M2 Generation Research Articles

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)

Agro-morphological evaluation of gamma ray-induced mutant populations and isolation of harder grain mutants in wheat (Triticum aestivum L.)

Abstract Gamma ray-induced mutations have been widely used to improve existing crop germplasm and create novel genetic variation. In the current study, a multi-year experiment was carried out to induce and isolate mutants with desirable agro-morphological traits and improved grain hardness through evaluation of induced mutant populations generated in soft-textured wheat variety HPW 89 irradiated with gamma ray dose of 250, 300 and 350 Gy. Mutagen sensitivity studies revealed a higher frequency of biological damage and seedling mortality for doses beyond 300 Gy in the M1 generation. However, the mutagenic treatments in the M2–3 populations significantly altered the magnitude of the biometrical traits. Results from the variability and association studies among traits showed that biological yield per plant, 1000-grain weight, spike length, grains per spike and plant height may be prioritized for higher genetic gain and could be used as selection criteria parameters. Multivariate analysis indicated induction of heterogeneity among mutant populations. Overall, 250–300 Gy doses were found ideal for a successful wheat mutation programme and 293 agro-morphologically superior wheat mutants were identified, out of which 108 had semi-hard grain texture based on single kernel characterization system. Among these, nine mutants were found to have the highest grain hardness index due to induced changes in one or both puroindoline genes. Hence, these mutants identified for several traits along with harder grain texture will serve as important genetic resource in future wheat-breeding programmes.

Development and Characterization of a New TILLING Population for Forward and Reverse Genetics in Barley (Hordeum vulgare L.).

Mutagenesis is an important tool in crop improvement and free of the regulatory restrictions imposed on genetically modified organisms. Barley (Hordeum vulgare L.) is a diploid species with a genome smaller than those of other members of the Triticeae crops, making it an attractive model for genetic studies in Triticeae crops. In this study, we report an ethyl methane sulfonate (EMS)-mutagenized population in the Chinese barley landrace TX9425, which is tolerant to both abiotic and biotic stress. A TILLING (Targeting Induced Locus Lesion in Genomes) population consisting of 2000 M2 lines was also constructed based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment. The mutant phenotypes of the M2 and M3 generations were scored and revealed the presence of a wide spectrum of morphological diversity. The population was evaluated by screening for induced mutations in five genes of interest. A detailed analysis was performed for the HvGLR3.5 gene and three mutations were identified by screening in 2000 M2 lines. Two of three mutations displayed tuft and yellow striped leaves compared to the wild type. Altogether, our study shows the efficiency of screening and the great potential of the new TILLING population for genetic studies in the barley crop model system.

Impact of electron beam and ethyl methane sulphonate on chlorophyll mutations in rice genotypes ASD 16 and Norungan

Induced mutagenesis facilitates the creation of novel gene combinations within a plant genome, preserving its basic structure. This study investigates the impact of electron beam radiation and ethyl methane sulphonate (EMS) on chlorophyll mutations in rice genotypes, ASD 16 and Norungan. The seeds were irradiated with five different doses of electron beam and EMS during rabi 2021-22. The M1 generation was assessed for seedling survival, seedling height and spikelet fertility followed by an identification of chlorophyll mutants in the M2 generation. At specific mutagen doses, ASD 16 and Norungan exhibited the genotypic difference for chlorophyll mutants. Various chlorophyll mutations, such as albino, chlorina, xantha, striata, viridis, albomaculata, alboviridis and xanthoviridis were observed. ASD 16 was more sensitive to both mutagens, while Norungan showed a broader response. EMS proved to be a more effective in inducing mutations than the electron beam. Lower and moderate mutagen doses demonstrated higher efficiency indicating the importance of optimizing mutagenic conditions. This study illuminates the significance of chlorophyll mutants genetic makeup varietal differences. The strong and diverse response observed in Norungan underscores its suitability for mutation breeding programmes. These findings contribute to the efficient utilisation of mutagenesis in improving rice traits providing practical implications for elevating crop quality and promoting genetic diversity in rice cultivation.

Genetic parameters of yield component and yield in M1 rice (Oryza sativa L.) generation irradiated with gamma-ray

High-yielding varieties are the primary determinant of the success of increasing Indonesia's rice production. Plant breeding for new high-yielding varieties is possible through mutation induction and selecting desired traits. The effectiveness and efficiency of selection require comprehensive genetic parameters. This study aimed to study the genetic diversity, heritability, genetic advance, and relationship between yield components and yield in the M1 generation of mutant rice irradiated with gamma ray. This research used a factorial randomized complete block design, involving gamma irradiation doses of 100 Gy, 150 Gy, and 200 Gy, as the first factor; and the second factor was the rice genotype: UnsoedBDBP, UnsoedBDIU, UnsoedBPIU, UnsoedIUBD, and UnsoedIBP. Each treatment was repeated three times. Results showed that traits with broad genetic variation were tiller number plant-1, grain number panicle-1, and grain yield plant-1. High heritability and genetic advance values were found in plant height, grain number panicle-1, and yield plant-1, indicating that improvement in these traits can be achieved simply through selection methods. Panicle number plant-1 and filled grain percentage panicle-1 showed a unidirectional relationship and directly affected high values on yield plant-1. Therefore, these traits can be considered selection indicators for breeding high-yielding mutant rice.

Evaluation of Morphological and Cytological Variations in Colchicine-Induced M1 and M2 Generations of Bambara Groundnut (Vigna subterranea L.)

Kacang bambara (Vigna subterranea L.) memiliki potensi dalam mengamankan pasokan pangan dalam menghadapi perubahan iklim karena memiliki manfaat dari aspek kandungan gizi, kesehatan, dan agronominya. Keterbatasan sumberdaya genetik menghambat pengembangan varietas unggul kacang bambara di Indonesia. Pemuliaan mutasi menggunakan kolkisin merupakan salah satu metode untuk meningkatkan keragaman kacang bambara. Penelitian ini bertujuan untuk mengevaluasi keragaman morfologi dan sitologi pada dua generasi kacang bambara yang diinduksi oleh kolkisin pada konsentrasi yang berbeda. Eksperimen dilakukan di dua waktu dan lokasi yang berbeda, yaitu Maret-Juli 2022 di lapangan percobaan Fakultas Pertanian Universitas Brawijaya dan Maret-Juli 2023 di rumah kaca di wilayah Joyo Agung, Lowokwaru, Kota Malang, Jawa Timur. Perlakuan kolkisin dilakukan dengan metode tetes pada pucuk apikal bibit dengan konsentrasi yang berbeda (0, 0,1, 0,2, 0,3, dan 0,4%). Pengamatan karakteristik morfologi dan sitologi dilakukan pada generasi M1 dan M2. Perlakuan kolkisin mempengaruhi perubahan morfologi dan sitologi tanaman kacang bambara pada generasi M1 dan M2. Pada generasi M2, perlakuan kolkisin konsentrasi 0,4% meningkatkan lebar daun sebesar 31%, panjang daun sebesar 32%, waktu berbunga sebesar 28%, lebar biji sebesar 29%, panjang biji sebesar 47%, bobot 100 biji sebesar 3%, lebar stomata sebesar 20%, dan panjang stomata sebesar 25% dibandingkan dengan kontrol. Namun, perlakuan kolkisin menurunkan jumlah daun sebesar 30%, tinggi tanaman sebesar 1%, jumlah biji pertanaman sebesar 58%, bobot biji pertanaman sebesar 56%, dan kerapatan stomata sebesar 15% dibandingkan dengan kontrol. Perlakuan induksi kolkisin menyebabkan peningkatan jumlah kromosom pada generasi M2 sehingga dapat digunakan untuk meningkatkan variasi dan mengatasi keterbatasan sumber daya genetik kacang Bambara.

Identifying mutation region of herbicide-resistant Mutant Quinoa (Chenopodium quinoa Willd.) lines by molecular characterization

Quinoa can be considered as an alternative plant with its features being compatible with unfavorable climate and soil conditions. Moreover, the nutrient content of its seed is great. But there is not a selective herbicide for weeds in quinoa fields. This is a significant problem in the quinoa cultivation. Therefore, it is necessary to develop herbicide-resistant quinoa lines In the study, Tititcaca, a variety of quinoa was used. Sodium azide was used 1.5 mM for mutagenesis. Herbicide-resistant plants were determined by applying herbicide belonging to the IMI group in the M3 generation. These were named ET-6, ET-7, OT-11 and T-103. The 4 mutant lines obtained as a result of seed mutagenesis, and mutant line OT-11 plants occurred in the form of replacement of the cytosine nucleotide with the adenine nucleotide (CA) in the ALS gene. Line ET-6 plants, the thymine nucleotide was replaced with a guanine nucleotide (TG). The mutation occurring in the OT-11 and ET-6 genotypes was evaluated as a transversion-type mutation. A transition type mutation occurred in mutant line T-103 plants as well, and this mutation occurred in the form of substitution of thymine nucleotide with cytosine nucleotide (TC) at nucleotide 1114. According to the results, weed control in agriculture can be done effectively by developing varieties resistant to IMI group herbicides in quinoa plants. Herbicide resistance studies should be continued in the next generations with the ET-6, OT-11, and T-103 lines.

Predictive Analytics in Genetic Engineering as an Optimization Problem

In genetic engineering, developing a breed with a desired trait is a search and optimization problem that sometimes requires many generations of field and laboratory experiments for an optimal solution to be found. The nature of the problem requires that a stochastic optimization algorithm be applied in the metaheuristic search rather than using a deterministic or mathematical approach. In the search for drought-tolerant cowpea, this study applied a genetic algorithm as a predictive analytics tool in the genetic engineering of three native cowpea landraces (Dan muzakkari, Gidigiwa, and Dan mesera) selected from Northern Nigeria (specifically from Kontagora in Niger State of Nigeria). The three cowpea species were subjected to mutagenic treatments using gamma irradiation and Ethyl Methane Sulphonate (EMS). Doses applied include 200, 400, 600, and 800 Gray of gamma irradiation and 0.372% v/v of EMS. Both treated and untreated cowpea landraces were planted and observed. Mutation-induced breeding aims to deepen the drought-tolerant trait of the cowpea mutants to survive conditions in drought-prone Northern Nigeria. The statistical analysis of the agro-morphological and yield parameters of the first mutant generation (M1 generation) indicates that mutagenic treatments have a positive impact on both the yield and the survival of the three landraces as all the treated landraces yielded better than the control, particularly the treatments combination of 600gray and 372% v/v of EMS. Also, the predictive outcomes of the computational simulation that was implemented in Python programming indicate that these local cultivars are developing drought-tolerant genetic variability. For the three computational experiments, the stochastic optimizer (genetic algorithm) converged at the 9412th, 9717th, and 14338th generations respectively. Such predictive analytics information is useful for guiding decision-making by researchers and breeders in the crop improvement program.

Induced mutagenesis for resistance to mungbean yellow mosaic virus and yield traits in mungbean

The M0 ѕeedѕ of mungbean variety DGGV 2 treated with different doѕeѕ of ethyl methane ѕulphonate (EMЅ) at 0.1%, 0.2% and 0.3% concentrations were assessed for Mungbean Yellow Mosaic Virus (MYMV) in M2 generation during summer 2023 at University of Agricultural Sciences, Dharwad. Considering the yield superiority over check (Mean + 2 Standard Deviation) and highly resistant response to MYMV disease, out of 962 M2 plants 17 superior M2 plants were identified. The seed yield plant-1 in these identified mutants ranged from 21.9 to 32.0 g which is higher compared to the average yield of check DGGV 2 (11.7 g) during that season. Also, the other yield contributing traits such as number of productive branches, number of cluster plant-1, number of pods cluster-1, and number of pods plant-1 were observed to be higher in these mutants in comparison to check variety.

Comparison of Mutations Induced by Different Doses of Fast-Neutron Irradiation in the M1 Generation of Sorghum (Sorghum bicolor).

Sorghum is an important C4 crop with various food and nonfood uses. Although improvements through hybridization and selection have been exploited, the introduction of genetic variation and the development of new genotypes in sorghum are still limited. Fast-neutron (FN) mutagenesis is a very effective method for gene functional studies and to create genetic variability. However, the full spectrum of FN-induced mutations in sorghum is poorly understood. To address this, we generated an FN-induced mutant population from the inbred line 'BTx623' and sequenced 40 M1 seedlings to evaluate the mutagenic effects of FNs on sorghum. The results show that each line had an average of 43.7 single-base substitutions (SBSs), 3.7 InDels and 35.15 structural variations (SVs). SBSs accounted for approximately 90.0% of the total number of small mutations. Among the eight treatment groups, FN irradiation at a dose of 19 Gy generated the highest number of mutations. The ratio of transition/transversion ranged from 1.77 to 2.21, and the G/C to A/T transition was the most common substitution in all mutant lines. The distributions of the identified SBSs and InDels were similar and uneven across the genome. An average of 3.63 genes were mutated in each mutant line, indicating that FN irradiation resulted in a suitable density of mutated genes, which can be advantageous for improving elite material for one specific or a few traits. These results provide a basis for the selection of the suitable dose of mutagen and new genetic resources for sorghum breeding.

Induced genetic diversity through mutagenesis in wheat gene pool and significant use of SCoT markers to underpin key agronomic traits

BackgroundThis research explores the efficacy of mutagenesis, specifically using sodium azide (SA) and hydrazine hydrate (HZ) treatments, to introduce genetic diversity and enhance traits in three wheat (Triticum aestivum L.) genotypes. The experiment entails subjecting the seeds to different doses of SA and HZ and cultivating them in the field for two consecutive generations: M1 (first generation) and M2 (second generation). We then employed selective breeding techniques with Start Codon Targeted (SCoT) markers to select traits within the wheat gene pool. Also, the correlation between SCoT markers and specific agronomic traits provides insights into the genetic mechanisms underlying mutagenesis-induced changes in wheat.ResultsIn the study, eleven genotypes were derived from parent varieties Sids1, Sids12, and Giza 168, and eight mutant genotypes were selected from the M1 generation and further cultivated to establish the M2 generation. The results revealed that various morphological and agronomical characteristics, such as plant height, spikes per plant, spike length, spikelet per spike, grains per spikelet, and 100-grain weight, showed increases in different genotypes from M1 to M2. SCoT markers were employed to assess genetic diversity among the eleven genotypes. The bioinformatics analysis identified a correlation between SCoT markers and the transcription factors ABSCISIC ACID INSENSITIVE3 (ABI3) and VIVIPAROUS1 (VP1), crucial for plant development, growth, and stress adaptation. A comprehensive examination of genetic distance and the function identification of gene-associated SCoT markers may provide valuable insights into the mechanisms by which SA and HZ act as mutagens, enhancing wheat agronomic qualities.ConclusionsThis study demonstrates the effective use of SA and HZ treatments to induce gene diversity through mutagenesis in the wheat gene pool, resulting in the enhancement of agronomic traits, as revealed by SCoT markers. The significant improvements in morphological and agronomical characteristics highlight the potential of mutagenesis techniques for crop improvement. These findings offer valuable information for breeders to develop effective breeding programs to enhance wheat quality and resilience through increased genetic diversity.

Molecular mechanisms of neutron radiation dose effects on M1 generation peas

The dose effect of radiation has long been a topic of concern, but the molecular mechanism behind it is still unclear. In this study, dried pea seeds were irradiated with 252Cf fission neutron source. Through analyzing the transcriptome and proteome of M1 generation pea (Pisum sativum L.) leaves, we studied the molecular rule and mechanism of neutron dose effect. Our results showed three important rules of global gene expression in the studied dose range. The rule closely related to the neutron absorbed dose at the transcription and translation levels is: the greater the difference in neutron absorbed dose between two radiation treatment groups, the greater the difference in differential expression between the two groups and the control group. We also obtained important sensitive metabolic pathways of neutron radiation, as well as related key genes. Furthermore, the overall molecular regulation mechanism of dose effect was revealed based on the main functional items obtained. Our research results can be applied to appropriate radiation dose estimation and agricultural production practice.

Keragaan Mutan M3 Padi Beras Merah (G16) Hasil Iradiasi Sinar Gamma 200 Gy

This study aims to determine changes in yield and correlation between quantitative traits of mutant plants of the M3 generation of G16 brown rice irradiated with 200 Gy gamma rays. The research method used was an experimental method which was carried out with field experiments and took place in paddy fields in Tatar Hamlet, Nyurlembang Village, Narmada District, West Lombok Regency, West Nusa Tenggara which was carried out from May to November 2022. The research was arranged using a Randomized Block Design (RAK) in a partitioned design. The treatment consisted of 33 genotypes resulting from gamma irradiation mutations and 3 comparators as test genotypes namely G16, Cigelis and Inpari 32. Each tester genotype was repeated 3 times, so that 42 experimental units were obtained. Observations were analyzed using analysis of variance and a 5% level of phenotypic correlation coefficient. Quantitative traits that show significant results were tested further using the Honest Significant Difference (BNJ) at the 5% level of significance. The results showed that 200 Gy of gamma irradiation did not change the yield of the M3 mutant strain. On the other hand, the criterion of the phenotypic correlation coefficient of the observed quantitative traits showed a significant difference with the results. Plant height and filled grain weight gave a very strong real correlation, while strong criteria were indicated by the longest panicle and number of filled grains, and panicle length and empty grain weight obtained a sufficiently significant correlation, and very weak criteria were obtained on flowering age, total tiller number, number of productive tillers, number of empty grain and 100 grain weight

Identification of Drought Tolerant Fenugreek Mutants at Seedling Stage Through Multi-Trait Genotype-Ideotype Distance Index (MGIDI)

The top-performing mutants from the M2 generation, induced by a combination of γ radiation and EMS treatment, underwent screening for drought tolerance at the seedling stage in the M3 generation under controlled laboratory conditions. This investigation aimed to assess their potential for drought tolerance. The top 15 high-yielding mutants were assessed under both normal (control) and induced drought conditions (10% PEG solution). Utilizing the MGIDI method, the evaluation provided valuable insights into selection traits and identified mutants with potential drought tolerance. Under normal conditions, mutants M3-181 and M3-149 exhibited superior performance in seedling growth traits, with M3-149 being the top performer under induced drought conditions, followed by M3-181. Their proximity to the red-cutline suggests promising potential that warrants further investigation. Furthermore, these findings indicate that mutant lines displayed superior multi-trait performance compared to the parent cultivar. Specifically, mutants M3-181, M3-149, and M3-29 ranked higher than the parent cultivar under normal conditions, while mutants M3-149, M3-181, and M3-168 surpassed the parent cultivar RMt-1 under induced drought conditions. This underscores the effectiveness of mutation breeding, particularly through the induction of mutations using γ radiation and EMS, as an efficient strategy not only for generating genetic diversity but also for producing variants superior to the parent cultivar. To the best of our knowledge, this is first report made on mutant selection in fenugreek for drought tolerance at seedling stage, using the MGIDI approach.

Isolation and evaluation of novel male sterile and self‐incompatible mutant lines of mungbean (Vigna radiata L.)

AbstractHeterosis has long been harnessed in crop production, and while crop fertility lays the groundwork for leveraging heterosis, there remains a dearth of comprehensive data regarding genic male sterility in mungbean. Mungbean (Vigna radiata (L.) Wilczek), being a self‐pollinating crop, exhibits significant hybrid vigour in F1 hybrid seed yield, suggesting the potential for developing hybrid varieties to propel mungbean yield beyond existing plateaus. A new male sterile mutant was induced, isolated, stabilized and evaluated, via gamma irradiation at a rate of 600 Gy, identified in the M3 generation of the GM 4 accession. A uniform male sterile line was crossbred with the wild‐type parental plant to explore the inheritance pattern of male sterility. Pollen sterility was validated through the acetocarmine test, while stigma viability was ascertained using the hydrogen peroxide. While all F1 plants displayed fertile flowers, the F2 generation showed a clear 3:1 segregation ratio for fertile to male sterile plants, indicating the control of male sterility by a single recessive gene, mms. Also, a novel existence of a truncated stigma nestled within the anther column inhibits the effective reception of pollen during anthesis, offering potential in reducing emasculation time by a slight cut on the flower bud and enhancing cross pollination during hybrid seed production. The isolation of these two mutants is poised to significantly advance the global mungbean hybrid breeding programme.

Cobalt 60 (60Co) Gamma Ray Irradiation for Genetic Improvement of Edamame Plant Growth and Yield (Glycine max. (L) Merril) M2 Generation

Cobalt 60 (60Co) Gamma Ray Irradiation for Genetic Improvement of Edamame Plant Growth and Yield (Glycine max. (L) Merril) M2 Generation

The effect of gamma ray re-irradiation on genetic variations in black rice based on RAPD and Bph gene resistance location based on SSR markers

Abstract The black rice variety Cempo Ireng M8 generation already has morphologically uniform characteristics and good productivity values. However, the black rice M8 generation is still susceptible to planthopper pests. Therefore, a re-irradiation process was carried out on M8 using 200 Gy gamma rays to obtain a black rice variety resistant to planthopper pests. This re-radiation treatment has produced the M2 generation. To determine the genetic variations between M8 black rice plants and the M2 generation that are formed, this research conducted molecular methods using six RAPD markers and three SSR markers to determine genes in the sample related to the Bph resistance gene. The sequenced SSR amplicons were analyzed using BLAST in NCBI. The results of the RAPD marker showed genetic variation in the seven black rice samples with an average polymorphism percentage of 92.85% and the Polymorphic Information Content (PIC) value for the six primers was between 0.25-0.5, which means the RAPD primers is informative. The analysis and sequence results of the RM5953 primer show that the primer is located in the Bph resistance gene (chromosome 4) and produces an amplicon at a band size of 129 bp.

Identification of Induced Chemical Mutants for Improving Yield and its Attributing Traits in Wheat (Triticum aestivum L.) against Changing Climate

Background: The enhancement of genetic traits in any crop relies on the existing variability within that particular crop. Through hybridization and traditional breeding methods, the variability within wheat crops has been nearly exhausted. Consequently, mutation breeding emerges as the sole viable and promising approach to introduce new variability for further improvements. Methods: An experiment was conducted during the Rabi seasons of 2020-21 (M1 generation) and 2021-22 (M2 generation) at the Crop Research Centre, Department of Genetics and Plant Breeding, ITM University Gwalior. The experiment assessed the impact of five different concentrations of Sodium Azide (0.02%, 0.03%, 0.04%, 0.05%, and 0.1%) and Hydroxyl Amine (0.2%, 0.3%, 0.4%, 0.5% and 0.6%), in addition to a control, on the wheat varieties MP-3382 and RVW-4106. It employed a randomized block design with three replications. Result: The findings revealed that Sodium Azide and Hydroxyl Amine, the most effective dosages were observed to be 0.1per cent and 0.4 per cent, respectively, in inducing genetic and phenotypic changes in seed quality parameters. Specifically, for the flag leaf area characteristic, the most effective concentrations were 0.03 per cent Sodium Azide and 0.4 percent Hydroxyl Amine. Plant height exhibited the greatest variability at 0.03 per cent Sodium Azide, 0.5 per cent Hydroxyl Amine and 0.1 percent Sodium Azide. A dosage of 0.5 per cent Hydroxyl Amine was found to be most effective in altering spike length. In terms of days to 50 per cent flowering, 0.05 per cent Sodium Azide was identified as the most effective dosage. From the study of traits in both the M1 and M2 generations, 12 mutants were confirmed, with seven validated for variety MP-3382 and five for variety RVW-4106.

Influence of gamma radiation and EMS on chlorophyll and macro-mutation in mungbean (Vigna radiata)

Present study was carried during rainy (kharif) season of 2021 (M1 generation), summer 2022 (M2 generation) and rainy (kharif) seasons of 2022 (M3 generation) at College of Agriculture, Swami Keshwanand Rajasthan Agricultural University, Bikaner, Rajasthan for creating genetic variability in mungbean [Vigna radiata (L.) R. Wilczek]. The experiment was conducted with 26 treatments and 2 popular archaic varieties of mungbean (Ganga-1 and GM-4), solitary application of gamma radiation (200, 400 and 600 Gy), and ethyl methane sulphonate (EMS) (0.1 and 0.2%) as well as their combinations. A wide spectrum of chlorophyll mutants (Chlorina, Xantha, Albina, Viridis and complex type) and morphological mutants (plant type, flower colour, pod type, leaf variation and maturity period) were identified at various crop growth stages. All mutagen treatments including solitary application of gamma radiation and EMS as well as their combinations were found effective for creating genetic variability. The lower concentration of EMS (0.1%) was found to be relatively more effective as compared to 0.2%. Similarly, the lower dose of gamma radiation (200 Gy) was also more effective than 400 and 600 Gy. Among all the treatment combinations, combined application of 200 Gy + 0.1% EMS was found most effective in generating chlorophyll and morphological mutants. Among two varieties of mungbean, Ganga-1 was found to be more sensitive to mutagens as compared to GM-4. Numerous mutants identified in the mutagenized population of both varieties exhibited stability and superiority in terms of agro- morphological traits over Ganga-1 and GM-4. A mutant (S-92 of GM-4) identified during M1 in mutagen treatment 0.1% EMS was very deep lobbed which altered the plant morphology and appearance. This novel unique mutant identified in M1 generation maintained its stability in M2 and M3 generations also. The leaf colour of this mutant was darker than the original leaf colour of GM-4 and found relatively more tolerant to soil moisture-deficit stress.

CO2 response screen in grass Brachypodium reveals the key role of a MAP kinase in CO2-triggered stomatal closure.

Plants respond to increased CO2 concentrations through stomatal closure, which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2 signal transduction mutants in grasses has yet to be reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2 control mechanisms of stomatal movements in grasses, we developed an unbiased forward genetic screen with an EMS-mutagenized B. distachyon M5 generation population using infrared imaging to identify plants with altered leaf temperatures at elevated CO2. Among isolated mutants, a "chill1" mutant exhibited cooler leaf temperatures than wild-type Bd21-3 parent control plants after exposure to increased CO2. chill1 plants showed strongly impaired high CO2-induced stomatal closure despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of sodium azide and CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, Mitogen-Activated Protein Kinase 5 (BdMPK5). The chill1 mutation impaired BdMPK5 protein-mediated CO2/HCO3- sensing together with the High Temperature 1 (HT1) Raf-like kinase in vitro. Furthermore, AlphaFold2-directed structural modeling predicted that the identified BdMPK5-D90N chill1 mutant residue is located at the interface of BdMPK5 with the BdHT1 Raf-like kinase. BdMPK5 is a key signaling component that mediates CO2-induced stomatal movements and is proposed to function as a component of the primary CO2 sensor in grasses.