A Noninvasive Gas Exchange Method to Test and Model Photosynthetic Proficiency and Growth Rates of In Vitro Plant Cultures: Preliminary Implication for Cannabis sativa L.

Supplemental sugar additives for plant tissue culture cause mixotrophic growth, complicating carbohydrate metabolism and photosynthetic relationships. A unique platform to test and model the photosynthetic proficiency and biomass accumulation of micropropagated plantlets was introduced and applied to Cannabis sativa L. (cannabis), an emerging crop with high economic interest. Conventional in vitro systems can hinder the photoautotrophic ability of plantlets due to low light intensity, low vapor pressure deficit, and limited CO2 availability. Though exogenous sucrose is routinely added to improve in vitro growth despite reduced photosynthetic capacity, reliance on sugar as a carbon source can also trigger negative responses that are species-dependent. By increasing photosynthetic activity in vitro, these negative consequences can likely be mitigated, facilitating the production of superior specimens with enhanced survivability. The presented methods use an open-flow/force-ventilated gas exchange system and infrared gas analysis to measure the impact of [CO2], light, and additional factors on in vitro photosynthesis. This system can be used to answer previously overlooked questions regarding the nature of in vitro plant physiology to enhance plant tissue culture and the overall understanding of in vitro processes, facilitating new research methods and idealized protocols for commercial tissue culture.

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study.

Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.