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 transcroption 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.
Our ongoing research in cacao (chocolate tree) combines transient expression, bioinformatics, and temporary immersion bioreactor design to propagate cacao as well as develop a better understanding of the genetic regulation of somatic embryos.
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 Weyerhauser).
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.
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).
Watermelon Shoot Proliferation in Scalable Low-Cost Bioreactors
This research has been supported by Nunhems, USA
Plant tissue culture provides a means to proliferate plants without the requirement of starting with seeds, 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 established the feasibility of utilizing temporary immersion bioreactors (TIBs) for watermelon shootsupplemented with benzylaminopurine and indoleacetic acid to induce shoot formation. The primary cotyledon explants developed much slower in TIBs as compared to solid media possibly due to the presence of a thick, waxy cuticle. In contrast, secondary shoots with less pronounced cuticles were successfully propagated in TIB culture. To enhance productivity, we explored the use of media additives to suppress tissue oxidation and increase the 'wetability' of the explants during liquid immersion. Consistent with our goal of implementing low-cost bioreactors, a next-generation temporary immersion bioreactor was designed and prototyped. The prototype consists of a disposable plastic bag suspended from a reusable headplate. This will be scaled up to a multiplexed system able to run a large number of reactors simultaneously. 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.
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 has been commercialized for many important economical crops, the mechanism behind this process is not completely understood. We plan to use cacao (chocolate tree) as a model to identify the regulators genes that control this SE with the goal to make it a more efficient and economical feasible process. By incorporating techniques such as DNA micro array and using the recently sequence cacao genome, we hope to isolate transcription factors in this process and transiently express them to achieve more and better quality embryos. Furthermore, we plan on using a low cost bioreactor design to allow this process to be scaled up to an industrial process.
In Fall 2010, we received an NSF grant entitled
where transient gene expression will be utilized to manipulate the physiology of plants in bioreactor systems.
This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports work to develop low-cost bioreactor systems that will enhance the ability to utilize tissue culture to improve agricultural plant productivity. In addition to the typical paradigm of optimizing the chemical and environmental conditions within the bioreactor, this research seeks to demonstrate the ability to control plant development using transcriptional factors that control the expression of multiple genes. These transcriptional factors will be delivered to the plant tissues using Agrobacterium, a bacteria that has been developed to transiently deliver DNA to cultured plant tissues (developed with previous NSF support; BCS-0003926). The genes associated with plant embryo formation will be identified by examining patterns of gene expression during somatic embryogenesis. The concept of inducing embryo formation will be implemented using Cacao, the source of chocolate, due to its commercial (and social) value. In addition to providing an instructional case study that demonstrates the use of interdisciplinary principles of bioreactor design and molecular biology, the effort will produce thousands of fungal resistant plants that will be distributed to smallholder farmers in South America. In addition, the low-cost bioreactor technology has potential applications to a broad range of food and biomass crops.
Researchers: Jeffrey Larsen, Ben Woolston, Matthew Curtis, Sergio Florez, Sydney Shaw, Kristin O'Neill, Tina Lai, Nate Hamaker, Lauren Andrews.
More Papers / Presentations / Patents:
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
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.
Curtis, Matt. Cloning of insecticidal BT genes for application to protection of mushrooms from fungus gnats. Dissertation. Penn State University, PA (2013).
Curtis, Matt. Novel temporary immersion bioreactor allows the manipulation of headspace composition to improve plant tissue propagation. Dissertation. 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. Penn State University: University Park, PA (2011).