Large-scale forward genetics reveals genetically separable components of plant cell proliferative capacity

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.