M2 Progeny Research Articles

Characterization of terminal flowering cowpea (Vigna unguiculata (L.) Walp.) mutants obtained by induced mutagenesis digs out the loss-of-function of phosphatidylethanolamine-binding protein

Cowpea (Vigna unguiculata (L.) Walp) is one of the major food legume crops grown extensively in arid and semi-arid regions of the world. The determinate habit of cowpea has many advantages over the indeterminate and is well adapted to modern farming systems. Mutation breeding is an active research area to develop the determinate habit of cowpea. The present study aimed to develop new determinate habit mutants with terminal flowering (TFL) in locally well-adapted genetic backgrounds. Consequently, the seeds of popular cowpea cv P152 were irradiated with doses of gamma rays (200, 250, and, 300 Gy), and the M1 populations were grown. The M2 populations were produced from the M1 progenies and selected determinate mutants (TFLCM-1 and TFLCM-2) from the M2 generation (200 Gy) were forwarded up to the M5 generation to characterize the mutants and simultaneously they were crossed with P152 to develop a MutMap population. In the M5 generation, determinate mutants (80–81 days) were characterized by evaluating the TFL growth habit, longer peduncles (30.75–31.45 cm), erect pods (160°- 200°), number of pods per cluster (4–5 nos.), and early maturity. Further, sequencing analysis of the VuTFL1 gene in the determinate mutants and MutMap population revealed a single nucleotide transversion (A-T at 1196 bp) in the fourth exon and asparagine (N) to tyrosine (Y) amino acid change at the 143rd position of phosphatidylethanolamine-binding protein (PEBP). Notably, the loss of function PEPB with a higher confidence level modification of anti-parallel beta-sheets and destabilization of the protein secondary structure was observed in the mutant lines. Quantitative real-time PCR (qRT-PCR) analysis showed that the VuTFL1 gene was downregulated at the flowering stage in TFL mutants. Collectively, the insights garnered from this study affirm the effectiveness of induced mutation in modifying the plant’s ideotype. The TFL mutants developed during this investigation have the potential to serve as a valuable resource for fostering determinate traits in future cowpea breeding programs and pave the way for mechanical harvesting.

Gamma-rays induced mutations increase soybean oil and protein contents.

Mutation breeding is one of the effective techniques used for improving desired traits such as yield quality and quantity in economic crops. The present study aims to develop oil and protein contents in addition to high yield attributes in soybean using gamma rays as a mutagen. Seeds of the soybean genotypes Giza 21, Giza 22, Giza 82, Giza 83 and 117 were treated with gamma rays doses 50, 100, 200 and 300 Gy. Plants were then scored based on morphological parameters correlated with yield quantity including plant height, seed weight and valuable protein and oil contents. Mutant lines exhibiting the highest yield attributes were selected and used as parents for M2 generation. The M2 progeny was further assessed based on their ability to maintain their yield attributes. Twenty mutant lines were selected and used as M3 lines. The yield parameters inferred a positive effect of gamma irradiation on the collected M3 mutant lines compared to their parental genotypes. 100 Gy of gamma rays gave the highest effect on the number of pods, branches and seeds per plant in addition to protein content, while 200 Gy was more effective in increasing plant height, number of pods per plant, and oil content. Six mutant lines scored the highest yield parameters. Further assessment inferred an inverse relationship between oil and protein content in most of the tested cultivars with high agronomic features. However, four mutant lines recorded high content of oil and protein besides their high seed yield as well, which elect them as potential candidates for large-scale evaluation. The correlation among examined parameters was further confirmed via principal component analysis (PCA), which inferred a positive correlation between the number of pods, branches, seeds, and seed weight. Conversely, oil and protein content were inversely correlated in most of yielded mutant lines. Together, those findings introduce novel soybean lines with favorable agronomic traits for the market. In addition, our research sheds light on the value of using gamma rays treatment in enhancing genetic variability in soybean and improving oil, protein contents and seed yield.

Effect of Mutation on Seed Yield per Plant and Chlorophyll Content in M3 Generation of Green Gram [Vigna radiata (L.) R. Wilczek

Aims: Mutation breeding used to induce quantitative variability in M3 generation of green gram of two excellent variety GM-4 and Meha with a view to study effect various mutagen and for the estimation of genetic variability parameters like GCV, PCV, Heritability and Genetic advance percent of mean for characters like as seed yield per plant and chlorophyll content.
 Place and Duration of Study: The Field experiment was conducted at Instructional Farm, COA, Junagadh Agricultural University, Junagadh during the Summer-2015.
 Methodology: The experimental material consisted of M2 derived M3 seeds which consists of sixty-six M3 progeny lines (64 mutant + 2 controls). The progeny lines were selected on the basis of yield and its associated characters from M2 generation.
 Results: The analysis of variance between family revealed that families differed significantly for chlorophyll content while non-significant for seed yield per plant. GCV and PCV were higher in all the mutagenic family with respect to control for both the characters as well as for both the variety. For both characters, phenotypic coefficient of variation was higher than genotypic coefficient of variation indicating that there was environmental influence on these traits. Heritability exhibited higher in all the mutagenic families and also higher genetic advance was observed. The combined results for heritability showed that the high estimates of heritability and genetic advance were scored for seeds per plant indicating that these characters were under the control of additive genetic effects.
 Conclusion: Mutagenic progenies with additive gene effect can be selected for improvement of desirable traits in mungbean.

Effect of Gamma Rays Irradiation to Cipedak Avocado Genetic Diversity

Avocado cv. Cipedak is a superior cultivar that match the preferences of Indonesian consumers. However, it has several weaknesses, such as low edible portion, thin skin, and short fruit shelf life. One effort to improve the character weakness is by mutation breeding using gamma ray irradiation. Induction of mutation by gamma ray irradiation at a dose of 10 Gy was conducted on Cipedak avocado shoots in October 2021 which produced 13 first generation avocado mutants (M1). Observation on leaves morphological characters and DNA analysis were needed to find out the changes level of genetical diversity. The research was conducted in Cukurgondang Research Station and Molecular Laboratory of Indonesian Tropical Fruits Research Institute from January to March 2022. The plant material used involved 13 M1 progenies and cv. Cipedak. The material used were 20 weeks old grafted plants. The observed variables were macro and micromorphological characters as well as DNA-RAPD analysis. The results showed that there were phenotypic and genotypic changes in 13 M1 avocados seedlings. The coefficients of differences on macro and micromorphological characters were up to 33% and 28%, respectively and the coefficient of difference for DNA-RAPD analysis was 74%.

Frequency and spectrum of M2 mutants and genetic variability in cyto-agronomic characteristics of fenugreek induced by caffeine and sodium azide.

Trigonella foenum graecum L. (Fenugreek) is a valuable medicinal plant cultivated for decades for its therapeutic characteristics. Still no pronounced improvement concerning wild form was accomplished as it is a self-pollinating crop. Induced mutagenesis is encouraged as a remarkable tool on this plant to circumvent the genetic bottleneck of cultivated germplasms. As a result, novel allelomorphic combinations for short-term agronomic attributes were developed. Fenugreek cultivar Pusa Early Bunching, selected for the present experiment, was mutagenized with five doses (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) of caffeine and sodium azide (SA) to evaluate its impact on the qualitative and quantitative traits of M1 and M2 generation conducted in a Complete Randomized Block Design (CRBD), replicated five times during 2019-2020 and 2020-2021, respectively. The frequency of induced phenotypic variations was assessed in M2 progenies, resulting in the identification and isolation of a broad spectrum of mutants with altered phenotypes. Mutagenic effectiveness and efficiency were found to be maximum at lower concentrations of the mutagen treatments and highest in SA, followed by caffeine. Various morphological mutants with modified characters were observed at different concentrations in M2 generation. The spectrum of mutations was wider in SA than in caffeine, as caffeine produced 51 while SA produced 54 individual mutants under seven major categories. The maximum frequency of morphological mutants was associated with leaf, followed by plant size, plant growth habit, pod, seed size, seed shape, and seed color. Morphological and structural variations in the guard cells of stomata and seeds were observed through scanning electron microscopy. The variations created in the economically important traits may enrich the genetic diversity of this plant species. Moreover, these morphological mutants may serve as a source of elite genes in further breeding programs of fenugreek.

Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat

ABSTRACT Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3’(2’), 5’-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

60Co gamma ray induced mutants of cowpea and assessment of genetic variability by SCoT marker

Mutagenesis is a well-known technique for introducing new variants into crop plants. In the present study, M2 populations were generated in the cowpea (Vigna unguiculata (L.) Walp.) variety CO7 using gamma irradiation. The M2 progeny were used to investigate the effectiveness of the gamma irradiation doses and examined for the agronomic traits. The genomic variation present in the mutants and their parents was analysed using five SCoT markers. Marker analysis revealed a total of 87 amplicons and among these, 20 amplicons showed polymorphism. The highest numbers of amplicons were observed at SCoT10 (39), while the lowest number of amplicons was produced by SCoT09 (07). The percentage of polymorphism ranged from 18.18% to 28.57%, with an average of 21.12%. Polymorphic information content (PIC) values ranged from 0.197 to 0.345. Analysis of Molecular Variation (AMOVA) showed 12% and 88% between the genotypes and within the genotypes respectively. The constructions of 4 clusters were identified through Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram tree based on the genetic distance deduced from SCoT marker analysis. Analysis of the genetic relatedness between parent and mutants through Principal Coordinate Analysis (PCoA) revealed two main groups. The present study concludes that the genetic variability induced by gamma irradiation and inherited in the next generations. This research investigation supports that gamma irradiation alters the growth and yield traits, which is helpful for generating the cowpea improvement.

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata.

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M1 progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

Induced Genetic Variability through Physical and Chemical Mutagens in M2 Generation of Greengram

Uniform and healthy seeds of green gram cv. Pratap (SG-1) were treated with two mutagens, gamma rays (physical mutagen) and EMS (chemical mutagen) alone and in combination. The two hundred forty M1 progenies were laid in randomized block design with three replications during Kharif, 2017 to raise M2 generation. Analysis of variance discloses significant differences among the treatments for all the eight characters studied. It was observed that in general, the combination dose, i.e., 200 Gy+0.2% EMS gave superior results in almost all the yield attributing characteristics. High heritability coupled with high genetic advance was recorded for seed yield per plant, which indicates the predominance of additive gene action. Number of pods per plant followed by pod length showed high significant positive correlation with seed yield per plant. The character number of pods per cluster had shown positive correlation with seed yield per plant, but path analysis revealed its negative direct effect on seed yield.

Efficacy of gamma rays, ethyl methane sulphonate and their combination treatment for resistance/tolerance against Fusarium oxysporum f. sp. ciceri

Chickpea (Cicer arietinum L.) is the third most important pulse crop grown throughout the world. About 65 per cent of the global area and 68 per cent of global production of chickpea is contributed by India. Fusarium oxysporum f. sp. ciceri is a soil-borne as well as seed-borne fungus. During the past few decades, modern techniques such as mutation breeding by radiation and chemical mutagens have been tried to identification/creation of novel genes that impart disease resistance/tolerance in cultivars of many crop plants. Present investigation entitled “Radiation induced mutation for resistance against Fusarium oxysporum f. sp. ciceri (Cicer arietinum L.)” was aimed at identification of suitable mutant or a combination of mutants influencing resistance/tolerance to Fusarium wilt in chickpea. The experimental material was consisted of the population of four selected cultivars of chickpea (JG 62, BDNG 798, JAKI 9218 and Vijay) grown in randomized block design at College of Agriculture Golegaon, VNMKV, Parbhani during Rabi 2018-19. Among 11Mutagenic treatments seven treatments (20 KR, 30 KR, 0.2% EMS, 0.3% EMS, 20 KR+0.2% EMS, 30 KR+0.2% EMS) showed significant impact on various morphological and biological parameters of the plant habit. Efficacy of Gamma rays, EMS and their combination treatment screened for resistance/tolerance in M2 progeny mutant chickpea against two isolates of Fusarium oxysporum f. sp. ciceri by using root dip inoculation technique. Present finding revealed that combination treatment of 30 KR+0.2% EMS showed resistance/tolerance against both the isolates of Fusarium oxysporum f. sp. ciceri in the three experimental genotypes i.e., BDNG 798, JAKI 9218 and Vijay whereas, 0.3% EMS shows resistance/tolerance against both the isolate of Fusarium oxysporum f. sp. ciceri for Vijay genotype.

Transgenerational activation of an autonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

Effect of Gamma-Rays on M1, M2 and M3 Progenies for an Egyptian Cotton Cross

Effect of Gamma-Rays on M1, M2 and M3 Progenies for an Egyptian Cotton Cross

Medicago lupulina lines with defects in the development of efficient arbuscular mycorrhiza

Background. The work is aimed to solve actual problems in biology of arbuscular mycorrhiza (AM). Currently, a lot of mutants had been obtained in various plant model objects with defects in genes controlling AM development, however, the mechanisms controlling development of effective AM symbiosis are still unclear.
 Materials and methods. The authors conducted a mutagenesis in Medicago lupulina, a new convenient model plant for molecular-genetic studies. High mycotrophic M. lupulina line have early and high response to mycorrhization, high seed production, as well as signs of dwarfism under conditions without of AM and low level of phosphorus available for plants. This method allows visually to identify plant lines with defects in AM symbiosis.
 Results. 14 modes for mutagenesis by ethylmethanesulfonate were conducted. Usage of 3 mutagenesis modes allowed to obtain: productive M1 progeny with high part of viable seedlings (73.3%86.0%); 1405 plants in M2 progeny.
 Conclusion. According to population analysis for mutant plants in M2 progeny (up to M9 generation) 15 plant lines were selected: one Myc plant line unable to form AM symbiosis, 4 Pen plant lines unable to form AM symbiosis, but characterized by appressoria formation; 3 Rmd plant lines forming low-activity ineffective AM symbiosis; 3 Rmd plant lines forming low-activity effective AM and 4 Rmd++ plant lines forming effective AM with high abundance of symbiotic structures (mycelium/arbuscules/vesicles) in the roots.

HorTILLUS-A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.).

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.

Identification of High Herbage and Oil Yielding Variety (Jor Lab P-1) of Pogostemon cablin (Blanco) Benth Through Mutation Breeding

Pogostemon cablin (Blanco) Benth, also known as patchouli is an aromatic herb mainly used for high quality perfumery, aromatherapy and fragrances. The study was conducted to identify high yielding mutant genotype of patchouli through gamma radiation. In this experiment 14 different doses of gamma adiation were given to rooted slips of patchouli variety Johor. All 482 M2 progenies were produced and their morphological, qualitative and quantitative oil data were recorded. Advanced progenies were developed and finally selected a single plant from each M4 progenies on the basis of high essential oil and high herbage yield. These 14 lines were planted in RCBD design along with the check variety with three replications. Significant differences were observed in most of the agronomic characters and oil quality parameters among all mutant progenies. Among all the mutant lines, line no. 9 was found to be superior with plant height, herbage yield per plant, essential oil percent and monopodia length in comparison with the check variety. The multi-location trials revealed that the average fresh herbage yield (960 g/plant), oil content (3.10 %), and patchouli alcohol content (37.94 %) were also found to be significantly higher when compared to the check variety.

STUDY OF EFFECT OF EMS, MMS AND GR ON YIELD CONTRIBUTING TRAITS IN URDBEAN, VIGNA MUNGO

The study of the effect of three well known mutagens, ethyl methane sulphonate (EMS), methyl methane sulphonate (MMS) and gamma rays (GR) on the yield contributing traits of the urdbean variety TPU-4 were carried out in the M3 generation. Effect of selected mutagenic treatments/doses of EMS (0.02, 0.03 and 0.04 M), MMS (0.0025, 0.05 and 0.01 M) and (GR) (30, 40 and 50 KR) on different yield contributing traits like plant height, plant spread, number of pods per plant, pod length, number of seeds per pod, seed yield per plant and 100 seed weight were analyzed in the M3 populations of the variety TPU-4. Seeds of M2 plants and control were harvested separately and sown to raise M3 population.. Genetic variabilty in the mutagen administered M3 progeny of the urdbean variety TPU-4 was analyzed by employing statistical methods. Data on mean values and shift in the mean of seven quantitative traits was evaluated on individual plant basis. The experimental findings revealed that concentrations / dose of the all these mutagens showed inhibitory effect on plant height, number of pods per plant, pod length and number of seeds per pod. Lower concentrations of mutagens exerted a promotory effect on plant spread, 100 seed weight and seed yield per plant while higher concentrations of these mutagens inhibited them to different extent.

The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity.

Stable inheritance of excellent agricultural traits induced by <sup>12</sup>C<sup>6+</sup> heavy-ions in lentil (Lens culinaris Medik.)

The lentil (Lens culinaris Medik.) belongs to the most important leguminous plants in Asia. It is a very popular and highly nutritious food. However, small pod size and low yield limit its agricultural use. Through irradiation mutagenesis of dry lentil seeds by 12C6+ ion beam, we found some lines with excellent agricultural traits in M2 progeny at doses of 90 Gy (grey), such as height increase and improvement of the number of branches and three-seed pods as well as seeds with increased grain yield. Six generations of irradiated lentils were screened for these traits. In the M8 generation, the grain yield of three high-yielding lentil lines (lines hyl-1, hyl-2, and hyl-3) reached 212.4%, 195.3% and 190.8%, respectively, of the non-irradiated controls. The results indicated that crop improvement was stable inherited across the generations. Statistical analysis revealed that the increase in grain yield was due to increased total pod number and seeds per pod. Fortunately, adverse effects of irradiation (e.g. reduced germination rates and poor pollen vitality) disappeared over the eight generations. In conclusion, we present a practical method for the improvement of lentils through radiation breeding, leading to high yielding cultivars, which could support the use of this crop in agriculture.

Random amplified polymorphic DNA analysis of salt-tolerant tobacco mutants generated by gamma radiation.

Salinity is one of the major problems limiting the yield of agricultural products. Radiation mutagenesis is used to improve salt-tolerant mutant plants. In this study, we aimed to improve salt-tolerant mutants of two oriental tobacco varieties. One thousand seeds of each variety (M₀) were irradiated with 100, 200, 300, and 400 Gy gamma rays by Cs-137 gamma. In the M₁ generation, 2999 single plants were harvested. The next season, these seeds were bulked and planted to obtain M₂ progeny. The seeds of 1900 M₂ plants were picked separately. Salinity tolerance was tested in the M₃ generation. Among M₃ plantlets, 10 salt-tolerant tobacco mutants were selected. According to the results of the selection studies, 100- and 200-Gy gamma radiation doses were the effective doses to obtain the desired mutants. Glutathione reductase enzyme activities of salt-tolerant tobacco mutants were determined biochemically as a stress-tolerance marker. The differences between control and salt-tolerant mutants belonging to the Akhisar 97 and İzmir Özbaş tobacco varieties were evaluated by random amplified polymorphic DNA analysis. The total polymorphism rate was 73.91%.