Reduction of pectin may decrease the embryogenicity of grapevine (Vitis vinifera) pro-embryonic masses after 10 years of in vitro culture

• The embryonic properties of grape pro-embryonic masses decreased after 10 years of subculture. • Enlargement of intercellular space in the cell wall of grape pro-embryonic masses subculture for a long time. • Methylated and de-methylated pectins reduction in long-term subculture pro-embryonic masses. Plant somatic embryos are the best materials for woody plant reproduction, germplasm preservation and genetic transformation. However, the embryonic regeneration potential of most woody plant embryogenic cultures is reduced or lost after long-term subculture, and the morphological and physiological changes associated with embryogenic cultures during long-term in vitro culture are largely unknown. In this study, we compared grapevine pro-embryonic masses (PEMs) after 10 years of constant tissue culture with newly induced PEMs. The results showed that the somatic embryo induction rate of PEMs decreased significantly after long-term subculture, and up to 92.2% of abnormal embryos appeared. Moreover, the newly induced grapevine PEMs were composed of uniform embryogenic cell clusters. However, the classic cell cluster structure almost disappeared and the space between adjacent cells increased in the long-term subcultured PEMs. Notably, pectin content decreased significantly in the cell walls of the long-term subcultured PEMs, along with a decrease in pectin methylesterase activity and an increase in polygalacturonase activity. Meanwhile, pectin in the cell wall was predominantly methylated pectin, with less or almost undetectable de-methylated pectin not only in the newly induced grapevine PEMs but also in the 10-year-old PEMs. Nonetheless, both types of pectin were present at lower levels in long-term subcultured PEMs. Therefore, we speculate that the decrease in methylated and de-methylated pectin in the long-term subcultured grapevine PEMs may have destroyed the cell wall mechanics, which affected the ability of PEMs to develop into embryos. This study provides new insight into the long-term subculture of grapevine PEMs.