Plant Tissue Culture

Introduction

As like plants their cells can grow. Simply, the potential of plant to grow is known as totipotency. Any parts of plant can be developed into a whole plant because of the totipotency nature. The different parts or cells of the plant can be developed into new plant artificially in the laboratory under a suitable environment. Tissue culture is the modern technique of growing a new plants with the help of cells tissues or organs of the plant in the laboratory under the suitable environment artificially.

Historic background

In the history of tissue culture Haberlandt was the first person to culture isolated vegetative cells of higher plant in simple nutrient solution in 1896.He was just able to maintain the nutrient media but unable to record the cell division. The first plant tissue which was done successfully in vito on artificial environment are embryos. In 1904,Hanning was the first person to bring positive result in vito who grew plants under a germs free condition.

In 1922, Robbins and Kotte was able to grow isolated root tips successfully and was able to breakthrough the field of plant tissue culture. Than after White(1934) grew tomato root tips for long period of twenty weeks using sucrose, inorganic salt and yeast extract. Than gradually process of development step ahead and now it has achieve a great position.

Types of tissue culture

Every parts of plants can be grow into the new plant. Parts like root, shoot, anther, bud, etc. can be used for the growth of new plants. On the basis of use of different parts of the plant tissue culture can be classified into following types:

1.Shoot culture –It is the technique to grow the new plant with help of various type of shoot such apical adventitious and axillary shoot in the suitable condition. Different shoots can be cultured onto the plant tissue to develop plant directly. It is also called clonal propagation.

2.Callus culture-It is the tissue culture in which large number of plantlets can be obtained from callus. Callus is defined as the unidentified and unorganized mass of cells. It is also known as the pre-matured unorganized cells. Usually callus is found around the cut edges of the plant segments when placed on the solidified culture medium. This technique can produce the large number of plantlets in the short duration of time. Here ,in this process new plant is formed from Calli.

3.Embryo culture-It is the type of plant tissue culture in which embryo are use for the culture of new plant. In 1922,Knudson was able to grow the orchid embryo into plantlets by culturing them on agar medium which contain sugar.

4.Meristem culture-It is the plant tissue culture in which the meristem of the plant is cultured in the laboratory in germs free condition. Meristem is the apical tip of plant which always goes on multiplying. As meristem is the diseases free part of the plant, it produce the germs free plants.

5.Anther culture-It is the technique of culture in which anther of the plant is use for the production of haploid plants. In this method pollen grain of the anther is utilized for the culture.This technique is also known as androgenesis. With the help of this a virus free plant can obtained .It can produce the hapliod plant.

6.Protoplast culture-Simply protoplast is the cell excluding the cell wall. It is the technique of plant tissue culture in which protoplasm is use to obtain new plant. Protoplasts can be obtain from various parts of plants such as leaves, stems, callus and from pollen grain too. Single proplast can produce a complete plant.

Methods of plant tissue culture

Tissue culture can be done in only two medium i.e. liquid medium and semi-solid agar medium. In case of liquid medium plant tissue should be fully immersed or partially immersed but in the case of semi-solid medium the plant tissue are just placed on the surface of the medium.

Tissue culture medium

Plant are the component of the nature and they grow in nature, get suitable temperature, light hormones and nutrients which are important for their development .When plants are grown in the vito, in artificial environment they only can use the nutrients that is available in the medium. The nutritional level of different plants may be different. So to over come the nutritional level of different plants, wide range of nutritional level is needed to maintain for plant tissue culture.

Composition of medium

All the plants get survive as well tissue or cell when they get proper amount of nutrients for their development. So tissue culture is possible when all the possible nutrients can be maintain in the medium. The medium should contain inorganic nutrients, organic nutrients and hormones which are required for their proper development. The composition of media are described below:

1. Inorganic nutrients: It is one of the essential nutrients for the normal growth and development of a plant. It includes 12 elements for the growth of plant. They are; nitrogen, calcium, potassium , sulphur magnesium, iron, maganese, copper, boron, zinc, phosphorus and molybdenum. Out of 12 , six macronutrients are required for the development in large amount (concentration more than 9.95 ) and other remaining six micronutrients is required in small amount(concentration ).
2. Organic nutrients: On the presence of organic nutrients like amino acids and vitamins cultured plants can grow and develop quick and better. Vitamin (Thiamine) is mostly used for the cultured plant. Vitamins like nicotinic acid, calcium pantothenate and pyridoxine are used for to improve the growth of the cultured plants. Other organic nutrients added in the medium are biotin acid and aminobenzonic acid. Sucrose is the most essential carbon source for the cultured plant. Other source of sugar are glucose, fructose, maltose, galactose, mannose and lactose.
3. Growth hormones: It is another composition required to culture the plants. Mostly growth hormones are use the tissue culture. The hormones used are auxins, cytokinins and gibberellins.
4. Among the growth hormones auxins is the hormone which in cell division and root differentiation. IAA (Indole acetic acid), IBAIndole-3-butyric acid), NAA( Naphthalene acetic acid) and 2,4-D(Dichlorophenoxy acetic acid) are the commonly used auxins. Cell division and differentiation of shoot is done by cytokinins. Some commonly used cytokinins are: BAP(Benzylamino acetic acid) and 2-isopentanyle.
5. Agar: It is the component only us in the solid medium. It is use at the concentration of 0.8-1%(w/v). When it is used in higher concentration, it makes the medium harder. It prevents the nutrients diffusion in the tissue.
6. pH: The pH of the medium should be maintain between 5 to 5.8 by adding NaOH or HCL.

Media preparation

Culture media can be prepared using commercially available powered media. It consists of all the essential nutrients and finally agar, sugar and other supplementary are added. Before the media is prepared it is very important of prepare the stock concentrated solution. For the preparation of the stock solution, it requires distilled water and a chemical with high purity. Stock solution should be prepare of different strength for different media component. The prepared stock solution are kept in plastic or glass at low temperature.

Media selection

As we know that different culturing plants required different medium. So suitable medium for the culturing plant should be select according to their necessicity. In the familiar medium cultured plant be grow properly. So to select a suitable medium for an untested system,board spectrum experiments may help.

Refrence:

Arvind K. Keshari,Kamak K. adhikari. A Text Book of Higher Secondary Biology. Kathmandu: Vidyarthi Pustak Bhandar, 2014.

Agrawal, sarita. principle of biology. 2nd edition . kathmandu: Asmita book Publication, 2068 ,2069

Mehta, Krishna Ram. Principle of biology. 2nd edition. kathmandu: Asmita, 2068,2069.

Jorden, S.L. principle of biology. 2nd edition . Kathmandu: Asmita book Publication, 2068.2069.