1988 Fiscal Year Final Research Report Summary
Transformation of basidiomycetes protoplasts by electroinjection of plasmid DNA
| Project/Area Number |
62560161
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
林産学
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| Research Institution | Hokkaido University |
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Principal Investigator |
MIURA Kiyoshi Faculty of Agriculture, Hokkaido University, 農学部, 助手 (40165957)
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| Project Period (FY) |
1987 – 1988
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| Keywords | DNA / Protoplasts / Cell fusion / Basidiomycetes / AC-field / DC pulse / 単子菌 |
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| Research Abstract |
The aim of present study is to obatin basic information with regard to the basidiomycetes cell by electroinjection of plasmid DNA, through the experimental process of applying electrical stimulation to basidiomycetes protoplasts.
With regard to protoplalst fusion, the following observations were made. Adhesion to the electrode occurred at a frequency of 2 MHz in an AC-field with a field strength of 50 V/cm or more; pearl chains were generated at a field strength of 750 V/cm. At AC field strengths over 1.25 KV/cm. It became more difficult for the protoplasts to separate from the electrode. Adhesion between fifferent species of protoplasts was accomplished by first placing on species of protpolast into a fusion chamber, adhering them to the electrode with an AC-field strength of 125 V/cm, and then adding a different species of protoplast to the chamber and applying an ACfield of 250 V/cm. Under these conditions, it was possible to cause fusion with a DC pulse voltage of 10 KV/cm and a duration of 6 sec. The maximum fusion ratio was 10% with this procedure. The rotation of protoplasts, which prevents fusion, halted at voltages less than or equal to 250 V/cm.
The equipment used for the present study was of the author's own design, leaving room for improvement in its operability. In order to obtain the AC and pulse voltages required for fusion, the electrode in the fusion chamber must be extremely marrow (40-50 m), and this resulted in some difficulties with regard to the manufacture of the chamber as well as the fusion of rotoplasts. The greatest advantages of the completely closed fusion chamber are: (1) it allows the maintenance of sterile conditions in non-sterile atmosphere, and (2) adhesion between different species of protoplasts can easily be accomplished by varying the AC voltage.
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Research Products
(2 results)
