Unit 01 Biology Module 3 Tissue Culture Aliyah Amarsingh

Introduction

Micro propagation is the propagation or cloning, of plants by tissue culture. ‘Micro’ refers to the small size of material used, usually isolated cells or
small pieces of tissue. The material is grown in special tissue solutions, so the process is known as tissue culture. It is developed from experiments
which showed that plant tissues removed (excised) from plants could be stimulated to grow in solution by the addition of nutrients and certain plant
hormones, particularly auxins and cytokinins. The latter are needed for continual cell division.

Why use tissue culture?

1. Plants with desired characteristics can sometimes be multiplied rapidly, producing many identical copies of the same plant. This is not
easily done. Using conventional breeding methods which rely on sexual reproduction, particularly when plants are adapted for cross
pollination and outbreeding. The technique is important for certain crop and other commercially important plants. New varieties obtained
by plant breeding can be reproduced quickly. The technique therefore speeds up the development and introduction of new varieties.

2. Using tissue culture cells can be genetically modified (transformed) into new/transgenic plants.

3. Tissue culture uses up little space.

4. Plants are grown in disease free conditions.

5. Hybrids have been developed e.g. hybrids between species such as: plants, tomato.

6. The techniques may prove an efficient way of producing useful chemicals such as: pharmaceutical products from plant materials.

Techniques involved in Tissue Culture

1. The culture must contain the correct nutrients and hormones. A typical medium will contain the inorganic ions needed for plant growth such
as a source of N, Mg, Fe and k. Also a source of sucrose for energy and vitamins.

2. These chemicals are mixed with agar to form a jelly-like nutrient similar to that used for growing bacteria and fungi.

3. The tissue culture is grown on the surface of the agar in flasks or petri dishes.

4. N.B. auxins and cytokinins are two essential hormones. Auxins stimulate root growth and cell elongation and cytokinins stimulate shoot
growth and cell division. Differences in the amount of auxin relative to cytokinins affect the way unspecialized cells develop.

5. The culture is grown in special growth rooms or cabinets to control temperature, light intensity, light quality, day length and humidity.

6. The procedure is conducted under sterilized conditions because bacteria and fungi can grow in the culture and outcompete the plants in
these conditions.

Definitions

• Explant is the piece taken from the stock.

• Meristem is a region where cell division is still taking place. E.g. root tip, shoot tip. Meristematic tissue from apical or auxillary buds are
commonly used.

• Another method is to produce a callus from non-meristematic tissue. A callus is an undifferentiated mass of cells. Roots and shoots can be
stimulated to grow from a callus or from non-meristematic tissue by the addition of auxins or cytokinins.

Aliyah Amarsingh-Mohammed
 Tissue culture (often called micropropagation) is a special type of asexual propagation where a very small piece of tissue (shoot apex, leaf
section, or even an individual cell) is excised (cut-out) and placed in sterile (aseptic) culture in a test tube, petri dish or tissue culture container
containing a special culture medium.

The culture medium contains a gel (agar) with the proper mixture of nutrients, sugars, vitamins and hormones, which causes the plant part to grow
at very rapid rates to produce new plantlets. It has been estimated that one chrysanthemum apex placed in tissue culture could produce up to
1,000,000 new plantlets in one year. Thus, tissue culture is used for rapid multiplication of plants. A very specialized laboratory is required for
tissue culture. All the procedures are done in a laboratory and special ventilated cabinet that is as sterile as an operating room.

Steps in Tissue Culture

Explant: Cut-out Plant Tissue and Place in Tissue Culture Container
The first step is to obtain what is called an explant. This means to simply cut-out a very small piece of leaf or stem tissue, or even isolate individual
cells, and place them in a tissue culture container. The tissue has to be sterilized so it will not have any contaminating bacteria or fungus. It is then
placed inside the tissue culture contain on a gel called agar. In the agar is dissolved all the sugar, nutrients and hormones the plant needs.

Explants can be pieces of any part of the plant (leaves, stems, flowers, etc.),
or even individual isolated cells.

Multiplication: Tissue Grows and Produces Small Plants

The tissue will begin to grow. It may make a big blob of tissue called callus, or it may make new shoots directly from the explant tissue that was
inserted in the container.

A mass of callus tissue is formed that is just starting to make new plantlets.

Rapid Multiplication by Transfer of Cultures

Once the plantlets start developing, some can be removed and placed in new tissue culture containers. Thus, another "forest"' of plants is produced.
This results in a rapid multiplication of the cultures and many thousand of plants can be produced in a few months.
Some of the small plantlets can be removed and transferred to new tissue culture
containers. These will produce more shoots and fill the container.

Transplanting
When the plantlets are large enough, they can be removed from the tissue culture container and transferred into pots with potting soil. The young
plants are growth in a greenhouse just like you would any young seedling or cutting.

When the small plant clones are removed from the culture containers, they must be transplanted into some type of acclimation container or kept
under a mist system until the acclimate to the ambient environment.
After acclimation, the young plants can be transplanted
and grown in pots in a greenhouse to produce new plants.

Aliyah Amarsingh-Mohammed