Metabolic comparison of cryopreserved and normal cells from
<Emphasis Type="Italic">Tabernaemontana divaricata</Emphasis> suspension cultures |
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Authors: | Liliani?Suhartono Frank?Van?Iren Ward?de?Winter Sittiruk?Roytrakul Young?Hae?Choi Email author" target="_blank">Robert?VerpoorteEmail author |
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Institution: | (1) Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, Leiden, The Netherlands;(2) Section of Plant Cell Physiology, Institute of Biology, Leiden University, Leiden, The Netherlands |
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Abstract: | A cryopreservation protocol for Tabernaemontana divaricata suspension cell cultures (6 Div BW 101) was established. Cells were precultured in MS medium supplemented with 0.5 and 0.33 M mannitol for 2 or 3 days following with incubation in MS media with a mixture of 1 M sucrose, 0.5 M glycerol, 0.5 M DMSO, and 0.04 M L-proline as cryoprotectant in an ice bath for 20 min. The cells were transferred into 2 ml cryogenic vials and then, the vials were put into the cryogenic container prior to placing at a −80 °C freezer for 4 h followed by rapid immersion in liquid nitrogen. The cells were transferred without washing a MS medium solidified with 7% (w/v) agarose. Cells that were precultured 3 days after subculturing in MS medium supplemented with 0.5 M mannitol for 3 days, showed growth recovery. Metabolic profiling of control and cryopreserved Tabernaemontana divaricata cells was performed by 1H-NMR spectroscopy combined with PCA, GC, and HPLC. Differences of metabolic accumulation were found in the level of several amino acids, carbohydrates, and fumaric acid. However, the levels of the main alkaloid precursor tryptamine did not change. |
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Keywords: | cryopreservation1H-nuclear magnetic resonance spectroscopy metabolic profiling suspension cell cultures Tabernaemontana divaricata |
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