Metabolic comparison of cryopreserved and normal cells from Tabernaemontana divaricata suspension cultures

• Published in: Plant cell, tissue and organ culture Vol. 83; no. 1; pp. 59 - 66
• Main Authors: Suhartono, L, Iren, F. van, Winter, W. de, Roytrakul, S, Choi, Y.H, Verpoorte, R
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01-10-2005
• Subjects: 2,4-D; amino acids; Biological and medical sciences; Biotechnology; carbohydrates; cell suspension culture; cryopreservation; culture media; dose response; Eukaryotic cell cultures; fumaric acid; Fundamental and applied biological sciences. Psychology; germplasm conservation; medicinal plants; metabolism; metabolites; methodology; Methods. Procedures. Technologies; micropropagation; Miscellaneous; Plant cells and fungal cells; quantitative analysis; Tabernaemontana divaricata; tryptamine; Cell culture; Suspension culture; Vegetals; Cryopreservation; suspension cell cultures; NMR spectrometry; Metabolism; 1H-nuclear magnetic resonance spectroscopy; metabolic profiling; Comparative study; Tabernaemontana divaricata
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-005-3869-8

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.