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The advancement of banana betterment by conventional genteelness methods has been slow due to narrow familial variableness ensuing from low female birthrate ; hence, in vitro somaclonal fluctuation caused by mutagenesis interventions can be considered to develop variableness. Different ethyl methanesulphonate ( EMS ) interventions were applied to look into their effects on proliferating shoot tips and to measure their possible to make variableness among banana cultivars ; ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ . The per centum of vertexs lasting ranged from 88.71, 81.10 and 90.62, at 150 millimeter for 30 min to 37.78, 34.44 and 31.03 at 250mM for 60 min in ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally. In ‘Rastali ‘ 150mM concentration for 60 min intervention caused the highest per centum of hibernating explants ( 31.05 ) but in ‘Berangan Intan ‘ and ‘Berangan ‘ , EMS at 150 and 250 millimeter for 60 min continuance intervention gave the highest per centum of hibernating explants production. Percentage of shoot tips renewing shoots ranged from 78, 75.67 and 74 with 150 millimeters of EMS solution for 30 min intervention to 30.63, 33.33 and 23.5 at the 60 min/250 millimeter intervention for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally. Based on the proliferation rate of ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ LD50 was 189.8, 177.58 and 152.04 for 30 min continuance intervention and 154.17 millimeter, 148.21mM and 141.83 millimeter for 60 min continuance intervention severally. The recommended interventions were adjudged to be 60min/200mM and 30min/250mM of EMS for all cultivars tested in this survey, which resulted in per centum of phenotypic fluctuations of 10.74 % and 11.40 % severally for ‘Berangan Intan ‘ , 12.42 % and 12 % severally for ‘Berangan ‘ , 13.17 % and 18.26 % severally for ‘Rastali.

KEYWORDS: Abnormality index, Banana, Ethyl methanesulphonate, Mutation initiation, Proliferation rate, Somaclonal fluctuation

Abbreviations: EMS, ethyl methanesulphonate ; DMSO, dimethylsulphoxide ; BAP, benzyl aminopurine.

Introduction

Mutagenesis is depicted as the exposing of every sort of biological stuff to a physical or chemical mutagen to heighten the frequence of mutant above the natural rates ( Benfey et al. , 1990 ; Kodym & A ; Afza, 2003 ) . As comparison with physical mutagens, chemicals may increase cistron mutants ( indicate mutant ) instead than chromosomal alterations ( Ikushima, 1987 ; Van Harten, 1998 ; Okagaki et al. , 1991 ; Jabeen & A ; Mirza, 2002 ; Toker et al. , 2007 ) . Ikushima ( 1987 ) asserted that the bulk of bodily mutant caused by ionising radiations arises through chromosomal alterations but chemical mutagens result in point mutants. One of the most critical attendings in mutant initiation processs is the choice of an effectual mutagen agent with a suited concentration for a definite period of clip at a peculiar temperature defined as efficient dosage for mutating of biological stuffs hence, the measuring of LD50 for chemicals is determined by changing the concentration and continuance of intervention ( Predieri, 2001 ; Kodym & A ; Afza, 2003 ) . The efficiency of mutagenic agent depends on both belongingss of chemical and genotype ( Kodym & A ; Afza, 2003 ) . An addition in concentration of EMS causes heightening mutant, but makes tissue harm or a lessening in endurance while the continuance of the intervention should be long plenty to allow hydration and extract of mutagen to the mark tissue. Temperature does non straight affect the rate of diffusion, but it influences the rate of hydrolysis of the mutagen solution as with lessening of temperature the hydrolysis rate is increased and the mutagen remains more stable which accordingly ensures responsiveness with the cells of mark tissue ( Kodym & A ; Afza, 2003 ) . Application of in vitro mutagenesis schemes systems particularly among vegetive propagated harvests such as banana and murphy along with in vitro choice have significantly improved the efficiency of mutant techniques in engendering plans ( Cassells, 2002 ; Cassells & A ; Doyle, 2003 ) .

Among chemicals, Ethyl- methanesulphonate ( EMS ; CH3SO2OC2H5 ) , belonging to the group of the alkylating agents, has been reported as a really effectual and efficient mutagen for making of somaclonal fluctuation in works harvests such as banana, pipeline, Piper nigrum, sweet murphy, petunia, rose and chrysanthemum ( Omar et al. , 1989 ; Bhagwat & A ; Duncan, 1997 ; Van Harten, 1998 ; Nonomura et al. , 2001 ; Predieri, 2001 ; Jabeen & A ; Mirza, 2002 ; Kodym & A ; 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 mutant. In this survey the sensitiveness of pedicels to EMS was shown as LD50 which was equal to 0.82 % ( v/v ) . Their consequences showed the efficiency of EMS to bring on mutant of chrysanthemum through in vitro civilization. In Capsicum annuum, EMS was applied to increase the familial variableness and to find the optimum clip demand for the familial variableness. The consequences of this survey demoing the dose of EMS intervention below the toxic degree which suggested being 0.5 % for 6 hours, could be used to heighten the familial variableness ( Jabeen & A ; Mirza, 2002 ) . Mutation initiation 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 & A ; Duncan, 1997 ; Predieri, 2001 ) . The best response of shoot tips to EMS was assumed as 24.69 millimeter and 3 hours of intervention. The effectual function of dimethyl sulfoxide ( DMSO ) as a bearer agent in this survey was clearly observed ( Omar et al. , 1989 ) . Bhagwat and Duncan ( 1997 ) besides used chemical mutagens in banana ( Musa spp. AAA group ) to bring forth mutations exposing opposition against fusarium wilt ( Fusarium oxysporum f. sp. cubense ) . They determined 200 millimeter of EMS for 30 min as an optimum dosage and continuance intervention. In vitro mutant initiation for salt tolerance utilizing EMS was besides reported in sweet murphy ( Luan et al. , 2006 ) .

The purpose of this survey was to find the optimum dose scope of ethyl- methanesulphonate ( EMS ) for subsequent mutant initiation surveies in banana cultivars to make fluctuation and to take the most suited dose based on in vitro growing and proliferation rate decrease by measuring the dose that consequences in a 50 % decrease of growing and proliferation ( LD50 ) .

Material and methods

Micropropagated civilizations of banana cultivars ; ‘Berangan Intan ‘ , ‘Berangan ‘ ( AAA group ) and ‘Rastali ‘ ( AAB group ) were used as the beginning of stuffs for the excising of shoot tips. Micropropagation medium consisted of the MS medium ( Murashige and Skoog, 1962 ) amended with 22.2 AµM BAP. After three months of civilization to let farther proliferation, the shoot vertexs were separated from shoot bunchs and so trimmed to a size of about 5 to 7 millimeters by taking several sheathing foliages and deletion with minimal basal corm tissues. Aqueous solution incorporating 1 M of ethyl methanesulphonate ( EMS ) along with 1 % v/v dimethylsulphoxide ( DMSO ) as a bearer agent was prepared in the deionized H2O and kept in 4oc. this solution was diluted with 0.1 M phosphate buffer ( pH 7 ) utilizing filter – sterilisation under sterile status to give working solutions of mutagen as the concluding concentrations of EMS were: 150, 200 and 250 millimeter. Sterile deionized H2O and unfertile phosphate buffer were besides prepared as controls. Excised shoot vertexs were submerged in mutagen solutions and controls as 1ml/apex for different periods of 30 and 60 min for each concentration. In the instance of control solutions merely period of 60 min was considered. All interventions were carried out at 25oc under room temperature. Fallowing EMS intervention shoot vertexs were rinsed three times with unfertile H2O and allowed to be dry for a piece as it has been reported that prolonged station – intervention lavation, followed by drying, consequences in a decrease in biological harm without a lessening in mutant output ( Benfey er al. , 1990 ) . Afterwards, EMS treated Explants were inoculated in 300 milliliter capacity jars ( 6apices/jar ) consisting of 60 milliliters MS basal salts and vitamins and sucrose ( 30 g/L ) , solidified with 2.8 g L-1 gelrite amended with 22.2 AµM of benzyl aminopurine ( BAP ) . Cultures were kept under a controlled environment at 28oc A± 2oc with 16 h photoperiod supplemented with cool white florescent visible radiation for 6 months to let farther proliferation and during this period subcultures were achieved at 45 yearss interval. After the first period of 45 yearss, the fresh weights of proliferating shoot tips for each replicated intervention were recorded and so subculture was carried out to the same proliferation medium as above for a farther 45 yearss. After the 2nd period of 45 yearss, informations were collected from all replicated interventions to document the per centum of vertexs renewing shoots, the mean figure of shoots per treated shoot tips, the figure of shoot tips which showed no farther growing but were alive which in this survey called as hibernating shoot tips and the per centum of shoot tips lasting. When shoot tips are used for chemical mutagenesis, a protocol will be needed to back up their in vitro growing and to bring forth the maximal proliferation of shoots after mutant initiation intervention hence, the per centum of in vitro growing of EMS treated shoot tips was calculated utilizing the undermentioned expression harmonizing to Musoke et Al. ( 1999 ) :

The per centum of in vitro growing = A- 100

Then the LD50 was calculated as the dosage of EMS which reduces the per centum of in vitro growing among treated shoot tips to 50 % of untreated control shoot tips based on leaner arrested development. LD50 computation on the capacity of the proliferation rate was besides worked out as the dosage of EMS that reduces the figure of shoots regenerated per treated shoot tips to 50 % of untreated shoot tips based on leaner arrested development harmonizing to Predieri and Virgilio ( 2007 ) . After three months of civilization, informations were recorded for phenotypic fluctuations comprised: foliage colour alterations, nanism and deviant morphology production such as ; hyperhydricity and abnormalcy among entire shoots regenerated from treated and untreated shoot tips, so the per centum of fluctuations among shoots regenerated from treated shoot tips compared to the untreated control shoot tips was assumed as a standard for efficiency of the different EMS interventions as a mutagen to bring on mutant. The per centum of phenotypic fluctuation was calculated harmonizing to Bhagwat and Duncan ( 1997 ) with alteration as fallow:

Percentage of phenotypic fluctuations = A-100

The experiments were arranged in a wholly randomized design with six replicates and the informations collected and calculated were analysed utilizing SAS and MSTATC computing machine plans and comparing of agencies were tested for significance, utilizing LSD trial, at 0.05 degree of chance.

Consequences

Different concentrations of EMS and intervention times of incubation were applied to look into their effects on proliferating shoot tips and to measure their possible to make morphological variableness among banana cultivars ; ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ . After the 2nd period of 45 yearss intervention with EMS, the per centum of vertexs lasting ranged from 88.71, 81.10 and 90.62, at 150 millimeter for 30 min to 37.78, 34.44 and 31.03 at 250mM for 60 min in ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally, as with increasing the dose of EMS, endurance of vertexs significantly decreased for both continuance interventions ( Table 1 ) . Nevertheless, non all lasting vertexs gave rise to shoot and a big figure of them remained hibernating whereas they represented no farther growing even after six month of civilization following EMS interventions ( Fig. 1 ) . Dormant shoot tips ( Table 1 ) were taken topographic point at all EMS interventions except controls. The happening of hibernating shoot tips was observed to be cultivar dependent responses to different concentrations and continuance interventions of EMS, as in ‘Rastali ‘ 150mM concentration for 60 min intervention caused the highest per centum of hibernating explants ( 31.05 ) but in ‘Berangan Intan ‘ and ‘Berangan ‘ , EMS at 150 and 250 millimeter for 60 min continuance intervention gave the highest per centum of hibernating explants production ( Table 1 ) . There were besides important differences in the per centum of vertexs renewing shoot with changing the concentration and continuance of EMS ( Table 1 ) as around 13 hebdomads after Treatments with EMS, while the control interventions caused about 100 % of shoot tips regenerate shoot, per centum of shoot tips renewing shoots ranged from 78, 75.67 and 74 with 150 millimeters of EMS solution for 30 min intervention to 30.63, 33.33 and 23.5 at the 60 min/250 millimeter intervention for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally ( Table 1 ) . Therefore, as the concentration of EMS increased the per centum of shoot – regenerating vertexs decreased and noticeable was the similarity of the tendency in decrease at both intervention periods ( Table 1 ) . The mean figure of shoots per explants declined significantly from the controls to the highest dosage and continuance of EMS as the lowest figure of shoot regeneration was occurred at the 60 min/ 250 millimeter intervention for all cultivars ( Table 2 ) , nevertheless cultivar ; ‘Rastali ‘ showed the highest potency of shoot regeneration in all EMS interventions ( Table 2 ) . Statistical analysis of the weights of shoot tips 45 yearss after interventions revealed important differences between the controls and all EMS interventions ( Table 2 ) ; nevertheless important differences among shoot tip fresh weights were depended on the mutagen concentration every bit good as continuance intervention which are presented in Table 2. Mentioning to the concentration and continuance intervention, the lowest value of shoot tip weight were recorded at the highest concentration ( 250 millimeter ) for 60 min incubation of EMS ( 1.68 g, 1.38 g and 1.89 g for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ , severally ) ( Table 2 ) . The dosage of EMS that resulted in a 50 % decrease of proliferation and growing ( LD50 ) was assessed based on the figure of regenerated shoots achieved in the 2nd subculture ( Table 3 ) and based on the in vitro growing decrease ( Table 4 ) calculated in the first subculture after intervention. The EMS dose bring oning LD50 was calculated with the equation ( Tables.3 and 4 ) , by replacing ( 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 continuance intervention and 154.17 millimeter, 148.21mM and 141.83 millimeter for 60 min continuance intervention severally ( Table 3 ) . In the instance of in vitro growing decrease, LD50 was estimated 302.3 Mm, 334.64Mm and 217.16 millimeter for 30 min continuance intervention and 175.77 millimeter, 166.99 millimeter and 167.79 millimeter for 60 min continuance intervention in ‘Beragan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally ( Table 4 ) .

Data collected for phenotypic fluctuation observed among regenerated shoots three months after intervention of shoot tips with EMS are presented in Table 5. The phenotypic fluctuations were observed as leaf coloring material alteration, short interval among foliages as nanism, hyperhydricity and deviant morphology production ( Fig 2 ) . A noticeable was all leaf coloring material alterations which were observed as a narrow violet chevrons on the foliage blades ( Fig 2 ) . Entire per centum of phenotypic fluctuations 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 millimeter of EMS for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally ( Table 5 ) . The highest phenotypic fluctuation was obtained by the highest degree of EMS intervention ( 60 min / 250 millimeter ) for all cultivars tested in this survey ( Table 5 ) , nevertheless, the lowest per centum of endurance and capacity of proliferation among such treated shoot tips for all cultivars renders this intervention unserviceable. In ‘Berangan Intan ‘ , 60min/200mM and 30min/250mM of EMS intervention resulted in 59.87 % and 57.67 % endurance every bit good as 45.33 % and 41.20 % shoot regeneration severally from explants ( Table 1 ) . In ‘Berangan ‘ , 60min/200mM and 30min/250mM of EMS intervention resulted in 48.33 % and 43.89 % endurance every bit good as 48.67 % and 42.33 % shoot regeneration severally from explants ( Table 1 ) . For ‘Rastali ‘ , 60min/200mM and 30min/250mM of EMS intervention resulted in 42.78 % and 57.22 % endurance every bit good as 39.67 % and 46.67 % shoot regeneration severally ( Table 1 ) . Therefore the recommended interventions were adjudged to be 60min/200mM and 30min/250mM of EMS for all cultivars tested in this survey, which resulted in per centum of phenotypic fluctuations of 10.74 % and 11.40 severally for ‘Berangan Intan ‘ , 12.42 % and 12 % severally for ‘Berangan ‘ , 13.17 % and 18.26 % for ‘Rastali ‘ ( Table 5 ) .

Discussion

Different EMS interventions revealed a gradual decrease in endurance, growing and regeneration capacity of the treated shoot tips among all cultivars tested in this survey as the concentration and continuance intervention of EMS increased which is besides in understanding with pervious findings in banana, chrysanthemum, and soya bean ( Omar et al. , 1989 ; Bhagwat & A ; Duncan, 1997 ; Latado et al. , 2004 ; Hofmann et al. , 2004 ) . Omar et Al. ( 1989 ) reported an obvious decrease in the figure 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 millimeters following 3 hours of incubation in mutagenic solution moreover, their consequences besides revealed the indispensable function of DMSO as a bearer agent to speed up the consumption of EMS into the explants. Sing their offer based on lower concentrations of DMSO to forestall Browning of explants and heighten soaking up to show better consequences, we applied merely 1mM of dimethylsulphoxide ( DMSO ) as a bearer agent in mutagen solution interventions. Consequences of this survey showed that endurance of explants besides decreased with the addition of concentration and clip of incubation in EMS. The consequences of this probe evidently indicate that mutant frequence additions with increasing of dose and clip continuance of EMS intervention which is in understanding with the findings reported by Bhagwat and Duncan ( 1989 ) , but sing the important decrease of endurance and regeneration capacity of treated shoot tips, the efficiency of mutagen besides decreases. Therefore, an optimal dosage of EMS should be chosen based on some factors such as per centum of endurance and proliferation rate every bit good as decrease of regeneration capacity by 50 % ( LD50 ) . Novak et Al. ( 1987 ) developed in vitro shoot tip civilization as efficient system for mutant initiation in banana and plantain. Novak et Al. ( 1990 ) besides stated the suited dosage of gamma irradiation on shoot tips based on one that reduces growing and regeneration to 50 % of untreated control ( LD50 ) . Harmonizing to Novak et Al. ( 1990 ) , our consequences showed that the suited dosage of EMS on the base of 50 % proliferation rate decrease could be 189.80 millimeter, 177.58 millimeter and 152.04mM for 30 min continuance intervention and 154.17 millimeter, 148.21 millimeter and 141.83 millimeter for 60 min continuance intervention for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally, and based on in vitro growing decrease, the suited dosage of EMS was assumed to be 302.30 millimeter, 334.64 millimeter and 217.16mM for 30 min continuance intervention and 175.77 millimeter, 166.99 millimeter and 167.79 millimeter for 60 min continuance intervention for ‘Berangan Intan ‘ , ‘Berangan ‘ and ‘Rastali ‘ severally. However, Bhagwat and Duncan ( 1997 ) pointed out that a 50 % growing decrease was non needfully the best standard for finding optimal intervention conditions for chemical mutagens which in the instance of 50 % in vitro growing decrease we are besides agree with them, but sing the immense necessity of recovery and extremely celerity of cloning discrepancies after mutant initiation, we concluded that look of LD50 based on the proliferation rate should be an of import standard for finding of the optimal dosage of chemical mutagen intervention. Furthermore, Khawale et Al. ( 2007 ) utilizing in vitro induced mutant with chemical mutagens in grape, expressed the LD50 on the footing of in vitro endurance of their treated explants. Latado et Al. ( 2004 ) estimated the LD50 for the mutagenesis of chrysanthemum pedicels with EMS to be 82 % ( v/v ) and they chose 0.77 % ( 0.077 M ) of EMS to handle the explants. In the instance of cultivars tested in our survey, EMS at 200 millimeter for 60 min and 250mM for 30 min was considered as the most efficient intervention based on comparatively high per centum of phenotypic variableness with the per centum of vertexs renewing shoot runing from 41 % to 48 % and endurance of 42 % to 60 % depending on cultivars. Bhagwat and Duncan ( 1997 ) offered 200 millimeter of EMS for 30 min as the best intervention for banana shoot tips as they recorded 5.8 % phenotypic fluctuation, with endurance of 80 % and regeneration from 31.6 % of treated explants. The differences between per centum of endurance and regeneration could be due to different cultivar responses to EMS interventions. The visual aspect of shoot tips exposing no farther growing and staying hibernating even after six months of civilization could be due to the repressive consequence of EMS harmonizing to old studies ( Bhagwat & A ; Duncan, 1997 ) , nevertheless, frequence of this in vitro response to EMS concentrations and continuance interventions was non the same among three cultivars tested in this survey. Relatively, the higher frequence of fluctuations observed among regenerated shoots from all cultivars tested in this probe comprise of foliage colour alterations, deviant morphology and nanism than that obtained by Bhagwat and Duncan, ( 1997 ) who reported 5.8 % as the highest frequence of phenotypic fluctuation among regenerated shoots, enable us to presume EMS as an efficient intervention to make variableness in banana cultivars. Furthermore, all leaf colour alterations observed in this probe was as a narrow violet strips on foliage blade which has ne’er been reported before.

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