Integrated transcriptome and physiological analysis reveal the molecular mechanism of the osmotic-responses induced by cryoprotectants in Norway spruce embryogenic tissue

Cryoprotectants, such as sorbitol and dimethyl sulfoxide, are widely used to stabilize the culture solution in cryopreservation, which is necessary for the application of somatic embryogenesis technique in conifer vegetative propagation. However, limited information is available about the molecular mechanism of the osmotic-responses induced by cryoprotectants in conifer species. Use Norway spruce (Picea abies (L.) Karst.) embryogenic tissues (ETs), cryoprotectant pretreatment (CPT) methods T3 and T6 (T3 with low regrowth rate; T6 with high regrowth rate) were selected out of eight CPTs for further analyses based on their similar solution thermal stability but distinct survival rate after cryopreservation. High dose of initial sorbitol (>=0.3 M) induced G1 to G2 cell cycle shift. While, extensive necrosis was detected after low dose of initial sorbitol (=<0.2 M) CPT. 27 RNA-seq libraries (untreated control, T3 or T6 pretreated 4 h, 24 h, 48 h and before freezing) were sequenced using Illumina sequencing platform. The RNA-seq data emphasized the importance of the early molecular responses induced by cryoprotectants. Differently expressed (DE) genes (DEGs) were enriched in cell wall modification, cell cycle regulation and oxidation reaction categories. DE transcription factors (TFs) in T3 vs T6 were enriched in MYB, AP2/ERF, NAC and WRKY families. PaMYB11, one of the hub genes in the early stage, was knockdown by RNA interference (RNAi) to verify our RNA-seq data. The PaMYB11 up-regulation was shown to be necessary for the survival of the ETs under CPT. Our study provides novel knowledge of the stimulus molecular responses to CPT in spruce ETs, which could guide the development of more efficient procedure for cryopreservation in conifers and further expand their industrial applications.