An Analysis of the Methods for Creating 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 proteinase 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.

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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 achieved 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 transgene at a single locus.

To assess the mortality rate among different insects, both petri dish assays and whole plant assays were performed. In petri dish assays, mortality rates were as follows: European corn borer = 85-95% Yellow stem borer = 100% Striped stem borer = 100% Cnaphalocrocis medinalis (leaffolder) = 67% Marasmia patnalis (leaffolder) = 55% In whole plant assays, no surviving insects were found on any BT expressing plants, although insects still survived on the control plants or non expressing BT plants.

In addition to this recent insertion of the BT gene into Indica rice, a similar procedure was conducted on Shuahggei 36, a variety of Indica rice. Transgeneicy of Shuahggei 36 was achieved by taking plasmid P410RH, which contained the coding region of the BT gene with the marker CaMV 35S-HPI-NOS plus 1.0 kb of DNA fragment, and inserting it into the pollen tube pathway. More specifically, the plasmid DNA was applied at the cut ends of rice florets from one to four hours after pollination. Next the seeds that were harvested were germinated under hygromycin B resistance.

However only 3% of the plants survived hygromycin resistance. After this, the seedlings from the second generation were again segregated for hygromycin resistance. From these seeds, seventy plant lines were screened for transgeneicy and fifteen displayed the BT protein. These results and the inheritance of the BT gene into offspring were confirmed by Southern blotting. Nevertheless, the question remains whether the BT gene was really integrated into the genome or whether it was expressed only as a plasmid.

The use of the arcelin gene is another experimental method of creating transgenic rice plants. The arcelin gene is a translationally enhanced Bacillus thuringiensis toxin construct that is effective on the rice water weevil. The rice water weevil or RWW is the major pest of the Texan rice crop. Previously, the RWW was combated by granular carbofuran, an insecticide that kills the RWW but has deleterious effects on water fowl that live in the crop area. So environmentalists have forced the cessation of the use of granular carbofuran and therefore, new methods have to be developed. One of the major genes that confer resistance to the RWW is the arcelin gene. Arcelin is a lectin that was originally discovered in the seeds of bean cultivators that showed resistance to the Mexican bean weevil. Next, researchers isolated a genomic clone encoding arcelin from the bean seed and then placed it under regulation of a rice actin promoter.

Then the clone with the rice promoter was introduced into rice protoplasts. Transgeneicy and inheritance was then confirmed by genomic DNA blots and immunochemical blots. In two separate experiments, six transgenic rice plants were subjected to RWW infestation under controlled conditions. The results of the first experiment are that similar numbers of RWW larvae were recovered from each set of six plants, but the size of those from arcelin expressing plants were significantly smaller. In the second experiment, although many normal larvae were recovered from control plants, only three small larvae came from arcelin expressing plants. This would indicate the benefits of inserting the arcelin gene into rice plants for RWW resistance.

Another experimental method of creating transgenic rice plants that are insect resistant includes the use of snowdrop lectin or galanthus nivallis agglutinin (GNA). Snowdrop lectin helps to control the sporadically serious pest the brown planthopper (BPH), which has developed a resistance to many pesticides. Luckily for the environment, snowdrop lectin provides high levels of toxicity to BPH but not to other animals. BPH is a member of the order Homoptera and feeds by sucking the phloem sap from the stems of rice plants. The major problem with combating BPH is that rice plants can not be engineered for BT toxin resistance against this pest because BT toxins that effect Homopterans have not yet been discovered or reported.

Therefore, other types of genes had to be manipulated in order to produce insect resistance against BPH. The best plant protein that provides resistance to BPHs turns out to be snowdrop lectin, and this was first confirmed by artificial diet bioassays. To create the transgenic rice plants, embryonic cell suspension cultures were initialed from mature embryos from two Japonica rice varieties, Taipei 309 and Zhonghua 8. Next, the protoplasts isolated from these cell suspension cultures were transformed by using the plasmid pSCGUSR, containing the nos-npt II gene as a selectable marker. Plasmid uptake was then induced by the PEG process and geneticin was used as a selection agent. Geneticin was added to the protoplast-derived colonies during the four and eight cell stages. From this, more than fifty putative transgenic plants have been regenerated from one thousand resistant colonies.

Another way of combating the brown planthopper is by producing phloem-specific promoters. These promoters are necessary because phloem is the exact site of feeding for the BPH. Although the CaMV promoter is active in phloem tissue, the possibility exists to institute a promoter from a gene that is specifically expressed only in phloem. This would be advantageous if there are other parts of the plant that may be negatively affected by the promoter and in this scenario, they would be unaffected. Recently, a phloem specific promoter has been obtained from the rice sucrose synthase gene RSs 1. RSs 1 promoter was used to drive the snowdrop lectin or GNA protein. The results were confirmed by the use of immunological assays and they indicated that not only is the gene being expressed in the phloem tissues, but that the protein product has been successfully transported to phloem sap.

Unfortunately, RSs 1 is heavily expressed in the seeds of rice plants, so an alternative promoter called PP2 is currently under study. So far, PP2 has been purified and partially sequenced. Also, a full cDNA library has been created for the gene and it has been used to probe a genomic library to obtain the corresponding gene. The promoter region form the PP2 gene is now being assayed.

One final method of creating insect resistance in rice plants is the use of the SBTI gene. SBTI gene is a trypsin inhibitor that acts against pests such as the yellow stem borer and the gall midge. Greater insect resistance can be created by introducing the Kunitz soybean trypsin inhibitor (SBTI) gene into varieties of Indica rice plants. First, a PCP product corresponding to the protein was isolated by oligonucleotide primers. Then, the resulting fragment was cloned, sequenced and expressed in E. coli cell cultures. The results were a recombinant SBTI gene that effectively fought off gall midges and yellow stem borers. Presently, the SBTI gene is being cloned into vectors and is being used to transform other types of embryos using the particle gun technique.

In conclusion, through the use of new technologies such as the introduction of potato proteinase inhibitor II gene, the establishment of the Bacillus thuringiensis toxin gene and the experimental methods of using the arcelin gene, the snowdrop lectin/GNA (galanthus nivallis agglutinin) protein, and phloem specific promoters and finally the SBTI gene, rice plants have become almost completely resistant to insects that used to destroy much of the crop. This has been an important step in biotechnology because the improvement of rice plants is a major concern that could potentially effect almost all of the populations of the world. Biotechnology has become an increasingly accepted method of solving some of the major problems in agriculture, medicine, and industry. Potentially, with the advancements of many techniques, almost whenever people eat, drink, take medicine, or go to work, they will be touched in some way by the many complicated processes of biotechnology, that are striving to make our world a better place to exist in.

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