Fundamental Studies on Environments in Plant Tissue Culture Vessels

(2) Effects of Stoppers and Vessels on Gas Exchange Rates between Inside and Outside of Vessels Closed with Stoppers

Abstract

The growth and development of a plantlet in vitro may be affected by the gas environment in a vessel. The gas micro-environment in the vessel may be in turn influenced not only by the generation and absorption of gas by the plantlet and the culture medium, but also by the gas exchange between the room air and the air in the vessel. The gas exchange rate between the room air and the inside air may be varied with the combination of a vessel and its stopper.
This paper describes a method for estimating the number of air changes per hour and the coefficient of gas exchange for various vessels closed with various stoppers. The measured values are also given for various vessels closed with various stoppers.
In the measurement, carbon dioxide gas was used as tracer gas. The gas concentration inside and outside the vessel was measured by using a gas chromatograph at a certain time interval.
Three kinds of stoppers were tested. Namely, aluminium foil cap C_A, plastic formed cap C_P, and silicon foam rubber plug P_S. Six kinds of vessels were tested. Three of them were glass test tubes and the rest were glass Erlenmeyer flasks (see Tables 1, 2 and Fig. 1).
The number of gas changes per hour estimated by using carbon dioxide gas as tracer gas, E_c, was estimated by the following equation:
E_c=-1/T⋅lnK-K_ou/K₀-K_ou
where T is the time interval from time 0 to time t, K the gas concentration at time t, K₀ the gas concentration at time 0, K_ou the gas concentration outside the vessel.
The coefficient of line carbon dioxide gas exchange, q_cc, and the coefficient of area carbon dioxide gas exchange, q_ac were, respectively, estimated by the following equations:
q_cc=V⋅E_c/C, q_ac=V⋅E_c/A
where V is the inside air volume of the vessel, C the inside circumference of the vessel, and A the inside area at the lip.
q_cc is used for the vessel closed with the stopper with no gas permeability, such as aluminium foil cap and plastic formed cap. q_ac is used for the vessel closed with the stopper with a certain degree of gas permeability, such as silicon foam rubber plug.
The number of gas changes per hour estimated by using carbon dioxide gas as tracer gas, E_c, for various vessels closed with various stoppers is shown in Table 3. The variations of E_c for different vessel-stopper combinations were considerable. As for the vessels closed with the same material of stopper, E_c was inversely proportional to the inside air volume of the vessel. As for the same vessel closed with different caps, E_c was the largest for C_P, and the smallest for C_A.
The coefficients of carbon dioxide gas exchange for various vessels closed with various stoppers, q_cc and q_ac, are given in Table 4. q_cc and q_ac showed a rather constant value for different vessels closed with the same stopper, regardless of the large variations of the inside air volume of the vessels. q_cc of C_P was about 10 times larger than that of C_A for the same vessel.
It is shown that the number of gas changes per hour and the gas exchange rate between the room air and the inside air are considerably affected by the vessel-stopper combination. Therefore, the concentration of a gas, and hence the growth and development of a plantlet in vitro may be affected by the vessel-stopper combination.
The method proposed in this paper may be applied to any gas such as ethylene gas, water vapor, oxygen gas etc., in principle, if the corresponding gas is used as tracer gas.