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Basic techniques and tools in Plant Tissue Culture.

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: 

  1. Sterilisation:

    1. Ensures the environment, tools and plants tissues are free of contaminants. 

    2. Common methods: autoclaving, filtration and using sterilising agents like ethanol and sodium hypochlorites. 

  2. Preparation of culture media: 

    1. Nutrient rich media provides necessary minerals, vitamins and hormones for plant growth. 

    2. Murashige and Skoog medium is widely used for general plant tissue culture. 

  3. Inoculation and Transfer:

    1. Plant tissues (explants) are placed on the culture medium in a sterile environment, usually inside a laminar flow hood.

    2. Explants may include leaves, stems, roots or meristems depending on the culture goal.

  4. Callus Culture:

    1. This involves including undifferentiated cell masses (callus) from explants using growth hormones. 

    2. Callus Culture is essential for plant regeneration and  transformation of studies. 

  5. Organogenesis and somatic embryogenesis: 

    1. Organogenesis: The development of organs ( shoots or roots ) from callus or directly from explants. 

    2. Somatic Embryogenesis: Developments of embryos from somatic (non-reproductive) cells, which can grow into whole plants. 

  6. Micropropagation: 

    1. A technique for producing large numbers of identical plants (clones) quickly. 

    2. Involves stages like shoot multiplication, rooting and acclimatisation for growth outside the lab. 

  7. Protoplast Culture: 

    1. Involves isolating the cells without the cell walls (protoplasts) and culturing them to regenerate into whole plants. 

    2. User for genetic engineering and hybridisation studies. 



Tools 

  1. Laminar Flow Hood: Provides a sterile environment to handle plant tissues and prepare cultures.

  2. Autoclave: Sterilizes media, tools, and containers using high-pressure steam to kill all microorganisms.

  3. Petri Dishes and Culture Tubes: Used for holding and growing plant cultures.

  4. Scalpel and Forceps: For handling and cutting plant tissues under sterile conditions.

  5. Growth Chamber/Incubator: Controls temperature, light, and humidity to create optimal growth conditions for cultures.

  6. pH Metre: Ensures the nutrient medium has the appropriate pH, usually around 5.5-6.0 for most plant cultures.

  7. 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.