Using CarboTrace 480 to detect protoplastation in pigment deficient mutant of Chlorella sorokiniana

Abstract

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

Plant phenotyping relevance

微細藻類の細胞壁状態とプロトプラスト形成を蛍光マーカーで識別する方法の開発・検証が研究の中心であり、植物状態の取得手法に該当する。

Code and data availability

The supplied blocks contain no public phenotype/trait datasets, microscopy images, analysis code, or trained models with author-deposited availability statements; only the article text, figures descriptions, and cited references are present.

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