Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO MUTAGENESIS IN BANANA 545
A typical banana improvement programme (Figure 1) would primarily depend on the
availability of elite and diverse germplasm (genetic variability or the gene-pool). It is
obvious that in the case of banana, it would be essential to generate additional genetic
variability facilitating the selection for desirable traits. In this regard, use of cellular and
molecular techniques viz., plant regeneration via tissue/cell cultures, in vitro mutagenesis,
genetic transformation and molecular markers has greater relevance (Jain &
Swennen, 2004; Smith et al., 2005). Mutation induction and selection of desired traits
in combination with in vitro techniques offer several advantages over conventional
methods such as mutagenizing the plant parts of minute size, handling large number
of samples in short time span, rapid production of large populations to separate
chimeras, uniform mutagen treatment and facilitating in vitro selection (van Harten,
1998). For the induction of mutations, both chemical as well as physical mutagens
have been employed, and amongst the physical mutagens, gamma rays have higher
penetrance and energy than any other ionizing radiations, and unlike neutrons, do
not result in secondary radioactivity. Gamma rays have also practically proved to be
better as evidenced by the release of more than 55% of the total released mutant
varieties (Maluszynski et al., 1995). The present article describes a generalized
protocol for in vitro mutagenesis in banana (Musa spp.) using gamma irradiation
of shoot-tip multiples (shoot-tip or multiple shoot cultures) and cell suspension
cultures.
2. ESTABLISHMENT OF IN VITRO CULTURES
In banana, in vitro regeneration can occur through two main pathways: shoot-tip
culture (direct organogenesis or shoot morphogenesis; Vuylsteke, 1998) and somatic
embryogenesis (indirect morphogenesis via callus and cell suspensions). Shoot apices
containing shoot apical meristems give rise to plantlets following the inhibition of
apical dominance. Indirect morphogenesis involves the initial culturing of male flower
buds or in vitro proliferating shoot-meristems, to produce callus (an undifferentiated
mass of cells) and somatic embryogenesis (Strosse et al., 2003).
The first step in conducting in vitro mutagenesis experiments is to establish the
profusely proliferating tissue/cell cultures with high frequency regeneration potential.
These can be used for inducing the mutations using ionizing radiations such as
gamma rays. Details of all the culture media compositions are provided in Table 1.
2.1. Shoot-tip Culture Pathway
2.1.1. Initiation of Aseptic Shoot-tip Cultures
The shoot apices either from side-suckers (Figure 2A) or male inflorescences are used
as the source materials for the establishment of in vitro shoot-tip cultures. The suckers
collected from the field growing plants are thoroughly washed with tap water to remove
adhering soil, after removing leafy top and the roots. These are trimmed to a size of
approximately 3–4 cm in length and 2 cm in diameter, by removing several sheathing
leaves and excision of a part of the corm tissue.