Plant tissue culture
Purpose: To demonstrate the totipotency of plant cells.
Introduction:
Totipotency is the ability of a single cell to divide and produce all the
differentiated cells in an organism, including extraembryonic tissues. Totipotent
cells formed during sexual and asexual reproduction include spores and zygotes.
Zygotes are the products of the fusion of two gametes (fertilization). In some
organisms, cells can dedifferentiate and regain totipotency. For example, a plant
cutting or callus can be used to grow an entire plant.
Human development begins when a sperm fertilizes an egg and creates a
single totipotent cell (zygote). In the first hours after fertilization, this cell divides
into 8 identical totipotent cells. Approximately four days after fertilization and
after several cycles of cell division, these totipotent cells begin to specialize.
Totipotent cells have total potential. They can specialize into pluripotent cells that
can give rise to most of different type of cells, but not all, of the tissues necessary
for fetal development. Pluripotent cells undergo further specialization into
multipotent cells that are committed to give rise to cells that have a particular
function. For example, multipotent blood stem cells give rise to the red cells,
white cells and platelets in the blood.
Importantly, totipotent cells must be able to differentiate not only into any cell in
the organism, but also into the extraembryonic tissue associated with that
organism. For example, human stem cells are considered totipotent only if they
can develop into any cell in the body. Unlike animal cells, most plant cells remain
totipotent throughout the life of the plant. Differentiation is irreversible in animal
cells but many differentiated in plant cells, therefore it can develop into a
complete new plant as they have ability to regenerate identical whole plant clone
from root, stem or leaf cuttings.
Hence, totipotency of plant cells allows plants to be reproduced using plant
tissue culture. Plant tissue culture is a practice used to propagate plants under
sterile conditions, often to produce clones of a plant. Explants are one of example
which sterilized the small pieces of plants and then placed on a solid agar medium
to allow it to grow. Plant tissue culture may offer certain advantages over
traditional methods of propagation: (1) the production of exact copies of plants that
produce particularly good flowers, fruits, or have other desirable traits. (2) The
regeneration of whole plants from plant cells that have been genetically modified.
(3) The production of plants from seeds that otherwise have very low chances of
germinating and growing. (4) The production of plants in sterile containers that
allows them to be moved with greatly reduced chances of transmitting diseases,
pests, and pathogens. (5) To quickly produce mature plants. Also, Plant tissue
culture is so important in plant biology research, plant breeding, genetic
modification of plants and in the conservation of endangered plants.
My hypothesis:
I think all the explants will grow healthy with a new stem, new leaves but it
will stop to grow later on as the agar provides nutrients and other needs for them,
however the nutrients are limited from agar.
Plan:
The controlled of plant tissue culture are light and temperature. Light and
temperature are the main factors for photosynthesis, therefore plants can be growth.
However the compounding of it is nutrients. As we cant control how much nutrients
can be provided by agars to the explants. Different volume or size of agars may
contain different quantity of nutrients.
Equipment:
Equipment
The reason for choice
Agar powder
It is used for providing
nutrients to explants
A tray of small plants
Obtain the seedling
( explants) from it
beaker
It is a utensil for agar
powder and water
Glass rod
stirring the agar solution
when it is heated
Set up of Bunsen
burner( e.g tripod, wire
gauze, heat proof mat,
Bunsen burner)
It is used for heating to
dissolve to agar powder into
water
5 Mc Cartney bottles
It is a utensil for dissolved
agar solution
A pair of scissors
It is used for cutting the
seedling from the shoot
apex of small plants
Methods:
1 Sprinkle some seeds of white mustard (Sinapsis alba) or rapid cycling brassica (Brassica
rapa) onto a damp sponge placed in a plastic tray. Cover with transparent cling film and
place in a warm, light place to germinate. When the seedlings have just started to unfold
their cotyledons (seed leaves), they are ready to culture.
3
2 Measure out 2.5 g of agar powder and add to 250 cm of distilled water. Heat and stir
gently until the agar dissolves.
3 Whilst the agar is still molten, pour about 2 cm depth into several short-necked test tubes
or McCartney bottles. Allow to cool and solidify.
4 With a sharp pair of scissors cut the tops off the seedlings just below the shoot apex
(growing tip). These are the explants. Leave the hypocotyls (the early stem) and roots
behind on the sponge.
5 Carefully push the cut end of each explant into the agar. Put one explant into each test
tube or bottle. Make sure the cotyledons do not touch the agar.
6 Cover the tubes with cling film or a clear lid. On each tube or bottle mark the numbers
and names. Place the tubes in a rack under a light bank or on a sunny windowsill. Do not
open the tubes again.
7 Observe the progress of your explants daily, and record when anything of note develops.
Try to observe over a period of 10 days or so.
possible risk:
Possible risk
How the risk will be
minimized
A pair of scissors is so
sharp
Use it carefully and
follow teachers’
instructions
During heating the agar Stir it gently and
solution, it is so hot and wearing goggle
starts to boil, the
solution may spill out
Precautions:
1. Make sure the cotyledons do not touch agar when put an explant into each
McCartney bottles because if the cotyledons touched the agar , the agar is solidify
and the leaves would be stuck in order to prevent it to grow upwards.
2. Cover the tubes with a transparent lid because light can get in and prevent the
microorganisms to grow as they will also obtain nutrients from agar. So the
explants can’t get enough nutrients for growth.
3. Cannot open the tubes again once set them up because agar normally contains
bacteria and it can prevent to increase the rate of growth of bacteria.
4. To be sure that agar powder completely dissolved into the hot water, the solution
should be clear and no any precipitate.
Result:
The records of the growth of explants in 2 weeks
Time / day
1
2
3
4
5
Tues
12:30pm
L: 3mm
G: 2
H: 3
L: 1mm
G: 4
H: 4
L: 5mm
G: 1
H: 1
L: 3.5mm
G: 1
H: 2
L: 3mm
G: 4
H: 4
Wed
L: 4mm
L: 1mm
L: 5mm
L: 6mm
L: 4mm
12:30pm
G: 3
H: 3
G: 4
H: 4
G: 2
H: 4
G: 3
H: 3
G: 4
H: 4
Thurs
1:10pm
L: 4mm
G: 4
H: 3
L: 1mm
G: 4
H: 5
L: 6mm
G: 3
H: 3
L: 6mm
G: 3
H: 4
L: 4mm
G: 3
H: 3
Fri
9:00pm
L: 4mm
G: 4
H: 4
L: 2mm
G: 5
H: 5
L: 6mm
G: 3
H: 3
L: 6mm
G: 3
H: 4
L: 3mm
G: 3
H: 3
Mon( after a
week)
L: 0mm
G: 5
L: 1mm
G: 5
L: 1mm
G: 4
L: 6mm
G: 3
L: 1mm
G: 5
10:45am
H: 5
H: 5
H: 5
H: 4
H: 5
Note: L – length of explants
G – colour of leaves 1 to 5 ( 1 green, 3 yellow, 5 brown)
H – healthy of explants 1 to 5( 1 most healthy, 5 most unhealthy)
Analysis and discussion:
We did five explants for the Plant Tissue Culture experiment. Most of them have
healthy greenish leaves at the beginning. After a week, the result showed that all
explants grew up which back my hypothesis that the explants grew up. However, they
got weaker and weaker. Their leaves were turn from green to brown Particularly the
last day, some of explants had even wilted, so we could not measure their length such
as the explants(1). Therefore the result opposes my hypothesis that they are not as
healthy as I think and they did not grow any new leaves or other new features. And
the reasons of the explants became weak and wilt as they were no longer to obtain
enough nutrients and water for growth from agar, so agar cannot provide enough
needs for explants to grow in a long term and it can be only used temporary. Also it is
lack of growth regulators.
Nevertheless, the explants had been grown up, thus it is proved that the shoot apex
contains totipotency of its plant cells and it has ability to regenerate identical plants
clones.
Evaluation:
In this experiment, I think the result of the record of last day is not reliable as the data
has a big different between the data was recorded before a week. We should record the
data everyday to check the change of explants. Also we should repeat the experiment
so I can obtain more accurate results but the time is limited. Moreover as the poor
growth of explants, it disturbed me to get an accurate result. I have considered to use
the growth regulators ( auxins and cytokines). It could be able to help the explants to
grow better. However, the errors still cannot reduce as the explants grow may be due
to other factors but not exactly affected by totipotency in plant cells. Furthermore, I
think it should be used other substances or things to replace agars, therefore it can
extends provision of water and nutrients for the explants. In addition, I used a lid
instead of a plastic sheet to cover on the McCartney bottle, it may improve the
accuracy of the result as it is stronger than plastic sheet to prevent the growth of
bacteria
During doing the experiment, I had poured the agar solution into the McCartney
bottles when I realized that the powder had still not completely dissolved it into hot
water so the time had been wasted. Therefore if I repeated the experiment, I would
check more carefully whether the solution is it ready. Moreover some of data are still
reliable as it can prove that the plants still grow up without seeds to be related to
totipotency.
Reference:
1. http://en.wikipedia.org/wiki/Totipotency ----- totipotency
2. http://www.fao.org/docrep/t0831e/t0831e00.htm ---- plant tissue culture
3. http://aggie-horticulture.tamu.edu/tisscult/tcintro.html --