Non Technical Summary
This project is funded topurchase a Barocycler 2320EXT manufactured byPressure BioSciences, Inc., South Easton, MA 02375. The bench top instrument uses the Pressure Cycling Technology (PCT) MicroTubes. The PCT platform allows for a high degree of safety, speed, reproducibility, and convenience in modern sample preparation protocols.The instrument will be used to conduct research activities funded by NIFA and other federal agencies at1890 universities. These projects aims to advance the scientific understanding of plant reproduction systems in tomatoes, and root development under soil stress for switchgrass;to identifynew molecular tools (such as small peptides) for improving nitrogen fixation efficiency in legumes;and to develop new ways (such as the use of essential oils, and plant-based materials) for extending shelf life of fragile fruits such as strawberries.Funding this proposal will allow 1890faculty and students to overcome the challenges in the extraction of biomolecules (DNA, RNA, proteins, metabolites) from small amount of tissue samples.The instrument will also be used by undergraduate and graduate students to conduct capstone projects/thesis/dissertation projects. Thecurrent TSU research infrastructure is comprised of sets of instruments that are essential for conducting theBarocycler 2320EXT enabled research projects. The installation of the instrument will immediately improvethe efficiencyof conducting 3-5 ongoingresearch projects in the PI's labs.The expected outcomes from this project include: 1)Providing the capacity for TSU faculty to be more productive, in particularly for projects involving identification of trait-related genes and biomarkers from single cell/single cell type tissues, and targeted analysis from complex biological tissues samples,2)A more effective platform to support student research and learning.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1460	1050	30%
201	7410	1060	30%
201	1122	1050	20%
201	1620	1050	10%
201	1640	1050	10%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1460 - Tomato; 1620 - Warm season perennial grasses; 1640 - Alfalfa; 1122 - Strawberry; 7410 - General technology;

Field Of Science
1060 - Biology (whole systems); 1050 - Developmental biology;

Goals / Objectives
This project supports the purchase of a Barocycler® 2320EXT. The instrument has a demonstrated capacity in extraction of biomolecules from small amount of tissue samples, precisely and consistently, under customized temperature conditions. It can also be used for enzymatic digestion in proteomics analysis. PIs of the project have identified the need of the instrument in both short and long-term research activities. The primary goal is to enhance the instrument capacity to support research and teaching activities.The two objectives include: 1)Utilize the instrument to extract proteins, RNAs, and small peptides from very small amount of plant tissues samples (pollen, roots, fruits, etc). These activities are part of ongoing research projects;2)Train new users from faculty, graduate and undergraduate students.

Project Methods
To accomplish the two major objectives, the instrument will be used to conduct research projects in three PI's laboratories and offering training sessions to new users including faculty and students. For the research activities, Dr. Zhou's lab will focus on developing the technical procedures for protein/DNA/RNA extraction from single cell-type samples of pollen, and root tissues using the instrument. The homogenous cell samples are collected using laser capture microdissection.These samples will be used for study pollen development in response to heat stress, root development under various soil and soil-less conditions.Dr. Roy's lab will focus on identification of peptide receptors in Medicago truncatula membrane fragments using biotinylated peptide hormones. The lab will use of the Barocycler to complete protein extraction from a small amount of tissue samples (<50 roots in 30 µL) in a single step. Success of this protocol will greatly increase protein extraction efficiency by replacing the protein extraction method using multiple steps of mortar and pestle grinding followed by in-buffer extraction. The final goal is to identify newsignaling componentssuch as small peptidesand their receptorsfor improving Nitrogen fixation efficiency in legumes.The Dr.Taghavi's lab at Virginia State University (VSU) leads the research on post-harvest of strawberry. Specifically, the lab is looking for very specific cell types that contribute to the cellular and molecular events happening during fruit expansion, ripening and fungal contamination to understand the physiological and molecular developmental stages and the plant host interaction. In collaboration with Dr. Zhou, the Dr. Taghavi's lab is developing the technology to study the epidermal layers of cells of strawberry fruits during fruit softening process. The Barocycler will be used for extraction of RNA/protein/metabolites from the LCM harvested homogeneous tissue samples. Data generated will be used in the strawberry projects at Virginia State University and help to develop a solid foundation for building a strong research consortium among the 1890 universities.For training new users, training sessions will be offered periodically based on the need from faculty and students. Faculty and graduate students are encouraged to use the instrument to conduct research projects.For undergraduate student training, the extraction of biomolecules can be very tedious and problematic for Biotech related experiments, especially for those with no lab experiences. The major advantage of the instrument is the capability of automatically running through the extraction procedures. Using the Barocycler 2320EXT, the extraction procedure becomes simple and easy to handle. Students only need to put samples into the MicroTubes, place sample in the instrument, and start the program. The extraction step will complete in an hour and the student can start the analysis experiments (such as running protein gels, PCR, DNA gels, etc.). Undergraduate students will be encouraged to conduct their senior and capstone projects using the instrument. We will also integrate learning to use instrument in the lab modules in graduate and undergraduate courses.