Mutagenic response of rhodes grass (Chloris gayana Kunth.) to gamma rays. II. Studies on second (M2) generation parameters

Summary The effect of gamma rays on parameters such as chlorophyll mutation frequency and spectrum, mutagenic effectiveness and efficiency and viable mutation frequency and spectrum were studied in M₂ generation of Rhodes grass employing nine doses of gamma rays. The chlorophyll mutation frequency increased in a linear fashion at low and medium doses and was erratic at higher doses. The chlorophyll mutant spectrum ineluded albina, xantha, chlorina, viridis, tigrina, striata, albo-xantha and albo-viridis. The frequency of these mutants varied with treatments. A dose rate of 60 krad was found to be effective as well as efficient both on M₁ and M₂ plant basis in inducing chlorophyll mutations. The highest frequency of viable mutations was found in 50 krad treatment. Viable mutants found were: nodal variant, peduncle variant, lax ears, coxcomb ears, earhead proliferation, level of fingers, incurved fingers, gappy ears, apical sterility, partial sterility and high sterility.     

EMS Induction of Early Flowering Mutants in Spring Rape (Brassica napus)

Studies were undertaken to induce early flowering mutants by ethyl methanesulphonate (EMS) treatments of Brassica napus seeds. EMS treatments for 12 h of a highly inbred B. napus line TBS had adverse effects on M¹ plant development and fertility only when concentrations were greater than 1%. However, an EMS concentration of 1.5% did not reduce M₁ plant fertility to an extent which significantly reduced production of M₂ seeds. Genetic changes induced by EMS treatment and affecting flowering time were of three main types: (1) Changes within a polygenic system reflected by increased variation in flowering time among M₂ families. As the increase in variation was due primarily to a higher frequency of later flowering plants, these polygenic changes would be of little value in developing better-adapted cultivars. (2) Induction of a recessive mutation at a major gene locus which caused M₃ plants homozygous for the mutant gene to flower at least 20 days earlier than the parental line TB8. (3) Induction of a dominant mutation at a major gene locus which affects flowering time by causing a substantial reduction in vernalization requirement. M₂ plants carrying the mutant gene flowered as early as 59 days before the parental line. These major gene mutations could be rapidly exploited in the development of agronomically superior cultivars for short-season, lower rainfall environments in Western Australia.     

The problem of induced sterility: A comparison between EMS and X-rays in Arabidopsis thaliana

Summary The problem of mutagen-induced sterility has been especially studied in barley by Gaul, and in Arabidopsis by Müller and by van der Veen. Their results and conclusions are essentially confirmed and extended by the present Arabidopsis experiment, which compares EMS with X-rays.For a given treatment, M₂-fertility is correlated with M₁-fertility, but mutant frequency in M₂ is equal for the different M₁-fertility classes. It follows that efficiency in mutation breeding can be increased by pre-selection for fertility in M₁, and by using doses which do not make fully fertile M₁-sectors too rare.EMS is more efficient than X-rays by a factor 4 at equal M₁-fertility, and a factor 3 at equal M₂-fertility. As a result of difference in fertility spectrum, fertility improvement from M₁ to M₂ is better for X-rays than for EMS.A considerable load of diplontic sterility and of embryonic lethals (non-germinating seeds) is simply the toll one has to pay for the higher mutant frequency after EMS.Fortunately, part of the diplontic sterility can be pre-selected against in M₁, and maternally conditioned embryonic lethals can be selected against by discarding seedlots from individual M₁-sectors which show reduced germination. (M₂-line selection may also be practised against recessive embryonic lethals and seedling lethals.)     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

Response to selection for plant height in X-ray treated population of jute (Corchorus capsularis L.) cv. JRC 212

Summary X irradiation of dry seeds of Corchorus capsularis L. cv. JRC 212 induced variations in plant height as evidenced by the high values of genotypic variance and heritability estimate in M₃ generation. Selection on the basis of line mean of plant height in M₃ generation was effective in increasing plant height as well as fibre yield in M₃ generation. A higher response for plant height rather than for fibre yield itself was observed in lower irradiation dose while responses for these two traits were almost similar in higher dose. An average of 12% increase in fibre yield was achieved in M₆ generation when all top five lines were selected. The top two lines, had 17% and 16% higher fibre yield than that of control.     

Dwarf-twisted: An induced mutation in Triticum durum Desf

Summary A mutant phenotype, mainly characterized by undulated leaf surface, dwarfism and sterility, has been repeatedly induced by physical (X-rays; thermal and fast neutrons) and chemical (diethylsulphate and ethylmethanesulphonate) mutagenic agents in different varieties of durum wheat. This phenotype is easily detectable at the seedling stage. Neutron treatments are the most effective method to induce this phenotype and a maximum of 0.6% mutations per M₁ spike-progeny was induced after the highest dose of thermal neutrons. On the average 0.23 mutations per 100 M₁ spike progenies and 0.55 mutant seedlings per 1000 M₂ plants were found after all treatments.Genetic analysis of different dwarf-twisted phenotypes shows that this character is conditioned by one recessive gene with an average segregation ratio of 22.9%. As the dwarf-twisted phenotypes segregate in highly fertile M₁ spikes and no evident chromosome changes have been detected in this caryotype, the hypothesis of a point mutation has been proposed.Contribution no. 171 from the Laboratorio Applicazioni in Agricoltura del C.N.E.N., C.S.N. Casaccia, S. Maria di Galeria, Roma, Italy.     

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.     

Preliminary results of using in vitro axillary and adventitious buds in mutation breeding of Chinese gooseberry

Summary Aseptically cultured shoots of Chinese gooseberry exhibited growth disorder and morphological aberrances, and some died after being exposed to sufficient gamma-ray irradiation. The death rate was dose dependant and the LD₅₀ was 80–90 Gy and 50–60 Gy respectively for cv. Hayward and clone 4. All petiole explants irradiated with gamma-ray could form calli as the control, but the rate of differentiation of adventitious shoots of the petiole explants decreased and was dependant on dose. Sensitivity of the shoot or petiole explants to gamma-ray irradiation varied with species. Gamma-ray irradiation did not deter either the 2-node segments from producing axillary shoots M₁, M₂, and M₃ or the advantitious shoots originating in the petiole explants and the M₃ shoots from forming advantitious roots. Therefore, using aseptically cultured axillary or adventitious buds for mutation breeding of Chinese gooseberry is feasible. A bacterium surviving in the explants lessened the efficiency of these two in vitro techniques in mutation breeding of Chinese gooseberry.Abbreviations IAA 3-indole acetic acid - IBA -indole butyric acid - MS Murashige & Skoog (1962)     

Increasing mutagenic efficiency of gamma-radiation in rice by irradiation of seeds at extremely low temperature

Summary Dry seeds of rice, Oryza sativa L., containing about 13 percent moisture, were exposed to ⁶⁰Co gamma raysat extremely low temperature (–196°C). M₁ damage on survival rate, seedling height and seed set were found to become much smaller in the cooling treatment than in the not-cooled control. Chlorophyll and heading date mutation frequencies in the M₂ generation were not affected by the decreased M₁ damage caused by the cooling treatment, and the mutation frequencies could be significantly increased by applying higher doses combined with the cooling treatment. Frequency of sterile or semi-sterile mutants in the M₂ generation was found to be significantly lower in the cooling treatment than in the not-cooled control.It is assumed from the experimental results that extremely low temperatures during irradiation could reduce the amount of physiological damage and gross chromosome aberrations. This results in an increased efficiency of producing minute deletions and/or point mutations.     

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.     

Identification of low lectin mutants in soybean

Lectin is one of the known antinutritional factors that deteriorate the soybean protein quality and development of cultivars with low lectin content will help to improve nutritional quality of soybean [Glycine max (L.) Merrill]. Therefore, attempts were made to induce mutations for low lectin content in the cultivar ‘MACS 450’. Soybean cultivar ‘MACS 450’ was subjected to combination treatments of γ‐rays and ethyl methane sulphonate (EMS) with an objective to induce variability for low lectin content. The treatments of different combinations of γ‐rays and EMS were 50 Gy + 0.2% EMS, 50 Gy + 0.4% EMS, 100 Gy + 0.2% EMS and 100 Gy + 0.4% EMS. Of the 3200 treated M₁ seeds sown, 16 400 M₂ plants were raised. In M_2, 72 plants were identified for low lectin content [<40 × 10⁵ haemagglutination unit (HAU)/mg] and were carried up to M₅ generation. In M₅ generation, lectin content in ‘MACS 450’ was 39.23 to 50.0 × 10⁵ HAU/mg, and was compared with the nine true breeding lines identified having low lectin content, ranging from 2.3 × 10⁵ to 27.46 × 10⁵ HAU/mg. Three mutants were found to possess very low lectin content (ranging from 2.0 × 10⁵ to 3.0 × 10⁵ HAU/mg). Thus, the identified mutant lines with low lectin content will greatly improve soybean protein quality, thereby reducing financial burden on the soybean industry for processing soybean meal and also making it suitable for human consumption. All the mutants showed normal seed development, having soluble protein content similar or higher than that in the parent (32.0 mg/ml). This indicates that the change in lectin content does not have any negative impact on the plant growth and protein content.     

Increasing erucic acid content in Ethiopian mustard through mutation breeding

Ethylmethane sulphonate (EMS) was applied to seeds of the Ethiopian mustard (Brassica carinata A. Braun) line C-101. Bulk samples of M₃ seeds from 8331 M₂ plants were evaluated for the fatty acid composition of their oil by near-infrared reflectance spectroscopy (NIRS) and by further gas chromatography on selected samples. A putative mutant, N2-6230, showing very low oleic acid content (4.7% vs. average of 8.6% in C-101) and erucic acid content within the range of variation of the line C-101 (40-49.3%) was identified. The M₃ progeny of this mutant showed a wide segregation for erucic acid content (39.1-57.9% vs. 41.8-50.3% in C-101), and maintained levels of oleic acid lower than in line C-101. Selection for high erucic acid content in the M₃ and M₄ generations led to the fixation of this mutation in the M₅ generation (52.2-59.3% vs. 39.0-47.6% in C-101). This is the first high erucic acid line obtained in Brassica species through mutation breeding. Its utility in future programmes to develop very high erucic acid lines is discussed.     

Breeding for Modified Fatty Acid Composition by Induced Mutations in Linseed (Linum usitatissimum L.)

Seeds of linseed (Linum usitatissimum L.) cultivars ‘Raulinus’ and ‘Bionda’ were treated mutagenically with ethyl methanesulfonate (EMS) in order to broaden the variation of fatty acid composition in the seed oil and to select mutants with reduced linolenic acid (C18: 3) content. The M₂ generation was evaluated for fatty acid composition of the oil. Genetic variation for unsaturated fatty acids was demonstrated to be extended in the M₂ generation as compared to the controls. Finally in the M₅ generation of cv. ‘Raulinus’, lines with reduced C18:3 (< 40% vs. 55%) and correspondingly increased linoleic or oleic acid contents, respectively, could be selected. These mutants proved to be genetically stable as demonstrated by parent-offspring (M₄/M₅) regression.     

An induced brachytic mutant of chickpea and its possible use in ideotype breeding

Mutations were induced in chickpea (Cicer arietinum L.) cultivar ‘JG 315’ through treatment of seeds with ethyl methane sulphonate (EMS). One of the mutants, named JGM 1, had brachytic growth (compact growth), characterized by erect growth habit, thick and sturdy stem, short internodal and interleaflet distances and few tertiary and later order branches. It was isolated from M₂ derived from seeds treated with 0.6% EMS for 6 h. Segregation analyses in F₂ progenies of its crosses with normal chickpea genotypes (JG 315, ICC 4929, and ICC 10301) suggested that a single recessive gene controlled brachytic growth in JGM 1. This gene was not allelic to the br gene for brachytic growth in spontaneous brachytic mutant E100YM. Thus, the gene for brachytic growth in JGM 1 was designated br2 and the br gene of E100YM was redesignated br1. Efforts are being made to use JGM 1 in development of a plant type with short internodes and erect growth habit. Such plant type may resist excessive vegetative growth in high input (irrigation and fertility) conditions and accommodate more plants per unit area.     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. 5. The S-genotype of tetraploid potato cultivar gineke

Summary Investigations of the genetics of self-compatibility and self-incompatibility in dihaploids and diploid derivatives from cv. Gineke revealed the presence of S ₁, S₂ and S ₃ at the S-locus of Gineke and in addition an S ₁-allele on a translocation. By means of a complete tester set involving the S-alleles S ₁, S₂ and S ₃ (all from Gineke) and S ₄ (from Black 4495) it was demonstrated that some Gineke dihaploids were compatible with all six testers. This indicated a fourth S-allele in Gineke, which differs from those in the tester series and was therefore assigned S ₅. Additional evidence was obtained from an analysis of F₁'s from crosses of two S ₅-bearing dihaploids and one of the testers. So the S-genotype of cv. Gineke was identified as S ₁S₂S₃S₅/S₁, the second S ₁ being the S-allele on a translocated fragment.     

A new source of cytoplasmic male sterility in sunflower

Summary Interspecific substitutions of the nucleus of Helianthus annuus (2n=34) cv. Saturn into the cytoplasm of H. petiolaris (2n=34) by successive backcrossing, resulted in progenies with indehiscent anthers containing white, rather than normal yellow, pollen. Further backcrossing by cv. Saturn failed to restore pollen shed, suggesting that the male sterility was cytoplasmic. In vivo germination tests of pollen from 23 such plants from eight BC₅ lines, indicated complete pollen sterility for 14 plants, but normal seed set, suggesting that female fertility was not affected. Meiosis in all plants examined was normal.Crosses between seven sources of pollen-fertility restorer, one collection of wild H. annuus, and an existing source of cytoplasmic male sterility, resulted in a high frequency of plants with normal pollen shed in all F₁ progenies. However, no normal pollen shed was evident in F₁ progenies for similar crosses between BC₅ male-steriles and three of the seven restorer sources, nor for the single wild H. annuus evaluated. The foregoing suggests that the backcross substitution lines are a new source of cytoplasmic male sterility. The inheritance of restoration of pollen shed was complex and not fully elucidated. Some data suggested that two independent, complementary, dominant genes were required, but others indicated two to three independent, dominant genes.     

Induction and selection of mutations for resistance against bacterial leaf blight in rice

Summary Dry seed lots of a rice cultivar, Harebare, susceptible for bacterial leaf blight (BLB), were exposed to thermal neutrons with and without pre-treatment of seed for boron-enrichment, and to gamma-rays. M₁ plants of each of the treatments were grown and their seeds were separately harvested. M₂ populations were raised in rice fields of a farmer in a region where BLB is epidemic every year, and M₂ and control plants which expressed resistant reactions to BLB were selected. M₃ and control lines as plant progenies of the selected M₂ plants were raised in the same rice fields as in M₂ generation in order to investigate their BLB resistance and other agronomically important traits. Variances for disease severity of M₂ populations were significantly larger than those of the control population. whereas their means were not singificantly different from that of the control population, suggesting induction of mutations toward both resistance and susceptibility to BLB. Mean disease severity values of the M₃ lines from selected M₂ plants were significantly smaller than those of the control lines, indicating gains of selection in M₂ for quantitative resistance against BLB. Thermal neutrons, especially with boron-enrichment pre-treatment were effective to induce mutations for resistance against BLB. Some M₃ mutant lines with quantitatively enhanced resistance against BLB were not modified in other agronomic traits from those of the original cultivar. Significance of the induced quantitative resistance in breeding programmes for BLB resistance is discussed.     

Present aspects of induced mutations in plant breeding

The present status of the utilization of induced mutations in plant breeding is briefly reviewed. It is concluded that with induced mutations in principle successes can be expected similar to those with the conventional breeding methods. Owing to the relatively small yield of progressive mutations the efficiency of mutation breeding, however, is rather poor at present. Greater efficiency may be expected with increased knowledge of both control of original mutation production and of selection.Control of mutation production seems to be possible in at least three ways, (a) by raising the total mutation frequency (b) by changing the relative frequency of chromosome versus point mutations and (c) by altering the spectrum of point mutations. The possibilities of such control through the diverse action of different radiations given with or without modifying agents is reviewed and various mutagenic chemicals are mentioned.Control of mutation selection may be achieved in two ways, (a) by a better understanding of diplontic selection (intrasomatic selection) of mutated cells and (b) by developing appropriate screening methods. A working hypothesis concerning the diplontic selection is briefly outlined. The one initial cell theory means that the greatest efficiency of mutation production can be expected after radiation of primordia or young buds with only one or a few initial cells which will form the tissue of interest. It is hoped that by this the intercellular competition is restricted and a reduced elimination of mutated cells will result.Procedures for selection of mutants, at least in barley, can already start with M₁-spikes. It was shown that completely fertile M₁-spikes possess the same frequency of point mutations (chlorophyll mutations) as those with disturbed fertility. Selection of fertile M₁-spikes should, therefore, eliminate to a large extent the undesirable chromosome mutations and in this way increase the efficiency of screening for progressive mutations. Maximum mutation frequencies of fertile M₁-spikes can, however, only be achieved if the tillering is reduced.It is suggested that more emphasis be put on screening of small mutations, which may generally be expected to have a greater importance for practical purposes than drastic deviations. Usually screening of micro vital-mutations will be advisable in the M₃-generation. Indicator characters may be found through mass selection methods which by their pleiotropic gene action also effect properties eventually of breeding value.Samenvatting Na een kort overzicht van de perspectieven voor toepassing van kunstmatige mutatie bij de plantenveredeling wordt nader ingegaan op enige problemen die thans de aandacht hebben. Voor het beheersen van de kunstmatige mutaties is het van veel belang de frequentie van mutatie te kunnen vergroten en te kunnen bevorderen dat relatief minder chromosoom- en meer gen-mutaties tot stand komen. Vooral van de micromutaties verwacht schrijver vooruitzichten voor de plantenveredeling. Het is echter thans nog zeer moeilijk daarop te selecteren.(Lecture delivered at the organizational meeting of the section Mutation and Polyploidy of EUCARPIA in Lund and Svalöf, July 9–11, 1958).     

Two powdery mildew resistance mutations induced by ENU in Pisum sativum L. affect the locus er1

The alkylating compounds and strong mutagens methylnitrosourea (MNU) and ethylnitrosourea (ENU) were used to widen the genetic variability of pea (Pisum sativum L.) via experimental mutagenesis. Amongst multiple mutations of agronomical interest, for the first time two mutations conferring broad-range resistance to powdery mildew (Erysiphe pisi Syd.) were induced in pea. Mutagenic treatments were carried out on seedlings using a technique that ensures very high mutagenic efficiency. Two-hour exposure of cv. Solara seedlings to chemical mutagens resulted in almost non noticeable lethality and sterility in the M1 generation and very high mutation rates: ~39% families with visible mutations in the M2 generation. The influence of the cell cycle phase on the process of mutagenesis was studied in cv. Frilene using a previously developed technique for synchronization of shoot apical meristem cells. The cell cycle phase at which cells were treated apparently did not influence the lethality and sterility in the M1 generation, while the visible mutation rate, assessed in the M2 generation, showed a clear cell cycle dependency. Seedlings treated at the G2 and M phases gave rise to progenies exhibiting the highest mutation rate, over 50% of M2 families with visible mutations. The powdery mildew resistant (PMR) mutant S(er1mut1) was induced by treatment of cv. Solara seedlings for 2 h with ENU, while the PMR mutant F(er1mut2) was induced by treatment of cv. Frilene seedlings with the same chemical mutagen for 1 h during the G2 phase of the cell cycle. The genetic analysis of the novel PMR mutant lines showed that both resistances are inherited as monogenic recessive traits. The performed genetic complementation analyses revealed that both mutations affect the same locus—er1, which determines most of the natural sources of PMR in pea. A project aiming at the isolation of the powdery mildew resistance mutated gene via map based cloning is currently under way.     

Variation and covariation in Vicia faba L. populations of Mediterranean origins

Summary Variation and covariation in 15 Vicia faba L. ssp. major populations from Italy. Greece and Egypt were analyzed. Ten quantitative morphobiological characters of agronomical and nutritional interest were observed. Differences among countries of origin were highly significant for all characters except for lowest podded node. Phenotypic distances among populations, both within and among origins, estimated on canonical variates evidenced that the Egyptian populations from a distinct group and that a relevant degree of similarity exists among the Greek and the Italian meterial. The covariation structure within populations, which was studied by means of factor analysis, was comparatively simpler in the Egyptian material than in that form the other two Mediterranean countries. The differences evidenced can be attributed to different gene associations between characters and suggest the possibility of obtaining, through recombination, genotypes agronomically more favorable.Research work supported by C.N.R., Italy, special grant. Sub-Project 1, Paper No. 299.