M2 Generation Research Articles

Responses of EMS on growth and yield of French bean (Phaseolus vulgaris)

An experiment was conducted during 2017-18, to find out the effect of EMS on growth and yield of French bean. The germination percentage in M1 generation decreased with the increased in concentration of EMS both in laboratory and field conditions. The varieties Arka Suvidha and Falguni produced several heat tolerant mutants during February and March, sown for days to flowering, number of pods per plant and seed yield per plant. The maximum numbers of mutants were observed in lower EMS concentration (0.15%) during February and March as compared to higher EMS concentration (0.25 and 0.35%), thus application of lower concentration of EMS has been recommended for crop improvement purposes in French bean through mutation breeding.

Early rosette mutant plants of safflower (Carthamus tinctorious L.)

Many high yielding safflower varieties with maturity duration of 140-145 days or more have rosette periodranging from 22-40 days. It was thought to convert high yielding varieties of safflower in to less rosette periodgenotypes, without reducing yield and oil content. For this if hybridization were applied it affects many other important traits. Therefore, in high yielding varieties/genotypes radiation were applied to create variation and genotypes with reduced rosette period. The mutants with less rosette period as compare to normal genotypes were selected in M2 generation and tested for their stability in M3 generation. In present study in A-1 and other genotypes, mutants were obtained having 15-20days less rosette than normal genotypes.

Evaluation of mutants for bold seed and high yield in niger (Guizotia abyssinica L.)

Two varieties RCR 18 and DNS 17 of niger were irradiated with different doses of gamma rays viz., 150Gy, 175 Gy, 200 Gy, 225 Gy, 250 Gy and 275 Gy. The M1 generation along with control was evaluated during rabi 2016. The LD 50 dose was found to be 250 Gy for DNS 17 and 275 Gy for RCR 18. Mutants showed variations for plant height, number of branches/plant, number of capitula/plant in both the varieties. Control showed maximum seed weight/plant (1.7360 g) in DNS 17. But in RCR 18, mutants irradiated with 150 Gy showed maximum seed weight/plant (3.92280 g) followed by 250 Gy (3.1334 g).

Estimation of Morphological and Molecular Variability in induced Mutant Fennel Lines

In the present study, M3 mutant lines generated from gamma radiation of widely adopted fennel cultivar Rajasthan Fennel-125 (RF- 125) was used. High variability was recorded for time of flowering, number of primary and secondary branches, number of umbels and umbellate/plant, number of seeds/umbel, plant height, days to maturity and seed yield/plant. Total of 24 diverse mutants were further selected for molecular characterization based on morphological diversity. The Random Amplified Polymorphic DNA (RAPD) analysis showed broad genetic diversity among the mutant lines. Total 73 loci were amplified in all 24 mutants and 23 loci were found polymorphic. Dendrogram showed similarity coefficient value in range of 0.00 to 0.88. The dendrogram divided the 24 mutants into four main clusters. The morphological and molecular study revealed that the mutants selected in the M2 generation showed variability at the genetic level for different essential traits of fennel.

Effect of chemical mutagens on growth of Okra (Abelmoschus esculentus L. Moench)

The purpose of the present study was to evaluate the effects of mutagens i.e. Ethyl methanesulfonate (EMS) and Sodium azide (SA) on the morphological growth of Okra. Seeds of two Okra varieties Subzpari and Pahuja were first pre-soaked in distilled water and then treated with 0.0 (Control), 0.1, 0.2 and 0.3% Sodium azide (SA) and Ethyl methanesulfonate (EMS) separately. The results showed that the germination rate was significantly decreased with increased lethality (%) and mutation frequency (%) in M1 of both varieties with increasing concentrations of EMS and SA against control. The results showed that different concentrations of EMS and SA affected morphological attributes of both okra varieties as the increase of mutagen concentration showed variable results in M1 generation compared with control, but M2 generation showed useful improvement in various traits. Such as the M2 generation of Subs pari from 0.1% SA treatment showed a decrease in flowering time while an increase in the number of fruits per plant, fruit size, seeds yield (g) per plant and 100 seeds weight. Keywords: Ethyl methane sulfonate; Growth; Induced mutagenesis; Okra; Sodium azide http://dx.doi.org/10.19045/bspab.2020.90116

Characterization of Induced High Yielding Cowpea Mutant Lines Using Physiological, Biochemical and Molecular Markers

Cowpea, Vigna unguiculata (L.) Walp. is an important grain legume grown in the dry agro-ecologies of the tropics with considerably low yield due to lack of improved varieties, aggravated by prevalent narrow genetic base. Thus, induced mutagenesis was employed using sodium azide and gamma rays to increase genetic variability in cowpea genotypes that resulted in isolation of eleven high yielding mutant lines at the M4 generation from the genetic background of cowpea varieties Gomati VU-89 and Pusa-578. In order to analyze the induced genetic divergence among the mutant lines and parent genotypes, biochemical and molecular characterization was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), simple sequence repeat (SSR) and CAAT box derived polymorphism (CBDP) markers. Activity of nitrate reductase (NR) and content of chlorophyll, carotenoid, protein and mineral were found to be significantly high in the selected mutant lines compared to their respective parent genotypes. SDS-PAGE profile of seed proteins generated 54 and 28 polymorphic bands and a total polymorphism of 62.06 and 41.17% in Gomati VU-89 and Pusa-578, respectively. SSR primers amplified a total of 16 and 24 polymorphic bands with an average polymorphism of 20.69 and 50.74% in Gomati VU-89 and Pusa-578, respectively. CBDP markers, used for the first time in mutagenized population, generated 175 bands with 77 bands being polymorphic in Gomati VU-89 and 121 bands with 59 bands being polymorphic in Pusa-578. Physiological, biochemical and molecular profiling of the selected promising mutants lines showed that Gomati VU-89-G and Pusa-578-C are genetically most diverged high yielding genotypes with significant increase in protein and micronutrient content, therefore, could be recommended for further research considerations. Thus, the favorable combination of genes induced in the novel cowpea mutants selected through the present study are valuable to correspond farmers requirements for new improved cultivars (direct or hybrids).

Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10−7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

Salt Tolerance of Barak Cenana Rice (Oryza sativa L cv. Barak Cenana) EMS Derived M1 Putative Mutants

Barak Cenana (Oryza sativa L cv. Barak Cenana) is a native red rice cultivar of Tabanan regency, Bali. We have previously created first-generation putative mutants (M1) lines of Barak Cenana using EMS. In this study, we aim to evaluate M1 lines tolerance to salt stress. Three-leaf seedlings of WT Barak Cenana and M1 lines were cultured in hydroponic system containing Yoshida solution and 100 mM NaCl for 7 days. The salt tolerance level of each seedling was evaluated using SES standard and the survived seedlings were transferred into the pot-containing soil growth for maturation. All WT were died when treated with 100 mM NaCl for 7 days. In contrast, some M1 lines were survived where 0.5% were highly tolerance (II2A-4) and 4.06% (1F-4, 1F-3, II2A-8, IID-1, IID-4, IID-6 and 1B-6) were tolerance to salt stress. This variation of salt tolerance level among M1 lines is likely due to the random mutation caused by EMS. Furthermore, all survived mutants were fertile and able to produce mature seeds. As characters in M1 generation are not stable, future studies are required to establish stable mutant lines.
 Keywords: Rice, EMS, Mutants, Barak Cenana, salt tolerance

Induced Mutagenesis Enhances Lodging Resistance and Photosynthetic Efficiency of Kodomillet (Paspalum Scrobiculatum)

The present research was focused in the development of photosynthetically efficient (PhE) and non-lodging mutants by utilizing ethyl methane sulphonate (EMS) and gamma radiation in the kodomillet variety CO 3, prone to lodging. Striking variations in a number of anatomical characteristics of leaf anatomy for PhE and culm thickness for lodging resistance was recorded in M2 (second mutant) generation. The identified mutants were subjected to transcriptomic studies to understand their molecular basis. Expression profiling was undertaken for pyruvate phosphate dikinase (PPDK), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen—(NADPH) and NADP-dependent malate dehydrogenase (NADP-MDH) in the mutants CO 3-100-7-12 (photosynthetically efficient) and in CO 3-200-13-4 (less efficient). For lodging trait, two mutants CO 3-100-18-22 (lodged) and CO 3-300-7-4 (non-lodged) were selected for expression profiling using genes GA2ox6 and Rht-B. The studies confirmed the expression of PPDK increased 30-fold, NADP-ME2 ~1-fold and NADP-MDH10 was also highly expressed in the mutant CO 3-100-7-12. These expression profiles suggest that kodomillet uses an NADP-malic enzyme subtype C4 photosynthetic system. The expression of Rht-B was significantly up regulated in CO 3-300-7-4. The study highlights the differential expression patterns of the same gene in different lines at different time points of stress as well as non-stress conditions. This infers that the mutation has some effect on their expression; otherwise the expression levels will be unaltered. Enhancement in grain yield could be best achieved by developing a phenotype with high PhE and culm with thick sclerenchyma cells.

Individual selection of roselle plant m4 generation (Hibiscus sabdariffa l.) based on anthocyanin, vitamin c and water content

Mutation of a plant is included to be one of the most effective methods to create a high genetic diversity in plants. This study aimed to get the selected individuals of M4 generation of Roselle (Hibiscus sabdariffa L.) in rows through gamma-ray irradiation based on the flower calyx production, the content of anthocyanin and vitamin C. This research used Roselle seed of Roselindo 2 variety (control) as many as 16 numbers selected selection. The study was conducted at the Agrotechnology field Universitas Sumatera Utara (± 25 m asl) starting from December 2018 until April 2019 with the germination date of December 7, 2018. The data obtained were tested using a t-test analysis and presented descriptively. Results showed that the observation of all the average dry weights of plants−1 did not show any significant effect between one and other plants (control and number of selected plants). The average wet weight of plant−1 showed a significant effect in plant numbers of R141 and R148 compared to the other sample numbers. Levels of plants−1 parameters of anthocyanin and vitamin C showed a significant effect between one plant to another in all plant numbers (control and number of selected plants).

İleri Kademe Ekmeklik Buğday (Triticum aestivum L.) Mutant Hatlarının Verim ve Kalite Parametrelerine Göre Seleksiyonu

In the presented research, conventional gamma ray mutagenesis was used to develop new mutant line(s), which have such characters moderate and/or high yielding, semi dwarf and early maturing. Thus, Selimiye cultivar, TCI2066 and three sisters’ line of TCI2021 were irradiated with 200 Gy gamma rays and segregated up to sixth generations. 15 advanced mutant lines from M6 generation and their parent and five commercial wheat cultivars, which have superior characters than others were evaluated according to some agronomic parameters, such as grain yield (GY), thousand kernel weight (TKW), test weight (TW), protein content (PRT), hardness (HARD), days of heading and plant height, under field condition during 2012-2014 growing years in Edirne, Turkey. As a result of study, the highest grain yield was determined from the mutant lines derived from Selimiye wheat cultivar than commercial cultivars. The mutant lines of TCI2021-21M4-2 and TCI2021-23M5-2 were given the highest value in terms of protein content, and the highest hardness values were determined from the mutant lines of TCI2066-9M2-3, TCI2066-9M2-1 and TCI2021-21M4-2, respectively. The mutant line of TCI2021-12M3-8 was measured the shortest one among them. Additionally, a strong positive correlation was determined between GY and TKW, and TW in both growing season. In conclusion, gamma ray mutagenesis had efficiently used in local bread wheat improving programmes to develop mutant lines with superior characters, and TKW and TW could be used as selection criteria for maintaining grain yield under divers rainfall conditions of Trakya Region.

Generational Analysis of Generations of M2 and M3 of Green Beans crop (Vigna radiata L.) Gamma-ray Radiation Results

The objective of this research was to determine phenotypic variability of mung bean M2 and M3 Mutant populations resulted from gamma ray irradiation. The Field experiment was conducted by using a completely randomized design in which three populations including M2, M3 and the parent (M0) were planted in 2017. Each population was planted in a plot of 3 m x 2 m with planting space of 30 cm x 20 cm to obtain 100 individual plant per plot. Every population was repeated 5 times so to get 500 plants per population. Observations were collected on all individuals in the population including plant height, number of stem internodes, length of internode, number of primary branches, age of flowering plants, date of harvest, length of pods, number of filled pods, number of seeds per pod, number of seeds per plant, weight of seeds per plants, and weights of 100 seeds. The results showed that gamma ray irradiation produced mung bean plants with a similar date of harvest. It could be seen from mean values ​​which were not significantly different, the values ​​of diversity, and range were almost equal which indicates the diversity of M2, M3 and M0 populations was relatively similar to the control. M2 and M3 generation of mung bean have large and high quality seeds weighing more than 6.5 g/100 seeds. The homogeneity of variance (HOV) analysis showed that variance of all population of all parameters observed were not homogeny except for plant height, number of stem internode and weight of 100 seeds.

Morphological diversity of faba bean (Vicia faba L.) M2 mutant populations induced by gamma radiation and diethyl sulfate

Induced mutagenesis is one of the most widely used breeding methods for rapidly increasing genetic diversity in many crops. This study used gamma radiation and diethyl sulfate (DES) to increase the genetic diversity in two faba bean genotypes. The genetic diversity assessment was based on 12 qualitative traits. The purpose of this study was to assess the genetic diversity in the M2 faba bean mutant populations and to select promising mutant. A total of 5271 M2 seeds generated from the M1 generation of two genotypes—Hassawi 2 and ILB4347—were used in this study. The seeds of M1 were obtained by gamma-ray exposure (25 and 50 Gy) and treatment of 0.01, 0.02, and 0.05% DES. A total of 4333 (82.2%) seeds germinated, of which 4102 plants survived and were screened for morphological diversity. A total of 36 mutant types are presented in this study. Determinate plant type, white flower color, white hilum, and black seed coat color were some of desirable traits that were considered for further generations. Multivariate-based principal component analyses showed that high genetic diversity was induced by gamma radiation and DES in faba bean mutant populations. This study revealed the potency of gamma radiation and DES on increasing genetic diversity and demonstrated the successful program of induced mutagenesis in the faba bean.

Studies on induction of genetic variation through seed mutation in cowpea (Vigna unguiculata L. Walp.) by gamma irradiation

The present study was carried out to induce seed mutants in cowpea for getting desired mutants for increased yield, bold seed size. Dry seeds of cowpea varieties were exposed to gamma irradiation from cobalt 60 source at 250 Gy dosages. The non-irradiated parent was used as the control. Identification and selections were carried out at the second generation (M2). The treated and untreated plants were self-fertilized for five generations to observe different morphological characters in M6 generation. Several unique and interesting mutants were isolated in thisinvestigation. Cowpea mutants were screened for useful agronomic traits. These independent mutants have different phenotypes from control. The most distinct mutants included were different pod size, pod shapeshape and coat color of seeds were also observed. The mutants selected from 250 Gy population included plants with increased vegetative growth of plants, increased number of pods, increase in size of pods, curved pods, early maturing plants with broad leaflets, Plants resistant to thrips, black seed colourand bold seed size. The selected mutants were found to possess useful agronomic traits capable of conferring on them selection advantage for increased yield, easy harvesting and insect tolerance.

Mutagenic effectiveness and efficiency of gamma rays and EMS in Glycine max (L.) Merill

Mutation has been successfully employed in breeding of several food crop varieties. The aim of this study was undertaken to assess the effectiveness and efficiency of EMS and gamma rays in two varieties of soybean. Genetically pure, uniform and dry seeds were treated with different doses/ concentrations of gamma rays (10KR, 20KR, 30KR and 40KR) and EMS (0.1%, 0.2%0.3% and 0.4%). Mutagenic effectiveness and efficiency of gamma rays and EMS was calculated on the biological damage (lethality and seedling injury) in the M1 generation and both chlorophyll and morphological mutations observed in the M2 generation. The results indicated that, mutagenic effectiveness and efficiency decreased with increase in mutagenic treatments.

Biological response, efficiency and effectiveness of gamma irradiation as mutagen on Pusa Navbahar cluster bean

Cluster bean is a highly self- pollinated vegetable with a very narrow genetic base. Hence, mutation breedingis a viable option for generating variation. In the present study, effect of mutagen was studied in Pusa Navbaharvariety of cluster bean using gamma rays from 50 Gy to 600 Gy. Preliminary evaluation at M1 generationfor germination and seedling growth parameters revealed at lower doses of 100 to 200 Gy germination andgrowth parameters showed improvement over control. Further pollens showed steady increase in sterility withincreasing dose and mutated lines showed less survivability compared to control. Thus, 100-200 Gy gammairradiation was found useful to improve the seed germination and seedling growth parameters in cluster bean.While evaluating M2 generation, total 9 types of mutants (dark green, branched, dwarf, white flower, sterile, longpod, short pod, curve pod, small leaf) were observed and the mutation frequency was high at higher doses,whereas both mutagenic effectiveness and efficiency showed decreasing trend with increasing dose. Highestmutation effectiveness was observed at 50-100 Gy. Mutation efficiency based on pollen sterility was maximumat 50 Gy and based on lethality, it was maximum at 150 Gy.

Starch, amylose and amylopectin levels of M5 and M6 generations of black rice irradiated by gamma Co60 ray

M5 and M6 of black rice generation that were the product of radiation Cobalt-60 on 300 Gy showed phenotype changed, one of which was the improvement of nutrition content. A polysaccharide is a kind of black rice nutrition from amylose and amylopectin polymers. This research’s purpose was to know the amount of starch, amylose, and amylopectin from M5, M6, and non radiated black rice. Content of starch was measured by the Luff Schoorl method, amylose by the Iodine-Colorimetric method, and amylopectin levels were obtained from the difference between starch and amylose content. The results showed that the starch content of M5 and M6 lower than non-radiation, i.e., M5 was 73.77%, M6 was 62.20%, and non-radiated was 83,28%. The amylose in irradiated black rice was 71.08% (M5) and 59,33% (M6), which was lower than non-irradiated (4,85%). Irradiated black rice has lower amylopectin levels than to non-irradiated black rice. Non-radiation black rice was 8.43%, M5 was 2.69%, and M6 was 2.86%.

Эффероцитоз усиливает экспрессию аргиназы-1 и тирозинкиназы Mer ГМ-КСФ-дифференцированными макрофагами человека

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK.

Nitrate reductase activity of black rice (oryza sativa l.) cempo ireng cultivar strain 13 and 46 as the result of plant breeding using 60Co gamma ray on drought stress variation

Black Rice (Oryza sativa L.) has been developed as a functional foodstuff due to its higher content of anthocyanins and nutrients. Black rice productivity is relatively low, while current drought stress phenomena may decline crop productivity. The breeding effort with gamma-ray irradiation is done to improve the plant’s ability to be more tolerant against drought stress and increase productivity. Some researches mentioned the use of gamma-ray radiation could initiate morphological, physiological, and biochemical changes in plants. Nitrate reductase activity can be used as a method of crop yields estimation due to its positive correlation with crop productivity. The study aims to examine the nitrate reductase activity and growth responses of the M5 generation from Cempo Ireng Black Rice strain 13 and 46 results of 60Co gamma-ray irradiation compared to black rice without radiation on drought stress variation. The research was conducted with a completely random design factorial. Drought has become a factor I with 3 levels of spacious capacity (100%, 75%, and 50%). Factor II is the black rice strain (13, 46, and no radiation). The observed variables are growth parameters, nitrate reductase activity, and chlorophyll content. The data obtained were analyzed using ANOVA continued with Duncan test at a significance level of 5%. The results showed that there was a variation of growth, chlorophyll content, and nitrate reductase activities from black rice crops irradiated by gamma rays, which is also showed a different response to drought stress. The black rice strain of 46 is the rice, which more resistant to drought stress evaluated from the growth parameters, the content of chlorophyll, and its nitrate reductase activity is higher than black rice without radiation.

Farklı Gamma Işını Dozlarının İki Sıralı Arpada (Hordeum vulgare L.) Bazı Tarımsal Özellikler Üzerine Etkisi

Bu çalışmada, Tarm-92 iki sıralı arpa çeşidi tohumlarına uygulanan farklı dozlarda gama ışınının (200, 300, 400 ve 500 Gy) M2 bitkilerinde bazı tarımsal özellikler üzerine etkisi incelenmiştir. Araştırma Isparta‘da 2016-2017 vejetasyon döneminde yürütülmüştür. M1 generasyonunda, her dozda başak ve bitki görünümüne göre normal ve anormal bitkiler ayrı hasat edilerek 9 farklı [200, 200 A, 300, 300 A, 400, 400 A, 400 A (çift başak oluşumu ve başakta dallanma eğilimi gösteren bitkiler Ç.B.) 500 ve 500 A gray] grup oluşturulmuştur. M2 generasyonunda kontrol (ışın uygulanmamış) ile toplam 10 parsel yer almıştır. Araştırma sonucunda gama ışını uygulaması bin tane ağırlığı, hektolitre ağırlığı, kavuz oranı ve protein içeriği üzerine istatistiksel olarak önemli etkisi belirlenmiştir. Gama ışını uygulaması bin tane ve hektolitre ağırlığını önemli seviyede azaltmış, kavuz oranı ve protein oranını arttırmıştır. Araştırmada her gruptaki tüm bitkilerde bitki boyu, başak uzunluğu, başakta tane sayısı ve başakta tane ağırlığı özellikleri tek bitki üzerinde incelenmiş diskriminant analizi yapılmış ve gruplar arasındaki uzaklık sonuçları belirlenmiştir. M2 generasyonuda incelenen özellikler yönünden kontrole benzeyen bitki oranı 400 Gy %7,83, 200 Gy %10, 300 Gy %19,60 ve 500 Gy %22,22 olarak hesaplanmıştır. Doğrusal ayırma fonksiyonlarını içeren katsayılar incelendiğinde en etkin özelliğin başak uzunluğu olduğu belirlenmiştir.