Evidences for differential expression of miR167d-5p, target, positional nucleotide preference, and its role in somatic and different stages of regenerating calli of Oryza sativa

The incorporation of important value-added features in rice by Agrobacterium-mediated transformation of scutellum-derived calli is an established biotechnological procedure. The regeneration potential of rice is genotype, phytohormone and synthetic media sensitive. Hence, it is essential to identify the biomarkers associated with plant regeneration for rapid assessment. Earlier reports on microarray data suggest higher expression of miR167 in differentiated calli. In this study, the expression of miR167 is standardized by stem-loop RT-PCR under different conditions, of Oryza sativa calli in regeneration were phytohormonal concentrations, age responses were compared with the somatic embryos. Non-recalcitrant, moderately recalcitrant and recalcitrant indica rice varieties were also investigated for miR167d-5p expression. Position weight matrix analysis of miR167-5p sequences reported (181 sequences) throughout the plant kingdom was analyzed by DAMBE. Targets were identified using psRNATarget followed by functional characterization. Evidences for the expression of target of miR167, the transcription factor Auxin response factor8 (ARF8) shows lower levels in regenerating calli. New target, NBS–LRR (nucleotide-binding site leucine-rich repeat disease resistance protein) showed a different response. In this work, it is quite evident that miR167d-5p shows 30-fold higher expressions in green regenerating calli as compared to non-green calli. This is in contrast to the expression of ARF8 where down-regulation by 2.5-fold is seen. Thus, miR167d-5p is key miRNA associated with plant regeneration capacity and this could be used for rapid screening of regenerating calli of O. sativa .