Plant Biotech

PLANT BIOTECHNOLOGY

Recent Publications:

1. Ogden, Aaron J.; Bhatt, Jishnu J.; Brewer, Heather M.; Kintigh, Jack; Kariuki, Samwel M.; Rudrabhatla, Sairam; Adkins, Joshua N.; Curtis, Wayne R. 2020. "Phloem Exudate Protein Profiles during Drought and Recovery Reveal Abiotic Stress Responses in Tomato Vasculature" Int. J. Mol. Sci. 21, no. 12: 4461.

2. Ogden, Aaron J.; Boukari, Wardatou; Nava, Alba; Lucinda, Natalia; Sunter, Garry; Curtis, Wayne R.; Adkins, Joshua N.; Polston, Jane E. 2020. "Characterization of Local and Systemic Impact of Whitefly (Bemisia tabaci) Feeding and Whitefly-Transmitted Tomato Mottle Virus Infection on Tomato Leaves by Comprehensive Proteomics" Int. J. Mol. Sci. 21, no. 19: 7241.

3. Shires, Morgan E., Florez, Sergio L., Lai, Tina S., Curtis, Wayne R. (Nov 2017) Inducible somatic embryogenesis in Theobroma cacao achieved using the DEX-activatable transcription factor-glucocorticoid receptor fusion. Biotechnology Letters 39 (11) 1747-55.

4. Florez, Sergio L., Curtis, Matthew S., Shaw, Sydney E., Hamaker, Nathaniel K, Larsen, Jeffrey S. A Temporary Immersion Plant Propagation Bioreactor with Decoupled Gas and Liquid Flows. Biotech. Prog. 32 (2): 337-345. (Online Dec 23, 2015, DOI: 10.1002/btpr.2221)

5. Florez, Sergio L., Erwin, Rachel L., Maximova, Siela N., Guiltinan, Mark J., and Curtis, Wayne R. Enhanced Somatic embryogenesis in Theobroma cacao using the homologous BABYBOOM transcription factor. BMC Plant Biology. (Accepted March 23rd, 2015)

6. Maximova SN, Florez S, Xiangling Shen X, Niemenak N, Zhang Y, Curtis WR, Guiltinan MJ (2014) Genome-wide analysis reveals divergent patterns of geneexpression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree. BMC Plant Biology 2014, 14:185 doi:10.1186/1471-2229-14-185.

7. Larsen, J.S.; Curtis W.R. (2012) RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots. BMC Biotechnology 2012, 12:21.

8. Woolston BM, Schlagnhaufer C, Wilkinson J, Larsen J, Shi Z, Mayer K, Waters D, Curtis W, Romaine C (2011) Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus. PLoS ONE 6(12): e28412, doi:10.1371/journal.pone.0028412

9. O’Neill KM, Larsen JS, Curtis WR (2008) Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture, Biotechnol. Prog. 24 (2), 373-376.

Research Overview

The plant biotechnology efforts of the Curtis Lab initially focused on secondary metabolites, fungal elicitation of plant defense compounds, and scale-up of plant cell suspension and hairy root cultures in bioreactor systems.

Protein expression in plant tissue culture included developing strains of Agrobacterium that would not overgrow plant culture (auxotrophs) and using both transient expression in leaves, as well as scaled up in bioreactor systems. More recently, we have utilized transient expression to deliver transcription factors to activate the developmental process of somatic embryos (plant embryos that develop without fertilization).

Bioreactors for plant tissue culture have been an ongoing theme of the laboratory ranging from traditional principles of mixing, pressure drop, residence time distribution in stirred tanks, trickled beds, and, for plant propagation, temporary immersion reactors. An important aspect of reactor design is minimizing cost (such as plastic bag bioreactors) and 'smart' operational strategies for delivering gas and extraction of secondary metabolites.

Projects that developed as a result of our plant biotech expertise include protein expression in mushrooms, bioplastics production from squid, plant expressed animal vaccines, and pine tree propagation (from Weyerhaeuser).

Plant Propagation

Plants can be propagated asexually (without seed) as a means of rapidly proliferating superior plant phenotypes. We have assisted in propagation of chocolate trees, Pines, (with Weyerhaeuser), and most recently yams (Dioscorea). This work includes genetic tools such as transcription factors and embryogenic proteins as well as bioreactor systems to facilitate commercial scaleup.

Bioreactor Systems

Temporary Immersion Bioreactors

Plant tissue culture provides a means to proliferate plants without the requirement of starting with seeds via somatic embryogenesis (Pictured right: carrot suspension culture undergoing SE from 1990), thus enabling the rapid propagation of superior plants such as sterile hybrids or disease resistant varieties. We are developing this technology for increasing the productivity of cultivated plants including fungal resistant Theobroma cacao via embryogenesis and seedless watermelon via organogenesis. This work with seedless watermelon meristem culture demonstrated ability to remove sugar from plant propagation bioreactor media using CO2 supplementation and light – a major advance towards scaleup with reduced contamination risk. See more about application of this project in CurtisLab's Food Security research projects.

Consistent with our goal of implementing low-cost bioreactors, a next-generation temporary immersion bioreactor was designed and prototyped over a period of five years per details in 2015 paper noted above by Florez et. al. The essential design consists of a disposable plastic bag suspended from a reusable headplate. Gravity, rather than gas pressure is used to transfer the media between the reactor and reservoir vessels. The overall design from materials to operation is evaluated in terms of scalability, reliability, and economic feasibility relative to typical rigid-vessel designs.

Genetic Tools

The process of somatic embryogenesis (SE) allows for the propagation of identical superior plants from non-embryonic tissue. Traits such as disease resistance or higher productivity can be maintained in all the plants generated through SE; resulting in a greater economic value. While the process of SE occurs naturally in some plants and has been commercialized for many important economical crops, the mechanism behind this process is not completely understood. In turn, this limits the technical reliability with which commercial SE can be reproduced in other crops that are vital for food and economic security.

The high-value commodity/luxury crop, Theobroma cacao (chocolate), enabled research using various molecular tools (e.g. microarray, genome sequencing) to provide greater insight into these mechanisms. Specifically, they helped us to identify several transcription factors and homologs of embryogenic proteins; first, in Maximova et. al., we published on successful transiently expression of BABYBOOM (BBM) transcription factors. Next, we demonstrated the ability to induce embryos using a transgenic DEX-activatable LEC2 transcription factor on young chocolate leaves--rather than only the diminutive chocolate tree flowers. In contrast to traditional somatic embryogenesis, this LEC2 (transcription factor)-method makes more plant mass viable as embryogenic tissue but also makes SE both much less time consuming and labor intensive.

Embryogenic proteins, which can be added exogenously (i.e. via drip solution) to enhance SE, have also been a focus in CurtisLab's work with T. cacao where they have been shown to dramatically increase embryo yields (overall 3-4-fold). Successful demonstration in T. cacao has enabled continued research into the efficacy of similar (homologous) proteins for other food staples including yam, banana, and rice.

Synergistically, CurtisLab plans to use low-cost bioreactor design to allow this process to be scaled up commercially.

Plants as Protein Production Platform

Transgenic Plant Vaccine Production

Transient protein expression in plants was a project developed in the mid-1990s as a means to rapidly express proteins in plant tissue culture without having to execute stable transformation. This was initially funded by Monsanto (2000), and then a joint Monsanto/NSF GOALI project with Monsanto acting as the industrial partner. The work continued with unrestricted funding from Merck, who came to the PI in large part recognizing his contributions in teaching and preparation of students to work in the pharmaceutical industry.

The overall goal was to develop a transient protein expression system where DNA is delivered to plant cell cultures grown in bioreactor systems to allow production of protein in a time scale of several days rather than months/years required for transgenic plants. Agrobacterium auxotrophs were generated that would not overgrow plant cell culture; 5000 transposon insertion mutants were screened for their compatibility in tissue culture and ability to express heterologous proteins from transiently delivered DNA. O'Neill et. all (see above) demonstrates expression of intimin, an adhesion protein of food-contaminant, pathogenic E. coli O157:H7, towards oral cattle vaccine production.

Transgenic plants expressing viral replicases were also generated to provide a complementary (ultra-safe) viral expression system where the sub-genomic virus only becomes functional when delivered to its ‘partner’ transgenic plant. The unique interaction of this ethanol-inducible transgenic replicase with oxygen in germinating seeds was used to characterize oxygen mass transfer in germinating seed embryos, which ultimately gave rise to a research program with Weyerhaeuser in propagating clonal tree embryos. That technology was transferred to Weyerhaeuser after developing bioreactor systems capable of producing hundreds of thousands of superior tree embryos (fully funded by Weyerhaeuser and still unpublished). Transient protein expression work went beyond its initial scientific goals to demonstrate scale-up of expression at a 60L scale in our pilot plant facility.

This work complemented low-cost bioreactor design development undertaken by undergraduates and a visiting scientist. The work generated 1-PhD, 2-MS and included nearly 30 undergraduates including 10 honors theses. As a profound example of broader impacts, a group of 3 undergraduate students took the principles of plant cell culture bioreactor operation (and equipment available from the work) and applied these to turbulent disruption of pig blood cells that has resulted in statistically substantiating new theories of turbulent disruption of blood cells (and two of the students are now medical doctors).

Expression of proteins in 'Mushroom' Plants

The H1N1 influenza pandemic of 2009 underscored the inadequacy of current commercial-scale manufacturing systems to

Other Applied Plant Biotechnology Work

Grants

NSF CBET: 103572 Low-cost Plant Propagation Bioreactor Development using Cacao (2011-15)

Plant Biotech Team

Researchers: Jeffrey Larsen, Ben Woolston, Matthew Curtis, Sergio Florez, Sydney Shaw, Kristin O'Neill, Tina Lai, Nate Hamaker, Morgan Shires, Chandler Thomas, Katie Legenski, Lauren Andrews.

Collaborators: Mark Guiltinan, Morufat Balogun.

More Products

Papers / Presentations / Patents:

Nate Hamaker, Tina Shing Li Lai, Kristen Fisher, Joanna Hofstaedter. Saffron Project. 2015 Ag Springboard Competition: University Park, PA. April 25, 2015. [2nd place & $2500]

Wayne Curtis and Jeff Beringer. Advances in Plant Propagation. 2015 SIVB Meeting: Tucson, AZ. May 31, 2015. [Co-convened session]

Sergio Florez. "Temporary Immersion Bioreactor & Somatic Embryogenesis in Micropropagation". 2015 SIVB Meeting: Tucson, AZ. May 31, 2015. [Invited Talk]

Sergio Florez. "Improving the Process of Somatic Embryogenesis Using Transient Expression of Transcription Factors and the implementation of a Novel Bioreactor Design". SIVB Meeting Oral Poster Competition. June 17, 2013. [Third Place]

Sydney Shaw. "Bioreactors for Cloning Plants: Promoting plant abstinence!", American Institute of Chemical Engineers Regional Student Conference Paper Competition. University Park, PA. April 9, 2011. (Oral Presentation)

Jeffery S. Larsen* and Wayne R. Curtis " RNA virus-amplified heterologous protein expression in plant tissue culture", Society of In Vitro Biology (SIVB) Meeting, St. Louis, MO, June 7-9, 2010.

Sydney Shaw*, Jeff Larsen, Wayne Curtis " Bioreactor Design for Plant Propagation: Enabling tissue culture productivity enhancements", Society of In Vitro Biology (SIVB) Meeting, St. Louis, MO, June 7-9, 2010. (Poster Presentation)

Jeffrey S. Larsen* and Wayne R. Curtis " Reducing Batch-to-Batch Variability of Agrobacterium-Mediated Transient Protein Expression in Plant Tissue Culture", AIChE Annual Meeting, Philadelphia, PA, Nov. 18, 2008.

O’Neill KM, , Larsen JS, Curtis WR (2007) Scale-up of Agrobacterium-mediated transient protein

expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture, Biotechnol. Prog. 24 (2), 373-376.

Collens JI, Mason HS, Curtis, WR (2007) Agrobacterium-mediated viral vector-amplified transient gene expression

in Nicotiana glutinosa plant tissue culture. Biotechnol Prog. May-Jun;23(3):570-576.

Andrews, Lauren B., Curtis, W.R. Comparison of transient protein expression in tobacco leaves and plant suspension culture. Biotechnol. Prog. 21(3):946-952, 2005.

Curtis WR (2005) Protein Production in Transgenic Plants. In: Encyclopedia of Chemical Processing, vol. 4 (Lee S, ed.), Taylor & Francis, NY, pp. 2489-2500.

Curtis, W.R., Growing cells in a reservoir formed of a flexible sterile plastic liner, U.S. Patent # 6,709,862, March 23, 2004.

Theses:

Florez, Sergio. Improved Plant Propagation through Manipulation of the Genetic & Physical Environment. Dissertation, Ph.D. Penn State University, PA (2015).

Erwin, Rachel. Methods for enhancing somatic embryogenesis in Theobroma cacao. Dissertation, B.S. Penn State University, PA (2015).

Curtis, Matt. Cloning of insecticidal BT genes for application to protection of mushrooms from fungus gnats. Dissertation, B.S. Penn State University, PA (2013).

Curtis, Matt. Novel temporary immersion bioreactor allows the manipulation of headspace composition to improve plant tissue propagation. Dissertation, B.S. Penn State University: University Park, PA (2013).

Shaw, Sydney. An improved temporary immersion bioreactor design for plant tissue culture propagation. Dissertation. Penn State University: University Park, PA (2012).

Woolston, Ben. Development of Agaricus bisporus as a platform for heterologous expression of biopharmaceuticals. Dissertation, B.S. Penn State University: University Park, PA (2011).

Tuerk, Amalie. Transient expression of therapeutic proteins and oxygen transport limitations in plant tissue culture. Dissertation. Penn State University: University Park, PA (2005).