High efficiency of protoplast preparation for artificially cultured Ulva prolifera(Ulvophyceae,Chlorophyta)

Protoplast isolation was relevant for gene manipulation in U lva, and universal protocols have been proposed based on evaluation for various wildly collected species. However, only clonal laboratory cultures were practical for genetic transformation, and whether applicability of such universal protocol existed for these artificial cultures has never been investigated. In this research, samples in different physiological states or developmental stages were tested in U. prolifera. The results proved that the protoplast yields were strongly dependent on the characteristics of samples. Neither F_v/F_m value nor chlorophyll content exhibited an ideal correlation with the protoplast yields. Alternatively, specific growth rate, coupled with developmental stage, could serve as an ef fective combined index to determine the right time for protoplast isolation. According to this instruction, here we reported the highest yields of protoplast((31.5±1.9)×10~6 cells/g f. wt.) in U. prolifera, following comparison between protocols, and further optimizations on enzyme content, incubation period, starting biomass and pretreatment. This specified protocol for artificially cultured clonal samples could meet the need for protoplast-mediated genetic transformation in U. prolifera.