FACULTY OF INDUSTRIAL SCIENCES & TECHNOLOGY

INDUSTRIAL BIOTECHNOLOGY

BSB3492 PLANT AND MAMMALIAN CELL

TECHNOLOGY LABORATORY
SEMESTER 2 2017/2018

LAB 1: INTRODUCTION TO EQUIPMENT AND ASEPTIC TECHNIQUES IN PLANT TISSUE

CULTURE
Instructor : DR NATANAMURUGARAJ GOVINDAN

Group Members : BEH YU LING SB15008

HU SHIAN HUAN SB15010
NURIN HAZIMAH BINTI RAMLE SB15018
SHERENE CHIN XIN YI SB15034
NURIN HAZIMAH BINTI RAMLE SB15018
SHARMILAA SEVAN SB15068
WAHYUNI WULANSARI SB17133

Practical Date : 28th February 2018

Submission Date : 28th March 2018

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INTRODUCTION
Plant tissue culture is the in vitro growing of parts of a plant such as organs,
tissues or cells on an artificial nutritional medium. This culture medium is also an
excellent resource for the growth of contaminates such as fungus and bacteria, and the
growth of the contaminates easily outpaces that of the cultured plant cells. Therefore,
general procedures of microbiology are applied to avoid contamination. Basically,
anything that may have the chance to make a direct contact with the culture medium
or explants has to be sterilized and manipulations should be designed such that there
is no direct link between the culture and its nonsterile surroundings.
Tissue culture practice usually uses three levels of containment to preserve
sterile conditions during culture manipulations. First is using sterilized medium, tools,
containers and explants. Second, working under a sterilized environment. Third,
practicing sterilizing techniques. Autoclaving, surface treatment and filtration are three
means to achieve sterilization of the tools and materials used. (Grout, 2017)
Contaminate may come from air flow, bench surface and others. Therefore, all
manipulations involving the cultures or culture media should be done in a laminar flow
hood. The laminar flow hood is a cabinet which provides unidirectional flow of filter-
sterilized air over a non-porous work surface, prevents non-sterilized air from entering
the workspace and creates a barrier against air-born contaminants, thus providing
protection for the cultures.
Autoclaving is a treatment of a high temperature and high pressure. The
standard conditions for autoclave sterilization are 121。C for 15 minutes under 1.05
kg/cm2 pressure. Longer times may be necessary for larger volumes. Most inorganic
compounds are heat stable and thus may be autoclaved safely. Organic compounds,
such as some plant growth regulators, amino acids and vitamins, may be degraded
during autoclaving. For instance, the breakdown of sucrose into fructose and glucose
during autoclaving will not reduce their nutritional value but increase the osmotic
potential of the medium. However, the breakdown of vitamins will cause the loss of
their biofunctions. Compounds that are heat-unstable, and thus should not be
autoclaved include, ABA, ascorbic acid, crystine, citric acid, folic acid, gibberellic acid,
IAA, IBA, kinetin, nicotinamide, pantothenic acid, pyridoxine, 2-iP, thiamine and zeatin.

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Tools and containers can be wrapped with aluminum foil and autoclaved.
(Hutson, Brazenor, Vaughan, & Trujillo-González, 2018)
Explants are the plant tissues to be cultured. Plant tissues that consist of
meristematic cells, have less cell walls or fibers, or are rich in cytoplasm are usually
good candidates for explants. Examples are root tips, shoot tips, young floral organs,
immature embryos, young leaves and young stems. Explants need to be surface
sterilized. This is usually done by soaking the explants in 10% bleach (hydrocloride)
solution for 5-10 minutes. Longer soaking time or higher bleach concentration usually
causes cell damage to the explants and thus reduces the chance for a successful culture.
A dip in a 70% ethanol may be done before soaking in bleach to reduce the surface
tension so that bleach can easily make contact with the explant’s surface. After
sterilized in a bleach solution, the explants are rinsed three times in autoclaved water
to remove bleach residue. Large explants such as leaf blades and stems should be cut
into smaller pieces to increase culture efficiency. Explant sterilization and preparation
need to be done in a laminar flow hood. (Grout, 2017)

1. Grout, B. (2017). General Principles of Tissue Culture. In B. Thomas, B. G. Murray,

& D. J. Murphy (Eds.), Encyclopedia of Applied Plant Sciences (Second Edition)
(pp. 437-443). Oxford: Academic Press.

2. Hutson, K. S., Brazenor, A. K., Vaughan, D. B., & Trujillo-González, A. (2018).

Monogenean Parasite Cultures: Current Techniques and Recent Advances. In
Advances in Parasitology: Academic Press.