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United States Department of Agriculture

Agricultural Research Service

Title: Using the Versflour Fluorometer to Quantify Gus Expression in Plant Tissuesbombarded with Biolistic Pds-1000/he Particle Gun.

item Tucker, Mark
item Lysenko, Nicholay

Submitted to: Survey Notes
Publication Type: Trade Journal
Publication Acceptance Date: May 1, 1999
Publication Date: N/A

Interpretive Summary: The purpose of this project is to identify and characterize DNA fragments that are responsible for regulating the accumulation of protein during leaf, flower and fruit abscission (organ separation). Our goal is to use these DNA fragments as part of a gene construct to genetically manipulate organ separation to benefit agricultural productivity (e.g., increase yield and reduced harvesting costs). We have described in this brief application note the procedure we use to characterize these DNA fragments. The methodology reported in this application note will be useful to other researchers interested in characterizing regulatory elements in other plant genes.

Technical Abstract: Abscission is the process by which plants shed organs. Cellulase accumulates tissue-specifically in induced abscission zones of bean. We have used transient GUS expression in particle bombarded tissue to identify regulatory regions in the gene promoter for a bean abscission cellulase. The VersaFluor fluorometer, when equipped with the simple to install 360 excitation and 460 emission filters, has the necessary sensitivity to accurately measure fluorescent methylumbelliferone, the product of GUS in plant tissues transformed by particle gun bombardment. An example of the approach used and results obtained is described. In addition to quantification of GUS activity, we use the VersaFluor fluorometer equipped with the same set of filters to quantify the DNA used in the particle gun bombardment experiments. The dye used for DNA quantification is Hoechst 33258 from Bio-Rad.

Last Modified: 4/18/2015
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