Long-distance transport or systemic silencing effects of exogenous biologically active RNA molecules in higher plants have not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid–protein nanocomplexes. In our experiments with tobacco and poplar plants, we have successfully demonstrated systemic gene silencing effects of cBSA/dsGUS RNA nanocomplexes when we locally applied the nanocomplexes from the basal ends of leaf petioles or shoots. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both the conditionally inducible DR5-GUS gene and the constitutively active 35S-GUS gene in leaf, shoot, and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants and potentially for in planta gene editing.

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock ‘Carrizo’ citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.

Thermal asymmetric interlaced-polymerase chain reaction-based and whole-genome sequencing-based T-DNA localization approaches have been developed for the recovery of T-DNA integration sites (TISs). Nevertheless, a low-cost and high-throughput technique for the detection of TISs, which would facilitate the identification of genetically engineered plants, is in high demand for rapid crop breeding and plant synthetic biology. Here, we present Tn5 transposase-based T-DNA integration site localization (TTLOC), a Tn5-based approach for TIS localization. TTLOC employs specialized adaptor-assembled Tn5 transposases for genomic DNA tagmentation. TTLOC library construction is straightforward, involving only six steps that requires two and a half hours to complete. The resulting pooled library is compatible with next-generation sequencing, which enables high-throughput determination. We demonstrate the ability of TTLOC to recover 95 non-redundant TISs from 65 transgenic Arabidopsis (Arabidopsis thaliana) lines, and 37 non-redundant TISs from the genomes of transgenic rice (Oryza sativa), soybean (Glycine max), tomato (Solanum lycopersicum), potato (Solanum tuberosum), and from the large hexaploid wheat (Triticum aestivum) genome. TTLOC is a cost-effective method, as 1 to 2 Gb of raw data for each multiplexing library are sufficient for efficient TIS calling, independent of the genome size. Our results establish TTLOC as a promising strategy for evaluation of genome engineered plants and for selecting genome safe harbors for trait stacking in crop breeding and plant synthetic biology.

Agrobacterium-mediated transient gene expression is a powerful technique for rapidly evaluating gene expression in higher plants. This study aimed to improve transient expression levels of T-DNA genes in citrus by investigating the effects of various factors, including seedling age, tissue treatments, Agrobacterium incubation medium and duration, hormone combinations, methylation inhibitors, antioxidants, and more. These parameters were optimized and tested, and significant increases in transient gene expression in juvenile epicotyls of Carrizo citrange and mature stem tissues of Pineapple sweet orange, Valencia orange, and Washington navel orange were observed. The present results demonstrated up to a six-fold increase in transient GUS gene expression, highlighting the effectiveness of these simple and inexpensive treatments. The juvenile and mature citrus explants used in this study displayed high levels of transient expression, which may provide a valuable tool for studying phloem-associated diseases such as Huanglongbing (HLB), facilitating rapid analysis of gene expression involved in Candidatus Liberibacter asiacticus (CLas) pathogenicity. This optimized method may also offer a promising tool for advancing genetic studies and improving the efficiency of Agrobacterium-mediated transgene-free editing in citrus and other economically significant perennial crops.

Long-distance transport of exogenous biologically active RNA molecules in higher plants has not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid-protein nanocomplexes. Using tobacco and poplar plants, we have shown effective uptake and long-distance transport of cBSA/dsGUS RNA nanocomplexes from basal ends of leaf petioles to leaf blades as well as from basal ends of shoots to their apexes and apical leaves. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both conditionally inducible DR5-GUS gene and constitutively active 35S-GUS gene in leaf, shoot and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants, and potentially for in planta gene-editing.