MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm-breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, researchers from the Chinese Academy of Forestry constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. The researchers predicted 110 target genes using bioinformatics, and validated 9 of them by 5′ RACE. 11 conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stages 5–8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cotyledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.
Morphology of embryogenic calli and embryos during the eight sequential developmental stages of larch SE
Stage 1 and stage 2 are embryogenic calli after 1–5 and 15 days of sub-culture, representing PEM I and PEM III, respectively. Stage 3, stage 4, and stage 5 are 1–5, 5–14, and 14–21 days post-differentiation cultures, when the calli of PEM III have developed into early, middle, and late single embryos, respectively. Stage 6, stage 7, and stage 8 are 21–28, 28–35, and 35–42 days post-differentiation cultures, when the late single embryos have developed into early and middle cotyledonary embryos, and fully mature embryos, respectively. The embryogenic–suspensor masses were stained by acetocarmine and observed using an inverted microscope at ×40 and ×10 magnification; the images of somatic embryos were obtained under an anatomical lens
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References
Zhang J, Zhang S, Han S, Wu T, Li X, Li W, Qi L. (2017) Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236(2):647-57. [abstract]