DIY Plant Tissue culture and Engineering

Plant Tissue Culturing

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

Plants from Test Tubes is a great book to get started. Download here: File:Plants From Test Tubes Complete.pdf

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 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

Some more links

Kitchen culture kit: http://www.kitchenculturekit.com/ Archive

discussion on maillist

growing fungi: http://makeprojects.com/Project/Home-Mycology-Lab/242/1

general vocabulary: http://mansfield.osu.edu/~sabedon/biol4035.htm#petri_dish

http://edgeqld.org.au/2013/01/plant-cell-culture/

http://www.scq.ubc.ca/your-guide-to-plant-cell-culture/

How to make slants and stabs:

• In a 400 ml beaker, make about 100 ml of Nutrient agar (but use only about half of the powder that the instructions on the label call for. Heat this until it boils (microwaving is the preferred method but you must watch it closely as it will sudden burst into foam). If heating it in a pan on a hotplate, swirl constantly while heating (pretend it is gravy, which you don't want to burn). It seems best to then pour the stuff from the pan into the beaker, and then pour from that into screw-topped culture tubes - fill the tubes about 1/4 full. Put on the caps almost tight and then autoclave to sterilize.

• Slants: Take tubes immediately out of the autoclave and find the right place to set them on a rather flat incline so that the proper 'slant' is achieved for them to solidify. Once cool and solid, screw caps on tightly to prevent evaporation. Longevity: cultures in slants last for several years if they are stored in the dark and do not dry out.

• Stabs: fill tubes 1/3 full, cap and sterilize. Stand them vertically to solidify. When cool, screw caps on tightly. To inoculate these, stab an inoculated loop down into the agar for about 2/3 way to bottom. Stabs preserve most bacteria for years and years in the dark if the caps are airtight and evaporation doesn't occur.

• Always store cultures in the dark as UV damage accrues from fluorescent lights and sunlight.