Nodulation and nitrogen fixation mutants of pea,Pisum sativum

Summary The 36 mutants which did not nodulate and 24 mutants which formed inefficient nodules with no or very low acetylene reduction activity were isolated among 86,000 M₂-seedlings of Finale pea, Pisum sativum L., after treatment with chemical mutagens. One mutant was found for approximately every 50 chlorophyll mutants. Most mutations were induced by ethyl methanesulfonate; some by diethyl sulfate, ethyl nitrosourea and acidified sodium azide. Putative mutants were selected as nitrogen deficient plants, yellowing from the bottom and up, when M₂ seedlings were grown in sand with a Rhizobium mixture and PK fertilizer. The mutants were verified in the M₃ generation by acetylene reduction assay on intact plants.     

Recovery from ethyl methanesulphonate-induced genetical damage in barley. Independence of sodium azide treatment

Barley seeds were treated for 3 h at 25°C with 240 mM ethyl methanesulphonate (EMS), washed for 18 h, treated with various concentrations of unbuffered sodium azide (pH 6.7–7.3) for 3 h at 25°C, re-dried to 30% water content and either sown immediately or stored at 25°C for 12 days and then sown. The synergistic action of sodium azide post-treatment has been demonstrated only for the EMS-induced M₁ germination reduction, while the EMS-induced M₁ sterility and the yield of M₂ chlorophyll mutants were unaffected. The ?storage” recovery from EMS-induced mutagenic effects was insensitive to sodium azide post-treatment. The 12 day-seed storage at 25°C brought about an improvement of M₁ germination, M₁ survival, M₁ fertility and a decrease in the amount of M₂ mutants, regardless of whether sodium azide post-treatment was applied or not.     

Production of doubled haploids through anther culture of M<Subscript>1</Subscript> rice plants derived from mutagenized fertilized egg cells

To produce stable mutants from Mankeumbyeo, a japonica rice (Oryza sativa L.) variety, we estimated the mutation efficiency of ethyl methane sulfonate (EMS) and N-methyl-N-nitrosourea (MNU) on fertilized egg cells using doubled haploids (DHs) derived from anther culture of M₁ plants. M₁ seed production and germination were higher in 1 mM MNU than in 94.2 mM EMS. A total of 68 DHs (35.4%) were regenerated by anther culture of M₁ plants. Twenty-one DHs (30.9%) were stable mutants, 14 DHs (20.6%) were unstable mutants, and the remainder (48.5%) were normal. The frequencies of stable mutants following EMS and MNU treatments were 20.7% (three semidwarfs, one early maturation and one glabrous line) and 38.5% (three semidwarfs, two early maturation, four glabrous and one long grain line), respectively. In a field trial of seven stable mutants for yield potential, five mutants did not show a significant difference in yield as compared with the original variety. Among these five, three glabrous mutants (MK-MAC 1, MK-MAC 4 and MK-MAC 26) with a smooth leaf and hull may be considered to be improved mutant lines because of the health benefits (reduced skin damage and generation of less dust compared to the original variety) to farmers handling the plant materials. MK-MAC 26, a glabrous mutant, had also less shattering resistance than that of the original variety. These stable mutants could be used as new breeding materials.Communicated by P.P. Kumar     

Subject index

Barley seeds were treated with ethyl methanesulphonate (EMS) for 5 h, washed for 18 h, exposed for 3 h to caffeine, re-dried to 30% water content and either immediately sown or stored for 10 days at 25°. The caffeine post-treatment considerably potentiated the EMS-induced M₁ germination and the M₁ seedling height reduction, but had no influence on the EMS-induced M₁ sterility or percentage of M₂ chlorophyll mutants. The caffeine post-treatment also had no significant influence on the “storage” recovery from the EMS-induced genetic effects.     

Activity of delta-endotoxin protein crystals in Bacillus thuringiensis mutants when influenced by UV treatments

The ability of some isolates of Bacillus thuringiensis to produce dark brown pigment was measured as an indicator to UV resistance. M₅ as an indigenous Egyptian isolate was used as wild type to improve its resistance to UV. It was exposed to UV irradiation for different periods ranging between 1 and 10?h. The induced mutants were examined morphologically by phase contrast microscope. One hundred and forty four mutants were obtained; 10 of them were selected and tested for their toxicity against Spodoptera littoralis. The results showed that mutants 62, 65 and 85 were the most toxic ones. These three mutants and the wild type were examined by transmission electron microscope. Crystal proteins with bipyramidal shape and active against Lepidopteran insects were detected in all the selected mutants.     

Methylammonium-resistant mutants ofNicotiana plumbaginifolia are affected in nitrate transport

This work reports the isolation and preliminary characterization ofNicotiana plumbaginifolia mutants resistant to methylammonium.Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up byNicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

Nature and type of gene action governing resistance to Helminthosporium turcicum leaf blight in maize (Zea mays L.)

Six generations, consisting of three resistant parents, three susceptible parents, their 15 possible F₁ crosses, 15 F₂'s, 15 BC₁'s (F₁ x resistant female parent) and 15 BC₂'s (F₁ x susceptible male parent) were analysed following Hayman (Heredity 12: 371–390, 1958) to evaluate the nature and type of gene action governing resistance to H. turcicum. The results showed that all types of gene effects, viz., additive, dominance and epistasis (i.e., additive x additive, additive x dominance and dominance x dominance) were operating in one cross or the other in controlling resistance. However, it was additive gene action and dominance x dominance type of epistasis with duplicate nature that were important in controlling resistance in most crosses. Depending upon the final objectives, one of the breeding methods, viz., recurrent selection, heterosis breeding, back-cross method or full-sib selection (bi-parental mating) may be followed.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Novel resistance to the Bymovirus BaMMV established by targeted mutagenesis of the PDIL5-1 susceptibility gene in barley

The Potyviridae are the largest family of plant-pathogenic viruses. Members of this family are the soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5-1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5-1-based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time-consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR-associated endonuclease technology to induce site-directed mutations similar to those described for PDIL5-1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene-free homozygous M₂ mutants were produced in spring barley. A variety of mutants carrying novel PDIL5-1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in-frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus-resistant mutants showed no adverse effects in terms of growth and yield.     

I. THE EXPRESSION OF INSTABILITY IN N. TABACUM × N. PLUMBAGINIFOLIA

Moav , Rom (Hebrew U., Jerusalem), and D. R. Cameron . Genetic instability in Nicotiana hybrids. I. The expression of instability in N. tabacum × N. plumbaginifolia. Amer. Jour. Bot. 47(2): 87—93. 1960.—N. tabacum (n = 24) and N. plumbaginifolia (n=10) are distantly related species both from morphological and cytological points of view. Hybrids of these species with various genome dosages have exhibited somatic variegation when plumbaginifolia dominant characters were superimposed on an appropriate tabacum genetic background. Five loci were studied in this respect: Wh and Tg—for flower coloration; Ws—for chlorophyll production; Kl—for pollen abortion and Bs—for black shank resistance. All 5 were found to be unstable. Backcross progenies of the sesquidiploid hybrid (tbc-tbc-pbg) to tabacum showed a marked increase in intensity of variegation. This has been attributed to the breaking up of the plumbaginifolia genome into individual chromosomes. The evidence indicates that variegation was due to somatic chromosomal aberrations which probably characterized all the plumbaginifolia chromosomes. An hypothesis regarding the heterogeneity of F₁ hybrids of distantly related homozygous species is outlined and the occurrence of instability due to hybridization in other Nicotiana hybrids is discussed.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

A pleiotropic mutation results in cross-resistance to auxin, abscisic acid and paclobutrazol

Summary Mutant lines of Nicotiana plumbaginifolia resistant to the synthetic auxins 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) were isolated as germinating seedlings on selective medium. In each case, resistance was conferred by a single recessive nuclear mutation at one of 3 loci designated iba1, iba2 and iba3. Labelling studies with ¹⁴C NAA suggest that resistance was not due to changes in the uptake or metabolism of NAA. Plants homozygous for the iba1 mutation exhibit a syndrome of atypical germination and growth suggestive of a defect in the biosynthesis, metabolism or localization of abscisic acid. Wild-type seeds treated with gibberellin exhibit the same syndrome, including resistance to NAA and IBA. On the basis of these observations, we propose that auxin toxicity in seeds may be mediated by a block in gibberellin biosynthesis.Abbreviations ABA abscisic acid - GA gibberellic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - p-cell protoplast-derived cell - 2,4-D 2,4-dichlorophenoxyacetic acid     

Characterization of three hormone mutants of Nicotiana plumbaginifolia: evidence for a common ABA deficiency

Summary Various auxin-resistant Nicotiana plumbaginifolia mutants have already been isolated, including 1217 which shows cross-resistance to paclobutrazol. Recently, a cytokinin-resistant mutant, CKR1, has been characterized and has been shown to be affected in abscisic acid (ABA) biosynthesis. We have isolated a new mutant, Esg152, which was selected on the basis of its early germination. In each of these mutants, resistance is due to a recessive nuclear mutation at a single locus. Complementation analysis indicated that mutants I217, CKR1 and Esg152 belong to the same complementation group. They have a similar phenotype, which includes a reduction in seed dormancy and an increased tendency to wilt. These mutants display an increased auxin tolerance and enhanced root formation when leaf or hypocotyl sections are cultivated on auxin. By immunoenzymatic methods, we show that the endogenous levels of ABA are significantly lower than in the wild-type. We have assigned the symbol aba1 to the recessive alleles of the locus affected in the three mutants. The complexity of hormonal interactions is discussed briefly emerging from a consideration of this class of mutants.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Temperature-sensitive plant mutants isolated in vitro

Summary Conditional-lethal, temperature-sensitive plant mutants have been isolated using a simple protoplast cloning method. The leaf protoplasts used were obtained from sterile, haploid shoot cultures of Nicotiana plumbaginifolia. Recessive mutations are described at three loci: ts1, ts2 and ts3. The mutations are lethal when either tissue cultures or plants are incubated at 33°C but not at 26°C.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

Storage of ethyl methanesulphonate-treated barley seeds with 15 per cent moisture. Influence of treatment and washing conditions

Barley seeds were treated with ethyl methanesulphonate (EMS), washed for 24 h, redried to 15 per cent moisture and stored at 25°C. The criteria used for expressing the effect of storage were chromosomal aberrations in root tips, M₁ germination, M₁ seedling height, M₁ survival and the frequency of M₂ chlorophyll mutants. The increase of the M₁ biological injury due to storage was not influenced:

1. by applying EMS solutions at pH 2, pH 7 and pH 10,
2. by lowering the EMS concentration and increasing the treatment time,
3. by different variations of washing with water and by washings with 0.005 N NaOH, 200 mM cysteine or 200 mM thiourea. The rate of the increase of the M₁ injury due to storage depends on the EMS dose. With a decrease in the EMS dose the storage effect is more delayed.

     

Inheritance of components of partial resistance of barley to Rhynchosporium secalis with particular reference to race specificity

Six spring barley cultivars with no known genes for resistance to specific virulences but varying in partial resistance to Rhynchosporium secalis, were crossed in all combinations (6 × 6 diallel including reciprocals). In addition to seeds from naturally selfed plants, seeds of all parent cultivars were also produced by artificial selfing (emasculation followed by pollination using pollen from the same cultivar). This ensured comparability between seeds of parents and F₁. Both sets of parents, F₁ and F₂ families were grown in the field as single spaced plants and inoculated at Zadoks growth stage 49 with spore suspensions (2 × 10⁶ spores ml^-1) of three races (pathotypes) of R. secalis (Zadoks, Chang & Konzak, 1974). Components of partial resistance, incubation period (ICP), infection frequency (IF) and spore production per lesion (SP/L) were assessed on each plant. There were highly significant differences for all three components of partial resistance in both sets of parent cultivars but rank order in both sets was similar as evidenced by correlation coefficients, r= 0.96 for ICP and IF and r= 0.87 for SP/L. All three components of partial resistance were strongly correlated with NIAB (National Institute of Agricultural Botany, Cambridge, UK) resistance ratings. Means of F₁ and F₂ families were correlated with mid-parent values for ICP and IF but not SP/L. No difference in aggressiveness was found between races but for each component of partial resistance there was a significant interaction between race and parent cultivar (artificial selfs) and, for IF and ICP, a significant interaction between race and F₁ family. There was no evidence of interaction between parent (natural selfs) and race nor between race and F₂ family. Examination of genetic control of resistance showed evidence of strong additive effects (combining ability) in both F₁ and F₂ for ICP and IF but not for SP/L. There was no evidence for maternal or reciprocal differences, but there was evidence for dominance effects although their nature differed between components of partial resistance and between F₁ and F₂ generations. In the F₁, but not the F₂ generation, several elements of dominance (direction, distribution of dominant genes between parent cultivars, specific combining ability) showed for ICP or IF (but not SP/L) significant interaction with race.