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␤-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase. These enzymes are encoded by phbA, phbB, Alfalfa (Medicago sativa L.) is primarily used as a source of forage and phbC, respectively (Slater et al., 1988). The monoand animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate meric composition of PHA polymers is determined by the potential of alfalfa as a source of industrial materials, we employed the substrate specificities of ␤-ketothiolase and PHB a genetic transformation approach to produce a biodegradable plastic, synthase and by the availability of acetyl-CoA and propoly-␤-hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three pionyl-CoA. In the case of 4-and 5-carbon PHA precurgenes from Ralstonia eutropha (formerly Alcaligenes eutrophus ) sors, when ␤-ketothiolase is encoded by phbA, a homoencoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and polymer of PHB is produced. When ␤-ketothiolase is a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrateencoded by bktB, a copolymer of PHB/V is produced co-hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for (Slater et al., 1998). Because the PHB/V copolymer has plastid targeting were introduced into alfalfa by Agrobacterium-medibetter physical properties than PHB, it is preferred for ated transformation. DNA and RNA blot analyses of transgenic plants most commercial applications (Anderson and Dawes, indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants 1990). Presently, the bacterially produced polymers are ranged from ≈0.025 to 1.8 g kg Ϫ1 dry weight (DW). Agglomerations not cost-competitive with nonbiodegradable plastic of PHB granules 0.2 to 0.4 m in diameter, similar to bacterial PHB, polymers derived from petroleum. By genetically engiwere located in the chloroplasts of transgenic plants, demonstrating neering crop plants to produce PHA, a less expensive that phb gene products were targeted into the plastids of transgenic source of these polymers could become available. alfalfa. Transgenic plants exhibited growth similar to untransformed PHB production in plants was first demonstrated in plants, suggesting that expression of PHB biosynthetic pathway genes transgenic Arabidopsis thaliana (L.) Heynh. (Poirier et at current levels and accumulation of product in the plastids had no al., 1992). Poly-␤-hydroxybutyrate is tolerated by plants deleterious effect on growth and fertility. F 1 hybrid progeny, obtained if it is synthesized in chloroplasts and PHB levels ranging from crosses of PHB transgenic plants with elite alfalfa germplasm, between 0.1 and 14% DW have been reported with exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly minimal effects on plant growth (Nawrath et al., 1994).inherited trait.Although various approaches for producing PHB in plants have been reporte...

Plants native to boreal, temperate, and subtropical regions are subjected to large seasonal variations in temperature. In these regions, woody perennial plants have evolved annual growth cycles that promote long-term survival and growth. These annual growth cycles involve alternations between active shoot growth and vegetative dormancy (endodormancy) that are closely timed with seasonal changes in the local climate. Dormancy, in general, is defined as the temporary suspension of visible growth of any plant structure containing a meristem (Lang, 1987). Endodormancy, which develops in the fall, is characterized by a requirement for sustained exposure to low, near-

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

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‐β‐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.