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CELL BIOLOGY & MOLECULAR GENETICS

Production of a Biodegradable Plastic Polymer, Poly-ß-Hydroxybutyrate, in Transgenic Alfalfa

^a Dep. of Plant Pathology, 495 Borlaug Hall
^b Biological Process Technol. Inst., 240 Gortner Laboratories
^c Dept. of Agronomy and Plant Genetics, 411 Borlaug Hall
^d USDA-ARS-Plant Science Research Unit and Dep. of Plant Pathology, 495 Borlaug Hall, Univ. of Minnesota, St. Paul, MN 55108-6030

* Corresponding author (debbys{at}puccini.cdl.umn.edu)

Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa could be achieved by 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-ß-hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha (formerly Alcaligenes eutrophus) encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrate-co-hydroxyvalerate, or 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. Polyhydroxybutyrate content in the leaves of transgenic plants ranged from 0.025 to 1.8 g kg^-1 dry weight (DW). Agglomerations of PHB granules 0.2 to 0.4 µm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants, demonstrating that phb gene products were 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 and accumulation of product 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 line, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.

Abbreviations: DW, dry weight • GC, gas chromatography • GC-MS, gas chromatography–mass-spectrometry • 3-HB, 3-hydroxybutyrate • ¹H-NMR, proton-nuclear magnetic resonance spectrometry • 3-HV, 3-hydroxyvalerate • NPTII, neomycin phosphotransferase II • PCR, polymerase chain reaction • PHA, polyhydroxyalkanoates • PHB, poly-ß-hydroxybutyrate • PHB/V, polyhydroxybutyrate-co-hydroxyvalerate • RT-PCR, reverse transcriptase-polymerase chain reaction

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