Plant Tissue Culture
Plant Tissue Culture Introduction:
Plant tissue culture - Plants are inevitable in one’s life. They are essential in our routine. Right from our foods to medicine they have vital role.
So, with growing demand for plants how they will be produced in large number. Conventional breeding practices could not satisfy the growing demand. This is where plant tissue culture comes into picture.
Plant tissue culture definition: It is defined as culturing/growing plants using its seeds, organ, tissues, explants, cells, protoplasts on a chemically defined synthetic nutrient media under controlled conditions to produce multiple copies of the plant.
In other words, it is the invitro cultivation of plant cell.
Gottlieb Haberlandt , an Austrian botanist is the first person to study plant tissue culture. He defined the possibilities of culturing the tissues. He is known as the father of plant tissue culture.
Before elaborate discussion of plant tissue culture, we must know concept of totipotency. Totipotency is the ability of single cell to grow, differentiate into an entire organism.
All cells don’t have this property, also it varies by genotype, tissues etc., but plant cell have this totipotent property.
Laboratory Requirements:
The main element for a tissue culture technique is laboratory facilities with washing facility for glassware and ovens for drying
There should be separate room for media preparation, with autoclave for sterilization along with pH meter, automatic shaker and weighing balance.
Transfer area must have laminar air flow chamber.
Incubation area with controlled conditions such as proper light setting, temperature and air flow plays a crucial role.
Plant Tissue Culture:
There are plant tissue culture techniques that are to be followed carefully for the successful tissue culture. The following are steps involved in plant tissue culture.
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Sterilization:
The first and foremost requirement for tissue culture is sterile area. Right from the culture room to the scalpels to be used must be sterilized
Glasswares used for the tissue culture should be autoclaved at 121 ⁰C for 15-30 minutes, followed by drying in hot air ovens. Scalpels are also sterilized by 70% ethanol followed by flaming and cooling.
Preparation Of Media:
The critical step of a tissue culture process is media preparation. Composition of a medium along with growth regulators is a must for successful development of plants. Not all plants use same composition of a nutrient medium, it varies according to the type of plant involved.
MS (Murashige and Skoog) medium is commonly used. White medium, Nitsch medium, B5 medium are other commonly used mediums.
Each differ in concentration. But their composition is similar. They all will have Macronutrients, micronutrients, minor nutrient, iron, growth hormones and vitamins.
The medium may be in solid, liquid or semi solid state. For solidification (the process in which a liquid becomes a solid), agar is added.
Culture Conditions:
Temperature: Optimal temperature is 25°C ± 2°C
Light: 16 hours of white light illumination
Aeration: For liquid medium, aeration can be done with the shakers and sometimes sterilized air is passed through the medium.
Micropropagation :
Micropropagation is a method of plant propagation using extremely small pieces of plant tissue from the mother plant. It is a vegetative propagation of new plants in tissue culture. Tissue culture is a starting step of micropropagation. It is an important plant tissue culture technique.
Micropropagation Has Five Steps:
- Selection of mother plant: A mother plant should be selected for initiation. It should be disease free. Required part (shoot, stem, leaves). The collected part is called explant. It is a part of the plant by which a whole plant can be produced through plant tissue culture technique.
- Initiation step: The collected explant is disinfected to avoid contaminants. Sodium hypochlorite or commercial bleach is used for this. The collected explant is rinsed in the disinfectants followed by rinsing them in the water. This step can be repeated few times. And the explant is placed in suitable nutrient media under aseptic conditions. This is the stage where induction of callus is done. Callus is a mass of unorganized cells.
- Multiplication stage: The callus differentiates into somatic embryos, these are sub cultured in nutrient mediums to produce plantlets. The sub cultured callus is induced to produce vegetative shoots by adding cytokinin in the medium.
- Preplant stage: This is where the roots are induced. Auxin is used to induce the roots. But this step is skipped if the explant is obtained from easily rooting plants.
- Hardening: The growing plantlet with developed shoot, root from invitro is transferred to greenhouse and then gradually subjected to normal environment.
Suspension cultures: In multiplication stage instead of mass cells, separate cells are put into liquid suspension culture, which is continuously agitated using rotary shaker. This continuous rotation keeps the cells separate. After the production of sufficient cells, subculturing is done.
Types Of Plant Tissue Culture:
Based on the type of explants, there are few types of plant tissue culture.
Organ culture: Anthers, ovaries, leaves, roots, shoots, flowers, seeds. Example: Orchid is grown under organ culture.
Meristem culture: When a meristematic tissue is the explant, it is meristem culture. Example: Banana.
Protoplast culture: Protoplasts are cells without cell wall. It regenerates into whole plant and into somatic hybrids. Example: Rice, lettuce.
Application Of Plant Tissue Culture:
Disease free plants can be produced. Healthy plants are recovered form disease infected mother plant.
Tissue culture is used to obtain the clones of a plant through micropropagation
Transmission of pest, virus, disease can be eliminated when grown under aseptic conditions
Plant tissue culture helps in conservation of endangered plant species, thus plays a crucial role in biodiversity
Ornamental plants can be produced in large numbers. Horticulture is truly benefitted by this
Unlike other conventional methods, plant tissue culture can be performed throughout the year
Primary and secondary metabolites can be produced using suspension cultures
Transgenic/genetically modified plants can be produced in masses, just by a single modified cell.
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