Liquid Culture Systems for in vitro Plant Propagation

Development of photoautotrophy in Coffea 329
in doubling of the embryo frequency in zonal geraniums. The positive
influence of a dark treatment to induce bud break, and thus increased in vitro
multiplication, was reported for evergreen azaleas (Hsia and Korban, 1998).
However, growing somatic embryos in dark or at low light intensity
significantly inhibits or delays the development of photosynthetic pigments.
In coffee, we observed that placing the somatic embryos under a relatively
high light intensity (PPF: 100 µmol m -2 s -1 ) for 14 days resulted in the
development of photosynthetic pigments, functional stomata and subsequent
enhancement of photosynthetic ability. Placing the embryos under high light
intensity increased the chlorophyll concentrations significantly for all stages
of embryos (Afreen et al., 2002a). In general, light-pre-treated embryos
exhibited higher stomatal density than those without light pre-treatment.
Well-developed stomata were observed in the converted embryos
irrespective of light pre-treatment. Both the pre-treated and non-pre-treated
converted embryos photosynthesized, but in the cotyledonary stage embryos,
CO2 assimilation was recorded only in the pre-treated ones (Afreen et al.,
2002a). From these findings we concluded that light pre-treatment of the
somatic embryos at high PPF (100 µmol m -2 s -1 ) increased the photosynthetic
ability in almost all embryonic stages, but the cotyledonary stage embryo is
the earliest stage to grow photoautotrophically.
4. Photoautotrophic culture of different stage coffee somatic
embryos
Pre-treated somatic embryos (torpedo, precotyledonary, cotyledonary and
converted) were cultured in plastic petridishes containing MS medium
excluding sugar and under CO2 enrichment. Overall greater performance in
terms of growth occurred in the embryos grown under photomixotrophic
conditions compared with photoautotrophic (Afreen et al., 2002a). After 60
days of culture, under photoautotrophic conditions, torpedo and
precotyledonary stage embryos lost at least 25 and 20% respectively, of their
initial dry mass. The most likely reason for this loss could be that the low
photosynthetic ability of the plant materials coupled with continuous
respiration, probably led the plantlets to depend completely on their own
reserve food material. On the contrary, under photomixotrophic conditions,
the dry mass of each of the torpedo and precotyledonary stage embryos
increased by upto 190 and 200% respectively, of their initial dry mass.
Under photoautotrophic conditions in the later stages i.e. cotyledonary and
converted embryos, the dry mass of each of the embryos was increased by
upto 10% and 50%, respectively, of their initial dry mass (Afreen et al.,
2002a). The probable reason for the dry mass increment in the later stages