Studies On Nutritional Physiology,Protoplast Preparation And Hybridization Breeding Of Two Microalgae

Both HamatOcoccus pluvthe and Chtorella vuharis are single cell, OPticalenergy atotroPhic, fresh Waer microalgae belongs to Chlorophyta. Because of theirhigh value of nutrient and photosynthesis caPability they two are payed more andmore atteIltion to. From aPplication research to cotnmercial production, the a1galstrain need imProve relatively Cell fusion has been proved a simple, direct and rapidhybridization method of single-cell algae breeding.There's much differeflt between Hpluvialis and Cvuharis in nforitionalphysiology Hpluvltrlis couldn't grow in the medium cont8ined urea as the nitrogensource or acetate as the carbon source, while Cvulgaris could grow very well insuch mediuxn.The preparation of two kinds of protoplasts were canied through with enzymaticmethod. According to the difference of the cel1 wal1 constitation, we used differefltenzyInes were used to get the protoplasts. After the Hpluvialis cells were treatedwith 0.06% proteinase K, pH7.8 Tris-HCl, 35aC for 1 hong we could get the highestprotOplast forming rate of 48.70% and highest protoplast regeneration rate of60.32% on the regeneration plate using the polyethylene glycol(PEG) maintainingthe osmotic pressure. The best condition of Cvuhoris protOPlaSt preparation wasdecided after a series of experiments to be: preculturing in the medium containing 2-deoxy-d-glucose, 2DG) fOr 24 hours, then treating wtth 2% cellulase, 2% pectinase,2% hemicellase, pH5.5 PBS, 30OC for l4 hours, resulting in gaining highestprotoplast forming rate of 37.l4% and highest protoplast regenefation rate of56.67% on PEG regeneration plate.TO make the fusion of the tWo kinds of Protoplasts, we combined two method,treating wth PEG and high calcium concentration along with high pH value: aftermixed twO kinds of protoplasts, added 20%PEG, 30oC for 30 millules, then addedthe fusion scrub solution (with high calcium concentration and high pH) for tWicefirst 10 miflules and second 20 milllles. After the treatmeni, inoculatCd the miXtueon the PEG regeneration plate.Due to the great difference between the two algal cells, we distinguished thefusor Which seemed 1ike Chlorella, but larger than ChlOrella cell. To comfirm thefusot, we analysed the fat acid content of Hpluvialis, C vuharis and the fusor as anindicatory'tg. Results proved that the fusor had some characters either Hpluvialis orCvuhoris had, or some Which two parents had not.