Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm⁻¹ to the autospores yielded transformants at a frequency of >10⁻⁶ per input cell for strain KJ and >10⁻⁷ per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.

单细胞绿藻，球孢藻。Obi和KJ菌株能够在较宽的pH范围内生长，并且在贫氮条件下以高于干细胞重量50％的水平积累脂质。需要适用于这两种菌株的有效遗传转化方法以对其进行遗传改良，以促进其在生物燃料的商业生产中的应用。经过长时间的反复试验，我们偶然确定了通过电穿孔转化菌株所需的条件。首先，将菌株在等渗介质中的生长例如，需要含0.6 M山梨糖醇或甘露糖醇的最低盐度培养基。第二，在明/暗周期下的生长至关重要。在周期的黑暗时期，细胞含有两个或四个被母细胞壁包围的内生孢子。光照期开始后半小时内，母细胞壁被劈开，具有电感受态的单细胞自孢子被释放。将10 kV cm ^-1的电场强度的单个电穿孔脉冲施加到自生孢子上，产生的转化体的频率为每个输入单元> 10 ^-6（菌株KJ）和> 10 ^-7每个输入单元的Obi菌株。该转化方法相对简单，并且比我们先前开发的生物弹轰击方法提供了几到几十个更高的转化频率。本研究还提供了可促进微藻遗传转化系统发展的见识，微藻通过内生孢子形成繁殖。