Expanding the application of an eGFP as a visual reporter for optimizing transformation and assessing pollen-mediated gene flow in alfalfa (Medicago sativa L.)

Abstract Efforts are still required for establishing an efficient and standard transformation procedure in alfalfa, an important worldwide forage crop, due to the unstable repeatability of the published genetic transformation. The objectives of this study were to optimize and establish an effective genetic transformation system in alfalfa (Medicago sativa L.) using eGFP as a visual reporter, and test the feasibility of established procedure in other Medicago species. Comparisons of percent callus induction using three different explants of the four alfalfa cultivars showed that hypocotyl explant had greater callus induction (70-98.6% across four cultivars) than that of cotyledon (17.1%-73.2%) and cotyledonary node (39.7%-70.2%). Explants of ‘Qingshui’ showed the greater mean value of callus induction (80.6%) compared to other cultivars (mean: 46.9-55.5% across three cultivars). Using hypocotyl of ‘Qingshui’, the optimal callus induction and bud differentiation mediums were determined. The established procedure showed the transformation rate of eGFP to ‘Qingshui’ was about 72% with the time taken 85-120 days. Fluorescence and PCR detections evidenced the integration of the eGFP in regenerated plant genome. The procedure also yielded the transformation rate > 64% in M. falcata and M. polymorpha. The eGFP-tagged pollens produced by regenerated alfalfa could be an enabling tool for tracking pollen movement (by wind or insect) to study the pollen distribution patterns and pollinator behavior, thus assess potential risks of transgene flow in genetically modified alfalfa. Additionally, the established procedure would serve a useful approach for introducing agronomically important traits into alfalfa, and thus contribute to breeding program of Medicago species.