Plant Tissue Culture is the process of growing isolated plant cells or organs in an artificial nutrient media outside the parent organism.
In other words, it is an in vitro culture of plant cells or tissues on an artificial nutrient media under aseptic conditions, in glass containers.
This is a technique by which new plants can be raised by the use of plant parts or cells. These small parts can be pollen, leaves, seed, root tip, embryo, etc.
Since all the above organs or cells contain the same genetic material as that of parent plants, a new plant of the same variety can be grown.
This capacity of plant parts or cell to grow into a full plant is termed as “totipotency”.
Media for tissue culture
Nutrient media plays an important role in tissue culture. It is very vital for the proper and timely growth of cells and their multiplication.
Since nutrient media is the only source of nutrition, it should supply all the basic requirements. These include carbohydrates, amino acids, minerals, hormones, and salts, etc at proper proportions. It should be sterile and be non-toxic to the tissue or cell under culture.
All the ingredients of the media are to be sterile hence one can use autoclave or membrane filters based on their thermal resistance power. Tissue culture media preparation should be done in aseptic rooms and conditions.
Potassium supplement: As KCl or KNO3 or KH2PO4.
Calcium supplement: CaCl2.2H2O, Ca (NO3)2. 4H2o.
Magnesium source: MgSO4.
Phosphorous source: NaH2PO4.H2O, KH2PO4, NaCl.
manganese supplement: MnCl2,
Zinc source: ZnSO4.
Copper source: CuSO4.
Aluminum source: AlCl3.
Other micronutrients like bismuth, molybdenum, nickel are also added.
Carbon source– Sucrose/glucose 2-4%
Thiamin HCl (o.o1mg/lit)
Nicotinic acid (o.5mg/lit)
Folic acid (0.5mg/lit)
Pantothenic acid (0.5mg/lit).
Amino acid source: Arginine, aspartic acid, glutamic acid, glutamine, methionine.
Indole-3 Acetic acid.
Naphthalene acetic acid.
2,4 D (2,4-dicho phenoxy acetic acid).
Kinetin, zeatin, benzyl adenine.
coconut water, yeast extract, malt extract, casein hydrolysate.
Also one can include activated charcoal to adsorbs impurities from media. the pH of 5.2 to 5.6 is best with a temp of 25o c.
Tissue culture equipment like Complete air-conditioned lab, laminar airflow, autoclave, BID incubators, Shakers are also needed.
You can find more at Plant Tissue Culture: Techniques by American horticulture society.
Steps in tissue culture
1. Tissue or cell of an interesting plant is selected and sterilized (disinfected) by mercuric chloride or alcohol.
Sterilization of cells
The cells taken for tissue culture are to be surface sterilized. This helps the cell wall and tissue surfaces to be free from any bacterial or fungal infections. Care should be taken during their handling, transfer, etc. to keep them free from infection.
Since tissue has no direct mechanism to take up oxygen, oxygen supply has to be provided. The gas should be free from contamination and also aseptic. The rate and pressure of the flow of gas into the chamber of tissue culture should be optimal.
3. The tissue or cell multiplies and then forms plant-lets.
4. This can be transplanted to the greenhouse. Tissue culture plants are highly sensitive to tolerate natural environment conditions. They have to be slowly adapted to a normal atmosphere. So first they are to be grown in greenhouses.
Tissue growth curve
When a cell or tissue is incubated in nutrient media, it shows phase difference in growth. There are four phases of tissue growth and when a graph is plotted with growth versus curve, we obtain tissue growth curve. This growth curve has
a) Lag phase
This is the first phase of the cell or tissue in the artificial nutritional media. As you can see in the above graph, the phase shows a horizontal line parallel to X-axis.
In this phase, the cell or tissue tries to adopt and get adjusted to the new environment out of the mother plant. Here the cells just grow in size but don’t multiply. Hence, the weight of tissue slightly increases but the number of cells in the media is the same.
b) Exponential phase (log phase)
Here the cells multiply profusely and grow in numbers. The line in the graph shows a prominent rise from the lag phase.
In this phase, the physical size of the callus (mass of the tissue) in the media grows enormously. This is a useful phase as the desired bi-products are produced in large quantities into the media.
c) Decline phase
The curve suddenly shows a decline in the graph of tissue growth. In this phase, the nutrients in the media are exhausted. Due to this, the multiplication of cells slows down. The size of the callus too starts to decrease than the previous phase. Even the production of bioproducts too decrease
d) Stagnant phase
This is the final phase of tissue culture. The curve in the graph shows a straight horizontal line.
The cells here remain in fewer numbers without further multiplication. This is due to the lack of nutrients, accumulation of toxins, etc.
The decline phase and the stagnant phase can be seen in batch cultures but not in continuous cultures.
Plant Tissue Culture Techniques
There are mainly two major techniques in plant tissue culture.
a) Static culture (Solid-agar Medium)
It can also be called as callus plant tissue culture. In this procedure, the plant-tissue is grown on a solid agar medium and always gives rise to tissue mass called a callus. This callus culture technique is easier as it is easier and even convenient for the initial maintenance of cell-lines, and also for carrying out the investigation studies related to organogenesis i.e organ formation.
b) Suspension cultures (Liquid media)
Here the cell aggregates or even single cells are grown in liquid culture. The cells are kept suspended by using agitators/shakers/ impellers. The actual growth rate of the liquid-suspension cultures is much higher in comparison to those grown solid-agar medium. Besides, this technique provides much superior control over the growth of biomass as the cells are always surrounded by the nutrient medium completely.
For more techniques, you can read Plant Tissue Culture: by American horticultural society.
Types of suspension culture:
1) Batch suspension cell culture
Here the cells or tissues are grown in a fixed volume of nutrient medium. Once the cells reach the exponential phase, the entire culture is replaced with a new one. It is a closed type of culture.
2) Continuous suspension culture
Here there is the continuous addition of nutrition media. The dilution rate is such that an equivalent volume of media is removed out proportional to the inflow from the top. The cells are always kept in the exponential growth phase.
I) Open type: Here the system is kept continuous with constant addition and removal of cultured cells.
II) Closed type: Here cell proliferates till completion of the exponential phase. Then there is a fresh addition of nutrient media & culture media.