Plant tissue culture is a set of methods to grow plant cells, tissues, organs or a whole plant under a sterile condition on a nutrient culture medium. Here are some of the essential tools and techniques:
Techniques:
Sterilisation:
Ensures the environment, tools and plants tissues are free of contaminants.
Common methods: autoclaving, filtration and using sterilising agents like ethanol and sodium hypochlorites.
Preparation of culture media:
Nutrient rich media provides necessary minerals, vitamins and hormones for plant growth.
Murashige and Skoog medium is widely used for general plant tissue culture.
Inoculation and Transfer:
Plant tissues (explants) are placed on the culture medium in a sterile environment, usually inside a laminar flow hood.
Explants may include leaves, stems, roots or meristems depending on the culture goal.
Callus Culture:
This involves including undifferentiated cell masses (callus) from explants using growth hormones.
Callus Culture is essential for plant regeneration and transformation of studies.
Organogenesis and somatic embryogenesis:
Organogenesis: The development of organs ( shoots or roots ) from callus or directly from explants.
Somatic Embryogenesis: Developments of embryos from somatic (non-reproductive) cells, which can grow into whole plants.
Micropropagation:
A technique for producing large numbers of identical plants (clones) quickly.
Involves stages like shoot multiplication, rooting and acclimatisation for growth outside the lab.
Protoplast Culture:
Involves isolating the cells without the cell walls (protoplasts) and culturing them to regenerate into whole plants.
User for genetic engineering and hybridisation studies.
Tools
Laminar Flow Hood: Provides a sterile environment to handle plant tissues and prepare cultures.
Autoclave: Sterilizes media, tools, and containers using high-pressure steam to kill all microorganisms.
Petri Dishes and Culture Tubes: Used for holding and growing plant cultures.
Scalpel and Forceps: For handling and cutting plant tissues under sterile conditions.
Growth Chamber/Incubator: Controls temperature, light, and humidity to create optimal growth conditions for cultures.
pH Metre: Ensures the nutrient medium has the appropriate pH, usually around 5.5-6.0 for most plant cultures.
Microscope: Allows examination of cultures, particularly useful for observing cellular changes and checking contamination.
Plant Growth Regulators (PGRs)
Auxins (e.g., IAA, NAA): Promote rooting and callus formation.
Cytokinins (e.g., BAP, Kinetin): Encourage shoot formation and multiplication.
Gibberellins: Promote cell elongation, used in some specific growth stages.