The ever-increasing food requirement with globally growing population demands advanced agricultural practices to improve grain yield, to gain crop resilience under unpredictable extreme weather, and to reduce production loss caused by insects and pathogens. To fulfill such requests, genome engineering technology has been applied to various plant species. To date, several generations of genome engineering methods have been developed. Among these methods, the new mainstream technology is clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases. One of the most important processes in genome engineering is to deliver gene cassettes into plant cells. Conventionally used systems have several shortcomings, such as being labor- and time-consuming procedures, potential tissue damage, and low transformation efficiency. Taking advantage of nanotechnology, the nanoparticle-mediated gene delivery method presents technical superiority over conventional approaches due to its high efficiency and adaptability in different plant species. In this review, we summarize the evolution of plant biomolecular delivery methods and discussed their characteristics as well as limitations. We focused on the cutting-edge nanotechnology-based delivery system, and reviewed different types of nanoparticles, preparation of nanomaterials, mechanism of nanoparticle transport, and advanced application in plant genome engineering. On the basis of established methods, we concluded that the combination of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies can accelerate crop improvement efficiently in the future.

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

Citrus reticulata ‘Chachi’ (CRC) has long been recognized for its nutritional benefits, health-promoting properties, and pharmacological potential. Despite its importance, the bioactive components of CRC and their biosynthetic pathways have remained largely unexplored. In this study, we introduce a gap-free genome assembly for CRC, which has a size of 312.97 Mb and a contig N50 size of 32.18 Mb. We identified key structural genes, transcription factors, and metabolites crucial to flavonoid biosynthesis through genomic, transcriptomic, and metabolomic analyses. Our analyses reveal that 409 flavonoid metabolites, accounting for 83.30% of the total identified, are highly concentrated in the early stage of fruit development. This concentration decreases as the fruit develops, with a notable decline in compounds such as hesperetin, naringin, and most polymethoxyflavones observed in later fruit development stages. Additionally, we have examined the expression of 21 structural genes within the flavonoid biosynthetic pathway, and found a significant reduction in the expression levels of key genes including 4CL, CHS, CHI, FLS, F3H, and 4′OMT during fruit development, aligning with the trend of flavonoid metabolite accumulation. In conclusion, this study offers deep insights into the genomic evolution, biosynthesis processes, and the nutritional and medicinal properties of CRC, which lay a solid foundation for further gene function studies and germplasm improvement in citrus.

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