Forced Ventilation Systems With Large Culture Vessels

As described above, the method of using gas permeable filter discs attached to the lid or the sidewalls of the culture vessel or increasing the gaseous concentrations in the culture room is a simple way to effectively increase the CO2 concentration during the photoperiod and decrease the relative humidity and ethylene concentration inside a culture vessel. However, the CO2 concentration and other gaseous concentrations in the culture vessel with natural ventilation are interrelated with a number of factors such as the metabolic activity of the plants in vitro, the plant size and leaf area, the number of air exchanges of the culture vessel and the culture room environment. Thus, the gaseous concentrations in the culture vessel with natural ventilation are often unpredictable and uncontrollable. Furthermore, it is difficult to provide a high number of air exchanges for a large culture vessel (Kozai and Nguyen, 2003).

Forced ventilation is a method involving the use of mechanical force generated by an air pump or an air compressor to flush a particular gas mixture directly into the culture vessel (often through microporous filters to prevent microorganisms from entering the vessel). In this system, the gaseous composition (CO2, water vapor, etc.) of incoming air and ventilation rate and/or air current speed inside the culture vessel can be controlled relatively precisely by use of a needle valve, a mass flow controller or an air pump with an inverter (Aitken-Christie et al., 1995; Jeong et al., 1995). With a proper control of the gaseous composition in culture vessels, the growth of plants in vitro with forced ventilation can be enhanced significantly compared with natural ventilation.

Photoautotrophic micropropagation makes it possible to use large culture vessels with minimum risk of microbial contamination. In photoautotrophic micropropagation using a large culture vessel with forced ventilation, the labor cost could be reduced by nearly fifty percent as compared with that in conventional, photomixotrophic micropropagation (Xiao et al., 2000).

In 1988, Fujiwara et al. developed a large culture vessel with a forced ventilation system for enhancing the photoautotrophic growth of strawberry (Fragaria x ananassa Duch.) explants and/or plants in vitro during the rooting and acclimatization stages, where CO2 gas was mixed with air and pumped into the vessel. This was a kind of aseptic micro-hydroponic system with a nutrient solution control system.

Kubota and Kozai (1992) described a forced ventilation system using a polycarbonate vessel containing a multi-cell tray with rockwool cubes for photoautotrophic growth of potato (Solanum tuberosum L.) plants in vitro. The net photosynthetic rate of potato plants was significantly greater than plants grown in conventional (small) culture vessels with natural ventilation. Another forced ventilation micropropagation system was demonstrated by Heo and Kozai (1997). The photoautotrophic growth of sweetpotato plants cultured in vitro with this system was several times greater than the photomixotrophic growth of plants cultured in small culture vessels with natural ventilation.

However, in both of the forced ventilation systems mentioned above, the growth of plants in vitro in the culture vessel was not uniform. The plants were larger near the air inlet and comparatively smaller near the air outlet.

A new type of large culture vessel with air distribution pipes for forced ventilation was developed by Zobayed et al. (2000) with a major aim to provide an air current pattern that enables uniform distribution of CO2 concentration and relative humidity as well as air current speed in the culture vessel, and consequently, the uniform growth of plants in vitro. The feasibility of this forced ventilation system has been tested for Eucalyptus camaldulensis (Zobayed et al., 2000) (Figure 10). A large culture vessel with a nutrient supply unit makes it possible to measure and control the pH, composition and volume of nutrient solution in the culture vessel. Moreover, the control of plant growth rate is relatively easy in such vessels with a nutrient control unit (Zobayed et al., 2000). Compared with eucalyptus plants in vitro cultured photomixotrophically in Magenta-type vessels, the plants cultured photoautotrophically in the large vessel with forced ventilation had a higher net photosynthetic rate, normal stomatal closing and opening, and significantly higher epicuticular leaf-wax content. Plants cultured in the large vessel with forced ventilation were, therefore, considered acclimatized in vitro, and their transpiration rates and percent water loss remained lower than those of conventional plants when transplanted to ex vitro conditions (Zobayed et al., 2001a, b).

Coffea arabusta plants in vitro, when cultured in a forced ventilation system, had significantly higher fresh and dry weights, shoot length and leaf area than those cultured with natural ventilation (Nguyen et al., 2001). The study included 40 days in the in vitro stage and 10 days in the ex vitro stage (Figure 11). Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. With the forced ventilation method in photoautotrophic micropropagation, the CO2 concentration inside the culture box could be easily adjusted to promote the net photosynthetic rate of plants.

Micropropagation Lab
Figure 10. Scaled-up photoautotrophic culture vessel (picture taken after removal of the lid). The vessel was 610 mm long, 310 mm wide and 105 mm high (volume approx. 201) (Zobayed et al., 2000).

Natural F. low rate F. high rate F. low rate F. high rate Low PPF Low PPF Low PPF High PPF High PPF

Figure 11. Coffee (C. arabusta) plants on day 10 of the ex vitro stage as affected by ventilation methods in the in vitro stage. In the treatment legends, Natural, Low rate and High rate denote natural and forced low and high rate ventilations (Nguyen et al., 2001).

Natural F. low rate F. high rate F. low rate F. high rate Low PPF Low PPF Low PPF High PPF High PPF

Figure 11. Coffee (C. arabusta) plants on day 10 of the ex vitro stage as affected by ventilation methods in the in vitro stage. In the treatment legends, Natural, Low rate and High rate denote natural and forced low and high rate ventilations (Nguyen et al., 2001).

The growth of shoots and roots of coffee (C. arabusta) somatic embryos cultured photoautotrophically in a large vessel (volume approx. 3 l) with nutrient supply system (TRI-bioreactor) were greater with forced ventilation than with natural ventilation (Afreen et al., 2002b). The percent survival was 98 percent for plants ex vitro derived from somatic embryos grown in the TRI-bioreactor with a forced ventilation system, while it was only 30 percent for those grown in the Magenta-type vessel system with natural ventilation. Furthermore, when cultured photoautotrophically, the cotyledonary embryos proved the highest embryo-to-plantlet conversion percentage in a TRI-bioreactor compared with those grown in Magemta vessel or modified RITA-bioreactor (Afreen et al., 2002b). The embryo-derived plants in the group grown in the TRI-bioreactor had the highest percent of survival and a faster growth when transferred to the ex vitro condition, followed by those in modified RITA-bioreactor and Magenta vessel.

Figure 12. Effect of different types of ventilation on the growth of Paulownia (Paulownia fortunei) plants in vitro (Nguyen and Kozai., 2001).
Figure 13. Growth enhancement of bamboo (Thyrsostachys siamensis Gamble) plants cultured photoautotrophically in forced ventilation vs. natural ventilation systems on day 25.

Another forced ventilation system for studying the photoautotrophic growth of Paulownia fortunei using a large vessel (volume approx. 17 l) was also developed (Nguyen and Kozai, 2001). The CO2 uptake rate increased with the increase in the airflow rate over the culture period of 28 days, resulted in the increase in net photosynthetic rate. The growth of single-node leafy cuttings of Paulownia was significantly greater under photoautotrophic condition with forced ventilation than under photomixotrophic condition with natural ventilation (Figure 12).

The forced ventilation system using a large vessel (volume approx. 13 l) has been recently proved to have a better effect on the photoautotrophic growth of bamboo (Thyrsostachys siamensis Gamble) plants in vitro for 25 days (data not shown). Increased dry weight, shoot and root lengths, percent of plants having roots and number of new shoots per explant were significantly greater in the forced ventilation treatment with Florialite-based medium compared with those in natural ventilation treatments with Florialite or agar-based medium in Magenta-type vessels. in the vessel with forced ventilation, bamboo plants developed a great number of lateral roots resulted in the growth enhancement of bamboo plants in the in vitro stage (Figure 13).

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