Studies On The Isolation, Purification, Identification And Protoplast Preparation Of Tricholoma Matsutake

The method of combining morphological character and molecular biological techniques was developed for identifying mycelial isolates of Tricholoma matsutake with their basidiocarps in this paper. The method of isolated, cultivated and prepared protoplast of Tricholoma matsutake was also studied in this paper.In the study, five kinds of tissues of fresh Tricholoma matsutake were isolated, which were the lamellae, the pileu, the boundary between pileu and stipe, the middle part and the below part of the stipe, then cultured separately on PDA, PDA+VB₁+VB₃, PDA+Jew's-ear-extract+wheat bran, BT, BT+VB₁, BM and BM+VB₃ mediums for germination. The result showed that only the tissue from lamellae can bourgeon mycelium after 25d. The mycelia bourgeon percent was the highest when being cultured on PDA+ Jew's-ear extract + wheat bran medium.The morphological character was compared by observing the mycelia of Tricholoma matsutake and the isolated with microscope, and it was primary confirmed that the isolated is the mycelia of Tricholoma matsutake; then the relations between Tricholoma matsutake and the isolated were analyzed by the RAPD technology. The RAPD-PCR conditions were optimized, and 17 of 40 arbitrary decamer nucleotide primers were also screened with good implication and repetition. The PCR reaction adopted the 17 kinds of random primers. The clear and steady DNA fingerprints were obtained. The SIMQUAL process of the NTSYS-PC software was used to count DNA fingerprints similarity. The result showed that the DNA fingerprints similarity coefficient between basidiocarps and their mycelia were 1.00 by constructing the UPGMA tree chart. So it was concluded the isolated mycelia are the mycelia of Tricholoma matsutake.By mensurating the rate, of growth, the better medium, pH and temperature for the mycelia of Tricholoma matsutake were filtered. The results showed that PDA medium is the best for culturing mycelia, the second is PDA+VB₃ medium, KH₂PO₄+ MgSO₄+ maltose+glucose +VB₁+ ammonia tartaric acid medium and BT medium, the last is PDA+ Jew's-ear extract +wheat bran medium; The best pH for culturing Tricholoma matsutake is 5.5～6.0 and the best temperature is 23℃. By mensurating the output of mycelia of Tricholoma matsutake, the best liquid culture medium for culturing Tricholoma matsutake was screened out. The result showed that the mycelia on PDA medium without agar medium grow the best. The data were analyzed by the Multiple Comparisons. The best compound of liquid-medium is potato 400g/L and dextrose 30g/L.Lywallzyme and snail enzyme were adopted to prepare the protoplast of Tricholoma matsutake in the condition of 30℃, pH 6.5 and 0.6 mol/L MgSO₄ qua osmotic pressure stabilize. The result indicated that the affection of lywallzyme enzyme is better than that of snail enzyme. When the time of lywallzyme enzyme is 3 hours, the output of Tricholoma matsutake protoplast is 2.65×10⁷ which is the biggest. Under the same condition, different kinds and concentration of osmotic pressure stabilizer to prepare the protoplast of Tricholoma matsutake was screened. The result shown: 0.4mol/L mannitol is the best.