Effects of Medium Supplements on Somatic Embryo Maturation and DNA Methylation in Pseudotsuga gaussenii Flous, a Species under Protection

Abstract

Pseudotsuga gaussenii is a forest species under protection in China. Propagation via somatic embryogenesis is efficient and needed for recovering this species. In this research, effects of medium supplements, i.e., methylglyoxal-bis (guanylhydrazone) (MGBG), polyethylene glycol (PEG), and maltose, on somatic embryo (SE) development were studied. More mature SEs developed with the pre-maturation treatment medium supplemented with 50 μM or 100 μM MGBG. During SE maturation, when the medium was supplemented with both PEG and maltose, the over-proliferation of embryogenic tissue was suppressed. When maltose was eliminated from the maturation medium, SEs were slightly smaller but developed better than other treatments due to the supply of lower carbon source, whereas when PEG was taken out from the maturation medium, no mature SE was obtained, only the heavily-proliferated plant tissue. Meanwhile, DNA methylation levels differed in these SE maturation cultures at the late culture stage of SE maturation. The level increased sharply with no maltose treatment (20.28%) while decreasing without PEG supplement (10.35%). DNA methyltransferase genes MET1-1, MET1-2, MET1-3, CMT3, DRM1, and DRM2 were partially cloned in this study to detect their expression level via qPCR. Expression of these genes, except MET1-3, in the culture of no PEG was higher significantly than the cultures of other treatments at weeks 1, 3, and 10. Our results suggested that MGBG and/or PEG play an important role in stimulating SE development and maturation. Furthermore, sugar supplements at a lower level benefited SE maturation in Pseudotsuga gaussenii.