Liquid Culture Systems for in vitro Plant Propagation

Temporary immersion system: a new concept 189
of axillary shoots were formed closer to the upper part of the container,
hence far from the agar and the surface of the liquid, where humidity was
higher and where the liquid remained on the surface of the needles.
Coffea arabica microcuttings revealed little or no hyperhydricity with
immersion times of 15 min every 6 hours, but longer immersion times
caused it to occur (Berthouly et al., 1995). For such immersion times, Coffea
canephora cuttings were glassy. In this species, which is more susceptible to
this problem, immersion times had to be reduced to 1 min every 6 hours.
Somatic embryos of Citrus grown in a temporary immersion system did
not reveal any hyperhydricity, unlike those obtained in suspensions or even
more so, those grown on an agar medium (Cabasson et al., 1997). Rubber
tree somatic embryos produced in a temporary immersion system did not
reveal any hyperhydricity symptoms (Etienne et al., 1997b). However, a
glassy appearance was found in germinating material placed in a plant
conversion medium, but that was probably due to the excessive immersion
times used (15 min immersion every 6 hours). Just as was seen with coffee,
hyperhydricity risks are greater in the later phases of somatic embryogenesis
(i.e. germination and conversion into plants). Short immersion times need to
be used to avoid this problem. We found that vitrification of C. arabica
somatic embryos was primarily caused by the immersion times, but not by
the frequency if short immersions (1 min) were used. Several 15 min
immersions per day led to embryo populations that were mostly glassy.
Using temporary immersion therefore, generally reduces hyperhydricity
problems, making it possible to control them by adjusting immersion
frequencies and times.
5.3 Acclimatization of material produced in a temporary
immersion system
Losses during the acclimatization stage can severely handicap
conventional micropropagation procedures. Indeed, the physiological
condition and hyperhydricity of plants grown on an agar medium or in a
liquid medium make this a problematic stage. Temporary immersion systems
have resulted in more successful acclimatization. Ex vitro acclimatization
and rooting of pineapple shoots (Escalona et al., 1999) and sugarcane shoots
(Lorenzo et al., 1998) produced in a temporary immersion system under
semi-industrial conditions has proved efficient and routine. In pineapple, the
survival rate increases linearly with shoot size. Shoots measuring over 6 cm
can be grown directly in the greenhouse, with 90 to 100% successful
acclimatization (Escalona et al., 1999). Smaller shoots require a longer
culture period prior in the bioreactor to acclimatization. Similarly, coffee
microcuttings (C. arabica and C.�canephora) obtained by temporary