Principles of Plant Genetics and Breeding

TISSUE CULTURE AND THE BREEDING OF CLONALLY PROPAGATED PLANTS 183
culture medium to manipulate growth and development.
Auxins (e.g., naphthalene acetic acid, indole-3-butyric
acid, and 2,4-diclorophenoxyacetic acid (2,4-D)) are
used to induce rooting, while cytokinins (e.g., kinetin,
benzylaminopurine) are used to induce shoot formation.
In actuality, it is the ratio of cytokinin to auxin that
has the morphogenetic effect, a higher ratio promoting
shoot formation, while a higher auxin to cytokinin ratio
promotes rooting. Some plant materials have appreciable
endogenous levels of hormones, needing only exogenous
amounts of cytokinin for optimal shoot multiplication.
Micropropagation
Seed is the preferred propagule for use in the propagation
and cultivation of most agronomic and forest
species. This is so because they are easy to handle before
and during the production of the plant. However, a
number of major food crops and horticultural species
are vegetatively propagated as a preferred method
because of biological reasons (e.g., self-incompatibility)
and the lack of uniformity in seed. Micropropation
is the in vitro clonal propagation of plants. It is
used for commercial propagation of ornamentals and
other high-priced horticultural species, rather than for
agronomic species. Micropropagation can utilize preexisting
meristems or non-meristematic tissue. The
method of micropropagation commonly used may be
divided into three categories: axillary shoot production,
adventitious shoot production, and somatic
embryogenesis.
Micropropagation can be summarized in five general
steps:
1 Selection of explant. The plant part (e.g., meristem,
leaf, stem tissue, buds) to initiate tissue culture is
called the explant. It must be in good physiological
condition and be disease-free. Factors that affect the
success of the explant include its location on the plant,
age, or developmental phase. Explants that contain
shoot primordia (e.g., meristems, node buds, shoot
apices) are preferred. Also, explants from younger
(juvenile) plants are more successfully used in micropropagation.
2 Initiation and aseptic culture establishment. The
explant is surface sterilized (e.g., with Chlorox®,
alcohol) before being placed on the medium. Small
amounts of plant growth regulators may be added to
the medium for quick establishment of the explant.
3 Proliferation of axillary shoots. Axillary shoot proliferation
is induced by adding cytokinin to the shoot
culture medium. A cytokinin : auxin ratio of about
50 : 1 produces shoots with minimum callus formation.
New shoots may be subcultured at an interval of
about 4 weeks.
4 Rooting. The addition of auxin to the medium
induces root formation. Roots must be induced on
the shoot to produce plantlets for transfer into the
soil. It is possible to root the shoot directly in the soil.
5 Transfer to the natural environment. Before transferring
into the field, seedlings are gradually moved
from ideal laboratory conditions to more natural
conditions by reducing the relative humidity, and
increasing the light intensity, a process called hardening
off.
Axillary shoot production
Pre-existing meristems are used to initiate shoot culture
(or shoot tip culture). The size of the shoot tip ranges
between 1 and 10 mm in length. Cytokinin is used
to promote axillary shoot proliferation. Some species
(e.g., sweet potato) do not respond well to this treatment.
Instead, shoots consisting of single or multiple
nodes per segment are used. These explants are placed
horizontally on the medium and from them single
unbranched shoots arise that may be induced to root to
produce plantlets.
Shoot tips are easy to excise from the plant and are
genetically stable. They contain preformed incipient
shoot and are phenotypically homogeneous. These
explants have high survival and growth rates. Axillary
and terminal buds have the advantages of shoot tips,
but they are more difficult to disinfect. On the other
hand, meristem tips contain preformed meristems and
are genetically stable and phenotypically homogeneous,
but are more difficult to extract from the plant. Further,
they have low survival rates.
Adventitious shoot production
Adventitious shoots originate from adventitious meristems.
Plant growth occurs at specific regions called
meristems where cells are undifferentiated (no specific
assigned roles or function). Non-meristematic tissue
can be induced to form plant organs (e.g., embryos,
flowers, leaves, shoots, roots). Differentiated plant cells
(with specific functional roles) can be induced to dedifferentiate
from their current structural and functional
state, and then embark upon a new developmental
path to produce new characteristics. This method of
micropropagation also goes through the stages previously
discussed. Adventitious shoot production through