Biomedical Engineering Reference
In-Depth Information
need to induce root and shoot formation on artificial
media. Immature pollen or microspores can also be
induced to form vegetative cells, producing haploid
callus or embryoids. Such cells can be persuaded to
undergo diploidization by treatment with mitotic
inhibitors.
The ease with which plant material is mani-
pulated and interconverted in culture provides many
opportunities for the development of techniques
for gene transfer and the recovery of transgenic
plants (Fig. 12.4). DNA can be introduced into most
types of plant material - protoplasts, cell suspen-
sions, callus, tissue explants, gametes, seeds, zygotes,
embryos, organs and whole plants - so the ability
to recover fertile plants from such material is often
the limiting step in plant genetic engineering rather
than the DNA transfer process itself. It is also pos-
sible to maintain transformed plant cell lines or
tissues (e.g. root cultures) producing recombinant
proteins or metabolites, in the same way that cul-
tured animal cells can be used as bioreactors for
valuable products.
Fig. 12.3
Photomicrograph of tobacco protoplasts.
of phytohormones. However, only cytokinin is
required for shoot culture and only auxin for root
culture, therefore increasing the level of cytokinins
available to the callus induces shoot formation and
increasing the auxin level promotes root formation.
Ultimately, plantlets arise through the development
of adventitious roots on shoot buds or through the
development of shoot buds from tissues formed by
proliferation at the base of rootlets. The formation of
roots and shoots on callus tissue is known as
organo-
genesis
. The culture conditions required to achieve
organogenesis vary from species to species, and
have not been determined for every type of callus.
The adventitious organogenesis of shoots and roots
can also occur directly from organized plant tissues,
such as stem segments, without first passing through
a callus stage.
Under certain conditions, cell suspensions or
callus tissue of some plant species can be induced
to undergo a different development process known
as
somatic embryogenesis
. In this process, the cells
undergo a pattern of differentiation similar to that
seen in zygotes after fertilization, to produce
embry-
oids
. These structures are embryo-like but differ from
normal embryos in being produced from somatic
cells and not from the fusion of two germ cells. The
embryoids can develop into fertile plants without the
Overview of gene-transfer strategies
Gene transfer to plants is achieved using three differ-
ent methods. The first exploits the natural ability of
certain bacteria of the genus
Agrobacterium
to natur-
ally transfer DNA to the genomes of infected plant
cells. This generally results in the stable transforma-
tion of the infected cell, and the transferred DNA
behaves as a new genetic locus. Initial limitations
with respect to the host range of
Agrobacterium
prompted research into alternative methods based
on direct DNA transfer. These include the chemic-
ally assisted transformation of protoplasts and the
bombardment of plant material with DNA-coated
microprojectiles. Such strategies can be used for
both transient and stable transformation. Finally,
plant viruses can been used as vectors for gene deliv-
ery. The viruses of plants never integrate into the
genome and are not transmitted through seeds, so
stable transformation cannot be achieved. However,
plant viruses often cause systemic infections, result-
ing in the rapid production of high levels of recombin-
ant protein throughout the plant, and they can be
transmitted through normal infection routes or by
grafting infected scions on to virus-free hosts.
