Liquid Culture Systems for in vitro Plant Propagation

A new approach for automation 417
(110 rpm) at 25°C and under light intensity of 5 µmol m -2 s -1 with a 16 h
photoperiod.
Coffea canephora (var. Robusta) L.: For coffee, young leaves collected
from greenhouse-grown trees (clone FRT105) were disinfected for 30 min in
a 4 % calcium hypochlorite solution then rinsed 3 times in sterile water. The
explants were placed on the callogenesis semi-solid medium described by
Yasuda et al. (1985). It was composed of 1/4 strength macronutrients and
half strength micronutrients of Murashige and Skoog (1962), B5 vitamins
(Gamborg et al., 1968), supplemented with 1.1 mg l -1 benzylaminopurine
(BAP), 30 g l -1 sucrose and 8.0 g l -1 Bacto-Difco agar. To establish
embryogenic cell suspensions, friable and yellowish calli were selected and
transferred into liquid medium of the same composition. The cell
suspensions were subcultured every 2 weeks at an inoculation density of 10
gl -1 . For the embryo production, 0.1 g of biomass was transferred into 100
ml of liquid production medium contained in 250-ml flasks. The production
medium (Ducos et al., 1999) contained macro and micronutrients of
Murashige and Skoog (1962), B5 vitamins (Gamborg et al., 1968), 1.1 mg l -1
BAP and 30 g l -1 sucrose. Adjustment of pH, autoclaving and culture
conditions were the same as previously described for carrot.
2.2 Embryo desiccation
Somatic embryos were dehydrated as reported by Florin et al. (1993). To
induce dehydration tolerance, an osmotic pretreatment was performed by
subculturing the embryo suspensions in a liquid medium containing 0.4 mol
sucrose during the last week of the embryo production phase. Then the
embryos were transferred onto paper disks (Whatman GF/C, diameter 55
mm) and placed into 370-ml glass jars with a 43 % constant relative
humidity (R.H.) created by the presence of a saturated solution of K2CO3.
The jars were placed at 24° C and in darkness.
For the sorting experiments, somatic embryos were bulk collected on
400-µm nylon sieves then placed by layer onto the paper disks for
desiccation (approximately 1,500 embryos per disk).
For the sowing experiments, torpedo-shaped somatic embryos were
manually selected (0.5-1.5 mm and 1.5 to 3.0 mm, for carrot and coffee
respectively) before being placed on the paper disks (150 embryos per disk)
for desiccation.
2.3 Sorting
Bulk dehydrated embryos were placed at the base of a seed selector (Mag
Process, diameter 5 cm, height 50 cm), which divides the seeds according to