Plant Tissue Culture

Introduction

Plant tissue culture is the art of science to grow new plant with the help of different parts of plants. A small part of the plant can produce the whole plant. Plant are totipotency in nature which means that every parts have capacity to grow in new plant. This technique can be used for the enhancement of the different rare plants. With the help of different parts of rare plant, they can be produce in large number.

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Source:en.wikipedia.org
Fig:Tissue culture

Sterilization

Grems free enviroment is known as the sterilized zone. The plant tissue culture is sensative type of experiment in which persence of germs may harm the experiment. So the media which is use for the plant tissue culture should be sterilized before placing the tissues. Though the plant may be grown but the resulting plant may not be healthly. It can be the dangerous for the other too. So, it ismost important to sterilized the everything that involve in the tissue culturre.

Source of contamination

There are many sources that may contaminate the medium. Some of the sources are described below:

1.Medium- Firstly medium itself can the sourece of contamination because there may be the presence of microbs at the very beginning. Everythings that are included in the medium should be sterilized. Every vessels that are included in the medium must be sterilized in steel autoclaves or in pressure cooker at 120 degree celcius for 15-20 minutes.

2.Culture vessels and instruments- All the metal vessel and instrument which are required for the plant tissue culture must be sterilized by pleceing them in the oven at 160-180 degree celcius for 2-3 hours . Some instrument like forceps, needle, etc. are needed to be dipped in 95% ethyl alcohol and flamed before use.

3. Plants materials- Those plants whose tissue is going to be cultured must be sterilized before their tissue is taken . Generally, calcium or sodium hypochlorite solution is use for the sterilization process of the plant surface.Generally, calcium or sodium hypochlorite solution is use for the sterilization process of the plant surface.

Micropropagation

Generally reproduction in plants take place by two means,either by vegetative means or by apomixis( seed that develop without meiosis and fertilization). Vegetative reproduction is the natural process which produce genetically identical plants. It is mostly used for the propagatino and multiplication of different important plants. This process produce genetically identical plants by asexual means which is also called clonal propagation. When clonal propagation is done in vitro, it is called micropropagation.

Micropropagation has following merits over conventional method of clonal propagation:

  1. In short period of time, a large number of plant can be produce by a single explant.
  2. Germs or diseases free plants can be obtained with process.
  3. This process can be done through the year without the problems of seasonal variation.

Micropropagation techniques

When clonal propagation is done in vitro, it known as micropropagation. There are three stages of micropropagation which are described below:

A.Shoot multiplication

Shoots involve following three main approach in vitro:

  1. Multiplication through callus culture- In this culture callus is used and large number of plantlets can be obtained from callus either through shoot-root or somatic embryogenesis. This is only one method from which a large number of callus followed by shoot regeneration.
  2. Multiplication by adventitious shoots- Adventitious shoots develop naturally on the plant at a place other than normal axial regions which show the structure like stem and leaf. Stems, tubers, bulbs, corms and rhizomes are the structure usually included in adventitious shoots. The vegetative propagation of adventitious bud formation is commercially practiced. For example, the development of shoots on the leaves of Begonia and some others ornamental plants. On the leaves and stems cuttings adventitious buds can also be induced even those species which are normally not propagated vegetatively. Adventitious buds can be produced from a piece of shoot apex. Within 3-4 months more than 8000 grapes plant can be obtained from a excised shoot tip.
  3. Multiplication by apical and axillary shoots - Apical shoots are those types of shoot which occupy the growing tip of shoots. But axillary shoots are those shoots which develop from their normal position on the plant in axial of leaf. Shoot can be develop from the bud and each bud has capacity to develop a shoot .The shoot culture is more affective on the medium containing cytokinin, since the rate of shoot multiplication can be enhanced by culturing shoot tip.In such cases a mass of branches of cultured plant can be obtained. Sub-culturing shoot multiplication cycle may be repeated and round the year its cultures may be maintained. 1-15years many species have been maintained in Proliferating shoots.

B. Rooting of in vitro shoots

Roots are absent in the culture formed by adventitious and axillary shoot. So, they most be placed to a root initiating medium which differs from shoot multiplication medium which contain growth hormone composition . Before planting in the mixing plot, the basal cut is treated with the root initiating powder which helps in the formation of root. All cytokinins inhibit rooting. Instead of using BAP in final stage of multiplication, use of kinetin improves rooting. Improvement of root can be done by lowering the concentration of macro salts to a half and the concentration of sucrose from 2 or 3% to 0.5 or 1%.

C. Transplantation

When the rooting is finished the plants are removed from the medium to the soil carefully. It is necessary to maintain humidity for 10-15 days. The plot can be covered with polythene having holes for aeration to maintaining humidity .The suitable time for transplanting is when roots begin to emerge out and the leaves are in photosynthetically self-supporting phase.

Application of micro propagation

  1. The rapid cloning of desirable plants can be done with the help of vitro propagation .So, it is the most powerful and attractive tool for cloning.
  2. Plant tissue culture is suitable for the growth of plants which cannot be multiply by seeds.
  3. Most of the vegetatively propagated crops are suspected to be infected by virus. So, virus free plants can be produced by the technique of meristem tip culture.
  4. The production of hybrid seeds of some crops is very expensive. Some of examples of plants that can be propagated in vitro and required parents multiplied for the production of hybrid seed are ;Broccoli, Cabbage and cauliflower.
  5. As a potential system of germplasm storage, micro propagation has great value.
  6. Commercial nurseries groups different plant for the maintenance of stock plants and it can be minimize by the help of tissue culture.

Meristem tip culture:

Simply, it is an art of science of culturing meristem aseptically in an artificial medium in the laboratory condition to get plants. Meristem is the apical growing part of plants which is always multiplying and diseases free. This culture is capable of producing virus free plants. Therefore, this technique has its major application in eliminating various diseases.

Application of plant tissue culture in agriculture

  1. Micropropagation-It is a important for agriculture because with the help of this technique large number of off spring or plantlets can be obtain within short time.
  2. Production of disease free plant – Tissue culture from shoot tip and meristem tip can produce diseases free plants where as roots, bulb, tuber, etc. are infected by pathogens.
  3. Androgenic haploid- This is the technique in which anther is used for tissue culture to produce haploid embryos. These sorts of plants are important from the research point of view.
  4. Protoplast culture and fusion- Protoplast culture can be used for several activities such as genetic manipulation and somatic cell fusion in the plants. Different non-related species hybrid can be obtained.
  5. Development of resistant plants- Varities of plants can be obtained with the help of plant tissue which can resist several diseases and germs.

Tissue Culture Laboratories in Nepal

Some of the names of tissue culture laboratory in Nepal are as follows:

  • Plant Research Division, Godawori, Latipur
  • Himalayan Floratech, Jawalakhel
  • Nepal Biotech Nursery, Bhaisepati
  • Botanical enterprises

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.

1. Adventitious shoots develop naturally on the plant at a place other than normal axial regions which show the structure like stem and leaf.

2. This process produce genetically identical plants by asexual means which is also called clonal propagation.

3. Generally, calcium or sodium hypochlorite solution is use for the sterilization process of the plant surface.

4. Every vessels that are included in the medium must be sterilized in steel autoclaves or in pressure cooker at 120 degree celcius for 15-20 minutes.

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