Optimization of Selection Agent Concentrations and Expanding G418 Utility for Gentamicin Resistance in Marchantia...

Genetic transformation of plants is pivotal for advancing biotechnology, with success depending largely on effective selection methods. Marchantia polymorpha has emerged as a model plant due to its evolutionary importance, ease of manipulation, and simple genetic structure. However, inconsistent antibiotic performance and limited studies on optimal selection agent concentrations have posed challenges. This study aimed to optimize selection agent use in M. polymorpha genetic transformation. We assessed the effects of five antibiotics (hygromycin, kanamycin, G418, neomycin, and gentamicin) and the herbicide chlorsulfuron on M. polymorpha gemmae growth. For each agent, we identified the minimum lethal concentration for nontransgenic plants and safe thresholds for transgenics, balancing false-positive prevention with reduced toxicity. Hygromycin, G418, and chlorsulfuron exhibited broad selective concentration ranges, enabling efficient transformant selection. Notably, we observed cross-activity of the gentamicin resistance enzyme with G418, a phenomenon also seen in tobacco. This study effectively determined optimal concentrations of selective agents for M. polymorpha gemmae transformation. Additionally, the unexpected cross-activity underscores the need for careful marker selection and highlights potential for strategic antibiotic use. Our findings enhance transformation protocols for M. polymorpha and possibly other plant species.