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.

Picea mongolica, a native species with excellent industrial wood quality and strong sand-fixing capacity, may be utilized in construction of urban green spaces in arid areas in China. However, now the sustainability of the ecosystems where this species grows is at serious risk due to a lack of natural regeneration. In this study, we developed an efficient regeneration system and comprehensively analyzed various factors affecting somatic embryogenesis (SE) using zygotic embryos as explants. We identified the optimal plant growth regulators (PGRs) performance and the best donor trees (k81) for the generation of somatic embryos (SEMs). Additionally, we confirmed that the positive developmental window of SEMs initiation was at the end of July to early August, which is when zygotic embryos was at the late embryogeny. In this time period, specific transcripts associated with the regulation of epigenetic modifications, plant hormone-related genes, and embryonic development-related transcription factors play important roles for early SEMs initiation. These results may provide a valuable resource for vegetative propagation of Picea mongolica. Our results may help to establish a reliable protocol for plantlet regeneration, which may facilitate urban greening applications and industrialization in arid areas.

Rose (Rosa rugosa) petals are rich in diverse secondary metabolites, which have important physiological functions as well as great economic values. Currently, it remains unclear how saline and/or alkaline stress(es) influence the accumulation of secondary metabolites in rose. In this study, we analyzed the transcriptome and metabolite profiles of rose petals under aline-alkali stress and uncovered the induction mechanism underlying major metabolites. Dramatic changes were observed in the expression of 1363 genes and the abundances of 196 metabolites in petals in response to saline-alkali stress. These differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) are mainly associated with flavonoid and terpenoid metabolism and the reconstruction of cell walls. Of them, TERPENE SYNTHASE 31 (TPS31) overexpression in tobacco leaves driven by its own promoter resulted in significant alterations in the levels of various terpenoids, which were differentially influenced by saline-alkali stress. An integrated analysis of metabolomic and transcriptomic data revealed a high correlation between the abundances of flavonoids/terpenoids and the expression of the transcription factor MYB5. MYB5 may orchestrate the biosynthesis of sesquiterpenoids and proanthocyanidins through direct regulation of TPS31 and ANR expression under aline-alkali stress. Our finding facilitates improving the bioactive substance accumulation of rose petals by metabolic engineering.

Population genetic diversity contributes to the protection and utilization of germplasm resources, especially via genetic breeding. In the present study, start codon targeted polymorphism (SCoT) molecular markers were used to study the genetic diversity of 278 individuals from 10 Prunus sibirica L. populations in Inner Mongolia. A total of 289 polymorphic bands were amplified with 23 SCoT primers, showing a polymorphism percentage of 98.87% and an average of 12.6 polymorphic bands per primer. The SCoT21, SCoT32, and SCoT53 primers amplified up to 17 bands, and the polymorphism percentage was 100%. The minimum number of bands amplified by SCoT25 was 9, and the polymorphism percentage was 90%. Therefore, SCoT molecular markers were shown to be highly polymorphic and suitable for genetic diversity studies of P. sibirica in Inner Mongolia. The analysis of molecular variance showed that 39% of the observed genetic differentiation occurred among populations and 61% occurred within populations, indicating that the genetic differentiation within populations was greater than that among populations. The results of the unweighted pair-group method with an arithmetic cluster analysis, principal coordinate analysis and STRUCTURE analysis were basically the same and divided the 278 individuals from the 10 populations into 2 groups. The results indicated that the efficient SCoT molecular marker-based genetic diversity analysis of P. sibirica in Inner Mongolia can provide a reference for P. sibirica variety breeding and resource development.

Picea mongolica is a rare tree species in China, which is of great significance in combating desertification and improving the harsh ecological environment. Due to the low rate of natural regeneration, high mortality, and susceptibility to pests and cold springs, Picea mongolica has gradually become extinct. At present, somatic embryogenesis (SE) is the most effective method of micro-proliferation in conifers, but the induction rate of embryogenic callus (EC) is low, and EC is difficult to differentiate from non-embryonic callus (NEC). Therefore, the EC and NEC of Picea mongolica were compared from the morphology, histological, physiological, and transcriptional levels, respectively. Morphological observation showed that the EC was white and transparent filamentous, while the NEC was compact and brownish-brown lumpy. Histological analyses showed that the NEC cells were large and loosely arranged; the nuclei attached to the edge of the cells were small; the cytoplasm was low; and the cell gap was large and irregular. In the EC, small cells, closely arranged cells, and a large nucleus and nucleolus were observed. Physiological studies showed significant differences in ROS-scavenging enzymes between the EC and NEC. Transcriptome profiling revealed that 13,267 differentially expressed genes (DEGs) were identified, 3682 were up-regulated, and 9585 were down-regulated. In total, 63 GO terms had significant enrichment, 32 DEGs in plant hormone signal transduction pathway were identified, and 502 different transcription factors (TFs) were characterized into 38 TF families. Meanwhile, we identified significant gene expression trends associated with somatic embryo development in plant hormones (AUX/IAA, YUCCA, LEA, etc.), stress (GST, HSP, GLP, etc.), phenylpropanoid metabolism (4CL, HCT, PAL, etc.), and transcription factors (AP2/ERF, MYB, WOX, etc.). In addition, nine genes were chosen for RT-qPCR, and the results were consistent with RNA-Seq data. This study revealed the changes in morphology, histology, physiology, and gene expression in the differentiation of NEC into EC and laid the foundation for finding the key genes to promote EC formation.

Zygotic embryogenesis is a critical process during seed development in gymnosperms. However, knowledge on the genome-wide transcriptional activation that guides this process in conifers is limited, especially in Picea mongolica. This tree species is endemic to semiarid habitats of Inner Mongolia in China. To extend what is known about the molecular events underpinning its zygotic embryogenesis, comparative transcriptomic analyses of gene expression in zygotic embryos were performed by RNA sequencing in P. mongolica. Our results showed that most changes in transcript levels occurred in the early embryonic pattering determination and formation of mature embryos. Transcripts related to embryogenic competence, cell division pattern, hormones, and stress response genes were identified during embryogenesis. Auxin is essential for early embryo patterning and pre-cotyledon embryonic formation. However, ABA is a major regulator of embryo maturation. Moreover, we found that methylation-related gene expression is associated with activation of early-stage embryos, late embryogenesis abundant proteins, and storage/energy-related genes with late and mature embryos. Furthermore, network analysis revealed stage-specific and multistage gene expression clusters during embryogenesis. WOX, MYB, AP2, and HLH transcription factors seem more relevant to embryogenesis in different stages. Our results provide large-scale and comprehensive transcriptome data for embryo development in P. mongolica. These data will lay a foundation for the protection and utilization of P. mongolica resources.