M

-»A ♦ -»■A - -

Plantlet height

m ♦ Mi i

C02 concentration outside the vessel □ 130 jimol mol"1

♦ 1890 (¿mol mol"1 ■ 2570 jo.mol mol"1

C02 concentration inside the vessel [jj.mol mol"1] (b)

0 1000 2000 3000

C02 concentration inside the vessel [jj.mol mol"1] (b)

Figure 6. CO2 concentration profiles in a test tube type vessel at (a) different PPFs and (b) different CO2 concentrations outside the vessel (Ohyama and Kozai, 1997). The vessel contained a sweetpotato plantlet on sugar containing MS agar medium.

2.1.4. Ethylene (C2H4) concentration in the vessel with natural and forced ventilation

Ethylene (C2H4) gas, a phytohormone produced by plantlets, accumulates in an airtight vessel. In turn, C2H4 concentration affects the C2H4 production rate of the plantlet and the growth and development of plantlets in vitro. The number of air exchanges of the vessel also significantly affects C2H4 concentration in the vessel. Figure 7 shows the C2H4 concentration over time in the vessel with natural and forced ventilation, containing Lagerstroemia thorellii plantlets 30 days after transplanting. The vessels were uncapped and flushed with sterile air and then recapped at 0 h on day 30. C2H4 concentration in the vessel was then measured during the following 30 h. At the end of measurement (30 h), C2H4 concentrations in the vessels capped with a silicone rubber bung, cotton plug and polypropylene film were, respectively, about 1.20, 0.10, and 0.02 ^mol mol-1. On the other hand, it was undetectable (lower than 10 nmol mol-1) for the vessel with forced ventilation. The numbers of air exchanges of the tested vessels were, respectively, 0.2, 1.1, 1.8 and 6.0 h-1. C2H4 gas released from the vessels into the culture room does not raise the ambient C2H4 concentration because the air volume of culture room is more than

1000 times that of culture vessels. C2H4 gas is diluted with the culture room air resulting in a C2H4 concentration lower than one 0.01 |imol mol1.

Figure 7. Time courses of C2H4 concentrations in the vessels containing Lagerstroemia speciosa plantlets during the photoperiod, as affected by the number of air exchanges of the vessel (Zobayed, 2000). Vessels were capped with silicone rubber bung (open circle), cotton bung (open square), polypropylene film (solid square) and forced ventilation (solid triangle). The numbers of air exchanges of those vessels were, respectively, 0.2, 1.1, 1.8 and 6.0 h-1.

Figure 7. Time courses of C2H4 concentrations in the vessels containing Lagerstroemia speciosa plantlets during the photoperiod, as affected by the number of air exchanges of the vessel (Zobayed, 2000). Vessels were capped with silicone rubber bung (open circle), cotton bung (open square), polypropylene film (solid square) and forced ventilation (solid triangle). The numbers of air exchanges of those vessels were, respectively, 0.2, 1.1, 1.8 and 6.0 h-1.

2.1.5. Oxygen concentration in the vessel

Oxygen concentration of atmospheric air is about 210,000 ^mol mol-1 or 21% (volume/volume) which is approximately 60 times higher than the CO2 concentration of atmospheric air. Decrease or increase in O2 concentration in the vessel due to the respiratory and photosynthetic activities of plantlets is associated with the same degree of increase or decrease in CO2 concentration in the vessel. A change in CO2 concentration in the vessel from 100 ^mol mol-1 (0.01% volume/volume) to 10 mmol mol-1 (1%) results in a change in O2 concentration from 21 to 20%. This 1% decrease in O2 concentration does not affect the respiratory and photosynthetic activities of plantlets. in an extreme case, CO2 concentration in the vessel can reach 2-4% resulting in an O2 concentration of 19-16%. Even so, this 2-

4% decrease in O2 concentration does not affect the respiratory and photosynthetic activities of plantlets significantly.

Reducing O2 concentration as low as 10% (volume/volume) reportedly increased the net photosynthetic rate and growth of photoautotrophically cultured Chrysanthemum plantlets (C3 plants) (Tanaka et al., 1991). This is due to the reduced photorespiration rate by lowering O2 concentration. In vitro O2 concentration can be strategically controlled by forcedly ventilating the vessels with air of desired O2 and CO2 concentrations.

2.2. Relative humidity

Figure 8 shows the relative humidities and air temperatures in the vessels over time with and without gas permeable film, and with the absence or presence of potato plantlets in vitro 2 days and 24 days after transplanted on to the culture medium. Relative humidity of culture room air during the photoperiod is often relatively low (around 40%), because the room air is dehumidified (water vapor of the room air being condensed as liquid water) on a cooling panel of the air conditioner. It reaches 80-90% during the dark period, when the air conditioner is operated only intermittently.

On days 2 and 24, relative humidity in a vessel during the dark period is higher than 98% in all treatments. Inside a completely airtight vessel, the relative humidity during the dark period is, theoretically, a little lower than 100%. This is because the water potential of air should be the same as that of culture medium. The water potential of culture medium ranges between -300 kPa and -800 kPa, which is equivalent to relative humidity of 99.8-99.5%. Relative humidity in a vessel that is not completely airtight would be lower than 99.5% due to the exchange of air from inside the vessel with air outside the vessel where relative humidity is lower than inside. During the photoperiod, however, relative humidity in the vessel with gas permeable film and plantlets is around 80% on day 2 and 90% on day 24.

It is often believed that relative humidity is always nearly 100% in a conventional vessel. However, in Figure 8, the relative humidity during the photoperiod is around 90% even in a vessel without gas permeable film and with plantlets. As shown in Figure.8, air temperature is about 2 degrees Centigrade higher in the vessel than in the culture room because the light is absorbed by the vessel walls, lid, medium and plantlets, and thus heat is generated in the vessel. In this case, the vessel wall temperature is lower than the dew-point temperature of the inside air, and water is condensed on the inside surfaces of the vessel walls and lid. This condensation of water does not indicate that the relative humidity in the vessel is 100%. On the contrary, it indicates that water vapor in the air is dehumidified by the vessel walls and lid, and that the relative humidity is lower than 100%.

The transpiration rate of plantlets is proportional to the saturation deficit of water vapor and, at relative humidities higher than 80%, the saturation deficit is almost proportional to the difference between 100 and the actual relative humidity. Thus, the transpiration rate at relative humidity of 80% (100-80 = 20) is 10 times higher that those at relative humidity of 98% (100-98 = 2) at the same air temperature.

Effects of relative humidity on physiological and anatomical characteristics are described in a later chapter (Chapter 6).

Loss of water from the medium in the vessel with high number of air exchanges is generally significant and the medium tends to dry up when the culture period is longer than one month. This problem can be solved by two simple methods. One is to simply supply more volume of medium in the vessel. Another is to keep the relative humidity of culture room near 80% during the photoperiod. When the relative humidities of the room air is either 40% or 80%, and the relative humidity in the vessel is 90%, the difference in relative humidity between inside and outside the vessel are 50% and 10%, respectively. Consequently, the amount of water loss from the vessel in culture room air at 80% RH is approximately 20% of a vessel in culture room air at 40% RH. Relative humidity of 70-80% in the culture room can be achieved by using a humidifier and/or by raising the surface temperature of cooling panels of air conditioners to reduce condensation of water on it.

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