constructed so that they contain left and right plastid-targeting regions (LTR and RTR),
which are 1–2 kb in size and homologous to a chosen target site (Fig. 7.23).
The design and construction of plastid vectors that allow the simultaneous expression of
several genes in an operon will be particularly useful in the engineering of agronomically
important traits, as described earlier. Transgene integration has been achieved at 16 inde-
pendent sites distributed across the plastid genome, ensuring that the positional effects,
which are often associated with DNA integration events in the nuclear genome, are elimi-
nated. Since there are 10–100 plastid genomes per plastid and approximately 10–100 plas-
tids per cell, as many as 10,000 transgene copies can be generated in a single cell, resulting
in highly abundant transgene transcription, producing as much as 46% of the total soluble
protein in a cell. As with nuclear genome transformation vectors,loxPsites in plastid
vectors can be engineered to flank the marker gene and excised when no longer required
using the Cre site-specific recombinase. Plastid transformation technology does not yet
extend to major crops, but has been demonstrated in soybean, carrot, and cotton through
species-specific chloroplast vectors, and plant regeneration through somatic embryogenesis.
7.7 Prospects
Recombinant DNA technology, vector design, and construction form the foundations on
which advances in modern plant biotechnology are built. The development of tools for
the rapid amplification and manipulation of DNA sequences are essential if we are to
keep pace the ever-increasing wealth of genetic information that results from the analysis
of plant, animal, bacterial, and viral genomes. To exploit this information fully, functional
studies must be conducted to determine the potential uses of such sequences, identifying the
elements required to control gene expression and the genes required to ensure the high crop
yields needed to sustain the planet’s expanding population. Understanding the elements
required for the efficient expression of genes in plants has already facilitated the develop-
ment of new crop varieties. Novel genetic engineering approaches resulting from recombi-
nant DNA technologies will provide the solutions to many of our future industrial,
pharmaceutical, and sustainable fuel requirements. This evolving technology forms the
basis of a new “Green Revolution.”
188 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION