Plant material, growth medium, and growth conditions.

Protea cynaroides microshoots, ≈10 mm in length with three nodes, were taken from in vitro–established stock plants that had been previously subcultured twice. The microshoots were cultured on full-strength woody plant medium (Lloyd and McCown, 1981) supplemented with 0.5 mg·L⁻¹ IBA, 0.01 mg·L⁻¹ kinetin, 100 mg·L⁻¹ myo-inositol, 100 mg·L⁻¹ silver nitrate, 200 mL·L⁻¹ coconut water, and 9 g·L⁻¹ agar. Two sucrose concentrations were used: 10 and 30 g·L⁻¹. The pH of the growth medium was adjusted to 5.5 before autoclaving. The growth medium was dispensed into Plantima^® (A-Tech Bioscientific Co., Ltd., Taipei, Taiwan) culture vessels (250 mL/vessel) and autoclaved at 121 °C and 104 kPa for 30 min. Each culture vessel was placed on a PlanTower^® shelf (A-Tech Bioscientific Co., Ltd.), which provided 50 μmol·m⁻²·s⁻¹ PPF. The PlanTower^® with the cultures were placed in a CO₂-enriched growth chamber (≈1000 μmol·mol⁻¹) with the temperature and photoperiod adjusted to 26 °C ± 2 and 16 h, respectively.

Ventilation treatments.

To set up a system that ensures efficient gaseous exchange between the inside and outside of the culture vessel, a novel approach was developed using modified Plantima^® containers, which are normally used for culturing explants in temporary immersion systems. Plantima^® culture vessels are usually separated into an upper section (where explants are placed) and a lower section (for the growth medium). In this study, the component that was used to partition each Plantima^® container into the upper and lower sections was removed. The container was then left with a single growing space where P. cynaroides microshoots were grown on semisolid medium. The inlet and outlet valves that were originally on the culture vessel served as the entry and exit points for gas exchange to take place, either by natural ventilation or forced ventilation. Natural ventilation took place through the natural diffusion of air between the inside and outside of the culture vessel. Forced ventilation was regulated by the system’s air-flow controller, whereby CO₂-enriched air was pumped at set intervals via silicon tubes first through a sterilant solution (Cu⁺⁺) and then through a 0.22 μm microfilter attached to the inlet valve and into the Plantima^® container (Fig. 1). This process allowed the air in the headspace inside the culture container to be renewed, with the forced pressure escaping through the outlet valve, which also had a 0.22 μm microfilter attached to prevent microbes from entering the vessel. Three ventilation treatments were tested: natural ventilation (control), and air pumped every 2 h for 2 min or every 4 h for 2 min (forced ventilation).

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Determination of chlorophyll content and chlorophyll fluorescence.

An SPAD-502 Plus Chlorophyll Meter (Konica Minolta, Tokyo, Japan), which is a portable diagnostic device that takes nondestructive measurements of the greenness or relative chlorophyll concentration of leaves, was used to measure chlorophyll content. The mean of three readings was obtained from the uppermost unfolded leaf of each microshoot. Determination of chlorophyll fluorescence (Fluoropen FP100; Photon Systems Instruments, Drásov, Czech Republic) of P. cynaroides microshoots was carried out according to Molero and Lopes (2012). Briefly, microshoots were dark-adapted by placing them in a dark room for 24 h. The youngest unfolded leaf on the uppermost shoot was selected to take chlorophyll fluorescence readings. The leaf was placed into the sensor head, making sure that the leaf completely covers the aperture of the sensor. The same leaves that were used to take the dark-adapted readings were used to take the light-adapted readings. The mean of three readings was obtained for each microshoot. The F_v/F_m ratio obtained was used to interpret the photosynthetic efficiency of the microshoots.

Statistical analysis.

One microshoot per culture vessel was used with seven replications per treatment. Data for the number of shoots, shoot length, leaf area (Version 1.6; Image J, Bethesda, MD), chlorophyll content, and F_v/F_m value were collected after 100 d in culture. Data were subjected to analysis of variation and mean comparison using SPSS version 17 for Windows.

Shoot and leaf growth.

Significant interaction effects were found between ventilation and sucrose in the number of shoots formed, whereas no significant interaction effects were found in the other growth parameters (Table 1). When cultured on growth medium with 10 g·L⁻¹ sucrose, microshoots force-ventilated for 2 min/2 h produced significantly higher number of shoots than those naturally ventilated or force-ventilated for 2 min/4 h (Table 2). However, in the same sucrose treatment, no significant differences in shoot numbers were found between the natural ventilation and 2 min/4 h treatments. In the 30 g·L⁻¹ sucrose treatment, no significant differences in shoot numbers were observed among all ventilation treatments (Table 2). Thus, these findings indicated that when cultured on low sucrose concentration (10 g·L⁻¹), high frequency ventilation (2 min/2 h) is required to induce shoot formation, whereas in the 30 g·L⁻¹ sucrose treatments where growth media have been supplemented with sufficient carbon and energy source, explants are able to develop similar number of shoots regardless of the type of ventilation treatment. According to Zobayed et al. (1999), high frequency ventilation will result in the relative humidity of the headspace in the culture vessel to be reduced to the point where the growth medium starts to lose water content, i.e., water loss from the growth medium caused by high ventilation rate (2 min/2 h) resulting in changes to its composition, most notably the sucrose concentration. As a result, it is probable that when P. cynaroides microshoots were force-ventilated for 2 min/2 h on medium containing 10 g·L⁻¹ sucrose, the sucrose concentration increased to a level where it promoted the formation of shoots, leading to an increase in the number of shoots formed. By contrast, the low number of shoots that formed under natural ventilation and 2 min/4 h treatments could be due to insufficient CO₂ diffusion into the culture vessels combined with low sucrose content. Several studies have stated that in growth media with reduced sucrose levels, either the CO₂ concentration or the light intensity needs to be increased to ensure that a positive carbon balance is achieved (Hdider and Desjardins, 1994; Kozai, 1991). Under natural ventilation, a significantly higher number of shoots was produced on growth media containing 30 g·L⁻¹ sucrose than those supplemented with 10 g·L⁻¹ sucrose (Table 3). This demonstrated that when naturally ventilated, a higher sucrose concentration (30 g·L⁻¹) is required to induce the formation of shoots in P. cynaroides microshoots, which is to be expected because higher sucrose concentrations are generally needed in the absence of increased CO₂ levels. On the contrary, under 2 min/2 h ventilation, microshoots cultured with 10 g·L⁻¹ sucrose produced significantly more shoots than those in the 30 g·L⁻¹ sucrose treatment. As previously mentioned, this could be because of the reduced sucrose concentration (10 g·L⁻¹) increasing to more optimal levels as a result of water loss in the growth medium caused by high frequency ventilation, which at the same time, might have raised the 30 g·L⁻¹ sucrose concentration to suboptimal levels, leading to lower shoot numbers. When the forced ventilation frequency was reduced, as in the 2 min/4 h treatment, the amount of sucrose in the growth medium did not significantly affect the number of shoots formed (Table 3). This suggests that at this ventilation frequency, conditions for the formation of shoots were similar, regardless of the sucrose concentration.

Table 1.Interaction effects of ventilation and sucrose concentration on the vegetative growth, chlorophyll content, and F_v/F_m values of Protea cynaroides microshoots.

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Table 2.Effects of different types of ventilation on the number of shoots formed in Protea cynaroides microshoots cultured under different sucrose concentrations.

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Table 3.Effects of different sucrose concentrations on the number of shoots formed in Protea cynaroides microshoots cultured under different ventilation treatments.

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With regard to shoot length, different sucrose concentrations within the same ventilation treatment did not significantly affect the growth of microshoots (Table 4). These results are consistent with findings reported by Wojtania et al. (2015b). In their study, no significant differences in shoot length were found between Pelargonium ×hortorum explants grown in 15 and 30 g·L⁻¹ sucrose. Similarly, no significant differences in the length of Magnolia ×soulangiana microshoots were observed between different sucrose concentrations in non-ventilated conditions (Wojtania et al., 2015a). By contrast, in the 10 g·L⁻¹ sucrose treatment, P. cynaroides microshoots in the 2 min/4 h ventilation treatment produced significantly longer shoots than those in the other two ventilation treatments. Very few studies have examined the effects of different ventilation frequencies and durations on explant growth. Nevertheless, the results of our study are in agreement with the findings by Rahayu and Habibah (2015) where Feronia limonia explants cultured on reduced sucrose concentration led to significant increases in shoot growth. Similarly, Capellades et al. (1991) stated that culturing explants on reduced sucrose concentrations and increased gaseous exchange improved the growth rate of explants.

Table 4.Effects of sucrose concentration and ventilation treatment on the growth of Protea cynaroides microshoots after 100 d in culture.

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Microshoots force-ventilated for 2 min/4 h produced the largest leaves, irrespective of the sucrose concentration. This is shown by the significantly higher mean leaf area of microshoots grown in the 2 min/4 h force ventilation treatment in both the 10 and 30 g·L⁻¹ sucrose treatments (Table 4). These results suggest that natural ventilation and 2 min/2 h forced ventilation represent the two extremes of the ventilation treatments, in which the ventilation conditions inside the culture vessel became suboptimal for leaf growth. It is likely that natural ventilation provided insufficient air exchanges, whereas the high ventilation frequency in the 2 min/2 h treatment caused excessive water loss in the growth medium. Goncalves et al. (2007) found that the higher water loss in ventilated culture vessels led to changes in media characteristics and concentration, which consequently affected plant growth. In particular, negative effects on vegetative biomass would become more pronounced with longer term culture (Mohamed and Alsadon, 2010). The results of the present study clearly indicated that this might have been the case because 100 d of frequent ventilation (2 min/2 h) most likely altered the media characteristics to the point where it adversely affected leaf growth, resulting in significantly lower leaf area. On the other hand, these findings illustrated that P. cynaroides microshoots force-ventilated for 2 min/4 h were optimal for leaf growth and development.

Chlorophyll content and F_v/F_m value.

Within each ventilation treatment, the chlorophyll content of microshoots grown on 30 g·L⁻¹ sucrose was significantly higher than those cultured on 10 g·L⁻¹ sucrose (Table 4). These results are in agreement with those reported by Abbott and Belcher (1980) and Langford and Wainwright (1987). However, contradictory findings have also been reported with regard to the effect of sucrose on chlorophyll content. According to Grout and Donkin (1987), a high exogenous supply of sucrose is not required for the normal development of chlorophyll. In the in vitro culture of walnut explants, the chlorophyll content of explants cultured on low sucrose concentration (15 g·L⁻¹) were higher than those cultured on 30 g·L⁻¹ sucrose in similar ventilation treatments (Hassankhah et al., 2014). Similarly, the chlorophyll content of Billbergia zebrina explants decreased as the sucrose concentration in the growth medium increased (Martins et al., 2015). These reports concluded that lower concentrations of sucrose in the growth medium stimulate chlorophyll production in explants cultured in vitro. By contrast, chlorophyll content in potato plantlets was not significantly affected by different sucrose concentrations; instead, significant effects were observed between different ventilation treatments (Mohamed and Alsadon, 2010). It is probable that the contradictory results of the relationship between sucrose concentration and chlorophyll content are associated with nitrogen levels, among other factors, in the growth medium. It has been suggested that because chlorophyll contains nitrogen in its structure, fluctuations in nitrogen levels in the growth medium would affect chlorophyll content in leaves (Richardson et al., 2002). Therefore, it can be concluded that in addition to sucrose, chlorophyll content of leaves is affected by interactions of several different factors.

F_v/F_m values of dark-adapted plant tissues are directly proportional to the quantum efficiency of PSII photochemistry (Butler, 1978), and are an excellent indicator for the PSII quantum yield, which provides a good indication of the photosynthesis efficiency of plants (Gouk et al., 1999). This in turn provides evidence as to whether a plant is stressed or not. A reduction in F_v/F_m values is associated with thermal damage of PSII reaction centers (Gouk et al., 1999). Plants that have F_v/F_m values less than 0.6 are considered stressed (Ritchie, 2006). In this study, P. cynaroides microshoots that had F_v/F_m values greater than 0.6 were cultured in 10 g·L⁻¹ sucrose with 2 min/2 h forced ventilation, 30 g·L⁻¹ with 2 min/2 h forced ventilation, and 30 g·L⁻¹ with 2 min/4 h forced ventilation, whereas the lowest F_v/F_m value was observed in microshoots grown in the natural ventilation treatment containing 30 g·L⁻¹ sucrose (Table 4). Although sucrose concentration in the growth medium has been shown to affect photosynthetic rate (Hdider and Desjardins, 1994; Mosaleeyanon et al., 2004), this was not observed in the present study. Instead, our findings showed significant differences in F_v/F_m values between forced ventilation (2 min/4 h) and natural ventilation in the 30 g·L⁻¹ sucrose treatment. These results are consistent with several studies that demonstrated significantly high photosynthetic efficiency of explants cultured in forced ventilated treatments (Heo and Kozai, 1999; Xiao et al., 2005; Zobayed et al., 1999). The positive effects of forced ventilation are due to the presence of consistently high CO₂ levels in culture vessels, which improves the photosynthetic performance of plantlets (Zobayed et al., 1999). Our study shows that forced ventilation providing sufficient CO₂ is the overriding factor in determining in photosynthetic capacity of P. cynaroides microshoots.

In conclusion, sucrose and ventilation were found to have interaction effects in the number of shoots formed. Overall, compared with microshoots grown on 30 g·L⁻¹ sucrose with natural ventilation, the use of 30 g·L⁻¹ sucrose in combination with 2 min/4 h ventilation produced significantly higher leaf area, chlorophyll content, and F_v/F_m values. The modified temporary immersion culture vessels described in this study are suitable to be used as a forced ventilation system for culturing P. cynaroides microshoots. Further studies are needed to investigate the effects of these treatments on the rooting of P. cynaroides microshoots, as well as their acclimatization to ex vitro conditions.