Method for Selecting Anthocyanin-Producing Cells by a Cell Sorter*

Abstract

We found a novel method to select anthocyanin-producing cells by a new means of cell staining. After staining with 0.2 ppm fluorescein isothiocyanate (FITC), the protoplasts from Aralia cordata cultured cells, which were composed of high anthocyanin-producing cells and non-producing cells, were observed to be distinguishable under a fluorescence microscope. The green fluorescence of FITC from the non-producing cells was clearly ascertained by the naked eye. On the other hand, the fluorescence of the anthocyanin-producing cells was completely missing. This phenomenon resulted from compensation of the green fluorescence (λ_max 525 nm) of FITC and the green light absorption (λ_max 530 nm) of anthocyanin. Although investigations of the flow cytometric method were developed on the basis of this phenomenon, the histograms of these protoplasts expressed the distinction between the anthocyanin-producing cells and the non-producing cells. In order to prove the technique, a highly productive cell line was selected from the heterogeneous populations of anthocyanin-producing or non-producing protoplasts from A. cordata cultured cells. The number of the sorted protoplasts containing anthocyanin was counted by the naked eye, the anthocyanin-producing protoplasts were approximately 90% in the whole protoplasts and the viability was 60 - 70%. Anthocyanin concentration the sorted protoplasts was 3-fold higher as compared with the protoplasts before sorting. The anthocyanin content of the cell line selected by this method was 7.7 ± 0.6%/g dw. Its value is comparable to that (7.3 ± 0.5%/g dw) of the high anthocyanin-producing cell line selected by small-cell aggregate selection in the dark and the time for the establishment of the stable cell line was reduced to one half or one third. Our flow cytometric method developed on the basis of nonradiative resonance energy transfer (RET) is widely applicable to the selection of plant pigment-producing cells which do not possess autofluorescence.

Key words

Anthocyanin - Aralia cordata - Araliaceae - protoplast - flow cytometric analysis - nonradiative resonance energy transfer