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

Foil

Agar

Ionized water

Sucrose

Thiamine

Murashige + Skoog Packets

pH Meter

Autoclave

NAA (naphthaleneacetic acid)

BAP (benzyladenine)


For sterilizing the leaves:

Sterile Hood

Sterilized Water

Empty Sterilized Petri Dishes

Beakers

Bleach

Liquid Detergent (Household OK)

70% Alcohol

Flame

Forceps

Tweezers

Sterile gloves

Parafilm

Freshly cut Nicotiana Tabacum Leaves

Protocol

Preparing the plates

One packet of Murashige and Skoog (for 1L)

20g of Sucrose

100ml of Thiamine

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 5.6

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

Add 5mg/liter of NAA and 10mg/liter of BAP and stir with a magnetic stirrer

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


Immerse the cut leaves in the bleach solution for 5 minutes

Dip in 70% alcohol for 15 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