Studies On The Isolation And Culture Of Protoplasts And Explants From Solieriaceae

Solieriaceae seaweed is the main raw material for producing the carrageenan andis widely used in the textile and food industy. Besides, it also has high medicine,economic value and important ecological significance,and is badly demanded indomestic and international market. At the present, most of Solieriaceae seaweeds arethe wild species which are decreasing, and in China Solieriaceae seaweeds are stillmainly dependent on imports from Southeast Asian countries. Carrying the work ofSolieriaceae seed breeding technology and genetic improvement research has becomethe urgent need of carrageenan industry development in our country.Isolation and culture of protoplasts is an important plant somatic cell breedingtechnology. The protoplast doesn’t have the cell wall, and is an important tool forbiological research materials, which makes it widely used in cell biology, somatichybridization, genetic transformation and gene positioning study. The isolation of theprotoplasts is usually carried out by the enzymes methods and fluorescent brightenerstaining,and Evans Blue staining methods are used to determine the vitality ofprotoplasts. In this study, we chose the young frond of the three algae fromKappaphycus alvarezii, Eucheuma denticulatum,and with snail enzymes, abaloneenzymes and cellulase, protoplasts were successfully isolated from the chosen threespecies. The combination of20%abalone enzymes,12%cellulases can yield105/gprotoplasts, which showed good applicability for the experimental goal algae. For thedensity of protoplasts,(64.20±2.80）×104/ml、(76.80±3.20）×104/ml and (67.80±2.40）×104/ml were achieved for these species.And the optimal enzymic hydrolysisconditions were primarily established. Kappaphycus alvarezii was treated with2.0mol/L glucose,30℃in darkness for4h, as a result, for the survival rate ofprotoplasts,(49.34±0.12)%was achieved for Kappaphycus alvarezii.Seaweed cells are totipotent. Under the control of the appropriate artificialconditions, the algal tissues can rapidly propagate into a new individual. This opens up new avenues for the study of algal morphogenesis, genetic breeding and lifehistory control problems. Meanwhile, somatic cell and tissue rapid propagationtechniques also can produce economic algal seeds. Through the research into theregeneration of protoplasts from Kappaphycus alvarezii， two developmentalpathways were observed in the study, that is the protoplasts of Kappaphycus alvareziicould form not only cell mass but also regenerated plantlets.This article carried out regeneration culture research on the tissues which were dealedwith enzymes and explants of Kappaphycus alvarezii, and respectively summarizedthe developmental pathways of differentiation. After dealing with the enzymes, twodevelopmental pathways were found, one was that the tissues were cultivated for65days before young buds were found; another way was that in75days culture cellmass were observed. The regeneration process of explants could also observe twomethods, that was forming cell mass and new buds tissues, in which about65%ofexplants could form new young buds.