|Samac, Deborah - Debby|
Submitted to: North American Alfalfa Improvement Conference
Publication Type: Abstract Only
Publication Acceptance Date: 7/17/2000
Publication Date: N/A
Technical Abstract: Plants can be used as living factories to produce industrial raw materials from renewable resources. Alfalfa, with its high biomass production, is an ideal candidate for producing a biodegradable plastic as a value-added product. In this study, alfalfa was engineered to produce biodegradable polymers, polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co hydroxyvalerate (PHBV). The homopolymer PHB is somewhat brittle, while PHB copolymers have more flexibility and therefore are suitable for many commercial applications. Three genes from Ralstonia euthropha encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and PHBV (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium-mediated transformation. Presence of transgenes was assayed by PCR, and DNA and RNA blot analyses confirmed the integration and expression of PHB biosynthetic pathway genes. Gas-chromatographic analysis of leaf extracts from 128 plants showed that synthesis of PHB in the leave ranged from 8 to 350 ug/g of fresh weight representing 0.024% - 0.2% of the plant dry weight. Only one out of the 95 transformants for PHBV synthesis actually contained hydroxyvalerate monomers. Transmission electron microscopy of plant sections revealed that PHB accumulated as agglomerations of electron-lucent granules 0.2 - 0.5 um in diameter that were located in the chloroplasts. Immuno-gold labeling of ultra-thin sections showed preferential binding of gold particles to the chloroplast structures including some to the surface of PHB-granules. Transgenic alfalfa with the highest concentrations of PHB were cross pollinated with plants being developed for biomass production and analysis of PHB content in F1 plants are currently under investigation.