Genetic dissection of bacterial speck disease resistance in tomato

The bacterial speck disease of tomato has been developed as a model system to elucidate the molecular basis of specificity in plant-bacterial interactions and to study signal transduction events involved in the expression of plant disease resistance. We have employed a mutagenic approach to define the steps involved in the expression of disease resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato (Pst) Eleven disease susceptible mutants have been identified and characterized twith altered recognition of Pst strains that express the avirulence gene avrPto. Genetic analysis of these mutants has revealed that they fall into two complementation groups. Five of the mutants map at the Pto locus, while six map a new locus that we have termed Prf. Further characterization of these mutants has revealed that the mutants that map at Pto are still sensitive to the insecticide fenthion, while the prf mutants are altered in resistance and also are insensitive to fenthion. Genetic mapping has also determined that the Prf locus maps near Pto. We are currently employing a map-based cloning strategy to isolate the Prf locus.     

The characteristics and stability of a range of Cox's Orange Pippin apple mutants showing different growth habits

Summary Seven hundred and fifty gamma-irradiated scions of Cox's Orange Pippin apple were grown to produce a V₁ generation and were then multiplied to produce a V₂ of 13 158 individual trees. 272 obvious vegetative mutants, mainly dwarf or compact types, were found in this population and classified according to growth habit. These were propagated to produce clones of the mutant types (V₃) and a study of these clonal mutants as compared with their original (V₂) characteristics showed that while the vast majority of the selected mutants produced dwarf or compact clones, no clear indication of final cropping performance could be drawn from the original phenotype of the selected V₂ mutants.The majority of mutants produced were not of commercial value, and the main reasons for the rejection of V₃ clones depended, in many cases, on the phenotype of the V₂ selection. Thus while all types of V₂ mutant produced approximately the same proportion of acceptable trees, the reason for the rejection of the V₃ clones varies with the V₂ phenotype. Therefore selection can be carried out at an early stage in a mutation breeding programme to reduce the proportion of certain unwanted types such as mericlinal chimaeras that otherwise are carried forward to yield trials. From the orchard trials of 82 mutant clones, 24 were short-listed for possible commercial introduction. All were derived from the less extreme mutant types.     

A mutant of a mutant of a mutant of a...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthemum morifolium Ram.

Summary Radiation-induced sports in Chrysanthemum morifolium Ram. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers.One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or radiation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut flowers.     

Reaction of cultivars of populus spp. to radiation induced virulent mutants of melampsora medusae

Summary Infection Type (IT) and Uredinial Number per Leaf Disk (ULD), induced on eleven cultivars of poplar in vitro, were employed to compare five radiation induced, mono-uredinial mutant lines of Melampsora medusae Thum. with the wild type race 5A from which they were derived. IT produced by the mutants was higher than (eight cultivars), similar to (two cultivars) and pronouncedly less than (IT from 4 to 1 in P. deltoides cv. 7–2) that of race 5A. Although the five mutants produced a uniform IT within a particular cultivar. ULD varied significantly between mutants on individual cultivars and there was a significant differential interaction of the mutant lines with cultivars. The ranking of mutant lines for aggressiveness on cultivars was not consistent while the differences among the mutants in mean ULD over all cultivars were not associated with the dosage level of irradiation from which they were isolated. The implications of the results in the interaction of leaf rust with poplar cultivars are discussed.     

Specific developmental stages of gametogenesis for radiosensitivity and mutagenesis in rice

Summary Developmental stages during gametogenesis of rice were histologically examined in the period from differentiation of reproductive organs to anthesis. Plants were exposed to acute X-rays of 20 Gy. Radiosensitivity and mutation frequency were investigated in relation to the developmental stages of reproductive organs. The most radiosensitive stage, as measured by reduction of the M1 pollen-and seed-fertilities, was the last premeiotic interphase. Mutations induced at different developmental stages were scored in M3 strains. Sterility mutants and short-culm mutants were most frequently observed. Grain shape, panicle morphology, heading-date and endosperm character mutants were induced at a relatively low frequency. The overall mutation frequency varied with the developmental stage at the time of irradiation. The highest overall mutation frequency was observed when radiation was applied 10 days before anthesis, the late tetrad stage of microspores. Radiation exposure of florets at the late tetrad stage was found to be a more efficient method of inducing a large number of mutations than radiations applied to seeds and fertilized egg cells.     

Unfecund, gigantic mutant of oats (Avena sativa) shows fecundity overdominance and difference in DNA methylation properties

Summary The fecundity heterosis of the gig/+oats (Avena sativa) was examined in a population showing segregation of the gene under the long photoperiods in S Finland. Segregating isolines of cv Hannes and of cv Kyrö were raised at two plant densities and the grain yield of heterozygotes and nonmutant homozygotes were evaluated. The heterozygotes in mean outyielded the homozygotes. The overdominance of grain number explains the persistance of this mutant at a relatively high frequency in oats in Finland, though the fecundity of the extremely late homozygous mutant is zero. Previous and current data indicate that the heterozygotes show heterosis of fecundity depending on genetic background, season or other environmental conditions. Grafting of cut stems of nonmutants onto wounded nodes of mutants did not transfer any observable florescence inductor. Hypomethylation of DNA, as revealed with HpaII digestion, occurred in topmost leaves of non-mutants, being absent or occurring to a lesser extent in the comparative leaves of mutants.     

The role of mutations in intraspecific differentiation of groundnut (Arachis hypogaea L.)

Summary Based on morphological diversity, cultivated groundnut (Arachis hypogaea L.) is classified into two subspecies (fastigiata and hypogaea) and further into four botanical types (Spanish bunch, Valencia, Virginia bunch and Virginia runner). In a cross between two Spanish cultivars belonging to ssp. fastigiata, a true breeding variant (Dharwad early runner) sharing some characters of both the subspecies was isolated. The variant, on mutagenesis with ethyl methane sulphonate (EMS) yielded a very high frequency of mutants resembling all four botanical types. Some of the mutants produced germinal reversions to Dharwad early runner in later generations indicating genetic instability. While most of the revertants bred true, some of the mutants continued to segregate, wherein each botanical group of mutants produced all other botanical types. A detailed analysis of the breeding behaviour of mutants revealed several unusual features (such as homozygous mutations, mutation outbursts, segregation distortions, somatic mutations and multiple character mutations) that could not be explained through conventional mutation theory. In the light of these findings, the role of mutations in evolutionary differentiation of the crop and the probable mode of their origin have been discussed.     

Studies on the amylose content and gelatinisation temperature in certain local cultivars and induced grain shape mutants in rice

Summary Ninety local cultivars and 124 induced grain shape mutants were screened for their amylose content and gelatinisation temperature as indexed by the alkali disintegration patterns. The amylose content was high in most of the local cultivars and ranged from 19.2 to 32.0% and from 12.3 to 33.2% in the mutants. Wide variation was also observed for the alkali disintegration values and most of the varieties had low alkali values. A positive but low correlation between alkali values and amylose content and a negative correlation between alkali values and protein content was observed. The preliminary studies suggest that the amylose content at different milling levels increased with an increase in the level of milling. Some of the local cultivars and induced mutants were found to be superior with high amylose and high gelatinisation temperature and could be utilized in breeding programmes directed towards improving the cooking quality of high yielding varieties.     

Differences in partial resistance of barley to powdery mildew (Erysiphe graminis DC F.SP. hordei Marchal) after chemomutagenesis. II. Reaction of mutants to pathotypes

Summary Seedlings of 45 barley mutants with partial resistance in the field and their parental cultivars Asse, Bomi, and Vada were exposed to six local and two foreign races of powdery mildew, in climatically controlled cabinets. Infectability, pustule size, infection grade, and infection type were estimated. No mutant did demonstrate strong race-specific reactions although some race-specific interactions of moderate grade could be detected. The results of infection of mutants with single races confirmed the quantitative character of their change in resistance determined in the earlier field assessments. The 20 investigated mutants with increased resistance expressed a lower level of disease with each of the used races. Out of the 10 mutants with higher susceptibility in the field, 4 mutants again exhibited higher degrees of infection over all the different races, while the remaining 6 mutants were not distinguishable from the original cultivar under the given growing conditions. Out of the tested 14 developmentally resistant mutants, only the 5 genotypes with different seedling reaction could be analyzed for race-specificity in this study. In all the above cases as the result of one gene mutation, quantitative shifts in level of infection were recorded over all the 8 races.Qualitative estimates of infection type were supported by data on the frequency of chlorotic or necrotic lesions and green islands on the infected leaves. But quantitative methods of assessment, e.g. infection frequency and pustule size, were more effective in studying partial resistance of the mutants. Infection grade, estimated visually 14 days after inoculation, was in agreement with the quantitative parameters accurately measured 7 days after inoculation. Therefore, its careful use can be recommended to speed the screening by narrowing down the materials. Differences in virulence level of the races were observed and their influence on race-specificity studies was discussed.     

Breeding for resistance to Phialophora cinerescens (Wr.) van Beyma in glasshouse carnations (Dianthus caryophyllus L.)

Summary A breeding programme is described, aimed at introducing resistance to Phialophora cinerescens in glasshouse carnations of both the standard and the spray type. Four resistant clones, selected from seed-grown garden cultivars, served as resistant parents; the susceptible parents included the major glasshouse cultivar William Sim and some of its mutants and a few other American, French and Italian cultivars.In three generations of breeding, with extensive screening of young seedling populations, an adequate level of resistance had been reached. Selection procedure was henceforth changed to allow for primary selection on quality and productivity, followed by clonal resistance tests.Inheritance of resistance appears to be mainly additive; at least two gene pairs are likely to be involved. Under prolonged high infection pressure a significant non-additive variance can be demonstrated.     

Heavy-ion-induced mutants in sweet pepper isolated by M1 plant selection

We examined the effects of heavy-ion bombardment on mutagenesis in sweet pepper (Capsicum annuum L.). Dose–response studies indicated that 10 Gy irradiation of ¹²C or ²⁰Ne ions on dry seeds is suitable for inducing mutations in plants. From ²⁰Ne-irradiated M₁ plants, putative mutants included two dwarf plants and one plant whose pericarp was yellow were isolated. Phenotypes of their M₂ progeny were similar to those of the M₁ plants and did not segregate. F₁ plants resulting from reciprocal crosses between the mutants and wild-type plants showed the wild-type phenotype, but phenotypes of F₂ and BC₁F₁ segregated at 1:3 (mutant:wild) and 1:1, respectively. These crossing experiments indicate that the three mutants have monogenic recessive mutations in nuclear genes. In light of these data, we discuss the effectiveness of using heavy-ion bombardment to mutate sweet peppers.     

Genetics of induced dwarf mutants in pearl millet,Pennisetum americanum (L.)Leeke

Summary Inheritance pattern of seven dwarf mutants revealed each of them to be controlled by a single recessive gene. Tests for allelism indicated existence of two groups of dwarfs, group 1 comprising six mutants and group 2 a single dwarf. The dwarfs of group 1 were found to be allelic to D₂ of the dwarf types already known in this crop and the dwarf of group 2 was allelic to D₁ of these types.Botany Department, R.V.V.N. College, Dharanikota, India.     

Induced mutations – A new paradigm in plant breeding

The use of ionizing radiation, such as X-rays, gamma rays and neutrons and chemical mutagens for inducing variation, is well established. Induced mutations have been used to improve major crops such as wheat, rice, barley,cotton, peanuts, and beans, which are seed propagated. Since the establishment of the Joint FAO/IAEA Division of the Nuclear Techniques in Agriculture, more than 1800 cultivars obtained either as direct mutants or derived from their crosses have been released worldwide in 50 countries. In vegetatively propagated plants, many of mutants were derived from irradiating rooted stem cuttings, detached leaves, and dormant plants. According to the FAO/IAEA database, of the 465 mutants released among the vegetatively propagated plants, most are in the floricultural plants and a few in fruit trees. These include chrysanthemum, Alstroemeria, dahlia, bougainvillea, rose, Achimenes,begonia, carnation, Streptocarpus, and azalea. The irradiation of in vitro cultured date palm, apple, potato, sweet potato and pineapple now provides a means to treat large populations which would not have been possible before. Irradiation of micropropagated plants, axillary and adventitious buds, apical meristems, regenerative callus cultures, anthers and microspores, and somatic embryos provides a miniaturized version of trees and seeds in the Petridish instead of the field. During the last decade, the use of radio-actively labeled probes in recombinant DNA research for cloning and mapping plant genes and transgenesis, particularly for RFLP, micro satellite based DNA fingerprinting, has become a routine procedure. Many homeotic mutants that change floral development have been isolated in Arabidopsis, Petunia, Antirrhinum and Lycopersicon. Mutants of Arabidopsis are being used to analyze genes, which determine response to auxins, cytokinins, gibberellin, abscisic acid and ethylene in plant growth, floral development and senescence, fruit formation and ripening. These mutants are facilitating the isolation, identification and cloning of the genes, which would ultimately help in designing crops with improved yield, increased stress tolerance, longer shelf-life and reduced agronomic inputs. The identification and analysis of mutants by using molecular techniques of DNA fingerprinting and mapping with PCR based markers, such as RAPDs, AFLP and STMS, and mutant tagging shall bring a new dimension in gene technology. Already, mutations can be linked to changes in DNA sequences for some plant traits and to establish molecular maps in structural and functional genomics of crop plants. These in turn would lead to a rapid enhancement of crop yields and quality. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of wheat mutants with increased resistance to powdery mildew from small induced variant populations

Small variant wheat populations created by induced mutagenesis (n = 69) or adventitious regeneration (n = 66) were intensively screened for an altered response (compared to the parent variety ‘Guardian’) to the causal pathogen of powdery mildew in wheat, Blumeria graminis f. sp. tritici. Intensive field screening following natural infection of replicated plots of wheat lines over two years revealed a total of 13 mutants exhibiting significantly greater resistance than ‘Guardian’: eight from induced mutagenesis (11.6%) of the M2 population and five from adventitious regeneration (7.6%). Complete resistance was identified in two lines, (one (M66) developed following induced mutagenesis, and the other (SC240) by adventitious regeneration). The complete resistance in the induced mutant was stable over two generations and was associated with a high frequency of leaf flecking, and consequently a low grain yield. Resistance in SC240 proved to be unstable; SC240 exhibited complete resistance to powdery mildew in the SC₂ and SC₃ generations, but only 20% of the SC₄ plants were completely resistant, while the remainder were indistinguishable in mildew response to ‘Guardian’. The mildew response of all the SC₅ generation of SC240 was not significantly different from ‘Guardian’. Yield analysis of the thirteen mutants with increased resistance in the presence of powdery mildew indicated that eleven exhibitedgrain yields at least as high as that of ‘Guardian’, while the mutant M19 exhibited a yield significantly higher than that of ‘Guardian’. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic male sterility in the pea (Pisum sativum L.)

Summary Genetic male sterility has been described in the pea (Pisum sativum L.), but no comprehensive effort has been made to study the phenomenon. A preceding companion paper reported the inheritance, allelism and linkage relations of thirteen male sterile mutants obtained from seed mutagen treatments. In the present study, the same male sterile mutants were investigated cytologically to determine the cause of sterility. Normal microsporogenesis and microgametogenesis was compared to that of the mutants. The ms-2, ms-3, and ms-4 mutants exhibited meiotic abnormalities similar to those described by Gottschalk and colleagues except that ms-3 had a high degree of female sterility. Chromosome clumping and spindle abnormalities leading to formation of coenocytic microspores and degeneration were characteristic of ms-2 and ms-4. The ms-2 and ms-4 mutants were previously found to be allelic, and were nearly identical cytologically in the present study. The ms-3 mutant exhibited a lack of chromosome condensation in meiosis I, and a lack of spindle formation in both meiotic divisions. Two mutations (ms-6 and ms-10) affected meiosis, with univalents at metaphase, and asynchronous divisions during meiosis II. Microspores of ms-6 completely degenerated whereas those of ms-10 showed some development. Sticky chromosomes, bridges and fragments, tripolar spindles, and lack of a second division were characteristic of ms-10. The ms-10 mutant also showed reduced female fertility. Two male steriles (ms-5 and ms-9) had abnormalities associated with premature degeneration of the tapetum. Three others (ms-7, ms-8, and ms-11) aborted pollen during meicrogametogenesis. Pollen grains of ms-11 had thinner walls than normal and lacked sculptured exine. The ms-10 mutant, and those affecting microgametogenesis (ms-5, ms-7, ms-8, ms-9, and ms-11) produced some stainable and viable pollen.     

Barley mutants with increased tolerance to aluminium toxicity

Acid soil and associated aluminium toxicity are considered as the number one abiotic factor limiting crop production. Over 2 billion hectares of acid soils exist world-wide, both in tropical and moderate climatic zones. In Poland acid soils represent up to60% of arable land. At soil pH < 5.0 Al ions become soluble in water and toxic to plants. Genetic improvement of Al tolerance in crops is the only alternative to soil liming, a traditional but short term and expensive agricultural cure to raise soil pH. Of the various cereals, barley is the most sensitive to Al toxicity. The known sources of Al tolerance in barley are limited to old cultivars and landraces. While they represent multiple alleles of a single locus, there is no potential to improve Al tolerance through recombination of non-allelic additive genes. In the Department of Genetics, Silesian University we have employed induced mutations for rapid creation of variability for Al tolerance in barley. Thirteen mutants with increased levels of tolerance to Al toxicity have been selected in M₃ generation after mutagenic treatment of four barley varieties with N-methyl-N-nitroso urea (MNH) and sodium azide. Six further Al tolerant mutants were identified in the collection of semi-dwarf mutants of the Department. All selected mutants confirmed Al tolerance with the use of three different methods of screening, i.e., root re-growth, root tolerance index and hematoxylin staining. Fourteen mutants exhibited significant root re-growth after 48 hour incubation with 3 ppm Al⁺³ and two of them, namely RL819/2 and RL820/6 were tolerant even to 6 ppm Al⁺³. Crosses of two selected mutants with their respective parent varieties indicated that Al tolerance in each mutant was controlled by a single recessive gene. Out of three methods tested, the root re-growth method facilitated by hematoxylin staining proved to be the most reliable technique for large scale testing. Double treatment with MNH or combined treatment with sodium azide and MNH and 6hinter-incubation germination between treatments were the most successful treatment combinations for induction of aluminium tolerance in barley. This revised version was published online in August 2006 with corrections to the Cover Date.     

The identification of faba bean (Vicia faba L.) trisomics by translocation tester set

Summary Five subtelocentric primary trisomics of Vicia faba L. were isolated from crosses between asynaptic mutants and normal plants or translocation tester stocks. The extra chromosome of each trisomic was identified cytologically by critical multivalent association at metaphase I of meiosis.     

Monogenic control of resistance in the tomato to the tobacco flea beetle: Probable repellance by foliage volatiles

Summary In comparisons made under typical summer conditions in the field at Davis, California, the foliage of the anthocyanin-deficient mutant af proved to be much more susceptible to attack by Epitrix hirtipennis (Melsheimer) than that of the isogenic normal line, normal cultivars, or any other tested mutant. Since a relatively uniform level of damage was observed in leaves of various ages that contacted the soil, the interaction is probably of a preference rather than antibiotic nature. A comparison with nine other anthocyaninless mutants proved that anthocyanin deficiency is not responsible for susceptibility. The severe reduction of glandular hairs and consequent lack of foliage aroma—hitherto unsuspected pleiotropic effects of af—probably account for the high susceptibility of this mutant. The evolutionary significance of the distinct foliage aromas characteristic of each tomato species is discussed.     

The potential of ripening mutants for extending the storage life of the tomato fruit

Summary The effect of the fruit ripening mutants rin, gr, nor and Nr on storage life and pigmentation was investigated in homozygous material, in heterozygous F₁ combinations between the mutants with the colour mutant hp and with the normal cv. Kewalo. Crosses with nor showed a 3-to 5-fold increase in storage life in comparison with the normal cv. or with hp. Maximum pigmentation of the fruits of crosses with nor was pale-red with vineripened fruit and pink with fruit harvested at the breaker stage and ripened on the shelf. The ripening inhibitory effect of rin in the different F₁ combinations was less pronounced than that of nor, and the colour of the fruits was improved. Fruits of the F₁ cross between rin and nor showed greatly improved storage life and developed pink or pale red colour. Most heterozygotes with hp showed improved pigmentation. Problems anticipated in utilizing ripening mutants in breeding for improved keeping quality are discussed.     

Occurrence, inheritance and use of reproductive mutants in alfalfa improvement

The widespread occurrence of 2ngametes (i.e. gametes with the somatic chromosome number) in the Medicago sativa-coerulea-falcata complex supports the concept that gene flow from diploid to tetraploid species occurs continuously in nature and plays a key role in alfalfa evolution. Breeders realized early that gene transfer between ploidy levels via 2n pollen and 2n eggs would have had potential use in cultivated alfalfa improvement. Cytological investigations provided insights into the types of meiotic abnormalities responsible for the production of 2n gametes. Alterations were defined as genetically equivalent to first (FDR) or second division restitution(SDR) mechanisms. For breeding purposes,data have proven that 2n gametes of the FDR type are more advantageous than those obtained by SDR for transferring parental heterozygosity and retaining epistatic interactions. The use of diploid meiotic mutants that produce 2ngametes is now recognized as one of the most effective methods available for exploiting heterosis and introgressing wild germplasm traits into cultivated tetraploid alfalfa via unilateral (USP) and bilateral sexual polyploidization (BSP) schemes. Both2n egg and 2n pollen producers could be used for direct gene transfer from wild diploid relatives into cultivated alfalfa by means of 2x-4x and4x-2x crosses. Although data have shown that forage yield improvement can be achieved when plants are sexually tetraploidized, problems related to reduced plant fertility and seed production remain largely unexplained. Apomixis has the potential of cloning plants through seed and thus provides a unique opportunity for developing superior tetraploid cultivars with permanently fixed heterosis and epistatic effects. A main goal in alfalfa breeding could be the introduction of functional apomixis (i.e. Apomeiosis and parthenogenesis) in cultivated alfalfa stocks. In the future, the efficiency of alfalfa breeding programs based on the use of reproductive mutants could be improved by direct selection at the genotype level using RFLPs and PCR-based markers. Suitable DNA markers and detailed linkage maps of alfalfa mutants should help to discover apomictic mutants and address basic genetic issues such as the extent of genomicre combination in polyploid hybrids and the effect of sexual polyploidization on heterosis. Molecular markers have recently been used in alfalfa for studying the inheritance of 2n gamete formation and identifying polymorphisms associated to genes involved in meiotic abnormalities. Molecular tagging of 2n egg and 2n pollen formation not only should explain the genetic control and regulation of these traits, but may also be an essential step towards marker-assisted selection of 2n gamete producers and implementation of USP and BSP breeding schemes. Future perspectives include strategies for the map-based cloning of genomic DNA markers,and screening of EST mini-libraries related to flowers at different developmental stages from meiotic mutants and wild-type scan lead to the identification of mRNAs and thus of candidate genes that control 2n gamete formation in alfalfa. This revised version was published online in July 2006 with corrections to the Cover Date.