Somatic embryogenesis (SE) is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156-SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156-SPL module in SE remains unclear. In this study, we found that the transcription factors (TFs) CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes CsSPLs and reduction of SE efficiency. In the STTM-miR156a overexpressed callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated; csi-miR172d was down-regulated, whereas the target genes CsTOE1.1 and CsTOE1.2, which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs, thus to alter starch accumulation level in callus cells and regulate SE in citrus. This study elucidates the pathway of miR156-SPLs and miR172-TOEs mediated regulation of SE, and provides clues to enhancing SE capability in citrus.

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Citrus nucellar poly-embryony (NPE) is a mode of sporophytic apomixis (SA) that asexual embryos formed in the seed through adventitious embryogenesis from the somatic nucellar cells. NPE allows clonal propagation of rootstocks, but it impedes citrus cross breeding. To understand the cellular processes involved in NPE initiation, we profiled the transcriptomes and DNA methylomes in laser microdissection (LMD) captured citrus apomictic cells. In apomictic cells, ribosome biogenesis and protein degradation were activated, whereas auxin polar transport was repressed. Reactive oxygen species (ROS) accumulated in the poly-embryonic ovules, and response to oxidative stress was provoked. The global DNA methylation level, especially that of CHH context, was decreased, whereas the methylation level of the NPE-controlling key gene CitRWP was increased. A C2H2 domain-containing transcription factor (TF) gene and CitRWP co-expressed specifically in apomictic cells may coordinate to initiate NPE. The activated embryogenic development and callose deposition processes indicated embryogenic fate of nucellar embryo initial (NEI) cells. In our working model for citrus NPE initiation, DNA hyper-methylation may activate transcription of CitRWP, which increases C2H2 expression and ROS accumulation, triggers epigenetic regulation, and regulates cell fate transition and NEI cell identity in the apomictic cells.

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