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.
Highlights
Pseudotsuga gaussenii has been listed as an endangered species, under national protection of the secondary level in China, due to the long-term utilization and the extremely low reproductive rate as well as the difficulties in cutting propagation
MGBG could inhibit the proliferation of embryogenic tissues via negatively regulating S-adenosyl-methionine decarboxylase (SAMDC), which is the key enzyme during polyamine biosynthesis [5]
FW, and the tion of maltose from the mature was(Mat) number of immature SEs (ISE) almost reduced to a half (Figure 4b). These results suggested that polyethylene glycol (PEG) was an important supplement of the mature medium and the lower sugar source by elimination of maltose from the mature medium was better for somatic embryo (SE) maturation
Summary
Pseudotsuga gaussenii has been listed as an endangered species, under national protection of the secondary level in China, due to the long-term utilization and the extremely low reproductive rate as well as the difficulties in cutting propagation. An optimized SEis technology is urgently needed for the large-scale propagation of endangered species, while SEis has not been reported in Pseudotsuga gaussenii. Multiple factors may affect the development and maturation of somatic embryos (SEs) in higher plants. In addition to plant growth regulators, culture medium supplements, such as methylglyoxal-bis (guanylhydrazone) (MGBG), polyethylene glycol (PEG) 4000, and maltose are among these factors in conifers [2,3,4]. The functions of PEG 4000 in promoting SE development and maturation, improving desiccation tolerance of SEs have been reported in various coniferous species, such as loblolly pine [8], white spruce [9], and maritime pine [10]. The influence of MGBG/PEG/maltose on SE development was not confirmed in Pseudotsuga gaussenii
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