Ns

*

*

Somatic embryos were developed and grown under a PPF of 30 ^mol m-2 s-1 for 14 weeks followed by 2 weeks pre-treatment at high PPF (100 ^mol m-2 s-1). Factor A (different conditions, photoautotrophy or photomixotrophy) and factor B (different stages of somatic embryos); *P< 0.05; **P < 0.01; ***P < 0.001; NS, non-significant according to the Tukey test. Each value represents a mean ± s.e. of 10 replicates (after Afreen et al., 2002a).

These results are in agreement with previous findings that cotyledonary and germinated embryos show stomatal development, scavenge CO2 and have high chlorophyll contents. In the case of cotyledonary and germinated embryos, the first true leaves to unfold were observed after 4-6 weeks of culture in both PA and PM. Precotyledonary stage embryos grown either under PM or PA conditions did not produce any true leaves; however, well-developed unfolded cotyledons were observed in somatic embryos after 6-7 weeks of culture in PM conditions. When grown photoautotrophically, about 54% somatic embryos also produced unfolded cotyledons, but these were smaller in size than those produced in the PM.

Table 4. Effects of different supporting media on the growth of cotyledonary stage coffee somatic embryos under photoautotrophic conditions.

Number of true Total fresh

Treatments leaves mass (mg)

Total dry mass Percentage

(mg) rooting Length of roots (mm)

Each value represents a mean ± s.e. of ten replicates. Means within a column followed by different superscripts are significantly different at P < 0.05 by the least significant difference test. All parameters except leaf number were significant at P < 0.01 (ANOVA) (after Afreen et al., 2002a).

Figure 10. Dry mass of coffee plantlets developed photoautotrophically from cotyledonary stage somatic embryos grown under different PPF (50, 100 and 150 ^mol m-2 s-1) and CO2 concentrations [400 ^mol mot1 (closed circles) and 1100 ^mol mol-1 (open circles)]. Embryos were initially developed and grown under a PPF of 30 fimol m-2 s-1 for 14 weeks followed by 2 weeks pre-treatment at high PPF (100 p.mol m-2 s-1) (after Afreen et al., 2002a).

Figure 10. Dry mass of coffee plantlets developed photoautotrophically from cotyledonary stage somatic embryos grown under different PPF (50, 100 and 150 ^mol m-2 s-1) and CO2 concentrations [400 ^mol mot1 (closed circles) and 1100 ^mol mol-1 (open circles)]. Embryos were initially developed and grown under a PPF of 30 fimol m-2 s-1 for 14 weeks followed by 2 weeks pre-treatment at high PPF (100 p.mol m-2 s-1) (after Afreen et al., 2002a).

In general, normal formation of leaves was observed in embryos grown photoautotrophically; however, under PM, hyperhydricity of leaves was quite obvious (Figure 8). Interestingly, under PM, some torpedo and precotyledonary stage embryos produced new embryos from the base. Under PM, roots did not develop in torpedo or precotyledonary stage embryos, whereas 33 and 58% of cotyledonary and germinated embryos, respectively, produced roots. When embryos were grown under PA, root formation was not observed in torpedo and precotyledonary stage somatic embryos. Some cotyledonary (15%) and germinated embryos (25%) produced roots. Taking into account the growth results, it becomes increasingly apparent that coffee somatic embryos can be grown under photoautotrophic conditions. Results also indicate that cotyledonary and germinated embryos show growth increments (compared with initial growth) when grown under photoautotrophic conditions. The values of both potential activity of PSII (OpMAX) and the actual photochemical efficiency of PSII (Op) measured after 60 days of culture of different stage embryos were not significantly different between the photoautotrophic and photomixotrophic conditions (Table 3). In general among the different stages the values were greater in cotyledonary and germinated embryos in both the conditions. Again, the chlorophyll a and b contents of different stage embryos grown photoautotrophically were nearly the same with their photomixotrophic counterparts (Table 3).

5.4. Optimization of growth under photoautotrophic conditions

Attempts were made to culture somatic embryos under photoautotrophic conditions and to optimize different environmental conditions to maximize growth. Cotyledonary stage somatic embryos were grown photoautotrophically in three different types of supporting media: (1) agar (8 g l-1; Kanto Chemical Co.); (2) vermiculite; and (3) Florialite [a mixture of vermiculite and cellulose fibre (described by Afreen et al., 2000; Nisshinbo Industries, Inc. Japan)]. The greatest total fresh and dry mass were recorded in the Florialite treatment, with values being 1.3 times those of the agar treatment. With the exception of rooting percentage, there were no significant differences among growth parameters between the Florialite and vermiculite treatments (Table 4). In agar-grown plantlets, percentage rooting was low; only 26% of embryos produced roots and those roots produced were very short. Conversely, in Florialite and vermiculite, 69 and 56% embryos, respectively, produced roots (Table 4).

To optimise the PPF and the CO2 concentrations, cotyledonary stage somatic embryos were cultured under the following PPF and CO2 concentrations:

(1) 50 ^mol m-2 s-1 PPF and ambient CO2 concentration of 400 ^mol mol-1

(5) 100 ^mol m-2 s-1 PPF and 1100 ^mol mol-1 CO2 and

in general, the dry mass of cotyledonary stage embryos was enhanced when Co2 was enriched (Figure 10). Increasing PPF further (150 ^mol m-2 s-1) did not lead to any change in the dry mass of embryos. In contrast, in low PPF (50 ^mol m-2 s-1) treatments there was hardly any increase in the dry mass (compared with initial dry mass). Therefore, results suggest that high PPF (100-150 ^mol m-2 s-1) and an

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