Cloned cauliflower - National Centre for Biotechnology Education

PRACTICAL
BIOTECHNOLOGY
Cloned
cauliflower
ADAPTED FROM AN ORIGINAL PRACTICAL DEVELOPED BY TONY STORR
PRACTICAL EXPERIENCE of plant tissue culture is called
for in many biology and some science syllabuses. The plant most
commonly used for such work in schools and colleges is
cauliflower. This material is ideal for plant tissue culture,
because it is readily available, robust enough to withstand the
rigours of being handled by students and grows rapidly. Growth
can be seen after 10 days and plantlets ready for transplantation
are formed within 12 weeks.
Materials
For each student or group of students:
Cauliflower curd (the white part)
Sterile distilled water, 100 cm 3
70% ethanol, 50 cm 3
20% Domestos solution (Chlorate I solution with
added detergent), 100 cm 3
Test tubes, each containing 2–3 cm 3 of plant tissue
growth medium as described below, 3
Sterile Petri dish
Metal forceps and scalpel
Non-absorbent cotton wool and aluminium foil
To make 1 litre of the plant tissue growth medium:
Granulated sugar, 20 g
Agar, 10 g
Murashige and Skoog (M&S) medium, 4.7 g
Kinetin stock solution, 25 cm 3
From this point on, quick, aseptic operations are
important to prevent contamination.
4. Rinse the explants in three successive beakers of
sterile distilled water. Use flamed, cooled forceps to
do this — the correct way to flame forceps and other
instruments is to dip them in alcohol, then to pass
them briefly through a flame to ignite the ethanol. As
the ethanol burns off, it heats the surface of the
instruments to 70°C, killing any contaminating
organisms. Do not heat forceps and scalpels until red
hot, and remember to keep ethanol away from
exposed flames.
5. The explants can be left in the final beaker of sterile
water (covered with a Petri dish lid) until required.
6. Take the first tube of growth medium, withdraw the
cotton wool plug, then briefly flame the neck. Use
flamed, cooled forceps to pick up an explant and
quickly drop it into the tube. Return the forceps to the
ethanol beaker. Flame the neck of the tube once more,
then replace the plug.
7. Repeat Step 6 with the two remaining explants and
two fresh tubes of growth medium.
8. The tubes should be kept in a warm, light place.
Growth should be visible within 10 days.
Contamination, if it has occurred, should also be
visible by this time. Failure of anything to grow
usually indicates that the bleach has not been rinsed
from the plant tissue.
Safety
Plastic gloves should be worn when handling kinetin.
Students should wear safety goggles when using bleach
solution.
ADDITIONAL
INFORMATION
Plant tissue
culture by Tony
Storr (1985)
Experimenting
with Industry
No.13, Association
for Science
Education.
ISBN: 0 86357 031 3.
This excellent and
inexpensive book
has for many
years been the
guide to plant
tissue culture
work in schools.
The kinetin stock solution contains 0.1 g kinetin in 1 litre of
distilled water. Kinetin does not readily dissolve in water;
the addition of one or two pellets of sodium hydroxide helps
the dissolution process. Stock solution should be stored in a
’fridge at 4°C.
To prepare the growth medium, dissolve the sugar, M&S
medium and agar in 725 cm 3 distilled water. Mix in the
stock kinetin solution, then dispense into test tubes. About
2–3 cm 3 will be needed per tube. Plug the tubes with nonabsorbent
cotton wool and cap them with aluminium foil.
Autoclave at 121°C for 15 minutes in a pressure cooker.
When cool, the tubes may be refrigerated until they are
needed.
M&S medium and kinetin are available from school science
suppliers e.g. Philip Harris Limited.
Practical details
1. Swab the working area with 70% ethanol. Keep the
ethanol away from exposed flames!
2. Cut out a small piece of cauliflower curd; roughly the size
of a cherry. Working on a clean Petri dish, divide the curd
into three.
3. Place the pieces (explants) in bleach e.g. Domestos solution
for 10 minutes to surface sterilize the tissue.
Ethanol used for sterilizing working surfaces should
be kept away from naked flames.
Photograph kindly supplied by students of St. Felix School, Suffolk.

Cloned
cauliflower
1. Cut out a small piece of
cauliflower curd (the white
part).
Sterile test tube
containing
growth medium
with added kinetin
Sterile cotton
wool plug
2. Cut the curd into three
small pieces (explants) on
a clean Petri dish.
Explant
transferred using
sterile forceps
7. Repeat Step 6 with the
two remaining explants.
Label the tubes with your
name and the date.
3. Drop the
explants into
fresh bleach
solution, and
leave them to
surface sterilize
for ten minutes
only.
Leave the explants in
the rinsing water for at
least a minute.
5. Rinse the
explants twice
more in the
same way,
each time
making sure
that the
forceps are resterilized.
Foil
cover
foil, placedAluminium 8. Look at the tubes
after ten days, and
again after a fortnight.
Chlorate
I
solution
4. After ten minutes, transfer
the explants into some sterile
water. Use flamed, cooled forceps to do this.
6. Aseptically transfer the
explant into a test tube
containing growth medium
with added kinetin.
over the cotton wool,
helps to reduce moisture
loss.
© National Centre for Biotechnology Education, 1995