Plant Tissue culture - Basics

Hi,
I know a bit about Plant tissue culture and I would like to share it here.

Plant Tissue culture Rack

Plant Tissue culture, to be simple, it is growing plants in laboratory (invitro). To be more simple, you grow plants in test tubes and bottles by providing them light, nutrient by mimicking the natural conditions.
It is effectively used as tool for producing GM(Genetically Modified) plants.

Don't confuse "plant tissue culture" with "HORTICULTURE"

The basic of Plant Tissue Culture :


Totipotency: The ability of a single plant cell to grow into an entire plant is generally referred as totipotency.

Plasticity: The ability of plants to survive in extreme conditions by modifying their metabolism is generally referred as plasticity.

Things you need to do plant tissue culture
1) Laminar air hood
2) Plant growth chamber
3)Chemicals for preparing medium
4)Autoclave
5)pH meter
6) Test tubes or Bottles with caps
7) Cotton plugs
8) Scalpels, Forceps, petriplates

I think, that's it. With the above mentioned things you can do plant tissue culture.

STEP 1: Preparation of Glasswares:
Wash your bottles, tubes, scalpels, forceps, petriplates with soap solution.
Acid wash them again.

Preparation of acid solution:
For 1 liter of water add 100 grams of Potassium dichromate.
Dissolve it well.
To this solution add 100 ml of  sulphuric acid along sides.
Note: Be very careful, else, you would get hurt with hot acid vapors.

After acid wash, wash them with double distilled water and rinse well.

STEP 2: Preparation of medium:
Generally MS(Murashige Skoog) medium is used. After dissolving the appropriate amounts of macro and micro nutrients in  sterile distilled water, set pH about 5.6   to 5.8. The pH must be strictly maintained for proper growth. If needed you can add coconut water to your medium after filter sterilising it. Coconut water is a rich source of plant growth hormones like Auxins, cytokinins etc.,

After this, autoclave your medium and pour it into bottles or test tubes in which you are going to grow your plant.

Adaxial surface of leaf

STEP3: Sterilisation of inoculum:
Inoculum is what you are going to inoculate. It may be seed,
If you are going to inoculate seeds. Wash them thoroughly with tap water. Then with mild detergent .Then with distilled water (to remove excess detergent). Then rinse with Mercuric chloride solution of 0.1 % and then wash again with Sterile distilled water for 5 times (each time 5 minutes) to remove traces of mercuric chloride.

Now wipe the seeds and let them dry for sometime. From the washing step with mercuic chloride, carry out in laminar hood using beakers.

If you are going to use leaf or shoot extract. Wash it in tap water thoroughly to remove dust particles. Soak in drinking water for 10 minutes. Then, soak in a beaker containing detergent and shake well for 10 minutes. Then, wash with sterile water. Then, use 0.1% of mercuric chloride for 3minutes and rinse well. Then, wash the explants with sterile distilled water for 5times (five minutes each time) Then, remove excess water with sterile tissue paper.
From the washing step with mercuic chloride, carry out in laminar hood using beakers.

STEP4: Inoculation of explant material:
Scalpels, Forceps, petriplates - the things used for inoculation must be STERILIZED ones. The seeds must be taken with the forceps and inoculate (gently place over the surface of the already prepared MS medium.
The leaves, roots or shoot explant which were surface sterilized as mentioned above - cut them using scalpels and inoculate using forceps. The leaf explants must be placed such that adaxial(upper) side is down.

Growth after incubation - Plant Tissue Culture

STEP4: Incubation:
Incubate in plant growth chamber or Tissue culture rack at the optimum temperature and light intensity for the plant you are using for culture. And provide appropriate dark and light period.

You can find your inoculum growing after a few days :) :)

Happy culturing :)


Note: I have just started learning Plant Tissue Culture techniques. So, there may be mistakes with the above mentioned procedure.