RNA-seq profiling of primary calli induced by different media and photoperiods for japonica rice ‘Yunyin’

The induction of embryogenic calli plays a vital role in the genetic transformation and regeneration of rice (Oryza sativa L.). Despite progress in rice tissue culture, the molecular mechanisms of embryogenic callus induction remain unknown. In this study, gene expression profiles associated with calli were comprehensively analyzed during callus induction of japonica rice ‘Yunyin’. We first confirmed that NMB medium with 24 h of light and 0 h of dark (NMB-L) was the optimal condition for ‘Yunyin’ callus induction, while J3 medium with 0 h of light and 24 h of dark (J3-D) was the worst condition. After transcriptome analysis, 33,597 unigenes were assembled, among which we identified 6,063 DEGs (Differentially Expressed Genes) related to media and seven DEGs related to photoperiod. Phenylpropanoid biosynthesis, plant hormone signal, and starch and sucrose metabolism were the top three pathways affected by media, while the circadian rhythm-plant pathway was associated with photoperiod. Furthermore, we identified two candidate genes, Os01g0965900 and Os12g0555200, affected by both medium and photoperiod. Statistical analysis of RNA-seq libraries showed that the expression levels of these two genes in J3-D calli were over 2.5 times higher than those in NMB-L calli, which was further proved by RT-qPCR analysis. Based on FPKM (Fragments Per Kilobase of transcript Per Million mapped reads), unigenes belonging to the NMB-L group were mainly assigned to ribosome, carbon metabolism, biosynthesis of amino acids, protein processing in endoplasmic reticulum, and plant hormone signal transduction pathways. We transformed Os12g0555200Nip and Os12g05552009311 into ‘Nipponbare’ calli and observed their effects on the growth and development process of rice calli using TEM (Transmission Electron Microscopy) and SEM (Scanning Electron Microscopy). Observations showed that Os12g05552009311 was more disadvantageous to rice callus growth than Os12g0555200Nip. Our results reveal that the Os12g0555200, identified from transcriptomic profiles, has a negative influence during ‘Yunyin’ callus induction.