RESUMO
Using both biolistic and Agrobacterium-mediated DNA delivery, we have investigated the transformation and regeneration capacity for five Nordic elite cultivars of barley. Transformation was followed as transient expression of the uidA or gfp gene in barley callus. Callus formation and regeneration of transformed callus were evaluated based on callus induction frequency, growth rate, callus appearance, and shoot formation frequency. From the accumulated results, one of the elite cultivars has been selected for our ongoing work in molecular breeding of barley.
Assuntos
Agrobacterium tumefaciens/genética , Hordeum/genética , Brotos de Planta/crescimento & desenvolvimento , Regeneração , Transformação Genética , Biolística/métodos , Técnicas de Cultura/métodos , Expressão Gênica , Hordeum/fisiologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Luz , Brotos de Planta/genéticaRESUMO
The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the sul mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, isol, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.
Assuntos
Genes de Plantas , Hordeum/enzimologia , Hordeum/genética , Isoamilase/genética , Amido/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/crescimento & desenvolvimento , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Sacarose/farmacologia , Triticum/enzimologia , Triticum/genética , Triticum/crescimento & desenvolvimentoRESUMO
Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.
Assuntos
Biolística , Genoma de Planta , Plantas/genética , Rhizobium/genéticaRESUMO
The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5' portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (beta/alpha)8-barrel topology of the alpha-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.