Aging peach palm (Bactris gasipaes Kunth) cultures lose embryogenic potential and metabolic cellular function due to continuous culture in hypoxic environments

Abstract

Formerly-embryogenic 8-year-old and 2-year-old embryogenic peach palm (Bactris gasipaes Kunth.) cultures were treated with 0, 4, 16, or 64 µM 5-azacytidine, an inhibitor of cytosine methylation, to evaluate its ability to restore embryogenic potential to old cultures and observe its effects on younger embryogenic tissue. Either 16 or 64 µM 5-azacytidine restored the ability of a small proportion of older cultures to produce somatic embryos, but the same concentration also caused somatic embryos from the 2-year-old embryogenic culture line to lose regulation, resulting in non-embryogenic cultures and fast-growing yellow callus. Two-year-old embryogenic tissue had slightly lower global methylation, but only 4 µM 5-azacytidine caused a drop in global methylation. Two-year-old and 5-azacytidine-treated 8-year-old embryogenic cultures both showed an up-accumulation of proteins involved in a number of cellular functions: cellular redox control, anaerobic fermentation, protein degradation, NO-related synthesis and storage, and several forms of epigenetic regulation, including the highly-conserved Argonaute 4 protein; and untreated 8-year-old cultures showed increased amounts of proteins involved in cell wall formation and rearrangement, defense-related peroxidases associated with the cell wall, and phospholipidase D. All three tissues showed proteins involved in hypoxia response. HPLC analysis of 8-year-old cultures with embryogenic and non-embryogenic 2-year-old cultures revealed that 8-year-old cultures lacked any detectable carbohydrates, whereas the younger ones contained measurable amounts of arabinose, ribose, and sucrose. Aging of in vitro cultures is, therefore, likely related to loss of metabolic ability due to constant hypoxia, leading to low vigor, inability to adapt to stress, and loss of embryogenic potential. Embryogenesis loss in ageing peach palm cultures is associated with hypoxia, leading to loss in detectable carbohydrates and down-accumulation of proteins associated with metabolism, nitric oxide storage, and epigenetic regulation