Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies

Cheatgrass ( Bromus tectorum L.) is one of the most invasive annual grasses in the western United States. It has spread extensively across rangelands and cropping systems and increasingly expresses herbicide resistance. Despite its ecological and agricultural significance as a major invasive weed, functional genetic studies in cheatgrass have been hindered by the absence of a reproducible transformation system. Here, we report the first successful protocol for Agrobacterium tumefaciens –mediated genetic transformation of cheatgrass. Mature and immature embryos were evaluated as explant sources to optimize callus induction and regeneration. Callus formation from immature embryos began earlier than from mature embryos, which required a longer culture period. Hygromycin-resistant calli expressing green fluorescent protein (GFP) were obtained from both explant types, confirming successful genetic transformation. Following Agrobacterium transformation, calli derived from immature embryos regenerated green, viable shoots, whereas those from mature embryos frequently produced bleached, chlorophyll-deficient tissues. PCR amplification of GFP and hygromycin phosphotransferase (HPT) confirmed stable integration of transgenes into the cheatgrass genome. Our work presents the first reproducible immature embryo–based transformation system for cheatgrass, enabling molecular genetics and functional genomics studies in weedy grasses.