Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the beta-glucosyl Yariv reagent (beta glcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of beta glcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with beta glcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (> 80%). However, without transfer to the AC medium, beta glcY at concentrations higher than 20 mu g ml(-1) inhibited cell division. No effect on cell survival nor cell division was observed with the alpha-galactosyl Yariv reagent. Staining of beta-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of beta-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without beta glcY, while little or no beta-1,3-glucan was stained by AB in protoplasts cultured with beta glcY. These results suggest that AGPs and beta-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.