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Figure 14. Chlorophyll a (A) and b (B) contents, chlorophyll fluorescence (C and D), stomatal density (E) and stomatal length (F) of leaves of 45-d-old coffee plantlets grown from cotyledonary stage embryos underphotoautotrophic conditions (after Afreen et al., 2002b).

Figure 15. Growth parameters of 30-d-old coffee plantlets after transplanting ex vitro. A, Leaf number; B, leaf area; C, leaf fresh mass; D, stem fresh mass; E, root fresh mass; F, total increase of fresh mass after transplanting ex vitro. Significant difference between treatments at P <0.05 indicated by a, b, c was determined by Student—Newman—Keuls test ( after Afreen et al, 2002b).

Figure 15. Growth parameters of 30-d-old coffee plantlets after transplanting ex vitro. A, Leaf number; B, leaf area; C, leaf fresh mass; D, stem fresh mass; E, root fresh mass; F, total increase of fresh mass after transplanting ex vitro. Significant difference between treatments at P <0.05 indicated by a, b, c was determined by Student—Newman—Keuls test ( after Afreen et al, 2002b).

The result clearly shows that in this treatment, the forced ventilation system provided the best conditions throughout the experiment for the assimilation of CO2. As a result, the net photosynthetic rate, which is a closer reflection of normal in vitro metabolism, was greater than those of plantlets grown in the modified RITA-bioreactor and in Magenta vessels (Figure 13B). Moreover, growing plantlets under the optimum physical, chemical and most importantly the environmental conditions are also helpful to increase the chlorophyll concentration, which is essential to maximize the photosynthesis process.

Microscopy highlighted the differences among treatments with respect to stomatal density (Figure 12e-g), which was highest in leaves of plantlets grown in the TRI-bioreactor (8.3 mm-2 leaf area) followed by those of plantlets from the modified RITA-bioreactor (7.5 mm-2 leaf area) (Figure 14E). Compared with the other treatments, stomatal density was lowest in leaves of plantlets grown in Magenta vessels (5.9 mm-2 leaf area) (Figure 14E). Average stomatal length was nearly the same in leaves of all three treatments (Figure 14F). The most noticeable feature was that some stomata that developed in the leaves of plantlets grown in the modified RITA-bioreactor were open wide (Figure 12G), while others were distorted or still morphologically immature. It is possible that these stomata may not function properly, although no specific attempt was made to investigate this in the present study.

The results showed that for the plantlet conversion from cotyledonary stage embryos under photoautotrophic conditions, Magenta vessel and modified RITA-bioreactor resulted in the lowest growth regime. Our results also highlighted that for the embryo-to-plantlet conversion under photoautotrophic conditions the use of modified RITA-bioreactor was less effective at promoting shoot and root growth compared with the newly developed TRI-bioreactor system. This is most likely to be because in the modified RITA-bioreactor after every immersion of the plant material with nutrient solution, the entire plant became wet and, because the relative humidity inside the vessel is normally high (95-99%), the plant material either is never completely dried out or takes a long period to dry out. Thus, this thin layer of water surrounding the plant material acts as a liquid boundary layer, which impedes the exchange of gases between the plant and the surrounding environment and possibly prevents the CO2 fixation in the chlorophyll-containing zones - clearly a key factor for the photoautotrophic growth of embryos. In case of conventional photomixotrophic systems, the media contain sugar and therefore the lack of air exchanges may not be as serious a consequence as it is for the plantlets which completely depend on CO2 in the atmosphere for their photoautotrophic growth.

Again, it is emphasized that the RITA-bioreactor system has not been developed for culturing plantlets under photoautotrophic conditions. Also, in this study, the RITA-bioreactor was modified by attaching three gas permeable filter membranes on the lid, as was done for Magenta vessels. Thus, a completely different result can be expected if the original RITA-bioreactor with sugar-containing nutrient solution was to be used.

After transplanting under glasshouse conditions, a similar trend was noted in terms of survival percentage and growth (Figure 12M-O). Ex vitro survival, which was recorded on day 15 of transplanting, was highest (89%) in plantlets grown in the TRI-bioreactor. Plantlets grown in Magenta vessels had a survival percentage of 67%, although their growth was much slower than that of plants grown in the TRI-bioreactor. When plantlets from the modified RITA-bioreactor were transferred ex vitro only 33% survived. In terms of ex vitro growth, it was noticeable that plants from the TRI-bioreactor exhibited much faster growth (Figure 12M) and, as a consequence, after 30 d of transplanting almost all the growth parameters were significantly greater than those of plants grown in modified RITA-bioreactors and

Magenta vessels (Figure 15). The leaf number and leaf area of TRI- bioreactor grown plants were 2.7 and 2.8 times greater, respectively, than those of plants from the modified RITA-bioreactor, and 2.0 and 2.7 times greater, respectively, than those of plants from the Magenta vessel on day 30 after transplanting (Figure 15A and B). Similarly, leaf and root fresh mass were also enhanced and were 69 and 19 mg per plant, respectively, in the TRI-bioreactor grown plants, compared with 19 and 7.3 mg per plant, respectively, in the Magenta vessel grown plants and 25 and 2.4 mg per plant, respectively, in plants from the modified RITA-bioreactor (Figure 15C and E). During the present study, it became increasingly apparent that the vigorous growth (Figure 15F) and higher survival percentage observed in plants from the TRI-bioreactor could be the result of many environmental and physiological conditions during the in vitro culture period: for example, the relative humidity under forced ventilation was lower (85-90%) than that in the modified RITA-bioreactor (95-99%) or in Magenta vessels (95%). Smith et al. (1992) suggested that reducing the relative humidity in the culture headspace could improve resistance to wilting of micropropagated grapevine. In a previous experiment (Zobayed et al., 2000), we found that lowering the relative humidity in the culture headspace by introducing forced ventilation can increase the deposition of epicuticular wax on the leaf surface, which can, in turn, prevent water loss after transplanting and thus increase the chance of survival and subsequent growth.

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