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The decrease in CO2 concentration in the vessel after the onset of the photoperiod shows that chlorophyllous cultures have photosynthetic ability. The resulting low CO2 concentration during the photoperiod shows that photosynthetic activity of chlorophyllous cultures is restricted mainly by low CO2 concentration. By definition, net photosynthetic rate of plantlets in vitro is zero at a CO2 compensation point even when other environmental factors are favourable for photosynthesis of in vitro plantlets.

Figure 3. A diurnal change in CO2 concentration in a culture vessel containing Ficus lyrata plantlets (Fujiwara et al., 1987). Dark period was from 6 to 14 h, and photoperiod was from 0 to 6 h and from 14 to 24 h. PPF at the culture level and air temperature in the culture room were 65 ¡xmol m'2 s'1 and 25 C, respectively.

Figure 3. A diurnal change in CO2 concentration in a culture vessel containing Ficus lyrata plantlets (Fujiwara et al., 1987). Dark period was from 6 to 14 h, and photoperiod was from 0 to 6 h and from 14 to 24 h. PPF at the culture level and air temperature in the culture room were 65 ¡xmol m'2 s'1 and 25 C, respectively.

Figure 4. Diurnal changes in CO2 concentration in the vessels containing strawberry plantlets cultured for 20 days as affected by the number of air exchanges of the vessel, N, and PPF on the empty culture shelf (Kozai and Sekimoto, 1988). Low N: N=1.5 h-1, High N: N=2.7 h-1, Low PPF: 34 /nol m'2 s'1, High PPF: 133 /mol m'2 s'1. Photoperiod: 8-24 h, Dark period: 0-8 h. Vessel air volume: 46 ml (test tube type vessel). Gas permeable film was attached on the lid for the high N treatment.

Figure 4. Diurnal changes in CO2 concentration in the vessels containing strawberry plantlets cultured for 20 days as affected by the number of air exchanges of the vessel, N, and PPF on the empty culture shelf (Kozai and Sekimoto, 1988). Low N: N=1.5 h-1, High N: N=2.7 h-1, Low PPF: 34 /nol m'2 s'1, High PPF: 133 /mol m'2 s'1. Photoperiod: 8-24 h, Dark period: 0-8 h. Vessel air volume: 46 ml (test tube type vessel). Gas permeable film was attached on the lid for the high N treatment.

Decrease in CO2 concentration in the vessel after the onset of photoperiod is more remarkable at lower number of air exchanges of the vessel and at higher net photosynthetic rates of cultures per vessel. Figure 4 shows the daily changes in CO2 concentration in the vessel as affected by the number of air exchanges of the vessel and PPF. Steady-state CO2 concentration in the vessel during the photoperiod is lower at a lower number of air exchanges of the vessel, higher PPF and on day 20 than on day 5. This is because the net photosynthetic rate per vessel is higher at higher PPF and as a result of the greater leaf area of in vitro plantlets at later stages. High net photosynthetic rate of plantlets in vitro lowers the steady-state CO2 concentration in the vessel and the low CO2 concentration restricts the photosynthesis and growth of plantlets in vitro.

Figure 5 shows time courses of steady state CO2 concentration during the photoperiod in vessels containing potato (Solanum tuberosum L.) plantlets cultured on sugar-free medium during a 15 d photoautotrophic culture period, as affected by the number of air exchanges of the vessels and the number of plantlets per vessel. In all the treatments, the steady state CO2 concentration during the photoperiod decreased with the passage of days. It was lowest in LS treatment throughout the culture period because the number of air exchanges of the vessel was 0.75 h-1 in LS treatment, compared with 5.0 h-1 in LM and LL treatments. Steady state CO2 concentration is lower in LM treatment than in LL treatment (although the numbers of air exchanges of the vessel are the same, 5.0 h-1), because the number of plantlets per vessel is 2 in LL treatment and 4 in LM treatment. Net photosynthetic rate per vessel was greater in LM treatment than in LL treatment.

Days of treatment

Figure 5. Time courses of steady state CO2 concentration during the photoperiod in Magenta type vessels (air volume: 370 ml) culturing potato plantlets for the 15 d photoautotrophic culture period (Niu and Kozai, 1997). PPF: 105 /umol m'2 s'1 with a photoperiod of 16 h'1. CO2 concentration in the culture room: 1300 /umol mo!1. Number of air exchanges: 0.75, 5.0 and 5.0 h'1 in LS, LM and LL treatments, respectively. Number of plantlets per vessel: 2, 4 and 2, respectively.

Days of treatment

Figure 5. Time courses of steady state CO2 concentration during the photoperiod in Magenta type vessels (air volume: 370 ml) culturing potato plantlets for the 15 d photoautotrophic culture period (Niu and Kozai, 1997). PPF: 105 /umol m'2 s'1 with a photoperiod of 16 h'1. CO2 concentration in the culture room: 1300 /umol mo!1. Number of air exchanges: 0.75, 5.0 and 5.0 h'1 in LS, LM and LL treatments, respectively. Number of plantlets per vessel: 2, 4 and 2, respectively.

In order to keep the CO2 concentration in the vessel during the photoperiod at a constant level over the culture period, either the CO2 concentration in the culture room or the number of air exchanges of the vessel or both must be increased with the passage of days. This is because, in general, net photosynthetic rate per vessel increases with increasing leaf area per vessel, especially at higher PPF. Mathematical relationships among the CO2 concentrations inside and outside the vessel during the photoperiod, the number of air exchanges of the vessel, PPF and photosynthetic characteristics of plantlets in vitro are described in detail by Fujiwara and Kozai et al. (1995a) and Niu and Kozai (1997). Effects of CO2 concentration on net photosynthetic rate and growth are described.

2.1.2. CO2 concentration profile in a test tube type vessel

In a relatively small but long culture vessel like a test tube, the air movement due to free convection in the vessel is significantly restricted and thus a significant vertical variation of CO2 concentration in the vessel is observed. Figure 6a shows the CO2 concentration profiles during the photoperiod in a test tube with 25 mm in diameter and 120 mm in height, containing a sweetpotato (Ipomoea batatas (L.) Lam.) plantlet on sugar-containing MS agar medium at different PPF. The CO2 concentration outside the test tube is 360 ^mol mol-1. At higher PPF, the vertical gradient of CO2 concentration is steeper due to the higher net photosynthetic rate per vessel. CO2 concentration just below the plantlet height was 180-190 ^mol mol-1 lower than that just below the lid of test tube. Figure 6b shows the CO2 concentration profiles during the photoperiod in the test tube at PPF of 100 ^mol m-2 s-1 and different CO2 concentrations outside the vessel. At higher CO2 concentration outside the test tube, the vertical gradient of CO2 concentration is steeper due to the higher net photosynthetic rate per vessel. These CO2 concentration profiles show that restricted air movement resulting in limited diffusion of CO2 gas in the vessel reduces the flow of CO2 from the air into the plantlet, thereby reducing net photosynthetic rate. The vertical gradient of CO2 concentration is not observed in a box type vessel like the Magenta vessel in which air movement due to free convection is more enhanced than in the test tube type vessel.

2.1.3. CO2 concentration in the culture vessel with forced ventilation

Photoautotrophic micropropagation makes it possible to use a large vessel with an air volume of 10-100 L. On the other hand, it is difficult to achieve more than 3-5 h-1 air exchanges using gas permeable film for enhancing natural ventilation, because the number of air exchanges of the vessel per hour, N, is expressed by:

where R is hourly ventilation rate and V is the air volume of the vessel (note the similarity in form between Equations 1 and 3). In the case of a vessel with natural ventilation, R is almost proportional to the area of gas permeable film. As the V value increases, the area of gas permeable film must be increased at the same proportion to obtain the same value for N. This is difficult because V value increases with 3rd power of the vessel size. Thus, we need to employ a forced ventilation system using an air pump to achieve a relatively high number of air exchanges of the vessel easily. Therefore, to practically achieve a relatively high number of air exchanges for the vessel, it is necessary to employ a forced ventilation system using an air pump.

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