Abstract

The objective of this study was to improve metabolite accumulation of grape cells by manipulating culture and environmental conditions. Response surface methodology (RSM) was used to design and conduct the study with the independent variables being: percent depletion of KH₂PO₄ or NH₄NO₃ and light intensity. Wounded grape leaves (Bogazkere cv.) were used for the initiation of callus formation. Grape calli were placed on the modified Murashige and Skoog (MS) media (from 0 to 75% KH₂PO₄ or NH₄NO₃ depleted) under various light regimes (from 30 to 120 μmol m^-2s^-1), and incubated in a controlled environment for three weeks to stimulate secondary metabolite biosynthesis. The anthocyanin - total phenolic (TP) contents of the calli were determined using chemical analyses and the antioxidant capacity of the cultures were determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Higher KH₂PO₄ depletion and 75 μmol m^-2 s^-1 light intensity favored the biosynthesis of anthocyanins and the other phenolic compounds and resulted in elevated antioxidant capacity in the cultures. However, an increase in KH₂PO₄ or NH₄NO₃ depletion resulted in a significant decrease in cell weight. Similar to that, increase of light intensity was correlated with decrease of cell weight. Further elevation of light intensity and KH₂PO₄ -NH₄NO₃ depletion was fatal to grape cells and resulted in formation of non-growing watery-brownish cultures. Individual phenolic compounds found in the grape cell cultures were identified using the gas chromatography-flame ionization detector (GC-FID) system. Catechin and epicatechin were identified as the most abundant simple phenolic compounds present in the cultures.         

Key words: Antioxidant capacity, callus, grape, phenolic compounds, response surface methodology.

Abbreviation

Abbreviations: RSM, Response surface methodology; HPLC, high performance liquid chromatography; NAA, 1-naphthaleneacetic acid; BA, 6-benzylaminopurine; 2,4-D, 2, 4- dichlorophenoxy acetic acid; DPPH, 1-diphenyl-2-picrylhydrazyl; TP, total phenolic; GC, gas chromatography; MS, Murashige and Skoog; Chs, Chalcone synthase.