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Micropropagation - Plant Tissue Culture - Role of Growth Regulators
Plant tissue culture is a biotechnology application that utilizes a commercial nutrient culture medium to produce clones of plant cells, tissues, seeds or organs under sterile conditions. Plant tissue culture took off in 1962 when Murashige and Skoog discovered the first reliable artificial medium. Thereafter, major discoveries took place in the advancement of tissue culture;

a.) 1992 - Habertlandt attempted to grow leaf palisade cells from different plants. However, these cells did not divide.
b.) 1934 - White established the need to supplement the medium with additives. He grew meristematic cells of tomato on a medium supplemented with yeast extracts, Vit B (thiamine, pyridoxine, and nicotinic acid), salts and sucrose.
c.) 1953 - Skoog and Miller discovered kinetin. This is a cytokine that plays a central role in organogenesis.
d.) 1959 - Orchids were discovered by Morel. These orchids were set free from viral diseases by Dahlias in 1960
e.) 1960's - Murashige and Skoog cloned plants invitro after discovering and publishing a recipe for Murashige and Skoog (M&S) medium.
f.) 1970's and 80’s marked the genesis of genetic engineering in tissue culture.

There are two main facts that scientists rely upon in plant tissue culture. Plants are totipotent and have ability to produce callus. Totipotency is the ability of a cell to develop into a whole plant or a plant organ when subjected to the right conditions. However, not all plant cells are totipotent. A callus is a mass of actively dividing undifferentiated cells produced by a plant tissue explant- an isolated portion of a plant that is used to initiate a culture (an inoculum)

Plants are sessile in nature and have long life span thus have developed plasticity. Plasticity is the ability to survive and adapt predation and extreme conditions than animals. They alter their metabolic processes and growth to suit the environment.

In plant tissue culture, ability of plants to initiate cell division from most of the tissues of a plant and the developmental responses to stimuli has been of a major interest. This plasticity allows one type organ of a plant to be produced from another hence regeneration of a whole plant when exposed to correct stimuli.

Plant tissue culture requirement
In order to initiate plant tissue culture, one needs; explants, suitable culture/growth media, aseptic conditions to curb growth of microorganisms, water, growth regulators (auxins and cytokinins), and frequent sub culturing to avoid accumulation of waste metabolite and enhance nutrition.

Culture medium
Plant tissue culture medium that is meant for cultivation of plant cells in vitro should contain mineral ions or essential elements in form of a mixture of complex salts, a source of carbon which is usually sucrose and organic supplements that supply vitamins and amino acids.

Organic supplements
Organic supplements supply vitamins and amino acids. The two main vitamins essential for in vitro tissue culture are myoinositol and thiamine while the most important amino acid is glycine.

Carbon source
Sucrose is the preferred carbon source essential in a culture medium. It is easily available, cheap, easily assimilated and stable.

Essential elements/nutrients
Essential elements are classified as macronutrients, micronutrients and an iron source. A combination of these elements is necessary for tissue culture.

Macronutrients are those elements that are supplied in large amounts for plant growth and development. They include; magnesium, sulphur, calcium, nitrogen, potassium, phosphorus and carbon which supplied separately. All these elements comprise more than 0.1% of plants’ dry weight. Nitrogen is mainly supplied as nitrate ions or ammonium ions. However, high concentrations of ammonium ions lead to acidification of the medium and increase vitrification.

Microelements are those elements that are needed in trace amounts in tissue culture media. However, they have diverse functions in plant growth and development. These elements include; iron, molybdenum, Manganese, iodine, cobalt, copper, boron and zinc.

Role of growth regulators in tissue culture

Due to the totipotency and plasticity of plant cells, certain manipulations to culture media are essential to determine certain developmental pathways of a plant cell. Plant hormones and their respective synthetic analogues are used as plant growth regulators. These growth regulators include;

Auxins- promote cell division and growth in explants. They support callus induction hence growth. However, high levels of auxins suppress organized growth promoting growth of meristem-like cells. The mostly used type of auxin for tissue culture is called 2, 4-Dichlorophenoxyacetic acid (2, 4-D).

Cytokinins- these are purine derivatives that support cell division. The two main types of cytokinins used in tissue culture are benzylaminopurine (BAP) and kinetine.

Gibberellins - these are naturally occurring compounds that are used in regulating plant cell elongation. GA3 is the most commonly used type of gibberellin in this technique.

Abscisic acid (ABA) - inhibits cell division in plants. It is mostly used in somatic embryogenesis to promote specific developmental pathways.

Auxins and cytokinins, as growth regulators, have basic roles to play in plant tissue culture. Often, they are used together but with different concentration rations which subsequently determine the type of culture regenerated. A high cytokinin to auxin ratio supports formation of shoots, whereas a high auxin to cytokinin ratio favors formation of roots. A balanced ratio favors production of callus.

Micropropagation procedure
1. Selection of an explant from a ‘mother plant’ that is healthy and vigorous. Usually, apical buds are preferred as explants but any other tissue can be used.
2. Establishment of this explant in a plant culture medium. A medium supports growth and cell division. Depending on the plant requirement, different types of media are used for specific types of plants.
3. Multiplication. In this stage, the explants give rise to a callus.
4. Differentiation and
5. Organogenesis

Culture types
Cultures are produced from ‘explants’. There are different types of cultures produced from explants depending on the conditions availed. They include;

This is an unorganized, growing and actively dividing mass of cells produced when both auxins and cytokinins are present in a culture medium, a procedure carried out in the dark to discourage differentiation. During formation of callus, there is morphological and metabolic dedifferentiation. Dedifferentiation results into inability of these cultures to photosynthesize hence attain a different metabolic profile from the ‘mother plant’. This feature precipitates addition of other culture components.

Manipulation of auxin to cytokinin ratios dictates root, shoot and somatic embryo development from which plants are produced. Callus cultures are classified as either compact or friable. Callus formation plays a central role in plant biotechnology.

Cell- suspension cultures
These cultures are produced from friable callus placed in a liquid medium and agitated. This releases single cells into the medium which under correct conditions, grow and divide to produce cell-suspension cultures. These cells are maintained as batch cultures in flasks.

These are plant cells without cell walls. Removal of cell walls can be done either mechanically or by use of enzymes. The former method results in poor quality yields while the latter yields high and pure cells. The liquid medium used is not agitated to avoid damaging the protoplasts. However, the medium is put maintained under high osmotic pressure and shallow to allow aeration. Organogenesis or somatic embryogenesis can be used to produce whole plants on solid media. Many transformations are done through this method.

Embryo culture
Embryos are used to produce either a callus culture or a somatic embryo. An immature embryo from an embryogenic callus is the most recommended for regeneration of monocot plants.

Other culture types include; microspore culture, root cultures and shoot tip and meristem cultures. These cultures give rise to plant regeneration.
Advantages of Micropropagation

1. Extremely high multiplication rates e.g. 10,00,000 plants/year from single explants. As a result this technique is highly suitable for rapid multiplication of rare genotypes and of plants having low multiplication rates.
2. Very small explants can be used for micropropagation, which is impossible with conventional techniques. Thus the technique is very valuable when only limited tissue is available.
3. During micropropagation, fungi and bacteria are mainly eliminated due to rejection of contaminated cultures. Therefore, the plants are obtained are clean.
4. Plants can be maintained in vitro in a pathogen free state and used for micropropagation. Such plants area easy to export since there is no quarantine problem and their packaging is easier due to smaller size.
5. It is possible to produce plants of one sex in case of dioecious species. This is done by selective multiplication of plants of desired sex.
6. In case of forest trees, mature elite trees can be identified and rapidly cloned by this technique.
7. Micropropagation can be carried out throughout the year independent of seasons.
8. In case of many ornamentals, tissue culture plants give better growth, more flowers and less fall out.

Limitations of Micropropagation:

1. High production cost has limited the application of this technique to more valuable ornamental crops and some fruit trees.
2. Suitable techniques of micropropagation are not available for many valuable species e.g. mango, coconut etc. Further research efforts are necessary to resolve many of the problems encountered, particularly with woody plant species.
3. Somaclonal variations may arise during in vitro culture, especially when a callus phase is involved. But in some species, e.g. banana, even shoot bud proliferation may lead to variation. But in most cases, including banana, the problem has been minimized by technique refinement and visual elimination of variants. In some ornamentals, the variants have been used as new varieties.
4. Vitrification may be a problem in some species.

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