DIY Plant Tissue culture and Engineering

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== Plant Tissue Culturing ==

For the purpose of creating newly grown leaves from leaf cuttings, in-vitro

Materials and Equipment Needed

For making the plates:

Petri dishes

Erlenmeyer Flasks

Graduated Cylinders

Scale and Measuring Accessories

Magnetic Stirrer

Pipettes and Tips



Ionized water



Murashige + Skoog Packets

pH Meter


NAA (naphthaleneacetic acid)

BAP (benzyladenine)

For sterilizing the leaves:

Sterile Hood

Sterilized Water

Empty Sterilized Petri Dishes



Liquid Detergent (Household OK)

70% Alcohol




Sterile gloves


Freshly cut Nicotiana Tabacum Leaves


Preparing the plates Medium for 1L

4.3g of Murashige and Skoog w/Gamborgs Vitamins

20g of Sucrose

1% weight/volume of agar

1L of water

Pour into a flask or beaker that can hold that much medium without boiling over

Place on a magnetic stirrer until everything is dissolved

Adjust the pH to approximately 5.7

Autoclave for 20 min in a slow-exhaust liquid cycle. Let cool 15-20 minutes

Add 100 µl Naphtalene Acetic Acid (NAA) [1mg/ml] and 1 ml 6-Benzacyl Aminopurine (6-BAP) [1mg/ml]

You may wish to add antibiotics and antimycotics at this time

Thoroughly wash hands, put on sterile gloves and in a sterile hood:

Make sure to gently swirl the flask before pouring, to keep the agar from settling

Pour plates

Let cool with sterile air blowing over it and the lids off. Once the agar has set, place the lids and leave in a sterile hood until its time to place the leaf tissue

Sterilizing the leaves

In the sterile hood:

250ml beaker containing 100ml of 70% concentration alcohol

Sterile petri dishes for rinsing sterilized leaf cuttings

Sterile petri dish for holding sterilized leaves ready to be placed

500ml beaker containing 200ml bleach and one drop of liquid detergent

500ml beaker containing autoclaved sterile water

Matchbook to light the flame

Agar plates

Parafilm or Micro-pore tape

Immerse the cut leaves in the bleach solution for 5 minutes

Swirl in 70% alcohol for 10 seconds

Rinse in sterile water

Transfer to a second sterile water rinse

Use the sterile forceps and tweezers to place the leaf cuttings onto your agar plates.

Cover and seal with Parafilm/Micro-pore tape