Progress 07/01/23 to 02/29/24

Outputs
Target Audience:The findings from this work aimed to advance practical knowledge in plant cell culture for applications in biomanufacturing as well as traditional applications such as propagation, breeding, seed production, and conservation. Biomanufacturing is an increasingly important prospect for the production of hard-to-access or hard-to-scale plant-based goods. Cell and tissue culture in plants is vital in these emerging bioproduction applications, but also in established fields of propagation, breeding, seed production, and conservation. However, in order for plant cell culture to be industrially relevant, innovative approaches are needed to quickly expand its utility across the 99% of plant species that remain inaccessible by this cultivation format.The developed database and methodologies are intended to be leveraged to rapidly advance plant cell culture processes in the 99% of plant species for which little to no culture knowledge exists today. Changes/Problems:This SBIR-funded project experienced only minor adjustments to the proposed work plan. Challenges encountered during project execution were surmountable, but did result in additional resource allocation to accomplish the proposed objectives. Seasonal impacts on callus initiation studies Challenge: Initial experimental designs intended to examine trends in culture requirements for callus initiation across a commercially relevant taxa. However, due to grant timing, studies were set to launch in the late fall and early winter when the proposed species under examination were heading into dormancy. Resolution: Studies were adjusted to examine trends in culture requirements for callus maintenance rather than callus initiation as maintenance experiments were not influenced by season of experiment launch. With the proposed adjustments, results more reliably indicated culture response to prepared media conditions by cutting out external factors like dormancy and weather. Limits in the availability of quality data for the database build Challenge: In phase I of the proposed work, a plant cell culture database was developed and loaded with data sourced from peer-reviewed literature. Variability in reporting styles and types of data introduces unwanted noise to the datasets. Resolution: A sizable quantity of data was collected, often with several data points per species, in order to partially address noise in the data. In subsequent database expansion, the organization will focus on massivein-house data generation to improve data accuracy and standardization. The resulting collections of robust and standardized data will furtherimprove the resolution of database analyses. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement: Global forest resources are under strain in the face of changing climate, increasing pace and intensity of natural disasters, and a growing demand for tree-sourced goods. Innovations in tree-based supply chains are needed to ensure continued access to critical products while enabling the retention of forests. Biomanufacturing, powered by plant cells, can overcome this production paradox by supplying tree-free equivalents of tree-sourced goods. Despite much promise, the adoption of cell-based biomanufacturing approaches has been impeded by long, costly process development timelines. This work introduces a database-driven platform designed to accelerate the design of biomanufacturing processes in new plant species, thereby improving utility of plant cell culture across species and applications. A first-of-a-kind plant cell culture database was developed. Data on 150+ plant species, gathered from peer-reviewed reports, were standardized, processed, and integrated into the database framework. Statistically significant trends and predictors of culture requirements were identified from the compiled data. Analytical trends were mirrored in preliminary empirical tests conducted on cell cultures of four tree species. Goal: develop a first ever database compiling species characteristics, culture information, and inter-species relationships. Objective 1a/b: Data on over 150 species spanning angiosperms and gymnosperms were compiled and standardized. A relational database was created and collected data incorporated. A relational backbone was developed inspired by phylogenetic data to allow for trend analysis across the dataset. The database represents a first-of-a-kind resource for consolidating knowledge on plant cell culture in a structure that enables predictive modeling to improve successful process development for unstudied species. Goal: characterize culture trends across species to show that culture behavior and requirements exhibit predictable patterns Objective 2a/b: A series of analyses including clustering and regression models were deployed to evaluate trends across the collected data. Statistically significant trends were identified from compiled data, and phylogenetic relatedness was an informative predictor of culture requirements. Initial analysis focused on macronutrient components found in the basal salt mixtures. Using a normalized data as the "component space", the distance between recipes was calculated using a city block metric. Mean distance was evaluated from a base recipe in question to two sets 10 recipes (randomly selected versus nearest neighbors). Comparing means, a two sample t-test with unequal variance shows a 95% confidence interval of [-0.4, -0.15] (notably excluding 0) with a p-value of <0.0001. Objective 3: A final report elaborating on database analysis was prepared and submitted to the appropriate recipient overseeing the performance of this SBIR grant. Goal: validate trends in culture behavior empirically and to demonstrate potential for prediction models to improve culture outcomes in poorly studied species. To complement the database build and subsequent analysis, in-house experiments were conducted to further evaluate whether phylogenetic positioning could be an informative predictor of successful callus culture requirements. ** Experimental objectives were modified slightly from those initially proposed. Experiments were redesigned to interrogate conditions for callus maintenance rather than initiation. This adjustment was made to mitigate the effect of uncontrollable conditions (e.g., collection season) on experimental outcomes, as these factors could have impacted the quality and usefulness of resulting data. **Objective 4 [Updated]: Successfully develop cell cultures for four selected species. One species will represent a "base" species and the remaining species will be selected on the basis of increasing evolutionary distance from the base species. Four tree species from the class Magnoliopsida were introduced to and stabilized in callus cell culture using tried and tested media specific to each species. Populus trichocarpa was selected as a "base" species or node for the subsequent comparisons of results. A second species from the same genus, Populus balsamifera, and two more distantly related species (from the same class, but different orders), were also selected for testing. Callus was successfully established and stabilized for all four species. **Objective 5 [Updated]: Design a media panel to evaluate culture growth for two closely related species and two increasingly distant relatives. Media formulations were designed to span a range of critical conditions (Table 5). Selected media recipes were based on one of three basal salt mixtures: Gamborg's B5 medium (Gamborg, 1968), Murashige and Skoog medium (Murashige, 1962), and Lloyd and McCown woody plant medium (Lloyd, 1981). Types and levels of auxin, cytokinin, and other organics were varied across the panel. All formulations were formulated with 30g/L of sucrose and 3 g/L of gellan gum (Gelrite, Sigma-Aldrich). **Objective 6 [Updated]: Apply proposed culture protocols to the selected species and monitor culture response by tracking growth rate and collecting phenotypic data Calli of the four selected species were cultivated on the designed treatments for 60 days. Biomass accumulation and phenotypic changes were monitored throughout. Biomass measurements were taken at twelve day increments as calli were subcultured to fresh plates. Initially, there were no significant differences in biomass accumulation across the tested species and media formulations. However, at 24 days of growth, significant differences in biomass accumulation became apparent. Both P. trichocarpa and P. balsamifera (same genus) showed similar responses to media formulations. Both Populus species had highest growth on M4 and M5 media (MS based). In both Populus species, callus growth on formulations M1, M2 and M6 media was comparable and significantly slower than growth on MS-based media. Despite similarities in relative performance of Populus species across the media panel, absolute growth did vary between P. trichocarpa and P. balsamifera. In more distant species, performance across the panel varied, with equal performance across MS and WPM-based media. Overall, the MS-based media seemed to support enhanced growth across the tested orders. Phenotypic data was collected alongside biomass measurements at twelve day increments. Media formulation was shown to impact both growth and phenotype of the callus over time. **Objective 7 [Updated]: Analyze culture response (e.g., growth rates and phenotypic data) in relation to species' evolutionary proximity. Database-driven analyses indicate that phylogenetic positioning can be an informative predictor of callus culture requirements. Results indicate that functional media formulations for two species are more likely to be similar when the respective species are closely related. Likewise,lab results indicated that culture response to a panel of media formulations was similar for the two most closely related species and less similar for more distant species. The majority of database recipes available for Populus and the other examined genera were formulated on MS salts and organics, suggesting that MS had been the most consistently tested and/or highest performing basis for media in these taxa. In follow-up experiments, tested MS-based formulations tended to yield consistently high biomass accumulation over a 60-day culture period.

Publications