Chlorophyll Measurement as a Quantitative Method for the Assessment of Cytokinin-Induced Green Foci Formation in Tissue Culture

Tissue culture systems have long been exploited to study the process of organogenesis. In response to externally applied cytokinins, pluripotent cells proliferate into green calli and subsequently regenerate shoots. Conventionally, the cytokinin-induced greening phenotype has been evaluated by counting numbers of green foci or to present photographic evidence of morphological changes. However, because the structure of calli is disorganized and the development of pigmentation takes place gradually from pale white through yellow to green, adequately defining and counting green foci remains difficult. In this study, we employed chlorophyll measurement as an alternative method to statistically assess the greening phenotype in tissue culture material. We found that N,N-dimethylformamide was the most effective solvent for the extraction of chlorophylls from callus tissue and that bead disruption of the structured tissue improved solvent penetration and the consistency of results. The sensitivity of the method facilitated the quantification of chlorophylls in single-cultured root explants and the use of a spectrophotometer increased the efficiency of measuring multiple samples. Our measurements showed that chlorophyll contents from calli of wild-type and altered cytokinin response mutants (cre1; ahk3, or cytokinin hypersensitive 2 (ckh2)/pickle (pkl) were statistically distinguishable, validating the method. Our proposed procedure represents gains in efficiency and precision and leads to more robust standardization than the conventionally used counting of green foci.