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Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase II | Award Amount: 500.00K | Year: 2010

This Small Business Technology Transfer (STTR) Phase II project seeks to identify new and improved promoters to create enhanced genetically modified crops. Plant biotechnology relies on the insertion of promoter-gene constructs into plants. The promoter is the portion of DNA that controls when and where a gene is expressed. The relatively few plant promoters in use today have significant limitations including inconsistent effects across different growing conditions and a lack of predictability. This project involves developing and implementing a novel pipeline for promoter discovery that starts with a sophisticated bioinformatics analysis to identify high confidence promoter candidates. Using fluorescent reporters and confocal imaging, these candidates are assessed in transgenic plants for cell-type-specific expression, developmental-stage-specific expression, and responsiveness to environmental stimuli. This pipeline was validated in the Phase I component of the project where four novel and patentable constitutive promoters were identified. The broader impacts of this research are the development of superior genetically modified crops. Genetically modified plants already play an important role in world agricultural production and will play a central role in averting widespread food shortages in the future. In addition, substantial research is being conducted to improve bioenergy crops though genetic engineering. Genetically enhanced bioenergy crops are predicted to play a key role in reducing our dependence on fossil fuels and in cutting greenhouse gas emissions. A critical innovation that will facilitate advances in all of these areas will be the introduction of new and enhanced plant promoters.

Busch W.,Duke University | Busch W.,Gregor Mendel Institute of Molecular Plant Biology | Moore B.T.,Duke University | Martsberger B.,Duke University | And 12 more authors.
Nature Methods | Year: 2012

To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions. © 2012 Nature America, Inc. All rights reserved.

Ingram P.,University of Chicago | Ingram P.,GrassRoots Biotechnology | Dettmer J.,University of Helsinki | Dettmer J.,Vlaams Institute for Biotechnology | And 2 more authors.
Plant Journal | Year: 2011

We have identified a gene, Lateral Root Development 3 (LRD3), that is important for maintaining a balance between primary and lateral root growth. The lrd3 mutant has decreased primary root growth and increased lateral root growth. We determined that the LRD3 gene encodes a LIM-domain protein of unknown function. LRD3 is expressed only in the phloem companion cells, which suggested a role in phloem function. Indeed, while phloem loading and export from the shoot appear to be normal, delivery of phloem to the primary root tip is limited severely in young seedlings. Abnormalities in phloem morphology in these seedlings indicate that LRD3 is essential for correct early phloem development. There is a subsequent spontaneous recovery of normal phloem morphology, which is correlated tightly with increased phloem delivery and growth of the primary root. The LRD3 gene is one of very few genes described to affect phloem development, and the only one that is specific to early phloem development. Continuous growth on auxin also leads to recovery of phloem development and function in lrd3, which demonstrates that auxin plays a key role in early phloem development. The root system architecture and the pattern of phloem allocation in the lrd3 root system suggested that there may be regulated mechanisms for selectively supporting certain lateral roots when the primary root is compromised. Therefore, this study provides new insights into phloem-mediated resource allocation and its effects on plant root system architecture. © 2011 Blackwell Publishing Ltd.

Ingram P.A.,GrassRoots Biotechnology | Zhu J.,GrassRoots Biotechnology | Shariff A.,GrassRoots Biotechnology | Davis I.W.,GrassRoots Biotechnology | And 3 more authors.
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2012

Nitrogen (N) and phosphorus (P) deficiency are primary constraints for plant productivity, and root system architecture (RSA) plays a vital role in the acquisition of these nutrients. The genetic determinants of RSA are poorly understood, primarily owing to the complexity of crop genomes and the lack of sufficient RSA phenotyping methods. The objective of this study was to characterize the RSA of two Brachypodium distachyon accessions under different nutrient availability. To do so, we used a high-throughput plant growth and imaging platform, and developed software that quantified 19 different RSA traits. We found significant differences in RSA between two Brachypodium accessions grown on nutrient-rich, low-N and low-P conditions. More specifically, one accession maintained axile root growth under low N, while the other accession maintained lateral root growth under low P. These traits resemble the RSA of crops adapted to low-N and -P conditions, respectively. Furthermore, we found that a number of these traits were highly heritable. This work lays the foundation for future identification of important genetic components of RSA traits under nutrient limitation using a mapping population derived from these two accessions. © 2012 The Royal Society.

Zhu J.,GrassRoots Biotechnology | Ingram P.A.,GrassRoots Biotechnology | Benfey P.N.,GrassRoots Biotechnology | Benfey P.N.,Duke University | Elich T.,GrassRoots Biotechnology
Current Opinion in Plant Biology | Year: 2011

Plant root system architecture (RSA) is plastic and dynamic, allowing plants to respond to their environment in order to optimize acquisition of important soil resources. A number of RSA traits are known to be correlated with improved crop performance. There is increasing recognition that future gains in productivity, especially under low input conditions, can be achieved through optimization of RSA. However, realization of this goal has been hampered by low resolution and low throughput approaches for characterizing RSA. To overcome these limitations, new methods are being developed to facilitate high throughput and high content RSA phenotyping. Here we summarize laboratory and field approaches for phenotyping RSA, drawing particular attention to recent advances in plant imaging and analysis. Improvements in phenotyping will facilitate the genetic analysis of RSA and aid in the identification of the genetic loci underlying useful agronomic traits. © 2011 Elsevier Ltd.

A plant growth array device includes an aerial growth chamber configured to receive aerial shoot portions of a plurality of plants and a root growth chamber configured to receive root portions of the plurality of plants. A dividing member is between the aerial growth chamber and the root chamber and has a plurality of apertures for receiving the plurality of plants therein. The plurality of apertures are configured so that the root portions grow substantially in a common orientation.

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 367.71K | Year: 2012

DESCRIPTION (provided by applicant): It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely recognized that advances in agricultural biotechnology will be required to meet the world's future nutritional needs. In nature, gene expression is regulated at multiple levels including transcription, alternative splicing, transcript stability, and translation. In contrast, the agricutural biotechnology industry has relied primarily on constitutive transcriptional control to regulate the expression of traits introduced into crops. This has been sufficient for the relatively simple trais that have been commercialized to date, but the complex traits that will be needed to meet future food demands are expected to require increased expression specificity. Thelong term goal of this project is to assemble a portfolio of modular expression elements that can be deployed in a combinatorial fashion to provide predictable and tunable expression solutions to the agricultural biotechnology industry. The goal of the Phase I component of this project is to demonstrate proof-of-concept that heterologous miRNA binding elements and pre-mRNA splicing elements can be used to increase the expression specificity of transcriptional promoters in the model plant Arabidopsis. As a specific product concept, Phase I research will focus on designing a root stele-specific expression cassette useful for regulating traits that target soybean cyst nematode. Our specific aims are to: 1) validate a modular post-transcriptional regulatory element that enhances stele expression specificity, and 2) create stele-specific expression cassettes that are combinatorially controlled by transcriptional promoters and post-transcriptional regulatory elements. Selection of miRNA binding elements will be based on recently published data demonstrating cell type-specific expression patterns of specific miRNAs that are inversely correlated with the expression patterns of their respective targets. Alternative splicing events wil be identified through bioinformatic analysis of cell type-specific RNA-seq and microarray expression data. Identified posttranscriptional regulatory elements will be validated in the context of a constitutive promoter and then tested for their ability to increase the expression specificityof a stele-enriched promoter. After demonstrating proof-of-concept of combinatorial control, Phase II research will expand this approach to crops including economically important cereals like corn. PUBLIC HEALTH RELEVANCE: It is currently estimated that 15% of the world's population is undernourished and 5 million childhood deaths a year are attributable to malnutrition. The ability to feed the world in the future will be even more difficult due to population growth, climate change, water scarcity, and competition for land. It is widely acknowledged by experts that increased agronomic productivity through biotechnology will be needed to meet future food demands. The goal of this project is to facilitate biotechnology approaches that lead to the production of more crops on less land with fewer resources.

Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 80.00K | Year: 2009

The USDA has established research on renewable energy as a high priority. Cellulosic ethanol from perennial grasses has the potential to become an important component of America's effort to reduce its dependence on foreign oil and alleviate the buildup of greenhouse gases. For energy crops to become viable biofuels, they must become cost-competitive with foreign oil and provide environmental benefits. This will require improvements in refining technologies to efficiently convert cellulose to ethanol, improvements in agronomic productivity, and maximal greenhouse gas reduction. GrassRoots Biotechnology is working to create enhanced energy crops by focusing on improved root architecture. In this project, we are focusing on two main components of root architecture: 1) greater deep root extension to increase drought resistance and carbon sequestration; 2) maintaining sufficient shallow root spread for nutrient acquisition from topsoil.

Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 400.00K | Year: 2010

America's dependence on foreign oil has severe economic, national security and environmental consequences. The development of alternative fuels and renewable energy can alleviate these problems. The USDA has established research on renewable energy as a high priority. Cellulosic ethanol from bioenergy crops like switchgrass has the potential to become an important component of America's effort to reduce its dependence on foreign oil. As an additional benefit, replacing fossil fuels with biofuels will lessen the buildup of greenhouse gases. To gain access to commercial markets, biofuels must become cost-competitive with fossil fuels. This will require improvements in refining technologies to efficiently convert cellulose to ethanol along with improvements in the agronomic productivity of biofuel crops, particularly under conditions of limiting nutrients and water. Roots play a critical role in the growth and development of all plants. In addition to providing anchorage, roots are the primary site of nutrient and water acquisition. To perform these tasks, the primary roots extend into soil and produce additional branching roots that originate from internal tissues. The network of the different types of roots on a single plant is known as its root system architecture. It is well established that plant root system architecture is correlated with agronomic productivity under limiting conditions. This goal of this proposal is to create enhanced biofuel crops by improving their root architecture in order to increase agronomic productivity under limiting conditions.

Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2013

Sorghum is a major crop for food, feed and industrial processes in the US and on the global market. It is a particularly relevant crop under the current pressures of climate change and food security because it is adapted to cultivation under low water and nutrient conditions. However, compared to other major crops, sorghum productivity is relatively low, and historic increases in sorghum yield have been minimal. Recently, it has been established that root architecture plays a critical role for crop yield potential and stability, especially under limiting environments, such as drought and low nutrient availability. Our preliminary analysis of root architecture traits among sorghum varieties has revealed a potential for genetic improvement of root architecture for sorghum agronomic productivity in sustainable agriculture. Current challenge remains in identification of root architecture features and their genetic control which have been limited by the phenotyping technologies available, as well as the application of emerging and potentially useful genome-wide association studies of root architecture. GrassRoots Biotechnology will apply a novel, high through-put and accurate imaging and analysis platform, RootXpose, for imaging roots targeting three dimensions and for measuring complex root traits for 242 sorghum genotypes of a minicore collection from 58 Countries from ICARISAT. Well also develop genome-wide association studies tools in sorghum to determine the genetics of root traits, and identify root traits that correlate with agronomic performance. Collectively, this project will set foundations to enhance sorghum productivity through modifying its root architecture traits for sustainable agriculture and increase the profitability and market share of this crop.

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