Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata

Abstract

During in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.