Liquid Culture Systems for in vitro Plant Propagation

Mass propagation of tropical crops 207
et al., 1997a; Cabasson et al., 1997; Berthouly and Etienne, 1999; Vilches et
al., 2002).
Standard bioreactors are also efficient for somatic embryo mass
production, allowing a precise control of environmental factors inside the
culture vessel (e.g. Preil, 1991). The factors limiting their practical
application in plant propagation are frequently associated with the poor
quality of the embryos produced and the lack of efficient germination
protocols, which usually involve the use of semisolid medium, thus
complicating the automation of the propagation process. However, the
combination of bioreactor technology for somatic embryo production and
embryo germination in TIS offers several advantages over conventional
germination in semisolid medium, opening new possibilities for developing
automated systems.
In our lab at the Institute of Plant Biotechnology, Cuba, experiments
were conducted in order to improve the germination of sugarcane somatic
embryos produced in bioreactors and to reduce manual labour costs.
Embryogenic cell suspensions from the cultivar C 8751 were directly
initiated in liquid medium from leaf segments of shoots derived from
axillary bud proliferation medium, according to the protocol described by
Freire (2001). Bioreactor culture was performed in 2-litre bioreactors with
bubble free aeration systems and the oxygen concentration was controlled by
gas blending. The effect of two partial oxygen pressures (40 and 80 %) on
somatic embryo production was studied. The highest production of somatic
embryos was obtained in the bioreactor with 80 %DO2. However, only
embryos developed under 40 %DO2 regime germinated when transferred
either to semisolid or liquid medium in TIS (1-litre twin flasks). In a second
set of experiments DO2 was controlled at 80 % from day 1 to 14 and
afterwards at 40 % from day 15 to 22. A total of 31,400 somatic embryos
was obtained, with a fresh weight of 44.9 g l -1 (Figure 5). The embryos
germinated when transferred to semisolid germination medium or liquid
germination medium in 1-litre TIS flasks, with an average germination of
20.6 % and 18.4 %, respectively (Figure 6).
Regenerated plants were successfully transferred for acclimatization and
a total of 23,200 plants were transplanted to the field. Comparative field
trials were conducted with plants regenerated from bioreactor culture, plants
propagated via axillary bud proliferation, plants regenerated from somatic
embryos from callus culture and traditional seed stalks from growers. Plants
from all in vitro culture methods showed an increase in the number of stems
per square meter and a decrease in stem diameter compared to plants from
traditional seed stalks. No statistical differences were observed in sugar
content (brix) as it was previously described by Jiménez (1995). Further
improvements of the procedure are necessary in order to increase embryo