Mutations In Plant Breeding Research Articles

Induction of flower-colour mutation by synchrotron-light irradiation in spray chrysanthemum

Chrysanthemum is one of the most important ornamental plants. Mutation breeding is an important tool in breeding of chrysanthemum, and many cultivars have been produced through mutation breeding by using radiation such as gamma ray, etc. Although ion-beam irradiation is now widely used as a mutagen in the mutation breeding of plants, synchrotron light, another form of radiation, has not been used as a mutagen. Therefore, this study was conducted to clarify whether synchrotron light can be used as a new mutagen in chrysanthemum breeding. Five cultivars of spray chrysanthemum were subjected to synchrotron-light irradiation. As a result of the irradiation, the survival rate decreased as the absorbed dose increased. Flower-colour mutations were observed in three cultivars. There were varietal differences in survival rates and the frequencies of flower-colour mutation. Flowers of the mutants showed various colour patterns such as a deeper and paler colour than the original flowers. In the mutant of a cultivar with a marginal picotee, the proportions of picotee changed. A moderate absorbed dose of synchrotron-light irradiation for flower-colour mutation was found to be 11 to 23 Gy. These results indicate that synchrotron light can be used as a new mutagen in the mutation breeding of chrysanthemum.

Effect of proton beam irradiation on survival and seedling growth parameters of Indian rice (Oryza sativa L.) variety ‘Indira Barani Dhan 1’

Rice crop, a staple food for a majority of population across the world, holds a significant role to play in alleviating the global hunger problem. Enhancement of genetic diversity of rice will serve to overcome constraints in sustained and ecologically effective improvement in the production of quality rice, challenged by erratic patterns of global climate, changing pest dynamics, resource availability in proportion to growing population etc. Proton ion beam has sprung up as a promising novel mutagen in the mutation breeding of crop plants, by virtue of its higher LET values, causing higher frequency of mutations. Dose optimization is pre-requisite to obtain a range of phenotypic mutants, without drastic reduction in survival and growth subsequently. This study was undertaken to standardize proton beam irradiation dose rate post the evaluation of in-vitro germination, growth and survival of rice variety IBD-1, with doses ranging from 0–500Gy. Doses between 152.47Gy253.53Gy were found suitable for mutagenesis, without drastically impairing growth and survival.

Identification of Substitutions and Small Insertion-Deletions Induced by Carbon-Ion Beam Irradiation in Arabidopsis thaliana.

Heavy-ion beam irradiation is one of the principal methods used to create mutants in plants. Research on mutagenic effects and molecular mechanisms of radiation is an important subject that is multi-disciplinary. Here, we re-sequenced 11 mutagenesis progeny (M3) Arabidopsis thaliana lines derived from carbon-ion beam (CIB) irradiation, and subsequently focused on substitutions and small insertion-deletion (INDELs). We found that CIB induced more substitutions (320) than INDELs (124). Meanwhile, the single base INDELs were more prevalent than those in large size (≥2 bp). In details, the detected substitutions showed an obvious bias of C > T transitions, by activating the formation of covalent linkages between neighboring pyrimidine residues in the DNA sequence. An A and T bias was observed among the single base INDELs, in which most of these were induced by replication slippage at either the homopolymer or polynucleotide repeat regions. The mutation rate of 200-Gy CIB irradiation was estimated as 3.37 × 10−7 per site. Different from previous researches which mainly focused on the phenotype, chromosome aberration, genetic polymorphism, or sequencing analysis of specific genes only, our study revealed genome-wide molecular profile and rate of mutations induced by CIB irradiation. We hope our data could provide valuable clues for explaining the potential mechanism of plant mutation breeding by CIB irradiation.

Efficiency of energy conversion and growth of gamma irradiated embryos and young seedlings of Triticum monococcum L. cultivar Einkorn

The study was conducted to determine whether the efficiency of energy conversion into growth can be used as an indicator for the determination of the optimal gamma irradiation dosage for mutation breeding. To meet this objective, embryo growth, shoot growth, root growth, mobilization of food reserves, respiration and energy conversion were studied in gamma-irradiated wheat Triticum monococcum L. cultivar Einkorn kernels. Kernels were exposed to 50, 150, 250 and 350 Gy and germinated. Kernels were collected 12 h after onset of imbibition and then every 12 h until 168 h. Irradiated seed demonstrated retardation in all parameters, which increased as the gamma irradiation dosage increased. For the most, dosage and time, as well as dosage by time interaction were highly significant. Root growth appeared to be the most sensitive to gamma irradiation, followed by shoot growth, mobilization of food reserves and efficiency of energy conversion. Full recovery of the efficiency of energy conversion took place at 50 Gy, with an increase in inefficiency with an increase in dosage. The point where full recovery of efficiency of energy conversion into growth gives way to incomplete recovery (100 Gy) is in line with the suggested dosages for practical mutation breeding in Triticum monococcum L. by the FAO/IAEA and is therefore an ideal indicator for predicting the dosage that will be optimal for plant mutation breeding.

Flow cytometry determination of ploidy level in winged bean [<i>Psophocarpus tetragonolobus</i> (L.) DC] and its response to colchicine-induced mutagenesis

Ploidy determination and mutation breeding of crop plants are inseparable twins given that mutation breeding is hinged majorly on polyploidization of crop’s chromosome number. The present research was aimed at determining the ploidy level of 20 accessions of winged bean (Psophoscarpus tetragonolobus) using known diploid standard as control and also subjecting two accessions to colchicine mutagenesis. For flow cytometric analysis, approximately 50mm2 of young fresh intact leaves of 8 weeks post-seedling emergence from the 20 accessions were used. Crude isolation of nuclei was done by chopping the leaf in 1ml of ice cold buffer from DNA kit (cysteine UV Partec GmbH Germany) using a razor blade. The crude suspension was then filtered through a 30μm pore diameter nylon mesh and stained with 4,6- diaminodino-2-phenylindole (DAPI). Fluorescence intensity of isolated nuclei was measured using a partec PAII flow cytometry equipped with an argon ion laser at 488nm.For mutation induction, 20 seeds from accessionsTPt26 and TPt154 were soaked in three different concentrations of colchicine (5,10, 15mg/l) for 24, 48, and 72 hours, while the control group was soaked in water. A plot of land measuring 10X10 meters in the University of Calabar Experimental farm was used. Four beds were made and three seeds were sown in per hole using a Randomized Complete Block Design (RCBD) in a 2X3X4 factorial experiment layout with 5 replications. At the end of the field experiment, phenological, yield and yield-related traits were collected and subjected to analysis of variance using Predictive Analytical Software (PASW) version 20. Results obtained showed that there were significant effect of colchicine on phenological traits except on germination. However, there were significant increasing effects on yield and yield-related traits, which was colchicine concentration, exposure time as well as accession-specific. Exposing winged bean seeds to 15mg/l of colchicine for 24 and 48 hour duration gave encouraging results and should be considered for further tests and exploitation.Keywords: Ploidy determination, flow cytometry, mutation breeding, colchicine, Psophoscarpus tetragonolobus

TILLING, high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.

Improvement of Ornamental Plants - A Review

Ornamental crops provide better income from a unit area with higher profitability. There is huge untapped flower production potential in our country which could benefit a large segments of the weaker sections of the society. Both the domestic market and the export potential of flowers and ornamentals are tremendous. Besides, earning foreign exchange and improving the national income, the floriculture business being labour intensive generates gainful employment to rural youth. Ornamental plants are appreciated by their ability to please the eye of consumers as garden or pot plants or when sold as cut material. The main emphasis in ornamental plant breeding is to improve variety traits, novel colour, form, size, number of flowers, flower vase life, repeat blooming, disease resistance, nutrient uptake capacity and growth habit. Various types of new varieties of ornamental crops have been produced for many years by cross hybridization and mutation breeding techniques, separately or in combination. Many ornamental plants have been originated from inter-specific and inter-generic crosses, which leads to high degree of heterozygosity in the resulting hybrids, often polyploidy and aneuploidy also occur. In the present review, origin, modes of reproduction, introduction, selection, hybridization, mutation and polyploidy breeding of various ornamental plants are discussed in details.

Effects of ion beam irradiation on size of mutant sector and genetic damage in Arabidopsis

Size of mutant sector and genetic damage were evaluated in Arabidopsis to further our understanding of effective ion beam use in plant mutation breeding. Arabidopsis seeds, heterozygous for the GLABRA1 (GL1) gene (GL1/gl1-1), were irradiated with 15.8MeV/u neon ions (mean linear energy transfer (LET): 352keV/μm), 17.3MeV/u carbon ions (113keV/μm), or 60Co gamma rays. The frequency and size of glabrous sectors generated because of inactivation of the GL1 allele were examined. The frequency and overall size of large deletions were evaluated based on the loss of heterozygosity of DNA markers using DNA isolated from glabrous tissue. Irrespective of the radiation properties, plants with mutant sectors were obtained at similar frequencies at the same effective dosage necessary for survival reduction. Ion beams tended to induce larger mutant sectors than gamma rays. The frequency of large deletions (>several kbp) increased as the LET value increased, with chromosome regions larger than 100kbp lost in most large deletions. The distorted segregation ratio of glabrous plants in the progenies of irradiated GL1/gl1-1 plants suggested frequent occurrence of chromosome rearrangement, especially those subjected to neon ions. Exposure to ion beams with moderate LET values (30–110keV/μm) is thought effective for inducing mutant sectors without causing extensive genetic damage.

Plant Mutation Breeding with Heavy Ion Irradiation at IMP

<p>The Heavy Ion Research Facility in Lanzhou (HIRFL) is one of the ion-beam acceleration facilities intensively used at IMP, founded as national laboratory and opened for user in world from 1992. Since then, a lot of experiments irradiated by heavy ion beam have been carried out in the HIRFL, including plant mutation breeding. In this review, the biological effects induced by heavy ions and their corresponding mechanisms were reported from the point of view of cytological, morphological and molecular levels. To date, a large number of mutants were isolated using heavy ion irradiation IMP, such as early maturity, flower color and shape, high yield and disease resistant. In conclusion, heavy ion beam irradiation is an efficient mutagen and has significant phenotypic variations in plant. Our research will be further focused on transformation of scientific and technological achievements and mutagenic mechanism of heavy ion beam on high plant at the molecular level in the recent future.</p>

Damage-repair model for mutagenic effects of plant induced by ionizing radiation

The plant mutation effects induced by ionizing radiation involve a rather complex process which is composed of physical, chemical, biochemical and biological stages. Nowadays, although ionizing radiation has been widely used in plant mutation breeding, the theoretical explanations for the mechanism of the ionizing radiation caused plant mutation effects are insufficient. Especially, a saddle shape relationship between the plant survival rate and radiation dose is found in the mutagenis effect of ionizing radiation on plants. The underlying mechanism of the saddle shape relationship remains unclear and challenges to all extant models.To explain this relationship, a damage-repair model for the plant mutation effects induced by ionizing radiation is proposed in the present work. Our model is based on the rate theory of ionizing radiation in which the cell damage and repair are taken into account simultaneously together with the micro-and macro-biological mutation effects of plant caused by ionizing radiation. The states of the radiated plant individuals are grouped into three categories: normal, damaged and lethal categories in our model. The evolution dynamics of the relative concentrations of the three categories are determined by a set of coupled equations which are mathematically the same as the Crow-Kimura equations in species evolution theories. With the numerical solution of our model in its steady state, the relative steady state concentration distributions of different categories of the radiated plants with increasing radiation dose are obtained. It is shown that without the plant repair effect, the relationship between the plant survival rate and radiation dose appears to be a conventional shoulder type one. With the plant repair effect, our model gives a saddle shape survival-dose relationship which has been observed commonly in the experiments on the radiated plants by ionizing radiation. To further test the model, the experimental data on the inbred lines of maizes radiated by heavy ion 7Li are used to determine the parameters of the model. It is shown that the theoretical results are basically consistent with the experimental ones. In addition, the mutation characteristic of the survival plants also appears to be a saddle effect-dose relationship, for which the theoretical model could also give a reasonable explanation. Our damage-repair model explains the saddle shape relationship between the plant survival rate and radiation dose, which indeed illuminates its power. And it provides a theoretical basis and reference for studying the biological effect mechanism of plants induced by ionizing radiation and conducting ionizing radiation plant breeding.

Morphological and biochemical responses of Oryza sativa L. (cultivar MR219) to ion beam irradiation

Heavy ion beam, which has emerged as a new mutagen in the mutation breeding of crops and ornamental plants, is expected to result in the induction of novel mutations. This study investigates the morphological and biochemical responses of Oryza sativa toward different doses of carbon ion beam irradiation. In this study, the dry seeds of O. sativa were irradiated at 0, 20, 40, 60, 80, 100, and 120 Gy, followed by in-vitro germination under controlled conditions. Morphological and biochemical studies were conducted to investigate the morphological and physiological responses of O. sativa towards ion beam irradiation. The study demonstrated that low doses (10 Gy) of ion beam have a stimulating effect on the height, root length, and fresh weight of the plantlets but not on the number of leaves. Meanwhile, doses higher than 10 Gy caused reductions in all the morphological parameters studied as compared to the control samples. The highest total soluble protein content [(2.11 ± 0.47) mg/g FW] was observed in plantlets irradiated at 20 Gy. All irradiated plantlets were found to have 0.85% to 58.32% higher specific activity of peroxidase as compared to the control samples. The present study also revealed that low doses of ion beam (10 and 20 Gy) had negligible effect on the total chlorophyll content of O. sativa plantlets while 40 Gy had a stimulating effect on the chlorophyll content. Plantlets irradiated between 40 to 120 Gy were shown to be 0.38% to 9.98% higher in total soluble nitrogen content which, however, was not significantly different from the control samples. Carbon ion beam irradiation administered at low to moderate doses of 10 to 40 Gy may induce O. sativa mutants with superior characteristics.

Exclusive induction of G:C to A:T transitions by 3-azido-1,2-propanediol in yeast

Sodium azide is a strong mutagen which has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolized to azidoalanine, but further bioactivation to a putative ultimate mutagen as well as the nature of the induced DNA modifications leading to mutations remain elusive. In this study, mutations induced in the CAN1 gene of yeast Saccharomyces cerevisiae by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) have been sequenced. Analysis of the forward mutation spectrum to canavanine resistance revealed that AZG induced nearly exclusively G:C to A:T transitions. AZG also induced reversions to tryptophan prototrophy by base-pair substitutions in a dose-dependent manner. This unusual mutational specificity may be shared by other organic azido compounds.

Characteristics of Ion Beams as Mutagens for Mutation Breeding in Rice and Chrysanthemums

Mutation breeding is a useful method to improve crops. More than 20 years have passed since mutation breeding with ion beams started in Japan, since which time many mutant varieties have been produced with ion beams. However, ion beams have not been sufficiently characterized in terms of mutagens for plant mutation breeding. This review introduces the characteristics of ion beams as mutagens for mutation breeding; investigated with three objectives: to obtain useful mutants with limited plant damage by irradiation treatment, the width of the mutated sector produced by irradiation, and the mutation spectrum, compared to gamma rays using rice and chrysanthemums. In addition, the optimum dose of ion beams and the criteria for an optimum irradiation dose are shown, based on which the irradiation treatment for the success of mutation breeding with ion beams is also described.

Radio-Sensitivity of Some Groundnut (Arachis hypogaea L.) Genotypes to Gamma Irradiation: Indices for Use as Improvement

Mutation breeding in crop plants is an effective approach in improvement of crop having narrow genetic base such as groundnuts (Arachis hypogaea L.). Determination of effective irradiation dose is prerequisite for mutation breeding and development of genetic variability by induced mutation. Three groundnuts (Arachis hypogaea L.) genotypes (JL12, JL24 and Kimpese) were irradiated to the absorbed doses of 100, 200, 400 and 600 Gy for effective dose determination and to compare their sensitivity to different doses of gamma irradiation in a completely random design. It was found that, irradiation reduced significantly germination and survival percentages of seedlings in higher doses and this reduction was more pronounced in JL 24 cultivar. This sensitivity expresses himself distinctly to the rank of stem lengths and roots. Results show a

Targeted mutation breeding of horticultural plants

Mutation breeding can be enhanced by genetic selection for novel alleles. Through targeted mutation breeding, genotypes with induced or natural mutations in candidate genes are identified for cultivar development. For most horticultural plants, targeted mutation breeding may be a more economically feasible approach to trait development than through transgenic technology. Substantial progress has been made in applying targeted mutation breeding to horticulture and this review summarizes recently published work in this area. To date, at least 16 horticultural crops have been screened for natural or induced allelic diversity in over 100 candidate genes. This approach has resulted in traits of commercial use, such as longer shelf-life (tomato, melon), improved starch quality (potato), and virus-resistance (peppers, tomato). Advances in genome sequencing and genetic screening will facilitate the development of cultivars with value-added traits derived from candidate gene polymorphisms.

Effect of Gamma Irradiation on Morpho-Agronomic Characteristics of Soybeans (<i>Glycine max</i> L.)

Mutation breeding in crop plants is an effective approach in improvement of crop having narrow genetic base such as soybean. The main objective of the present study is to determine the effect of different doses of gamma irradiation on different morpho-agronomic characteristics. Agronomic traits that were analyzed included; grain yield, number of pods/plant, number of seeds/plant and weight of 100 seeds and numbers of days to 50% flowering. Morphometric characterization of the descriptive data included plant height, stem diameter, number of leaves/plant, leaflet length, leaflet width, number of ramifications/plant, and pod length and width at 3 lodge stage. The results of the present study revealed that the two gamma irradiation doses used (0.2 kGy and 0.4 kGy) decreased significantly most of agronomic and morphological traits evaluated in M1 populations. Different effects of 0.2 kGy and 0.4 kGy irradiation were observed in M2 populations with significant increase of grain yields and yield components in all the three soybean varieties. In general, a significant decrease or no changes of morphological traits were observed for the two irradiation doses in M2 populations. The levels of changes varied among varieties. Potential high yielding mutants were identified in progenies of irradiated seeds.

Development of an efficient mutagenesis technique using ion beams: Toward more controlled mutation breeding

Ion beams have been used as a mutagen to improve the eciency of plant mutation breeding. Mutation b reeding is sometimes perceived as a random process. In this review, we describe our recent progress in developing a more ecient mutagenesis technique using ion beam irradiation combined with sucrose pretreatment or subsequent re- irradiation. To shorten the time required for breeding new cultivars of cyclamen, we identied anthocyanin biosynthesis g enes and examined the eectiveness of PCR screening of irradiated deletion-mutant candidates at early growth stages. We believe this research is a step toward more ecient and controlled mutation breeding using ion beams.

Plant mutation breeding in agriculture.

Abstract A mutation is a sudden heritable change in the DNA in a living cell, not caused by genetic segregation or genetic recombination. Mutation breeding is the purposeful application of mutations in plant breeding. Unlike hybridization and selection, mutation breeding has the advantage of improving a defect in an otherwise elite cultivar, without losing its agronomic and quality characteristics. Mutation breeding is the only straightforward alternative for improving seedless crops. Since the first release of mutant cultivars that resulted from basic mutation research in Europe, mutation breeding has found a niche in plant breeding because of these advantages. Methodologies for mutation induction have been improved in main crops for both physical and chemical mutagens, and selection methodologies for mutant populations have been described. New mutagenic agents such as ion-beam radiation and cosmic rays are being investigated and a hitherto undescribed wide spectrum of mutations has been observed. Nevertheless, alkylating agents and ionizing radiation are still popular. The development of robust in vitro techniques for many crop species has contributed to improving the efficiency of mutation breeding. The ability to handle large mutagenized populations in a confined space, faster progeny turnover in vegetatively propagated species and the ability to screen for several biotic and abiotic stress factors in the culture environment make in vitro approaches very efficient. Mutant screening has undergone revolutionary changes in the past decade with reverse genetic approaches taking precedence. Therefore, integration of mutation techniques with molecular approaches is providing exciting opportunities for modern plant breeding.

Theoretical studies on population development design in mutation breeding of allogamous plants

The “Crossing-within-Spike-Progeny (CSP) method” is a method to efficiently screen for mutants in allogamous (cross-fertilizing) plants. This paper presents a theoretical analysis of how to minimize the total number of plants in the screening generation (T) and the total cost (Tc) required to detect one or more mutants with a given probability when using the CSP method. Tc depends on the number of plants per line in the screening generation (n), the number of plants per hill plot in the M2 generation (h), the ratio of the cost needed in non-screening generations to that in the screening generation (W), and the mutation rate per cell (p1). T was lowest when n = 1 (T = 47.93 × p1−1), irrespectively of h. On the other hand, the value of n which gave minimum Tc (designated n*) was not constant, but varied with h. n* was 6 when h = 2, increased as h increased, and was 15 when h = 48, when W = 10. Unlike in the case of autogamous species, the presence of chimeras in the M1 inflorescences greatly increased both T and Tc. A method for estimating the mutation rate per cell is also presented.

Studies on Biological Effects of Ion Beams on Lethality, Molecular Nature of Mutation, Mutation Rate, and Spectrum of Mutation Phenotype for Mutation Breeding in Higher Plants

Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.