells existing in a state of
cell development characterized by isodiametric cell shape, very little or no vacuole and a
large nucleus. These cultures lack specialized structures or functions.

Clone - A group of plants propagated only by vegetative or asexual means, all members
of which have been derived from a single individual. It is also used to refer to making
many copies of a gene and to reproduce an animal from somatic cell.

Organogenesis - A process of differentiation by which plant organs are formed from

tissue or callus or de novo differentiation of organs as separate entities, i.e., roots and
shoots.

Somatic embryogenesis - An in vitro plant regeneration process from somatic cells that
involves differentiation via a somatic embryo which mimics a zygotic embryo.

Protoplast - A single plant cell from which the cell wall has been removed (usually by
use cell wall degrading enzymes).

Plant protoplasts
Histology study of plant embryo

What Makes Plant Tissue Cultures Grow?

1. Abiotic factors
1. Light - The lights in r culture room operate at a 23:1 hour light to dark
photoperiod. A combination of soft white and grow-light bulbs are used on
each shelf. Different photoperiods are used for different plant species or to
trigger different developmental programs.
2. Temperature - A constant temperature of 23 degrees C (74 degrees F) is
maintained through cooling units and fans. Again, different temperatures
may be used in order to obtain specific plant growth and developmental
responses.
2. Biotic factors
1. Plant tissue type - Plant species cultured, explant type, age and condition
of tissue and combinations of these factors affect the ability of the tissue to
regenerate into a plant.
2. Media - the components and type (liquid or solid) have effects on the
ability of the tissue to regenerate into a plant.
Components of Tissue Culture Media

Amino acids, minerals and vitamins - Plant tissue cultures, as well as all living organisms
require a specialized balance of basic nutrients to support life. Depending on the type and
age of the plant culture, these components must be supplied in different concentrations.

Sugars - Since most plant tissue cultures are not able to produce their own energy
through photosynthesis, an outside source of energy (sugars) must be supplied.
Depending on the type and stage of plant tissue development, different types or
combinations of sugars are added to the media.

Hormones - Phytohormones are added to tissue culture media to induce the cultures to
produce calli, embryos, shoots or roots. These are generally of the classes auxin and
cytokinins and the concentrations and balances of the two types are varied to obtain
various growth and differentiation responses.

Antibiotics - Antibiotics are added to media for control of Agrobacterium (used in some
transformation protocols). Another use of antibiotics is as a selective agent for
transformed plants. If an antibiotic resistance gene is transferred into a cell, then its
clones will survive exposure to an otherwise lethal dose of an antibiotic.

Agar-based agent - For use in all solid media, an agar-based gelling agent is used to
provide a strong, clear and flexible substrate on which the tissue culture can grow.

Media pH - The pH of the media must be matched with growth stage of the tissue culture.
Low pH will often result in a bleaching effect on younger tissue cultures.
 Soybean shoots on solid media
Soybean cultures in liquid media

Applications of 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
• 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)
• Tissue culture allows fast selection for crop improvement - explants are chosen
from superior plants, then cloned
• Tissue culture clones are ‘true to type’ as compared with seedlings, which show
greater variability