[RajniSahni]

Plant Tissue Culture: A field of possibilities

I would like to share my knowledge of Plant Tissue Culture here in a way that might help a newbie/layman to understand what Plant Tissue Culture is; and what are the possibilities associated with this field of extra-ordinary biotechnical talent.

Let's start with: What is Plant Tissue Culture ?

Plant Tissue Culture refers to the art of growing "plant cells or tissues" inside the confinements of a laboratory in glass tubes/petridishes/bioreactors using synthetic growth media and plant hormones (growth regulators). Whenever an experiment is performed in glass apparatus (typical of a laboratory set-up), we call it an "in-vitro" approach. Thus, the art of growing plant cells/tissues in-vitro, may be referred to as "Plant Tissue Culture".

Following is a photographic summary of what plant tissue culture looks like:

Now, let's shift to: How Plant Tissue Culture is carried out?
(i) Explant: Plant Tissue Culture often starts with the 'choice of Explant'. Now, "What is an Explant?" Explant refers to the part/tissue of the plant which is used to start tissue culture. For example, if you take plant leaves as the part for initiating the tissue culture, then explant in that case is Leaf tissue. Whereas in most plants, the tissue culture can be initiated from explant obtained from any part of leaf/stem/root/shoot/flowers, in some plants, explants obtained from different parts exhibit different growth rates. It is thus advisable to choose explant carefully. Not only this, your choice of explant can greatly decide the haploid/diploid nature of the tissue grown (anther used as explant will lead to haploid culture).

(ii) Sterilization of Explant: The next step towards plant tissue culture involves sterilization of the explant. Sterilization is very important for following reasons:
a) Explant exposed to natural conditions is expected to be contaminated with plethora of microbes and fungi.
b) The contaminants (microbes/fungi) have very high growth rate as compared to plant cells, thus they can easily overgrow on the media on which plant cells are expected to grow.

But, sterilization doesn't mean heating/autoclaving the explant (which would lead to death of the sensitive cells). Careful surface sterilization of the explant is carried out in a well defined protocol. Following is the protocol for Sterilization of Explant (assuming a leaf):

1. Wash your hands with soap and hot water. Excise several healthy leaves, both young and old, and discard the petioles.
2. Wash the leaf blades briefly in cool soapy water. Rinse them in running tap water, and prepare for aseptic procedures.
Aseptic procedure means that your entire working area inside the laminar hood should be aseptic (sterilized). Aseptic conditions can be maintained as follows:

• Ensure that you have washed your hands well with an anti-septic soap/solution before handling any cultures or reagents.
  
• Switch on the laminar flow hood (this should start the air flow from HEPA filters). DONOT switch on the UV light right now, while you are creating aseptic environment!
  
• Wipe down the sides, top and bottom with a clean paper towel and
  

• Wipe the surface of all materials you will need with 70% ethanol, before placing them into the hood.
  
  
• Bottles, pipettes, forceps, etc. should be placedtogether in a rear corner of the hood to avoid any obstructeions in the workspace (Be sure not to block the airflow!).
  
  
• To maintain sterility always work toward the back of the hood and try not to put your hands behind the cultures (this could blow spores from your hands into the culture).
  
  
• All apparatus should be well autoclaved (cutting blades, forceps, pipettes, scissors, empty flasks etc)
  
  
• Media transfers and preparations should be performed inside the hood in front of a flame.
  
  
• Culture bottles shouldn't be left open inside the hood.
  
  
• Tools like scoops, blades should be sterilized by flaming before reusing them.
  
  
• When finished, remove all materials from the hood then ethanol wipe all surfaces in the hood.
  

3. Dip the leaf blades in ethanol (70% v/v) for 5 seconds and rinse in sterile DDH2O  (Double Distillled Autoclaved water) in a 250-cm3 beaker.

4. Complete the surface sterilization by immersing the blades in the 10% hypochlorite solution for 10 min.

5. Make sure to handle the leaves with only sterile, flamed forceps (but not with hot blades/forceps!).

6. Each blade is transferred to a sterile Petri dish containing filter paper, and explants are prepared with forceps and scalpel. The filter paper will remove the excess moisture from the final DDH2O rinse.

7. The leaf tissue most effective in organogenesis is located in the central part, and the outer margins and leaf tip are relatively unproductive. Slice the blade into rectangles approximately 10-12 mm of a side, ensuring that each explant contains a portion of the mid-vein of the leaf.

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Keep watching this space for further information on this topic.

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References:

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http://passel.unl.edu/
wikimedia
Bhojwani and Razdan's PTC

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