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
Immature embryos and embryonic calli of six rice materials were bombarded with
tungsten particles coated with DNA of two plasmids containing the appropriate
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
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...