Plant Tissue Culture:

FROM THE CSIRO LAB AT SCITECH:
There is a program in the CSIRO Lab at Scitech called PLANTECH.
Another sterile technique of tissue culture, carried out in laminar flow cabinets, is being offered for high schools, called an embryo rescue. It is also used in conservation biotechnology at Kings Park, plus a huge range of other plant biotechnology and botanic activities.
Agar is not actually needed to do enquiries into the effect of plant hormones on growth.
Plant rooting hormones and potting mix or vermiculite can simply be used. The best way to measure roots is to harvest the plant, wash it out and use a grid that you lay the roots across and count the number of intersections of roots across the lines.
Hormones can be bought at gardening and hardware stores and probably at scientific suppliers.
AGAR CULTURE:
Low grade agar powder is fine.
Sugar needs to be in the agar, plus a complete fertiliser and M&S (Murashige and Skoog) basal medium
Sugar encourages fungal and bacterial growth, so the vials must be sterilised in an autoclave or pressure cooker. Be aware that some types melt, some don’t.
PLANT TISSUE CULTURE:
Prepare media for agar vial tissue culture at least one day before class
Mix in 2 litre beaker:
1 bottle MS (nutrients) 4 g (rinse out)
1 litre distilled or tap water
30 g sucrose. Sucrose aids photosynthesis but also promotes microbial growth
Adjust pH to 6 with sodium hydroxide solution or HCl (10%), added drop-wise with a pipette
8 g agar
Let it boil to mix agar (in microwave), approximately 5 minutes
Pour into polycarbonate vials ready to autoclave (sterilise)
Eg: 10 ml in 120 mL vial. Smaller containers mean that can get more out of the 1 litre agar.
Prepare tools for autoclave or pressure cooker.
Requirements:
Livingstone Mediplast-pressure tape (autoclave masking tape with colour change)
2 forceps, 2 scalpels or dissecting scissors
Sterilise laminar flow cabinets
Wipe down regularly with ethanol
Leave UV light on for at least 20 minutes
Or do this in a sterilised aquarium. This is not as good but much cheaper.
CLONING:
You can propagate plants from softwood cuttings.
Auxins (plant hormones) induce new root formation.
In horticulture, auxins, especially Indole-3-butyric acid and Indole-3-acetic acid, are commonly applied to stimulate root growth when taking cuttings of plants.
In a couple of weeks, plants will have adventitious roots.
Adventitious roots differ from the more usual root formation of branches of a main primary root, and instead originate from the stem, branches, leaves, or old woody roots.
LOOKING AFTER YOUR CLONED PLANT:
The cloned plant can be transplanted within three weeks, when it has started to develop adventitious roots. It can transplanted it into a pot with potting mix.
The new plant is a clone as it has the same genetic material (DNA) as the parent plant from which it was cut.
PLANT TISSUE CLONING:
What to do
1. Wash and dry a pair of scissors. Clean scissors are required to avoid infection and disease.
2. On the petri dish, cut a piece of shoot from a main stem of your parent plant making sure there is a node.
3. Remove most of the older leaves so that the plant doesn’t use too much water or die because of a lack of roots. Place unwanted plant material in the bin.
4. Keep only the young growing shoot. This called an explant.
5. Make sure that your explant is the right way up.
6. Dip the bottom end of the explant into the auxin (plant hormone).
7. Shake off any excess root hormone.
8. Remove the lid from your vial.
9. Push the explant into the agar.
10. Replace the lid.
It is important to shake off surplus auxin (plant hormone) as too much auxin will stop root growth. A small amount of root hormone will make adventitious roots grow.

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