Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-021-02210-3 ORIGINAL ARTICLE CO2 supplementation eliminates sugar-rich media requirement for plant propagation using a simple inexpensive temporary immersion photobioreactor Marena Trauger1  · April Hile1  · Krishnan Sreenivas1,2  · Eva Mei Shouse1,3  · Jishnu Bhatt1,4 · Tina Lai1,4  · Ramya Mohandass2  · Leena Tripathi5  · Aaron J. Ogden6  · Wayne R. Curtis1 Received: 23 September 2021 / Accepted: 1 December 2021 © The Author(s) 2022 Abstract In vitro plant propagation systems such as temporary immersion bioreactors (TIBs) are valuable tools that enable production of disease-free plants with improved traits. However, TIB systems can be expensive, difficult to implement, and prone to contamination due to sugar rich propagation media. Using rapidly growing chicory root cultures to expedite design-build- test cycles, we report here an improved, low-cost version of a previously reported Hydrostatically-driven TIB (Hy-TIB) that facilitates economical use of gas mixtures. Bioreactor improvements include decreased material costs, expanded modes of operation, and a horizontal orientation of a plastic film plant growth chambers that increase propagule light exposure. To take advantage of these improvements, we describe here experiments that evaluate the impacts of elevated C O2 on propaga- tion of cacao (Theobroma cacao) secondary embryos and nodal cultures of yam (Dioscorea spp.) during both phototrophic and photomixotrophic growth. Our experiments show that elevated C O2 during plant propagation significantly improved both cacao and yam propagule development and eliminated the need for supplemental sugars in tissue culture growth media. Thus, our improved Hy-TIB shows potential as a simple, low-cost, and scalable propagation platform with cost-effective gas composition control and reduced risk of contamination overgrowth. We provide detailed instructions for assembly of this Hy-TIB design and discuss the implications of its adoption in food-insecure regions of the world. Key message Elevated CO2 in a temporary immersion bioreactor facilitated removal of sugar from the propagation media, resulting in decreased contamination issues and improved plant root development in agriculturally relevant plant species. Communicated by Paloma Moncaleán. * Wayne R. Curtis wrc2@psu.edu 1 Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA 2 Department of Genetic Engineering, SRM University, Tamil Nadu, Kattankulathur 603203, India 3 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA 4 Intercollege Program in Plant Biology, The Pennsylvania State University, University Park, PA 16802, USA 5 International Institute of Tropical Agriculture, P.O. Box 30709, Biosciences Nairobi 00100, Kenya 6 Pacific Northwest National Laboratories, Richland, WA 99301, USA Vol.:(012 3456789) Plant Cell, Tissue and Organ Culture (PCTOC) Keywords Single-use bioreactors · Plant propagation · Theobroma cacao · Dioscorea spp. · Temporary immersion bioreactor · Hairy root culture Abbreviations a struggle (Matemilola and Elegbede 2017). Nigeria, the DBT Design-Build-Test most prolific exporter of yam, has suffered 60% deforestation FAO Food and Agriculture Organization of the since 1990 (Gates 2021), a trend that is unlikely to reverse United Nations course without technological intervention. Hy-TIB H ydrostatically-driven temporary immersion Indeed, modern and sustainable agriculture across Africa bioreactor and elsewhere would benefit from more robust and afford- H Horizontal orientation (describing Hy-TIB able plant propagation systems. In many developing coun- vessel) tries, cacao and yam both represent economic security crops. LA Labview-based automated operation Cacao is a small woody tree and a luxury crop that consti- M M anual operation tutes a major component of the GDP for several tropical McA Microcontroller-based automated operation countries. Similarly, yam is a carbohydrate-rich staple crop NO Nitric oxide supporting 60 million West African livelihoods at current PAR Photosynthetically active radiation production levels, with 70% of global supply produced in PTC Plant tissue culture Nigeria (Nanbol and Namo 2019). Despite the enormous PP Polypropylene economic importance, yam has not shown progressive pro- RI R efractive index ductivity gain in recent decades due to various production SE Somatic embryogenesis constraints. These constraints include the shortage of qual- SS S tainless steel ity seed yam of popular landraces and improved varieties, TIB Temporary immersion bioreactor as well as the high cost of planting materials. Robust agri- V V ertical orientation (describing Hy-TIB vessel) cultural production via a decentralized supply chain from WUB Wave and undertow bioreactor farmers is imperative to mitigate adverse climate impacts and enable widespread prosperity by supporting both Nige- ria’s domestic demand and income security by increasing Introduction production for foreign supply. To pragmatically address food security challenges, the The propagation of plants with desirable traits has been a plant propagation industry must always carefully consider cornerstone of societal development throughout history. Its advances in technology relative to the cost of implementa- current technological application spans from luxury crops tion. To that end, superior plant varieties (e.g. high yield, (e.g. ornamental flowers and plants, coffee, chocolate) to stress-tolerant, disease-free) can be clonally propagated in industrial agriculture. Commodity crops like seedless water- aseptic tissue culture, which is particularly useful for plants melon and grape illustrate our ability to take advantage of with long maturation cycles. Temporary immersion bioreac- the non-obvious characteristics of plant propagation. Fur- tor (TIB) systems facilitate aseptic plant propagation until ther, virus-indexed crops such as seed potatoes provide a plants are sufficiently mature for transition to soil. To date, disease-free supply chain that can otherwise result from plant propagation systems include single-use rocking reac- traditional vegetative propagation. In less developed agri- tors (e.g. Southern Sun Biosystems® Rocker (Kämäräinen- cultural cropping systems, the serial propagation of plants Karppinen et al. 2010), WAVE, Biowave, BIOSTAT® Culti- via seeds, tubers, and cuttings is part of the fabric of eve- bag), slug bubble (Valdiani et al. 2019), Wave and Undertow ryday life. As human population grows and climate change (WUB), Appliflex, CELL-tainer (Eibl et al. 2009), rotating worsens, plant propagation will invariably remain a criti- wall vessels (Valdiani et al. 2019) or rotating drum reactors cal component of modern agriculture. A report by the Food (e.g. roller bottles (Shetty 2005)), mist bioreactors or nutri- and Agriculture Organization of the United Nations (FAO) ent sprinkle reactors (Steingroewer et al. 2013), and Tempo- estimates that 10% of the world population has severe food rary Immersion Bioreactors (TIBs). Existing TIBs include insecurity exposure, affecting 750 million people globally. Temporary Root zone Immersion (TRI) bioreactors (Neu- This trend will likely be exacerbated by climate change, lim- mann et al. 2020a), Periodic Immersion Bioreactor (PIB), ited arable land and water, and increasing global popula- Plantform™ (Ruta et al. 2020), and the twin flask bioreactor tion, particularly for developing nations (FAO et al. 2020). system commercialized as the RITA®/SETIS™ (Georgiev Food production is not only being affected by biotic and et al. 2013). However, due to design constraints that limit abiotic stresses and climate change but contributes 19% of economic feasibility (Eibl et al. 2018) these solutions have global greenhouse gas emissions. Even in Nigeria, despite generally been relegated to research environments (Balogun having the continent’s highest GDP, food security remains et al. 2017) and commercial production of high-margin plant 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) medicinal products and luxury crops (Ducos et al. 2010). described (Li et al. 1998; Maximova et al. 2002). Tissue Thus, to achieve cost viability for low-margin fiber and food was kept at 25 °C in the dark on solid media per protocol staples, further economical advancements are required. utilizing secondary callus growth medium (SCG / E5B) Our laboratory previously presented a novel hydrostat- and embryo development (ED) media (Shires et al. 2017) ically-driven TIB (Hy-TIB) system that decouples gas and for ~ 6–8 weeks. Dioscorea cayenensis (yam) plantlets were liquid flows thereby facilitating economical use of gas phase provided by Morufat Balogun, University of Ibadan, Nige- mixtures that would otherwise be cost-prohibitive compared ria; D. rotundata (IITA accession no. Tdr2436) were pro- to air (Florez et al. 2016). This work demonstrated enhanced vided by Leena Tripathi, IITA-Nairobi, Kenya. Both species growth in tobacco (Nicotiana benthamiana) hairy root tis- were maintained based on the protocol optimized by Mano- sue cultures by elevating oxygen levels to 40%, and triploid haran et al. (2016). Briefly, nodal cuttings of approximately seedless watermelon (Citrullus lanatus) with elevated CO2, 25 mm in length were subcultured on solid Yam Callus Pro- likely due to changes in oxygen partial pressure that drive liferation Media (YCPM), a MS-based media supplemented mass transfer and suppression of ethylene signaling, respec- with picloram, casein hydrolysate, and proline (see Online tively. Our previous Hy-TIB design also implemented typical Resource S1A-1) for three weeks. Thereafter, they were sub- engineering automation software to control the height of cultured every 2–3 weeks onto fresh solid Yam Basic Media media reservoirs and fault monitoring. While effective, this (YBM) (see Online Resource S1A-2) for 2 months, in air, design was unnecessarily complicated for the operational without light, and at 25 °C. Next, explants were subcultured requirements of a TIB bioreactor. on the same YBM media into larger Magenta™ GA-7 cul- After extensive design-build-test (DBT) cycles, we pre- ture vessels and placed in a growth chamber with 16:8 h sent here a newly designed low-cost Hy-TIB that functions diurnal cycle, measured as PAR of 77 ± 2 μE m −2 s −1) for either manually or automatically via low-cost, open-source approximately 6–8 weeks or until plants reached ~ 95 mm platforms (e.g. Arduino). As shown in Florez et al 2016, and had substantial node growth. After several months of we demonstrate that our improved Hy-TIB design can be meristem proliferation, the process was re-initiated to ensure implemented using 40% elevated oxygen to improve chic- propagules maintained embryogenicity. ory (Cichorium sp.) heterotrophic root growth. We further show that C O2 supplementation significantly enhances root Horizontal (H) Hy-TIB vessel component fabrication development in cacao (Theobroma cacao) and survival rates in yam (Dioscorea spp.). Finally, we also demonstrate that H Hy-TIB version 3 (v.3) elevated C O2 during propagule development eliminates the need for sugar in the plant growth media, resulting in The cost analysis spreadsheet (Online Resource 2) out- both carbon capture and decreased contamination risk. We lines the source and costs of materials for both previously provide detailed instructions for assembly of the Hy-TIB reported vertical orientation (V) of Hy-TIB and horizontal designs to facilitate its adoption as an FTO (freedom to oper- orientation (H) Hy-TIBs of the current work. Intermediate ate) and open-source technology. materials, including high durometer PP tubing, larger inner and outer diameter (ID/OD) SS tubing in fluid distributor, that were used only in versions 1 (v.1) and 2 (v.2) have been Materials and methods excluded. Several of the components of the reactor were custom machined at Penn State’s Learning Factory includ- Plant tissue culture ing (1) stainless steel (SS) insert that had to be cut to size, sanded, perforated, and bent, (2) SS fluid distributor that Cichorium intybus (chicory) Agrobacterium-transformed had to be cut to size, perforated, and epoxied to insert, and root cultures were kindly provided from Premier Tech (3) SS gas connectors that had to be cut to size and lathed to (https:// www. premi ertech. com) and grown on solid B5 specification. CAD component drawings, PDFs, and machin- media without plant hormones. Serial cultures were main- ing specifications are included in Online Resource S3,1B, tained on liquid B5 medium (Gamborg et al. 1968) with and S1C, respectively. 25 g  L−1 sucrose and subcultured bi-weekly. Subculture involved transferring ~ 0.5 g fresh weight (FW) of tissue into H Hy-TIB infrastructure, assembly and inoculation 50 mL of fresh B5 media in wide-mouth 125 mL Erlenmeyer flask on a 120 RPM orbital shaker with a 2.5 cm stroke. Pre-sterilization assembly Root culture maintenance and bioreactor experiments were conducted at 25 °C in the dark. Theobroma cacao secondary Online Resource S1D and S4 provide instructions on the somatic embryo tissue cultures of PSU Scavina 6–1 were assembly of H Hy-TIB vessel via step-by-step pictorial established from greenhouse grown floral buds as previously instructions and video. In brief, the insert is placed inside 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) the polypropylene (PP) plastic bag (VWR 95-42-564) with implications for cost and performance and is particularly protruding fluid distributor-end inserted first (towards closed challenging for evaporation under enhanced light flux. end). Just below (~ ½”) the seam and in the center of the bag, the distributor is pushed through the PP bag, and the bag is Manual infrastructure and operation punctured to ensure flow. Similarly, gas tubing is attached to the bag with the press-fit swage as described in the Dis- For manual operation, the pulley system was replaced by cussion. A growth matrix (e.g. filter paper, cheesecloth, a two-tiered stand, each with two ‘arms’, to provide high fabric) is added onto the insert; this prevents propagules and low positions for the shelf of inverted media reservoirs. from flowing through perforations during operation. For in- Other configurations could employ a manual hoist like the line sampling (i.e. refractive index), a Luer tee connector automated (LA and McA) systems for scaled-up reservoirs. (Cole Parmer, EW-45500-56) and syringe port (MediDose, Online Resource S1F contains a diagram of Hy-TIB infra- IV2004) were installed into tubing connected between the structure setup, noting the height recommendations for rais- fluid distributor and media reservoir. ing and lowering of reservoir relative to Hy-TIB vessel to ensure immersion without propagule displacement. Post-sterilization inoculation Microcontroller-based infrastructure and operation H Hy-TIB reactors were pre-assembled without tissue and autoclaved. Explants (e.g. root fragments, cotyledons, nodal Raising and lowering of the media reservoirs was accom- cuttings) were aseptically distributed into the bag, sealed plished using the same infrastructure described by Florez in a laminar flow hood using a commercial heat sealer and et al. (2016) (adapted into CAD drawings; see (see Online inflated via the gas inlet filter with a Drummond pipettor to Resources S1B, S3), with the exception that operation of ensure asepsis (or ‘sterility’). Media was introduced post- the pulley system was accomplished via a microcontroller autoclaving by replacing the empty bottle with a sterile rather than computer-based LabView software. In brief, the media-filled counterpart, then clamping all tubing lines to inverted media reservoir shelf is raised and lowered by a avoid liquid getting into the air lines when the media bottle pulley system. In this case, the process was controlled by an was inverted into operational position (noting that air lines Arduino Uno (www. ardui no. cc) with the program provided must be above media level). in Online Resource S5. This program sent the stepper motor controller a (0/1) logic output to specify stepper motor direc- tion and a pulsing signal for rotational steps that generated Bioreactor operation and monitoring a linear translation rate of roughly 0.5 cm per second. The stepper motor is only powered on during the cycle, with the Gas composition and flow monitoring down position as default to minimize energy use. The gas delivery infrastructure including a low-cost humidi- Hy-TIB experimental conditions and data analysis fication setup and manifold was described previously (Flo- rez et al. 2016). Gas flow rates were targeted at 10 mL per Refractive index/sugar consumption minute per vessel. Oxygen and Carbon Dioxide Analyzer (Illinois Instruments, Model 3750) was used to confirm Refractive index (RI) was measured with a high-precision composition of supplemented gases. Because we required refractometer (Leica, Model AR600) to determine sugar extremely low overall gas flow rates, particularly for sup- consumption by the explants. For chicory roots, a syringe plementation gases (O2 and CO2), even the lowest-flow rota- port (Cole Parmer, EW-45503-04) was installed in the flow meters (Brooks Sho-Rate model 1355E, tube: R-2-15-AAA, path of fluid distributor such that 0.3 mL media samples spherical glass floats, nominal 47.1 mL  min−1) were insuf- could be taken via sterile syringe every 1 to 3 days. ficient for maintaining flow. Maintaining low gas flows that more closely match consumption through the TIB reactor Root tensile strength is desirable but challenging—particularly for inexpensive multiplexed gas delivery. Inlet gas flow resistance was main- The tensile strength of propagule roots impacts ease of tained based on back-pressure filters that were preceded by manipulation and explant survival. To evaluate differences a metal gas manifold with a small resistance heater (Florez in root tensile strength, a simple tensometer was fabricated et al., 2016). To prevent condensate from causing variable using two Hoffman tubing clamps (Online Resource S1I). gas flow resistance in exhaust filters, we describe alternative Both clamps were covered with a soft silicone tubing to pro- solutions in Online Resource S1E. This issue has important vide for a gentle clamping of both ends of a segment of root with the bottom clamp fixed to a spring nut and weight. The 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) force (weight) necessary to break the root was assessed by polyester fabric (JoAnn fabrics, Thermolam Pellon TP980) gently pulling upward on the upper clamp and video-taping that required manual submersion in media during inocu- the balance weight to observe the maximum weight loss on lation due to hydrophobic properties. All reactors were the balance at the time of root rupture. Only roots which maintained on the same media schedule over a period of ruptured away from the clamp position were considered a 4.5 weeks (31 days). Reactors were operated with 350 mL valid measurement. All plants with roots removed for tensile of media: 250 mL of ED media with an additional 100 mL strength measurement thrived after potting and did not affect containing 30 g  L−1 sucrose and 1 g  L−1 glucose. plant survival measurements. The photosynthetically active radiation (PAR) light inten- sity was measured to be 77 ± 2 μE  m−2  s−1 using a light Chicory root elevated oxygen study sensor (LICOR, LI-1400, Quantum PAR sensor) where uniformity was achieved by surrounding the TIB growth To assure actively growing roots, explant tissue was obtained area with reflective mylar film. The lighting was main- by combining several 1-week-old 125 mL flask subcul- tained 24/7 for the first 2 weeks post-inoculation followed tures, aseptically blotted and weighed, resulting in ~ 2 g by a 16:8 light:dark photoperiod thereafter. The reactors fresh weight (FW) tissue. The inoculum was incubated for were cycled twice daily (every 12 h) to submerge the tissue three-days in several flasks containing 50 mL of B5 media for ten minutes each cycle. Reactors were harvested after to overcome stress from recently cut tissue, reduce the lag 4.5 weeks (31 days) in a fashion similar to chicory roots to growth phase of roots, and identify any contamination. After obtain fresh and dry weight data. In addition, propagules incubation, the inoculum was distributed onto filter paper were photographed, and image analysis was conducted with (Sigma-Aldrich, WHA1001150) in the assembled autoclave open-source Semi-Automated Root Image Analysis (saRIA) sterilized H Hy-TIB reactors with additional 175 mL of fresh program to determine root volume (Narisetti et al. 2019). B5 media (total 225 mL liquid inoculum). Reactors were operated with either air or elevated (40%) oxygen in air. Both Yam trophism study the incubation periods and the reactor run were carried out at 25 °C under dark conditions. The reactor was manually A study of yam in McA-H Hy-TIB (v.3) involved growth cycled every 4 h (6 times a day), submerging plant tissue conditions as described above. The explants were estab- for ~ 10 min each cycle. lished through serial nodal cutting and propagated within Per design-build-test (DBT) cycle (see Results and Dis- Magenta™ GA-7 vessels. From these plantlets, approxi- cussion), Hy-TIBs were initially operated manually but once mately 1-inch nodes were excised, all appearing of compa- shown to be comparable with LA-V and Mc-A, different rable health (i.e. no visible overcrowding, excessively dry modes were used interchangeably as dictated by experimen- tissue, or necrosis) to provide 25 nodal explants for each tation. The heterotrophic root reactors were harvested after of eight bioreactor vessels. Twenty-five nodal explants approximately 2 weeks post-inoculation, or once sugar con- were also inoculated into the GA-7 vessel for the agar con- sumption plateaued for either treatment. Prior to harvesting, trol; notably, in a prior study of D. cayenensis (see Online a small sample of media was used to test for contamination Resource S1H), growth based on equal area distribution based on growth on highly permissive R2A media (van der (i.e. 25 nodes among 4 GA-7) showed comparable growth Linde 1999). Roots were carefully removed from the reac- to air/sugar Hy-TIB. The nodal explants were initially trans- tor and blotted on paper towels and weighed. To allow mass ferred to solid YBM plates and maintained at 25 °C in 16:8 balance closure due to evaporation, final media volume was light:dark cycles for three days to check for contamination meticulously assessed by weighing reactors, roots, and paper prior bioreactor inoculations. towels used to blot. For dry weight measurements, a 2 g All McA-H Hy-TIB (v.3) vessels were assembled and fresh weight (FW) sample of the root tissue was placed on sterilized with cheesecloth (grade 10) inoculated with pre-tared aluminum foil packets and dried at 70 °C until nodal cuttings and 250 mL liquid YBM (Online Resource consistent dry weight. 1A-2) with and without 30 g  L−1 sucrose based on experi- mental design of two reactor replicates for each treatment. Cacao elevated CO2 study Treatments consisted of air without sucrose, air with sucrose, 5% CO2 supplementation without sucrose and Growth of cacao in M-H Hy-TIBs v.1 were operated with 5% C O2 supplementation with sucrose. The agar control either air or elevated 2% C O2 in air, each with two repli- containing 50 mL solid YBM containing sugar was placed cates. Experimentation was conducted with 12 secondary on the same shelf as the reactors to ensure comparable somatic embryos per reactor (< 1 cm length, without root experimental conditions. Lighting setup and intensity was or leaf development). The total fresh weight of inoculum the same as cacao study above. Reactors were harvested per TIB was 1.53 ± 0.13 g. The growth matrix used was 6 weeks after inoculation. Fresh weights were obtained 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) by removing each plantlet from its reactor, blotting on a Results paper towel, and weighing. In addition, propagules were photographed, and image analysis was conducted with Enhanced Hy-TIB design and operational modes saRIA for determination of the fibrous root surface area. for reduced cost Dry weights were not taken since these explants contin- ued in a potted survival study. Figure 1 provides a diagram of the improved horizontal M-H Hy-TIB as well as a photo of M-H Hy-TIB in active operation (Fig. 1A) highlighting the design changes between Yam survival studies previously reported vertical LA-V Hy-TIB (Fig. 1B). Areas of modification of vessel orientation and mode of opera- Plantlets from harvested reactors were transferred to soil tion were motivated by the goal to (1) reduce component in 4 × 9 well plastic potting trays on 1020 greenhouse flat and operation costs, (2) increase light flux to the tissue to trays with plastic domes for a high-humidity environment improve phototrophic growth, and (3) improve reliability under LED lighting. Plants were treated as needed with by eliminating risk of breaking the media reservoir siphon. Gnatrol (Valent, WDG, Bacillus thuringiensis, subsp. The re-configuration from vertical (V) Hy-TIB to horizontal israelensis, strain AM 65-52 fermentation solids) to sup- (H) eliminated both the headplate and use of glass beads press fungus gnats. These plants were monitored and (Fig. 1B). The horizontal orientation nearly doubled the watered when soil appeared dry for 26 days. Photos were lighted growth area for each reactor as outlined in Table 1 taken of these explants at 2-day intervals to monitor their with an associated 90% increase in photon flux for the same progress. Survival fraction was calculated relative to the 8″ × 10″ polypropylene (PP) growth bag. Despite increased 25 nodes initially inoculated. growth area, media requirements were comparable (see Table 1). The change to a horizontal delivery allowed for reliable liquid fill and drain cycles by avoiding the siphon Fig. 1 Cartoon representation juxtaposing the designs of the (manual) of a headplate that reduces propagule light exposure. Both designs M-H and (automated) LA-V Hy-TIB designs. A The improved of H use a shelf (orange) to hold the media reservoir. In the manual M-H Hy-TIB design incorporates a pre-sterilized horizontally oriented Hy-TIB operation, the media reservoir is physically moved between plastic bag to house plant tissues to increase propagule light exposure low and high shelf locations to cycle media in and out of the plas- to tissues grown on a stainless-steel plate (A, right). B The previously tic bag. The newly developed automated McA-H Hy-TIB uses open- reported LA-V Hy-TIB incorporates unnecessarily complicated and source Arduino to drive the previously described stepper motor and costly reactor automation. Further, the LA-V Hy-TIB requires the use pully lift assembly Table 1 Hy-TIB cost and design Hy-TIB bioreactor LA-V Hy-TIB M-H Hy-TIB v.3 McA-H Hy-TIB v.3 specifications Cost per reactor $117.98 $27.80 $53.03 Min. media volume 220 mL 250 mL Lighted growth area 122 cm2 232 cm2 Cost estimates per reactor assumes 8 reactors operating simultaneously. The minimum required media and lighted growth area are included to highlight McA- and M-H Hy-TIB design’s cost effectiveness relative to previously reported LA-V Hy-TIBs 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) which would periodically break as experienced using the Other cost considerations are consumables that are equiv- vertical configuration (Fig. 1B). The cycle time of 10 min alent in all scenarios. These consisted of only two compo- remained the same and was empirically arrived at based on nents, including a PP bag ($0.73) and filter paper ($0.22), the flow resistance of the liquid delivery tubing and hydro- where a variety of less expensive tissue matrix can be used. static head of 7.6 cm. This cycle period is small relative to For the optional addition of a syringe port to the flow-path the 4–12 h drained cycles. to provide greater environmental control and/or in-line monitoring would cost $1.92. While media requirements are similar for all TIBs, the cost per plant is proportional to Inexpensive not disposable as a design goal lighted surface area within the bioreactor available for plant growth. The horizontal H Hy-TIB utilized 1.0 mL c m−2 as In developing plant propagation systems, the criticality of compared with 1.8 mL per cm2 for the previous vertical V cost conservatism cannot be understated due to the low value Hy-TIB, achieving a roughly 40% cost reduction for media. of plant propagules ranging between $0.20-$1 (Kozai and Based on significant costs of hormones, antibiotics, media Xiao 2006; Kozai 2008). A key aspect of Hy-TIB design additives, and nutrients—even at low concentrations—this is enabling economical use of gas composition control by would have significant operating cost implications. However, avoiding pneumatically-driven media flows. Previously it should be noted that plants are efficient at assimilating achieved cost reduction for mixed-gas use (Florez et al. nutrients from a dilute environment, and much lower nutri- 2016) is retained in the present designs. The focus for the ent levels that more closely match stoichiometric utilization improved design is overall implementation reliability and are likely feasible. bioreactor component cost reduction. Table 1 outlines the overall cost per bioreactor for its components control mod- Chicory root culture with elevated oxygen facilitates ules for the M-H and McA-H Hy-TIBs v.3 relative to LA-V rapid horizontal Hy-TIB prototyping Hy-TIB (complete cost breakdowns for each scenario are provided in Online Resource 2). Capital costs (e.g. heat To expedite Hy-TIB DBT cycles while evaluating alterna- sealer to seal PP bag, shelving, basic laboratory infrastruc- tive reactor configurations, we chose to use rapidly growing ture, gas delivery) that would be equivalent for all three chicory root cultures (C. intybus). This allowed us to con- reactor types were excluded. The use of epoxy in H Hy-TIB duct iterative experiments on the order of weeks, rather than v.3 was considered negligible. All calculations assume eight months while taking advantage of the mechanistic similarity bioreactors would run in tandem to maximize use of more gas transport limitations between oxygen-dependent heter- expensive components (e.g. humidification train, 4-way otrophic growth and C O2-dependent phototrophic growth. gas manifold). The previously described LA-V Hy-TIB’s Throughout DBT cycles of M- and McA-H HyTIB reac- requisite computer and LabView software were omitted tor versions 1-3 (v.1-v.3, Table 2), we repeatedly tested the due to cost variability but pragmatically would be fiscally impacts of ambient and elevated oxygen levels on chicory significant whereas the M-H Hy-TIB’s shelving would be root growth rate. Using the improved H-M Hy-TIB, elevated comparatively minimal. Therefore, these projected costs are oxygen resulted in more rapid and uniform root growth that conservative estimates. Relative to the previously reported eventually formed a solid mat (Fig. 2A). Further, elevated La-V Hy-TIB, these design modifications resulted in a 55% oxygen also alleviated visible hypoxia and reduced the pro- and 76% cost reduction for McA-H v.3 and M-H v.3, respec- duction of a secreted pink metabolite (Fig. 2A, right). tively. Notably, the use of a plastic bag to house propagules Sugar consumption was measured at regular intervals by suggests the H Hy-TIB culture vessel size can be increased refractive index as a proxy for biomass accumulation (Ram- with minimal cost increase. akrishnan et al. 1999). Bioreactors were harvested at 20 days Table 2 Chronological progression of Hy-TIB designs and modifications Hy-TIB version Description LA-V Hy-TIB Hy-TIB with Labview-based Automated Operation, Vertical vessel with headplate, glass beads (Florez et al. 2016) M-H Hy-TIB v.1 1st version of H Hy-TIB (headplate, glass beads removed) with perforated SS plate insert with fabric, perfo- rated PP media distributor, press-fit swage M-H Hy-TIB v.2 2nd version of H Hy-TIB that modified H1 Hy-TIB media inlet/outlet from PP to SS material, added ‘capil- lary condenser’ outlet string, various fabric substitutions M-H Hy-TIB v.3 3rd version of H Hy-TIB that modified H2 Hy-TIB media inlet/outlet to have smaller diameter SS tubing McA-H Hy-TIB v.3 Hy-TIB with Microcontroller-based Automated Operation, Horizontal vessel with perforated SS plate insert 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) Fig. 2 Chicory (Cichorium intybus) root tissue culture growth used to facilitate expedited Design-Build-Test (DBT) cycles for gas-phase composition control using the manual M-H Hy-TIB (v.1-3). A Elevated oxygen supplemented to 40% resulted in substantially healthier looking roots (left) relative to ambient air (right). B As a proxy for root growth, sugar consumption was moni- tored at regular intervals by refractive index. Chicory roots consumed sugar significantly faster when grown in elevated oxygen (p < 0.001, ANOVA with no left-out terms). C At time of harvest (21 days post inoculation into the reactor), chicory roots grown in elevated oxygen reached higher fresh- and dry-weights (represented on the left and right axes, respec- tively) relative to plants grown in ambient air. Scale bars in (A) represent 25 mm or earlier if sugar consumption plateaued due to depletion. flows, we executed experiments to evaluate the impacts of We observed that elevated oxygen resulted in a significant, elevated C O2 on cacao (T. cacao) propagule development nearly 50% increase in the rate of sugar consumption, ris- from secondary somatic embryos. Compared to rapidly ing from 0.38 g sugar  L−1  day−1 in ambient air to 0.57 g growing tissues, the slower growth of cacao and a 16:8 sugar  L −1  day−1 (Fig. 1B)(p-value < 0.001, ANOVA with no photoperiod lends itself to far fewer temporary immersions significant left-out terms). This enhanced growth is facili- (twice daily), and thus more practical manual operation. Pre- tated by increased oxygen availability in the root meristem vious LA-V Hy-TIBs successfully grew cacao cotyledonary (Asplund and Curtis 2001), which supports previous obser- embryos for 8 weeks, producing considerable leaf develop- vations of linear oxygen-transport limited root growth made ment under photoheterotrophic growth conditions (unpub- using Agrobacterium rhizogenes-transformed root cultures lished data, see Online Resource S1G-1). This suggested (Larsen and Curtis 2012; Florez et al. 2015). After 21 days Hy-TIBs have the potential to generate potable plants in less of growth in ambient air, chicory roots reached a final fresh- than 2 months. To evaluate the benefit of supplemented CO2 and dry-weight of 37.5 g and 3.15 g, respectively (bioreac- on cacao propagule development, a set of manually cycled tors n = 2). In contrast, plants grown under elevated oxygen M-H Hy-TIBs (v.1) were inoculated with cacao secondary reached a final fresh- and dry-weight of 64.2 and 4.4, respec- somatic embryos with either ambient air or air supplemented tively (bioreactors n = 3). (Fig. 2B, C). with C O2 followed by quantification of root growth by image analysis (Fig. 3A) (Narisetti et al. 2019). After 31 days of Cacao somatic embryo development is enhanced growth, elevated C O2 resulted in a significant increase in by elevated CO2 in manual horizontal Hy-TIB root volume relative to ambient air (Fig. 3B) (Mann–Whit- ney U-test, p = 0.01, U = 92). Figure 3A highlights this To further demonstrate the utility of our improved Hy-TIB observation with photos of median roots ranked by volume designs that take advantage of decoupled gas and media for each treatment; these were analyzed as 17.7 m m3 for 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) Fig. 3 Evaluation of cacao (Theobroma cacao) propagule root devel- median of the respective treatments. B Elevated CO2 resulted in a opment under elevated and ambient CO2 using M-H Hy-TIB (v.1). significant increase in cacao root volume (p = 0.01, Mann–Whitney A After 31  days of growth, root development was quantified by U-test). C While rare, additional observations included rapid devel- tracing and image analysis using saRIA for both elevated C O2 (top) opment of floral and leaf tissues in M-H Hy-TIBs using elevated and and ambient CO2 (bottom). The example plants shown represent the ambient C O2, respectively. Scale bars in (A) represent 5 mm CO2 and 1.2 mm3 for air. Significant abscission of roots and of sugar and elevated C O2, indicative of photomixotrophic cotyledons were noted in both treatments, with about half growth. Most importantly, growth with supplemental CO2 as much abscised tissue observed with C O2 supplementa- in the absence of sugar was greater than typical hetero- tion. This is consistent with carbon dioxide’s role as a plant trophic growth using sugar and ambient CO2. This is notable growth and stress regulator (Huang and Xu 2015). While because the elimination of sugar from reactor media drasti- rare, we also observed accelerated stages of plant develop- cally reduces the risk for contamination overgrowth. Struc- ment during growth in Hy-TIB reactors, including early leaf tural integrity of roots is expected to influence plant survival emergence and flower formation (Fig. 3C). While standard while transferring explants from reactors to soil). During the flowering generally occurs on mature trees over a period course of these experiments, the authors observed a two- of 3–4 weeks (Swanson 2004), this floral development fold increase in the tensile strength of yam propagule roots occurred in less than 31 days from the exposure of second- grown in Hy-TIB reactors using elevated CO2 and photo- ary embryos to light, and is to the authors’ knowledge this trophic conditions relative to those grown in ambient air is the most rapid observation of floral development reported, (Online Resource S1I). To evaluate explant survival, Hy- and notably took place in the absence of exogenous hormone TIB-grown yam propagules were transferred to soil and their supplementation. Epinastic growth was also noted through- survival was monitored 25 days post-transplantation. Plants out both treatments but not quantified (see Online Resource grown in Hy-TIBs with elevated CO2 and no additional sugar S1G-2). survived as well or better than other conditions (Fig. 4D). These data demonstrate that Hy-TIB reactors can be used Elevated C O2 enhances yam propagule development to propagate plants using elevated C O2 without sugar sup- and survival using improved Hy-TIBs plementation that survive at high frequency after transfer to soil. The enhanced root development and greater mechanical To further evaluate the impacts of controlled gas compo- strength of roots grown in elevated C O2 are consistent with sition using improved Hy-TIB reactors, experiments were greater investment of carbon resources to roots when grown designed to evaluate the impact of elevated C O2 on yam in an elevated CO2. (Dioscorea rotunda) propagule development. Specifically, yam propagules were grown for 42 days in McA-H Hy- TIBs supplied with (1) air, (2) air + sugar, (3) CO2, and Discussion (4) C O2 + sugar. Providing a supplemental carbon source resulted in substantially healthier plants (Fig. 4A). Growth In vitro propagation bioreactors allow control of the envi- under elevated CO2 resulted in a significant increase in ronment, nutrient, and hormone conditions of plant tissues. propagule fresh weight and root surface area both with In the development of this type of process system, it is and without supplemental sugar (Fig. 4B, C). The greatest important to recognize that ‘design’ and ‘operation’ provide accumulation of biomass was observed for the combination distinctly different opportunities for improvement and cost 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) Fig. 4 Yam (Dioscorea rotundata) propagule development from axil- CO2 significantly increased both fresh weight and surface area of lary buds in the Arduino micro-controller automated McA-H Hy- propagule roots relative to those grown using typical ambient air and TIB (v.3) with altered sugar and CO2 supplementation. A D. rotun- sugar media. D To evaluate robustness of horizontal H Hy-TIB grown data propagules grown in elevated C O2 with or without sugar in the propagules, the survival of explants was monitored after transfer to growth media resulted in substantially healthier plants compared soil. Propagules grown using elevated CO2 without growth media to ambient air or ambient air with sugar-rich media (top). Elevated containing sugar survived at similar rates as equivalent plants grown CO2 also resulted in substantially healthier propagule root develop- using sugar-rich media. Scale bars in (A) represent 25 mm ment (bottom). B, C Upon harvest after 42 days of growth, elevated reduction. Most efforts focus on the design or fabrication system developed here makes major strides toward that goal of the bioreactor device. Operation relates to how a device by making most components highly reusable or minimal is used such as continuous media addition, or in this work replacement cost (e.g. plastic bag vessel) as we have devel- control of the gas composition and flow rates. The following oped previously for large scale suspension culture (Curtis discussion emphasizes these differences. 2001). In this phase of DBT, we focused on achieving low- cost, reliable fluid connections, combatting contamination Significance of bioreactor design improvements and achieving phototropic growth. The heterotrophic chicory root growth data of Fig. 2 is Design-build-test the publishable result of the extensive DBT effort. However, this does not reflect the important outcome of incremen- The development of technology is an iterative process tal improvements and experience from the DBT process. commonly referred to as a design-build-test (DBT) cycle. Using the manual cycled bioreactor alleviated simultane- The key to this process is to rapidly achieve prototypes ous troubleshooting the technical aspects of automation but with baseline functionality while overcoming design flaws resulted in a very demanding effort (cycling 4–8 reactors and refining design for cost, ease-of-use, etc. The Hy-TIB at 4 h intervals, 6 times per day, 7 days per week). With a 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) roughly 3-week turn-around on reactors, this effort encom- was also crucial for fluid distributor and achieved from out- passed about 8 months, which would have taken more than side the bag with wire rather than pipet tip. 2 years if implemented with slower growing tissue. Among the issues of leaks, plugged filters, and contamination, was Significance of bioreactor operational an inability to collect root biomass data due to unanticipated improvements entangled growth into fabric tissue support matrix. These ‘rapid failures’ are an underappreciated success of the DBT Comparison to alternative technology process. Table 2 outlines the chronology of Hy-TIB designs, which were denoted sequentially as versions (v.) 1, 2, and Given a typical product value of less than a dollar per plant, 3. Online Resource S1C has a further detail of each version achieving economic feasibility for axenic tissue culture including specific fabrication details involved. methods is a tremendous challenge. Since the first report of a Temporary Immersion System, the Auxophyton by Cor- nell’s Steward Lab in 1952, significant productivity gains, Achieving reliable low-cost fluid connections and in turn fiscal costs (per plant), have been realized; an analysis of sugarcane in vitro production was reported to Simplification of the TIB to a plastic bag culture vessel has reduce production costs by 46% in 2002 (Neumann et al. been enabled by using an inexpensive means of attaching 2020a, b). Nonetheless, in vitro production of pineapple in tubing directly to a polymer film. Making simple adhesive- a Periodic Immersion Bioreactor (PIB) reported in 2003 was free, autoclavable connections emphasized cost-cutting still cost-prohibitive with a 500-fold increase in production while preserving reliability. The solution for the tubing con- despite a mere 35% cost increase. Thus, to move beyond nection to the bag is depicted Fig. 5 using a ‘press-fit swage’, exceptions like ornamentals (e.g. orchids, sterile hybrids) a small SS reducing connector that facilitates reliable con- and high-value, long-lived specialty crops (e.g. coffee, choc- nections simply by deforming (i.e. pressing to fit) the PP bag olate), the contribution of the bioreactor to productions costs over it, with only snugly fit elastic tubing to hold it in place. must be near-zero as we have achieved with the plastic bag The press-fit swage was customized from a SS tube, which culture vessel. was turned on a lathe, cut in short pieces and polished on a Gaining an economically feasible basis for using gas buffing wheel (see Online Resource S3, S1B). These connec- mixtures other than air was the driving force behind the tions are surprisingly robust and did not experience failure or design of the LA-V Hy-TIB that decoupled gas and liquid leakage in any of the DBT or implementation test efforts. In flows (Florez et al. 2016). With the elimination of pneu- contrast, failure to sufficiently puncture the stretched plastic matic operation of media flow, the Hy-TIB provided an resulted in several operational failures. As depicted in Fig. 5, important scale-up advantage over other TIB systems like this puncture was reliably achieved by pipette tip insertion RITA™, Plantform™, and Temporary Root Zone Immersion through the fitting. The utility of silicone tubing is especially system. Estimated costs of 2.5% CO2 supplementation at important, where its elasticity and non-thermosetting nature scale (depending on scale of operation, i.e. pure or industrial could achieve these connections with only appropriate sizing C O2) were between $0.01–$0.10 US per week per bioreac- (i.e. no additional clamps). The connection of the SS insert’s tor (Shaw 2012; Florez et al. 2016), made possible by gas fluid distributor was similarly stretched by deforming the usage rates that are orders of magnitude lower than most bag and connected by silicone tubing. Puncture of the bag that operate based on pneumatic movement of media. We Fig. 5 Achieving simple, reliable adhesive-free silicone tubing con- pipette tip. B The reusable press-fit swage tubing connectors are used nections to the plastic film bioreactor. A The press-fit swage con- to connect the bag culture vessel to the media reservoir and gas inlet/ nections are formed by first deforming the culture vessel plastic film, outlet lines pressing the tubing over the deformation, and piercing the bag using a 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) recognize opportunities for additional minor cost reduction Future improvements/challenges based on materials. 3D printing and the notable success of replacing microfluidic devices with coated, precision-lathed The target of Dioscorea spp. represents a crop that has wood (Andar et al. 2019) may provide both lower cost and favorable characteristics of value to smallholder farmers in carbon impact than SS used in insert, manifold, and its Luer- Africa, while solving issues of propagation of disease and NPT connectors. overcoming issues of reliability of year-to-year yam ‘seed’ propagation. We suggest that scaleup in natural lighting is the next major technical hurdle to advance the Hy-TIB Gas phase manipulation platform’s mission to achieve economic feasibility though we also recognize the potential role of microorganisms Having now worked with half a dozen plant species in TIB to enhance field performance, which would require rein- systems over the last decade, decoupling of the gas phase is troduction after proliferation in an aseptic tissue culture not only cost-saving, but precision control over gas phase environment. composition enables other advantages of environmental control in manipulating plant growth and development that Natural light are consistent with observations of others. Varying con- centrations of elevated oxygen were shown in coffee cell The challenge of increasing light levels to improve photo- suspensions to promote either multiplication or somatic trophic performance is managing the associated heat load. embryogenesis (SE). Notably, Nie et al. (2013) conducted a Those unfamiliar with plant tissue culture are often surprised metanalysis of 110 studies of CO2 supplementation, show- to learn that they are heterotrophic, and C O2 accumulates in casing general benefits of root development and carbon plant tissue culture (PTC) enclosed vessels (McKelvey et al. sequestration (Nie et al. 2013). Optimization of conditions, 1993). As an example, the root cultures used in this work are however, is often species-dependent and/or developmental oxygen transport-limited—typical of heterotrophic microbial stage-dependent with variability also enhanced by complex culture. Artificial lighting such as fluorescent and LED pro- interactions of stress, hormones, growth conditions, and vides considerable light for plant physiological functions at nutrient availability to name a few (Zhao and Guo 2011; minimal heat load. In contrast, natural light doubles the inci- Niu et al. 2016; Lahive et al. 2018). The TIB system lends dent energy based on the solar spectrum alone. For exam- itself not only to environmental control of the gas phase ple PAR levels at 100 µE m −2  s−1 corresponds to roughly but also monitoring to elucidate these complex interactions. 22 W  m−2 and natural sunlight can be over 1000 W  m−2. Ethylene, jasmonate and nitric oxide (NO) are among the The issue this represents for a TIB system is not only the volatile compounds known to be indicative of plant stress ‘greenhouse effect’ heating, but a proportional increase in (Yoo et al. 2009; Zhao and Guo 2011; Kazan 2015; Elhiti the water load of the exhaust gas which is an ongoing chal- et al. 2018) and the TIB system described here provides a lenge associated with blockage of the exhaust filters (see means of systematic study of gas-phase plant signaling. Online Resource S1E). Rapid advances in plastic and glass film coatings to exclude solar heat load will invariably be a component of the next generation plant propagation systems, Technology adoption/implementation which spans the transition from heterotrophic plant tissue culture to axenic phototrophic propagation and ultimately Combining low capital cost with considerations of minimal field introduction. operational costs (CO2 and light), the remaining hurdle for technological adoption is ease-of-use and reliability. Toward Microbial reintroduction that end, we have initiated efforts with a multidisciplinary “Learning Factory” design team to create both a platform The beneficial association of microorganisms with plants for bioreactor assembly and IoT-based system for remote is increasingly recognized (Santos et al. 2019) which needs monitoring (https://b it.l y/ 3siVY8 J) that would be similarly to be taken advantage of in the transition from the plant tis- low-cost and open-source. More critically, since the reported sue culture environment to the field. As noted above, photo- scale of propagule production is small (i.e. hundreds of prop- trophic growth provides an excellent platform to introduce agules), there is a clear need to scale up the system. The use microorganisms with greatly reduced issues of microbial of phototrophic growth conditions to minimize contamina- overgrowth. In our work with cultures of D. cayenensis, tion risk provides a clear path forward for a larger format we observed a slow-growing bacterial ‘contaminant’ which system and an implementation that can provide a substantial would tend to grow more aggressively during tissue culture supply chain for disease-free, superior performance plant stress. These putative endophytes were characterized by propagules. 16S ribosomal RNA gene sequencing (sequence in Online 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) Resource S1J) and were identified as methylotrophs (Shouse Author contributions MT, plant tissue culture (PTC: cacao, yam), bio- 2018)—Bacillus aryabatthai (Bhattacharyya et al. 2017), reactor experimentation, manuscript preparation; AH, PTC (chicory, Bacillus ginsengihumi (reclassified as Paenibacillus species) cacao, yam), bioreactor experimentation, USDA permitting, cost anal-ysis, manuscript preparation; ES, contaminant characterization; KS, (Ash et al. 1993; Lee et al. 2007), and Hyphomicrobium machining, PTC (chicory), bioreactor experimentation, contamination facile (Fesefeldt et al. 1997). Methylotrophs are common and DBT troubleshooting; JB, PTC (yam), bioreactor experimenta- endophytes, as plants produce methanol as a biochemical tion; TL, PTC (cacao), bioreactor experimentation; RM, manuscript byproduct of pectin formation, making it an ideal carbon preparation; LT, manuscript preparation; AJO, statistical analysis, manuscript preparation; WRC, project conceptualization, machining, source for endosymbiotic microorganisms. These putative instrumentation, PTC, bioreactor experimentation, statistical analysis, endophytes are also associated with nitrogen-fixing capabil- manuscript preparation. ity which would have obvious benefits to be added back to the plants before field planting. Funding This research was sponsored by NSF BREAD ABRDC grant #1543929 in conjunction with the Bill & Melinda Gates Foundation with partial support from the Defense Advanced Research Projects Agency (DARPA) under agreement HR0011-17-2-0055. The views, Role in plant improvement opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the With the advent of NextGen Sequencing and CRISPR-Cas9 Department of Defense, the National Science Foundation, or the U.S. Government. technologies, the barriers to development of transgenics have been lowered significantly. Moving forward, the more Data availability datacommons@PSU.edu is the repository for raw prominent barrier will be the recalcitrance of superior cul- CAD files: https://w ww.d ataco mmons.p su.e du/d ownlo ad/e ngine ering/ tivars to regeneration (Altpeter et al. 2016; Campos et al. PCTOC/ 2017), especially in (non-model) plants critical to food and economic security that are considered highly vulnerable Code availability Relevant code can be found in Online Resource files. like Musa spp. (Ordonez et al. 2015) and T. cacao (Evans 2016). Overcoming regeneration recalcitrance includes tak- Declarations ing advantage of genetic control via manipulation of embry- Conflicts of interest The authors declare no conflict of interest. ogenic or morphogenic transcription factors (Florez et al. 2015; Lai 2016; Shires et al. 2017) as a means of expand- Ethical approval Not applicable. ing those plants amenable to a tissue culture based supply chain for superior plants. The transient delivery of DNA Consent to participate Not applicable. into in vitro grown plant tissues can be implemented in TIB Consent for publication Not applicable. bioreactors—not only as a tool for next generation research of plant development—but also scaled in production systems Open Access This article is licensed under a Creative Commons Attri- in support of agronomic production. bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long Supplementary Information The online version contains supplemen- as you give appropriate credit to the original author(s) and the source, tary material available at https://d oi.o rg/1 0.1 007/s 11240-0 21-0 2210-3. provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are Acknowledgements We acknowledge Dr. Morufat Balogun for provi- included in the article's Creative Commons licence, unless indicated sion of yam cultures and project collaboration. We also acknowledge otherwise in a credit line to the material. If material is not included in assistance from Sergio Florez and Morgan Shires for LA-V Hy-TIB the article's Creative Commons licence and your intended use is not operation; Noah Willis and Haonan Xu for machining; John Driscoll, permitted by statutory regulation or exceeds the permitted use, you will Anna Fillipowski, David Krum, and Bill Muzika for CAD design and need to obtain permission directly from the copyright holder. To view a drawings; Moez Essajee for IoT and gas flow troubleshooting; Samwel copy of this licence, visit http://c reati vecom mons.o rg/l icens es/b y/4.0 /. Kariuki for Dioscorea advice, Andrew Sell for coding, setup and opera- tion; Aisa Sam, Brielle Hohne, Mariela Torres and Nathan Vorodi for tissue maintenance; Nadia Waterton for illustrations and digital media editing; Natalie Thompson for proofreading; Ben Geveke, Alyssa Grube, Lucas Nugent, Jake Scoccimerra Hamdan Almarzooqi and References Mariela Torres for preservation of bioreactor materials during labora- tory moves; Mark Signs and Kim Martin for support and access to Penn Altpeter F, Springer NM, Bartley LE et al (2016) Advancing crop trans- State Shared Fermentation Facility; and Penn State Learning Factory formation in the era of genome editing. Plant Cell 28:1510–1520. for lathing and milling access and training, sourcing materials. This https:// doi.o rg/ 10.1 105/t pc.1 6.0 0196 work was supported, in whole or in part, by the Bill & Melinda Gates Andar A, Hasan MS, Srinivasan V et al (2019) Wood microfluidics. Foundation [NSF BREAD ABRDC grant #1543929]. 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