Pod Mutant Research Articles

Mutagenic effectiveness and efficiency of gamma rays and combinations with EMS in the induction of macro mutations in blackgram (Vigna mungo (L.) Hepper)

Blackgram, an important pulse crop of India and other Asian countries, is a rich source of protein. Genetic improvement in this crop is generally slow-paced due to the lack of wide genetic variability available for selection. To create variability for important morphological traits and yield, induced mutagenesis was attempted in a popular blackgram cultivar CO 6 using gamma rays (γ rays-60Co) and a combination of ethyl methanesulphonate (EMS) and gamma rays. In M1 generation, the mutagenic treatments of 200 Gy γ rays and 200 Gy + 20 mM EMS showed a high percentage of viable and chlorophyll mutants. However, in M2 generation, 400 Gy + 20 mM EMS generated a higher proportion of viable mutations. A broad spectrum of viable macro mutants namely variation in growth habit, duration, size of leaf and pod, nature of pod and seed, seed yield and protruded stigma mutant were observed. The combination treatments exhibited a maximum frequency of viable macro mutations. Among viable macro mutations, the growth habit mutants recorded maximum frequency, followed by leaf mutants and pod mutants. The protruded stigma mutant was isolated at the higher dose of 400 Gy of γ rays. Studies on palynological traits among fertile and sterile plants using Scanning Electron Microscopy (SEM) revealed variations in pollen and anther length and shape among the mutants. The pollen lumina and muri shape in the fertile plant were observed as polygonal and narrow straight, whereas it was collapsed in the sterile plant. The inheritance pattern for most of the viable macro mutants was true-breeding which was further confirmed in the M3 generation. These viable mutants could be used for the blackgram improvement program.

Transcriptome analysis of pod mutant reveals plant hormones are important regulators in controlling pod size in peanut (Arachis hypogaea L.).

Pod size is an important yield-influencing trait in peanuts. It is affected by plant hormones and identifying the genes related to these hormones may contribute to pod-related trait improvements in peanut breeding programs. However, there is limited information on the molecular mechanisms of plant hormones that regulate pod size in peanuts. We identified a mutant with an extremely small pod (spm) from Yuanza 9102 (WT) by 60Co γ-radiation mutagenesis. The length and width of the natural mature pod in spm were only 71.34% and 73.36% of those in WT, respectively. We performed comparative analyses for morphological characteristics, anatomy, physiology, and global transcriptome between spm and WT pods. Samples were collected at 10, 20, and 30 days after peg elongation into the soil, representing stages S1, S2, and S3, respectively. The differences in pod size between WT and spm were seen at stage S1 and became even more striking at stages S2 and S3. The cell sizes of the pods were significantly smaller in spm than in WT at stages S1, S2, and S3. These results suggested that reduced cell size may be one of the important contributors for the small pod in spm. The contents of indole-3-acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) were also significantly lower in spm pods than those in WT pods at all three stages. RNA-Seq analyses showed that 1,373, 8,053, and 3,358 differently expressed genes (DEGs) were identified at stages S1, S2, and S3, respectively. Functional analyses revealed that a set of DEGs was related to plant hormone biosynthesis, plant hormone signal transduction pathway, and cell wall biosynthesis and metabolism. Furthermore, several hub genes associated with plant hormone biosynthesis and signal transduction pathways were identified through weighted gene co-expression network analysis. Our results revealed that IAA, GA, and BR may be important regulators in controlling pod size by regulating cell size in peanuts. This study provides helpful information for the understanding of the complex mechanisms of plant hormones in controlling pod size by regulating the cell size in peanuts and will facilitate the improvement of peanut breeding.

Enhanced Chlorophyll Degradation Triggers the Pod Degreening of "Golden Hook," a Special Ecotype in Common Bean (Phaseolus vulgaris L.).

To reveal genetic factors or pathways involved in the pod degreening, we performed transcriptome and metabolome analyses using a yellow pod cultivar of the common bean “golden hook” ecotype and its green pod mutants yielded via gamma radiation. Transcriptional profiling showed that expression levels of red chlorophyll catabolite reductase (RCCR, Phvul.008G280300) involved in chlorophyll degradation was strongly enhanced at an early stage (2 cm long) in wild type but not in green pod mutants. The expression levels of genes involved in cellulose synthesis was inhibited by the pod degreening. Metabolomic profiling showed that the content of most flavonoid, flavones, and isoflavonoid was decreased during pod development, but the content of afzelechin, taxifolin, dihydrokaempferol, and cyanidin 3-O-rutinoside was remarkably increased in both wild type and green pod mutant. This study revealed that the pod degreening of the golden hook resulting from chlorophyll degradation could trigger changes in cellulose and flavonoids biosynthesis pathway, offering this cultivar a special color appearance and good flavor.

Characterization of 60CO γ-ray induced pod trait of blackgram-A promising yield mutants

Purpose: Blackgram (Vigna mungo (L.) Hepper) possesses natural flower dropping character with self-pollination, which was highly challenging to create variability through hybridization. Gamma irradiation is a powerful tool to induce genetic alteration and improvement in crops with beneficial mutants. Hence, the present investigation focused on finding out the mutant characters related to the improvement of the agronomic characters and yield-related traits by using gamma irradiation. Pod mutant isolated with fruitful agronomic trait at M2 (Mutagenic) generation was identified and isolated by gamma irradiation is a central focus in the present study, which enhances the yield of the blackgram.Materials and methods: Viable and uniform size of blackgram Vamban-4 seeds were irradiated at a different dose of 60CO (Cobalt) γ-rays viz., 200, 400, 600, 800, 1000, and 1200 Gray (Gy). The further generations were screened and raised to M3 and M4 plants to select the stable mutants with yield traits.Results: In the M2 generation, the following viable and pod mutants were identified, such as tall, dwarf, multipod, small pod, bold pod, striata pod. Morphological traits, such as plant height (PH), number of branches per plant (NBP), number of leaves per plant (NLP), number of cluster per plant (NCP), number of pods per plant (NPP), number of seeds per pod (NSP), pod length (PL), a hundred seed weight (HSW), seed yield per plant (SYP), were measured. Among the mutants, the increased morphogenetic traits were recorded in tall mutants (600 Gy), and the enhancement of yield characteristics noticed in multipod (800 Gy) and bold pod mutants (600 Gy). Compared to other doses, 600 Gy shows enhanced quantitative traits for blackgram improvement.Conclusion: The 60CO γ-rays induced genetic variation in the yield and its related traits and obtained pod mutants, which are useful to enhance the commercial value of Vigna species and crop improvement.

A recessive high-density pod mutant resource of Brassica napus

In Brassica napus, pod number and pod density are critical factors to determine seed yield. Although the pod density is an essential yield trait, the regulation of yield formation in oil crops, as well as the genetic and molecular mechanisms, are poorly understood. In this study, we characterized a rapeseed high-density pod mutant (dpt247) from composite hybridization. To shed some light on the nature of this mutation, it was investigated morphologically, anatomically, physiologically, genetically and transcriptomically. The mutant plant showed noticeable phenotypic differences in comparison with the control plant, including reduced plant height and primary branch length, decreased number of primary branches, significantly increased number of pod on the main inflorescence, and more compact pod distribution. Besides, the mutant had higher levels of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in the shoot apical meristem (SAM). The dense pod trait was controlled by two major recessive genes identified in the segregating genetic populations of GRE501 and dpt247. RNA sequencing indicated genes participated in auxin, cytokinin and WUS/CLV signalling pathway in dpt247 were more active in the mutant. These results provide important information for understanding the regulation of yield formation and high yield breeding in rapeseed.

Molekularna karakterizacija mutanata leblebije izolovanih iz tretmana EMS i gama zracima

Conventional mutation breeding techniques have often been used to improve yield, disease and pest resistance in crop plants. In the present attempt, two well-known mutagenic agents ethyl methane sulphonate and EMS were employed in order to induce genetic variability variety of chick pea Co -4 obtained from Tamil Nadu Agricultural University. The seeds were treated with different concentrations of EMS (10-50 mM) and Gamma rays (20-60 kR). A total of 369 mutants which alter the height, leaf structure, colour of flower, seed, size of pod, duration of the plant were isolated and characterized. Among them, five true breeding mutants namely Early flowering mutant (EFM), Bold pod mutant (BPM), Bold seed mutant BSM), High yielding mutant (HYM) and High protein content mutant (HPC), which directly influenced the economic value of the crop were selected for further study. The results revealed a significant positive increase in the qualitative traits among the mutant lines. The RAPD profiling of isolated mutants revealed a total of 129 bands, among them 92 were polymorphic with an average of 70.99 percentage of polymorphism. Apart from 28 bands, all the bands seemed to be mutation. The presence of new bands and absence of existing bands might be the reason for quality improvement of chickpea. The RAPD is considered as an excellent marker to differentiate mutants from their parents, to assess genetic variation and phylogenetic relationship.

STMS (sequence tagged microsatellite site) molecular markers as a valuable tool to confirm controlled crosses in chickpea (Cicer arietinum L.) breeding programs

Chickpea (Cicer arietinum L.) is an annual, diploid and autogamous grain legume. Most breeding processes developed in this crop start with complementary crosses, but artificial hybridization is a tedious operation probably due to the injury of the pistil caused during emasculation due to the small size of the flowers. The success in pod formation after hybridization together with the possibility of testing the hybrid nature of F1 is a key point to optimize chickpea breeding. Morphological characters are not always adequate to distinguish between progeny, consequently molecular markers are needed to verify hybrid nature. In this study, we show our experience in crossing programs and the use of molecular markers to test hybridization rate in three different cases (A) obtaining advanced lines combining resistance to blight and fusarium wilt; (B) developing segregating populations for Orobanche foetida and (C) providing suitable materials to do genetic studies involving double pod mutants. A total of 2041 pollinated flowers produced 21.7, 16.8 and 8.7% of pods in each one of the three cases analyzed. Hybridization nature in F1 seeds was tested with STMS (sequence tagged microsatellite site) markers and ranged from 86 to 91% for the three cases. STMS markers were a valuable tool to detect hybrids, being simple to visualize and having a low cost. The confirmation of hybrid nature at the initial development stages of F1 plants is critical for reducing time and costs in breeding programs.

Genetic engineering of lignin biosynthesis pathway improved stem bending disorder in cut gerbera (Gerbera jamesonii) flowers

Gerbera (Gerbera jamesonii) is an important commercial species of cut flowers all around the world. Stem bending is a major disorder in cut gerbera flowers caused mainly by the lack of mechanical support in the flower stem. Lignin is the component of plant cell walls that provides strength. Three major enzymes comprise the pathway of lignin anabolism including peroxidase (POD), cinnamyl alcohol dehydrogenase (CAD), and cinnamoyl-CoA reductase (CCR). In this study, three overexpressed mutants for each of these major genes were generated and analyzed for their role in stem bending. Comparing the pod transgenic plants with ccr and cad mutants indicated that pod plants had the highest lignin content. Compared to wild-type (WT) plants, the highest vase life, membrane stability and solution uptake were obtained in gerbera pod mutants. Therefore, it appears that higher lignin contents can decrease stem bending and improve cell wall rigidity of cut gerbera flowers. These results also suggest that POD is a key gene in the biosynthesis of lignin in gerbera.

IN VIVO MULTISTEP MUTAGENESIS INDUCTION USING COLCHICINE ON COWPEA MUTANT 1 (Vigna unguiculata L. Walp)

In the previous research, we obtained three catagories of M1 mutant cowpea namely 1) purple pod mutant cowpea, 2) green pod mutant cowpea which has three pods in a stalk, and 3) green pod mutant cowpea which has two pods in a stalk. All those three catagories of M1 mutant cowpeas were treated again with colchicine in a multistep mutagenesis proses in vivo. Then, we found changes in number of leaflet in a petiole to 4 leaflets (quadrifoliate) and to five leaflets in a petiole (pentafoliate). These changes are indications that beside as a chromosome multipying alkaloid, colchicine causes other genetic changes as well. Through this mutagenesis research, we created gigas mutant cowpea that having longer leaves, longer pods and heavier seeds. The accurrence of two kinds of insect pests attack were found namely cowpea aphid Aphis craccivora and pod sucking bug Riptortus linearis.

Western Ambar and Western Dhamaka: High yielding Spanish bunch groundnut varieties for Maharashtra

In groundnut, a high yield potential variety having desired combination for Spanish bunch plant habit, large pod size, higher oil content, improved oil quality and early/medium crop duration is rare. Western Ambar is a large pod mutant of popular variety TG-37A, and Western Dhamaka is a recombinant selected from a cross between TG-37A and TPG-41. Both the varieties maintained higher productivity in station, multi-locations and adaptive trials compared to check varieties in respective trial during rabi/summer season. Western Ambar and Western Dhamaka recorded overall an average pod yield of 4892 kg/ha and 5269 kg/ha, and exhibited superiority over the best check TG-37A (4610 kg/ha) by 6.1 and 14.3 per cent, respectively. Further, in State Agricultural Universities trials in Maharashtra, Western Ambar and Western Dhamaka recorded average yield of 3028 kg/ha and 3356 kg/ha, and showed superiority over national check TAG-24 (2645 kg/ha) by 14.5 and 26.9 per cent, respectively. Both the varieties were found tolerant to major biotic stress, and have mediumcrop duration, higher oil content and improved oleic/linoleic (O/L) ratio. In consideration of superiority for pod yield and their important component and quality characters; Western Ambar and Western Dhamaka have been accepted by Department of Agriculture, Government of Maharashtra to include in license for their cultivation in Maharashtra.

Effect of Gamma Rays in Induced Morphological Mutants on M<sub>2</sub> Generation of Chilli (<i>Capsicum annu</i><i></i><i>um </i>L<i>.</i>) Var K<sub>1</sub>

The present study was carried out to induce morphological mutation in chilli (Capsicum annuum L.) var k1. The seeds were subjected to different treatment levels of gamma rays are 20, 30, 40, 50, 60 and 70Kr along with control. The treated and untreated seeds were raised in a randomized block design to observe the different morphological characters in M2 generation. Several unique and interesting mutants were isolated in this investigation, these independent mutants have differ from control, the most distinct mutants included tall, dwarf, bushy, flower, sterile and pod mutant, was observed in M2 generation.

Effect of Gamma Rays in Induced Morphological Mutants on M<sub>2</sub> Generation of Chilli (<i>Capsicum annu</i><i></i><i>um </i>L<i>.</i>) Var K<sub>1</sub>

The present study was carried out to induce morphological mutation in chilli (Capsicum annuum L.) var k1. The seeds were subjected to different treatment levels of gamma rays are 20, 30, 40, 50, 60 and 70Kr along with control. The treated and untreated seeds were raised in a randomized block design to observe the different morphological characters in M2 generation. Several unique and interesting mutants were isolated in this investigation, these independent mutants have differ from control, the most distinct mutants included tall, dwarf, bushy, flower, sterile and pod mutant, was observed in M2 generation.

Effect of EMS on Induced Morphological Mutants and Chromosomal Variation in Cowpea (<i>Vigna unguiculata</i> (L.) Walp)

Effect of EMS (ethyl methane sulphonate) on induced morphological mutants and chromosomal variation in cowpea was studied using five different doses of mutagen along with a control in randomized blocked design with three replications. The morphological mutants there are two types of viable and chlorophyll mutants. Viable mutant contains tall, dwarf, early maturity, late maturity, leaf mutants pod mutant and flower mutants. The frequency of chlorophyll mutant contains albino, xantha and viridis. This concentration can damage or modify important components of plant cells and have been reported to affect the morphology, anatomy, biochemistry and physiology of plants differentially depending on the concentration level. These effects include changes in the cellular structure and metabolism of the plants e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds. The morphological and chromosomal variation was found to be mutagen sensitive in somatic cells of cowpea. It was found to increase with increasing the concentration of EMS in Cowpea plants. The chemical mutagen like ethyl methane sulphonate induces high frequency of chromosomal changes like anaphasic bridge; anaphasic laggard, anaphasic bridge and clumbing of chromosome were including control plants also observed.

Effect of EMS on Induced Morphological Mutants and Chromosomal Variation in Cowpea (<i>Vigna unguiculata</i> (L.) Walp)

Effect of EMS (ethyl methane sulphonate) on induced morphological mutants and chromosomal variation in cowpea was studied using five different doses of mutagen along with a control in randomized blocked design with three replications. The morphological mutants there are two types of viable and chlorophyll mutants. Viable mutant contains tall, dwarf, early maturity, late maturity, leaf mutants pod mutant and flower mutants. The frequency of chlorophyll mutant contains albino, xantha and viridis. This concentration can damage or modify important components of plant cells and have been reported to affect the morphology, anatomy, biochemistry and physiology of plants differentially depending on the concentration level. These effects include changes in the cellular structure and metabolism of the plants e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds. The morphological and chromosomal variation was found to be mutagen sensitive in somatic cells of cowpea. It was found to increase with increasing the concentration of EMS in Cowpea plants. The chemical mutagen like ethyl methane sulphonate induces high frequency of chromosomal changes like anaphasic bridge; anaphasic laggard, anaphasic bridge and clumbing of chromosome were including control plants also observed.

Identification of RAPD Markers Linked to Five Marker Genes (blu, dgs, y, arg, and a Flat Pod Mutant) in Common Bean

The development of a complete linkage map, including both classical (visible) and molecular markers, is important to understand the genetic relationships among different traits in common bean (Phaseolus vulgaris L.). The objective of this study was to integrate classical marker genes into previously constructed molecular linkage maps in common bean. Bulked segregant analysis was used to identify 10 random amplified polymorphic DNA (RAPD) markers linked to genes for five classical marker traits: dark green savoy leaf (dgs), blue flower (blu), silvery [Latin: argentum] green pod (arg), yellow wax pod (y) and flat pod (a spontaneous mutation from round to flat pod in `Hialeah' snap bean). The genes for dark green savoy leaf (dgs) and blue flower (blu) were located in a previously constructed molecular linkage map. These results indicate that classical marker genes and molecular markers can be integrated to form a more complete and informative genetic linkage map. Most of the RAPD markers were not polymorphic in the two mapping populations used, and molecular markers from those mapping populations were not polymorphic in the F2 populations used to develop the RAPD markers. Alternative genetic hypotheses for the pod shape mutation in `Hialeah' are discussed, and the experimental difficulties of pod shape classification are described.

Polarity-Defective Mutants of Aspergillus nidulans

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the α subunit of yeast mitochondrial phenylalanyl–tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed.

Mapping of Five Morphological Markers (dgs, blu, rnd, Ib, and asp) in a Molecular Linkage Map of Common Bean

The classical genetic linkage map of common bean contains only a fraction of the total genes that has been reported. These genes can be mapped in a molecular linkage map so that genetic relationship among diferent genes can be better understood. The objective of this study was to identify RAPD markers linked to genes for five morphological traits using bulked segregant analysis and to map the markers to a molecular linkage map previously constructed in common bean. Five segregating populations were evaluated. Three BC2F2 populations with dgs (dark green savoy leaf), blu (blue flower), and rnd (round leaf), respectively, were developed with a Florida breeding line 7-1404 and 5-593 as the recurrent parent. One BC3F2 population with the asp (dull seed coat) was developed from a BC2F2 5-593 line and 5-593 as the recurrent parent. Finally, an F2 segregating population for Ib (flat pod) was developed from `Hialeah' flat pod mutant × `Hialeah'. The linked RAPD markers were mapped in a molecular linkage map previously constructed using recombinant inbred population of the cross PC-50 × XAN-159. The results of this study indicate that integrating morphological and molecular merkers can result in a more complete genetic linkage map in common bean.