Liquid Culture Systems for in vitro Plant Propagation

416 J.P. Ducos et al.
causes contaminations after transfer to a non-sterile environment (Molle et
al., 1993). Mainly due to this difficulty to control the asepsis during the
embryo development, this approach is difficult to implement. Consequently,
some authors have described another approach where somatic embryos are
embedded in sterilized plugs moistened with medium containing sucrose
then maintained in an aseptic environment until they become
photoautotrophic (Timmis et al., 1991, Gupta et al., 1993; Dupuis et al.,
1999). However, automation of embryo distribution from liquid medium
onto such plugs causes problems of aggregation and blockage inside the
delivery pipes.
As a post-harvest treatment, desiccation has been applied to many species
to enhance the embryo-to-plantlet conversion rate and/or the short term
storage (Florin et al., 1993; Attree and Fowke, 1993). We investigated
whether dehydrated somatic embryos can be handled using seed sowing
technologies and equipment. To evaluate this possibility, we used somatic
embryos of Daucus carota L. and Coffea canephora L., two species for
which large numbers of somatic embryos can be produced in stirred
bioreactors, up to 10 6 l -1 in Daucus and up to 0.4·10 6 l -1 in Coffea (Ducos et
al., 1993 a, b).
2. Materials and methods
2.1 Embryo formation
Daucus carota L.: Cell cultures were initiated from hypocotyl segments
of aseptic seedlings of a cytoplasmic male sterile genotype (Clause
Semences, Bretigny Sur Orge, France). Embryogenic tissues were developed
on semi-solid medium (8.0 g l -1 of Bacto-Difco agar) then multiplied in
liquid medium of the same composition. This medium contained the macro
and micronutrients of Murashige and Skoog (1962), the mixture of organic
substances of Halperin (1964), 0.1 mg l -1 2,4-dichlorophenoxyacetic acid
(2,4-D) and 20 g l -1 sucrose. The medium pH was adjusted to 5.8 prior to
autoclaving (1.1 bar, 20 min at 115° C). Cell cultures were maintained by
repeated subcultures every 12 days at an inoculation density of 10 g l -1 . For
embryo production, cultures were successively filtered through two nylon
sieves of 100 and 50 µm pore sizes. The cell clumps remaining on the latter
filter were washed three times in fresh basal medium lacking 2,4-D and then
transferred into the same medium at a density of 0.1 % (v/v) based on
packed cell volume. Embryo productions were achieved in 0.5 litre of
medium contained in 1-litre flasks. Cultures were grown on a orbital shaker