Liquid Culture Systems for in vitro Plant Propagation

Potential of flow cytometry 339
dissociate chimeras. This method was also applied on ECS to verify the
unicellular origin of somatic embryos. After colchicine treatment, the ploidy
of two cell lines were determined using flow cytometry before transfer to the
regeneration medium and after complete regeneration into plantlets.
3. Results
Abnormalities in DNA content could be detected at an early stage during
in vitro culture. Two non-regenerable banana suspensions, characterized by
their extremely fine structure and white colour were growing twice as fast as
compared to regenerable embryogenic cell suspensions. Flow cytometric
analysis revealed that these unusual suspensions were in fact polyploid. One
was hexaploid (6x) and the other decaploid (10x) compared to the normal
triploid (3x) cultivars from which the ECS were originating (Figure 1). Other
aneuploid banana ECS with 2n=28 could be detected at an early stage
(Figure 2). Cells from four Musa accessions were analyzed every two or
three days during 30 days of culture. Peaks on graphs displaying the ratio of
frequency of cells in G2 and G1 indicated waves of mitotic activity, i.e., a
certain degree of cell cycle synchrony. It seems that when the cells were
transferred to fresh medium, the cell cycle is stimulated in a relatively
synchronous manner. The highest proportion of cells in G2 phase was
observed 8 days after subculture in three of the four Musa ECS analysed
(Figure 3).
Nuclear DNA content distribution in ECS cultures of two cell lines
(WIL-124C and WIL-124T) was determined using flow cytometry before
transfer to the regeneration medium R1 (Figure 4) and after complete
regeneration into plantlets (Table 1). Even though the two cell lines (were
initiated from the same accession “Williams”, the effect of colchicine on
polyploidy induction was different in both cell lines. The response to
colchicine was clearer for cell line WIL-124C than for WIL-124T especially
when high concentrations are applied (0.1% and 0.2%). The proportion of
polyploidized cells increased to about 50% for WIL-124C, while this was
only 15% for WIL-124T. Among the regenerated plants, none were
mixoploid. This suggest that embryos are of single cell origin (Table 1).