Clip to close the vessel

Figure 4. Different components of an ideal large vessel for photoautotrophic micropropagation (Zobayed et al., 2004).

e) an autoclaveable multicell tray for growing plants in the culture vessel. in case the culture room is not enriched with CO2, each vessel should be connected directly to a CO2 gas cylinder or a gas mixture chamber from where a desired concentration of CO2 enriched air can be pumped into the culture vessel.

Sterilization of a large culture vessel is a major step for the successful establishment of aseptic mass propagation system in large vessels. For sterilization, autoclaving the vessel at 121C and 1.4 kg cm-1 for 20-40 min is a common procedure (Afreen et al., 2002, Nguyen et al., 1999a and b; Heo and Kozai, 1999; Heo et al., 2001; Wilson et al., 2001). When the vessel is too big to fit in the autoclave machine, for example, 125 L vessel used by Xiao et al. (2000) or when the vessel is made of non-autoclaveable plastics, such as acrylic sheet used by Zobayed et al. (1999c), the alternative procedure of sterilization is the surface sterilization by using disinfectants such as sodium hypochlorite solution, KMnO4, formaldehyde etc. The step by step sterilization procedure for a 125 L vessel, described by Xiao et al. (2000) is as follows. (1) wash the culture vessel with clean water, (2) wipe the culture vessel with 0.2% sodium dichloroisocyanurate (C3O3N3Cl2Na), a disinfectant, (3) stifle the culture vessel with KMnO4 (5 g m-3), formaldehyde (10 ml m-3) for 10 hours, and (4) spray the culture vessel with 70% ethanol before transplanting. Trays were cleaned with water, and sterilized by dipping them into 0.2% sodium dichloroisocyanurate a disinfectant solution for twenty minutes.

Planting density is an important factor for the successful commercial scale micropropagation. in a large vessel planting density can be increased noticeably without significantly reducing the dry mass of plantlets. Moreover, designing of a large culture vessel should also consider to increase planting density by manipulating nutrient supply system, selecting suitable substrate, lighting system and uniform CO2 supply. Recently, a number of reports showed that the planting density can be increased significantly in large vessels under photoautotrophic condition. For instance, 4600 potato (Xiao et al., 2000) 3000 Limonium latifolium (Xiao et al., 2000) and 2644 eucalyptus plantlets (Zobayed et al., 2000a) have been cultured per meter square culture area of large culture vessels. These planting densities are significantly higher compared to the conventional system of propagation in small vessels.

For photoautotrophic micropropagation in large vessels, the selection of a suitable supporting material is another important criterion not only for the optimum growth, easy to propagate and transplant ex vitro but also to make the system suitable for handling and sterilizing. There are evidences that roots growing in agar medium showed structural abnormalities of the tissues (Kataoka, 1994), often lacked root hairs and died shortly after transplanting, resulting in plantlets that ceased to grow (Afreen et al., 1999; Debergh and Maene, 1984). Perlite (Xiao et al., 2000) and vermiculite (Heo and Kozai, 1999) have been used for propagating plantlets in large culture vessels. However, these substrates are composed of fine granules, which are not easy to handle and thus may not be suitable as a commercial rooting substrate for using in large vessel. Polyester fiber cube (Fujiwara et al., 1988), Cellulose plug (Heo and Kozai, 1999), Rock wool cube (Kubota and Kozai, 1992) and Florialite (Zobayed et al., 1999c, 2000a,b and 2001a; Wilson et al., 2001; Afreen et al., 2002) all are available in block form of different sizes and therefore are easy to handle in large vessel. However, in case of Cellulose plug, inoculation of explants is often difficult and sometime, because of its spongy texture, the inoculated explants are uprooted (Afreen et al., 1999). Moreover, root growth and morphology and ex vitro survival were found to be better in Florialite compared to Cellulose plugs or even vermiculite and agar medium (Afreen et al., 1999).

Figure 5. Micropropagation of Eucalyptus plantlets in a large vessel under photoautotrophic conditions (for details see Zobayed et al, 2000a)

3.2.2. Designing of a large vessel: a chronological development

Probably the first system of large culture vessel was developed by Fujiwara et al., (1988) where, a large culture vessel of approximate volume of 19 L (58 cm long, 28 cm wide and 12 cm high) with an attached air pump for the forced ventilation of the vessel was used to enhance the photoautotrophic growth of strawberry (Fragaria x ananassa Duch.) explants and/or plants during the rooting and acclimatization stages. This was an aseptic micro-hydroponic system with a nutrient solution control system. Roche et al. (1996) developed a commercial-scale photoautotrophic micropropagation for potato micro-plants in which 100 nodal explants were cultured under natural ventilation in a stainless steel tray containing a block of polyurethane foam (85 x 300 x 25 mm) and enclosed with a polyethylene sleeve.

Figure 6. Coffee plantlets developed from somatic embryos are grown in a large vessel under photoautotrophic conditions (for details see Afreen et al., 2002).

Figure 6. Coffee plantlets developed from somatic embryos are grown in a large vessel under photoautotrophic conditions (for details see Afreen et al., 2002).

Kubota and Kozai (1992) grew potato plantlets under forced ventilation in a large vessel (2.6 L) containing a multi-cell tray. Heo and Kozai (1999) developed a similar type of system using forced ventilation attached to a large culture vessel (volume 13 L), where 20 sweet potato plantlets were cultured photoautotrophically. However, the disadvantage of these large culture vessels commonly faced is the variation in growth of the cultured plantlets mainly due to the un-uniform distribution of CO2 concentration and other environmental factors in the culture headspace. As CO2 is the sole carbon source in a photoautotrophic micropropagation system, uniform distribution of CO2 concentration in the culture headspace of a large vessel is an important factor to achieve uniform plant growth. In a large vessel system described above, CO2 concentration in the vessel is highest at the air inlet and is lowest at the air outlet especially, if there is only one inlet and one outlet. Consequently, large horizontal gradients in CO2 concentration between the inlet and outlet is created which resulted in a wide variation and lack of growth uniformity, with larger plants near the air inlet and comparatively smaller plants near the air outlet. This un-uniform growth is more obvious generally during the later growth stage of plantlets when the net photosynthetic rate per plantlet is high (Kozai and Kubota, 2001). The reason for this un-uniform growth was due to the failure to create uniform environmental conditions inside the large vessels using forced ventilation. Generally, the larger the vessel volume, the more difficult it is to achieve uniform distribution of CO2 in the vessel and thus the growth.

Zobayed et al. (1999c) engineered a forced ventilation system attached with a large vessel (volume 3.4 L; 40 plantlets per vessel) that supplies sterile nutrient solution throughout an extended culture period. The vessel was fitted with air distribution pipes (horizontally) inside the vessel for forced ventilation and the major aim of the system was to provide an air current pattern which enables uniform distributions of CO2 concentration and relative humidity as well as those of air current speeds, and thus the uniform plantlet growth. Heo et al. (2001) also developed a similar forced ventilation system with large vessel (13 L; 20 plantlets per box) horizontally fitted with air distribution pipes and successfully achieved uniform plantlet growth.

Figure 7. A photoautotrophic micropropagation system using large culture vessels attached with forced ventilation system applied for the commercial production of calla lily (Zantedeschia elliottiana) and China fir (Cunninghamia lanceolata) (Xiao and Kozai, 2004).

Zobayed et al. developed another large-scale micropropagation system by using a scaled-up vessel (volume 20 L; 500 plantlets per vessel; Figure 5a) with forced ventilation and air distribution chamber (Zobayed et al., 2000a). The bottom part of this large vessel has an air distribution chamber and the co2 enriched air was distributed through vertical pipes across the plug tray to obtain a uniform CO2 distribution over plantlets in the vessel and provide a uniform plantlet growth (Figure 5b). In a larger vessel (volume >20 L), without such an air distribution system, the distribution of CO2 concentration tends to be uneven over the plantlets even when air distribution pipes were used (Zobayed et al., 2000a). This was probably due to the significant pressure drop (thus reduce flow rate) in the air distribution pipe as the distance from the source (air pump) increased.

Afreen et al. (2002) designed a large culture vessel with forced ventilation system (temporary root zone immersion bioreactor) for the photoautotrophic mass-propagation of cotyledonary stage coffee somatic embryos (volume: 9 L; Figure 6). In this system an automatic nutrient supply system was attached with the vessel for temporarily immersing the root zone of the plantlets with the nutrient solution. This temporary immersion system ensured the exposure of the roots to the air and improved the root quality.

- Annona squamosa ■ Annona muricata

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