Growth Hormons Avocado

1. Bio.Kitchen
1
In Vitro Avocado propagation (cloning protocol)
Step one: preparation of plant growth hormones
There are two main groups of plant hormones (phytohormons) used for in vitro
propagation: Cytokinins and Auxins. They are used to induce shoot and root
growth. We will use Cytokinin: 6-Benzylaminopurine(BA) and Auxin: Indole-3-
butyric acid (IBA). To obtain 1mg/ml stock solution of IBA:
1. Take 100ml Duran bottle and add 5ml of 1N NaOH
2. Wight 0.1g of IBA and add to the bottle
3. Shake the bottle and add 95ml of distilled sterile water
To obtain 1mg/ml stock solution of BA:
1. Take 100ml Duran bottle and add 5ml of 1N NaOH
2. Wight 0.1g of BA and add to the bottle
3. Shake the bottle and add 95ml of distilled sterile water
In the next step the stock solution are added to plant growth media
Step two: plant growth media preparation
Plant growth media is a simulation of the soil. It is defined and controlled mixture
of macro and micro elements, vitamins and nutrients needed for plant growth,
free of contaminants or chemical suppressors. We will use MS or Woody Plant
Medium (WPM) and add phytohormones.
1. Take 500ml Duran bottle and feel them with 150ml of distilled sterile water
2. Wight 0.46g of WPM and add the powder to the Duran bottle with the
water
3. Add 2 tablets of Agarose and microwave for 2 minutes
4. Add 20ml of BA and IBA stock solution to the mixture to obtain final
concentration of 0.1mg/ml phytohormons in the medium
5. Measure pH with the pH meter and adjust it to 5.7
6. Sterilise the medium in the autoclave with standard program
Step Three: preparation of flasks with growth media
1. Work in the Biosafety cabinet
2. Pure 200ml of the sterilised growth medium in sterile flasks
3. Let is solidify

2. Bio.Kitchen
2
Step four: Preparation of plant material
1. Cut the fresh axillary shoot (explant) from the mother plant (3-5cm length)
2. Immerse the explant in 0.5M NaOH solution for 10min
3. With the sterile pinsetter transfer the explant in the sterile distilled water
4. With the sterile pinsetter transfer the explant in the 70% ethanol
5. With the sterile pinsetter transfer the explant in the sterile distilled water
again
Step five: cultivation of cloned explant
6. With the sterile pinsetter place the explant in the solidified growth media
7. Place the flask under the light on room temperature (25°C)
8. Monitor the growth for following two weeks
In case of successful sprouting cloned plant can be re-cultivated in the fresh
medium with additional hormones and supplements. Addition of certain elements,
such as peptone, activated charcoal, different phytohormons (IAA), antibiotic,
antiviral agents etc. or changing growth condition gives an opportunity to
manipulate and enhance the new plantlet.