Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO MUTAGENESIS IN BANANA 557
with isodiametric cells with dense cytoplasm studded with starch grains) and their
potential to develop into somatic embryos and plantlets is equally important.
• Knowledge about the LD50 dose helps in finalizing the actual irradiation-doses
for mutation induction. For example, if the LD50 is observed to be 30 Gy, the
•
in vitro cultures can be mutagenized by gamma-irradiation at 20, 30 and 40 Gy.
The doses above this range would possibly result in abnormal/detrimental
variations and the doses in lower range will result in production of excessively
large populations with low chances of recovering a desirable mutant.
It is well known that the irradiation-damaged cells can lose competence for
proliferation and hence either are suppressed or lost in competition with normal
ones. For this reason, no shoot-apical regions shall be discarded during subculture
of the mutagenized shoot-tip multiples.
• The dose rate (Gy/min) at the time of irradiation shall be recorded before
beginning with irradiation experiments.
• The cells for irradiation as well as the non-irradiated control shall be derived
from same cell line.
• The non-irradiated control populations are maintained in parallel with the
irradiated populations. Each dose is replicated at least 3 times.
• At the time of each subculture of irradiated and control cells, an aliquot of 1 ml
cells are plated for checking embryo development and plantlet regeneration.
• The number of cultures increases exponentially with each subculture cycle
warranting advance-planning and availability of the resources.
6. CONCLUSIONS AND PROSPECTS
Efficient in vitro regeneration system via direct organogenesis and somatic embryogenesis
is critical for studies on genetic manipulation using either mutagenesis or
genetic transformation. In order to be applicable, in vitro mutagenesis needs to be
extended to diverse banana cultivars. Estimation of radiosensitivity is important in
determining the actual irradiation doses. Shoot-tip multiples and cell suspensions can
be effectively used for mutagenesis; however both have their own advantages and
limitations. Shoot-tip cultures are easy to establish but pose a serious problem for
chimera separation. On the other hand, the cell suspensions have been reported to be
ideal for overcoming chimerism but still are not routine to establish and regenerate. So
far, there has been no published data in case of the mutant-populations arising from
mutagenesis of cell suspensions. Although there could be comparatively less probability
of having chimeric genotypes for field-testing, this needs to be validated with
experimental evidences.
The ability to culture and manipulate a large number of totipotent cells provides
a greater opportunity for in vitro selection of useful mutations at cellular level.
It is possible to select for cell lines resistant to biotic stress for example disease
resistance and tolerant to abiotic stress viz. aluminim tolerance, salt, drought and
frost tolerance. In banana, studies have been conducted on in vitro selection
following mutagenesis, mainly for fungal disease resistance. A complete operating
strategy should involve in vitro mutagenesis and selection, ex vitro confirmation of