Barley aleurone layer cell protoplasts as a transient expression system.
Protoplasts were prepared from barley aleurone layers using 'Onozuka' cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of alpha-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley alpha-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormone-responsive elements in hydrolase genes.
Gopalakrishnan
B
B
Department of Biochemistry, Kansas State University, Manhattan 66506.
Sonthayanon
B
B
Rahmatullah
R
R
Muthukrishnan
S
S
D000040:Abscisic Acid / Q000494:pharmacology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005875:Gibberellins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001467:Hordeum / Q000235:genetics* / Q000378:metabolism; D009029:Mosaic Viruses / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014170:Transformation, Genetic / Q000502:physiology; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D005875:Gibberellins; D011994:Recombinant Proteins; D000040:Abscisic Acid; C007842:gibberellic acid; D015500:Chloramphenicol O-Acetyltransferase; D000516:alpha-Amylases