Transformation of basidiomycetes protoplasts by electroinjection of plasmid DNA

本研究的目的是通过电刺激担子菌原生质体的实验过程，通过电注射质粒DNA获得有关担子菌细胞的基本信息。关于原生质体融合，进行了以下观察。在50v /cm或更强的交流电场中，在频率为2mhz时发生与电极的粘附；在750 V/cm场强下产生珍珠链。在交流场强大于1.25 KV/cm时。原生质体与电极分离变得更加困难。不同原生质体之间的粘附是这样完成的：首先将一种原生质体放入融合腔中，以125 V/cm的交流电场强度将其粘附在电极上，然后将另一种原生质体加入融合腔中，并施加250 V/cm的交流电场。在这些条件下，可以用10 KV/cm的直流脉冲电压和6秒的持续时间进行熔合。这种方法的最大熔合率为10%。原生质体的旋转会阻止融合，当电压小于或等于250 V/cm时，原生质体的旋转会停止。本研究使用的设备是作者自己设计的，在可操作性方面还有改进的余地。为了获得熔合所需的交流电压和脉冲电压，熔合室中的电极必须非常细（40-50米），这导致了关于腔室制造以及旋转体熔合的一些困难。全封闭融合室的最大优点是：(1)它允许在非无菌气氛中维持无菌条件；(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附。

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.