Plant Tissue Culture Techniques: 6 Methods & Protocols
Plant Tissue Culture is the process of growing an 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 artificial nutrient media under aseptic conditions, in glass containers.
This is a technique by which new plants can be raised on artificial nutrient media by 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 plant, a new plant can be grown.
This capacity of plant parts or cell to grow into a full plant is termed as “toti potency”.
Media for tissue culture: Nutrient media plays an important role in tissue culture. It is very vital for 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 ingredient 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.
Inorganic elements: Macro elements:
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 micro nutrients 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, aaspartic acid, glutamic acid, glutamine, methionine.
Indole-3 Acetc acid.
Napthalene 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. pH of 5.2 to 5.6 is best with temp of 25o c.
Tissue culture equipment like Complete air conditioned lab, laminar air flow, autoclave, BID incubators, Shakers are also needed.
You can finde 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.
Oxygen supply: 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 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 green house. Tissue culture plants are highly sensitive to tolerate natural environment conditions. They have to be slowly adopted to normal atmosphere. So first they are to be grown in green houses.
Tissue growth curve:
When a cell or tissue is incubated in nutrient media, it shows phase different 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: The phase of adjustment to new environment. Here the cells just grow in size but don’t multiply.
b) Exponential phase (log phase): Here the cells multiply profusely and grow in numbers. This is a useful phase to produce bi-products in large quantities.
c) Decline phase: Here the multiplication of cells slows down. Nutrients are exhausted.
d) Stagnant phase: The cells here remain in fewer numbers without further multiplication. This is due to lack of nutrients, accumulation of toxins etc.
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 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 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 are much higher in comparison to those grown solid-agar medium. Besides, this technique provide 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: Techniques and Experiments
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 exponential phase, the entire culture is replaced with new one. It is closed type of culture.
2) Continuous suspension culture: Here there is continuous addition of nutrition media. The dilution rate is such that an equivalent volume of media is removed out proportional to the in flow from top. The cells are always kept in 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 proliferate till completion of exponential phase. Then there is fresh addition of nutrient media & culture media.