Workshop on Biotechnology in Agriculture Summary Report, Jakarta, Indonesia, March 13-14, 1986 (1986) / Chapter Skim
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WORKING GROUP REPORTS
WORKING GROUP REPORTS
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PART II Working Group Reports
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These revelations opened the door to an unprecedented explosion of scientific knowledge, the most recent of which includes the biotechnological developments of recombinant DNA technology or gene splicing (altering heredity by transplanting genes from one organism into another)
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RECOMBINANT DNA AND HYBRIDOMA TECHNOLOGIES Recombinant DNA Technology Recombinant DNA technology, considered a modern-day form of genetic engineering, is not a single discipline in itself. Rather, it represents a fusing of ideas and techniques from biochemistry, molecular biology, genetics, and organic chemistry.
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Once the vector carrying the inserted foreign DNA molecule is placed into an organism such as bacteria or yeast, it will replicate to A RECOMBINANT DNA EXPERIMENT Vector DNA (plasmid) Insert DMA Racombinant Plasmid DNA FIGURE 1 Basic recombinant DNA experiment.
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In addition to recombinant DNA techniques, two developments, both in organic chemistry, have greatly facilitated progress in genetic engineering. The first involves chemical methods to synthesize genes or gene fragments de novo in an effort to modify or alter genes.
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Vaccines and Antitoxins One of the major and earliest ways in which recombinant DNA and hybridoma technologies will improve animal production is by providing the animal health care industry with efficacious vaccines and antitoxins that will reduce morbidity and mortality from Infectious disease. Of the 45 million cattle born last year in the United States, approximately 10 percent died of infectious disease.
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Monoclonal antibodies have also been generated for protection of newborn calves and swine against enteric collibacillosis, which is responsible for neonatal diarrhea or scours. Although both conventional and genetically engineered vaccines are available, the monoclonal antibody approach appears to be far superior to vaccination for two reasons.
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iNO FORMS RECOMBINANT CNA THE RECOMBINAHT PLASMIO IS INSERTED 9iCX INTO THE BACTERIUM. -WHICH DIVIDES AND REPLICATES, COPYING JTSELF AND THE RECOMBINANT DNA O O EXPRESSI0.\' OF BOVIXE PAPILLOMA VIRUS COAT PROTEIN C2NE PRODUCTION OP COVINE PAPILLOMA VIKUS COAT PROTEIN VACCINE BY FERMENTATIO;: papillomavirus vaccine by recombinant DNA
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GROWTH PROMOTANTS The development of natural growth hormones for livestock and poultry represents a major means of improving animal production, and genetic engineering techniques have made this development a reality for both logistical and economic reasons. Several groups have now cloned bovine growth hormone (bGH)
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Generally, engineered approaches to improving animal production appear to be directly applicable to hormones and other growth promotants where availability of the natural substance is limited and the costs of obtaining the natural hormone exceed reasonable marketing considerations. Further studies are required to determine the safety of recombinant bGH for both treated animals as well as the consumer of its milk.
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Recombinant DNA procedures have been utilized to isolate the zein gene and determine its precise biochemical structure, and this has resulted in deduction of the primary structure of the zein storage protein. With the availability of this information, it is now possible, employing genetic engineering approaches, to alter the zein gene structure in an effort to increase its lysine content and therefore its nutritional quality.
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Recombinant DNA will allow microorganisms to produce less expensive and more nutritious feed ingredients. Genetic engineering will eventually help increase crop yields, make possible more nutritious corn and other crops, and produce less expensive vitamins, amino acids, and single-cell protein.
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- 30 On the other hand, it carries the realizable potential of contributing significantly to the solution of the most difficult problems facing animal health and production today.
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PRESENT STATE OF ACTIVITIES IN INDONESIA Ongoing R&D Programs The very limited R&D activities in embryo transfer and animal production technologies carried out to date in Indonesia have been scattered in various institutions and universities. Host of the activities worth mentioning have been conducted at Bogor Agricultural University (IPB)
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Thus, Indonesian scientists working on embryo transfer and animal production biotechnologies are becoming increasingly isolated. FUTURE PLANS Future plans for the development of R&D on embryo transfer and animal production biotechnologies should focus on application of the latter.
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Based on the needs of Indonesia as well as the potential of achieving its goals, the following R&D programs in embryo transfer and animal production biotechnologies have been identified: o Increasing through embryo transfer the number of animals, and thereby the amount of available animal protein o Improving breeding stocks o Producing supporting materials for animal production -- for example, hormones, vaccines, and monoclonal antibodies. Cooperative R&D activities between Indonesian and U.S.
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Moreover, meetings between scientists working in embryo transfer and animal production biotechnologies should be encouraged while the centers of activities are being established.
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The ability to reproduce desirable species using plant cell and tissue culture techniques has resulted in their practical applications. Recent breakthroughs with recombinant DNA technology using bacteria has spawned tremendous interest in directly incorporating specific genes into plants using r-DNA techniques.
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It is unclear to what extent haploid culture will increase in importance. SOMOCLONAL VARIATION In addition to their usefulness in producing plant uniformity, plant cell and tissue culture techniques can enhance genetic variation.
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PROTOPLAST CULTURE No discussion of plant cell and tissue culture techniques is complete without mentioning protoplast culture. Because this technique enables one to make single cells from plants and then manipulate these cells to differentiate into embryos, this stage represents an opportunity to work with plant cells as if they were bacteria, and it is the probable target for substantial research to incorporate r-DNA into the plant genome.
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Since the primary target of most applications of plant cell and tissue culture techniques is crop improvement, it is essential to decide which plants have the highest priority. This decision relies on both a financial evaluation and a research consideration.
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Universities worldwide are possible resources, and companies offer exploitable opportunities as well because of their expertise and their orientation toward practical goals. CONCLUSION Over the past five years, the business sector has invested heavily in plant biotechnology, including plant cell and tissue culture, and has employed outstanding researchers to formulate and implement their plans.
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For example, it is being employed successfully for such varied purposes as rapid clonal propagation and virus elimination; varietal development, genetic modification, and crop improvement; and production of secondary substances, independent of environmental factors. Clonal Propagation The most advanced applications of plant cell and tissue culture have been in rapid clonal propagation.
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Secondary Metabolites In the early 1950s, it was discovered that plant cells, like microorganisms, could be grown in liquid medium. Thus, plant tissue culture has provided an alternative to the cultivation of whole plants as important sources of many useful compounds, including drugs, flavorings, enzymes, essential oils, and food colorings.
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Pimpinella. Sonchus Note: IPB, Bogor Agricultural University; ITB, Bandung Institute of Technology; UGM, Gadjah Mada University; LBN, National Biological Institute; BORIEC, Bogor Research Institute of Estate Crops; MARIF, Malang Research Institute of Food Crops; LEHRI, Lembang Horticulture Research Institute; BP3G, Central Research Institute for Sugarcane; UI, University of Indonesia.
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. Facilities Both the universities and research institutes have at least basic plant cell and tissue culture research facilities.
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FUTURE PLANS R&D Programs R&D programs must be established, with the following topics getting high priority: o Rapid clonal propagation -- Estate crops: oil palm, coconut, rubber, coffee, cacao, clove Industrial crops: dipterocarp, rattan, sandalwood, teak, eucalypts Horticultural crops: banana/plantain, melon, potato, strawberry, asparagus, garlic, pineapple, tropical fruits. o Crop improvement Food crops: rice and corn cultivars with a high nutritive value as well as a tolerance of salinity and heat Forage legumes: high nutritive value Horticultural crops: potato cultivars tolerant of high temperatures and salt concentrations as well as resistant to bacterial wilt; tomato cultivars tolerant of high salt concentrations and resistant to disease Industrial crops: highly productive, disease-resistant sugarcane cultivars.
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Periodic assessment of manpower needs at research institutes and universities is needed. Facilities Tissue culture laboratories in research institutes and universities should have at least the following facilities: preparation room, washing room, chemical storage room, sterile room, cold storage for media, and temperature-controlled culture room with illuminated shelves.
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Alternatively, can corn or similar crop plants be genetically engineered to form symbiotic root nodules with rhizobia? The development of biotechnology has been unexpectedly rapid with respect to solving the technical problems of transferring genes into crop plants and having these genes expressed appropriately.
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It is also attractive because it does not require either partner in the symbiosis to behave in an altruistic manner. This model will be of considerable importance in future attempts to improve the basic efficiency of nitrogen fixation.
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REGULATION OF NODULE FORMATION AND NITROGEN FIXATION It is becoming increasingly evident that the legume/rhizobia symbiosis is a highly evolved and regulated association. Several recent investigations have shown that nodule formation and nitrogen fixation are subject to various forms of feedback regulation.
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- 49 COMPETITION FOR NODULE OCCUPANCY A major practical problem for growers wishing to obtain maximum benefit from Rhizobium nitrogen fixation is that of getting inoculated rhizobia to generate enough nodules, particularly in the face of competition from native rhizobia already in the soil. Past research efforts have mainly concentrated on finding strains of rhizobia that nodulate and survive well in the soil.
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The latter has been made possible in part by implementation of a liming program for acid soils, which constitute a large part of the newly opened areas. The ability of soybean crops to obtain nitrogen through biological nitrogen fixation (BNF)
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A national program on biological nitrogen fixation was established in 1981 under the coordination of the Indonesian Institute of Sciences. Financial constraints, however, meant that scientists invited to participate in the program were able to meet only three times during a five-year period (the last meeting held in 1984)
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This in turn affects how widely BNF is applied to agricultural practices in Indonesia. FUTURE PLANS R&D Programs A core group of scientists undertaking BNF research that represents the relevant disciplines must be organized.
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Manpower Development More well-trained technicians and scientists are required to develop a BNF research capability. Because it is important that the appropriate curricula are offered to prepare university graduates for carrying out solid research in BNF, rapid development of the following scientific disciplines is recommended: microbial physiology and molecular genetics, general and plant biochemistry, plant physiology and breeding, plant molecular genetics, and soil sciences.
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It is also found as monomeric sugars or soluble oligomers in cassava syrup, molasses, and raw sugar juice. Biomass also occurs as lignocellulose in the form of wood chips, crop residues, forest and mill residues, urban refuse, and animal manures.
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This paper concentrates on the relatively low-cost, high-volume products labeled "primary petrochemicals" in Figure 1. The markets for "bioproducts" -- products that might be produced from renewable materials -- are listed in Table 2 (Busche, 1983b)
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Organic solvents and acids Amino acids 1,700 Antibiotics 1,625 Vitamins 667 Industrial enzymes 440 Steroids and alkaloids Polypeptides and hormones Nucleotides, nucleosides Medicinal enzymes 155 Biopolymers Polysaccharide gums 100 r»*
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For the free market economy of the United States, however, these dehydration processes suffer from inherently poor raw material stoichiometry, expressed earlier in the maxim: "Don't remove 1^0 from Ct^O." Hence, it seems more reasonable to expect that as fossil fuels become more expensive, both cellulose and starch could become increasingly important as cheap raw materials for oxychemicals that retain the oxygenated nature of the glucose monomer units of biomass. Against the background of the present synthetic organic chemicals industry, the 16 top oxychemicals listed in Table 3 have been, are being, or could be produced from renewable materials rather than fossil materials.
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Ethanol Ethylene 6,790 Butadiene 1,320 Octane enhancer 560 Industrial 380 Subtotal 9,050 Ethylene glycol 1,260 Adipic acid 1,030 Acetic acid 620 Isopropanol 500 Acetone 460 Acrylic acid 360 Glycerol 250 1,4-Butanediol 240 Propylene glycol 220 Methylethylketone 210 n-Butanol 200 Citric acid 190 Sorbitol 90 Propionic acid 35 Fumaric acid 25 TOTAL OXYCHEMICALS 14,180 Source: U.S. International Trade Commission, Washington, D.C.
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Companies entering these markets will need to develop both a manufacturing position and a raw materials position to be successfully involved. RAW MATERIALS SUPPLY As a product of solar energy, biomass depends on land dedicated to useful photosynthesis.
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. About 105 million annual dry tons of corn stalks and 180 million annual dry tons of cereal straw are available and could be collected if the demand warranted it.
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TABLE 7 U.S. Cellulosics Potential -- Forest Resources (Million Annual Dry Tons)
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Hardwoods are, however, preferred for chemical use since they contain less lignin and tacky dirt-collecting extractibles. Presumably, they could be used without seriously competing with the pulp and paper industry for raw materials.
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770 million dry tons, collectible supply. ACETIC ACIO SLYCEROL ACETONE n-BUTANOL ISOPROPANOL ADIPIC ACID OTHER OXTCHEHOLS SINGLE CELL PROTEIN HYDROGtNATION I PHENOLS AROMATICS \ DIBASIC AUDS \ OLEFINS \ FOOD GRADE SWEETENERS FIGURE 2 Feedstocks from renewable resources.
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. To evaluate the competitive advantages of any scheme developed from the "biorefinery" overview, one needs to examine the relative cost of raw materials and the conversion costs of the process alternatives.
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Corn @ $3.00/bu $1.05 Grains credit @ $140/t (.46) Steam -- alcohol recovery .10 -- grains recovery .06 Other conversion costs .23 Mill costs .97 Cost plus 30 percent pretax $1.42 10 I4.220l 10 3 Z *
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Thus, a trade-off occurs between the low cost of raw materials and the high investment needed for hydrolysis equipment. TABLE 11 Costs of Pretreated Wood Chips (Dollars per Dry Ton)
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The dilute acid process reduces acid-associated costs to about $.003 per pound of sugar, produced at a lignocellulose cost of $.03 per pound sugar (1985 dollars) ; however, power costs are high.
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. Research on new versions of concentrated acid hydrolysis has lagged behind that on dilute acid processes.
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: 1980, 1985, 1990 1980 1985 1990 Corn stover $.015 $.021 $.031 Whole tree wood chips .013 .018 .026 Pretreated wood chips .031 .050 .072 Biosugar ex lignocellulosics Enzyme/acid pretreat .080 .129 .193 Concentrated acid/recycle .081 .123 .181 Dilute acid/extrusion .088 .140 .209 Concentrated acid/once-through .126 .187 .268 Corn syrup (as glucose)
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Fermentation Costs Of all the fermentation parameters, product concentration has the greatest effect on conversion cost. Batch time, or dilution rate for continuous operation, is second in importance.
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Figure 5 also compares the conversion costs of three alternative routes to ethanol. The $.12 per pound ($.75 per gallon)
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In newer energy-efficient fermentation plants, total plant energy demand has been reduced to as low as 30,000-50,000 Btu/gal. Most of this is for the recovery operation.
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For example, acetic acid and water are relatively close in volatility. To recover glacial acid from a 1.5 wt% aqueous solution by simple distillation, as shown in Figure 6, would require a column operated at a very high 2.8 reflux ratio, at a steam load of 275,000 Btu/lb of acid recovered (E.
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The acid could then be recovered by the extraction process shown in Figure 7. In the case of a plant that was scaled to produce 250 million annual pounds of AGIO REFINER HEAT EXCHANGER Aqu«oui Recycle FIGURE 7 Acetic acid recovery via solvent extraction.
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. The energy demands for recovering acetic acid by various processes are summarized in Table 14.
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1983a. Recovering chemical products from dilute fermentation broths.
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1984. Acetic acid manufacture -- fermentation alternatives.
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1981. Partial acid hydrolysis of poplar wood as a pretreatment of enzymatic hydrolysis.
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1960. The Development of a Concentrated Sulfuric Acid Wood Hydrolysis Process in Japan.
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In the industrial sector, biotechnology will greatly help in the production of biomass from agricultural by-products, production of high-value chemical compounds as well as solvents, and treatment of industrial and municipal wastes. PRESENT UTILIZATION OF AGRICULTURAL BY-PRODUCTS Current R&D Programs R&D programs are now under way at various Indonesian universities and government and private research institutes.
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The few that exist are usually not well supplied with the current biotechnology literature or research results. FUTURE PLANS R&D Programs The amount of by-products and waste generated annually by the agricultural sector is approximately three to five times the product itself.
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Programs that seek to produce the following appear to be feasible and profitable: o Single-cell protein and biomass for animal feed o Industrial enzymes, especially hydrolases o Antibiotics -- tetracycline, penicillin, erythromycin, kanamycin o Steroid drugs o Vitamin Bj» through fermentation o Fish protein from fish scraps o Supercritical extraction-based products such as vitamin A/B carotene (crude palm oil) , coconut oil, soy oil, and decaffeinated coffee and caffein from coffee.
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/ ^ 1 Chemical engineering 1 I Industry | Production (ethanol, citric acid, MSG, HFS, FS, single-cell protein, antibiotics, vitamins, etc.) 1 1 FIGURE 1 Scheme of cooperation among various government R&D institutes, universities, and industry.
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- 84 Funding source Indonesia Information Manpower ' ^ Technology Biotechnological Institute (U.S.) Technology U.S.
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