Ventilation

For healthy growth, all parts of a plant must exchange their internal gases readily with those in the surrounding air. Nevertheless, in conventional micropropagation systems the exchange of gases between tissues and the air is frequently restricted (Kozai et al., 1991; Jackson et al., 1987). Many plant species when grown in vitro release a variety of substances, which may accumulate and have significant effects on growth and development of plants in vitro (Heyser and Mott, 1980). The most widely studied gaseous product from cultures is ethylene.

4.1. Ethylene

Ethylene is a volatile that has received considerable study as a regulator of plant growth. Ethylene is known to accumulate in the culture vessel especially with a very low number of air exchange and to be associated with various physiological responses of in vitro grown plants during growth and development such as inducing epinasty (Zobayed et al., 2001a), causing leaf (Armstrong et al., 1997; Lemos and Blake, 1994) and flower bud abscission (Zobayed et al., 2002), reducing photosynthesis (Zobayed et al., 1999b) and thus plant growth (Jackson et al., 1991). Plants in vitro can be affected by ethylene and result in, for example, poor cell differentiation (Miller and Roberts, 1984), an absence of somatic embryogenesis (Meijer and Brown, 1988; Purnhauser et al., 1987; Roustan et al., 1989), reduced shoot height and leaf area (Jackson et al., 1987 and 1991) and poor callus proliferation and growth (Adkins et al., 1990 and 1993).

The accumulation of ethylene in the culture vessels is also responsible for the hyperhydricity of a number of species (Jackson et al., 1991). The number of air exchanges of the vessel significantly affects ethylene concentration in the culture vessel and thus under forced ventilation, ethylene cannot accumulate in the culture vessel due to the continuous flushing out of air from the vessel.

Figure 8 shows ethylene concentrations over time in the vessel with natural and forced ventilation, containing Annona squamosa L. plantlets (Zobayed et al., 2002). The vessels were uncapped and flushed with sterile air and then recapped at 0 h on day 46. Ethylene concentration in the vessel was then measured during the following 48 h under airtight system, natural (polypropylene disc) and forced ventilation.

Ethylene concentrations increased rapidly in the airtight vessel and reached peak levels (1.3 pmol mof1 in A. squamosa and 1.15-1.17 pmol mof'in A. muricata) within only 24 - 48 h of culture. For both the species, the pattern of the changes in ethylene concentration under natural ventilation was similar to that in the airtight vessels (Figure 8), and the peak concentration was only ca. 0.1 pmol mof1 for both the species. Under forced ventilation, no ethylene accumulation was observed in the culture vessels, probably because any ethylene (or other toxic gases) produced by the plants was flushed out by the forced ventilation. It is interesting that even in the airtight vessels ethylene concentrations reached equilibrium: in this case net production also ceased rather abruptly and this strongly suggests the switching-in of a negative feedback mechanism.

4.2. Carbon dioxide

Where cultures are grown under a day: night regime, in a conventional micropropagation system with limited air exchange, CO2 concentrations fluctuate due to the respiration and photosynthesis of the plants. During the dark period, when photosynthesis is not occurring, CO2 concentrations increase through respiratory metabolism of glucose by glycolytic and tricarboxylic acid pathway (Lowe et al., 2003). During the photoperiod the photosynthetic activity of chlorophyllous plants in the culture vessel results in a decline in CO2 concentration. Depletions of CO2 concentration during the photoperiod is a common phenomenon as reported by many authors (Desjardins et al., 1988; Kozai et al., 1987; Kozai and Iwanami, 1988; Solarova et al., 1989; Zobayed et al., 1999a; Kozai et al., 1999). In airtight vessels the concentration may drop to levels that are generally considered to be limiting (Buddendorf-Joosten and Woltering, 1994 and 1996). During dark period CO2 concentration in the culture vessel may remain much higher than the ambient in airtight vessel (De Proft et al, 1985; Fujiwara et al., 1988; Jackson et al., 1991). However the concentrations of CO2 strongly depend upon the ways in which the culture vessels are capped. Jackson et al. (1991) demonstrated that Ficus lyrata cultures with loose, intermediate and tightly sealed vessels, contained respectively 0.5. 3.4 and 8.5% CO2 in the dark period. As shown in Figure 9, in A. squamosa, the CO2 concentration in the headspaces of airtight vessels rose rapidly during any dark period and fell rapidly at the onset of any subsequent photoperiod (Zobayed et al., 2002). At the end of the dark period, the CO2 was very high (approximately 1.5% or 15000 pmol mof'), while in the photoperiod it decreased approximately 40 pmol mof1 and, because gas exchange with the outside atmosphere was restricted, this represents the CO2 compensation point for these plants. In natural ventilation (capped with polypropylene disc) the Co2 concentration decreased during the photoperiods to 88 pmol mof1. It should be noted that the rate of net photosynthesis at 88 pmol mof1 is significantly less than at atmospheric CO2 concentration. With forced ventilation, during the photoperiod, the CO2 concentrations were sustained at even higher levels (200 pmol mof1) than in the natural ventilation system despite an appreciably higher rate of consumption (i.e. higher photosynthetic rates). The difference reflects the impedance to gas exchange of the polypropylene disc in the natural ventilation system. In the dark period, the CO2 concentration in the natural ventilation system did not rise as steeply as in the airtight vessels but attained a concentration of 480 pmol mof1, and again reflecting the impedance to gas exchange of the polypropylene disc. However, the more effective gas-exchange induced by forced ventilation prevented the CO2 concentrations from exceeding atmospheric levels. Thus, concentrations during the dark period with forced ventilation were 0.7* and 0.02* those in the natural ventilation and airtight systems, respectively. It has also been reported that CO2 concentration in the culture vessel can be increased significantly during the photoperiod. Woltering (1989) found 1.3% and 13% CO2 with Gerbera jasmesonii in semi-closed and tightly sealed containers respectively during the photoperiod. Righetti et al. (1988) found 20% CO2 inPrunus shoot cultures grown in the light in (airtight) jars. Jackson et al. (1991) also reported 8.5% CO2 in the dark in airtight vessels containing Ficus lyrata and this decreased to 0.2% and 1% at the end of the photoperiod with loose and intermediate sealing of the culture vessels respectively. While cauliflower plantlets were grown in vitro in an airtight vessel, Zobayed et al. (1999b) found high concentration of CO2 in the culture vessel during the photoperiod, which was just below atmospheric in well ventilated vessels. To observe this occurrence, we cultured cauliflower cuttings in a relatively small culture vessel (50 ml) and capped with silicone rubber bung (airtight system) or attached with forced ventilation (flow rate 5 ml min-1 throughout the culture period). in the airtight vessel, from days 4 to 7 when the new leaves were unfolded and beginning to photosynthesis, the CO2 concentration in the culture vessel decreased to nearly compensation point, 40 ^mol mol-1 (Figure 10a) during the photoperiod. Around day 13, the concentration increased to reach 7200 ^mol mol-1. This increase of CO2 concentration was thought to have been due to the accumulation of ethylene (Figure 10a) in the culture vessel, which impeded photosynthesis (Figure 10a), so that respiratory CO2 accumulated; this also probably indicated the first stages of ethylene-induced premature senescence of the tissues. It should be mentioned that cauliflower is known to very sensitive to accumulated ethylene (Zobayed et al., 1999a and b). In case of the forced ventilation system the CO2 concentration in the culture vessel was just below atmospheric (290 - 300 ^mol mol-1) during the first 14 days of culture and was reduced to 270 ^mol mol-1 at the end of the experiment. No ethylene was accumulated in the culture vessel and the net photosynthetic rate (per plantlet) was gradually increased with time (Figure 10b).

4.3. Relative Humidity

Relative humidity in the culture vessel is an important environmental factor that affects the water relations of cultured tissues (Jeong et al., 1995). Relative humidity is normally high in the culture vessel and may have some deleterious effects on cultured plantlets. Several studies have demonstrated that lowering relative humidity in the culture vessel improved the resistance of tissues to water loss (Wardle et al., 1983; Smith et al., 1990 and 1992). It has also been reported that the growth of in vitro grown plantlets may be improved by manipulating the relative humidity inside culture vessels (Wardle et al., 1983; Smith et al., 1990). The improvement in gas exchanges under forced ventilation might have been expected to reduce the relative humidity in the culture vessel which can enhance transpiration. Thus, an improved growth obtained under forced ventilation, may also, at least partially result in the increased transport of minerals from the culture medium. Loss of water from the medium in the vessel under forced ventilation system with high number of air exchanges is generally significant and the medium tends to desiccate when the culture period is longer than one month. This problem can be solved by any of the following three methods: i) supply more volume of medium in the vessel (Kozai and Kubota, 2001); ii) keep the relative humidity of culture room 70-80% during the photoperiod (Fujiwara and Kozai, 1995) or iii) supply humid air in the culture vessel by the forced ventilation system (Zobayed et al., 1999a and b, Armstrong et al., 1997).

Other volatile substances released in vitro are ethane, ethanol, methane, acetylene and acetaldehyde (Thomas and Murashige, 1979a and b). Rice (Oryza sativa L.) callus culture modified the atmosphere of the culture vessel by producing CO2, ethylene and ethanol, while utilizing oxygen (Adkins et al., 1990). These changes in the gaseous atmosphere of the culture vessel can suppress the growth of cultured explants and promote necrosis (Adkins, 1992). A well ventilated vessel can overcome all of these problems and improve the growth and quality of the plantlets significantly.

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