Principles of Plant Genetics and Breeding

184 CHAPTER 11
organogenesis occurs by one of two pathways: indirect
or direct.
Indirect organogenesis
The indirect organogenetic pathway goes through a
stage in which a mass of dedifferentiated cells (callus)
forms (i.e., the explant forms a callus from which adventitious
meristems are induced and from which plant
regeneration is initiated). The callus consists of an
aggregation of meristem-like cells that are developmentally
plastic (can be manipulated to redirect the morphogenic
end point). The negative side of this method is
that the callus phase sometimes introduces mutations
(some clonal variation, making this not always a 100%
clonal procedure). The callus phase also makes it more
technically challenging than shoot tip micropropagtion.
Direct organogenesis
Direct organogenesis bypasses a callus stage in forming
plant organs. The cells in the explant act as direct precursors
of a new primordium. This pathway is less common
than the callus-mediated pathway.
Somatic embryogenesis
As previously discussed, a zygote is formed after an egg
has been fertilized by a sperm. The zygote then develops
into an embryo (zygotic embryo). In vitro tissue culture
techniques may be used to induce the formation
of embryos from somatic tissue (non-zygotic embryo
or somatic embryogenesis) using growth regulators.
Somatic embryos arise from a single cell rather than
budding from a cell mass as in zygotic embryos. This
event is very important in biotechnology since transgenesis
in plants may involve the manipulation of single
somatic cells. However, without successful regeneration,
plant transformation cannot be undertaken. Somatic
embryogenesis has been extensively studied in Apiaceae,
Fabaceae, and Solanaceae. Embryo development, zygotic
or somatic, goes through certain stages: globular, scutellar,
and coleoptilar stages (in monocots), and globular,
heart, torpedo, and cotyledonary stages (in dicots). It is
generally difficult to obtain plants from somatic embryos.
Other tissue culture applications
There are other tissue culture-based applications besides
micropropagations, such as the following.
Synthetic seed
Somatic embryogenesis has potential commercial applications,
one of which is in the synthetic seed technology
(production of artificial seeds). A synthetic seed
consists of somatic embryos enclosed in protective coating.
There are two types currently being developed:
1 Hydrated synthetic seed. This kind of seed is
encased in hydrated gel (e.g., calcium alginate).
2 Desiccated synthetic seed. This kind of seed is coated
with water-soluble resin (e.g., polyoxethylene).
Synthetic seed technology is currently very expensive.
To develop synthetic seed, it is critical to achieve a
quiescent phase, which is typically lacking in somatic
embryogenesis (i.e., without quiescence there is continuous
growth, germination, and eventually death). The
application will depend on the crop. Lucerne (Midicago
sativa) and orchardgrass (Dactylis glomerata) are among
the species that have received significant attention in
artificial seed development. Potential application of
artificial seed is in species that are highly heterozygous
and in which conventional breeding is time-consuming.
Trees can be cloned more readily by this method. In
some typical species that are seed propagated but have
short duration of viability, artificial seed production
could be economic, because of the high economic value
of these crops (e.g., cocao, coconut, oil palm, coffee).
Also, hybrid synthetic seed could be produced in species
in which commercial hybrid production is problematic
(e.g., cotton, soybean).
Production of virus-free plants
Viral infections are systemic, being pervasive in the
entire affected plant. Heat therapy is a procedure that is
used for ridding infected plants of viral infections. After
heat treatment, subsequent new growth may be free of
viruses. More precisely, meristems dissected from leaf
and shoot primordia are more often free of viruses even
when the plant is infected. Tissue culture technology is
used to nurture the excised meristematic tissue into full
plants that are free from viruses.
The process starts with detection (e.g., by enzymelinked
immunosorbent assay, ELISA) of the presence of
a viral infection in the plant. Once confirmed, the meristems
on the shoots are aseptically removed and sterilized
(dipped in 75–99% ethanol or 0.1–0.5% sodium
hypochlorite or household bleach for a few seconds or
minutes). The explant is submitted to tissue culture as