BGY 3001

CELL AND DEVELOPMENTAL BIOLOGY

PRACTICAL 4:
TISSUE CULTURE

Name : Joanne Ch’ng Yu Rou

Matric : 156205
No
Course : B Sc. (Hon) Microbiology
Tutor : Noor Azrizal Bin Abdul Wahid
Date : 12/08/2010
Experiment Number: 4
Title: Tissue Culture

Objective:
To learn how to sterilize the explants
To learn how to extract the cotyledon from the seed
To learn how to perform tissue culture
To learn about the stages involved in tissue culture

Introduction:

Tissue culture refers to growing more cells from an existing sample using a
nutrient medium in an uncontaminated, closed environment. It is a method of
biological research in which fragments of tissue from an animal or plant are
transferred to an artificial environment in which they can continue to survive and
function. Examples of tissues which can be cultured are embryos, tissues and most
cells.
Culturing plant tissue is beneficial. The plants grown from tissue culture will
retain all traits of the mother plant. Plants with good traits can essentially be cloned
with this method, ensuring that the desirable traits will persist through further
generations of the plant. Example: Trees which produce sweet fruit may be cultured
and cloned to ensure that sweet fruit will be produced.
Plant tissue culture is easier to carry out compared to cross breeding,
fertilization and hybridization. If successful, the seedlings, can mature in 2 to 4
weeks. The expedite growth of the plantlets have commercial potential as they are
easier to store and transport compared to grown plants.
This method is also useful in preserving rare species of flora. Plants that have
medicinal properties can be mass produced (cultured in huge quantities) for research
purposes. Many plants can be produced in the absence of seeds or necessary
pollinators. Almost extinct plants can be regenerated from the plant cells used.
The production of plant tissue culture in sterile mediums greatly reduce the
chances of contamination. There is less chance of transmitting diseases, pests or
pathogens from one plant to another.
 Hard-to-grow plants like orchids or nepenthes can be grown with this method
for commercial or horticulture purposes.
MSO is also known as Murashige and Skoog medium. It is the most
commonly used medium in plant tissue culture. This media contains the following:
Ammonium nitrate, Boric Acid, Calcium chloride, Cobalt chloride, Magnesium,
Sulphate, Cupric sulphate, Potassium phosphate, Gerrous Sulphate, Potassium Nitrate,
Manganese sulphate, Potassium iodide, Sodium molybdate, Zinc sulphate and
Na2EDTA · 2H2O. Other ingredients include: i-INositol, Niacin, Pyridoxine,
Thiamine, IAA, Kinetin, Glycine, Edamine, Sucrose and Agar.
Petai seeds are used in this experiment because of their rapid growth factor.
When placed in suitable media, they will germinate quickly, enabling easy
observation of the results within a week. Other types of plants may take several weeks
to germinate. This is very time consuming. Petai is also known as Parkia speciosa.
Plant hormones play a very important part in the growth of a plant. For
example, the hormone Auxin promotes cell elongation and cell expansion. Auxin is
usually present at the tips of the roots which are elongating towards water to soak up
as much moisture as possible. It enhances embryogenesis in suspension culture and is
a good addition to the medium
Cytokinins are also another important plant hormone. In high doses, they help
speed up the formation of shoots and auxiliary shoots.
Other plant hormones include Abscisic acid, ethylene and Gibberellins.
Abscisic acid is the hormone which causes leaves to fall in the autumn. Ethylene
promotes fruit ripening and Gibberellins promote flowering.

Apparatus:

Measuring cylinder, forceps, 50ml conical flasks, parafilm, 10ml pipette and rubber
safety ball, aluminium foil, liquid waste container, scalpel, cotton, petri dishes with
covers

Material:
Sterilized distilled water, Clorox, dishwasher liquid, sterilized MSO media, sterilized
Petri dishes, alcohol.
Method:
All the apparatus to be used are sterilized. This is done by soaking cotton in
alcohol and then using the cotton to wipe the apparatus. The cotton should not be used
more than one time and forceps are used to hold the cotton to prevent contamination
from the oil and bacteria found on hands or fingers.
The petai seeds are separated from the skin. The seeds are washed with tap
water for about 20 minutes. The petai seeds are swabbed with dishwasher liquid then
rinsed with distilled water. The petai seeds are immersed in Chlorox (15%) + tween
20 (1 drop). The petai seed is placed in the petri dish, covered and then shaken for 15
minutes.
The petai seeds are washed 3 times with sterile distilled water. The seeds are
placed in a sterile petri dish. After that, they are cut horizontally with a sterilized
scalpel. The side with the embryo is chosen. It’s is cut horizontally to the left and
right. A little bit of the cotyledon is left with it. Care is taken not to damage the
embryo.
The embryos are placed into the MSO media. The mouth of the conical flasks
are covered first with aluminium foil then parafilm. This part must be done quickly
and efficiently to prevent contamination of the medium or of the embryos from
airborne bacteria or viruses.
The flasks are labelled and left for a week to grow.
After a week, the flasks are observed and the results are recorded.
Results:

Figure 4.1: MSO media before embryos are placed inside.

Figure 4.2: MSO media after embryos are placed inside.

Figure 4.3: MSO media after one week. (Germinating Embryo)

Figure 4.4 Germinating Embryo in Flask 1

Figure 4.5 Germinating Embryo in Flask 2

 Figure 4.6 Front view of Flask 1 and Flask 2

Figure 4.7 Close up of Embryos in MSO media

Discussion:
Both embryos placed in the MSO media germinated. Figure 4.4, 4.5, 4.6 and
4.7 show the seedlings that are growing. The absence of bacterial colonies or yeast
colonies on the MSO media show that the environment is sterile. The exercise is a
success.
If the MSO media is not kept sterile, contamination might occur. Fungi or
bacteria will grow on the media and compete with the embryo for nutrients, thus
stunting its growth or causing it to not grow at all. This is why it is important for the
media to be kept clean.
There are five stages in plant tissue culture. First is the preparation of culture
medium, followed by the selection and preparation of the mother plant, then the
sterilization of all apparatus and materials. After that is the growth and rooting of the
explants. The process ends with the transfer of the explants.
In the first stage, the culture media in which the plant is to be grown has to be
prepared. The culture media should contain all of the following: salts, essential
elements, mineral ions, vitamins, amino acids, carbon source (sugar/sucrose), gelling
agents, plant growth regulators and antibiotics. The salts, essential elements, mineral
ions, vitamins, amino acids and carbon source are the nutrients needed for the plant to
grown. The gelling agent is so that the culture media will become semi solid and not
remain in liquid form. The plant growth regulators help stimulate the growth of the
explants whereas the antibiotics prevent infection from bacteria.
The second stage of plant tissue culture is the selection and preparation of the
mother plant. The selected tissue is called an explant. It is important to select a
healthy mother plant for the tissue culture. The plantlets will inherit all the
morphological traits of the mother plant so it’s imperative that a mother plant with
desirable traits is chosen to be cultured. A healthy mother plant will increase the
chances of a successful culture as well as ensure that the plantlets will be resistant to
herbicides, diseases and pests.
Sterilization is the third step of the plant tissue culture. The plant has to be
cleaned with alcohol, chlorox and distilled water in other to ensure it is completely
clean. The fast evaporating properties of alcohol and chlorox will dessicate any
bacteria or fungi on the plantlet. The distilled water washes all alcohol and chlorox
residue off the plantlet. In addition to that, all the apparatus used to prepare the tissue
culture must be sterilized either by alcohol or by heating.
 For certain plant tissue cultures, the explant must be transferred from old
media to new ones. Each new media contains different plant hormones and different
nutrients in order to promote the growth of the different tissues of the plants. This
transplantation will be done at intervals until the explants forms a plantlet.
The growing plantlet will be transferred to a farm in the last stage. The
explants that have grown sufficient roots and shoots will be removed from the media
and then cleaned with distilled water. They will be placed in clean polybags and left
under shade for 12 to 16 weeks in order for them to acclimatize or get used to the new
environment. After the acclimatization period, they will be transferred to the farm.

Conclusion:
Embryos can be grown in MSO media. Care must be taken not to contaminate
or damage the embryo in any way. Otherwise, it may stunt or inhibit the growth of the
seedlings.

References:
http://www.accessexcellence.org/LC/ST/st2bgplant.php, by Lydiane (Ann) Kyte (7.56pm
24/08/2010)
http://www.liv.ac.uk/~sd21/tisscult/what.htm, by University of Liverpool (8.27pm
24/08/2010)
http://www.cipotato.org/csd/materials/tissue/capitulo4.pdf, by International Potato Center
(8.53 pm, 24/08/2010)
Indra K. Vasil, Trevor A. Thorpe (1994) Plant Cell and Tissue Culture. Kluwer Academic
Publishers (593p)
Paul F. Kruse Jr. M. K. Patterson Jr. (1973) Tissue Culture, Academic Press, Inc. (868p)
E. F. George, M. A. Hall, and G.-J. De Klerk (2008). Plant propagation by tissue culture.
Volume 1. The background. 3rd edition Springer Dordrecht (501 p)