Progress 05/01/24 to 04/30/25

Outputs
Target Audience:Undergraduate and graduate students, plant biology researchers in academia, and scientists in biotechnology industries focus on oilseed improvement, photosynthesis, and renewable energy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided undergraduate and graduate students a training opportunityduring the reporting period. How have the results been disseminated to communities of interest?Publications Deo, B., Muthan, B., Cruise, T.L.A., Mukherjee, T., Allen, D.K., & Sanjaya, S.A. (2025). Flue Gas Desulfurized Gypsum as a Sustainable Amendment for Coal Mine Soil Reclamation and Bioenergy Crop Production. Science of the Total Environment (In Review). Sanjaya, S. (2025). How biotechnology can revolutionize agriculture: feeding the world and powering the future in the face of climate change. In proceedings of an International Conference on Future perspectives in plant science (ICFPPS-2025). PSG College of Arts and Science, Coimbatore, India. pp-24. OSBN 978-81-980915-0-5. Poster Presentations: Muthan, B., Khatiwada, S., Jayaraman, K and Sanju Sanjaya (2024). Genetic engineering of switchgrass biomass for bioenergy production. ASPB meeting, Honolulu, Hawaii, June 2024. Bagyalakshmi Muthan, Jie Wang, Ruth Welti, Dylan K. Kosma, Linhui Yu, Bikash Deo, Subhiksha Khatiwada, Vijaya K.R. Vulavala, Kevin L. Childs, Changcheng Xu, Timothy P. Durrett, Sanju A. Sanjaya (2025). Lipid metabolism in duckweed under heavy metal stress conditions. Gordon Research Conference on Plant Lipids: Structure, metabolism and functions, Pomona, CA, USA. Oral Presentations: Sanjaya, S. (2024). Understanding How Plants Tolerate Heavy Metal Stress: The molecular Mechanisms. East Tennessee University, TN, USA. Sanjaya, S. (2025). Lipid metabolism in duckweed under heavy metal stress conditions. Gordon Research Conference on Plant Lipids: Structure, metabolism and functions, Pomona, CA, USA. Sanjaya, S. (2025). Molecular mechanisms of heavy metal stress tolerance in Spirodela polyrhiza. Plant and Animal Genome Conference 32 (PAG32), San Diego, CA, USA. Sanjaya, S. (2025). Molecular Mechanisms of Plant Tolerance to Heavy Metal Stress. North Carolina A & T State University, Greensboro, NC, USA. Sanjaya, S. (2025). Genetic Transformation of Recalcitrant Upland Energy Crop Switchgrass for Enhanced Oil Yield. Plant and Animal Genome Conference, Asia, New Delhi, India. Sanjaya, S. (2025). How biotechnology can revolutionize agriculture: feeding the world and powering the future in the face of climate change. PSG College of Arts and Science, Coimbatore, India. Sanjaya, S. (2025). Potentials of Biotechnology to Produce Renewable Hydrocarbons in Plants for Dealing with the Effect of Climate Change. University of Mysore, India. Sanjaya, S. (2025). Metabolic engineering of hydrocarbons in plants for bioenergy production. PES University, Bangalore, India Sanjaya, S. (2025). Can the application of biotechnology techniques help to feed and fuel the world? M.S. Ramaiah College of Arts, Science & Commerce, Bangalore, India. What do you plan to do during the next reporting period to accomplish the goals?We will continue with a comprehensive analysis of differentially expressed genes identified in control and stressed plants. This will be followed by validation of selected gene expression patterns using quantitative RT-PCR. Concurrently, we will proceed with Agrobacterium-mediated transformation of soybean to generate transgenic plants through tissue culture-based methods. Planned downstream work includes molecular genotyping of transgenic events to confirm transgene integration and expression. Additionally, we will conduct detailed phenotypic evaluations, focusing on growth and yield performance, photosynthetic efficiency, and key agronomic traits under both normal and stress conditions.

Impacts
What was accomplished under these goals? We investigated the effects of abiotic stress on Arabidopsis seedlings using three homozygous stable transgenic lines expressing our candidate gene. The results indicated that transgenic plants grown on media supplemented with salt and heavy metals exhibited improved growth and overall health compared to wild-type controls. Notably, transgenic seedlings developed significantly longer roots and accumulated higher biomass under stress conditions. To further explore the physiological mechanisms underlying these observations, ongoing experiments are assessing antioxidant levels and lipid peroxidation in both transgenic and control lines. Based on preliminary stress assays, we selected the best-performing transgenic lines for comprehensive genomics and lipidomics studies under both normal and stress conditions. For transcriptomic analysis, time-course samples were collected from control and transgenic plants subjected to salt and heavy metal stress. We successfully isolated total RNA from samples. RNA quality and integrity were confirmed using the Qubit assay kit and the Agilent TapeStation system. Subsequently, mRNA libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit. Sequencing and downstream bioinformatics analyses are currently in progress. In parallel, we conducted lipidomics analysis on the same set of samples (control and transgenic plants, with and without stress treatments). Lipids were extracted using a protocol optimized in our laboratory. Lipidomics experiments have been completed, and data analysis is underway. In addition to functional characterization in Arabidopsis, we cloned our candidate genes--individually and in combination with known transcription factors and terminal enzymes involved in fatty acid and triacylglycerol biosynthesis--into binary vectors under the control of constitutive and seed-specific promoters. Cloning was confirmed by DNA sequencing, and the constructs were subsequently mobilized into Agrobacterium tumefaciens. We have initiated Agrobacterium-mediated transformation of soybean using a protocol developed and refined in our laboratory. Transformation and regeneration experiments are ongoing.

Publications

• Type: Other Status: Under Review Year Published: 2025 Citation: " Deo, B., Muthan, B., Cruise, T.L.A., Mukherjee, T., Allen, D.K., & Sanjaya, S.A. (2025). Flue Gas Desulfurized Gypsum as a Sustainable Amendment for Coal Mine Soil Reclamation and Bioenergy Crop Production. Science of the Total Environment (In Review).
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: " Sanjaya, S. (2025). How biotechnology can revolutionize agriculture: feeding the world and powering the future in the face of climate change. In proceedings of an International Conference on Future perspectives in plant science (ICFPPS-2025). PSG College of Arts and Science, Coimbatore, India. pp-24. OSBN 978-81-980915-0-5.