Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO MUTAGENESIS IN BANANA 553
In an earlier study, Kulkarni et al. (1997) subcultured the irradiated populations for
four cycles (including rooting). Subsequently it was decided to test the adequacy of
subculturing up to M1V4 for chimera dissociation, by studying the variability at the field
level (Kulkarni et al., unpublished results). An experiment was hence designed to
test the 5 populations derived from 5 suckers (originating from a single selected
mutant plant) for existence of inter-population variability. Detection of significant
differences between all the 5 populations indicated that the “selected initial plant”
was possibly chimeric at the time of field testing, and thus additional in vitro
multiplication cycles were necessary for proper chimera separation. It was also
possible that the populations differed because of the somaclonal variations during
in vitro passages. The differential expression could as well be an integral effect of
these and other unknown causes. In view of this, it is suggested that the subculturing
shall continue till M1V5 – 7 (Novak et al., 1990; Rao et al., 1998) or even more as the
case may be.
3.2. Cell Suspensions
The induction and establishment of ECS in a specific banana genotype of interest is still
not a routine practice, and thus, only a few reports are available on gamma-irradiation
of ECS (Kulkarni et al., 2004a; Roux et al., 2004). The following procedure is suggested
for mutagenizing the banana cell suspensions using gamma irradiation.
3.2.1. Radiosensitivity Estimation
About 0.5–1.0 ml aliquot of the ECS containing about 25–50 mg fresh weight cells in
the exponential (G1) growth phase are transferred to Eppendorf type plastic tubes of 1.5
ml capacity and centrifuged at 5000 rpm for 5 min (Kulkarni et al., 2004a). Alternatively,
these cells can also be irradiated in the sterile Petri dishes (Roux et al., 2004).
Supernatant medium is discarded and the tube with pelleted cells is exposed to
60 Co gamma rays (0–100 Gy, in steps of 10 Gy). Each treatment shall contain
minimum 3 replications. Immediately after irradiation, the cell pellets are transferred
to 20 ml of fresh liquid medium in 100 ml capacity conical flasks. The growth of the
cells during the subcultures is determined by recording fresh and dry weights in 1 ml
aliquot. The cells are plated (as described above) for embryo development and
plantlet regeneration (as given in the earlier section). The plantlet regeneration data
is compared with the non-irradiated control cells and the LD50 is estimated.
Kulkarni et al. (2004a) observed that the growth of cells reduced with increasing
dose up to 30 Gy, and a dose of 40 Gy and beyond was observed to be completely
lethal (Figure 5). Accumulation of cell weight (with each successive subculture) was
maximum in control, and reduced with increasing dose. The gain in fresh and dry
weights of the cells with increasing irradiation dose was very gradual during first
two subculture cycles, but became evident in the 3rd subculture and was maximum
in the 4th one.