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Title:   Author  item
                                            SARUUL, PUREVIN - UNIVERSITY OF MINNESOTAitem
                                            SRIENC, FRIEDRICH - UNIVERSITY OF MINNESOTAitem
                                            SOMERS, DAVID - UNIVERSITY OF MINNESOTAitem
                                            Samac, Deborah

                                                         - *Debby*
  Submitted to: Crop Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/19/2001
                                    Publication  Date: 5/1/2002
                                    Citation: Saruul, P., Srienc, F., Somers, D.A., Samac, D.A. 2002. Production of a biodegradable plastic polymer, poly-B-hydroxybutyrate, in transgenic alfalfa. Crop Science. 42:919-927.Interpretive Summary: Many organisms, including humans, produce small amounts of a compound called polyhydroxybutyrate (PHB) that has physical and thermal properties that allow it to be formed into plastic. The plastic is completely biodegradable by microorganisms. It can be used to make packaging for consumer goods and for medical applications. Bacterial cultures have been  used to produce this plastic, however such production is not cost competitive with petroleum-based plastics. We have tested whether PHB can be produced in alfalfa plants, which would be economical to grow for plastic production. We introduced three genes from a bacterium that are needed for plastic production into alfalfa plants. We found that the genes functioned correctly and that plants accumulated PHB in their leaves. PHB production had no observed deleterious effect on the plants. The ability to produce PHB was inherited in offspring of the original plants and the  offspring had PHB accumulation comparable to the parents. At least 2 grams of PHB were produced per kilogram of dry alfalfa leaves. The PHB extraction procedures available likely removed only a small portion of the PHB from leaves; more efficient extraction methods are needed to obtain the full amount of PHB produced by these plants. These alfalfa plants could be integrated into a system in which leaves were extracted for PHB and stems were used for energy production through synthesis of ethanol or burning to produce electricity. Such a system would greatly enhance the value of the alfalfa crop for the grower and provide an economical source of biodegradable plastic.
                                Technical Abstract: Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa likely requires developing new uses of this perennial crop. To investigate the potential of alfalfa as a source of industrial materials, we employed a genetic transformation approach to produce a biodegradable plastic, poly-beta-  hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (PHB/V) (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium-mediated transformation. DNA and RNA blot analyses of transgenic plants indicated integration and expression of the PHB biosynthetic pathway genes. PHB content in the leaves of transgenic plants ranged from approximately 0.025 to 2.0 g kg-1 dry weight. Agglomerations of fPHB granules 0.2 - 0.4 mm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants demonstrating that phb gene products were correctly targeted into the plastids of transgenic alfalfa. Transgenic plants exhibited growth similar to untransformed plants suggesting that expression of PHB biosynthetic pathway genes at current levels in the plastids had no deleterious effect on growth and fertility. F1 hybrid progeny, obtained from crosses of PHB transgenic plants with elite alfalfa germplasm, exhibited leaf PHB levels similar to the transgenic parental lines, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.


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