Cell and tissue culture

Cell and tissue cultureBy,Abhinava J VUniversity of Agricultural Sciences, Dharwad

Tissue culture is a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to survive and function.

Plant tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time.

Setting up of a tissue culture labMedia preparation room

Aseptic transfer area

Culture room

Analytical room

Acclimatization room

STERILIZATION

Physical method

Media PreparationPlant tissue culture media should generally contain some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, source(s) of carbon, undefined organic supplements, growth regulators and solidifying agents.According to the International Association for Plant Physiology, the elements in concentrations greater than 0.5 mM.l-1 are defined as macroelements and those required in concentrations less than 0.5 mM.l-1 as microelements.

MacronutrientsBesides C, H and O, macroelements includes: Nitrogen (N)Phosphorus (P) Potassium (K)Calcium (ca)Magnesium (mg) Sulphur (S)

MicronutrientsIron (fe)Manganese (mn)Zinc (zn)Boron (B)Copper (cu) Molybdenum (mo). Chlorine (Cl)Cobalt (Co)Iodine (I)

CarbohydratesSucroseD-MannitolD-Sorbitol

VitaminsVit B complexAdenineCholineFolic AcidRiboflavinThiamine

Growth regulatorsAuxinsCytokinninsGibberellinsAbscisic acid

AntibioticsAmpicillinCefotaximeGentamicin sulphatePolymyxin B

Amino acidsL-alanineL-arginine

Solidifying agentsAgarAgarose Gellan gum

Stages in Tissue Culture

Tissue must be sterile - completely free of any microoganisms; done using aseptic technique .Starting tissue is called an explant: differentiated cells (these cells have developed to be part of specialized tissue (root, leaf, stem, ovary, cotyledon, etc.).Explants are plated on a sterile petri dish containing hormones and nutrients that promote the explant cells to develop into Callus ( a mass of undifferentiated cells)

Steps involved in Tissue Culture

Individual cells (or clumps of cells) of the callus are transferred aseptically to a different bottles containing sterile medium that encourages the undifferentiated callus cells to become shoots.

From the callus cells multiple shoots were developed then it transferred to other bottles for development into shoots and roots.

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These plantlets were then moved for hardening process.The plantlets were washed free off agar medium and taken to the micropots containing soilrite and then shifted to growth tunnels for further establishment.During primary and secondary hardening process the plants get adjusted to the environment. These plants were then ready to planting in the field.

Application