Dose Of Ethyl Methanesulfonate Research Articles

Determination the effective dose of mutation in pepper (Capsicum annum L.)

This study was carried out to determine the "Effective Dose of Mutation” (EMD50) for mutation breeding study on Çermik, Kandil and Üçburun pepper varieties grown intensively in Diyarbakır. For this purpose, pepper seeds were exposed to chemical mutagen source Ethyl Methane Sulfonate (EMS) at doses of 0%, 01%, 0.2%, 0.3%, 0.4%, 0.5%, 0.75% and 1% and to irradiation with physical mutagen source Co60 at doses of 0, 50, 100, 200, 300, 400, 500 and 600 Gy. In irradiated and chemically exposed seeds, on the 45th day following seed sowing date, germination percentage (%) for each dose was calculated and seedlings’ lengths were measured to investigate the effects of different doses and EMD50 value was calculated. According to the data obtained as a result of the study; it was found that the germination rate in pepper seeds of all three varieties-genotypes gradually decreased with the increases of doses in application of EMS on pepper seeds. While the decreases were especially evident in the seeds of Kandil and Üçburun varieties subjected to 0.3% EMS dose and 9 hours of application; in Çermik genotype the germination rate started to decrease with 0.5% EMS dose due to local population. It was also found that germinations decreased in all three varieties depending the application of 300 Gy CO60. In 6-hour EMS application, EMD50 values were respectively determined as 0.67%, 0.97% and 1.08%. for Kandil, Üçburun and Çermik. And for 9-hour EMS application, EMD50 values were determined as; 0.52%, 0.77%, 0.89%. Kandil, Üçburun and Çermik varieties. According to the results obtained in the study, the usage of both physical and chemical mutagens in pepper varieties; increasing of doses and durations of mutagens, decreased germination rates and also caused to decrease the plant heights. As a result, it seems possible to obtain variations in peppers with the usage of physical and chemical mutagens to get new varieties in plant breeding.

Effects of ethyl methanesulfonate on growth and yield parameters of wheat and tolerance to imazamox

In the present study objective bread wheat (Triticum aestivum L.) and durum wheat (Triticum durum Desf.) lines resistant to Imazamox herbicides and effective lethal ethyl methanesulfonate (EMS) doses for plants. During first year (2019/2020), 1000 seeds of bread wheat and durum wheat treated with 11 different EMS doses were sown under field and greenhouse conditions. During second year (2020/2021), M1 (first generation) cereal seeds were grown as M2 (second generation) in field experiments. Herbicide with Imazamox active ingredient at 125 ml da-1 was applied to the approximately 15 cm tall plants in the M2 generation. In bread and durum wheat lines, emergent plants were observed up to a dose of 60mM EMS. The effective EMS doses for the wheat species were determined as 80 mM for bread wheat and 50 mM for durum wheat. Bangladesh J. Bot. 53(3): 487-493, 2024 (September)

Improving French bean yield potential through induced mutagenesis using EMS and SA

IntroductionFrench bean (Phaseolus vulgaris L.) holds global significance as one of the most consumed legumes, with commercial value surpassing that of all other legume crops combined. In India, the consumption of French beans has grown steadily, especially in the North Eastern region, driven by heightened consumer interest in its nutritional benefits. Considering these factors, we initiated an induced mutagenesis program to enhance the genetic diversity of locally grown French bean genotypes, traditionally cultivated for their superior adaptability.MethodsTo achieve this, we initiated an induced mutagenesis program. Seeds from the village seed stock were subjected to treatments with varying doses of ethyl methane sulfonate (EMS) ranging from 0.1% to 0.4% and sodium azide (SA) from 0.1% to 0.4%. The objective was to increase yield potential and enhance genetic diversity.ResultsThe treatment with EMS and SA led to a non-specific, dosage-independent reduction in biophysiological characteristics in French bean mutants. Notably, the 0.4% SA treatment significantly inhibited germination and fertility, causing a decrease in chlorophyll (10.02 mg. g-1 FW) and carotenoid (1.57 mg. g-1 FW) levels. This suggests a disruption in genes associated with chlorophyll and carotenoid synthesis. However, in the M2 generation, the mutagenic treatments substantially improved yield and associated traits. The highest pod yield per plant was recorded at 79.50 gm for the 0.2% EMS treatment. A character association study revealed strong correlations (0.217 to 0.995) between pod yield and other agronomic traits.DiscussionThe results indicate that selecting mutants based on these traits in populations treated with EMS and SA can significantly increase crop yield. The 0.2% SA and 0.2% EMS M2 mutant populations exhibited the highest induced variability, making them ideal for selecting higher-yielding mutant lines for further breeding generations. The increased yields in these mutant lines, derived from a local cultivar, show promise for meeting the growing demand for French bean production through their widespread cultivation.

Mutagenic sensitivity, effectiveness and efficiency of gamma rays and ethyl methane sulfonate on soft and semi-hard bread wheat (Triticum aestivum L.) varieties in the north-western Himalayan climate

Purpose The North-western Himalayan region requires unique varietal traits for the cultivation and quality of grain produced. Wheat varieties released for this zone in the past remained very popular among the farmers. However, with the passage of time certain traits such as the appearance of pathogenic rust races and grain softness have become threat to the fecundity of these genotypes and needs immediate improvement in this region. Mutation breeding facilitates improving one or two traits of a popular cultivar and to generate variability for most of plant traits upon which selection can be imposed. The purpose of this study is to evaluate the mutagenic sensitivity, effectiveness and efficiency of physical and chemical mutagens in four bread wheat varieties with differential grain texture. Materials and methods Four bread wheat varieties; HS 490, HPW 89, HPW 360 and HPW 251 were irradiated using six doses of gamma rays (γ-rays) ranging from 175 to 300 Gy; Co60 source (BARC, Mumbai, India) and six doses of ethyl methane sulfonate (EMS) ranging from 0.3 to 1.3%; EMS (Sigma-Aldrich, Bangalore, India) to assess their mutation sensitivity, effectiveness, efficiency and spectrum of induced macro-mutations in M1 and M2 generation. Results Based on mutagen sensitivity tests, both gamma rays and ethyl methane sulfonate had similar effects as the doses/concentrations increased in all four varieties. Ethyl methane sulfonate had a discernible effect on seed germination and growth parameters as compared to gamma irradiated treatments. Pollens viability studies confirmed the differential effects of both mutagens on germination and plant survivability. The LD50 and LC50 values varied between 290–315 Gy for gamma rays and 0.90–1.35% for EMS under controlled laboratory conditions, however, the range substantially differs for gamma rays (240–290 Gy) and for EMS (0.50–1.1%) under field conditions, irrespective of the variety treated. The frequency of chlorophyll mutations was low and showed a linear correlation with the doses/concentrations of the mutagen. A total of 117 putative mutants with desirable agro-morphological characteristics were also isolated. Mutagenic effectiveness and efficiency results showed that gamma irradiation doses of 250–300 Gy and ethyl methane sulfonate of 0.7–1.3% were most potent for an effective mutation breeding programme in wheat crop. Conclusions It was found that semi-hard textured varieties showed higher sensitivity to chemical mutagens as compared to soft-textured varieties. Gamma irradiation dose of 250–300 Gy and ethyl methane sulfonate concentration of 0.7–1.3% were found to be most effective and efficient across four bread wheat varieties and can be used in large scale mutagenesis programmes.

Effect of ethyl methanesulfonate mediated mutation for enhancing morpho-physio-biochemical and yield contributing traits of fragrant rice.

Chemical mutagenesis has been successfully used for increasing genetic diversity in crop plants. More than 800 novel mutant types of rice (Oryza sativa L.) have been developed through the successful application of numerous mutagenic agents. Among a wide variety of chemical mutagens, ethyl-methane-sulfonate (EMS) is the alkylating agent that is most commonly employed in crop plants because it frequently induces nucleotide substitutions as detected in numerous genomes. In this study, seeds of the widely consumed Basmati rice variety (Super Basmati, Oryza sativa L.) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1.0%, and 1.25% to broaden its narrow genetic base. Sensitivity to a chemical mutagen such as ethyl methanesulfonate (EMS) was determined in the M1 generation. Results in M1 generation revealed that as the levels of applied EMS increased, there was a significant reduction in the germination percent, root length, shoot length, plant height, productive tillers, panicle length, sterile spikelet, total spikelet, and fertility percent as compared to the control under field conditions. All the aforementioned parameters decreased but there was an increase in EMS mutagens in an approximately linear fashion. Furthermore, there was no germination at 1.25% of EMS treatment for seed germination. A 50% germination was recorded between 0.50% and 0.75% EMS treatments. After germination, the subsequent parameters, viz. root length and shoot length had LD50 between 05.0% and 0.75% EMS dose levels. Significant variation was noticed in the photosynthetic and water related attributes of fragrant rice. The linear increase in the enzymatic attributes was noticed by the EMS mediated treatments. After the establishment of the plants in the M1 generation in the field, it was observed that LD50 for fertility percentage was at EMS 1.0% level, for the rice variety. Hence, it is concluded that for creating genetic variability in the rice variety (Super Basmati), EMS doses from 0.5% to 0.75% are the most efficient, and effective.

Utilization of Mating Behavior as a Parameter to Understand Adaptive Response in <i>Drosophila melanogaster</i> using Ethyl Methanesulfonate and Methyl Methanesulfonate

Monofunctional alkylating agents, Ethyl Methanesulfonate (EMS) and Methyl Methanesulfonate (MMS) were used to understand adaptive response utilising mating behaviour as a parameter in D. melanogaster. Selected conditioning and challenging doses of EMS (0.5mM and 15mM) or MMS (0.1mM and 3mM) by larval feeding were tested employing different combinations of crosses. The results have revealed that both EMS and MMS affected courtship elements significantly in different combinations of crosses. Nonetheless, significant increases in orientation, tapping, wing vibration and licking were observed when both males and females were treated with a challenging dose of MMS compared to EMS (p<0.05). On par with this, were also the results of female rejection elements in both the tested chemicals. When conditioning and challenging doses were given after 2 hours of time lag between them to 48±4h or 72±4h aged larvae of D. melanogaster, the results showed that male and female courtship elements significantly reduced compared to the additive effect of respective agents. Similarly, the courtship latency and copulation latency were significantly decreased in contrast to copulation duration which was significantly increased (p<0.05). Thus the results demonstrate the presence of adaptive response in D. melanogaster using courtship elements and the authors opine that mating behaviour can be used as a parameter to analyze adaptive response in D. melanogaster within a short period of time compared to other test procedures.

Assessment of Mutagenic Sensitivity and Lethal Dose of EMS in Papaya (Carica papaya L.) variety CO 7

Background: Mutagenesis is the process through which an organism’s genetic makeup is irreversibly altered. The objective of this study was to determine the optimal ethyl methanesulfonate (EMS) dosage to create desirable genetic variations in the papaya variety CO 7. Methods: The pre-soaked papaya seeds were exposed to five different concentrations of EMS (0.2%, 0.4%, 0.6%, 0.8% and 1.0%) for three hours. Non-treated seeds were used as a control. Data was recorded for germination per cent, seedling survival per cent, seedling length, girth, number of leaves, leaf length and width, petiole length and girth. Result: A declining trend in germination and growth of seedlings with the increase in EMS doses was observed. The probit curve analysis based on the seed germination percentage revealed that the LD50 value was found to be 0.55% EMS which was fixed as an optimal dose for large-scale mutagenesis experiments in papaya variety CO 7. The R2 value ranged from 0.73 to 0.99 and the growth reduction percentage (GR50) varied from 0.69 to 1.16 for different seedling traits studied. Since the optimization of mutagen dose would be expected to create desirable mutations with nominal biological damage, this study might assist further mutagenesis experiments in papaya.

Ethyl Methanesulfonate (EMS)-Mediated Fruit Mutants Of Bitter Gourd (Meghna-2): A Popular Landrace Of West Bengal

Ethyl methanesulfonate (EMS) is a stable and effective chemical mutagen. Bitter Gourd (Meghna-2) seeds were treated by 0.3, 0.5, and 1% EMS for 24 h and 48 h to optimize EMS mutagenesis. Median lethal dose of EMS was obtained at 0.3% EMS treated for 48 h. After treated by 0.3% EMS for 48 h, 250 M1 plants were grown in field for phenotype determination. In comparison to the control, a decline in germination, leaf shape, fruit size, fruit shape, vine length, and emergence was seen in the M1 generation when the concentration of applied EMS was increased. The fertility of M1 bitter gourd was very low and only 24 lines produced seeds after self crossing. Of the mutant 24 lines in the plant growth, five were dwarf, two were tall, four had retarded fruit size, and 13 had abnormal branching and flowering. Only four lines (G 6, G 9, G 14 and G 15) raised from M2 generations of Meghna-2, were screened critically and observed no significant reduction in seed germination and pollen viability but the fruit size was significantly decreased. Interestingly, the short fruit type and round shape of Meghna-2 (G 9) was chosen amongst the four due to farmer’s acceptability, which was grown further to confirm the stability.

ASSESSMENT OF SEED GERMINATION IN BLACKGRAM (VIGNA MUNGO L. HEPPER) UNDER EMS INDUCED MUTAGENESIS

The present investigation was carried out to estimate the mutagenic sensitivity of blackgram accession IC 436524 with EMS (Ethyl methane sulfonate). The seeds of IC436524 were treated with four different concentrations of EMS viz., 0.2%, 0.3%, 0.4% and 0.5%. Thirty seeds of each concentration were placed in Petri plates in three replication along with untreated seeds for germination test, shoot length(cm) and root length(cm). The germination was observed on the 5th day after treatment while shoot and root length of seedling was recorded on the 10 day. The results showed that the germination% was recorded highest (86.67%) at 0.2% of EMS followed by (80.00%) at 0.3% of EMS and lowest seed germination% was recorded at 0.4 and 0.5% of EMS. The reduction percentage of seed germination with control (untreated seeds) at 0.2% of EMS was 6.66% followed by 13.33% at 0.3% of EMS, 53.33% at 0.4% of EMS and 80% at 0.5% of EMS respectively. The results also showed that the highest shoot and root length (cm) was recorded at 0.2% of EMS (8.60cm and 5.50cm) followed by at 0.3% of EMS (6.50cm and 3.83cm). Thus 0.4 and 0.5% dose of EMS seemed very effective in reducing percent seed germination in black gram. The results excerted that the blackgram accession IC436524 could be germinated up to 0.3% EMS concentration to create variability in blackgram which are further useful for crop improvement programs.

The Results of Mutations Made with Specific Ems Dose on Chickpea (Cicer arietinum L.) Germination Properties

The aim of creating induced mutations is to bring about genetic changes that increase the variation in the desired properties. The selection of appropriate mutagens depends on their availability and effectiveness at certain doses to achieve the necessary genetic modifications. This research was performed at the Dicle University Faculty of Agriculture Biotechnology Laboratory to determine the effects of 10 specific doses of EMS (Ethyl Methane Sulfonate) on mutation on the chickpea cv. Gökçe of germination characters. Seed was handled with an EMS of 0 (control), 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM. In the germination cabinet, EMS applied seeds were left to germinate with 3 replications according to the completely randomized design with 25 seeds in each petri dishes. In the study, germination rate, coleoptile length, root length, coleoptile fresh weight, root fresh weight, coleoptile dry weight and root dry weight were measured. It was decided that there was a significant difference in all the germination features tested between the doses applied. Due to the rise in EMS doses, a reduction was defined for all features and the lowest values were obtained from the dosage of 100 mM. As a result of the study, it was determined that increasing the dose of EMS in chickpea reduces the germination rate, the seedling characteristics and the healthy growth rate of the seeds and that the effective mutation can be obtained from 50-60 mM doses.

Impact of chemical mutagenesis using ethyl methane sulphonate on tepary bean seedling vigour and adult plant performance

Tepary bean is an important food legume. The genetic improvement of the crop is limited by a narrow genetic base but this genetic base can be broadened through induced mutagenesis. This study was designed to determine the (i) effects of chemical mutagenesis using ethyl methane sulfonate (EMS) on M1 seedlings (ii) the adult plant performance of M2 - M4 generations and (iii) the relationships between growth attributes at the seedling and adult plant stage. Mutagenized seed with varying doses of EMS was germinated at room temperature in order to raise M1 seedlings. There were highly significant (P < 0.01) differences due to dose effects among the seedlings. The highest LD50 (3.37 % EMS v/v) was observed for ‘Genotype 3’. Under field conditions, all the three factors influenced the plant performance. The results demonstrated the potential of EMS to induce genotypic variation and desirable agronomic traits in tepary bean.

Effect of Ethyl Methanesulfonate on Induced Morphological Variation in M3 Generation of Chrysanthemum indicum var. aromaticum

Mutation breeding is considered to be economic and efficient in plant improvement, and the use of chemical mutagens such as ethyl methanesulfonate (EMS) can potentially address plant breeding challenges. The aim of this study was to induce morphological mutants in C. indicum var. aromaticum using EMS treatments with different doses, and to analyze the morphological and physiological traits of obtained mutants in expectation of finding favorable mutants. Results revealed significant effects of EMS doses on seed germination. The sample germination rate significantly decreased with increasing of EMS doses. The obtained morphological mutants were two viable types, containing leaf and stem mutants. Overall leaf size was significantly larger as a result of EMS treatments. And the height of mutant plants was significantly higher. Anatomical characteristics exhibited changes in both leaves and stems of the mutant plants. The puncture strength of the bent stem from the mutant plants was low, with weak penetration resistance. The total lignin and cellulose contents of mutant plants stem decreased significantly as a result of the EMS treatments. These results demonstrate the efficiency of EMS to induce mutations in C. indicum var. aromaticum, and this method can be useful in the future to assist breeding of this plant.

The Effect of Single and Combined Use of Gamma Radiation and Ethylmethane Sulfonate on Early Growth Parameters in Sorghum.

Success in inducing genetic variation through mutagenic agents is dependent on the source and dose of application. The objective of this study was to determine the optimum doses of a single and combined use of gamma radiation and ethylmethane sulfonate (EMS) for effective mutation breeding in sorghum. The study involved two concurrent experiments as follows: in experiment I, the seeds of four sorghum genotypes (‘Parbhani Moti’, ‘Parbhani Shakti’, ‘ICSV 15013′, and ‘Macia’) were treated using gamma radiation (0, 300, 400, 500 and 600 Gy), EMS (0, 0.5 and 1.0%), and gamma radiation followed by EMS (0 and 300 Gy and 0.1% EMS; 400 Gy and 0.05% EMS). In experiment II, the seeds of two genotypes (‘Macia’ and ‘Red sorghum’) were treated with seven doses of gamma radiation only (0, 100, 200, 300, 400, 500 and 600 Gy). Overall, the combined applied doses of gamma radiation and EMS are not recommended due to poor seedling emergence and seedling survival rate below LD50. The best dosage of gamma radiation for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively. The EMS optimum dosage ranges for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia were between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively. The above dose rates are useful to induce genetic variation in the tested sorghum genotypes for greater mutation events in sorghum breeding programs.

Genetic Amelioration in lentil (Lens culinaris L.) using Different Doses of Ethyl Methane Sulphonate

BackgroundLentil is an important source of food not only locally but globally. The importance of this legume crop is highlighted in light of the transport of strategic food sources such as wheat, maize, and rice due to environmental challenges. So, the process of genetic improvement of this crop has become imperative.ResultsThe final results confirmed that the three lentil cultivars Giza 9 (G1), Giza 29 (G2), and Giza 51 (G3) were exhibited unparalleled superiority for all growth, germination, and other traits under study when exposed to the three doses of ethyl methane sulfonate (EMS) compared to the control. Moreover, the three doses of this mutagen exceeded on the control experiment and this superiority were most severe especially at the third dose. The higher limit of positive percentages values of induced mutagenesis in M2 lentil generation (the second mutant generation) was observed in all studied traits especially at the third dose of EMS (0.3). This fact confirmed the success of the genetic improvement process in all lentil traits mentioned above.ConclusionThe three lentil cultivars (Giza 9, Giza 29, and Giza 51) were recorded highly mean values of all studied in M2 generation for the three doses of EMS. The third dose of mutagen (0.3) was come in the first rank followed by the second dose (0.2) then the first dose in this regard.

Mutagenic effect of ethyl methane sulfonate (EMS) on barnyard millet (Echinochloa frumentaceae) variety CO(KV)2

Barnyard millet variety CO (KV) 2 was treated with 2 different doses of EMS (Ethyl Methane Sulfonate) (ie. 0.2%, 0.4%, 0.6%, 0.8% and 1.0%). Six days after treatment germination percentage was calculated and LD50 value were fixed as 0.51%EMS. M1 population was raised with two treatments 04% and 0.5%. Seedlings with normal growth and development were observed in 0.4% EMS dose and very poor establishment, stunted growth and 80% spikelet sterility was observed in 0.5% EMS treatment. In M2 generation macro-mutants such as plant type, earliness, study stem, bigger panicle size and higher plant yield (28g/plant) were obtained.

Inducing genetic mutation on selected vernonia accessions using predetermined ethylmethanesulfonate dosage, temperature regime and exposure duration conditions

Vernonia is a novel, underutilised oilseed crop that has suffered genetic erosion and neglect from previous classification as a weed. Mutations of a single or few genes possessing target traits are invaluable in crop improvement programs, and the use of chemical mutagens like ethylmethanesulfonate (EMS) can potentially address agronomic challenges in vernonia. This study was conducted to induce genetic variation using predetermined EMS treatment conditions in two vernonia accessions. Two accessions (Vge-1 and Vge-4) were treated using previously determined optimum mutagenesis conditions (0.372% EMS dose for one hour at 35 °C for Vge-1, and 0.372% EMS dose for two hours at 32.5 °C). These were planted under rainfed conditions along with untreated controls in a randomised complete block design. Data on time to emergence, emergence percentage, time to heading, number of chloroplast mutants, percentage of sterile plants, time to flowering, time to maturity, number of seeds per head, plant height, thousand seed mass and plot yield was recorded. Treatment with EMS treatment significantly delayed phenology, reduced plant height, yield and yield related parameters. Observation of chloroplast mutants confirmed EMS mutagenesis in vernonia. Harvested mutants will be used in further breeding programmes to address agronomic challenges in vernonia.

Inducible protective processes in animal systems XV: Hyperthermia enhances the Ethyl methanesulfonate induced adaptive response in meiotic cells of grasshopper Poecilocerus pictus

PurposeTo understand the role of hyperthermia in adaptive response, Ethyl methanesulfonate (EMS) an anticarcinogenic agent, adapted meiotic cells of Poecilocerus pictus was used. Materials and methodsBased on the pilot toxicity study, the effective higher temperatures of 40°C and 45°C for 15 or 30min were chosen. P. pictus were treated with conditioning (L) or challenging (H) doses of EMS and 2h time lag (TL) between these doses (L-2h-H) was employed. Different treatment schedules were used to analyze the influence of hyperthermia on EMS induced adaptive response namely (i) pre treatment; (ii) inter treatment; (iii) post treatment and (iv) cross adaptation. After each treatment schedule, animals were sacrificed at 12, 24, 36 and 48h recovery times, testes were processed for meiotic chromosome preparations and anomalies were analyzed. ResultsThe frequencies of anomalies induced by both conditioning and challenging doses of EMS were significantly higher (p<0.05) compared to those of the control and hyperthermia groups. The combined treatments resulted in 44–50% reduction compared to additive effect of EMS. The pre, inter, post and cross adaptation treatments with hyperthermia significantly reduced the frequencies of chromosomal anomalies compared to the challenge and combined treatments with EMS at all recovery times (p<0.05) tested. ConclusionThere is a protection against EMS induced anomalies by hyperthermia in in vivo P. pictus. As far as our knowledge is concerned, this is the first report to demonstrate that hyperthermia enhances the EMS induced adaptive response in in vivo meiotic cells.

Physiological and biochemical changes in pre-bearing mutants of Kinnow mandarin (C. nobilis Lour × C. deliciosa Tenora)

Impact of gamma (γ) irradiation and ethyl methanesulfonate (EMS) on leaf physio-biochemical parameters were studied in pre-bearing induced mutants of Kinnow mandarin budded on uniform Jatti Khatti rootstock. The mutants were developed by treating Kinnow bud sticks with different doses (5, 10, 15 and 20Gy) of gamma rays and EMS (0.05, 0.1, 0.2 and 0.5%). The Survival rate (%) of the mutants were recorded minimum at 20Gy (60%) and 0.5% EMS concentration (70.0%). The surviving mutants 24-month-old for each mutagenic dose were assessed for different parameters. Mutagenic treatments significantly reduced the leaf area in the surviving mutants at 20Gy and 0.5% EMS treatments. The mutants at these doses, however maintained high relative water content (RWC) with lower leaf membrane injury. Maximum chlorophyll breakdown in the mutants were observed due to 20 and 15Gy doses. The induced biochemical changes revealed enhanced proline accumulation in the leaves at 0.2% EMS followed by 0.5% EMS treatment, while elevated phenol contents were recorded in the mutants at 20Gy followed by 15Gy and 0.5% EMS doses. Mutagenic treatments at 20 and 15Gy upregulated superoxide dismutase, catalase and peroxidase enzyme activities. The study highlights that both physical and chemical mutagens at higher doses have the potential to generate much needed variation in ‘Kinnow’ mandarin with respect to physio-biochemical traits including antioxidant enzyme and should thus help in evolving desired mutants for economic traits including seedlessness and subsequent use in future breeding programmes.

Effect of mutagens on regeneration and growth of in vitro grown epicotyl segments of rough lemon seedlings (Citrus jambhiri Lush.)

The present study on the effect of mutagens on regeneration and growth of in vitro grown epicotyls segments of rough lemon seedlings (Citrus jambhiri Lush.) was carried out during the years 2009- 2010 and 2010- 2011 in the Tissue Culture Laboratory, Department of Fruit Science, Punjab Agricultural University, Ludhiana. Developmental characteristics of the in vitro grown epicotyls segments on regeneration media were treated employing gamma radiation in Gray(Gy) at 0, 5,10,15, 20,25,30,35,40 and 45 Gy; the alkylating agent ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) each at 0, 0.1, 0.2, 0.3, 0.4,0.5 and 0.6% (v/v) were evaluated. Epicotyl segments from one month old in vitro grown seedling were cultured in regeneration medium (MS+BAP 1.0 mglit-1) under controlled laboratory conditions (25±2? C, 16 hr photoperiod, 2000 lux light). LD50, the dose required to kill half of the tested population corresponded to 35Gy for gamma radiation, 0.3% each for EMS and MMS treatments. Number of days taken for regeneration increased with increasing dose of gamma irradiation, EMS and MMS. Percent regeneration, number of buds, number of shoots, shoot length, number of leaves, internodal length, primary root length and number of secondary roots decreased with increasing dose of gamma radiation , EMS and MMS. The study would be beneficial to induce desirable variations in plant growth characteristics of rough lemon by the use of mutagens treatment.

Mutagenesis of in vitro cultures of Miscanthus × giganteus cultivar Freedom and detecting polymorphisms of regenerated plants using ISSR markers

Giant Miscanthus (Miscanthus × giganteus; M × g) is a key candidate energy crop for use in biomass to liquid fuel production. It is a naturally seed sterile triploid lacking genetic variation, the basis for selection. Therefore, induced mutation techniques are particularly important in M × g. The objective of this research was to induce variation in Freedom, an elite cultivar, through in vitro chemical mutagenesis. A previously-optimized in vitro propagation protocol for Freedom was used following the in vitro mutagenesis procedure. Immature inflorescence explants (2–3mm) and calli (2–3mm3) were treated with five mutagenic dose treatments [0.01%, 0.1%, 0.5%, 1%, and 3% (v/v) of ethyl methanesulfonate (EMS) for 90min] along with 2% (v/v) dimethyl sulfoxide (DMSO) as a carrier agent to determine the optimum mutagen dosage. The dose at which 50% of the calli/explants recovered (RP50) after the mutagen treatment was used as the optimum EMS dose. Calli and explants of M × g were treated with RP50, 2×RP50, and 3×RP50 EMS dosages, and subsequent regenerants that arose from explants/calli were transferred to soil. Inter simple sequence repeat (ISSR) markers were used to identify variants in the regenerated plants. Results showed variability among regenerants originating from EMS mutagenic treatments. Putative mutants of M × g may be useful for bioenergy research and functional genomics.