Transgenic Rice Plants

For centuries, rice has been
one of the most important staple crops for the world and it now currently feeds
more than two billion people, mostly living in developing countries. Rice
is the major food source of Japan and China and it enjoys a long history of
use in both cultures. In 1994, worldwide rice production peaked at 530 million
metric tons. Yet, more than 200 million tons of rice are lost each year to
biotic stresses such as disease and insect infestation. This extreme loss
of crop is estimated to cost at least several billion dollars per year and
heavy losses often leave third world countries desperate for their staple food.
Therefore, measures must be taken to decrease the amount of crop loss and
increase yields that could be used to feed the populations of the world. One
method to increase rice crop yields is the institution of transgenic rice plants
that express insect resistance genes. The two major ways to accomplish insect
resistance in rice are the introduction of the potato proteinas
e inhibitor
II gene or the introduction of the Bacillus thuringiensis toxin gene into the
plant\'s genome. Other experimental methods of instituting insect resistance
include the use of the arcelin gene, the snowdrop
lectin/GNA (galanthus nivallis
agglutinin) protein, and phloem specific promoters and finally the SBTI gene.

The introduction of the potato proteinase inhibitor II gene, or PINII,
marks the first time that useful genes were successfully transferred from a
dicotyledonus plant to a monocotyledonous plant. Whenever the plant is wounded
by insects, the PINII gene produces a protein that interferes with the insect\'s
digestive processes. These protein inhibitors can be detrimental to the growth
and development of a wide range of insects that attack rice plants and result
in insects eating less of the plant material. Proteinase inhibitors are of
particular interest because they are part of the rice plant\'s natural defense
system against insects. They are also beneficial because they are inactivated
by cooking and therefore pose no environmental or health hazards to the human
consumption of PINII treated rice.
In order to produce fertile transgenic
rice plants, plasmid pTW was used, coupled with the pin 2 promoter and the
inserted rice actin intron, act 1. The combination of the pin 2 promoter and
act 1 intron has been shown to produce a high level, wound inducible expression
of foreign genes in transgenic plants. This was useful for delivering the
protein inhibitor to insects which eat plant material. The selectable marker
in this trial was the bacterial phosphinothricin acetyl transferase gene (bar)
which was linked to the cauliflower mosaic virus (CaMV) 35S promoter. Next
the plasmid pTW was injected into cell cultures of Japonica rice using the
BiolisticTM particle delivery system. The BiolisticTM
system proceeds as
follows:
Immature embryos and embryonic calli of six rice materials were
bombarded with
tungsten particles coated with DNA of two plasmids containing
the appropriate
genes.
The plant materials showed high frequency
of expression of genes when stained
with X-Gluc. The number of blue
or transgenic units was approximately 1,000.
After one week, the transgenic
cells were transferred onto selection medium
containing hygromycin
B. After two weeks, fresh cell cultures could be
seen on bombarded
tissue. Some cultures were white and some cultures were blue.
Isolated cell
cultures were further selected on hygromycin resistance. However,
no
control plant survived.
Then twenty plates of cells were bombarded with
the PINII gene, from which over two hundred plants were regenerated and grown
in a greenhouse. After their growth, they were tested for PINII gene using
DNA blot hybridization and 73% of the plants were found to be transgenic.
DNA blot hybridization is the process by which DNA from each sample was digested
by a suitable restriction endonuclease, separated on an aragose gel, transferred
to a nylon membrane, and then finally hybridized with the 1.5 kb DNA fragment
with pin 2 coding and 3\' regions as the probe. The results also indicate that
the PINII gene was inherited by offspring of the original transgenic line,
that the PINII levels were higher among many of the offspring and that when
PINII levels rose in wounded leaves, the PINII levels in unwounded leaves also
rose. However, the PINII gene is not 100% effective in eliminating insects
because it does not produce an insect toxin, just a proteinase inhibitor.
Yet, greater insect resistance can be achiev
ed by adding genes to produce
the Bacillus thuringiensis or BT toxin.
Bacillus thuringiensis is an entomocidal
spore-forming soil bacterium that offers a way of controlling stem boring insects.
Stem borers such as the pink and striped varieties are difficult to