Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis

Abstract

AbstractBackgroundBarley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive.ResultsThis study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-threeIAA, fourteenARF, eightSAUR, threeYUC, and fourPINgenes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) onIAAgenes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation.BBM,LEC1, andPLT2exhibited a significant increase in expression levels in the IME system but were not activated in the ME system.WUSshowed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, includingSUVH3A,SUVH2A, andHDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression ofHvBBMandHvWUSonAgrobacterium-mediated barley transformation. The transformation efficiency in the group expressing thePLTPpro:HvBBM + Axig1pro:HvWUSconstruct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity.ConclusionsWe identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.