Optimization and establishment of Agrobacterium-mediated transformation in alfalfa (Medicago sativa L.) using eGFP as a visual reporter

• Published in: Plant cell, tissue and organ culture tissue and organ culture, 2024, Vol.156 (1), p.7, Article 7
• Main Authors: Gao, Yang, Zhang, Youxin, Wu, Hui-Zhen, Min, Xueyang, Zhang, Bing, Kim, Do-Soon, Yan, Xuebing, Zhang, Chuan-Jie
• Format: Article
• Language: English
• Subjects: Agrobacterium; Alfalfa; Biomedical and Life Sciences; Breeding; Callus; Cultivars; Distribution patterns; Explants; Genetic modification; Genetic transformation; Insects; Life Sciences; Medicago; Medicago sativa; Optimization; Original Article; Plant breeding; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Plants (botany); Pollen; Pollinators; Risk taking; Transformations; Transgenes; Transgenic plants
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-023-02659-4

Efforts are still required for establishing an efficient and standard transformation procedure in alfalfa, a prominent forge crop worldwide, due to 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 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. Established procedure showed the transformation efficiency of eGFP to ‘Qingshui’ was about 72% with the time taken 85–120 days. Fluorescence and PCR detections evidenced the presence of eGFP in regenerated transgenic plants. The procedure also yielded the transformation efficiency > 64% in M. falcata and M. polymorpha . The eGFP-tagged pollens produced by transgenic alfalfa plants 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, established procedure could serve a useful approach for introducing agronomically important traits into alfalfa, and thus contribute to breeding program of Medicago species. Key message We optimized and established an efficient Agrobacterium -mediated transformation using eGFP as a visual reporter in alfalfa and successfully applied this procedure to other Medicago species.