Preliminary Study On Protoplast Fusion Of Lactarius Delicious And Pleurotus Ostreatus

In order to create a new bacterial strain with new hereditary capacity, this paper uses the protoplasts treated by ultraviolet and heat as a mark to investigate the protoplast preparation, regeneration and fusion between Lactarius deliciosus and Pleurotus ostreatus.The results show as follow:The optimal conditions of protoplast preparation and regeneration of Lactarius deliciosus:Lactarius deliciosus protoplast8.36×107/ml were obtained under the Lactarius deliciosus mycelia cultured4th days as raw material, lywallzyme at concentration of2.5%, water bath in30℃for4hours,0.6mol/L KC1with pH6.0as osmotic pressure stabilizer, the regeneration rate of Lactarius deliciosus protoplasts was9.1×10-4on the optimal regeneration medium. The optimal conditions of protoplast preparation and regeneration of Pleurotus ostreatus:Pleurotus ostreatus protoplast7.75×10~7/ml were obtained under the Pleurotus ostreatus mycelia cultured for6days as raw material, lywallzyme at concentration of2.0%, water bath in30℃for3hours,0.6mol/L KC1with pH6.0as osmotic pressure stabilizer, the regeneration rate of Pleurotus ostreatus protoplasts was7.4×10-4on the optimal regeneration medium.This article studies protoplast’s preparation, regeneration and fusing of Lactarius deliciosus’by using ultraviolet-heating double inactivated marking method. The Lactarius deliciosus should be disposed by heat inactivation, with water bath in65℃for50minutes to get an inactivation ratio up to100%. The Pleurotus ostreatus should be disposed by ultraviolet inactivated, irradiated30minutes under the15W ultraviolet lamp about30cm, and its inactivation ratio was up to100%.The conditions of fusinf these two protoplasts are:admixing the two protoplasts as a rate of1:1, with30%Polyethylene Glycol (PEG4000) and buffer solution of Ca2+as fusogen, fused for30minutes under30℃, the fusion ratio was4.85×10-5. Pick6fusants from the regeneration medium by comparing the phenotype of colony, experiment of antagonism, determination of polyacrylamide gel electrophoresis (PAGE) with esterase isozymes and the whole protein.Investigation of the biological traits of the fusant shows, the highest amylase’s activity of fusants with11.81U/ml, CMCase’s activity1.13cm, laccase’s activity2.05U/ml, xylanase’s activity1.96U/ml. The biomass of fusant is obviously higher than their parents in the situation of same cultural time. The highest hypha’s wet weight of the fusants can reach48.1600g and the dry weight1.022g. Growth speed of fusant was faster than their parents. The heredity of fusant is stable, based on the test of genetic stability.