Application Of Protoplast Technique In The Rejuvenation,Breeding And Life Cycle Of Poria Cocos

In this study, we used protoplasts from strain T1 and L for experimental material in rejuvenation and fusion, combined with proper screening method, obtained the fusants and rejuvenation whose sclerotia yield was higher than original strain. Detailed results as follows:We prepared protoplasts from Wolfiporia cocos strain T1 which showed the evidance of degeneration after activation and amplification Culture. 25 regeneration strains grew faster in CYM medium were preliminarily selected from 136 regeneration strains; We also compared with growth rate in wheat medium and sawdust of pine medium; The cultivation experiment showed the average sclerotia yield of strains which had been preliminarily screening and secondary screening were both higher than T1 by statistics. So this methord was useful.In the test of inactivated protoplast fusion, the shortest UV-kill time and heat-kill time was 9min, 20min respectively. Heterofusants could be regenerated by used PEG6000. Selected from regeneration medium after one week. There are 47 regeneration strains in 118 reborn colonies harmony with T1 and disharmony with L, both antagonism appeared when 71 regeneration strains that could harmonious with each other cultured with parents. The 71 regeneration strains were fusants was determined preliminarily. Amplification results of ISSR primer P27 from 32 strains from which 71 regeneration strains by comparing growth rate showed, all of them had both specific bands of parents which proved them being fusants on molecular level.The hyphae antagonistic test showed, between monospore strains all grew together, the same result appeared in most protoplast regeneration strains; There are antagonistic lines between monospore strains and strain L; Only 5 protoplast regeneration strains that numbered as LP-13,LP-25,LP-62,LP-91 and LP-136 produced antagonistic lines on which Honeycomb or petal liked fruiting body could grow accidentally cultured with other protoplast regeneration strains. Based on ITS sequence analysis, the 4 strains LP-13,L,B5,T were proved to be poria cocos. Morphological Observation of fruiting body by paraffin sections and scanning electron microscope, we could see 4 stalks on each basidium, every spore connected one stalk. Spores with binuclei and hyphae with multinuclear were common with Fluorescent Staining.Above all, the technique of protoplasts regeneration combining with sereening method of growth rate could get renewed strains with good traits. fusants identified by ISSR for the first time in Inactivated poria cocos parents protoplast fusion test; paraffin sections and scanning electron microscope reveal Surface Morphology of fruiting body of poria cocos for the first time, the evidence may help us illustrate the life cycle of poria cocos.