Plant cell cultures offer a promising platform for producing valuable biomolecules, yet their use in bioreactors remains under-optimized. Compared to animal or microbial cells, plant cells grow more slowly, limiting productivity. A common bioprocess strategy to improve yields involves controlling dissolved oxygen (DO) levels. However, little research has focused on combining agitation and aeration to regulate oxygen in plant cell cultures within bioreactors. The aim of this study was to evaluate the impact of an oxygen cascade mixing agitation and aeration on plant cell growth in stirred-tank systems. By maintaining 30% DO through this approach, the specific growth rate (µ) increased from 0.082 d⁻¹ to 0.144 d⁻¹ on average in Nicotiana benthamiana cultures at the 2 L scale, decreasing batch lengths from 21 to 10 days. These conditions were successfully replicated in a 7 L stainless-steel pilot bioreactor using previous values of geometry, k_La and N_RE as scale-up criteria, obtaining a µ of 0.161 d⁻¹. These results demonstrate that plant cell cultures’ efficiency can be enhanced by using standard bioprocess parameters. While this work confirms the feasibility of cascade oxygen control for improvements in growth, further studies are needed to evaluate its specific impact on biomolecule production across different systems.

A la hora de realizar cultivos in vitro vegetales, hay que tener en cuenta diversos factores que pueden afectar a su crecimiento, como pueden ser: medio de cultivo, temperatura, luz, etc. Estos factores afectarán de forma diferente a cada tipo de cultivo, especialmente a diferentes especies vegetales. En este ensayo, se ha caracterizado cómo afectan la luz, el tamaño del inóculo y la disgregación del inóculo al crecimiento de cultivos en matraces de Nicotiana benthamiana. El factor que más influye en el crecimiento de este tipo de cultivo es el tamaño del inóculo: se requiere un mínimo porcentaje de inóculo para que el crecimiento del cultivo sea significante.

Summary: Plant cell cultures underpin regeneration, transformation and emerging applications in biotechnology and plant cell agriculture, yet the genetic determinants of proliferative performance remain poorly characterized. Here, we combined large-scale forward genetics with automated image-based phenotyping to quantify 27,648 calli representing 2,880 Arabidopsis thaliana T-DNA insertion lines. Of these, 2,456 lines produced calli smaller than the WT population mean, whereas only 190 produced larger calli, revealing a strongly asymmetric phenotypic distribution. Enhanced callus growth was not consistently associated with increased vegetative growth, indicating that these traits can be genetically uncoupled. Validation of selected high-growth mutants in suspension culture identified lines with enhanced biomass accumulation, while serial propagation revealed independent variation in sustained proliferative capacity. Thus, callus growth, whole-plant growth, short-term suspension biomass accumulation and sustained proliferation represent distinct, not necessarily coupled components of proliferative performance. These findings establish a scalable framework for identifying genetic determinants relevant to the development of improved plant cell production lines. Highlights: Established a high-throughput platform for quantitative phenotyping of plant callus growth. Screened 2,880 Arabidopsis T-DNA lines, revealing extensive genetic variation in callus proliferative capacity. Identified mutants with enhanced biomass accumulation or sustained proliferative capacity in suspension culture. Established a framework for the genetic improvement of plant cell production platforms. Graphical Overwiew of the study. Image created with Biorender.

A pesar de las ventajas que presentan los cultivos de células vegetales para expresión y producción de proteínas y otros metabolitos de interés en biorreactores frente a los cultivos microbianos, tienen algunas limitaciones. Dos de las más importantes son el alto porcentaje de inóculo que se necesita para que los cultivos sean viables y el estrés por cizalla que sufren por la agitación mecánica. El estrés por dilución, o bajo porcentaje de inóculo, y el estrés por cizalla dan lugar al proceso de muerte celular programada (MCP). En estudios previos se ha visto que un medio de cultivo condicionado puede proteger a las células de la MCP. En este trabajo se ha estudiado este efecto frente al estrés por cizalla y