Monostroma latissimum is one edible Chlorophyta species,and is a potential new species for economic seaweed cultivation by fishermen in China,for viable protoplasts could be potentially used as a source for seed material of macrophytic marine algae cultivation and for other applied phycological research,so the aim of this study was to develop a new breeding method by culturing the protoplasts of M.latissimum,to form seedlings quickly,to shorten the traditional period of germlings cultivation,to maintain a stock of seedlings in the laboratory for longer periods,and to provide abundant germlings for largescale commercial cultivation.Protoplasts were isolated respectively from different parts of the thalli of M.latissimumwhich are collected at different time by enzymatic method(optimal enzyme composition consisted of 4% pectinase and 2% cellulase).And the regeneration,differentiation and development pathway of different derived protoplasts are studied under uniform conditions of isolation and incubation.According to the cell morphology,such as the formation of rhizoids,whether having hyaline sheath,or the size and arrangement ways of the cell,and the eventual development and differentiation trend,the protoplast development can be divided into eight results,include forming somatic cells and reproductive cells.The somatic cells formation can be classified into cell mass which includes regular or irregular cell mass; abnormal seedlings which include rhizoidal,rhizoidallike,no rhizoidal and tubular abnormal seedlings; normal seedlings,3 types,7 results.The reproductive cells formation developed into gametangia firstly,and then developed into sporangia.Each development pathway is associated with age,size and location of algae.This is the first time to report that some protoplasts underwent repeated cell divisions and developed directly into thalli similar to the parent thalli,and the application and expectations of protoplast culture were also discussed.