Banana and plantains (Musa spp.) are important staple food crops for many people throughout the developing world and major source of nutrition for many people. Cultivar Tanduk is one of the popular banana varieties in Malaysia and widely cultivated by smallholders for production of banana used for the purpose of industrial processing. In banana production industry, the necessity to develop new commercially acceptable banana is still in a concern. Most of existing banana cultivars needs to be improved for some agronomical characteristics such as high yield, early flowering, better physical attributes and quality of fruit. However, it is not easy to fulfill the demands of both domestic and international markets without employing new approach. The conventional breeding of most commercially demanded banana is handicapped due to a complex genetic background, lack of seed formation, parthenocarpic nature and triploidy factors that hamper the improvement of this crop with the desired quality (Kumar, 2006). However, the development of plant cell, tissue culture and mutation breeding using in vitro mutagenesis technique over the last 20 years has made it possible to transfer part of the conventional breeding work from field to laboratory conditions. In vitro mutation breeding can support in overwhelming hurdles in the progress of new and superior banana cultivars for sustainable and continuous fruit production without disease spread (Javed et al. 2004). Through this technology a large number of improved mutant varieties have been released for commercial cultivation in many crops demonstrating the economic value of the mutation breeding technology (Kharkwal and Shu 2009; Jain and Suprasanna 2011).

Gamma rays are the most commonly used mutagenic agent in an in vitro mutagenesis experiment (Roux et al. 2004) since it has higher degree of accuracy, sufficient reproducibility and deep penetrating power into a biological matter that can cause a greater number of variation in physiochemical composition (Yamaguchi, 2003). Biological effect of gamma radiation depends on atoms or molecules interaction inside the cell, especially water, to produce free radicals (Kovacs and Keresztes 2002). These radicals could harm or adjust main components of plant cells and disturb the physiochemical and biochemical process of plants depending on the radiation level (Ashraf et al., 2004). Gamma rays could restructure plant cell and metabolism include thylakoid membranes dilation, modification in photosynthesis, adjustment of the anti-oxidative structure, and accumulation of secondary metabolite (Ashraf et al. 2004; Kovacs and Keresztes 2002; Mbaye, 2017).

In vitro mutagenesis refers to mutations induced by treating explants or in vitro cultures with a mutagen, followed by mutant screening and characterization. This process involves a sequence of essential steps: choice of proper target plant material (explants or cultures); choice of a mutagen and determination of a suitable dose, post-treatment management and a series of subculture, and regeneration of plants for mutant selection. Mutagenic treatments can be applied to explants or in vitro cultures such as multiple shoot cultures, callus, cell suspensions, protoplasts and microspores and a variety of explants can be also used such as apical meristems, axillary buds, roots and tubers. In a principle, all kind of plant material used for mutagenic treatment, whether from explants or in vitro cultures are needed to undergo a series of subcultures after being mutagenized to separate chimeras and produce plants for selection of desired mutants since mutation remains undetectable by chimeras in the first generation (Banerjee et al. 2015).

However, in the recent studies by Karmarkar et al. (2001), who had made an attempt to induce mutation using in vitro individual shoot, the researcher had exposed in vitro individual shoot to gamma radiation followed by sub culturing on rooting medium without undertaking sub-culture processes to examine the post-irradiation rooting response in the laboratory condition. However, he could not go further and observe the explants response to gamma irradiation in the nursery stage. The present study was conducted to fill this gap. Thus, the individual shoot was singled out from multiple shoot culture and mutagenized with gamma radiation and then immediately transferred to rooting medium without undertaking sub-culture. The post-radiation rooting response and growth performance were studied in the laboratory condition and nursery stage. Hence, the present study was conducted to study the effect of different dose of gamma rays on the in vitro individual shoot growth performance under in vitro and nursery condition.

Gamma exposure, culture condition and acclimatization of mutated plantlets

The sword sucker of banana cultivar Tanduk (25~35 cm) in height approximately 3~4 months old were used. Shoot tip culture were established and maintained until fourth (M₁V₄) sub-culture cycle using MS medium supplemented with 5 mg/l BAP. Individual shoot (1~2 cm in length) were isolated and used as explant (Fig. 1). The roots, necrotic tissue and traces of solidifying agent were removed from the explant and then placed in the sterile Petri dish wetted with distilled water. The individual shoot in the Petri dishes were irradiated with different doses of gamma (γ) rays (10, 20, 30, 40, 50, 60 and 70 Gy) using Cesium-137 source at a dose rate of 5.5 Grey/minute to study the radiosensitivity. The total samples of (140) individual shoot were irradiated and the final treatment were selected after this test. The treated explants were then washed thoroughly with sterilized distilled water prior to culture and then cultured aseptically on MS medium fortified with 2 mg/l activated charcoal to regenerate into complete plantlets. The basal medium contained inorganic salts of MS (Murashige and Skoog, 1962) plus Myo-inositol 100 mg/L, Nicotinic Acid 0.5 mg/L, Pyridoxine HCL 0.5mg/L, Thiamine HCl 0.5 mg/L, Glycine 2.00 mg/L and sucrose 30 g/L. The pH was adjusted to 5.7 prior to adding Agar (7 g/L). All cultures were incubated in the culture room at 25 ± 2°C and 55~65% relative humidity with photoperiod of 16 hours light under fluorescent light (1000 lux) and 8 hours dark. After 4 weeks, well grown rooted plantlets (7~9 cm in length) were carefully removed from the culture vessel and then the roots were washed thoroughly in tap water to remove the traces of nutrients and were transplanted in the greenhouse in polybags containing a ratio 2:1 mixture of peat moss and coco peat.

Figure 1.

Individual shoot explant.

Experimental observation and statistical analysis

The LD₅₀ values were determined based on the percentage of surviving of explants. Post-irradiation rooting responses of in vitro individual shoots was determined by recording days to root initiation, root number, root length (cm) and shoot length (cm), during and after 30 days of treatment In the case of hardened plants; plant height (cm), number of leaves and chlorophyll content by using (Minolta chlorophyll meter SPAD-502) (Spectrum Technologies, Inc., Plainfield, IL, U.S.) were measured. The experiment was laid out in a completely randomized design (CRD) with a 5 replication. The data were analyzed by one-way analysis of variance (ANOVA) with Duncan Multiple Range Test (p<0.05). The LD₅₀ calculation using survival rates (%) were analyzed by linear regression (Singh, 1990).

Radiosensitivity and determination of lethal dose (LD₅₀)

In the present study, the survival rate of irradiated explants was found to decrease linearly with the increasing of the gamma doses (Fig. 2). A remarkable decrease in survival rate was observed at 40, 50, 60 and 70 Gy of gamma dosage. Meanwhile, among the treated explants maximum survival rate of 71% was recorded at 10 Gy, followed by 69.5% at a dose of 20 Gy, 67 % at 30 Gy and minimum of survival rate was observed following the T7 (70 Gy) treatment, where remarkably dropped from 100% to 42% at the first week of culture and 15.5% at the fourth weeks of culture resulting in a total reduction of 74.5%. The non- irradiated explant recorded the highest survival percentage of 100%. The lethal dose (LD₅₀) was found to be 33 Gy based on the survival percentage, the estimation was carried out using linear regression analysis. Karmarkar et al. 2001 had made similar observations in cv. Basrai (AAA) which corroborate our finding.In principle, the most effective doses for mutation induction are thought to be the gamma doses lower than the LD₅₀. As a result of these measurements, a large quantity of individual shoot was finally treated with 10, 20 and 30 Gy doses of gamma radiation and then the explants were transferred on rooting medium.

Figure 2.

Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival rate of individual in-vitro shoot.

Effect of gamma irradiation on the growth of irradiated in vitro individual shoots under laboratory condition

Growth assessment of irradiated in vitro individual plantlets in the nursery condition

In conclusion, the result from the current experiment indicated that γ treatments induced morphological and physiological variations on banana cultivar Pisang Tanduk. Lower dose of γ irradiation induced positive impact on the growth of in vitro plantlets during the multiplication phase and rooting stage, whereas higher γ irradiation had caused a negative impact on the growth, in terms of higher mortality rate and reduction in growth.

Therefore, we suggested that γ radiation can be usefully employed in banana for induction of desirable mutations and further, work on field performance of mutants, in terms of early flowering, yield quantity, quality and other agronomical traits need to be looking next.

The author would like to acknowledge Islamic Development Bank Group (IDBG) for generous funding of this research.