Difference between revisions of "DIY Plant Tissue culture and Engineering"

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(Protocol)
Line 82: Line 82:
  
 
One packet of Murashige and Skoog (for 1L)
 
One packet of Murashige and Skoog (for 1L)
 +
 
20g of Sucrose
 
20g of Sucrose
 +
 
100ml of Thiamine
 
100ml of Thiamine
 +
 
1% weight/volume of agar
 
1% weight/volume of agar
 +
 
1L of water
 
1L of water
 +
  
 
Pour into a flask or beaker that can hold that much medium without boiling over
 
Pour into a flask or beaker that can hold that much medium without boiling over
 +
 
Place on a magnetic stirrer until everything is dissolved
 
Place on a magnetic stirrer until everything is dissolved
 +
 
Adjust the pH to approximately 5.6
 
Adjust the pH to approximately 5.6
Autoclave for 20 min in a slow-exhaust liquid cycle.  Let cool 15-20 minutes.
+
 
 +
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
 
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:
 
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.
+
 
 +
Make sure to gently swirl the flask before pouring, to keep the agar from settling
 +
 
 
Pour plates
 
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.
+
 
 +
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
 +
 
  
  
Line 102: Line 115:
  
 
In the sterile hood:
 
In the sterile hood:
 +
 
250ml beaker containing 100ml of 70% concentration alcohol
 
250ml beaker containing 100ml of 70% concentration alcohol
 +
 
Sterile petri dishes for rinsing sterilized leaf cuttings
 
Sterile petri dishes for rinsing sterilized leaf cuttings
 +
 
Sterile petri dish for holding sterilized leaves ready to be placed
 
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 200ml bleach and one drop of liquid detergent
 +
 
500ml beaker containing autoclaved sterile water
 
500ml beaker containing autoclaved sterile water
 +
 
Matchbook to light the flame
 
Matchbook to light the flame
 +
 
Agar plates
 
Agar plates
 +
 
Parafilm
 
Parafilm
 +
  
 
Immerse the cut leaves in the bleach solution for 5 minutes
 
Immerse the cut leaves in the bleach solution for 5 minutes
 +
 
Dip in 70% alcohol for 15 seconds
 
Dip in 70% alcohol for 15 seconds
 +
 
Rinse in sterile water
 
Rinse in sterile water
 +
 
Transfer to a second sterile water rinse
 
Transfer to a second sterile water rinse
  
 
Use the sterile forceps and tweezers to place the leaf cuttings onto your agar plates.
 
Use the sterile forceps and tweezers to place the leaf cuttings onto your agar plates.
 +
 
Cover and seal with Parafilm
 
Cover and seal with Parafilm

Revision as of 11:39, 23 November 2009

== DIY Designed Plant Tissue Culture ==

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

PLEASE NOTE:

<a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/3.0/us/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-sa/3.0/us/88x31.png" /></a>
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