| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Some factors affecting the isolation of protoplasts from mycelia and the regeneration and reversion of isolated protoplasts for A. auricula-judae and A. polytricha were investigated. Protoplast isolation was affected by enzyme system and mycelial age. Maximum yield of protoplasts was obtained by a combination of Novozym 234 1%, and chitinase 0.1%, and by using mycelia of 4-day-old culture for A. auricula-judae and that of 6-day-old culture for A. 'polytricha.
High protoplast reversion was obtained by using protoplasts isolated from mycelia of 4-day-old culture for A. auricula-judae and that of 6-day-old culture for A. polytricha.
In order to select hybrid cells by protoplast fusion, the protoplasts of both monokaryotic strains were treated with two different metabolic inhibitors, iodoacetic acid sodium salt(TAS)and diethylpy rocarbonate(DP). From the results of broken and reversion frequencies of protoplasts, the concentrations of IAS and DP were found to be 4% for each monokaryotic strain(Mo A 11 and Mo P 16).
The condition which gave the largest fusion frequency of protoplasts was a DC pulse of 10 /. tsee, 275 times with intervals of approximately I see, and a field strength 3 kv/em.
Fusants obtained by electrical protoplast fusion, were investigated in order to clarify their characteristics.
In paired cultures between monokaryotic strains(Mo A 11 and Mo P 16)and 5 fusants, all the fusants formed antagonistic lines against both Mo A 11 and Mo P 16.
All 5 fusants were found, by analyzing the electrophoretic isozyme, to possess both specific bands which were present in the monokaryotic strains. By using restriction endonuclease analysis, the molecular size of mitochondrial DNA of Fu 9 was confirmed to be 85.8 kbp. All 5 fusants had a mitochondrial plasmid-like DNA.
The result of pulsed field electrophoresis indicated that parent, monokaryotic, and fusant strains had 4 chromosomal DNA.
From these evidences, it is concluded that all 5 fusants are heterokaryon.
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