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Figure 1. A culture module of the photoautotrophic micropropagation system using large culture vessels (PAM system) in the left and a culture module of the photomixotrophic micropropagation system using small vessels with natural ventilation (PMM system) in the right.
Figure 2. The culture module of photoautotrophic micropropagation system using large culture vessels with forced ventilation.

2.3. Culture vessel unit

This consisted of a plexiglass box (115 cm wide x 52 cm deep x 20 cm high; air volume, 120 L; culture area, about 0.6 m2) with two air inlets (diameter: 5 mm) and six air outlets (diameter: 20 mm) for forced ventilation. The two inlets for providing air forcedly into the vessel were located on the sidewalls 8 cm from the bottom of vessel. Each air inlet was connected to an air-valve for controlling the number of air exchanges of the vessel, defined as the hourly ventilation rate of the vessel divided by vessel air volume. The six air outlets for discharging the vessel air naturally to the culture room were located at different points of the upper surface of the vessel (Figure 2). Locations of the air outlets are determined by trial and error to obtain a uniform air distribution in the vessel. Gas-permeable microporous filters (diameter: 20 mm, pore diameter: 0.5 ^m) were attached to the air outlets to prevent dust and microbes from entering. Three trays (48X36X7 cm) were placed in each vessel. The culture vessel had a door (45 cm wide and 13 cm high) at the front side for accessing the trays.

2.4. Forced ventilation unit for supplying CO2-enriched air

A forced ventilation unit for supplying CO2 enriched air consisted of a CO2 container with gas tubes, pressure gauges, airflow meters, an air pump and valves, an air disinfection and humidification tank, and a CO2 concentration controller (Figure 3). This unit was used for forced ventilation of all the modules.

Pure CO2 from the CO2 container passed through the gas tube with the CO2 pressure gauge and airflow meter into the disinfection and humidification tank containing 2% NaClO3 (w/v) solution. In addition, the culture room air was sent by the air pump with a microporous filter through a gas tube with an airflow meter into the disinfection and humidification tank in order to dilute pure CO2 in the container. The pure CO2 and culture room air were mixed in the gas tube before being sent into the disinfection and humidification tank. Finally, the disinfected CO2- enriched air was passed through the gas tubes with the airflow meter and valves into the culture vessel through the two air inlets of the vessel. The CO2 concentration of the mixed air was measured and adjusted by using a CO2 concentration controller.

2.5. Lighting unit

White fluorescent lamps were used as a light source. Six 36-W fluorescent lamps each with one switch were installed on each shelf to adjust PPF on the shelf at a desired level in a range between 50 and 200 |imol m-2 s-1. To increase the uniformity of PPF distribution over the shelf and the ratio of light energy received by the plantlets to the light energy emitted from the lamps, the vessel was surrounded by white reflective sheets. Two sheets (120X13 cm) were installed on the front and back of the vessel, and another two (50 X 13 cm) were installed on the left and right side of the vessel. The four sheets were connected with the culture shelf by hinges.

Figure 3. Schematic diagram of a forced ventilation unit for CO2 enriched air supply.

2.6. Sterilization

The culture vessels were sterilized as follows (Xiao et al., 2000): (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 a disinfectant solution with 0.2% sodium dichloroisocyanurate for twenty minutes. Substrate (vermiculite) in 5-L cloth bags and nutrient solution in 1000-mL bottles were autoclaved at 121 to 123 C for 40 min. The substrate was sprayed with water to get it wet before autoclaving for increasing its thermal conductivity. Sterilized nutrient solution was supplied to the substrate.

A 5-shelf unit of the PMM (photomixotrophic micropropagation) system was identical to the one used in the PAM, although the ventilated vessels, white reflective sheets and forced ventilation unit were not used.

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