A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD₆₀₀ of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

Salix psammophila, a sandy shrub known as desert willow, is regarded as a potential biomass feedstock and plays an important role in maintaining local ecosystems. However, a lack of genomic data and efficient molecular markers limit the study of its population evolution and genetic breeding. In this study, chromosome counts, flow cytometry and SSR analyses indicated that S. psammophila is tetraploid. A total of 6,346 EST-SSRs were detected based on 71,458 de novo assembled unigenes from transcriptome data. Twenty-seven EST-SSR markers were developed to evaluate the genetic diversity and population structure of S. psammophila from eight natural populations in Northern China. High levels of genetic diversity (mean 10.63 alleles per locus; mean H_E 0.689) were dectected in S. psammophila. The weak population structure and little genetic differentiation (pairwise F_ST = 0.006–0.016) were found among Population 1-Population 7 (Pop1-Pop7; Inner Mongolia and Shaanxi), but Pop8 (Ningxia) was clearly separated from Pop1-Pop7 and moderate differentiation (pairwise F_ST = 0.045–0.055) was detected between them, which may be influenced by local habitat conditions. Molecular variance analyses indicated that most of the genetic variation (94.27%) existed within populations. These results provide valuable genetic informations for natural resource conservation and breeding programme optimisation of S. psammophila.

Light is an important environmental factor for plant growth, and in higher plants, phytochrome A (phyA) is the predominant far-red photoreceptor, involved in various photoresponses. The FAR1/FHY3 transcription factor family, derived from transposases, is able to regulate plant development in response to multiple photosensitizers phytochrome. In total, 51 PtrFRSs were identified in the poplar genome, and were divided into 4 subfamilies. Among them, 47 PtrFRSs are located on 17 chromosomes. Upstream cis-acting elements of the PtrFRS genes were classified into three categories: growth and metabolism, stress and hormone, and the hormone and stress categories contained most of the cis-acting elements. Analysis of the regulatory networks and expression patterns showed that most PtrFRSs responded to changes in light intensity and were involved in the regulation of phytochromes. In this study, 51 PtrFRSs were identified and comprehensively bioinformatically analyzed, and preliminary functional analysis and prediction of PtrFRSs was carried out.

The NAC gene family is of great value for plant stress resistance and development. In this study, five NAC genes with a typical NAM domain were isolated from Salix psammophila, which is a stress-resistant willow endemic to western China. Two hundred sixty-two NAC genes from Salix psammophila, Salix purpurea, and Arabidopsis were used to construct the phylogenetic tree to examine the phylogenetic relationship. Five NAC genes in Salix psammophila were the focus of bioinformatics analysis and conserved structural domain analysis. The SpsNAC005 gene was overexpressed in Populus hopeiensis, and the transgenic lines were subjected to salt and simulated drought stress to analyze their phenotype changes and tolerance to stress. The results showed that transgenic poplar height and leaf area increased by 29.73% and 76.36%, respectively, compared with those of wild-type plants. Under stress treatment, the height growth rates and ground diameter growth rates of the transgenic lines were significantly higher than those of the wild-type, whereas their fresh weight and dry weight were decreased compared to those of the wild-type. The SOD activities, POD activities, and Pro contents of the transgenic plants were significantly increased, and the accumulation of MDA was significantly lower than that in the wild-type, and the transgenic lines showed clear tolerance to salt and drought. The expressions of the SOS1, MPK6, HKT1, and P5CS1 genes were downregulated in the transgenic lines. The expression of the PRODH1 gene was downregulated in the transgenic lines. These results indicate that overexpression of the SpsNAC005 gene in transgenic plants can promote plant growth and development and improve tolerance to salt and drought.

Salix psammophila, commonly known as the sandlive willow, is a vital shrub species within the Salicaceae family, particularly significant for its ecological role in regions susceptible to desertification and sandy soils. In this study, we assembled the complete S. psammophila mitochondrial genome using Pacbio HiFi third-generation sequencing data. The genome was found to be a typical single circular structure, with a total length of 715,555 bp and a GC content of 44.89%. We annotated 33 unique protein-coding genes (PCGs), which included 24 core mitochondrial genes and 9 variable genes, as well as 18 tRNA genes (5 of which were multicopy genes) and 3 rRNA genes. Comparative analysis of the PCGs from the mitochondrial genomes of S. psammophila, Populus deltoides, Populus simonii, Salix wilsonii, and Salix suchowensis revealed that these genes are relatively conserved within the Salicaceae family, with variability primarily occurring in the ribosomal protein genes. The absence of the rps14, which encodes a ribosomal protein, may have played a role in the evolution of stress tolerance in Salicaceae plants. Additionally, we identified 232 SSRs, 19 tandem repeat sequences, and 236 dispersed repeat sequences in the S. psammophila mitochondrial genome, with palindromic and forward repeats being the most abundant. The longest palindromic repeat measured 260 bp, while the longest forward repeat was 86,068 bp. Furthermore, 324 potential RNA editing sites were discovered, all involving C-to-U edits, with the nad4 having the highest number of edits. These findings provide valuable insights into the phylogenetic and genetic research of Salicaceae plants.