DIY Plant Tissue culture and Engineering

== DIY Designed Plant Tissue Culture ==

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

PLEASE NOTE:

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DIY Designed Plant Tissue Culture by Allison Kudla is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/3.0/us/">Creative Commons Attribution-Noncommercial-Share Alike 3.0 </a>.

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 Laser-cut or Die-cut Nicotiana Tabacum Leaves (still living)

Protocol

Preparing the plates, amounts are for 1L

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