Deciphering induced variability, character association and multivariate analysis utilizing gamma rays and ethyl methanesulfonate in bread wheat (Triticum aestivum L.) genotypes with differential grain texture.

PURPOSE: Sustainable wheat production and higher genetic gains can be realized by broadening the genetic base and improving the well adapted varieties. In the present study, a multi-year experiment involving induced mutagenesis was conducted to create genetic variation, assess trait associations and genetic divergence in four wheat varieties with differential grain texture treated with six doses of gamma rays and ethyl methane sulfonate using ten agro-morphological traits. MATERIALS AND METHODS: Healthy selfed seeds of four bread wheat varieties with differential texture were irradiated using six doses ranging from 175 Gy-300 Gy of gamma rays (Co60: BARC, Mumbai) and six concentrations of ethyl methanesulfonate (0.3-1.3%) (Sigma-Aldrich, Bangalore, India) to evaluate variability, character association and degree of genetic diversity induced among the mutagenic treatments of wheat varieties with differential grain texture. RESULTS: Significant inter-population differences were observed for almost all the traits. The sample mean of twelve mutant populations in each of the cultivar exhibited superior quantitative phenotypic traits and increased values of the genetic parameters. Based on association and variability studies, plant height, spike length, grain filling period, biological yield per plant and harvest index can be used as early generation criteria for maximum genetic improvement. Multivariate studies indicated the contribution of various traits towards divergence and indicated the efficiency of mutagens in generating variability. Gamma-irradiation dosages between 200-250 Gy and 0.5-1.1% EMS for soft-textured varieties, whereas doses between 225-275 Gy and 0.5-0.9% EMS were found to be most potent for semi-hard-textured varieties. CONCLUSIONS: Assessment of mutagen sensitivity showed that semi-hard wheat varieties were responsive to both mutagens, particularly EMS and generated higher variability and divergence than the soft textured varieties. Hence, gamma rays were proved to be more effective in generating higher variability than ethyl methanesulfonate. A total of 117 putative mutants were identified with desirable agro-morphological attributes. Among these, mutants with higher inter-cluster distance can be used as parents in hybridization programs and serve as important genetic resources in future wheat improvement programs.

Enhancing cold resistance in Banana (Musa spp.) through EMS-induced mutagenesis, L-Hyp pressure selection: phenotypic alterations, biomass composition, and transcriptomic insights.

BACKGROUND: The cultivation of bananas encounters substantial obstacles, particularly due to the detrimental effects of cold stress on their growth and productivity. A potential remedy that has gained attention is the utilization of ethyl mesylate (EMS)-induced mutagenesis technology, which enables the creation of a genetically varied group of banana mutants. This complex procedure entails subjecting the mutants to further stress screening utilizing L-Hyp in order to identify those exhibiting improved resistance to cold. This study conducted a comprehensive optimization of the screening conditions for EMS mutagenesis and L-Hyp, resulting in the identification of the mutant cm784, which exhibited remarkable cold resistance. Subsequent investigations further elucidated the physiological and transcriptomic responses of cm784 to low-temperature stress. RESULTS: EMS mutagenesis had a substantial effect on banana seedlings, resulting in modifications in shoot and root traits, wherein a majority of seedlings exhibited delayed differentiation and limited elongation. Notably, mutant leaves displayed altered biomass composition, with starch content exhibiting the most pronounced variation. The application of L-Hyp pressure selection aided in the identification of cold-resistant mutants among seedling-lethal phenotypes. The mutant cm784 demonstrated enhanced cold resistance, as evidenced by improved survival rates and reduced symptoms of chilling injury. Physiological analyses demonstrated heightened activities of antioxidant enzymes and increased proline production in cm784 when subjected to cold stress. Transcriptome analysis unveiled 946 genes that were differentially expressed in cm784, with a notable enrichment in categories related to 'Carbohydrate transport and metabolism' and 'Secondary metabolites biosynthesis, transport, and catabolism'. CONCLUSION: The present findings provide insights into the molecular mechanisms that contribute to the heightened cold resistance observed in banana mutants. These mechanisms encompass enhanced carbohydrate metabolism and secondary metabolite biosynthesis, thereby emphasizing the adaptive strategies employed to mitigate the detrimental effects induced by cold stress.

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.

A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Inducing mutation and ascertaining lethal dosage of in vitro cultures of banana cv. Ney Poovan to ethyl methane sulfonate.

In vitro mutation breeding in vegetatively propagated crops like banana offers a benefit in screening for beneficial variants in plant cells or cultured tissues. An attempt was made to induce mutants and determine the lethal dose, as it is the prerequisite to optimize the concentration and duration of the mutagen used to recover a larger population in mutation research. Shoot tip cultures were treated for 2 and 4 h at six different EMS concentrations ranging from 80 mM to 160 mM, whereas proliferating multiple shoots were exposed for 30 and 60 min at six different EMS concentrations ranging from 8 mM to 40 mM. Survival percentage, shoot length, and number of shoots reduced linearly and significantly as concentration and duration increased in both shoot tips and proliferating multiple buds. The probit curve-based analysis of mortality of treated explants revealed that the LD50 was 155.83 mM for 2 h and 113.72 mM for 4 h, respectively for shoot tip cultures, whereas for proliferating multiple buds, the LD50 value was adjusted to 39.11 mM for 30 min and 30.41 mM for 60 min. 160 mM EMS for 4 h resulted in a shorter shoot, a longer rooting duration, a lesser number of roots, and decreased root development. In proliferating multiple shoots, the smallest shoot, longest rooting duration, least number of roots, and shortest root were observed in 40 mM EMS for 60 min. Similar reductions in growth parameters were observed in proliferating multiple shoots at higher exposure to EMS for a longer duration.

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

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Improving desirable agronomic traits of M2 lines on fourteen Ethiopian Sesame (Sesamum indicum L.) genotypes using Ethyl Methane Sulphonate (EMS).

Sesame is an important oilseed crop cultivated in Ethiopia as a cash crop for small holder farmers. However, low yield is one of the main constraints of its cultivation. Boosting and sustaining production of sesame is thus timely to achieve the global oil demand. This study was, therefore, aimed at identifying mutant genotypes targeted to produce better agronomic traits of M2 lines on fourteen Ethiopian sesame genotypes through seed treatment with chemical mutagens. EMS was used as a chemical mutagen to treat the fourteen sesame genotypes. Quantitative and qualitative data were recorded and analyzed using analysis of variance with GenStat 16 software. Post-ANOVA mean comparisons were made using Duncan's Multiple Range Test (p≤ 0.01). Statistically significant phenotypic changes were observed in both quantitative and qualitative agronomic traits of the M2 lines. All mutant genotypes generated by EMS treatment showed a highly significant variation for the measured quantitative traits, except for the traits LBL and LTL. On the other hand, EMS-treated genotypes showed a significant change for the qualitative traits, except for PGT, BP, SSCS, LC, LH and LA traits. Mutated Baha Necho, Setit 3, and Zeri Tesfay showed the most promising changes in desirable agronomic traits. To the best of our knowledge, this study represents the first report on the treatment of sesame seeds with EMS to generate desirable agronomic traits in Ethiopian sesame genotypes. These findings would deliver an insight into the genetic characteristics and variability of important sesame agronomic traits. Besides, the findings set up a foundation for future genomic studies in sesame agronomic traits, which would serve as genetic resources for sesame improvement.

Detection of genetic divergence among putative ethyl methane sulfonate mutants of super basmati using microsatellite markers.

BACKGROUND: Seeds of super basmati were mutagenized with different ethyl methane sulphonate (EMS) doses for creating genetic variability. METHODS AND RESULTS: A total of 48 randomly selected putative EMS mutants of super basmati were analyzed to dissect the genetic diversity by using 25 SSR primers located on twelve chromosomes of rice. SSRs analysis revealed that wide-range of genetic diversity is present among mutants of super basmati. A sum of 91 alleles were identified, out of these, 82 alleles were polymorphic and the rest of nine alleles were monomorphic in nature. The range of allele number was 2-10 with mean of 3.64 alleles/locus. The value of polymorphic information content was range between 0.039 (RM5) and 0.878 (RM44) with mean of 0.439 for each locus. A number of polymorphic markers showed unique bands of various sizes ranges from 75 to 1000 bp, during genetic dissection of mutant population. Dendrogram divided whole mutant population into four major groups. Phylogenic analyses revealed that 40-96%genetic similarity is present among individuals of mutant population. CONCLUSION: It is concluded that EMS induced genetic variability and SSRs markers (RM44, RM154, RM1, RM252, RM334, RM487, RM110 and RM257) could be handy for the selection of rice mutants as parents for functional genomic and molecular breeding program.

A large-scale whole-exome sequencing mutant resource for functional genomics in wheat.

Hexaploid wheat (Triticum aestivum), a major staple crop, has a remarkably large genome of ~14.4 Gb (containing 106 913 high-confidence [HC] and 159 840 low-confidence [LC] genes in the Chinese Spring v2.1 reference genome), which poses a major challenge for functional genomics studies. To overcome this hurdle, we performed whole-exome sequencing to generate a nearly saturated wheat mutant database containing 18 025 209 mutations induced by ethyl methanesulfonate (EMS), carbon (C)-ion beams, or γ-ray mutagenesis. This database contains an average of 47.1 mutations per kb in each gene-coding sequence: the potential functional mutations were predicted to cover 96.7% of HC genes and 70.5% of LC genes. Comparative analysis of mutations induced by EMS, γ-rays, or C-ion beam irradiation revealed that γ-ray and C-ion beam mutagenesis induced a more diverse array of variations than EMS, including large-fragment deletions, small insertions/deletions, and various non-synonymous single nucleotide polymorphisms. As a test case, we combined mutation analysis with phenotypic screening and rapidly mapped the candidate gene responsible for the phenotype of a yellow-green leaf mutant to a 2.8-Mb chromosomal region. Furthermore, a proof-of-concept reverse genetics study revealed that mutations in gibberellic acid biosynthesis and signalling genes could be associated with negative impacts on plant height. Finally, we built a publically available database of these mutations with the corresponding germplasm (seed stock) repository to facilitate advanced functional genomics studies in wheat for the broad plant research community.

M2 plants derived from different tillers of a chemically mutagenized rice M1 plant carry independent sets of mutations.

Generation of mutant populations with high genetic diversity is key for mutant screening and crop breeding. For this purpose, the single-seed descent method, in which one mutant line is established from a single mutagenized seed, is commonly used. This method ensures the independence of the mutant lines, but the size of the mutant population is limited because it is no greater than the number of fertile M1 plants. The rice mutant population size can be increased if a single mutagenized plant produces genetically independent siblings. Here, we used whole-genome resequencing to examine the inheritance of mutations from a single ethyl methanesulfonate (EMS)-mutagenized seed (M1 ) of Oryza sativa in its progeny (M2 ). We selected five tillers from each of three M1 plants. A single M2 seed was selected from each tiller, and the distributions of mutations induced by EMS were compared. Surprisingly, in most pairwise combinations of M2 siblings from the same parent, ≥85.2-97.9% of all mutations detected were not shared between the siblings. This high percentage suggests that the M2 siblings were derived from different cells of the M1 embryo and indicates that several genetically independent lines can be obtained from a single M1 plant. This approach should allow a large reduction in the number of M0 seeds needed to obtain a mutant population of a certain size in rice. Our study also suggests that multiple tillers of a rice plant originate from different cells of the embryo.

Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population.

Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley.

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe, making it an attractive model for genetic studies in Triticeae crops. The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations. In this study, we report an ethyl methanesulfonate (EMS)-mutagenized population consisting of 8525 M3 lines in the barley landrace "Hatiexi" (HTX), which we complement with a high-quality de novo assembly of a reference genome for this genotype. The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase, depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis. We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING (targeting induced locus lesion in genomes) platform in barley. Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals. We discovered abundant allelic mutants for dozens of genes, including the barley Green Revolution contributor gene Brassinosteroid insensitive 1 (BRI1). As a proof of concept, we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy, demonstrating the value of this resource to support forward and reverse genetic studies in barley.

Generation of Mutants by Combined Treatment of Physical and Chemical Mutagens in Rice.

Mutants can be generated by physical or chemical mutagens. The procedure includes mutagenesis treatment, post-treatment, planting of M1, harvest of M1, planting of M2, phenotypic observation or mutation scanning in M2 plants. Here we describe the combined treatment of the rice seeds from the indica variety Tenuo with 137Cs gamma rays at 100 Gy and a chemical mutagen-ethyl methanesulfonate (EMS). Theoretically, the method can be used to study various plant biological processes which include plant gametogenesis in addition to the generation of mutants by straightforward physiological screens. As an example of the procedure, we screened for mutants resistant to imazethapyr, an herbicide which can kill grass weeds.

Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol.

One of the most promising avenues of biofuel research relates to using waste as a starting feedstock to produce liquid or gaseous energy carriers. The global production of waste glycerol by the refinery industry is rising year after year. The aim of the present study was to examine the effect of ethyl methane sulfonate (EMS) on the growth rates and intracellular lipid accumulation in heterotrophically-cultured Schizochytrium limacinum microalgae, grown on waste glycerol as the carbon source. The strain S. limacinum E20, produced by incubating a reference strain in EMS for 20 min, was found to perform the best in terms of producing biomass (0.054 gDW/dm3·h) and accumulating intracellular bio-oil (0.021 g/dm3·h). The selected parameters proved to be optimal for S. limacinum E20 biomass growth at the following values: temperature 27.3 °C, glycerol level 249.0 g/dm3, oxygen in the culture 26%, and yeast extract concentration 45.0 g/dm3. In turn, the optimal values for lipid production in an S. limacinum E20 culture were: temperature 24.2 °C, glycerol level 223.0 g/dm3, oxygen in the culture 10%, and yeast extract concentration 10.0 g/dm3. As the process conditions are different for biomass growth and for intracellular lipid accumulation, it is recommended to use a two-step culture process, which resulted in a lipid synthesis rate of 0.41 g/dm3·h.

Screening of an Ethyl Methane Sulfonate Mutagenized Population of a Wheat Cultivar Susceptible to Fusarium Head Blight Identifies Resistant Variants.

Fusarium head blight (FHB) primarily caused by Fusarium graminearum is a key disease of small grains. Diseased spikes show symptoms of premature bleaching shortly after infection and have aborted or shriveled seeds, resulting in reduced yields. The fungus also deteriorates quality and safety of the grain because of production of mycotoxins, especially deoxynivalenol (DON), which can result in grain being docked or rejected at the point of sale. Genetic host resistance to FHB is quantitative, and no complete genetic resistance against this devastating disease is available. Alternative approaches to develop new sources of FHB resistance are needed. In this study, we performed extensive forward genetic screening of the M4 generation of an ethyl methane sulfonate-induced mutagenized population of cultivar Jagger to isolate variants with FHB resistance. In field testing, 74 mutant lines were found to have resistance against FHB spread, and 30 of these lines also had low DON content. Subsequent testing over 2 years in controlled greenhouse conditions revealed 10 M6 lines showing significantly lower FHB spread. Seven and 6 of those 10 lines also had reduced DON content and fewer Fusarium-damaged kernels, respectively. Future endeavors will include identification of the mutations that led to resistance in these variants.

Enhanced characterization of the thyA system for mutational analysis in Escherichia coli: Defining mutationally "hot" regions of the gene.

We have extensively characterized base substitution mutations in the 795 base pair (bp) long E. coli thyA gene to define as many of the base substitution mutational sites that inactivate the gene as possible. The resulting catalog of mutational sites constitutes a system with up to 5 times as many sites for monitoring each of the six base substitution mutations as the widely used rpoB/Rifr system. We have defined 75 sites for the G:C -> A:T transition, 68 sites for the G:C -> T:A transversion, 53 sites for the G:C -> C:G transversion, 49 sites for the A:T -> G:C transition, 39 sites for the A:T -> T:A transversion, and 59 sites for the A:T -> C:G transversion. The system is thus comprised of 343 base substitution mutations at 232 different base pairs, all of which can be sequenced with a single primer pair. This allows for the examination of mutational spectra using a more detailed probe of known mutations, while still allowing one to compare the number of repeated occurrences at specific sites. We have examined several mutagens and mutators with this system, and show its utility by looking at the spectrum of cisplatin, that has a single hotspot, underscoring the value of having as large an array of sites as possible at which one can monitor repeat occurrences. To test for regions of the gene that might be hotspots for a number of mutagens, or "hot" (mutaphilic) regions, we have looked at the ratio of mutations per set of an equal number of mutational sites throughout the gene. The resulting graphs suggest that there are "hot" regions at intervals, and this may reflect aspects of secondary structures, of the higher order structure of the chromosome, or perhaps the nucleoid structure of the chromosome plus histone-like protein complexes.

NH787 EMS mutant of rice variety Nagina22 exhibits higher phosphate use efficiency.

Rice (Oryza sativa L.), a major dietary source, is often cultivated in soils poor in available inorganic orthophosphate (Pi), which is a key nutrient for growth and development. Poor soils are amended by phosphorus (P) fertilizer, which is derived from the non-renewable rock phosphate reserves. Therefore, there is a need for developing rice varieties with high productivity under low P conditions. At the ICAR-IIRR, ethyl methanesulfonate (EMS) mutagenized rice genotype Nagina22 (N22) were screened for high grain yield in Pi-deprived soil, which led to the identification of ~ 10 gain-of-function mutants including NH787. Here, detailed comparative morphophysiological, biochemical, and molecular analyses of N22 and NH787 were carried out in hydroponics and potting soil under different Pi regimes. Under Pi-deprived condition, compared with N22, NH787 exhibited higher root and vegetative biomass, the number of tillers, and grain yield. The augmented agronomic traits of NH787 were corroborated with significantly higher photosynthetic rate, pollen fertility, stigma receptivity, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Further, several genes involved in the maintenance of Pi homeostasis (GPH) were differentially regulated. The study thus revealed a wide-spectrum influence of the mutation in NH787 that contributed towards its higher Pi use efficiency (PUE).

Effectiveness and efficiency of electron beam in comparison with gamma rays and ethyl methane sulfonate mutagens in cowpea.

Cowpea is the poor man's crop that lacks variability due to its autogamous nature. Induced mutation serves as a potential source in the induction of variability in crops. On the other hand, the effectiveness and efficiency of mutagens will vary between species and even varieties. In the present study, a novel mutagen electron beam was used in cowpea for the first time along with commonly used mutagens gamma rays (physical) and ethyl methanesulfonate (chemical). The biological damages on eight quantitative characters in M1 generation and chlorophyll mutants in M2 generation were recorded. Two popular varieties viz., P 152 and VBN 1 constituted as the biological material of study. The rate of reduction in biological damage on quantitative characters was directly proportional to the dose of mutagen irrespective of the varieties and mutagens used. Physical mutagens showed the highest biological damage (EB- 37.5% and G- 37.3% overall reduction from control) than chemical mutagen (EMS- 30.4%). Comparing the physical mutagens at similar doses, 200 Gy or 300 Gy of electron beam showed more biological damage than 200 Gy or 300 Gy of gamma rays. Eleven different types of chlorophyll mutants were identified in the M2 generation. Xantha is the most occurred chlorophyll mutants (44.44%), while aurea and yellow viridis have least occurred mutants. Chemical mutagen (EMS) is considered to be the most effective (6.47%) and efficient mutagen (27.09%) based on the chlorophyll mutants and it was followed by an electron beam and gamma rays. Among the physical mutagens, electron beam showed the highest biological damage (37.50% overall reduction from control) and higher effectiveness and efficiency (3.80% and 23.38%) compared to gamma rays (1.87% and 13.38%). Hence, the electron beam can also be used as an effective mutagen in creating variation in cowpea and other crops as it is highly effective, cost less and safe mutagen.

Development and screening of EMS mutants with altered seed oil content or fatty acid composition in Brassica napus.

Brassica napus is an important oilseed crop in the world, and the mechanism of seed oil biosynthesis in B. napus remains unclear. In order to study the mechanism of oil biosynthesis and generate germplasms for breeding, an ethyl methanesulfonate (EMS) mutant population with ~100 000 M2 lines was generated using Zhongshuang 11 as the parent line. The EMS-induced genome-wide mutations in M2-M4 plants were assessed. The average number of mutations including single nucleotide polymorphisms and insertion/deletion in M2-M4 was 21 177, 28 675 and 17 915, respectively. The effects of the mutations on gene function were predicted in M2-M4 mutants, respectively. We screened the seeds from 98 113 M2 lines, and 9415 seed oil content and fatty acid mutants were identified. We further confirmed 686 mutants with altered seed oil content and fatty acid in advanced generation (M4 seeds). Five representative M4 mutants with increased oleic acid were re-sequenced, and the potential causal variations in FAD2 and ROD1 genes were identified. This study generated and screened a large scale of B. napus EMS mutant population, and the identified mutants could provide useful genetic resources for the study of oil biosynthesis and genetic improvement of seed oil content and fatty acid composition of B. napus in the future.

Novel Ergot Alkaloids Production from Penicillium citrinum Employing Response Surface Methodology Technique.

Ergot alkaloids are novel pharmaceutical and therapeutic agents synthesized in this study using fungal species Penicillium citrinum. To get the maximum yield of ergot alkaloids a statistical process of response surface methodology was employed using surface culture fermentation technique. Initially, the strain of Penicillium was improved using physical (ultraviolet (UV) and chemical (ethyl methane sulfonate (EMS) treatments to get the maximum yield of ergot alkaloids through surface culture fermentation technique. After improving the strain, survival rate of colonies of Penicillium citrinum treated with UV and EMS was observed. Only 2.04% living colonies were observed after 150 min of exposure of Penicillium citrinum in UV light and 3.2% living colonies were observed after 20 min of the exposure in EMS. The mutated strains of Penicillium citrinum were screened for their production of ergot alkaloids and after fermentation experiments, maximum yield was obtained from PCUV-4 and PCEMS-1 strains. After strain improvement, Plackett-Burman design (PBD) and Box-Behnken design (BBD) of RSM were employed and 10-fold yield enhancement (35.60 mg/100 mL) of ergot alkaloids was achieved. This enhancement in yield of ergot alkaloids proved the positive impacts of RSM and UV on the yield of ergot alkaloids. The study provides a cost effective, economical and sustainable process to produce medically important ergot alkaloids which can be used in various pharmaceutical formulations to treat human diseases.