Nanoparticle-Mediated Genetic Transformation in a Selaginella Species.

The genus Selaginella holds a key phylogenetic position as a sister species to vascular plants, encompassing desiccation-tolerant members. Some Selaginella species thrive in extremely arid conditions, enduring significant water loss and recovering upon rehydration. Consequently, Selaginella has emerged as a model system for studying desiccation tolerance in plant science. However, the absence of an efficient genetic transformation system has limited the utility of Selaginella species as a model. To address this constraint, we developed a nanoparticle-mediated transformation tool utilizing arginine-functionalized nanohydroxyapatites. This biocompatible system enabled the transient expression of the GFP, GUS, and eYGFPuv reporter genes in Selaginella moellendorffii. Establishing a stable genetic transformation technique for S. moellendorffii holds promise for application to other Selaginella species. This tool could be instrumental in identifying genetic resources for crop improvement and understanding genome-level regulatory mechanisms governing desiccation tolerance in Selaginella species. Furthermore, this tool might aid in identifying key regulatory genes associated with desiccation tolerance, offering potential applications in enhancing drought-sensitive crops and ensuring sustainable food production.

Overexpression of the Selaginella lepidophylla bHLH transcription factor enhances water-use efficiency, growth, and development in Arabidopsis.

Abiotic stresses have the greatest impact on the growth and productivity of crops, especially under current and future extreme weather events due to climate change. Thus, it is vital to explore novel strategies to improve crop plant abiotic stress tolerance to feed an ever-growing world population. Selaginella lepidophylla is a desiccation-tolerant spike moss with specialized adaptations that allow it to tolerate water loss down to 4% relative water content. A candidate basic helix-loop-helix (bHLH) transcription factor was highly expressed at 4% relative water content in S. lepidophylla (SlbHLH). This SlbHLH gene was codon-optimized (SlbHLHopt) and overexpressed in Arabidopsis for functional characterization. Overexpression of the SlbHLHopt gene not only significantly increased plant growth, development, and integrated water-use efficiency, but also significantly increased seed germination and green cotyledon emergence rates under water-deficit stress and salt stress conditions. Under a 150 mM NaCl salt stress condition, SlbHLHopt-overexpressing lines increased primary root length, the number of lateral roots, and fresh and dry biomass at the seedling stage compared to control lines. Interestingly, SlbHLHopt-overexpressing lines also have significantly higher flavonoid content. Altogether, these results suggest that SlbHLH functions as an important regulator of plant growth, development, abiotic stress tolerance, and water-use efficiency.

A nano-biomimetic transformation system enables in planta expression of a reporter gene in mature plants and seeds.

Plant genetic engineering will be essential to decipher the genomic basis of complex traits, optimize crop genomics, and enable plant-based production of recombinant proteins. However, established plant transformation approaches for bioengineering are fraught with limitations. Although nanoparticle-mediated methods show great promise for advancing plant biotechnology, many engineered nanomaterials can have cytotoxic and ecological effects. Here, we demonstrate the efficient uptake of a nano-biomimetic carrier of plasmid DNA and transient expression of a reporter gene in leaves of Arabidopsis, common ice plant and tobacco, as well as in the developing seed tissues of Arabidopsis, field mustard, barley, and wheat. The nano-biomimetic transformation system described here has all the advantages of other nanoparticle-mediated approaches for passive delivery of genetic cargo into a variety of plant species and is also nontoxic to cells and to the environment for diverse biotechnological applications in plant biology and crop science.