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