Background MiRNAs (microRNA) are 18–24 nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. Results This study used high-throughput sequencing technology for the first time in Larix olgensis , predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60 days, miR160, miR166 and miR396–2 exhibited the highest expression in leaves. At 120 days, most miRNAs in roots and stems decreased significantly. At 180 days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396–2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166–1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950–1, first decreased and then increased. Similarly, in the roots, miR950–2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. Conclusions The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis. To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.

Larix olgensis is one of the main coniferous tree species in northeastern China and has excellent timber properties and strong tolerance to stress. Thirteen HD-Zip family genes with a complete CDS region were identified on the basis of cambium transcriptome data from Larix olgensis. All 13 genes were analyzed via bioinformatics by their conserved domain protein sequence and amino acid composition, including their physicochemical properties and protein structure. The spatiotemporal expression and abiotic stress responses of these genes were analyzed by real-time quantitative PCR. The results showed that the 13 HD-Zip genes of Larix olgensis were expressed in the roots, stems, and leaves at different stages. The expression of three of these genes (LoHDZ2, LoHDZ11, LoHDZ13) was highest in nonlignified roots, indicating that they might be related to the secondary growth of Larix olgensis; in addition, three genes (LoHDZ5, LoHDZ9, LoHDZ10) were highly expressed in partially and completely lignified stems and leaves. These 13 genes were expressed specifically under drought stress. The expression of two of them (LoHDZ1, LoHDZ5) was obviously upregulated, and the expression of 6 genes (LoHDZ2, LoHDZ3, LoHDZ4, LoHDZ8, LoHDZ10, LoHDZ13) was significantly downregulated. The expression trends indicate that these genes could be involved in drought stress. The expression of all 13 genes was downregulated when the plants were treated with 0.2 M NaCl for 96 h, indicating that these genes are inhibited by salt stress. Overall, the results have significant implications for the study of the gene function of members of the LoHD-Zip transcription factor family.