Novel Temporary Immersion Bioreactor Allows the Manipulation of Headspace Composition to Improve Plant Tissue Propagation

Open Access
Author:
Curtis, Matthew Steven
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Michael John Janik, Honors Advisor
Keywords:
  • Plant Propagation
  • Somatic embryogenesis
  • Organogenesis
  • Aseptic design
  • Bioreactor
Abstract:
This thesis describes the iterative development of a bioreactor system to propagate plants. While a large focus of the effort was in instrumentation associated with gravity-driven liquid flows, the work also included extensive trouble-shooting associated with various difficulties of implementation as well as the techniques of maintaining a wide variety of tissue cultures. A temporary immersion bioreactor is tissue culture system in which the liquid nutrient medium is periodically flooded into the culture vessel to provide dissolved nutrients. This is particularly useful for culture plant tissues because constant submergence can lead to being water-logged and difficulties in providing oxygen from the gas phase. In addition to proving for control of liquid contacting, the TIB system allows for changing the nutrients and hormones in the medium. Of particular interest to this project, is the ability to facilitate the proliferation of plant shoots (termed meristem propagation) or the formation of embryos from somatic tissues (termed somatic embryogenesis). The specific work described within this effort involves the design of a stepper motor system to raise and lower media reservoirs that facilitates the desired intermittent exposure to media. This involved writing a LabVIEW computer program that interfaced to a stepper motor through a DIY stepper motor kit that was interfaced to the computer via a simple parallel computer (printer) port. The LabVIEW program included a high level of sophistication including text messaging for operational verification, warnings associated with externally mandated computer resets, and the capture of video images as 'time lapse' files to be monitored over the internet to verify proper bioreactor operation. The thesis also describes the evolution of various designs from basic design and materials of the bioreactor, reservoir, gear and pulley systems, to trouble-shooting of contamination issues where studies of vessel pressure levels using an integrated circuit pressure sensor that demonstrated unexpected internal bioreactor pressure changes that could become sub-ambient and compromise sterility of operation. A solution for gas delivery from an inexpensive manifold to multiple reactors while maintaining a positive pressure in the vessels is presented as part of the 'Design of Gas Handling' chapter of the thesis. Since the final performance runs are in progress, only brief descriptions of are included of the numerous studies carried out during the period of nearly three years; these include meristem propagation of watermelon, root culture growth rates under elevated oxygen. This work has led to establishing a collaboration with a Nigerian researcher who will be looking to scale up this technology to propagate yam, and the project continues as a bioreactor to be used to deliver transcription factors to manipulate the process of somatic embryo formation as a final illustration of the ability of a bioreactor system to facilitate the manipulation of the gene expression in propagated plant tissues.