Liquid Culture Systems for in vitro Plant Propagation

Temporary immersion system: a new concept 179
4.1 Immersion time
In culture systems with temporary tissue immersion, it is clear that the
immersion time is very important, since it governs nutrient uptake and
expression of hyperhydricity. The immersion times used for different work
vary considerably (Table 1). This is probably due to the large variety of
species, micropropagation processes and temporary immersion systems
used. Long immersion times (1 h every 6 h) prove to be efficient for potato
tuberization, whereas very short immersion times (1 min every 12 h)
stimulate somatic embryo production most in coffee and rubber (Etienne et
al., 1997a,b). Likewise, very frequent immersions (30 sec every 30 sec) can
prove to be highly efficient in tilting machines for grapevine shoot
propagation (Harris and Mason, 1983).
Krueger et al. (1991) showed the importance of immersion frequencies
for the proliferation of serviceberry shoots. Hyperhydricity was observed
with immersions for 5 min every 30 min, but was not seen with immersions
for 5 min every 60 min. On the other hand, the first combination is better for
the number of shoots obtained. In order to combine the advantages of both
combinations, the authors recommended using the first combination during
an initial proliferation cycle, then the second combination to maintain shoot
quality. They also revealed the existence of a period of adaptation, when
switching to the lowest frequencies. Stress resulted in partial desiccation of
the shoots, but the material recovered later. With radiata pine shoots grown
on agar medium, liquid nutrient replenishment at a frequency of twice a
week more effectively stimulated growth (FW x 1.2) and shoot quality (x
1.5) than frequencies of once every 2 or 4 weeks (Aitken-Christie and Jones,
1987).
Berthouly et al. (1995) showed with coffee microcuttings that the
immersion time substantially affected the multiplication rate, estimated by
the number of micronodes produced after 6 weeks. Indeed, immersion times
of 1, 5 and 15 min applied every 6 h gave multiplication rates of 3.5, 5.4 and
8.4 respectively in their experiment. In addition, the optimum immersion
time varied depending on the coffee species used. For instance, it was 15
min every 6 h for C. arabica microcuttings and only 1 min every 6 h for
C. canephora microcuttings.
In coffee, modifying the immersion time greatly affects somatic embryo
production. According to Berthouly et al. (1995), 15 minutes' immersion
every 6 hours led to successive development and germination of embryos,
whereas for an identical culture medium, immersions of 1 minute every 24
hours halted embryo development and stimulated the production of
secondary embryos.