Principles of Plant Genetics and Breeding

182 CHAPTER 11
Overview of the tissue culture environment
The most critical aspect of in vitro culture is the provision
of a sterile environment. A plant has certain natural
defenses against pathogens and the abiotic environment
in which it grows. Cells and tissues lack such protection
once extracted from the parent plant. An indispensable
piece of equipment in a tissue culture lab is an autoclave,
which is used for sterilizing some materials. All glassware
and other tools and plant materials are sterilized
before use. Chemicals such as ethanol and Chlorox®
(household bleach) are used in a tissue culture lab for
sterilizing the working areas and materials. Another key
piece of equipment for maintaining a sterile environment
is the laminar flow hood, which blows air horizontally
over the working area, towards the worker.
The growth environment for growing plants in the
soil under natural conditions should provide adequate
moisture, nutrients, light, temperature, and air. Plant
performance can be enhanced by supplementing the
growth environment (e.g., by fertilization, irrigation).
In tissue and cell culture, plant materials are grown in a
totally artificial environment in which the same growth
factors, plus additional ones (e.g., growth regulators),
are supplied. The cultural environment in tissue culture
may be manipulated by the researcher to control the
growth and development of the cultured material. For
example, the researcher may manipulate the hormonal
balance in the culture medium to favor only root or
shoot development.
Tissue culture is conducted under controlled environmental
conditions. The temperature is maintained at
about 25°C (slightly lower at night: 23 or 24°C). The
photoperiod is about 16 hours and is maintained by cool
white fluorescent tube lighting with an intensity of
about 3,000–5,000 lux.
In vitro culture medium
Growing plants in the field requires a medium (e.g.,
soil) containing nutrients and other growth factors for
success. The components of a tissue culture medium
may be categorized into four groups: a physical support
system, and mineral elements, organic compounds, and
growth regulators.
Support system
In vitro culture occurs in either liquid medium or on
solid medium, depending on the objectives of the
project. In liquid media culture (or suspension culture
as it is called), tissues or cells are cultured in water containing
nutrients and other growth factors. The liquid
medium has to be frequently agitated for good aeration.
Solid media are prepared by using gelling agents (e.g.,
agar and agarose). Agar is the most widely used gelling
agent. It is easy to prepare and handle. It is usually prepared
at a concentration of between 0.5% and 1.0%.
Agar is resistant to enzymes and does not react with
media components. Agar provides reduced contact of
the explant with the medium and is an additional cost to
the operation.
Agarose is a purer support material that is extracted
from agar. It is preferred by some researchers for its lack
of impurities (agaropectin and sulfate groups) found
in agar, and higher gel strength (thus requires smaller
amounts for preparing a solid medium). There are other
gelling agents, such as gellan gums (e.g., Phytagel®),
that provide clear gels (rather than translucent gels).
Nutrients
The basic components of a tissue culture medium are
inorganic salts, organic salts, amino acids, sugar, and
vitamins. A large variety of basic media have been developed
for various uses, the more common and broad-use
media including MS (after its developers, Murashige
and Skoog), Gamborg, and White media. These ingredients
supply both macronutrients and micronutrients.
One of the most popular media is the MS (Table 11.1).
In addition to these basic components, growth regulators
(auxin and cytokinins) are included in the tissue
Table 11.1 Murashige and Skoog (MS) medium salts.
Nutrient Source
Nitrate NH4NO3 KNO3 Sulfate MgSO4 .7H2O MnSO4 .H2O ZnSO4 .7H2O CuSO4 .5H2O Halide CaCl 2 .H 2 O
KI
CoCl 2 .6H 2 O
P, B, Mo KH2PO4 H3BO3 Na2MoO4 NaFeEDTA FeSO4 .7H2O NaEDTA