Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 224

has been addressed by attempting to increase its host range (Godwin, Fordlloyd
and Newbury, 1992) which has met with success, leading to improved transfor-
mation procedures (Le et al ., 2001). In essence, the wild type plasmid contains
genes which causes the transfer of a piece of DNA, 'T-DNA', into a plant cell.
This piece is bordered by sequences of 24 base pairs in length which are repeats
of each other. This structure is fairly common in DNA and is described as
a direct repeat. The T-DNA comprises genes which cause crown gall disease.
These genes may be cut out and replaced by DNA containing the gene of choice
to be introduced into the plant. There are many additional elements which may be
included in the construct. For example, if the aim is to express the gene, it is pre-
ceded by a strong promoter, most commonly the '35S' promoter of Cauliflower
Mosaic Virus.
In addition to the above, it is important to know if the 'foreign' gene is being
expressed and so frequently a 'reporter' gene described in the section above is
also included located close to the gene of interest. Recombination is not 100%
efficient, and so a method of selection is required such that only plants containing
the novel DNA grow. This is frequently a gene coding for weedkiller or antibiotic
resistance. On the grounds of size, this is usually introduced more successfully
on a second Ti plasmid during a co-infection with Agrobacterium carrying the
plasmid containing the gene of interest. The experiment can become somewhat
complicated at this stage, as other selector genes are introduced into the plasmids
to ensure that growth is only possible if all the desired elements are present in the
plant cell. This can involve infection with two or three cultures of Agrobacterium
each containing its own engineered Ti plasmid. A very detailed description of
the Ti plasmid is published elsewhere (Hughes, 1996).
Selected examples of developments in plant GE
The purpose of these examples is to illustrate the potential plant GE could hold
for practical applications in the field of environmental biotechnology. In some
cases the intention is to reduce the amount of herbicide and pesticides, or other
agricultural chemicals required to produce a given crop yield, in others it is to
improve tolerance of harsh conditions or to protect the plants from attack thus
reducing wastage. The intention is to note the technical details here, while the
effects such developments may have on the environment as a whole, feature
elsewhere throughout this topic.
Broad range protection
A general strategy to protect plants from various viruses, fungi and oxidative
damage by a range of agents, has been proposed using tobacco plants as a model.
The transgenics express the iron-binding protein, ferritin, in their cells which
appears to afford them far ranging protection (Deak et al ., 1999).