Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane（Saccharum officinarum L.）

Ashok A.Nikm，Rhyy M.Devrumth，Aksh Ahuj，Hrinth Bu，Mhdeo G.Shitole，Penn Suprsnn*

aTissue Culture Section，Vasantdada Sugar Institute，Manjari（Bk），Pune 412307，India

bMolecular Biology and Genetic Engineering Division，Vasantdada Sugar Institute，Manjari（Bk），Pune 412307，India

cDepartment of Botany，Pune University，Pune 411007，India

dPlant Stress Physiology&Biotechnology Section，Nuclear Agriculture&Biotechnology Division，Bhabha Atomic Research Centre，Trombay，Mumbai 400085，India

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane（Saccharum officinarum L.）

Ashok A.Nikama，Rachayya M.Devarumathb，Akash Ahujab，Harinath Babub，Mahadeo G.Shitolec，Penna Suprasannad，*

aTissue Culture Section，Vasantdada Sugar Institute，Manjari（Bk），Pune 412307，India

bMolecular Biology and Genetic Engineering Division，Vasantdada Sugar Institute，Manjari（Bk），Pune 412307，India

cDepartment of Botany，Pune University，Pune 411007，India

dPlant Stress Physiology&Biotechnology Section，Nuclear Agriculture&Biotechnology Division，Bhabha Atomic Research Centre，Trombay，Mumbai 400085，India

A R T I C L E I N F O

Article history：

form 11 September 2014

Accepted 28 September 2014

Available online 13 October 2014

Gamma-rays

Mutagenesis

Embryogenic callus

Saccharum officinarum L

Salt tolerance

Gamma ray-induced in vitro mutagenesis and selection for salt（NaCl）tolerance were investigated in sugarcane（Saccharum officinarum L.）.Embryogenic callus cultures were irradiated（10to80 Gy）andsubjectedtoinvitroselectionbyexposureofirradiatedcallustoNaCl（0，50，100，150，200，and 250 mmol L-1）.Increasing NaCl concentrations resulted in growth reduction and increased membrane damage.Salt-selected callus lines were characterized by the accumulation of proline，glycine betaine，and Na+and K+concentration.Higher accumulation of proline and glycine betaine was observed in NaCl stressed callus irradiated at 20 Gy.Na+concentration increased and K+concentration decreased with increasing salt level.Irradiated callus showed 50-60%regeneration under NaCl stress，and in vitro-regenerated plants were acclimatized inthe greenhouse，with 80-85%survival.A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions.Of these，18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%，number of millable canes，and yield.The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

?2014 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.All rights reserved.This is an open access article under the CC BY-NC-ND license（http：//creativecommons.org/licenses/by-nc-nd/3.0/）.

1.Introduction

Sugarcane（Saccharum spp.）is an important industrial crop，rankingamongthetenmostplantedcropsinthe world.Besides being the major sugar contributor，accounting for more than 70%of the world's sugar，sugarcane is important as the raw material for sugar-producing and allied industries［1］.Conventional breeding has contributed greatly to the development of agronomically improved varieties；but limitations such as a narrow gene pool，a complex genome，poor fertility，and a long breeding/selection cycle make it difficult to undertake further improvement.Agronomically improved sugarcane varieties endowed with tolerance to biotic and abiotic stresses are highly beneficial，as unfavorable environmental factors can challenge cultivation and crop productivity.Although crops tolerant to biotic and abiotic stresses have been selected by conventional breeding programs，speeding up the pace of breeding is essential for developing improved varieties.

Soil salinity has become a major limiting factor that adversely affectscropproduction［2］.Worldwide，itisestimatedthataround 800 million hectares of land are affected by salinity，with salinity levels ranging from 2 to 4 dS m-1［2］.Salinity affects plant cells，causing alterations in water relations，ionic and metabolic perturbations，generation of reactive oxygen species（ROS），and tissue damage［3］.Development of salt-tolerant cultivars by conventional，mutational，and biotechnological approaches can augment the utilization of salinity-affected regions.The availability and screening of large populations for mutagenesis are prerequisites to obtaining sufficient genetic variability.In vitro culture in combination with radiation-induced mutations has become an important method to induce genetic variability and rapidlymultiplytheselectedmutants［4-6］.Methodsofchemicaland/or radiation-induced in vitro mutagenesis have been successfully used to improve agronomic traits including salinity and droughttoleranceinseveralcropplants［7-10］.Determinationsof radiosensitivity and of optimal doses of ionizing radiation are important steps for undertaking induced mutagenesis for crop improvement.Their importance has been well demonstrated in plants such as rice［11］，groundnut［12］，sweet potato［10］，banana［13］，and Zoysia［14］.

Although studies of salt selection are available for diverse plantspecies，limited research has been conducted insugarcane. Sugarcane embryogenic callus has been shown to be sensitive to sodium chloride（NaCl）［15］and gamma radiation［16］.Saif et al.［17］reported the isolation of salt-tolerant mutants from irradiated sugarcane callus.Although these studies have demonstrated the application of mutagenesis and in vitro techniques to study radiosensitivity or isolation of mutants in sugarcane，little information on characterization of salt tolerant callus and progeny is available.Studies of the application of ionizing radiation for developing novel mutant germplasm in sugarcane will accordingly be beneficial for sugarcane improvement.The objective of the present study was to apply gamma ray-induced mutagenesis to isolate sugarcane mutants with improved tolerancetosalinity，followedbymorphologicalandagronomical characterization of selected mutants.

2.Materials and methods

2.1.Plant material and culture conditions

The commercial sugarcane variety Co86032 was used as the experimental material.The tops of mature canes were harvested from field-grown plants at the Vasantdada Sugar Institute，Manjari，Pune（India）.The explant material was washed first in tap water and then for 5 min in sterile distilled water at least three times.Surface decontamination was performed with 80%ethanol（v/v）for 5 min and mercury chloride（0.1%w/v）for 4-5 min，followed by three washes with sterile distilled water for 15 min each.After removal of the outer leaves，the innermost leaf segments were cut into 2-3 mm pieces and aseptically inoculated onto MS ［18］medium supplemented with 1 mg L-1thymine HCl，20 mg L-1inositol，3 mg L-12，4-D，10%coconut water and 2.0%sucrose. This medium is referred to as callus induction medium. Cultures were incubated in the dark at 25±2°C at relative humidity70-80%.After45 daysofculture，thecallus was subcultured onto modified MS medium with 1000 mg L-1casein hydrolyzate，1 mg L-1thymine HCl，20 mg L-1inositol，3 mg L-12，4-D，5%coconut water，and 2.5%sucrose to obtain embryogenic callus.This medium is referred to as callus maintenance medium.

2.2.Gamma ray radiation treatments

Embryogenic callus cultures were irradiated with 0，10，20，30，40，50，60，70，and 80 Gy gamma rays using Gamma Cell 220（a60Co source）at a dose rate of 9.6 Gy min-1.Post irradiation，callus cultures were transferred to freshly prepared MS medium supplemented with 3 mg L-12，4-D，1000 mg L-1casein hydrolyzate，1 mg L-1thymine HCl，20 mg L-1inositol，5%coconut water，and 2.5%sucrose.Survival of the irradiated callus was determined using relative growth rate after four weeks of radiation treatment.The surviving callus was then subcultured for at least four passages on maintenance medium.

2.3.Salt stress and its effects on irradiated callus

Irradiated and non-irradiated callus cultures were subjected to treatments with different concentrationsof salt（NaCl）stress（0，50，100，150，200，and 250 mmol L-1）to study salt stress effects andidentifythe optimal concentration of NaCl to be usedinthe selection medium.After four weeks of treatment，the response was recorded using several parameters：tissue water content（TWC），relative electrolyte leakage（REL），relative growth rate（RGR），accumulation of proline and glycine betaine（GB），protein content，and Na+and K+concentration.

Membrane damage was determined in terms of relative electrolytic leakage（REL）by the method of Sullivan［19］.For REL measurement，callus was incubated for 24 h in a test-tube（25 mm×150 mm）containing distilled water（25°C）and the initial electrical conductivity（EC1）was measured after the incubation period.Samples were then autoclaved for 15 min at 121°C to release the ions from the tissue，and the final electrical conductivity（EC2）was measured after cooling to room temperature.The REL was calculated as：（EC1/EC2）×100. TWC of the callus was determined as described by Lokhande et al.［20］.The percent tissue water content（TWC%）was determined using the following equation：TWC（%）=［fresh weight（FW）-dry weight（DW）/（FW）］×100.

2.4.Free proline concentration

Proline concentration was evaluated by the method of Bates et al.［21］with minor modifications.Callus was ground in 3% sulfosalicylicacidandcentrifugedat4°C.The filtratewasmixed withequalvolumesofacidninhydrinandglacialaceticacid，and thenincubatedat100°Cin ahotwater bathfor1 h.Thereactionwas terminated in an ice bath and allowed to cool at room temperature.The reaction mixture was extracted with 4 mL toluene and mixed vigorously with a stirrer for 10-15 s.The chromophore-containing toluene was aspirated from the aqueousphaseandwarmedtoroomtemperature.Theopticaldensity was measured at 520 nm using toluene as a blank.The amount of proline was determined from a standard curve usingL-proline and expressed as proline μmol g-1FW.

2.5.Glycine betaine（GB）concentration

GB concentration was determined by the method of Grieve and Grattan［22］.The callus was mechanically shaken with deionized water for 24 h at 25°C.Samples were filtered and the filtratewasdiluted（1：1）with2 mmol L-1H2SO4.Theextractwas cooled in ice and mixed with 200 μL of I2-KI reagent（a mixture of 20%potassium iodide and 15.7%iodine）.The tubes were gently mixed and stored at 4°C for 16 h followed by centrifugation at 10，000×g for 15 min at 0°C.Periodide crystals were dissolved in 9.0 mL of 1，2-dichloroethane，and after 2 h，absorbance was measured at 365 nm.GB concentration was determined from a standard curve prepared using standard glycine betaine and expressed as μg g-1FW.

2.6.Lipid peroxidation

Oxidative damage to membrane lipids was estimated by measurement of the concentration of thiobarbituric acidreactive substances（TBARSs），expressed as equivalents of malonyldialdehyde（MDA）.The amount of MDA was calculated as described by Hichem et al.［23］with some modifications. Callus was ground in 0.25%thiobarbituric acid（TBA）in 10% trichloroacetic acid（TCA）.The homogenate was centrifuged，and the absorbance was read at 532 and 600 nm with 10%TCA as a blank.The TBARS was expressed as μmol g-1FW.

2.7.Na+and K+analysis

Na+and K+analyses were performed by the procedure of Basu et al.［24］.Callus was dried at 80°C for 48 h and digested in nitric acid and perchloric acid（2：1，v/v）at 150°C for 4 h.The residue was dissolved in distilled water and the final volume of the solution was adjusted to 1 mL.It was used for Na+and K+measurement using a flame photometer.

2.8.Regeneration of plants from selected callus cultures

Embryogenic callus cultures were placed on 100 mmol L-1NaCl-supplemented MS medium and selection was performed for surviving callus.The irradiated and NaCl-selected calli were subjected to four passages on freshly prepared medium with 100 mmol L-1to select for surviving tolerant callus.All the cultures were transferred onto MS basal medium supplemented with1 mg L-1thymineHCl，20 mg L-1inositol，5%coconutwater，and 2.5%sucrose without hormones to induce plant regeneration.The cultures were initially kept in the dark for 10 days and then the culture bottles were incubated under 16 h light illumination.Plantletsofabout5-6 cminlengthweretransferred to half-strength MS medium with NAA（4.0 mg L-1）for root induction.Well-rooted plantlets were then transferred to poly bags in the green house for 40 days.Plant survival was determined after 45 days.

2.9.Agro-morphological characterization

Toassesstheagronomicperformanceofirradiatedand NaCl-selected plants，evaluation was performed in the control，non-saline soil in a ground nursery trial.The plantlets were planted at distance 1.2 m×1.0 m plant to plant along with parent variety Co86032.Selection of the promising mutants on the basis of stalk number per plant，diameter of canes，and Hand refractometer（HR）Brix%was recorded，and on the basis of high Brix%of cane at months 10 and 12 in comparison to the parent was used for selection of promising mutants.These selected mutants were field-transplanted for evaluation of agronomic performance in a clonal trial（augmented）under saline conditions.Data for cane and juice parameters were recorded in the months of 10 and 12 for total height of cane，millable height of cane，diameter of cane，number of internodes per cane，weight of single cane，Brix%，sucrose%，purity%，and commercial cane sugar（CCS%）and compared with parent and standard varieties.

Thebiological effectof the treatmentwas analyzed based on survivalandvariantcharacteristics（morphologicaland agronomical）.Mutationfrequencywascalculatedaspercentage of selected promising mutants from the M1 generation. Different morphological and agronomic characters were compared between mutants and the parent variety.Mutagenic effectiveness and efficiency were calculated following Walther［25］.

2.10.Statistical analysis

The experiment was laid out in a completely randomized design（CRD）.The analyses were repeated with three independentbiologicalsamples.Thedatawerestatistically analyzed by two-way analysis of variance（ANOVA）and Scott-Knott group significance at 5%probability［26］using Windostat software（version 8.5，http：//www.windostat.org/）. The results were presented as means with standard error of three replicates and different levels were compared by Scott-Knott group test，with significant group indicated by a horizontal line.A graphical presentation showed changes in colors of bar graphs，indicating significant differences at 5% probability by ANOVA.Field evaluations of cane and juice and biochemical analysis were analyzed by an augmented method.Data are reported as mean values，and standard check varieties and mutants with least significant difference（LSD）in CD（≤0.05）values are reported.Mutants were compared with parent and standard varieties and also with the moderately salt-tolerant CoM0265 sugarcane variety.

3.Results

3.1.Gamma irradiation and salt-stress response

Fig.1-Effects of radiation and salinity（NaCl）on callus growth，plant regeneration，and field plants of sugarcane variety Co86032.（a）Embryogenic control callus；（b）embryogenic callus irradiated with 20 Gy on 100 mmol L-1NaCl；（c）embryogenic callus irradiated with 30 Gy on 150 mmol L-1NaCl；（d）regeneration from callus irradiated with 20 Gy on 100 mmol L-1NaCl；（e）regeneration from callusirradiatedwith30 Gyon100 mmol L-1NaCl；（f）plantsfrom20 Gyand100 mmol L-1NaCltreatments；（g）plantsfrom30 Gyand 100 mmol L-1NaCl treatments growing in the field.

Fresh，actively proliferating embryogenic callus showed more sensitivity to gamma radiation than the control（Fig.1-a，b，c）. RGR decreased with increasing doses（10-80 Gy）of gamma rays（Fig.2）.Callus exposed to low doses of 10 and 20 Gy showed 50%reduction over control callus，whereas 70% reduction in RGR was observed in treatments with 30 to 40 Gy and＞70%reduction at higher doses（50 to 80 Gy）.The extent of browning also increased with radiation dose，and the callus turned completely brown at doses above 40 Gy. Exposure of embryogenic callus to 100-150 mmol L-1NaCl inhibited callus growth（Fig.3-a）.Lower RGR was observed with increase in salt concentration and radiation dose.A reduction in TWC was also observed with increase in radiation and NaCl treatment（Fig.3-b）.Significant reduction up to 81.9%or 77.8%was observed at 30，40 Gy，respectively，as compared to higher TWC （86.0%）at 20 Gy.Similarly，a significant reduction in TWC was observed in irradiated callus treated with 100 mmol L-1and higher NaCl（150，200，and 250 mmol L-1）.The pooled data of irradiated and NaCl stress callus showed a gradual reduction in percent tissue water content with increase in irradiation and NaCl concentration.

The increase in radiation dose and NaCl concentration resulted in significant increase in percent relative electrolyte leakage（Fig.3-c）.Pooled data of irradiated and control callus showed significantly higher RELs of 83.2%，84.1%，and 87.0%at NaCl concentrationsofrespectively 150，200，and 250 mmol L-1，with the differences showing group significance by Scott-Knott test.However，the 0 and 50 mmol L-1NaCl concentrations led to significantly lower REL than the 100 mmol L-1NaCl treated callus（Fig.3-c）.In contrast，40 Gy-irradiated callus showed maximum REL under NaCl concentration.

Fig.2-Effect of gamma radiation on relative growth rate（RGR）of Co86032 callus after 30 days of radiation.Error bars indicate SE and letters above bar indicate significant differences at 5%probability.

Fig.3-Effect of radiation and salinity on sugarcane callus：RGR（a），tissue water content（TWC%）（b），relative electrolyte leakage（REL%）（c），and lipid peroxidase（MDA）（d）.Number above bar indicates radiation dose in Gy：1（0），2（20），3（30），4（40），and NaCl（mmol L-1）：1（0），2（50），3（100），4（150），5（200），6（250）mmol L-1NaCl.Graph color change indicates significance at 5%probability level and horizontal line on X-axis indicates group significance level by Scott-Knott test.The error bars show standard errors of means.

Higher MDA content was observed in irradiated callus exposedtoNaClstress（Fig.3-d）.The30 Gyradiationtreatments showed significant higher MDA（53.5 μmol g-lFW）than other treatments and non-irradiated callus（Fig.3-d）.Irradiated callus exposed to 150 and 200 mmol L-1NaCl showed significantly higherMDAcontentsof55.8and 53.3 μmol g-lFW，respectively.

Pooled data of control and 20 Gy-irradiated callus showed significantly higherproline（4.9 and4.7 μmol g-1FW）than 30and 40 Gy-irradiated callus（3.1 and 2.9 μmol g-lFW）（Fig.4-a）.Irradiated callus on 100 mmol L-1NaCl showed the highest proline accumulation（5.5 μmol g-1FW）.Irradiated callus exposed to 50 and 150 mmol L-1NaCl stress showed group significance，whereas control and 250 mmol L-1NaCl-stressed callus showed the lowest accumulation of proline（Fig.4-a）.Glycine betaine（GB）accumulation was significantly higher in 30 Gy irradiated callus（10.8 μg g-lFW）.Higher levels of GB（13.9 μg g-1FW）were observed in irradiated callus grown under 150 mmol L-1NaCl stress.As in the case of proline，the lowest accumulation of GB wasobservedinthecontroland250 mmol L-1NaCltreatedcallus（Fig.4-b）.

The concentration of sodium（Na+）was significantly higher in gamma-irradiated and NaCl-treated callus than in control callus，and Na+concentration increased with NaCl concentration（Fig.4-c）.In contrast，K+concentration was significantly higher in control callus than in irradiated and NaCl-treated samples（Fig.4-d）.The maximum sodium（468.4 μmol g-1FW）and lowest potassium（44.4 μmol g-1FW）accumulations were observed in 250 mmol L-1NaCl-stressed callus.

3.2.Regeneration and selection for salt tolerance

Irradiated embryogenic callus cultures on NaCl（salt）selection media showed pronounced browning with increasing salt concentration，compared to control.Embryogenic callus placed on 100 mmol L-1NaCl-supplemented MS medium showed surviving callus，which was selected for further selection.The irradiated，NaCl-selected callus was subcultured for four passages on 100 mmol L-1and surviving tolerant callus was selected（Fig.1-c，d）.The regeneration response of the selected callus was affected by increase in radiation dose and NaCl treatment（Fig.1-d）.Irradiated callus showed 50-60%regeneration under NaCl stress（Fig.1-e）.Some（2-3%）albino plants were also observed during regeneration of irradiated callus in salt selected medium（data not shown）.The 30 Gy-irradiated callusshowedinductionofshootsafteraninitialnecroticeffect，but only slight proliferation was observed on incubation of selected cultures for a long period（50-60 days）on regeneration medium.Control，non-irradiated callus cultures showed no regenerationonmediasupplementedwithhigherNaClconcentration，whereas 50 and 100 mmol L-1NaCl-selected callus cultures showed good regeneration of plantlets.In contrast，irradiated callus selected on 150 mmol L-1NaCl regenerated few shoots even after prolonged culture.The in vitro regenerated plants were found to acclimatize with 80-85% survival in the greenhouse.A total of 138 plants that showed normal phenotypeand growth were subsequentlyfield-planted（Fig.1-f，g）.

Fig.4-Effect of radiation and salinity on sugarcane callus：proline（a），glycine betaine（b），sodium（Na+）（c），and potassium（K+）（d）concentrations.Number above bar indicatesradiationdoseinGy：1（0），2（20），3（30），4（40）and NaCl（mmol L-1）：1（0），2（50），3（100），4（150），5（200），6（250）.Graph color change indicates significance at 5%probability，and horizontal line on X-axis indicates group significance level by Scott-Knott test.Error bars show standard error of mean.

3.3.Field evaluation

Irradiated and salt-selected plants developed normally under field conditions，with few exceptions.One mutant clone developed under 20 Gy+NaCl stress showed improved cane growth，with some changes in phenotypic characters including broad leaf lamina，increased leaf length，and increased waxiness compared to control，whereas a few 30 Gy-irradiated and NaCl-selected clones showed stunted growth with narrow，spiny leaves and waxy leaf sheath（data not shown）.

Of138plantsscreenedforphenotypeandgrowthcharacters，18 mutants were selected and field-transplanted for evaluation ofagronomicperformance ina clonal trial of augmenteddesign under saline conditions.Agronomic data of the mutants were compared with those of the parent variety and standard check varieties（Tables 1 and 2）.Clone 8270 showed the highest germination（52.1%）compared to standard varieties（Table 1）. Clone 8151 showed higher tillering（11.1%），followed by 8188（9.0%）and 8149（8.1%），than the standard check varieties（5.3%） at 45 days after planting.The mutant clone 8151 showed a highernumberofmillablecanes（92，400 ha-1）thanthestandard varieties（62，800 ha-1）.There was a significant reduction in leaf length and leaf width in mutant clones（82.8 and 3.2 cm）in comparison to the standard varieties（114.0 and 4.8 cm）.The leaf length of the clones 8151，8182，8268，and 8271 was significantly greater，at 127.3，115.3，111.7，and 118.7 cm，respectively，than that of the parent Co86032（95.0 cm）under salt stress，whereas clones 8162，8168，8174，8188，8193，8197，8209，8210，8219，8236，and 8270 showed significant reductions in leaf length（Table 1）.

Clones 8147 and 8188 showed higher Brix%（22.0 and 20.2，22.0 and 21.4）at months 10 and 12，respectively（Table 2）. However，clones 8151，8209，8268，8270，and 8271 showedhigher Brix%（20.0，19.7，20.0，20.2，and 20.4）only at month 10 than the parent Co86032（19.0）.Clone 8188 and standard CoC671 showed the highest sucrose%（19.3 and 19.8，19.5 and 19.7）at months 10 and 12 respectively.Among the mutants，clone 8151 showed higher cane yield（101.9 t ha-1）and CCS（12.4 t ha-1）.

In general，mutagenic frequency increased with dose of gamma rays up to 30 Gy（Table 3）.An increasing trend in lethality percentage was observed with increasing dose of gamma radiation in sugarcane calli.The 30-Gy dose of gamma radiation produced the maximum（90.5）lethality.The biological damage efficiency observed was highest under the 20-Gy treatment.The dose giving maximum mutation efficiency for sugarcane calli of Co86032 may thus be considered as 20 Gy of gamma radiation.

Table 1-Field performance of sugarcane mutant clones of Co86032 under salinity stress.

4.Discussion

The application of radiation-induced mutagenesis with in vitro culture has proved effective in the induction of genetic variation，selection，and multiplication of mutant clones［6，9，27］.Assessment of radiosensitivity is a prerequisite for identifying a suitable dose for in vitro mutagenesis of a particularcultivar.Inthepresentstudy，sugarcaneembryogenic callus was exposed to different doses of gamma radiation and post-irradiation survival based on RGR showed significant reduction in growth rate with increasing doses of gamma radiation.The 20 Gy radiation dose was found to be the optimum LD50for Co86032 sugarcane callus.Taras et al.［28］reported 20 Gy as the LD50for Brassica and characterized radiosensitivity using LD50as the criterion.However，LD20or LD30are also used as optimum doses，as these levels of mutagensshowednotoxicitytoplanttissues［29］.Insugarcane，Patade et al.［16］suggested 20 Gy as the optimum dose for embryogenic callus and also observed reduced regeneration frequency with increasing gamma radiation.In our study，mutagenic frequency also increased with increasing dose of gamma rays（Table 3）and based upon the results，the dose giving maximum mutation efficiency for calli of Co86032 may thus be considered as 20 Gy of gamma radiation.

In this study，in vitro selection was applied to embryogenic callus culture by inclusion of growth-inhibitory levels of NaCl in the selection medium.Screening of mutagenized cultures during dedifferentiation and differentiation stages could be very useful for selection of salt tolerance，as described earlier［14］.The present study indicated a significant reduction in the growth rate of sugarcane embryogenic callus exposed to 100 to 250 mM NaCl stress.This reduction may have resulted from reduced water availability to callus cells due to increased NaCl stress in the medium.In sugarcane a significant decline in callus growth rate occurred with 150 mmol L-1NaCl［30］and 171 mmol L-1NaCl［16］.The results also showed that reduction in callus growth with increasing salt concentration resulted from lower percent tissue water content and membrane damage to cells.This effect presumably arises from dehydration of cells through low water potential or nutritional imbalance because of interference of salt ions with essential nutrients［31］.In our study，we observed an increase in REL and reduction in water content with increasing salt and radiation concentrations.Correlation of REL and water content has also been observed in salt-stressed wheat［31，32］，tobacco［33］，and Sesuvium［20］.NaCl stress results in oxidative damage to membranes and peroxidation of membrane lipids［34］.The degradation of membranes due to lipid peroxidation also leads to leaching of cellular electrolytes，a response used as an indicator of disturbance of membrane integrity.In soybean，MDA content increased significantly at 50 and 100 mmol L-1NaCl［35］.In our study，MDA rapidly increased under NaCl stress treatments（100 to 200 mmol L-1NaCl）as well as under higher doses of radiation.

Table 2-Field performance of sugarcane mutant clones of Co86032 under salinity stress.

To avoid oxidative damage，plants have evolved various defensive mechanisms to counteract the effect of reactive oxygen species in cellular compartments［36］.These defenses include modulated expression in the metabolic and defensive pathways and synthesis of osmolytes［37］.The results of this study revealed increase in proline concentration in irradiated and NaCl-stressed callus cultures.A change in proline concentrationhasbeencorrelatedwithitscapacitytotolerateandadapt to salinity conditions［38］.Gandonou et al.［15］reported that proline accumulation increases in salt-tolerant callus under salinity.Theaccumulationofprolineiswidelyusedasaselection criterion for salinity and drought tolerance［39］.Salt-sensitive barley plants synthesized more proline and glycine betaine than did salt-tolerant plants［40］and salt-tolerant rice cultivars accumulated less proline under NaCl stress［41］.Our results indicated significantproline accumulation in 100 mmol L-1NaCl stress callus and higher proline in 20 Gy-irradiated callus underNaCl stress，indicating the salt tolerance of irradiated callus. Significantly higher accumulation of GB was also observed in callus with 150 mmol L-1NaCl-stressed callus，and in 30 Gyirradiated callus exposed to NaCl，according to Scott-Knott group significance.Higher accumulation has been reported in salttolerant species，whereas moderately tolerant species accumulate intermediate levels and sensitive species accumulate low or no levels［34］.

Table 3-Mutagenic effectiveness and efficiency of gamma radiation in sugarcane callus of variety Co86032.

ThestudyofNa+andK+levelsrevealedthatthesodium（Na+）concentration increased in irradiated callus exposed to salt stress，a response that may be due to osmotic adjustment of cells.The reduction of callus growth may be due to nutritional imbalance resulting from interference by Na+.However，increasing amounts of Na+destabilize osmotic potential，creating a highly toxic environment to plant cells，even with the aid of defensemechanism ofantioxidant enzymes，andleaving callus slimy or dead［16，30］.Our results revealed that Na+concentration was higher in NaCl-treated callus than in control callus，in contrast to K+concentration，which was higher in control than in NaCl-treated callus.It is important to note that growth retardation is often associated with increase in Na+but decline in K+concentration，demonstrating the typical glycophyte nature of sugarcane［16，30］.The result indicates that the increase in accumulation of sodium in plant cells adversely reduced the uptake of potassium.The combined accumulation ofsalt ions（Na+and K+）and osmolytes（proline，glycine betaine，and MDA）may play an important rolein osmotic adjustment in sugarcane cells under NaCl stress.

Planttissueculturetechniqueshavebeenusedinconjunction with induced mutagenesis to create genetic variation，and gene mutations can occur more frequently in tissue-culture-derived plants［42］.In vitro mutagenesis of cultured explants，cells，and tissue cultures represents a feasible method for induction of genetic variation，which can be subjected at the cellular level to selection for desirable traits［43，44］.However，success of in vitro mutagenesis programs will depend on evaluation of mutant clones under field conditions to confirm their performance for the selected trait of interest.The performance of selected salt-tolerant genotypes of durum wheat under saline and non-saline field conditions indicated that genetic variation for traits such as number of grains per spike，grain weight per spike，1000-grain weight，number of spikes per m2，grain yield，and harvest index could be induced by mutagenic treatment［45］.In our study，agronomic traits showed a wide range of genetic variation for leaf length，tillering ratio，total height，number of millable canes/ha，CCS，Brix%，sucrose%，purity%，and CCS%（Tables 1 and 2）.Number of millable canes is the most important character，contributing directly to higher yield［46］.Number of stalks has also been consideredasa major contributing factor for cane yield［47］.We could obtain mutant lines（8151）with improved number of millable cane（NMC）（92.4）over the parent variety（64.4）and other standard check varieties.This is a significant observation，given that the mutant also had high tillering ratio and leaf length.Zhou［48］recommended a focus on the tiller development and leaf development parameters that influence cane yield or its components.Such studies are likely to be useful in identifying potentially high-yielding varieties during the early stages of sugarcane selection.In a study of genetically diversified sugarcane clones tested for yield stability，sugar yield showed significant positive correlation with tillers/plant，cane length，weight/stool，and cane yield［49］.In the present study，irradiated and salt-selected（mutant）clones were evaluated for their performance based on morphological and agronomic characters，resulting in the selection of 18 mutants.Among these，mutants，8151，8209，8268，8270，8188，and8271arethebest performingandpromisingforfurtherstudies.Theresultssuggest that in vitro-induced mutagenesis followed by in vitro selection can be applied to induce genetic variation for salt tolerance，besides improving agronomic characteristics in sugarcane.

5.Conclusion

This study has successfully demonstrated that in vitro mutagenesis and selection can be used to generate mutants and salt-tolerant lines in sugarcane and to study the physiobiochemical basis of salinity tolerance.Our results suggest that theaccumulation ofsaltions（Na+and K+）andosmolytes（proline and glycine betaine）plays an important role in osmotic adjustment in sugarcane cells under NaCl stress.Agronomically superior mutants can be useful in sugarcane improvement programs.

Acknowledgments

This study was performed with partial financial support from the Department of Atomic Energy-Board of Research in Nuclear Sciences（DAE-BRNS）project grant sanction No.2009/ 37/51/BRNS.We thank the Director General，Vasantdada Sugar Institute，Pune，India，for providing research facilities.We also thank Dr.Kapil Sushir，Scientist，Plant Breeding Section，VSI，for help in the observations of mutant plantlets in the field.

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18 June 2014

in revised

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E-mail addresses：penna888@yahoo.com，prasanna@barc.gov.in（P.Suprasanna）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.

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