Powdery mildew, caused by the fungus Erysiphe pulchra, is one of the most serious diseases affecting the popular ornamental tree flowering dogwood (Cornus florida). Employing gene editing techniques such as CRISPR to introduce powdery mildew resistance by inactivating the Mildew Locus O (MLO) gene requires an efficient genetic transformation system. This novel research will fill a critical gap in our knowledge of flowering dogwood biotechnology. Previous research efforts have genetically transformed embryogenic dogwood cultures, now we are aiming to produce transgenic plantlets. Recent research focused on using the RITA® temporary immersion bioreactor system for testing germination of somatic embryos and conversion to plants. The primary test was the impact of the plant growth regulator (PGR) gibberellic acid (GA3). For this experiment, we analyzed the impact of different environmental light exposures on dogwood embryo stress levels by observing anthocyanin production. The starting material was unwashed callus transformed with the GUS reporter gene and subjected to one of three RITA® treatments to examine the influence of varying light levels. Treatments included full darkness, continuous low light, and a combination of both light conditions for different durations of time. Few somatic embryos germinated from the treatment with 60 days of full darkness, but the anthocyanin stress was absent. We observed the same result for constant low light exposure except for exponential growth of the transgenic callus. The treatment in which the somatic embryos were in full darkness for 30 days and switched to low light for another 30 days showed a higher germination rate, but there were increased signs of anthocyanin stress. Implementation of this research will assist in the optimization of the production of plantlets from dogwood cultures transformed with a CRISPR-Cas9 construct that can inactivate the MLO gene to obtain powdery mildew resistance.
