Transformation of basidiomycetes protoplasts by electroinjection of plasmid DNA

本研究的目的是通过对担子菌原生质体施加电刺激的实验过程，通过电注入质粒DNA获得担子菌细胞的基本信息。在原生质体融合方面，做了以下观察。在场强为50V/cm或更高的交流场中，以2 MHz的频率附着在电极上；在场强为750V/cm时产生珍珠链。在交流场强大于1.25KV/cm时。原生质体从电极上分离变得更加困难。不同种类的原生质体之间的粘合是通过以下步骤完成的：首先将不同种类的原生质体放入融合室，以125V/cm的交流电场将它们附着在电极上，然后在融合室中加入不同种类的原生质体，并施加250V/cm的电场。在此条件下，可以用10KV/cm的直流脉冲电压和6秒的持续时间进行熔合。该方法的最大融合率为10%。阻止融合的原生质体的旋转在低于或等于250V/cm的电压下停止。本研究使用的设备是作者自己设计的，在可操作性方面留下了改进的空间。为了获得融合所需的交流电压和脉冲电压，融合室中的电极必须非常薄(40-50米)，这就造成了融合室的制造以及旋转体融合的一些困难。全封闭融合室的最大优点是：(1)它允许在非无菌环境中保持无菌条件；(2)通过改变交流电压，可以很容易地实现不同种类原生质体之间的粘合。

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.