Liquid Culture Systems for in vitro Plant Propagation

326 F. Afreen et al.
precotyledonary-stage embryos (Figure 1c), whereas well-developed
stomata were seen in the cotyledonary (Figure 1d) and converted
embryos (Figure 1e). Stomatal density was highest in the converted
embryos (Figure 2c). The absence of stomata in these stages was
reflected in the limited ability of these embryos to photosynthesize.
3. Chlorophyll which is essential for plant photosynthesis, uses light
energy to synthesise chemical energy. Low chlorophyll concentrations
were recorded in torpedo and precotyledonary-stage embryos. In the
cotyledonary stage embryos, chlorophyll concentrations were higher
compared with those in the torpedo or precotyledonary stage embryos;
chlorophyll a and b were highest in the converted embryos (Figure 2d, e)
4. The potential activity of PSII (�pMAX) as estimated in the dark was
low in the torpedo stage but increased in precotyledonary stage embryos
(�pMAX = 0.69) followed by cotyledonary stage embryos (�pMAX =
0.84) and was highest in converted embryos (�pMAX = 0.88) (Figure
2f). As expected from their low PSII activity, torpedo stage embryos
exhibited low electron-transport activity (�p = 0.1). An increase in the
quantum yield for electron-transport was observed in the cotyledonary
and converted embryos (Figure 2g).
The results revealed that the cotyledonary and converted embryos showed
photosynthetic ability, stomata did not fully develop in the precotyledonary
stage embryos and were absent in the torpedo stage. Low chlorophyll
concentrations were noted in the torpedo and precotyledonary stage
embryos, but the concentration increased in the cotyledonary and converted
embryos. Therefore, we suggest that the cotyledonary stage is the earliest
stage, which can be cultured photoautotrophically to develop into plantlets.
3. Technique to enhance photosynthetic ability of coffee
somatic embryos
To develop an optimised protocol for the regeneration of somatic
embryos from coffee leaf discs, cultures have to be incubated in darkness or
at a low light intensity (PPF: 20-30 µmol m -2 s -1 ) (Afreen et al., 2002a). This
is because endogenous plant growth regulator concentrations and/or the
sensitivity of cells to growth regulators are known to differ in dark or light
conditions and thus affect embryogenesis. Hutchinson et al. (2000) reported
that a continuous light treatment significantly reduced the amount of the
endogenous plant growth substances in geranium (Pelargonium x hortorum)
tissues with concomitant inhibition of somatic embryogenesis. Croke and
Cassells (1997) also observed promotive effect of dark incubation resulting