Progress 10/01/06 to 09/30/10
Outputs
OUTPUTS: During this project, we have continued to focus on two objectives: identification of homologs of candidate "pod-shatter" genes from Arabidopsis in canola, and development of a rapid regeneration tissue culture system and transformation protocol.We have identified several homologs and are also using this data and approach in other crops including camelina and fonio. We have also further characterized al the SCAMPS from Arabidopsis to determine if any of the homologs also affect abscission and shatter. Particular attention has been focused on characterization of DAB4 and DAB5 (SCAMP5) and interacting genes. This has contributed to our knowledge on these genes and the process of abscission. We have optimized the culture conditions of Brassica napus for the varieties Sunrise and UISC.00.1.3.5 provided by the University of Idaho Brassica breeding program. We have also continued to develop improved transformation methods and have begun evaluating expression of some delayed abscission genes in canola and camelina. Information has been shared through publications, presentations at National and International Meetings and through UW Madison seminars and outreach. It has led to an industry collaboration with Rohm and Haas supporting additional research. In addition, training of graduate student Amber Robertson Smith led to an outstanding University Teaching Award and completion of her PhD on the SCAMPs. PARTICIPANTS: Graduate Student: Amber Robertson; Undergraduates: Jordy Wells, Marilyn Elliot, Pamela Fife TARGET AUDIENCES: General agriculture participants and plant research scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes will increase our general knowledge of abscission and seed shatter as well as provide direct improvements to canola. These findings can be applied to many crops including both dicots (Canola, camelina and poplar) and monocots (fonio and rice).
Publications
- Sara E. Patterson, 2010 SCAMPS AND ABSCISSION. Acta Horticultura (in press)
- Pamela Fife, Sara Patterson and Amber Smith. 2010 Do Pectin Methylesterases (PMEs) Influence Dlayed Abscission in Arabidopsis thaliana Wisconsin SROP 2010 Summer Publication
- Amber R Smith. The Participation of Secretory Carrier Membrane Proteins in Floral Organ Abscission in Arabidopsis thaliana (PhD thesis December 2010 UW, Madison)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: In the past year, we have continued to focus on several objectives: transformation and characterization of candidate "pod-shatter" genes from Arabidopsis into selected canola lines, and further development of a rapid regeneration tissue culture system and transformation protocols.We identified homologous genes in B. napus using PCR based approaches and genome sequence. Sequence data was obtained from NCBI (http://www.ncbi.nim.nih.gov/) and Brassica Genome Gateway (http://brassica.bbsrc.ac.uk) databases. In addition, we identified related clones were found by performing a nucleotide BLAST search using Arabidopsis cDNA sequence to the Brassica database using the WU-BLAST server (http://blast.wustl.edu/). We have further characterized al the SCAMPS from Arabidopsis to determine if any of the homologs affect abscission and shatter and are currently evaluating microRNA lines in both Arabidopsis and canola. Recent work from collaborators Aalen and Liljigren supports the idea that secretory pathway-associated genes are critical in regulation of abscission. We are continuing to use Brassica napus varieties Sunrise and UISC.00.1.3.5 provided by the University of Idaho Brassica breeding program. PARTICIPANTS: Amber Robertson - graduate student; Sara Patterson - PI TARGET AUDIENCES: Results are being disseminated to the general scientific community including presentations at American Society of Plant Biology, invited talks around the US in Plant Biology departments and canola farmers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In general, the impact of this project will be both a better understanding of seed shatter and development of new cultivars of canola that will be more resistant to seed shatter. In addition, we have identified improved methods for transformation of canola. We also anticipate modifications of cultural practices based on learned information. These findings have the potential to be applied to many crops including both dicots and monocots.
Publications
- Binder,B., Patterson, S. (2009) Ethylene-dependent and independent regulation of abscission. Stewart Postharvest Review.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In the past year, we have continued to focus on two objectives: identification of homologs of candidate "pod-shatter" genes from Arabidopsis in canola, and development of a rapid regeneration tissue culture system and transformation protocol. Identification of homologous abscission-related genes in B. napus Homolog identification of delayed abscission genes DAB5 and DAB4 has been approached using sequence similarity to identify homologous genes in B. napus followed by cloning using PCR methods. Sequence data of B. oleracea and B. rapa was also be used in instances that B. napus data was sparse or not available. Sequence data for the Brassica species and Arabidopsis was taken from the NCBI (http://www.ncbi.nlm.nih.gov/) and Brassica Genome Gateway (http://brassica.bbsrc.ac.uk/) databases. Similar clones were found by performing a nucleotide BLAST search using Arabidopsis cDNA sequence to the Brassica database using the WU-BLAST server (http://blast.wustl.edu/). We have also further characterized al the SCAMPS from Arabidopsis to determine if any of the homologs also affect abscission and shatter. In addition, work in the lab on characterization of DAB4 and interacting gens has contributed to our knowledge on this gene. Optimization of tissue culture conditions We are continuing to optimize the culture conditions of Brassica napus for the varieties Sunrise and UISC.00.1.3.5 provided by the University of Idaho Brassica breeding program. We have also continued to develop improved transformation methods and have begun evaluating expression of some delayed abscission genes in canola. PARTICIPANTS: Amber Robertson - graduate student; Hongyu Rao - technician (now Post Doc in another department) TARGET AUDIENCES: General scientific community and canola farmers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Outcomes will increase our general knowledge of abscission and seed shatter as well as provide direct improvements to canola. These findings can be applied to many crops including both dicots and monocots.
Publications
- Robertson A.L., Rao H., Kusner K, Butenko M.A., Wang W.,Lindsey J.L., and Patterson S.E. 200X What are SCAMPs doing in plants A secretory membrane protein regulates cell separation in Arabidopsis resulting in delayed floral organ abscission. (under revision) (related pub to project)
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have evaluated three canola lines for regeneration and transformation from the University of Idaho (UISC.00.1.3.5, Sunrise, and Erika). We have identified three optimal tissue culture conditions using a balance of NAA and BA, and begun to characterize differences between cultivars. We have also cloned several caola genes that are associated with cell separation in Arabidopsis and are evaluating gene expression in canola.
PARTICIPANTS: Graduate Student: Amber Robertson Undergraduates: Jordy Wells Marilyn Elliot
TARGET AUDIENCES: General agriculture participants and plant research scientists
PROJECT MODIFICATIONS: none
Impacts
We have determinded alternative methods for generation of new calli and plantlets in canola with the cultivars that are currently popular breeding lines. This understanding will help lead to new genes inserted in canola and ultimately improved yield.
Publications
- Rao H, Kusner K, Butenko MA, Robertson AL, Wang W, and Lindsey JD*, Patterson SE. (2007). What are SCAMPs doing in plants? A secretory membrane protein regulates cell separation in Arabidopsis resulting in delayed floral organ abscission. (pending review in Plant Cell)
- Elliott M*, Robertson A, Patterson S. (2006). Cell Separtion in Arabidopsis and Canola. Wisconsin Summer Research Program (SRP) Research scholar publication
- Robertson A, Rao H, Wang, W, Patterson S. Secretory Carrier Membrane Proteins (SCAMPs) in Arabidopsis thaliana (2007). Abstract. American Society Plant Biology, Chicago, IL
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Progress 01/01/06 to 12/31/06
Outputs
In the past year we have focused on two objectives: identification of homologs of candidate pod-shatter genes from Arabidopsis in canola, and development of a rapid regeneration tissue culture system and transformation protocol. Homolog identification was approached using sequence similarity to identify homologous genes in B. napus followed by cloning using PCR methods. Sequence data of B. oleracea and B. rapa was also be used in instances that B. napus data was sparse or not available. Sequence data for the Brassica species and Arabidopsis was taken from the NCBI (http://www.ncbi.nlm.nih.gov/) and Brassica Genome Gateway (http://brassica.bbsrc.ac.uk/) databases. Similar clones were found by performing a nucleotide BLAST search using Arabidopsis cDNA sequence to the Brassica database using the WU-BLAST server (http://blast.wustl.edu/). The sequences with an E value lower than e-40 were considered a good match. The clone sequences were aligned to form a contig using
the EditSeq function in the DNAStar software package (DNASTAR, Inc.). Primers were designed from the contig and used to amplify the gene via PCR. Appropriate sized PCR products were excised from the gel and gel purified. The purified product was inserted into a Promega pGem-Easy cloning vector (Promega) for sequencing. The products were cloned into a vector first to ensure that only one PCR product would be sequenced. A Big-Dye reaction was performed followed by sequencing by the UW-Biotechnology Center (http://www.biotech.wisc.edu/). The sequence was subsequently analyzed using DNAstar and Bioedit (Hall, T., Ibis Theraputics) to construct a contig sequence. Once DAB5 was amplified, the 5' and 3' ends of the gene were determined using rapid amplification of cDNA ends (RACE). We are trying to optimize the culture conditions of Brassica napus for the varieties Sunrise and UISC.00.1.3.5 provided by the University of Idaho Brassica breeding program. We have used combinations of
concentrations of two cytokinins, BA and 2iP, and three auxins, IAA-aspartate, IAA, and NAA and found that combinations with 3-10 uM IAA-aspartate and 5 uM 2iP worked best. Using hypocotyl explants we found the callus would go directly to vegetative growth. The vegetative tissues were then transferred to rooting media. We will continue to develop improved transformation methods and have begun evaluating expression of some delayed abscission genes in canola.
Impacts
Early pod shatter of canola typically results in the loss of approximately 10% of the crop, and in some cases as much as 50%. By understanding the genes that regulate the process of pod shatter, we may be able to alter losses and improve quality. We are using the close relative Arabidopsis as the entire genome has been sequenced and there are many mutants available. Candidate genes are being identified and homologs found in canola with the ultimate objective of determining gene expression in canola and possible modification of expression.
Publications
- No publications reported this period
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