Maximum Yield Australia/New Zealand March/April 2018 | Page 38

tissue culture
The basic process of plant multiplication with tissue culture
starts with healthy, well-grown stock plants from which the
propagation material is taken from. In this example, we’ll
use African violet, as it is an easy-to-work-with plant used by
many to develop their culturing skills before venturing onto
more valuable species. The first step is to cut a mature leaf
from the parent plant and sterilise this material by immers-
ing it in a solution of diluted bleach. This kills any surface
microbes that would otherwise contaminate the plant mate-
rial inside the culture flask.
The leaf is then rinsed with sterilised water and further
prepared inside a sterile still air cabinet, or “clean box,”
which is used whenever working with open flasks and
transferring the plant material. In commercial tissue
culture labs, a laminar flow hood carries out this role
of preventing microbial contamination of the sterilised
flasks and media by passing filtered air from the rear of
the hood outward on a positive pressure gradient. In small
or non-professional settings, a countertop cabinet can be
made using a glass fish tank on its side, a large plastic
container, or even a do-it-yourself cabinet made from
plastic film stretched over PVC piping. These enclosed
transfer boxes work well provided they are regularly
sterilised by a misting spray of bleach solution or other
disinfectant. The still air cabinet needs to have sufficient
working space, and dimensions of 60x50x50 cantimetres is
usually adequate for small propagators.
“THE MOST commonly used hormones
in agar gel are cytokinins to stimulate
shoot development and auxins to
stimulate new root development.”
Top: A commercial tissue culture lab growth room.
Bottom: Tissue culture vessels with agar medium in their bases.
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Inside the clean box, the sterilized leaf is cut into one- to
two-centimetre pieces and transferred to the agar medium
inside a culture flask or jar (baby food jars are recommended
for this). The gel-like agar needs to be purchased pre-made or
prepared ahead of time. The agar-based medium is prepared
by mixing together the liquid medium, agar powder, and
other ingredients; placing the mixture into the flasks or jars;
and heat treated in either the microwave or a pressure cooker
for a certain length of time. All tools and utensils such as
scalpels, forceps, and knives must also be sterilised in this
way. Isopropyl alcohol or ethyl alcohol is also used to flame
cutting blades before they are used on plant material to
ensure no microbes contaminate the plant tissue.
Once the pieces of leaf have been placed into the culture
jars and sealed (the lid must be closed before the jar
is removed from the clean box cabinet), they are left to
generate new shoots. During this period, African violet
leaves first form small bumps. Over two to four weeks,
these develop into a mass of tiny shoots, a process that
is promoted by the hormones in the tissue culture agar
medium. The jars are often kept on shelves with lights
positioned 23-30 centimetres above them. Cool-white
fluorescent propagation lights are ideal, as they don’t
create a heat buildup inside the flasks. An ideal lighting
period of 16 hours per day and temperature control is also
important for this process.