Liquid Culture Systems for in vitro Plant Propagation

338 Nicolas Roux et al.
1. Introduction
The use of shoot-tip culture in Musa has allowed great progress in mass
propagation (micropropagation), conservation (medium-term conservation
and cryopreservation), elimination of virus diseases and exchange of
germplasm (Van den Houwe et al., 1995; Panis et al., 1996). Unfortunately,
the use of banana shoot tips as target tissues for genetic engineering
strategies such as in vitro mutagenesis and genetic transformation leads to
chimeric plants (Roux et al., 2001). Somatic embryogenesis is usually a
preferred mode of regeneration over organogenesis because of the presumed
single cell origin of the regenerants (Thorpe, 1988; Peschke and Phillips,
1992). The occurrence of off-types among regenerants has delayed the widespread
industry acceptance of micropropagated bananas (Smith and Hamill,
1993). Before adopting somatic embryogenesis as a new method to support
genetic improvement and mass propagation of bananas and plantains, further
studies need to be undertaken to better understand the phenomenon of
somaclonal variation. The aim of this study was to verify the ploidy level
stability of embryogenic cell suspensions prior to the formation of embryos
or plants.
2. Material and methods
Embryogenic cell suspensions (ECS) of four triploid (2n=3x=33) Musa
cultivars were kindly provided by the Laboratory of Tropical Crop
Improvement at the Katholieke University of Leuven, Belgium. The
suspensions were maintained in 100 ml Erlenmeyer flasks containing 15 ml
of liquid maintenance media (Dhed’a et al., 1991). Erlenmeyer flasks were
placed on an orbital shaker at 70 rpm. The medium was renewed every two
weeks.
For monitoring the genetic stability of ECS, flow cytometric analysis was
performed according to Dolezel et al. (1997). The flow cytometric assay
involved the use of nuclei isolated from chicken red blood cells (CRBC),
which served as internal reference standard. The gain of the instrument was
adjusted so that the Go/G1 peak of CRBC nuclei was positioned
approximately at channel 100. The relative DNA content of ECS was
expressed as a ratio of DNA content of CRBC and Musa (DNA index). By
flow cytometric analysis, it was also possible to measure the proportion of
cells in the G1 and G2 phase of the cell cycle since the DNA content during
G2 is doubled compared to G1 phase. Through a combination of mixoploidy
induction using colchicine and flow cytometry detection, Roux et al. (2001)
could monitor the effectiveness of three micropropagation techniques to