Barley aleurone layer cell protoplasts as a transient expression system |
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Authors: | Bhuvana Gopalakrishnan Burachai Sonthayanon Rownak Rahmatullah Subbaratnam Muthukrishnan |
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Affiliation: | (1) Department of Biochemistry, Kansas State University, Willard Hall, 66506 Manhattan, KS, USA;(2) Present address: Department of Biochemistry, Mahidol University, Bangkok, Thailand |
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Abstract: | 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 -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 -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes. |
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Keywords: | aleurone protoplasts barley hormone-responsive /content/p2w511g05756l72h/xxlarge945.gif" alt=" agr" align=" BASELINE" BORDER=" 0" >-amylase synthesis transient expression |
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