Natural Ventilation

To improve the air exchange and thus the growth, normality and quality of the plants, the vessel need to be ventilated i) naturally (natural ventilation) or ii) forcedly (forced ventilation). Natural ventilation is the energy-efficient process of bringing outer fresh air inside the culture vessel and extracting the same amount of air from the vessel. Natural ventilation generally takes place through the air gap between the vessel and the lid or through a gas permeable microporous film (pore diameter 0.2 - 0.5 ^m; Figure 1a) attached on the lid (Figure 1b) or on the wall of the vessel. Driving force for gas exchange in a tissue culture vessel under natural ventilation are i) the pressure gradient between the inner and the outer environment ii) the temperature gradient between the inner and the outer environment and iii) the velocity and current pattern of the air surrounding the vessels. Therefore, the shape of the vessel, orientation of the lids and vents, air current and environment around the vessels will affect the number of air exchanges of naturally ventilated vessels (Kozai and Kubota, 2001). Air current speed around a vessel is experimentally confirmed to enhance air exchange of the vessel (Ibaraki et al., 1992).

Natural ventilation through the air gap between the vessel and the lid is probably the simplest way to improve the air exchange between the outer environment and the in vitro culture vessel environment. Loosely fitted lids have been found to improve the growth and quality of micropropagated plants (Jackson et al., 1991). However, this type of natural ventilation can increase the risk of microbial contamination especially when sucrose is used as a sole carbon source in the culture medium. Generally, the mass of medium, plant material and the air itself in the vessel is a little warmer than the surroundings, and thus, compared to the photoperiod, the temperature in the vessel is usually lower by 1-3 C during the dark period. This can create a partial vacuum, which pulls surrounding air into the vessel, and is one of the causes of exogenous contamination. Therefore, there is a need to vent the culture vessel with a reliable integral submicron membrane or the gas permeable film, which is part of a well-closed vessel.

Figure 1. a) Scanning Electron micrograph of a Millipore filter disc; b) adhesive Millipore filter discs are attached on the lids of Magenta vessels to increase natural ventilation.

Currently, many types of gas permeable films are commercially available, for example, MilliSeal membrane (adhesive microporous filter disc, pore diameter 0.45 ^m; Milli-Seal, Nihon Millipore Ltd.,Yonezawa, Japan; Figure 1a, b), MilliWrap membrane seal (Microporous sheet; pore diameter 0.45 ^m; Millipore Corporation, USA), transparent polypropylene disc (thickness 25 |m; Courtaulds Films, Bridgewater, Somerset, UK), Teflon membranes (Vent Spots; pore diameter 0.5 ^m; Flora Laboratories; Australia), Suncap closer (Sigma, USA), TQPL discs (adhesive microporous filter disc; TQPL supplies, UK). Ready-to-use vented vessels or lids attached with porous films/membranes are also currently available, such as, Culture Pack (culture box made of gas permeable transparent films; 25 |m in thickness; Daikin Industries, Japan), LifeGurad Sealed Vessel System (microporous filters attached to a transparent vessels; Osmotek Ltd., Israel), LifeLine Vented Lids (microporous filters attached to a semi transparent lids; Osmotek Ltd., Israel) and Phytocap closure (capping system for test tubes with 20 mm or 25 mm diameter; Phytotechnology Laboratories, USA).

The diffusion rate of CO2 through these gas permeable films is proportional to the difference in CO2 and water vapour concentrations inside and outside the vessel and the gas conductance of the gas permeable film. The efficiency of these ventilation systems can be evaluated by measuring the time taken, (t50), for half of an injected standard sample of a marker gas, e.g. ethylene, to escape from the vessel (Jackson et al., 1991; 1994). Figure 2 shows the results of t50 measured in the culture vessels capped with various capping system. The t50 for the removal of ethylene from a 120 ml glass vessel by attaching one adhesive microporous filter discs (filter pore diameter 0.45 ^m; Millipore Corporation, USA) on the hole (8 mm) of the lid was only 10 min compared to 30 min for the Suncap closer (Sigma, USA), 147 min for the polypropylene disc, 195 min for the aluminium foil functioning diffusively and 285 min for the cotton bung. Microporous filter disc are available in different sizes and pore diameters and the ventilation rate can be varied with the size and shape of the culture vessels. Therefore, the key parameter to compare different types of vessel or the ventilation system is the number of air exchanges as proposed by Kozai et al. (1986). For example, a 25 mm test tube could have 8-10 numbers of exchanges from a 10 mm microporous filter, while the same filter on a Magenta vessel would be only 1-2 numbers of air exchanges. By increasing the number of filter disc attached on the vessel it is possible to increase the number of air exchange. Depending upon the plant species and number of plants, number of air exchange may be necessary to increase or decrease.

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