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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
1. Determine if unintended effects are produced in transgenic crops, using fruit ripening in tomato as a model system. 1A. Determine if unintended effects are produced in transgenic crops, using gene expression analysis as a monitoring tool. 1B. Determine if unintended effects are produced in the fruit of transgenic crops that affect fruit quality or composition, through metabolomic and proteomic profiling and an examination of agronomic trait performance. 2. Determine if unintended effects are reduced in transgenic plants through the use of promoters with tissue-specific expression.

1b.Approach (from AD-416)
1) Utilize genomic, metabolomic, proteomic and agronomic approaches to evaluate phenotypic difference between tomatoes. 1A) Utilize natural diversity between tomato cultivars, together with conventional breeding techniques, to capture a reasonable phenotypic range from diverse tomato germplasm. 1B) Utilize RNAi and artificial microRNA gene silencing technologies to adjust RIN gene expression levels and alter fruit ripening. Compare phenotypic effects of transgenes to the range observed with conventional cultivars..
2)Leverage research on fruit specific or ripening stage specific promoter sequences to further tailor the modulation of RIN gene expression in the target tissue. Assess the efficacy of tailored gene modulation on reducing unintended effects via genomic, metabolomic, proteomic and agronomic monitoring.

3.Progress Report
This project replaces 1907-21000-026-00D and began formally 1/29/2008. Transgenic modification of crop plants to improve product quality or agronomic performance is a commonly utilized tool for modern agriculture. However, consumer concerns regarding transgenic crops has restricted the use of plant transformation from many sectors of agricultural production. Part of the consumer aversion is due to fears of unintended effects on plant composition or development due to plant transformation or other stochastic factors. The proposed research will address unintended effects on crop growth, development and performance via monitoring of gene, protein, and metabolite expression and agronomic traits using tomato as a model. Tomato was selected as it is a food crop species with excellent molecular genetic and genomic resources, plant transformation is readily possible, and tomato is one of the most important fresh market vegetables in the US. In order to conduct the research, we initiated construction of a series of transgenic tomato varieties that modulate the expression of an agronomically important gene, ripening inhibitor (rin). A conventionally obtained mutant form of rin is in widespread use within the tomato breeding community to increase shelf life and delay ripening. Thus, the rin effect is widely accepted by consumers, producers and regulators as safe. The rin effect is due to inadequate amounts of a functional RIN protein, which is a transcription factor that potentiates ethylene dependent fruit ripening. Therefore, gene silencing via transformation is an appropriate strategy to replicate the rin mutant effect. We selected a widely used gene-silencing vector, pHellsgate12, to induce gene silencing. We have generated approximately fifty independent transgenic events using two different gene-silencing constructs by the end of the third quarter, FY2008. We have initiated characterization of these transgenic events. These experiments will continue into FY2009. We proposed to evaluate transgenic tomatoes using a variety of genomic, metabolomic and other methods. These results would then be compared with results obtained from conventional tomato cultivars, in order to estimate the boundaries of consumer acceptable phenotypic variation. These conventional cultivars were obtained from university collaborators and from commercial sources. Genetic crosses were made with normal and rin mutant tomatoes in the greenhouse so as to complete the creation of our conventional comparison panels. A pilot metabolomic study was conducted on greenhouse grown materials with the assistance of scientists at our location and at the ERRC. While we are still analyzing the data obtained from the NMR and LC-MS based methods, we are confident that we will be able to characterize the materials that will be generated from our summer research field.

1. Less than half of Americans polled consider transgenic crops to be safe. One of the concerns among consumers is that transgenically modified crops contain unintended changes in food composition or quality. To address this concern, we must first understand how composition and quality vary within stakeholder acceptable varieties. We have assembled a panel of diverse tomato varieties and are conducting compositional analyses. This information will help us to evaluate the significance of compositional changes in transgenic tomatoes and eventually reduce consumer concerns regarding transgenic crop improvement (NP302 Problem Statement 3A, Improve and Assess Genetic Engineering Technology).

5.Significant Activities that Support Special Target Populations
Significant activities that support special target populations The Hoekenga Laboratory hosted an undergraduate student intern from Xavier University of Louisiana, a Historically Black College (6/2/08 – 8/8/08). The internship was supported by a grant from the National Science Foundation Research Experiences for Undergraduates (NSF-REU). The intern learned valuable laboratory skills, read primary scientific literature to expand his basic scientific knowledge and improve problem-solving skills, and made an oral report at the end of the ten-week program. This NSF-REU program is directed at increasing participation in science by members of under-represented minorities.

6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings3
Number of Other Technology Transfer1

Last Modified: 9/3/2015
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