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Transgenic Rice Plants

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For centuries, rice has beenone of the most important staple crops for the world and it now currently feedsmore than two billion people, mostly living in developing countries. Riceis the major food source of Japan and China and it enjoys a long history ofuse in both cultures. In 1994, worldwide rice production peaked at 530 millionmetric tons. Yet, more than 200 million tons of rice are lost each year tobiotic stresses such as disease and insect infestation. This extreme lossof crop is estimated to cost at least several billion dollars per year andheavy losses often leave third world countries desperate for their staple food.

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Therefore, measures must be taken to decrease the amount of crop loss andincrease yields that could be used to feed the populations of the world. Onemethod to increase rice crop yields is the institution of transgenic rice plantsthat express insect resistance genes. The two major ways to accomplish insectresistance in rice are the introduction of the potato proteinase inhibitorII gene or the introduction of the Bacillus thuringiensis toxin gene into theplant’s genome.

Other experimental methods of instituting insect resistanceinclude the use of the arcelin gene, the snowdrop lectin/GNA (galanthus nivallisagglutinin) 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 adicotyledonus plant to a monocotyledonous plant. Whenever the plant is woundedby insects, the PINII gene produces a protein that interferes with the insect’sdigestive processes. These protein inhibitors can be detrimental to the growthand development of a wide range of insects that attack rice plants and resultin insects eating less of the plant material. Proteinase inhibitors are ofparticular interest because they are part of the rice plant’s natural defensesystem against insects. They are also beneficial because they are inactivatedby cooking and therefore pose no environmental or health hazards to the humanconsumption of PINII treated rice. In order to produce fertile transgenicrice plants, plasmid pTW was used, coupled with the pin 2 promoter and theinserted rice actin intron, act 1. The combination of the pin 2 promoter andact 1 intron has been shown to produce a high level, wound inducible expressionof foreign genes in transgenic plants. This was useful for delivering theprotein inhibitor to insects which eat plant material. The selectable markerin this trial was the bacterial phosphinothricin acetyl transferase gene (bar)which was linked to the cauliflower mosaic virus (CaMV) 35S promoter. Nextthe plasmid pTW was injected into cell cultures of Japonica rice using theBiolisticTM particle delivery system. The BiolisticTMsystem proceeds asfollows: Immature embryos and embryonic calli of six rice materials werebombarded with tungsten particles coated with DNA of two plasmids containingthe appropriate genes. The plant materials showed high frequencyof expression of genes when stainedwith X-Gluc. The number of blueor transgenic units was approximately 1,000.

After one week, the transgeniccells were transferred onto selection medium containing hygromycinB. After two weeks, fresh cell cultures could be seen on bombardedtissue. Some cultures were white and some cultures were blue.

Isolated cellcultures were further selected on hygromycin resistance. However,nocontrol plant survived. Then twenty plates of cells were bombarded withthe PINII gene, from which over two hundred plants were regenerated and grownin a greenhouse. After their growth, they were tested for PINII gene usingDNA 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 digestedby a suitable restriction endonuclease, separated on an aragose gel, transferredto a nylon membrane, and then finally hybridized with the 1.5 kb DNA fragmentwith pin 2 coding and 3′ regions as the probe. The results also indicate thatthe PINII gene was inherited by offspring of the original transgenic line,that the PINII levels were higher among many of the offspring and that whenPINII levels rose in wounded leaves, the PINII levels in unwounded leaves alsorose. However, the PINII gene is not 100% effective in eliminating insectsbecause it does not produce an insect toxin, just a proteinase inhibitor. Yet, greater insect resistance can be achieved by adding genes to producethe Bacillus thuringiensis or BT toxin.Bacillus thuringiensis is an entomocidalspore-forming soil bacterium that offers a way of controlling stem boring insects.

Stem borers such as the pink and striped varieties are difficult to controlbecause the larvae enter the stem of the plant shortly after hatching and continueto develop inside the plant, away from the toxins of sprayed insecticides.

Therefore, the stable institution of the BT gene into the rice plant’s genomewould provide a method of reaching stem borers with toxins that are expressedin the plant tissues themselves. Bacillus thuringiensis is comprised ofso-called cry genes that encode insect specific endotoxins. Recently somelower varieties of rice, such as Japonica, have been successfully transformedwith cry genes, but the real challenge lies in transforming Indica rice, anelite 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 usedbecause 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 attachedto a CaMV cassette for hygromycin selection of transformed tissues. Followingthe linkage of the cry IA and the CaMV 35S cassette, the DNA is delivered tothe embryonic cells by particle bombardment with a particle inflow gun. Morespecific transformation includes the following:Immature Indica rice embryoswere isolated for ten to sixteen days after pollination from other greenhouseplants 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 highlyosmotic medium containing a higher percentage of sucrose (10%), were incubated,and then were bombarded with plasmid pSBHI and gold particles by the particleinflow gun. After bombardment, the thin layer of 10% sucrose was placed onthe layer of 3% sucrose. This sandwich technique allowed continuous adaptationof the target tissue to the osmotic conditions, which was shown to be optimalfor callus induction. After twenty four hours, the 10% sucrose layer was removedand the embryos were cultured on the 3% sucrose layer. After one week, theywere transferred to a 3% sucrose medium that was selected for hygromycin Bresistance. After a further three to four weeks, regenerated plants were transferredto soil and placed in the greenhouse under appropriate conditions. The resultsof this process were eleven transgenic plants out of a total of thirty six.

Transgeneicy of the rice plants was confirmed by similar banding patternsin Southern blotting. The presence of the BT protein was also demonstratedin Western blot analysis, where a protein with the expected size of sixty-fivekilobases was found in all plants tested. Interestingly enough, the BT proteinlevels were higher in older plants than in younger plants, possibly questioningthe role of inheritance of BT gene. Yet, inheritance was determined by usingDNA blot hybridization, which revealed a segregation ratio of 3:1. This indicatesthe integration of all copies of transgene at a single locus. To assessthe mortality rate among different insects, both petri dish assays and wholeplant assays were performed. In petri dish assays, mortality rates were asfollows:European corn borer = 85-95%Yellow stem borer = 100%Striped stemborer = 100%Cnaphalocrocis medinalis (leaffolder) = 67%Marasmia patnalis(leaffolder) = 55% In whole plant assays, no surviving insects were foundon any BT expressing plants, although insects still survived on the controlplants or non expressing BT plants. In addition to this recent insertionof the BT gene into Indica rice, a similar procedure was conducted on Shuahggei36, a variety of Indica rice. Transgeneicy of Shuahggei 36 was achieved bytaking plasmid P41ORH, which contained the coding region of the BT gene withthe marker CaMV 35S-HPI-NOS plus 1.0 kb of DNA fragment, and inserting it intothe pollen tube pathway. More specifically, the plasmid DNA was applied atthe cut ends of rice florets from one to four hours after pollination. Nextthe 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 hygromycinresistance. From these seeds, seventy plant lines were screened for transgeneicyand fifteen displayed the BT protein. These results and the inheritance ofthe BT gene into offspring were confirmed by Southern blotting. Nevertheless,the question remains whether the BT gene was really integrated into the genomeor whether it was expressed only as a plasmid. The use of the arcelin geneis another experimental method of creating transgenic rice plants. The arcelingene is a translationally enhanced Bacillus thuringiensis toxin construct thatis effective on the rice water weevil. The rice water weevil or RWW is themajor pest of the Texan rice crop. Previously, the RWW was combated by granularcarbofuran, an insecticide that kills the RWW but has deleterious effects onwater fowl that live in the crop area. So environmentalists have forced thecessation of the use of granular carbofuran and therefore, new methods haveto be developed. One of the major genes that confer resistance to the RWWis the arcelin gene. Arcelin is a lectin that was originally discovered inthe seeds of bean cultivators that showed resistance to the Mexican bean weevil.

Next, researchers isolated a genomic clone encoding arcelin from the beanseed and then placed it under regulation of a rice actin promoter. Then theclone with the rice promoter was introduced into rice protoplasts. Transgeneicyand inheritance was then confirmed by genomic DNA blots and immunochemicalblots. In two separate experiments, six transgenic rice plants were subjectedto RWW infestation under controlled conditions. The results of the first experimentare that similar numbers of RWW larvae were recovered from each set of sixplants, but the size of those from arcelin expressing plants were significantlysmaller. In the second experiment, although many normal larvae were recoveredfrom control plants, only three small larvae came from arcelin expressing plants.

This would indicate the benefits of inserting the arcelin gene into rice plantsfor RWW resistance.

Another experimental method of creating transgenic riceplants that are insect resistant includes the use of snowdrop lectin or galanthusnivallis agglutinin (GNA). Snowdrop lectin helps to control the sporadicallyserious pest the brown planthopper (BPH), which has developed a resistanceto many pesticides. Luckily for the environment, snowdrop lectin provideshigh levels of toxicity to BPH but not to other animals. BPH is a member ofthe order Homoptera and feeds by sucking the phloem sap from the stems of riceplants. The major problem with combating BPH is that rice plants can not beengineered for BT toxin resistance against this pest because BT toxins thateffect Homopterans have not yet been discovered or reported. Therefore, othertypes of genes had to be manipulated in order to produce insect resistanceagainst BPH. The best plant protein that provides resistance to BPHs turnsout to be snowdrop lectin, and this was first confirmed by artificial dietbioassays. To create the transgenic rice plants, embryonic cell suspensioncultures were initiated from mature embryos from two Japonica rice varieties,Taipei 309 and Zhonghua 8. Next, the protoplasts isolated from these cellsuspension cultures were transformed by using the plasmid pSCGUSR, containingthe nos-npt II gene as a selectable marker. Plasmid uptake was then inducedby the PEG process and geneticin was used as a selection agent. Geneticinwas added to the protoplast-derived colonies during the four and eight cellstages. From this, more than fifty putative transgenic plants have been regeneratedfrom one thousand resistant colonies. Another way of combating the brownplanthopper is by producing phloem-specific promoters. These promoters arenecessary because phloem is the exact site of feeding for the BPH. Althoughthe CaMV promoter is active in phloem tissue, the possibility exists to institutea promoter from a gene that is specifically expressed only in phloem. Thiswould be advantageous if there are other parts of the plant that may be negativelyaffected by the promoter and in this scenario, they would be unaffected. Recently,a phloem specific promoter has been obtained from the rice sucrose synthasegene 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 indicatedthat not only is the gene being expressed in the phloem tissues, but that theprotein product has been successfully transported to phloem sap.Unfortunately,RSs 1 is heavily expressed in the seeds of rice plants, so an alternative promotercalled PP2 is currently under study. So far, PP2 has been purified and partiallysequenced. Also, a full cDNA library has been created for the gene and ithas been used to probe a genomic library to obtain the corresponding gene.

The promoter region form the PP2 gene is now being assayed. One finalmethod of creating insect resistance in rice plants is the use of the SBTIgene. SBTI gene is a trypsin inhibitor that acts against pests such as theyellow stem borer and the gall midge. Greater insect resistance can be createdby introducing the Kunitz soybean trypsin inhibitor (SBTI) gene into varietiesof Indica rice plants. First, a PCP product corresponding to the protein wasisolated by oligonucleotide primers. Then, the resulting fragment was cloned,sequenced and expressed in E. coli cell cultures. The results were a recombinantSBTI gene that effectively fought off gall midges and yellow stem borers. Presently, the SBTI gene is being cloned into vectors and is being used totransform other types of embryos using the particle gun technique.Inconclusion, through the use of new technologies such as the introduction ofpotato proteinase inhibitor II gene, the establishment of the Bacillus thuringiensistoxin gene and the experimental methods of using the arcelin gene, the snowdroplectin/GNA (galanthus nivallis agglutinin) protein, and phloem specific promotersand finally the SBTI gene, rice plants have become almost completely resistantto insects that used to destroy much of the crop. This has been an importantstep in biotechnology because the improvement of rice plants is a major concernthat could potentially effect almost all of the populations of the world. Biotechnology has become an increasingly accepted method of solving some ofthe major problems in agriculture, medicine, and industry. Potentially, withthe advancements of many techniques, almost whenever people eat, drink, takemedicine, or go to work, they will be touched in some way by the many complicatedprocesses of biotechnology, that are striving to make our world a better place to exist in.

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Cite this Transgenic Rice Plants

Transgenic Rice Plants. (2019, Jan 03). Retrieved from https://graduateway.com/transgenic-rice-plants/

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