Construction Of New Alcohol-producing Fusants By Trichoderma Koningii And Saccharomyces Cerevisiae Protoplast Fusion

The protoplast fusion technology is a way of microbial breeding.This method is simple and easy.But there are still many questions about selecting objective bacterial strain to be solved by this method.This paper is focused on Trichoderma koningii and Saccharomyces cerevisiae,protoplast fusion techonology and inherited characteristics of fusant are analyzed.To provide a suitable fungal material for protoplast-fusion and mutation-breeding of Trichoderma koningii.This research work for microbial breeding laid a foundation.Conditions for Trichoderma koningii protoplast preparation and regeneration including mycelium growth time,the content of cellulose,temperature and time for reaction,various compounding of osmotic stabilizers were examined.The optimal conditions for protoplast preparation of Trichoderma koningii were as follows:22h mycelium growth time,the content of cellulase was 2.0%,2.5 h reaction at 30℃,the optimal osmotic stabilizer of cellulase was 0.6 mol/L NaCl.Microscopical observation indicated that the protoplasts of Trichoderma koningii were largely released from the apex and hypha segment.A satisfied concentration of Trichoderma koningii protoplasts could be achieved for the cell fusion and the cell mutation induced in this research.The regeneration culture medium was researched , determining the optimal regeneration culture medium for Trichoderma koningii protoplast was 0.8 mol/L sucrose YPD.The research showed that 4.13×105 regeneration colonies were in this regeneration culture medium . One modes forming irregular outshoot was found to regulate regeneration of Trichoderma koningii.Fusants were got by two methods.Drug resistance genetic marker and the single heated died parent were used for selection marker in this experiment.The nystatin concentration was 750 U/mL,The alcohol concentration was 23%,the single heated died parent of Trichoderma koningii protoplast was heated up to 67℃,10 min.The 246 fusants were selectd by the two methods.The fusants through the preliminary selection for fermenting glucose to the alcohol were selected by TTC.Then the fusants through continuous selection for breakdown of glucose were selected by sodium carboxymethyl cellulose culture medium.The fusants have the strong ability for production alcohol.The fusants have the highest enzymic activity strain by TTC and sodium carboxymethyl cellulose culture medium.The selected fusants were to be tested for genetic stability.The results were that the four fusants were the genetic stability fusants.Individual figure,biochemical characteristic,specific genetic sequence and soluble protein were studied by this experiment.The results were that the fusants for B1,B2,B3 and B4 proceeded gene recombination,but the ability for enzymic activity and production alcohol were lower than the parents.But this experiment provided the foundation for protoplast fusion breeding.