Improved procedures to assess plant protoplast viability: Evidencing cytological and genomic damage

Abstract

Plant protoplasts are valuable in biotechnology, enabling from plantlet regeneration to the determination of gene function. Viability tests are required in protoplast applications to determine the rate of viable cells, allowing to decide on the most adequate isolation and purification procedures and to verify whether sufficient cells are available for subsequent steps. Fluorescence microscopy is usually employed for this purpose. However, obstacles have pointed out: the long time required to count a relatively small number of protoplasts, cell clumps preventing their observation, and the observers subjective visual perception of the fluorescence. Therefore, this study aimed to establish procedures for the viability test adapted for flow cytometry (FCM), Muse cell analyzer (Muse) and Comet Assay (CA). Capsicum annuum L. was chosen as a study species based on the recalcitrant morphogenic nature of its protoplasts. After isolation and purification, the FCM and Muse allowed assessing large numbers of protoplasts, and the CA the protoplast nuclei within a short period. Using the adjusted procedures, various cytological characters could be evidenced by FCM and Muse, and different levels of genomic damage evaluated by CA, allowing the discrimination and measurement of the viable protoplasts. Considering these results, the present study introduces improved quantitative procedures for viability tests. Moreover, aiming at plantlet regeneration, distinct applications should employ to measure the protoplast viability and thus define the most adequate isolation and purification procedures. Contributing with this purpose, guidelines present here to adequate and standardize the laboratory conditions for protoplast viability test using FCM, Muse and CA.