A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30 degrees C (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthocyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was decreased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influenced by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg.g-fresh cell(-1) was obtained on day 9 by a temperature-shift from 30 degrees C, after 3-d culture, to 15 degrees C. The highest anthocyanin yield of 318 mg.L-1 on day 12 was achieved when the temperature was shifted from 30 degrees C, after 5-d culture, to 20 degrees C. For a global optimization of both the yield and productivity, the optimum anthocyanin yield and productivity of 272 mg.L-1 and 30.2 mg.L-1.d(-1) on day 9 were achieved by a two-stage culture with a temperature-shift from 30 degrees C after 3 d to 20 degrees C.

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.