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United States Department of Agriculture

Agricultural Research Service

Title: Green Fluorescent Protein as a Tissue Marker in Transgenic Maize Seed

Authors
item Shepard, Colin - ISU
item Vignaux, Nathalie - ISU
item Peterson, Joan - ISU
item Johnson, Lawrence - ISU
item Scott, Marvin

Submitted to: Cereal Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 15, 2007
Publication Date: March 1, 2008
Citation: Shepard, C.T., Vignaux, N., Peterson, J.M., Johnson, L.R., Scott, M.P. 2008. Green Fluorescent Protein as a Tissue Marker in Transgenic Maize Seed. Cereal Chemistry. 85:188-195.

Interpretive Summary: Corn grain is composed of several different tissues with different properties. One way to capture value from grain is to mechanically separate it into fractions that are better suited to a particular use than whole grain. This process is called milling. The objective of this work was to develop corn varieties that can be used to monitor and improve milling efficiency. An important challenge in milling to determine the fate of each grain tissue in the fractionation process. In the work described here, we addressed this problem by producing several varieties of corn each containing a different grain tissue labeled with a fluorescent protein. We show that by fractionating grain from these varieties and examining the fluorescence in each fraction, it is possible to track a specific tissue through the fractionation process. With this information, it will be possible to improve milling proceedures so that tissues can be separated more efficiently. Improved milling efficiency will enable recovery of high value products from corn. This work will benefit the grain processing industry and make new and improved corn products available to consumers.

Technical Abstract: Seed tissues (endosperm, embryo, and pericarp) are often separated into tissue-enriched fractions by wet or dry milling methods for use in food, feed and industrial products. To optimize fractionation processes, seed tissue markers that are sensitive and readily quantifiable would be useful. To meet this need for tissue markers, we set out to produce and characterize different transgenic maize lines, each containing Green Fluorescent Protein (GFP) in either endosperm or embryo. We examined mRNA transcripts using expressed sequence tag (EST) profiles of several major seed proteins and selected several with strong seed tissue preferences. We tested the promoters of these seed proteins in a transient expression assay to confirm promoter activity. Stably transformed maize lines were produced and visual observation of fluorescence confirmed the presence of GFP in the desired tissues. To establish the utility of this grain for evaluating the effectiveness and/or separation efficiencies of fractionation processes, transgenic kernels were hand-dissected into pericarp, endosperm, and embryo fractions and the GFP concentration in each fraction was determined. The GFP distribution between fractions of each transgenic event was calculated from GFP concentration and mass balance, which enabled the determination of GFP yield based on the hand-dissection fractionation data and the amount of tissue contamination in each fraction. Our transgenic lines exhibited strong tissue preference for either embryo or endosperm. These lines should be useful for assessing separation efficiencies in maize fractionation processes.

Last Modified: 9/10/2014
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