ABSTRACT
The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.
Subject(s)
Acetyl-CoA C-Acyltransferase/metabolism , Acyltransferases/metabolism , Alcohol Oxidoreductases/metabolism , Arabidopsis/metabolism , Cupriavidus necator/enzymology , Polyesters/metabolism , Acetyl-CoA C-Acyltransferase/genetics , Acyltransferases/genetics , Alcohol Oxidoreductases/genetics , Arabidopsis/genetics , Cupriavidus necator/genetics , Homozygote , Models, Molecular , Molecular Structure , Plant Leaves , Plants/metabolism , Plants, Genetically Modified , Recombinant Proteins/metabolism , SeedsABSTRACT
A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.
Subject(s)
Cell Aggregation , Cells, Cultured/cytology , Cytological Techniques , Liver/cytology , Animals , Cell Survival , Cells, Cultured/ultrastructure , Humans , Liver/ultrastructure , Microscopy, Electron , Organelles/ultrastructure , Rats , Rats, Sprague-DawleyABSTRACT
The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency.
