Font Size: a A A

Protoplast Preparation And Tissue Culture Of Two Carrageenan-producing Seaweeds

Posted on:2014-01-28Degree:MasterType:Thesis
Country:ChinaCandidate:J P LiFull Text:PDF
GTID:2231330398499966Subject:Marine biology
Abstract/Summary:PDF Full Text Request
Kappaphycus alvarezii and Eucheuma denticulatum are the primary sources of thecommercially valuable hydrocolloid carrageenan, as well as the potential sources for bioethanolproduction. Despite the fact that there is a huge market demand for these valuable macroalgae,the development of the industrialized cultivation is slow. Long-term vegetative propagation of K.alvarezii and E. denticulatum results in serious degeneration of germplasm, which has hinderedthe development of the industry. Therefore, researches on strain breeding should be valuedhighly to protect the industry developing stably and sustainabilly. It is hard to clarify the sexualreproduction of these algae. In the meanwhile, the asexual reproduction has degeneratedseriously. This research focus on protoplast isolation and tissue culture of these species, with thepurpose of a study on cell fusion and hybridization bewteen different species, which aremethods for further breeding research program. The main results are as follow:1) A few protoplasts were obtained by digesting the cells of K. alvarezii and E.denticulatum with mixture of cellulase and pectinase. The cell walls of the majority of free cellshave not been digested even after enzyme digestion. The highest free cell yeilds of K. alvareziiand E. denticulatum were6.48106and6.80106Cells, when0.4g homogenate pellets weretreated with3ml enzymic extracts for48h, at30℃and pH6.0. The highest relative free cellyields of K. alvarezii and E. denticulatum were obtained when0.1g homogenate pellets wereincubated with3ml enzymic extracts for48h, at30℃and pH6.0. In the mean time, the resultsshowed that the more algal homogenate pellets were added in the tested range (0.1-0.4g freshweight), the higher the free cell yields and the lower relative cell yields per biomass wereobtained. The yields of free cells of both K. alvarezii and E. denticulatum were promoted as thetemperature increased in the certain range from20℃to30℃. The suitable pH range forenzymatic digestion was from6.0to8.0. In the range of12h to24h, the free cell yieldsincreased as the time of enzymatic digestion was extended.2) By digesting the homogenate pellets using the crude enzymatic extracts of viscus ofSiganus fuscessens, a certain amount of protoplasts of Kappaphycus and Eucheuma wereobtained. Callus could be induced by protoplast cultivation, and fused cells could be generatewhen the protoplast fusion were induced by PEG. The highest protoplast yeilds of Kappaphycus and Eucheuma were3.8103and4.15103Cells, when0.4g homogenate pellets were digestedwith3ml enzymic extracts for48h, at30℃and pH6.0. The highest relative protoplast yieldsof Kappaphycus and Eucheuma were obtained when0.1g homogenate pellets were treated with3ml enzymic extracts for48h, at30℃and pH6.0. Meanwhile, the preparation of protoplastsfrom Eucheuma was easier compared with Kappaphycus. The more the algal homogenate pelletswere added in the tested range (0.1-0.4g fresh weight), the higher the total protoplast yieldsand the lower relative protoplast yields per biomass were obtained. Extension the time ofenzymatic digestion could improve the total and relative protoplast yield. The favorable pH andtemperature for protoplast preparation of these seaweeds were pH6.0and25℃individually.3) New sprouts were induced to from branches of Kappaphycus and Eucheuma via tissuecultivation. The stem segments in PES medium survive better than that in F/2and ESS medium.The survival rates of the cultivation system with1mg/L NAA and0.5mg/L Zeatin weresignificantly higher than that with1mg/L IAA and0.5mg/L Zeatin and that without planthormones. The highest survial rates were emerged at100μmol m-2s-1between the light intensitygradient from50to200μmol m-2s-1. The branches at laying style grow better than that at uprightstyle and that laying style after rip cutting.4) In the experiments of preservation method, it can be seen that, the plant ofKappaphycus and Eucheuma could kept their vitality for five days, in dark, at17-20℃, and at70-100%RH.
Keywords/Search Tags:Kappaphycus, Eucheuma, Siganus fuscessens, protoplast, tissue culture
PDF Full Text Request
Related items