Abstract:

There are substantial challenges in the genetic improvement of alfalfa （Medicago sativa）， a globally important forage crop， because of the limitations of conventional transformation methods. These methods are time-consuming， genotype-dependent， and reliant on labor-intensive tissue culture processes. To address these issues， we developed a rapid， tissue culture-free transformation system for alfalfa. This system was developed and optimized using the alfalfa cultivar Longmu 806. The system employs Agrobacterium rhizogenes-mediated infection combined with a simple stem-pricking infiltration method. This innovative approach eliminates the need for callus induction and somatic embryogenesis， enabling the generation of transgenic chimeric alfalfa within just 14 days-a dramatic reduction compared with the 3-6 months typically required with traditional protocols. We used this system in two applications： 1） Rapid functional analysis of the MsRCI2D gene， which conferred enhanced salt tolerance in transgenic chimeric plants， as evidenced by increased antioxidant enzyme activities， decreased reactive oxygen species accumulation， and improved ion homeostasis under 200 mmol·L^-1 NaCl stress； and 2） Establishment of an efficient guide RNA screening platform using the RUBY reporter system， which achieved a 23.07% editing efficiency in transformed roots， providing a robust and visual tool for optimizing CRISPR gRNAs. This breakthrough transformation strategy addresses major bottlenecks in alfalfa genetic engineering-namely genotype dependence and prolonged timelines—and offers a powerful platform for high-throughput gene function studies， abiotic stress tolerance improvement， and precision genome editing. By integrating simplicity， speed， and high efficiency， our system holds transformative potential for both fundamental research and molecular breeding in alfalfa and other recalcitrant forage crops.

Key words: Medicago sativa, tissue culture-free transformation, transgenic chimeras, salt tolerance, gRNA screening