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in the culture vessel should contribute towards the high production efficiency and value-added product quality, thereby considerably expanding the application of plant micropropagation techniques. A list of micropropagated plant species along with summarize results benefited from different ventilation systems are presented in Table 1. Some of these benefits are discussed below.

a) By using ventilated vessels (natural or forced ventilation), CO2 depletion during the photoperiod can considerably be reduced, and depending on the plantlet size, CO2 concentrations can be maintained near to atmospheric concentration. CO2 concentration could be enriched above atmospheric very easily by increasing concentrations outside the vessels (Kozai et al., 1999) and thereby increasing photosynthesis and giving even higher yield (Zobayed et al., 2000a; Kozai et al., 1999; Kubota and Kozai, 1992). Natural or forced ventilation systems also adequately reduce the accumulation of CO2 in the dark period. Therefore, it is possible to optimize the growth of in vitro plants throughout the culture period by using forced ventilation system. However, the concentrations of different gases including CO2, water vapour, O2 and ethylene in the vessel headspace under natural ventilation can be manipulated by number of factors such as the photosynthetic efficiency of the chlorophyllous plants, metabolic activity of the plants, the size and leaf area of the plants, the culture room environment and most importantly the number of air exchanges of the vessel. Thus, the gaseous concentrations in vessels with natural ventilation are often unpredictable and uncontrollable (Figure 11a). In contrast, one of the most important features of the forced ventilation system is that the number of air exchanges can be controlled precisely during the growing cycle and thus almost all the gaseous concentrations including CO2 and water vapour concentration can be manipulated to maximize the biomass production (Figure 11b). Figure 11, shows different gaseous concentration in the culture vessel containing eucalyptus plantlets. In a natural ventilation system (Figure 11a) the number of air exchange was almost constant (uncontrollable) throughout the culture period and thus with increase of biomass and the photosynthetic activity of plantlets in the culture vessel the CO2 concentration was depleted during the photoperiod with time which seriously restricted the net photosynthetic rates (Figure 11a) and thus the growth. On the other hand in the forced ventilation system the CO2 concentration in the culture vessel during the photoperiod was constant throughout the growth period by increasing the flow rate of the ventilation system (thus the number of air exchange) every 3-4 days. As a result the net photosynthetic rates per plantlet increased with time followed by growth.

(b) Using t50's as indicators (Figure 2 and Armstrong et al., 1997) both forced and natural ventilation systems were found to maintain the vessels free from ethylene, and this also would probably apply to any other potentially toxic gases. Thus the need for the use of ethylene inhibitors, absorbents or antagonists can be eliminated.

(c) By providing appropriate ventilation the adverse effects of accumulated ethylene and unusually high relative humidity such as hyperhydricity of plants can be prevented. The symptoms of hyperhydricity in the relatively airtight vessels include malfunctioning of stomata, chlorophyll deficiency, cell hyperhydricity, hypolignification, reduced deposition of epicuticular waxes and changes in enzymatic activity and protein synthesis (Ziv, 1991a and b). Hyperhydrated plantlets appeared to be 'glassy' with thick, translucent, and brittle leaves, showing excessive basal growth and callus formation (Ziv, 1991b) and with little or no root development.

Figure 12. a, b) Stomata on the abaxial surface of the leaf of potato plantlets grown under photoautotrophic conditions with forced ventilation (a) and under photomixotrophic conditions with natural ventilation (b), photographs were taken during the dark period; c, d) Transverse sections of leaves of Eucalyptus plantlets grown under photoautotrophic conditions with forced ventilation (c) and under photomixotrophic conditions with natural ventilation (d).

Figure 12. a, b) Stomata on the abaxial surface of the leaf of potato plantlets grown under photoautotrophic conditions with forced ventilation (a) and under photomixotrophic conditions with natural ventilation (b), photographs were taken during the dark period; c, d) Transverse sections of leaves of Eucalyptus plantlets grown under photoautotrophic conditions with forced ventilation (c) and under photomixotrophic conditions with natural ventilation (d).

The physiological abnormalities and morphological disorders mentioned above are the major shortcomings of the conventional micropropagation system. The incorporation of ventilation in the culture vessel especially the forced ventilation system ensure the normality of plants and significantly increases the biomass production and net photosynthetic rate (Kubota and

Kozai, 1992; Zobayed et al., 1999b and c; Hoe and Kozai, 1999), increase the leaf area, shorten the multiplication and growth period. By using ventilation in the culture vessel, i) the occurrences of leaf hyperhydricity in cauliflower (Zobayed et al., 1999a and b), potato (Zobayed et al., 2001a) and eucalyptus (Zobayed et al., 2001b) has been avoided, ii) leaf epinasty in cauliflower (Figure 6b; Zobayed et al., 1999a) and leaf and flower-bud abscission inAnnona (Armstrong et al., 1997; Zobayed et al., 2002) was prevented, iii) failure of leaves to unfold in potato in low-ventilated system was improved (Zobayed et al., 2001a), iv) regenerated shoot maturation in Annona was increased (Zobayed et al., 2002) and carbohydrate accumulation in the plant tissues increased (Wilson et al., 2001). Along with photoautotrophic micropropagation system, forced ventilation ensures in vitro acclimatization of micropropagated plants during rooting stage (Kozai et al., 1999) and the ex vitro survival percentage increases significantly after transplanting even without any special ex vitro acclimatization.

(d) Various anatomical and physiological abnormalities common in the conventional micropropagation system can be prevented by applying ventilation. These abnormalities include the production of malfunctioning stomata, which remain permanently widely open even in the dark and unorganised palisade and mesophyll tissues in leaf. Figure 12 shows the abaxial surface of the leaf taken in the dark period. Stomata on the leaves grown under forced ventilation remained close during the dark period (Figure 12a; Zobayed et al., 1999d, 2000b and 2001c), while in the airtight system (natural ventilation), stomata remained fully open even in the dark period (Figure 12b). This suggested that ventilated plants had functional stomata. Transverse sections of leaves of the plants grown under forced ventilation showed normal, organized palisade and spongy mesophyll layers (Figure 12c), whereas, the airtight treatment showed thin and unorganised palisade and spongy mesophyll layers (Figure 12d).

(e) in conventional airtight system, accumulated ethylene result a low chlorophyll content in leaf; the chlorophyll contents can increase significantly by using forced ventilation and this, together with the co2 enrichment, no doubt responsible for the high photosynthetic rate followed by high yield observed in many plant species (Table 1).

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