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Paper Abstract: Technical discussion of genetic transformation of tomato plant to inhibit production of ethylene & modify ripening process to create longer shelf life. Paper Introduction: The shelf life of fruits and vegetables is economically important to anyone in the farming of these goods or in the grocery business. It is also important in terms of transportation of these goods. Timing of harvest and arrangement of transportation are dependent on the rate of ripening of produce items. For this reason, genetic engineering may become an important asset in the produce industry. This paper will look at the genetic transformation of the tomato plant to inhibit the production of ethylene, a chemical known to be involved in the ripening process. Genetic engineering has many applications, and it is beginning to play a significant role in agriculture. Introducing certain genes into plants can afford them protection against pests and against adverse climatic conditions. This impro Text of the Paper: The entire text of the paper is shown below. However, the text is somewhat scrambled. We want to give you as much information as we possibly can about our papers and essays, but we cannot give them away for free. In the text below you will find that while disordered, many of the phrases are essentially intact. From this text you will be able to get a solid sense of the writing style, the concepts addressed, and the sources used in the research paper. The Plant Cell, 3, pp. Klee, H. These reasons make the production oftransgenic plants an important economic factor in agriculture. The second line exhibited an ethylene reductionof 78 percent. F., Kishore,G,. 284-287. Since this method of transformation of tomato plants was effective inintroducing genes to control the rate of ripening, it may also be a usefulmethod by which to introduce genes which would protect the tomato plantfrom insect pests. These results indicate that inhibition of ethylene production infruit and vegetables by ACC deaminase will considerably extend the shelflife of fruits and vegetables, and thus is an important economic tool. These factors are of considerableeconomic importance to tomato growers, as these fruits deteriorate rapidlyunder normal circumstances and have a very limited storage and shelf lifeonce they have been harvested. Bacteria expressing this protein were capable of degrading ACC toequimolar amounts of alpha-ketobutyric acid and ammonia. (1991). While control plants progressed to full red in seven days, andexhibited a marked degree of softening within two weeks, the transgenictomatoes did not reach the full red stage until 24 days, and remained firmfor an extended period, longer than 4 days, and did not abcise, whereasthe control fruit had abscised after 14 days. Homozygous plants from the higher inhibition line were examined forphenotypic effects. Subsequent HindIII and Smal deletions narroweddown the ACC deaminase activity to 2.4-kb fragment. Thisfuses the ORF to a duplicated CaMV 35S promoter and a pea rbcS-E9 gene 3'.The plasmid vector was mobilized into the ABI strain of Agrobacterium bythe triparental conjugation system using the helper plasmid pRK2 13.Tomato UC82B leaf tissue was transformed using the procedure described byMcCormick et al (1986). J., Hayford, M. The 6G5 ORF was engineered to permit high-level expression inE. Seventeen of them matched the predicted sequence of the 6G5gene product. The pMON17 16 plasmid contains the gene 1 promoter from phage T7 adjacent to the cloning sites. M. Young leaf tissue from plants of the two lines expressing the highestlevels of ACC deaminase were used in ethylene generation assays. Introducing certain genes into plantscan afford them protection against pests and against adverse climaticconditions. (1986). 1187-1193. Tomato ripening is dependent on the phytohormone ethylene, which isalso responsible for many plant development processes such as senescenceand response to stress (Klee et al, 1991, p. Control of ethylene synthesis by expression of a bacterialenzyme in transgenic tomato plants. McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., &Fraley, R. Plant Cell Rep., 5, pp. J., Lycett, G. B., Kretzmer, K. This paper will look at thegenetic transformation of the tomato plant to inhibit the production ofethylene, a chemical known to be involved in the ripening process. A 1 17-bp EcoRV-Sacl fragment from pMON1 27 containing the ACCdeaminase ORF was cloned into a plant transformation vector pMON893. Itcan also be used to alter the maturation process in plants, delayingripening and prolonging fruit life. For this reason, genetic engineering may becomean important asset in the produce industry. Antisensegene that inhibits synthesis of the hormone ethylene in transgenic plants.Nature, 346, pp. 1187). ACC is an intermediate in the synthesis of ethylene from S-adenosylmethionine. The ACC deaminase was used to transform UC82B tomato plants under thecontrol of the cauliflower mosaic virus (CaMV) 35S promoter. Restriction endonuclease digestion patterns indicated that all cosmidclones contained an overlapping region of the 6G5 genome. Chemical inhibitors of ethylene synthesis or action completely blockthe ripening of fruits and the senescence of flowers in many plant species. Of those identified, 18 werechosen for further study. Ethylene induces the expression of many genes involved in ripening at themolecular level and induces the response of flowers, fruits, and vegetablesto pathogens. The line with the highest inhibition of ethylene productioncontained approximately .5 percent of the soluble protein in ACCdeaminase, and the second line contained approximately . Timing ofharvest and arrangement of transportation are dependent on the rate ofripening of produce items. However, anumber of phenotypically normal transgenic plants were obtained from thetransformation. 5 percent. DNA sequencing of this fragment revealed a single openreading frame of 1 17 nucleotides encoding a protein with a molecularweight of 36,8 . Control fruit was completelydessicated at this time. Ethylene generation was measured by enclosing whole leaves or fruit insealed containers and withdrawing 1. The shelf life of fruits and vegetables is economically important toanyone in the farming of these goods or in the grocery business. Visual observation of transgenic fruit stored at roomtemperature indicated a significant reduction in softening, with fruitremaining firm for longer than five months. Seeds from the transgenic plants germinated normally,and the resulting plants were indistinguishable phenotypically from controlplants. W., & Grierson, D. Genetic engineering has many applications, and it is beginning to playa significant role in agriculture. A., Barry, G. In the transgenic tomato fruitexpressing the gene encoding for ACC deaminase, there is a correlationbetween the degree of ethylene inhibition and the delay in the progressionof ripening. (199 ). The research group screened 6 soil bacteria for ACC-degradingenzymes by selecting for organisms capable of growing on minimal mediumwith ACC present as the sole source of nitrogen. This experiment showed that inhibition of ethylene production, whileevident throughout the plant, only exerted its effects at the fruit-ripening stage of plant development in tomatoes. Selection for growthon ACC led to identification of a 2.4-kb DNA fragment containing the ACC-degrading gene. Both thetransgenic line and the normal UC82B line fruit exhibited a peak ofethylene production at three days, but the transgenic fruit synthesizedonly 1 percent of the amount of ethylene synthesized by normal plants. It isalso important in terms of transportation of these goods. 81-84. While ACC deaminase reduces the production of ethylene in theseplants, it does not inhibit their capability of reacting to it, asexogenously applied ethylene induces normal ripening in the transgenicplants. Protein analysis was carried out onpolyacrylamide gels. The plants showed no delay in the onset of flowering or ripening,but they did show a difference in the progression of ripening. In theline best expressing ACC deaminase, ethylene was reduced by 9 percent. This improves crop yields, and is particularly useful indeveloping countries where growing conditions are particularly adverse. There were no effects onthe rest of the plant, or its development. Two pseudomonas specieswere identified and selected for further characterization, and one of them,named 6G5, was used for the production of transgenic tomatoes. coli. -mL gas samples after one hour.Ethylene was quantified by gas chromatography using an alumina column and aflame ionization detector. To confirm the identity of the 6G5 protein, ACC deaminase of thePseudomonas strain ACPC was analyzed and the first 21 amino acids wereidentified. References Hamilton, A. This appeared to confirm that 6G5 codes for an ACCdeaminase. A 1 .6kb.BamHHl-Xbal fragment that retained ACC deaminase activity was subcloned from oneof the cosmids into pUC118. The cDNA encoding for ACC synthetase and ACC oxidasehave recently been cloned, and when the cDNA is expressed in an antisenseorientation, it causes a decrease in ethylene synthesis and delays ripeningof excised tomato fruit (Hamilton, Lycett and Grierson, 199 ). Ina second experiment, this plant line exhibited ethylene reduction ofgreater than 97 percent. Klee et al (1991) examined the role of ethylene in delayingthe ripening of tomatoes, and the effect of introducing a gene coding for asubstance which irreversibly degrades the precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), thus inhibiting ethyleneproduction. For example, tomato fruits are often attacked by tomatocaterpillars and it may be possible to use transformation to produce atransgenic plant which is resistant to these and other pests Since the ACC deaminase gene is effective in inhibiting ethyleneproduction in the tomato plant, and since ethylene has shown to have thiseffect in many plant species, it will should be applied to other types offruit and vegetables for the same reasons: it will delay ripening, thusallowing for sufficient time to harvest and transport produce to storesnationwide, even worldwide, and still give them added shelf life. If it is possible toinhibit the development of this ripening agent in tomato and other plants,then this will provide a means to delay ripening and thus extent the periodof time from picking to edible ripeness, and thus increase transportationand shelf life of these produce items. The cosmid librarywas packaged into lambda phage particles and introduced into Escherichiacol, and cosmid-containing cells were then screened for their ability togrow with ACC as their only nitrogen source. A cosmidlibrary was constructed from genomic DNA in EcoRI-cut pMON17 16, aderivative cosmid vector pHc79. Enzymatic and protein gel blot analysis of plant tissueindicated that several of these plants expressed the ACC deaminase gene athigher levels, up to .5 percent of the total protein. Leaf disc transformation of cultivated tomato (L.Esculentium) using Agrobacterium tumefaclens. 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