The effect of the type of closure on the gas composition of the headspace and the growth of GF 677 peach × almond rootstock cell suspension cultures

Abstract

The influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO2 and C2H4 levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO2 increased up to 18%, with a net production of about 0.8 mmol CO2, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 µl liter−1 after 96 h. When aluminum foil closures were used, C2H4 and CO2 increased over the first 72 h, reaching concentrations of about 6 µl liter−1 and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 µl−1 and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs, slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most cells occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.