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Basics of Plant Tissue Culture
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Objectives



1.      To know the basics of plant tissue culturing.
2.      To know the production of callus from carrot.
3.      Measure the efficacy of root and shoot.

 

Theory



The theoretical basis for plant tissue culture was proposed by Gottlieb Haberlandt, German Academy of science in1902 on his experiments on the culture of single cell. The first true cultures were obtained by Gautheret from cambial tissue of Acer pseudoplatanus.

The term plant tissue culture (Micro propagation) is generally used for the aseptic culture of cells, tissues, organs and their components under defined chemical and  physical  conditions in vitro. The basic concept of the plant body can be dissected into smaller part termed as “explants” and any explants can be developed into a whole plant.  It is a central innovative areas of applied plant science, including  agriculture and plant biotechnology. This technique is effective because almost all the  plants cell are totipotent; In each cell possesses the genetic information and cellular machinery necessary to generate the whole organism. Since, this technique can be used to produce a higher number of plants that are genetically similar to a parent plant as well as to another.

Two concepts, plasticity and totipotency, are the central processes to understand the regeneration and plant cell culture. Plants, due to its longer  life span and sessile nature, have developed a greater ability to overcome the  extreme conditions. Most  of the processes inculed  in plant development and the growth,  adapt to environmental conditions. When the plant cells and tissues are cultured in vitro, most of them   are generally exhibit a very high degree of plasticity, which allows one type of organ or  tissue  to be initiated from another type. Like this way, the whole plant can be subsequently regenerated. These maintenance of genetic potential is called totipotency.

The plant tissue culture medium is an artificial nutrient supplement of organic and inorganic nutrients used for cultivation of plant tissue media. The appropriate composition of the medium largely determines the success of the culture. The culture media used for the in vitro cultivation of the plant cells are composed of three basic components.

1)      Essential elements (normal ions) supplied as a complex mixture of salts.

2)      An organic supplements providing vitamins and  amino acids.

3)      A source of fixed carbon which is usually supplied as sucrose.

When cultured in an appropriate medium having auxin and cytokinin, explants will give rise to an unorganized, growing and dividing mass of cells called callus. Callus cultures are initiated from a small part of an organ or tissue segment called the explants on a growth supporting solidified nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the explants. At the time of callus formation, there is some degree of dedifferentiation happens  both in morphology and metabolism. One of the major consequences of this dedifferentiation is that most plant cultures lose their ability to perform photosynthesis. The necessitates of  the addition of other components such as carbon and  vitamins source to the culture media, in addition to the unusual mineral nutrients.

 

Morphology of callus:

 

Callus varies considerably in appearance and texture, ranging from hard nodular cell masses to friable soft ones. They maybe white or creamish, orange, green  either in whole or part as a result of chloroplast development. The shape of individual cells within the callus mass ranges from the near spherical or markedly elongated.

A typical unorganized plant callus initiated from a new explants or piece of previously initiated calli has three stages of development.

 

  1. The induction of cell division.
  2. A period of active cell division during which differentiated cells lose specialized features they may have acquired and become         de–differentiated. Cell division usually occurs in the outer layer of the explants.
  3. Period when cell division slows down on ceases and when within the callus, there is increasing cellular differentiat

 

Callus culturing is performed in the dark while light can  be encourage  the  differentiation of the callus. At the time of  long term culture, the culture may loss the requirement for cytokinin and auxins. Manipulation of the auxins to cytokinin ratio in the medium can leads to the development of shoots, roots or somatic embryos from which the plant can be subsequently produced.

 

Callus culture is useful for many purposes.

   1  Callus is the starting material for the suspension culture which cells are separated.
   2. It helps in the production of secondary plant products.
   3. It is useful for the synthesis of starting compounds that are subsequently modified to yield the desired product.
   4. It is the starting materials for vegetative propagation of plants.

 

Based on the availability of the various invitro techniques, the dramatic increase in their application to various problems in basic biology, agriculture, horticulture, and forestry. The applications can divide conveniently   into five broad areas;

   1. Cell behavior.
   2. Plant modification.
   3. Germplasm storage and pathogen –free plants.
   4. Clonal propagation.
   5. Product formation.
   6. Improved varities.
 

 

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