Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana

Abstract

We found that embryogenic tissues of Picea balfouriana adopt different physiological pathways for long-term proliferation with 6-benzylaminopurine treatment and identified the miRNAs closely associated with proliferation. The long-term maintenance of somatic embryo production capacity in conifer embryogenic tissue (ET) is essential for the production of vigorous somatic seedlings. However, this ability is often lost after several months of proliferation in many conifer species including Picea balfouriana. Cytokinins are known to influence several important physiological processes during plant growth and development, including somatic embryogenesis (SE). In this study, we found that the 6-benzylaminopurine (BA) concentration influenced the yields of P. balfouriana somatic embryos and their germination response. Only ET of P. balfouriana proliferated on medium supplemented with 3.6 µM BA produced somatic embryos that germinated into normal plants. Most hormone levels increased in ET after prolonged proliferation. Moreover, antioxidant enzyme activities and polyamine contents were also significantly changed after 8 months of culture, which might be modulated by accumulated zeatin riboside (ZR). Finally, some selected microRNAs and their target genes were confirmed to be involved in the proliferation of ET of P. balfouriana and they also might be regulated by accumulated ZR. These findings may facilitate efforts to clarify basic physiological processes after the long-term proliferation stage of SE in conifers and delay the decreased production capacity of somatic embryos.