PRACTICAL BIOTECHNOLOGY

Poplar tissue culture

3. 4. Petri dish and wet them thoroughly with the kinetin solution. Place the twigs, cut surface uppermost, on the lter paper. Replace the Petri dish lid and secure it well, but not over-zealously, around the rim with adhesive tape. The aim is to reduce evaporation of the kinetin solution, without making it difcult to remove the lid to replenish the solution at intervals as necessary. Keep the Petri dishes in a warm, well-lit place. Examine them at weekly intervals. After a few weeks callus tissue will form. Buds may also grow shortly after this. If mould appears on the uppermost lter paper, just slip it out from under the twigs using forceps, leaving the uncontaminated paper discs below. Should the lter paper begin to dry out, simply re-wet it with distilled water.

PLANT TISSUE CULTURE experiments in school are often a problem: either the plant tissue is insufciently sterilized, or for some reason the tissue just refuses to grow. This is compounded by the high cost of both the tissue culture medium and plant growth substances, and the inordinate length of time it takes to prepare everything. Heres a practical which overcomes all of these difculties. It requires neither aseptic conditions or expensive growth media.

Materials
Freshly-sprouted twigs from poplar trees (Populus sp.) Aqueous solution of kinetin, about 1 cm3 Make up a concentrated stock solution of kinetin in distilled water, then dilute this further to obtain 0.002 g per litre. Kinetin does not dissolve readily in water unless the solution is made alkaline using, for example, a few pellets of sodium hydroxide. 9 cm diameter discs of Whatman No.1 lter paper, 23 Clean Petri dish Adhesive tape or Paralm Sharp knife or scalpel

5.

6.

7.

Safety
Plastic gloves should be worn whilst handling kinetin powder. Spills of kinetin should be washed up promptly, using plenty of water.

Practical details
1. Cut internodal lengths of poplar twigs about 12 cm long, and split them lengthwise using a sharp knife or scalpel. Put 23 lter paper discs into the base of the

Further activities
Students could investigate the effect of light, the concentration of kinetin, interaction with other plant growth substances (such as IAA) or the use of different plant species.

2.

ADDITIONAL INFORMATION
This investigation was suggested in Experiments in plant tissue culture (Second Edtn) by John Dodds and Lorin Roberts (1985) Cambridge University Press. ISBN: 0 521 31516 6. It has been adapted for schools use by John Schollar at the NCBE.

Poplar tissue culture

1. Split the poplar twigs lengthways (a scalpel is more convenient than an axe for this task).

2. Put two or three pieces of filter paper into a Petri dish and moisten them with kinetin solution.

3. Place the split twigs (cut surface uppermost) onto the moistened paper.

4. Seal the Petri dish lid loosely, with sticky tape.

(Dont fix the lid on too firmly, as you may wish to water the twigs from time to time.)

5. Keep the Petri dish in a warm, light place.

Cartoon by Colin Brown

3 weeks later ...

6 weeks later ...

6 months later ...

National Centre for Biotechnology Education, 1995