Transgenic Rice Plants

Transgenic Rice Plants The following form contents were entered on 15th Apr 97 Date = 15 Apr 97 23:58:50 subject name = Sarah Lenhardt email = publish = yes subject = Biology title = Transgenic Rice Plants Transgenic Rice Plants that Express Insect Resistance 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.

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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 control because the larvae enter the stem of the plant shortly after hatching and continue to develop inside the plant, away from the toxins of sprayed insecticides. Therefore, the stable institution of the BT gene into the rice plant’s genome would provide a method of reaching stem borers with toxins that are expressed in the plant tissues themselves. Bacillus thuringiensis is comprised of so-called cry genes that encode insect specific endotoxins.

Recently some lower varieties of rice, such as Japonica, have been successfully transformed with cry genes, but the real challenge lies in transforming Indica rice, an elite breeding rice that composes almost 80% of the world’s rice production. In order to transform Indica rice, the synthetic cry IA gene must be used because it is the only cry gene to produce enough of the BT protein. Next, the synthetic cry IA gene under the control of the CaMV 35S promoter is attached to a CaMV cassette for hygromycin selection of transformed tissues. Following the linkage of the cry IA and the CaMV 35S cassette, the DNA is delivered to the embryonic cells by particle bombardment with a particle inflow gun. More specific transformation includes the following: Immature Indica rice embryos were isolated for ten to sixteen days after pollination from other greenhouse plants and were plated on a solid MS medium containing sucrose (3%) and cefotaxime. After twenty four hours, embryos were transferred to a thin layer of highly osmotic medium containing a higher percentage of sucrose (10%), were incubated, and then were bombarded with plasmid pSBHI and gold particles by the particle inflow gun.

After bombardment, the thin layer of 10% sucrose was placed on the layer of 3% sucrose. This sandwich technique allowed continuous adaptation of the target tissue to the osmotic conditions, which was shown to be optimal for callus induction. After twenty four hours, the 10% sucrose layer was removed and the embryos were cultured on the 3% sucrose layer. After one week, they were transferred to a 3% sucrose medium that was selected for hygromycin B resistance. After a further three to four weeks, regenerated plants were transferred to soil and placed in the greenhouse under appropriate conditions.

The results of this process were eleven transgenic plants out of a total of thirty six. Transgeneicy of the rice plants was confirmed by similar banding patterns in Southern blotting. The presence of the BT protein was also demonstrated in Western blot analysis, where a protein with the expected size of sixty-five kilobases was found in all plants tested. Interestingly enough, the BT protein levels were higher in older plants than in younger plants, possibly questioning the role of inheritance of BT gene. Yet, inheritance was determined by using DNA blot hybridization, which revealed a segregation ratio of 3:1.

This indicates the integration of all copies of tra …