A temporary immersion plant propagation bioreactor with decoupled gas and liquid flows for enhanced control of gas phase

Abstract

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic-driven TIB (Hy-TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO2 supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ∼0.0002 atm) caused by flexing of non-rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337-345, 2016.

Inducible somatic embryogenesis in Theobroma cacao achieved using the DEX-activatable transcription factor-glucocorticoid receptor fusion

“…We recently reported on the ability to utilize transient expression of the transcription factor BABY BOOM in tissues of Theobroma cacao to enhance the formation of somatic embryos (Florez et al 2015b ). This work complements our ongoing efforts to develop bioreactor propagation technology to allow for improved plant development by manipulating the physical environment (Florez et al 2015a ). In parallel with this previously reported transient gene expression method, which achieves transcription factor expression from Agrobacterium T-DNA that is not chromosomally integrated, we also sought to develop a highly-controlled inducible system based on stable transformation to avoid the complexity of Agrobacterium presence during TF expression.…”

“…The ability to expand the source tissue for SE is particularly notable for our propagation studies of T. cacao , where scaled-up bioreactor work is severely limited by the extensive tissue that is required for such experiments. The ability to rapidly generate tissue for bioreactor studies will permit a greater focus on elements of bioreactor design to see if our observations of oxygen-enhanced heterotrophic growth (Asplund and Curtis 2001 ), or CO 2 -mediated reduction in plant stress and enhanced growth in sugar-free media (Florez et al 2015a ) can be applied to economically-relevant species such as T. cacao . We are currently transitioning our efforts from this ‘luxury crop’ to food staples.…”

CO2 supplementation eliminates sugar-rich media requirement for plant propagation using a simple inexpensive temporary immersion photobioreactor

“…Gas Composition & Flow Monitoring. The gas delivery infrastructure including a low-cost humidi cation setup and manifold was described previously (Florez et al, 2016). Gas ow rates were targeted at 10 mL per minute per vessel.…”

“…Maintaining low gas ows that more closely match consumption through the TIB reactor is desirable but challenging-particularly for inexpensive multiplexed gas delivery. Inlet gas ow resistance was maintained based on back-pressure lters that were preceded by a metal gas manifold with a small resistance heater (Florez et al, 2016). To prevent condensate from causing variable gas ow resistance in exhaust lters, we describe alternative solutions in Online Resource S1E.…”

“…Our laboratory previously presented a novel hydrostatically-driven TIB (Hy-TIB) system that decouples gas and liquid ows thereby facilitating economical use of gas phase mixtures that would otherwise be cost-prohibitive compared to air (Florez et al, 2016). This work demonstrated enhanced growth in tobacco (Nicotiana benthamiana) hairy root tissue cultures by elevating oxygen levels to 40%, and triploid seedless watermelon (Citrullus lanatus) with elevated CO 2 , likely due to changes in oxygen partial pressure that drive mass transfer and suppression of ethylene signaling, respectively.…”