Oligohistidine-functionalized single-walled carbon nanotube guided RNA delivery to improve plant callus shoot regeneration

Plant tissue culture is fundamental to genetic engineering and biotechnology, yet its efficiency remains limited by intrinsic genetic barriers. Here, we present a novel approach to enhance shoot regeneration in plant callus using single-walled carbon nanotubes (SWNTs) functionalized with oligohistidine peptide (His6-SWNT) for targeted RNA delivery. The His6-SWNT carrier exhibits enhanced pH sensitivity and controlled release of nucleic acids in the cytosolic environment, enabling effective delivery of short tandem target mimicry RNA (STTM396) to suppress miR396, a known inhibitor of shoot regeneration. We demonstrate that the His6-SWNT complex (STTM396-SWNT) maintains colloidal stability while achieving efficient RNA loading and release characteristics. The nanocarrier showed minimal impact on plant cellular processes, as evidenced by transcriptome analysis. When applied to Arabidopsis and tomato callus, STTM396-SWNT treatment resulted in 1.7- and 2.1-fold increases in shoot regeneration efficiency, respectively, compared to controls. This enhancement was accompanied by sustained suppression of miR396 and upregulation of its target GROWTH-REGULATING FACTOR (GRF) genes. Our findings establish a practical platform for overcoming genetic barriers in plant tissue culture through targeted, transient manipulation of regulatory RNAs, offering potential applications to wide-range of plant species especially those recalcitrant to regeneration, thereby advancing plant biotechnology and genetic engineering.