A Study On Protoplast Breeding And Distant Hybridization Breeding Of Pleurotus Nebrodensis

In order to select high-production,high-quality,short growth cycle and wide-adption new species of Pleurotus nebrodensis,protoplast regeneration clone,UV-mutagenesis breeding technology and distant hybridization between Pleurotus nebrodensis and Pleurotus eryngii were used to refine the Pleurotus nebrodensis strain.The results are as follows.1.We established an efficient preparation and regeneration system.The Pleurotus nebrodensis protoplasts preparation and regeneration conditions were studied by single factor and orthogonal experiment from kinds of media,enzyme concentrations,mycelium ages,enzymolysis temperatures,enzymolysis time,kinds of osmolarity stabilizers.The highest protoplasts yield with 37.75×106 mL-1 could be obtained on these conditions:mycelia were cultured in the enriched PD liquid medium for 7 days,then digested at 30 degrees Celsius for 2 h by 2%lywallzyme enzyme that was dissolved in 0.6 mol/L MgSO4·7H2O.And the best regeneration rate was 0.89%using sucrose as osmotic pressure stabilizer.2.By using protoplast regeneration clones and protoplast ultraviolet mutagenesis breeding technology,we obtained an excellent mutant strain"10s n29"which had better commodity,higher uniformity and production,shorter growth cycle than its parent strain.Then,it had proven to be an excellent mutant strains by RAPD technique.3.RAPD and ISSR and SRAP molecular markers for Pleurotus nebrodensis and Pleurotus eryngii strains were used to identify their affinity analysis and genetic diversity analysis.The results showed that the genetic differences between Pleurotus nebrodensis and Pleurotus eryngii strains were related to their affinity analysis.Pleurotus nebrodensis strains which were from the first genetic category had a high affinity with each type of Pleurotus eryngii strains,In addition,Pleurotus eryngii strains which were from the third genetic category had a high affinity with each type of Pleurotus nebrodensis.4.One-way nuclear migration and two-way nuclear migration could occur in Pleurotus nebrodensis and Pleurotus eryngii single spore strains.USing ITS-RFLP and EF1a-RFLP technology,we could know that Xingbailing strains which were the hybrids of the Pleurotus nebrodensis and Pleurotus eryngii were true hybrids from the level of cytoplasm and nuclei,But this method cannot tell the difference among these isonuclear alloplasm strains.5.It was found through cultivation that Xingbailing strains had three different traits:some were similar to Pleurotus nebrodensis,some were similar to Pleurotus eryngii.and others were somewhere in between.But all of their growth cycle were shorter than Pleurotus nebrodensis,so,they were the fast-growing strains we obtained.