Progress 01/01/12 to 12/31/12
OUTPUTS: During this year we have completed one of the two objectives of the project, which is genetic engineering of wheat. This success of this objective comes with a lot of hard work from the graduate student and undergraduate students especially Manali Shirke and Kim Buehner who put many untiring hours to the project. The wheat is a recalcitrant plant species for genetic manipulations by Agrobacterium tumefaciens for some unknown reasons. This is the reason most of the wheat transformations are done by gene gun. Three years ago we took this challenge and by performing calculative step-wise improvements in the protocol achieved this objective successfully. In this process we have developed the right strain of Agrobacterium, right type of media for transformation including the type of sugars, hormones, and concentrations of various salts. During the year of 2012, one graduate student (Manali Shirke), and three undergraduate students (Kim Buehner, Nina Kamrowski, and Sara Glisczinski) were trained for with hands-on for this technology. One undergraduate (Kim Buehner) student was able to achieve high distinction in her Honors College education because of this successful project. By achieving transformation capabilities in cereals, we have established collaboration with Dr. Justin Farris (USDA, North Dakota) and helped him with the production of transgenic rice plants with Tsn1 gene. We provided 6 transgenic lines for Tsn1 gene to him. Towards the second objective of the project, a graduate student (Moustafa Eldakak) and a visiting scientist (Dr. Ansuman Roy) worked on the proteomics aspect of this hatch project during this year. As a result we have identified 96 different proteins, whose expression levels have been affected by drought, heat, drough+heat and/or fusarium infection. These are interesting proteins for the downstream experiment to study further in greater details. This work has yielded us collaboration with Penn State University (Dr. Surinder Chopra). In this collaborative project we are studying molecular factors, especially proteins that are responsible for pericarp color change and bented stem phenotype by an epigenetic mutation in a wild type allele. Dr. Roy is in the process of writing his peer-reviewed research paper and for its submission to a peer-reviewed journal very soon. The graduate student is advancing the work on refining the transcriptomics, proteomic and metabolomics technologies, and to discover more genes that are responsive to biotic and abiotic stress environments. The overarching goal of this project is to use these genes in the improvement of South Dakota crop plant's for variable climate change. During the year 2012, we have completed one objective of the project and are continuing on the second objective with a great pace. Other than Manali and Moustafa, two graduate students (Prateek Tripathi and Achal Neupane) were also trained for transcription factor and MAPK studies. The results of the project are continually presented by student and faculty at state level scientific meeting and national/international level meetings. PARTICIPANTS: People involved on this project during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student), Achal Neupane (graduate student), Prateek Tripathi (graduate student), Kim Buehner (undergraduate student), Nina Kamrowski (undergraduate student) and Sara Glisczinski (undergraduate student). TARGET AUDIENCES: The target audiences of this project are US wheat, soybean and rice farmers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Transforming wheat by Agrobacterium was one of the great challenges because of its recalcitrant nature. During this year we have tested, 20 different genotypes of wheat along with four different Agrobacterium strains for their virulence to the wheat calli and by modifying the medium, growth, and infection conditions we were successful to hit the right Agrobacterium strain with right growth conditions and with the right Agrobacterium infection media. This knowledge has greatly enhanced our understanding for the wheat calli and Agrobacterium infection chemistry. The modification in all these parameters yielded to a successful Agrobacterium-mediated wheat transformation events. The next step was more interesting when we moved the regenerated transgenic plantlets to the soil, and for their histochemical and molecular analysis. Not all regenerated plants showed positive event for the genetic transformation, it do come with false positives. That means some regenerated plants were able to pass the selection pressure but were not transgenic plantlets. All the positive plants showed the presence of transformed gene during the histochemical assay. Since the plants were transformed with GUS gene, an enzyme whose substrate is X-Gluc. For this objective, we have used a constitutive promoter to drive this gene in plant system; as a result the gene should be expressed in all the plant tissues. For histochemical assays we used the leaves of regenerated transgenic plants for GUS staining. A number of transgenic plants show blue staining confirming the successful integration of T-DNA into wheat cells and the regeneration of a transformed plant from that particular cell, which expressed a positive GUS expression in regenerated plant's leaves. These plants were later subjected to re-confirmation by PCR technology. Since wild type plants lack that gene but the transgenic plants must have it, it was selectively amplified only in the PCR tube which contained the DNA from the positive transgenic plants. This independent experiment re-confirmed the success of the Agrobacterium mediated transformation of the wheat cells. These results have a great impact on wheat biotechnology industry. The data analysis of the experiments for second objective are in process but the preliminary data coming out is very exciting. By utilizing proteomics and mass spectrometry technology we have identified 7 Fusarium responsive genes. This data eventually will be very useful in tackling the wheat scab disease in the state. We are in the process of revalidating these genes. Towards the broader goal of generating new knowledge and enhancing the understanding of the drought tolerance, disease resistance mechanism in wheat and soybean, we are utilizing systems biology approach by inclusion of transcriptomics, proteomics and metabolomics platforms. We have generated a vast amount of data from these experiments, which is under analyses at this moment.
- Proteomic dissection of near isogenic lines for the discovery of scab responsive genes of wheat. Eldakak, M., A. Roy, Y. Zhuang, K. Glover, S. Ali, Y. Yen, and J.S. Rohila. 2012 Poster presentation at National Fusarium Head Blight Forum, Orlando, FL. pp 133.
- Shirke, M., S. Rohila, N. Kamrowski, S. Glisczinski, K. Buehner, A. Roy, M. Eldakak, and J. S. Rohila. 2012. Exploiting the potential of genetic engineering to improve wheat for South Dakota. Poster presentation at 7th Annual SD Biotechnology Summit, Sioux Falls, SD.
- Neupane, A., S. Piya, R. N. Reese, J.S. Rohila, S. Subramanian, and M. P. Nepal. Identification of Mitogen Activated Protein Kinase Family Members in Soybean. Botany 2012, Poster presentation at Botany 2012 - The Next Generation Conference July 7- 11. Columbus, OH, USA.
- Neupane, A., S. Piya, S. Subramanian, J. S. Rohila, R. N. Reese, and M. P. Nepal. A Nomenclatural Conundrum: Applying Existing Nomenclature to the Identification of Soybean (Glycine max) MAP Kinase Genes. Poster presentation at ASPB Midwestern Section Meeting 2012, March 24-25. Lincoln, NE, USA.
- Gibberrella zeae Chemotype Diversity on Moderately FHB Resistant Wheat Genotypes in South Dakota. Ali, S., M. Eldakak, P. Gautam, K. Glover, J.S. Rohila, J. Gonzalez, W. Berzonsky. In: Canty, S., A. Clark, A. Anderson-Scully and D. van Sanford (Eds.), Proceedings of the 2012 National Fusarium Head Blight Forum. Pages 115-117.
- The WRKY transcription factor family in Brachypodium distachyon. 2012. Tripathi, P. R.C. Rabara, T.J. Langum, A.K. Boken, D.L. Rushton, D.D. Boomsma, C. I. Rinerson, J. Rabara, R. N. Reese, X. Chen, J. S. Rohila, P. J. Rushton. BMC Genomics 13 (1), 270.
Progress 01/01/11 to 12/31/11
OUTPUTS: During the second year of the project two Ph.D. students, one visiting scientist and one undergraduate student were trained. The project was mainly focused on studying the drought stress responsive proteins at molecular levels and improving wheat transformation efficiencies. The results of the project were presented at one state level scientific meeting and at one international level meeting. PARTICIPANTS: People involved on this project in my lab during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student) and Kim Buehner (undergraduate student). Dr. Chopra and his lab members at Pennsylvania State University, University Park, PA were collaborators during this period. TARGET AUDIENCES: The target audiences of this project are US wheat and corn researchers. PROJECT MODIFICATIONS: Not relevant to this project.
During the second year of the project we have done preliminary proteomics experiment in corn and wheat. The focus of these experiments was to understand the drought responsive phenomenon and to discover biomarkers for drought tolerance in plants. We have identified a few differentially expressed proteins in wheat as a result of drought occurrence. In corn, our proteomic studies were in collaboration with Dr. Surinder Chopra (Pennsylvania State University) using Ufo1-1 mutant corn lines. This study leads to the identification of abiotic stress-associated proteins in corn. Revalidation of the proteins identified in corn is underway.
- Roy, A., Yang, Z., Robbins, M., Chopra, S. and Rohila, J.S. 2011. Proteome analysis of maize pericarp by DIGE and ITRAQ. In: 96th Annual Meeting of South Dakota Academy of Science at Oacoma, SD, USA.
- Yang, Z., Robbins, M., Chopra, S., Roy, A., Rohila, J.S. 2011. Pericarp proteome reveals that Unstable factor for orange1 plants exhibit altered expression of stress-associated genes. In: 53rd Annual Maize Genetics Conference at St. Charles, Illinois.
- Roy, A., Rushton, P.J. and Rohila, J.S. 2011. The Potential of Proteomics Technologies for Crop Improvement under Drought Conditions. Critical Reviews in Plant Sciences. 30:471-490.
Progress 01/01/10 to 12/31/10
OUTPUTS: During the first year of this project two Ph.D. students, one visiting scientist and one undergraduate student were trained for wheat transformation and wheat, soybean and rice proteomic technologies. For this period the project was mainly focused on student training, standardizing the protocols in the newly established lab and producing preliminary results. The results of the project were presented at two state level scientific meetings and one international level meeting. PARTICIPANTS: People involved on this project during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student) and Kim Buehner (undergraduate student). TARGET AUDIENCES: Target audiences for this project are plant biology students, researchers in the US and around the globe. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
During this first year of the project we have standardized the wheat transformation and proteomics protocols. Towards the goal of understanding the drought tolerance phenomenon we have performed a series of drought experiments in wheat and soybean plants. By performing proteomic studies we have found a number of differentially expressed protein spots on 2D gel electrophoresis as a result of regulated drought stress in wheat and soybean plants. The mass spectrometric identification of the novel proteins is underway. The findings of novel drought resistance genes will add immensely to our understanding of this crop yield reducing stress at molecular levels.
- 1. Roy, A. and Rohila, J. S. 2010. 2-DE profiling of total proteome changes in cereals and oil crops under abiotic stresses. In: 95th Annual Meeting of South Dakota Academy of Science. 2010. Spearfish, SD, USA.
- 2. Shirke, M., Buehner, K., Roy, A., Rohila, S. and Rohila, J.S. 2010. Establishment of an efficient callus induction and regeneration protocol from immature embryos of wheat. In: 95th Annual Meeting of South Dakota Academy of Science, April 9-10, 2010. Spearfish, SD, USA.
- 3. Liu, Z., Rohila, J. S., Liu, W., Wang, Q., and Yang, Y. 2010. A Nep1-like Protein Toxin from Magnaporthe oryzae Interacts with a Conserved, Ubiquitin-like Rice Protein and Elicits Plant Cell Death. In: The 5th International Rice Blast Conference, August 12-14, 2010. Little Rock, Arkansas, USA.