The progress of banana improvement by conventional breeding methods has been slow due to narrow genetic variability resulting from low female fertility; therefore, in vitro somaclonal variation caused by mutagenesis treatments can be considered to develop variability. Different ethyl methanesulphonate (EMS) treatments were applied to investigate their effects on proliferating shoot tips and to evaluate their potential to create variability among banana cultivars; 'Berangan Intan', 'Berangan' and 'Rastali'. The percentage of apices surviving ranged from 88.71, 81.10 and 90.62, at 150 mM for 30 min to 37.78, 34.44 and 31.03 at 250mM for 60 min in 'Berangan Intan', 'Berangan' and 'Rastali' respectively. In 'Rastali' 150mM concentration for 60 min treatment caused the highest percentage of dormant explants (31.05) but in 'Berangan Intan' and 'Berangan', EMS at 150 and 250 mM for 60 min duration treatment gave the highest percentage of dormant explants production. Percentage of shoot tips regenerating shoots ranged from 78, 75.67 and 74 with 150 mM of EMS solution for 30 min treatment to 30.63, 33.33 and 23.5 at the 60 min/250 mM treatment for 'Berangan Intan', 'Berangan' and 'Rastali' respectively. Based on the proliferation rate of 'Berangan Intan', 'Berangan' and 'Rastali' LD50 was 189.8, 177.58 and 152.04 for 30 min duration treatment and 154.17 mM, 148.21mM and 141.83 mM for 60 min duration treatment respectively. The recommended treatments were adjudged to be 60min/200mM and 30min/250mM of EMS for all cultivars tested in this study, which resulted in percentage of phenotypic variations of 10.74% and 11.40% respectively for 'Berangan Intan', 12.42% and 12% respectively for 'Berangan', 13.17% and 18.26% respectively for 'Rastali.
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KEYWORDS: Abnormality index, Banana, Ethyl methanesulphonate, Mutation induction, Proliferation rate, Somaclonal variation
Abbreviations: EMS, ethyl methanesulphonate; DMSO, dimethylsulphoxide; BAP, benzyl aminopurine.
Mutagenesis is depicted as the exposing of every kind of biological material to a physical or chemical mutagen to enhance the frequency of mutation above the natural rates (Benfey et al., 1990; Kodym & Afza, 2003). As compare with physical mutagens, chemicals may increase gene mutations (point mutation) rather than chromosomal changes (Ikushima, 1987; Van Harten, 1998; Okagaki et al., 1991; Jabeen & Mirza, 2002; Toker et al., 2007). Ikushima (1987) asserted that the majority of somatic mutation caused by ionizing radiations arises through chromosomal changes but chemical mutagens result in point mutations. One of the most critical attentions in mutation induction procedures is the selection of an effective mutagen agent with a suitable concentration for a definite period of time at a particular temperature defined as efficient dose for mutating of biological materials therefore, the measurement of LD50 for chemicals is determined by altering the concentration and duration of treatment (Predieri, 2001; Kodym & Afza, 2003). The efficiency of mutagenic agent depends on both properties of chemical and genotype (Kodym & Afza, 2003). An increase in concentration of EMS causes enhancing mutation, but makes tissue damage or a decrease in survival while the duration of the treatment should be long enough to permit hydration and infusion of mutagen to the target tissue. Temperature does not directly affect the rate of diffusion, but it influences the rate of hydrolysis of the mutagen solution as with decrease of temperature the hydrolysis rate is increased and the mutagen remains more stable which consequently ensures reactivity with the cells of target tissue (Kodym & Afza, 2003). Application of in vitro mutagenesis strategies systems especially among vegetative propagated crops such as banana and potato along with in vitro selection have significantly improved the efficiency of mutation techniques in breeding programs (Cassells, 2002; Cassells & Doyle, 2003).
Among chemicals, Ethyl- methanesulphonate (EMS; CH3SO2OC2H5), belonging to the group of the alkylating agents, has been reported as a very effective and efficient mutagen for creating of somaclonal variation in plant crops such as banana, grapevine, pepper, sweet potato, petunia, rose and chrysanthemum (Omar et al., 1989; Bhagwat & Duncan, 1997; Van Harten, 1998; Nonomura et al., 2001; Predieri, 2001; Jabeen & Mirza, 2002; Kodym & Afza, 2003; Hofmann et al., 2004; Latado et al., 2004; Luan et al., 2006; Khawale et al., 2007; Berenschot et al., 2008). Latado et al. (2004) obtained many new cultivars of chrysanthemum from induced mutation. In this study the sensitivity of pedicles to EMS was shown as LD50 which was equal to 0.82% (v/v). Their results showed the efficiency of EMS to induce mutation of chrysanthemum through in vitro culture. In Capsicum annuum, EMS was applied to increase the genetic variability and to determine the optimal time requirement for the genetic variability. The results of this study showing the dosage of EMS treatment below the toxic level which suggested being 0.5% for 6 hours, could be used to enhance the genetic variability (Jabeen & Mirza, 2002). Mutation induction in banana has been carried out by subjecting of shoot tips to EMS followed through regenerating of treated shoot tips (Omar et al., 1989; Bhagwat & Duncan, 1997; Predieri, 2001). The best response of shoot tips to EMS was assumed as 24.69 mM and 3 hours of treatment. The effective role of dimethyl sulfoxide (DMSO) as a carrier agent in this study was clearly observed (Omar et al., 1989). Bhagwat and Duncan (1997) also used chemical mutagens in banana (Musa spp. AAA group) to produce mutants displaying resistance against fusarium wilt (Fusarium oxysporum f. sp. cubense). They determined 200 mM of EMS for 30 min as an optimal dose and duration treatment. In vitro mutation induction for salt tolerance using EMS was also reported in sweet potato (Luan et al., 2006).
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The aim of this study was to determine the optimal dose range of ethyl- methanesulphonate (EMS) for subsequent mutation induction studies in banana cultivars to create variation and to choose the most suitable dosage based on in vitro growth and proliferation rate reduction by assessing the dosage that results in a 50% reduction of growth and proliferation (LD50).
Material and methods
Micropropagated cultures of banana cultivars; 'Berangan Intan', 'Berangan' (AAA group) and 'Rastali' (AAB group) were used as the source of materials for the excising of shoot tips. Micropropagation medium consisted of the MS medium (Murashige and Skoog, 1962) amended with 22.2 ÂµM BAP. After three months of culture to allow further proliferation, the shoot apices were separated from shoot clusters and then trimmed to a size of approximately 5 to 7 mm by removing several sheathing leaves and excision with minimum basal corm tissues. Aqueous solution containing 1 M of ethyl methanesulphonate (EMS) along with 1% v/v dimethylsulphoxide (DMSO) as a carrier agent was prepared in the deionized water and kept in 4oc. this solution was diluted with 0.1 M phosphate buffer (pH 7) using filter - sterilization under aseptic condition to give working solutions of mutagen as the final concentrations of EMS were: 150, 200 and 250 mM. Sterile deionized water and sterile phosphate buffer were also prepared as controls. Excised shoot apices were submerged in mutagen solutions and controls as 1ml/apex for different periods of 30 and 60 min for each concentration. In the case of control solutions only period of 60 min was considered. All treatments were carried out at 25oc under room temperature. Fallowing EMS treatment shoot apices were rinsed three times with sterile water and allowed to be dry for a while as it has been reported that prolonged post - treatment washing, followed by drying, results in a reduction in biological damage without a decrease in mutation yield (Benfey er al., 1990). Afterwards, EMS treated Explants were inoculated in 300 ml capacity jars (6apices/jar) consisting of 60 ml MS basal salts and vitamins and sucrose (30 g/L ), solidified with 2.8 g L-1 gelrite amended with 22.2 ÂµM of benzyl aminopurine (BAP). Cultures were kept under a controlled environment at 28oc Â± 2oc with 16 h photoperiod supplemented with cool white florescent light for 6 months to allow further proliferation and during this period subcultures were achieved at 45 days interval. After the first period of 45 days, the fresh weights of proliferating shoot tips for each replicated treatment were recorded and then subculture was carried out to the same proliferation medium as above for a farther 45 days. After the second period of 45 days, data were collected from all replicated treatments to document the percentage of apices regenerating shoots, the average number of shoots per treated shoot tips, the number of shoot tips which showed no farther growth but were alive which in this study called as dormant shoot tips and the percentage of shoot tips surviving. When shoot tips are used for chemical mutagenesis, a protocol will be needed to support their in vitro growth and to produce the maximum proliferation of shoots after mutation induction treatment therefore, the percentage of in vitro growth of EMS treated shoot tips was calculated using the following formula according to Musoke et al. (1999):
The percentage of in vitro growth = Ã- 100
Then the LD50 was calculated as the dose of EMS which reduces the percentage of in vitro growth among treated shoot tips to 50% of untreated control shoot tips based on leaner regression. LD50 calculation on the capacity of the proliferation rate was also worked out as the dose of EMS that reduces the number of shoots regenerated per treated shoot tips to 50% of untreated shoot tips based on leaner regression according to Predieri and Virgilio (2007). After three months of culture, data were recorded for phenotypic variations comprised: leaf color changes, dwarfism and aberrant morphology production such as; hyperhydricity and abnormality among total shoots regenerated from treated and untreated shoot tips, then the percentage of variations among shoots regenerated from treated shoot tips compared to the untreated control shoot tips was assumed as a criterion for efficiency of the different EMS treatments as a mutagen to induce mutation. The percentage of phenotypic variation was calculated according to Bhagwat and Duncan (1997) with modification as fallow:
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Percentage of phenotypic variations = Ã-100
The experiments were arranged in a completely randomized design with six replicates and the data collected and calculated were analysed using SAS and MSTATC computer programs and comparison of means were tested for significance, using LSD test, at 0.05 level of probability.
Different concentrations of EMS and treatment times of incubation were applied to investigate their effects on proliferating shoot tips and to evaluate their potential to create morphological variability among banana cultivars; 'Berangan Intan', 'Berangan' and 'Rastali'. After the second period of 45 days treatment with EMS, the percentage of apices surviving ranged from 88.71, 81.10 and 90.62, at 150 mM for 30 min to 37.78, 34.44 and 31.03 at 250mM for 60 min in 'Berangan Intan', 'Berangan' and 'Rastali' respectively, as with increasing the dosage of EMS, survival of apices significantly decreased for both duration treatments (Table 1). Nevertheless, not all surviving apices gave rise to shoot and a large number of them remained dormant whereas they represented no farther growth even after six month of culture following EMS treatments (Fig. 1). Dormant shoot tips (Table 1) were taken place at all EMS treatments except controls. The occurrence of dormant shoot tips was observed to be cultivar dependent responses to different concentrations and duration treatments of EMS, as in 'Rastali' 150mM concentration for 60 min treatment caused the highest percentage of dormant explants (31.05) but in 'Berangan Intan' and 'Berangan', EMS at 150 and 250 mM for 60 min duration treatment gave the highest percentage of dormant explants production (Table 1). There were also significant differences in the percentage of apices regenerating shoot with altering the concentration and duration of EMS (Table 1) as around thirteen weeks after Treatments with EMS, while the control treatments caused nearly 100% of shoot tips regenerate shoot, percentage of shoot tips regenerating shoots ranged from 78, 75.67 and 74 with 150 mM of EMS solution for 30 min treatment to 30.63, 33.33 and 23.5 at the 60 min/250 mM treatment for 'Berangan Intan', 'Berangan' and 'Rastali' respectively (Table 1). Therefore, as the concentration of EMS increased the percentage of shoot - regenerating apices decreased and noticeable was the similarity of the trend in decrement at both treatment periods (Table 1). The average number of shoots per explants declined significantly from the controls to the highest dose and duration of EMS as the lowest number of shoot regeneration was occurred at the 60 min/ 250 mM treatment for all cultivars (Table 2), however cultivar; 'Rastali' showed the highest potential of shoot regeneration in all EMS treatments (Table 2). Statistical analysis of the weights of shoot tips 45 days after treatments revealed significant differences between the controls and all EMS treatments (Table 2); however significant differences among shoot tip fresh weights were depended on the mutagen concentration as well as duration treatment which are presented in Table 2. Referring to the concentration and duration treatment, the lowest value of shoot tip weight were recorded at the highest concentration (250 mM) for 60 min incubation of EMS (1.68 g, 1.38 g and 1.89 g for 'Berangan Intan', 'Berangan' and 'Rastali', respectively) (Table 2). The dose of EMS that resulted in a 50% reduction of proliferation and growth (LD50) was assessed based on the number of regenerated shoots achieved in the second subculture (Table 3) and based on the in vitro growth reduction (Table 4) calculated in the first subculture after treatment. The EMS dose inducing LD50 was calculated with the equation (Tables.3 and 4), by substituting (y) with the value of 50% of control (0Mm of EMS). Based on the proliferation rate of 'Berangan Intan', 'Berangan' and 'Rastali' LD50 was 189.8, 177.58 and 152.04 for 30 min duration treatment and 154.17 mM, 148.21mM and 141.83 mM for 60 min duration treatment respectively (Table 3). In the case of in vitro growth reduction, LD50 was estimated 302.3 Mm, 334.64Mm and 217.16 mM for 30 min duration treatment and 175.77 mM, 166.99 mM and 167.79 mM for 60 min duration treatment in 'Beragan Intan', 'Berangan' and 'Rastali' respectively (Table 4).
Data collected for phenotypic variation observed among regenerated shoots three months after treatment of shoot tips with EMS are presented in Table 5. The phenotypic variations were observed as leaf colour change, short interval among leaves as dwarfism, hyperhydricity and aberrant morphology production (Fig 2). A noticeable was all leaf colour changes which were observed as a narrow violet stripes on the leaf blades (Fig 2). Total percentage of phenotypic variations among regenerated shoots ranged from 2.9, 1.87 and 3.66 at 30 min/150 mM to 12.22, 13.68 and 19.54 at 60 min/250 mM of EMS for 'Berangan Intan', 'Berangan' and 'Rastali' respectively (Table 5). The highest phenotypic variation was obtained by the highest level of EMS treatment (60 min / 250 mM ) for all cultivars tested in this study (Table 5), however, the lowest percentage of survival and capacity of proliferation among such treated shoot tips for all cultivars renders this treatment unusable. In 'Berangan Intan', 60min/200mM and 30min/250mM of EMS treatment resulted in 59.87% and 57.67% survival as well as 45.33% and 41.20% shoot regeneration respectively from explants (Table 1). In 'Berangan', 60min/200mM and 30min/250mM of EMS treatment resulted in 48.33% and 43.89% survival as well as 48.67% and 42.33% shoot regeneration respectively from explants (Table 1). For 'Rastali', 60min/200mM and 30min/250mM of EMS treatment resulted in 42.78% and 57.22% survival as well as 39.67% and 46.67% shoot regeneration respectively (Table 1). Therefore the recommended treatments were adjudged to be 60min/200mM and 30min/250mM of EMS for all cultivars tested in this study, which resulted in percentage of phenotypic variations of 10.74% and 11.40 respectively for 'Berangan Intan', 12.42% and 12% respectively for 'Berangan', 13.17% and 18.26% for 'Rastali' (Table 5).
Different EMS treatments revealed a gradual reduction in survival, growth and regeneration capacity of the treated shoot tips among all cultivars tested in this study as the concentration and duration treatment of EMS increased which is also in agreement with pervious findings in banana, chrysanthemum, and soybean (Omar et al., 1989; Bhagwat & Duncan, 1997; Latado et al., 2004; Hofmann et al., 2004). Omar et al. (1989) reported an obvious reduction in the number of regenerated shoots from treated explants with increasing concentration of EMS. They suggested that the best response of shoot tips to the EMS was achieved with 24.69 mM following 3 hours of incubation in mutagenic solution furthermore, their results also revealed the essential role of DMSO as a carrier agent to accelerate the uptake of EMS into the explants. Regarding their offer based on lower concentrations of DMSO to prevent browning of explants and enhance absorption to present better results, we applied just 1mM of dimethylsulphoxide (DMSO) as a carrier agent in mutagen solution treatments. Results of this study showed that survival of explants also decreased with the increase of concentration and time of incubation in EMS. The results of this investigation obviously indicate that mutation frequency increases with increasing of dosage and time duration of EMS treatment which is in agreement with the findings reported by Bhagwat and Duncan (1989), but regarding the significant reduction of survival and regeneration capacity of treated shoot tips, the efficiency of mutagen also decreases. Therefore, an optimum dose of EMS should be chosen based on some factors such as percentage of survival and proliferation rate as well as reduction of regeneration capacity by 50% (LD50). Novak et al. (1987) developed in vitro shoot tip culture as efficient system for mutation induction in banana and plantain. Novak et al. (1990) also stated the suitable dose of gamma irradiation on shoot tips based on one that reduces growth and regeneration to 50% of untreated control (LD50). According to Novak et al. (1990), our results showed that the suitable dose of EMS on the base of 50% proliferation rate reduction could be 189.80 mM, 177.58 mM and 152.04mM for 30 min duration treatment and 154.17 mM, 148.21 mM and 141.83 mM for 60 min duration treatment for 'Berangan Intan', 'Berangan' and 'Rastali' respectively, and based on in vitro growth reduction, the suitable dose of EMS was assumed to be 302.30 mM, 334.64 mM and 217.16mM for 30 min duration treatment and 175.77 mM, 166.99 mM and 167.79 mM for 60 min duration treatment for 'Berangan Intan', 'Berangan' and 'Rastali' respectively. However, Bhagwat and Duncan (1997) pointed out that a 50% growth reduction was not necessarily the best criterion for determining optimum treatment conditions for chemical mutagens which in the case of 50% in vitro growth reduction we are also agree with them, but regarding the huge necessity of recovery and highly speediness of cloning variants after mutation induction, we concluded that expression of LD50 based on the proliferation rate should be an important criterion for determining of the optimum dose of chemical mutagen treatment. Moreover, Khawale et al. (2007) using in vitro induced mutation with chemical mutagens in grape, expressed the LD50 on the basis of in vitro survival of their treated explants. Latado et al. (2004) estimated the LD50 for the mutagenesis of chrysanthemum pedicles with EMS to be 82% (v/v) and they chose 0.77% (0.077 M) of EMS to treat the explants. In the case of cultivars tested in our study, EMS at 200 mM for 60 min and 250mM for 30 min was considered as the most efficient treatment based on relatively high percentage of phenotypic variability with the percentage of apices regenerating shoot ranging from 41% to 48% and survival of 42% to 60% depending on cultivars. Bhagwat and Duncan (1997) offered 200 mM of EMS for 30 min as the best treatment for banana shoot tips as they recorded 5.8% phenotypic variation, with survival of 80% and regeneration from 31.6% of treated explants. The differences between percentage of survival and regeneration could be due to different cultivar responses to EMS treatments. The appearance of shoot tips displaying no farther growth and remaining dormant even after six months of culture could be due to the inhibitory effect of EMS according to previous reports (Bhagwat & Duncan, 1997), however, frequency of this in vitro response to EMS concentrations and duration treatments was not the same among three cultivars tested in this study. Comparatively, the higher frequency of variations observed among regenerated shoots from all cultivars tested in this investigation comprise of leaf color changes, aberrant morphology and dwarfism than that obtained by Bhagwat and Duncan, (1997) who reported 5.8% as the highest frequency of phenotypic variation among regenerated shoots, enable us to assume EMS as an efficient treatment to create variability in banana cultivars. Furthermore, all leaf color changes observed in this investigation was as a narrow violet strips on leaf blade which has never been reported before.