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.     

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.     

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.     

Mutation research in peas

Dry seeds of the pea variety Pauli were irradiated with 15,000 r X-rays, 18,000 r X-rays, 18,000 r rays and with neutrons; the latter treatment for 6 hours. In addition the chemical mutagen ethyl methane sulphonate (EMS) was used, in concentrations of 0.33% and 0.50% respectively.After raising the germlings in a glasshouse surviving plants were planted in the field. The germination capacity was only little disturbed after the irradiation; however, it was greatly reduced after the EMS treatment. A very large number of germlings did not grow further, their roots showing a deep brown discoloration, ultimately leading to death. Several other influences after planting in the field killed many other plants.The fertility of the M₁-generation was only little disturbed by the irradiation, but much by the EMS-treatment. In the M₂-generation, both after irradiation and after EMS-treatment, at higher dosages and concentrations lower percentages of mutated progenies were found than at lower dosages and concentrations. This was presumably due to the fact that the number of plants per M₂-line in the former case was on average twice as high. The percentage progenies with mutations and the percentage mutated plants was larger after EMS-treatment than after irradiation.Of normal looking plants with a good fertility, progenies were raised in non-replicated small plots. The average yield per plant and the distribution in the irradiations did not differ significantly from that of the untreated material. After EMS-treatment the average yield was significantly lower and the distribution significantly higher. The fertility disturbance caused by EMS, therefore, also became manifest in the M₃-generation.     

Induction of mutations for rust resistance in wheat

About 25,000 M₂ and M₃ seedlings of the bread wheat variety C. 591 were tested for resistance to 3 races of stem rust. A mutant with resistance to race 40 of stem rust was found in a family derived from treatment with 16,000r of X-rays. No other mutant was found. Albina seedlings were found to be totally free of rust infection. From the present study it seems that mutations for rust resistance can be induced in bread wheat by ionizing radiations but the frequency of such mutations appears to be very low.     

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.     

Frequency and spectrum of mutations induced by radiations in some varieties of bread wheat

1. 1.
   The frequency and types of mutations induced by different dosages of X-rays, fast and thermal neutrons and beta particles from P³² and S³⁵ were studied in two fully awned (C. 591 and H. 167) and two awnless strains (N.P. 809 and H. 389) of bread wheat.     
   
   

Improvement of seed yield in Vicia faba L. By using experimental mutagenesis. II. Comparison of gamma-radiation and ethyl-methane-sulphonate (EMS) in production of morphological mutants

Summary Gamma-rays and EMS have been used in two dosages to test their effectiveness in inducing mutations in one major (cv. Aguadulce) and one minor (cv. Manfredini) Vicia faba cultivar. The plants of the M₁ and M₂ generations showed reduced emergence, survival, and fertility. EMS was more effective than gammarays in inducing mutations giving a mutation frequency in M₁ and a frequency of M₂ mutants from two to four times higher. The two mutagenic agents do not seem to differ in mutation spectrum. The minor cv. Manfredini was more sensitive to the lower dosage of gamma-rays than the major cv. Aguadulce. Both mutagens proved to be effective in enlarging the morphological variation of both cultivars.Research work supported by C.N.R. special grant I.P.R.A.-Sub-project 1. Paper N. 515.     

Early Embryo-Sac Development in Faba Beans (Vicia faba L.)

Cuphea viscosissima Jacq. is an herbaceous annual dicot indigenous to the U.S. This species is being developed as a new oilseed crop and commercial source of medium-chain fatty acids (MCFAs). In this paper, the phenotypes and genetics of the mcm-1 and cpy-1 mutations of C. viscosissima are described. These mutations affect the percentages of fatty acids of seed storage lipids. They were induced by treating seeds with 0.04 M ethyl methanesulfonate for 5 h. Observed segregation ratios for these mutations were not significantly different from expected ratios for codominant genes. The mcm-1 mutation eliminates 95.0 % of wildtype MCFAs — caproic (C6: 0), caprylic (C8: 0), and capric (Cio: 0) acid percentages are decreased from 0.8 to 0.0 %, 20.7 to 0.5 %, and 68.6 to 6.7%, respectively. The cpy-1 mutation decreases C6: 0 and C8: 0 from 0.9 to 0.0 % and 19.8 to 3.9 %, respectively, whereas it increases C12: 0 and C14: 0 from 2.2. to 14.3 % and 0.8 to 7.4 %, respectively.     

Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annum L.)

In vitro regenerated sunflower (Helianthus annuus L.) plants (R₁) were self-fertilized and the R₂ generation was evaluated for qualitative traits. A broad range of phenotypic variation was observed and mutation frequencies were calculated. Some in vitro induced variant phenotypes were similar to known spontaneous or induced mutations in sunflower, while others were new. Chlorophyll and carotenoid deficiencies, chimaerical variegation, fasciated stem and capitulum, abnormal shoot development, and other morphological variations, were noted. Substitution of anthers with petaloid structures in a disk-floret mutant indicates a possible homeotic mutation induced by in vitro tissue culture.     

Effect of gamma radiation on growth and lignin content in Brachypodium distachyon

Brachypodium distachyon has been highlighted as a model monocot plant with small genome and short life cycle. Biofuels are being developed as renewable energy sources to replace fossil fuels. Bioethanol production is negatively correlated with lignin content. Here, Brachypodium was acutely or chronically irradiated at doses of 50, 100, 150, 200, and 250 Gy. The effect of radiation on plant growth and generation of mutant populations was explored. The lethal effect of radiation was higher in acutely irradiated M0 populations. A dose-dependent negative effect in plant height, tiller number, floral spikelet, and total seed number was observed, with a positive effect in days to heading. The phenotype of 1,773 M₁ plants was evaluated, with 417 plants being selected to construct the M₂ population. The 31 M₂ plants that showed the least staining with phloroglucinol were selected. These mutants could be useful materials for studies such as identification of nucleotide substitutions in genes involved in lignin biosynthesis pathway, monitoring of mutant physiological traits, and evaluation of fitness for bioethanol production. As biological resources, the M₂ populations generated in this work will contribute to studies of functional genomics of Brachypodium and to the breeding of grass crops.     

水稻类病变突变体c5的遗传分析与目标基因的精细定位

水稻类病变突变体c5是由粳稻品种中花11种子经化学诱变剂EMS (甲基磺酸乙酯)诱变处理得到的。该突变体叶片在三叶期开始出现近似圆形褐色斑点，经DAB染色和台酚蓝染色显示这些斑点积累了过多的H₂O₂并引起程序性细胞死亡。与野生型相比，突变体c5的成熟期株高从110.4 cm减少到74.6 cm，有效分蘖数和每穗着粒数分别减少23.7%和28.5%，千粒重和结实率都显著降低，此外，c5还表现出对白叶枯病菌的广谱抗病性，对10个菲律宾生理小种都有强烈的抗性反应。遗传分析表明，c5的突变性状受单隐性核基因控制。利用c5和明恢86配组形成的包含6269个单株的F₂群体和18个分子标记，将c基因限定在水稻第5染色体长臂STS标记S41和S47之间大约102 kb的遗传距离内。序列分析发现该区间内其中有11个编码基因，且它们与现已报道的类病变基因都不同，暗示c5可能是一个新型类病变性状控制基因。     

All hermaphrodite progeny are derived by self-pollinating the sunrise papaya mutant

Self‐pollination of a hermaphroditic cultivar normally gives a ratio of 2 : 1 hermaphrodite to female papayas with genotypes M₂m and mm, respectively. Much effort has been dedicated to marking the sexual types of papaya at the seedling stage to distinguish hermaphroditic from female papayas. A hermaphroditic papaya mutant (SR*) has been obtained, derived from the ‘Sunrise’ papaya cultivar mutant. Self‐pollination of the mutant resulted in all progenies being hermaphroditic. The genotype of the female was lethal, as a result of a lethal gene being linked to the mm female gene complex in this case. However, a 3 : 1 segregation ratio was obtained from the progeny of the hermaphroditic cultivar ‘Thailand’ crossed with SR*, indicating that all genotypes survived. Homozygous genotypes (M₂M₂) would be lethal according to Storey's model. Randomly selected F1 plants of the ‘Thailand’ SR* combination were self‐pollinated to obtain an F₂ generation. The F₂ segregation ratio suggested that the SR* mutant had a different form of the M₂ allele, now designated as M_@, which allowed the dominant M_@M₂ to survive in cross combinations. Genetic study has proved that SR* has the M_@ml genotype, a new mutant. It is capable of producing all hermaphroditic papaya progenies.     

对一个新发现的棉纤维突变体的鉴定及特性分析

在农杆菌介导的转基因组织培养再生后代中发现了一个无绒有絮的纤维发育突变体,通过自交选择T₃代获得其纯合体,命名为CM突变体。尽管CM突变体从转基因后代中发现,但和转基因插入位点无关,推测是在组织培养过程中产生的点突变所致。通过与陆地棉遗传标准系TM-1,海岛棉军海1号,以及新乡小吉无绒有絮(XinFLM),新乡小吉无绒无絮(XinWX),徐州142无绒无絮(XZ142WX),显性光子N1N1,隐性光子n2n2、SL-7-1、MD17及T586等一系列纤维发育突变体分别配制F₂组合进行突变基因的遗传及等位性分析,结果表明CM突变体与纤维发育正常的TM-1和军海1号杂交,F₁表型均为无绒有絮,F₂表现无绒有絮和有绒有絮3∶1分离,说明该突变体与纤维发育正常材料相比,在短绒发育方面存在一个位点的差异,该突变性状由单显性基因控制。等位性测验及分子定位均表明, 控制该突变体短绒的基因与控制N1N1显性光子的N1基因等位。扫描电镜进一步证明该基因突变会造成纤维起始突起延迟。与N1N1突变体相比,CM突变体的衣分比N1N1显著高,而百粒重比N1N1极显著低。推测CM突变体中的突变基因与显性N1基因为复等位基因。     

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.     

Somatic Embryogenesis in Maize and Comparison of Genetic Variability Induced by Gamma Radiation and Tissue Culture Techniques

Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F₁ and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with ⁶⁰Co gamma radiation in situ. Mature caryopses were harvested and assigned as M₁ material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R₁ generation, while regenerants from irradiated explants were considered as M₁R₁ generation. The genetic variability was evaluated in the M₂, R₂ and M₂R₂ generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M₂ populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M₂R₂ generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R₃ and M₃R₃ generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.     

EMS-induced mutation of an endoplasmic reticulum oleate desaturase gene (<Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">2</Emphasis>) results in elevated oleic acid content in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The development of rapeseed cultivars (Brassica napus L.) with high oleic acid and low linolenic acid is highly desirable for food and industrial applications. In this study, the Korean rapeseed cultivar Tamla was used for ethyl methanesulfonate (EMS)-induced mutagenesis and seed oils were screened up to generation M₇ for high oleate mutants. Two mutant populations (M₇) with an average of approximately 76% oleic acid content were isolated. Yield components between two mutant populations and untreated Tamla plants were not substantially different, although the mutants in the vegetative stage were slightly smaller in size than Tamla. Genomic analyses of six fatty acid desaturase (four FAD2 and two FAD6) genes revealed that the elevated oleic acid content in the mutants is the result of single gene mutations. Changes in DNA sequence were observed in two genes out of six fatty acid desaturase (four FAD2 and two FAD6). FAD2-2 exhibited a 2-bp deletion in the upstream region of the gene in the two mutants, resulting in a severely truncated polypeptide (57 aa instead of 469 aa), while six point mutations in the other gene did not result in changes in the amino acid sequence. Based on these results, FAD2-2, an endoplasmic reticulum (ER) oleic acid desaturase, is affected in the mutants, resulting in a ~ 7% increase in oleic acid content in comparison to untreated Tamla plants. The induced mutants could be utilized for the development of high oleic oil rapeseed varieties and for regulatory studies of lipid metabolism in seed oils.     

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.)     

Productivity and Cytogenetic Analysis of an Unbranched Mutant in Hyoscyamus niger L.

An unbranched mutant (Ub) with marginal arrangement of large leaves substantially altered floral and reproductive features in H. niger was isolated in M₂. It bred true for all mutant characteristics in M₂ generation. Appearance of restricted branching (Rb mutant) in some M₃ families seems 10 be a transient phase. Erect growth habit Coupled with non-overlapping leaf-disposition is useful for high crop density to obtain higher biomass field and, in turn, more crude drug production per unit area. Improvement in alkaloid content is an additional advantage augmenting its economic viability. Seed setting was, however. Poor due to pollen-polymorphism accruing from desynapsis and lack of organized alignment of chromosomes at Ml and subsequent abnormal disjunction at Al.     

New mutants of winter rapeseed (Brassica napus L.) with changed fatty acid composition

Seeds of the winter oilseed rape (Brassica napus L.) line PN 3756/93 were treated with ethyl methanesulphonate to induce mutations in the fatty acid biosynthetic pathway. The seed mutagenic treatment was repeated in the M₂ generation. After treatments, individual seed and plant selections were made for changes in fatty acid composition during several generations of inbreeding. Self‐pollinated plants with changed fatty acid compositions were inbred to obtain genetically homozygous and stable mutant lines. Two mutants, M‐10453 and M‐10464, with increased levels of oleic acid (approximately 76%) and reduced linoleic and linolenic acid contents (8.5% and 7.5%, respectively) were selected. Gene or genes controlling desaturation of oleic acid were probably mutated in these plants. The third mutant, M‐681 had a very low linolenic acid content (approximately 2.6%) and increased linoleic acid content (approximately 26%). This would suggest the occurrence of mutations in genes controlling linoleic acid desaturation. The results of selection work during several generations showed that the environment had substantial influence on the composition of seed oil. This made the search for mutants with modify fatty acid compositions difficult. The induced mutants are not directly usable as new varieties, but can be used as parents in crosses for the development of high quality rapeseed varieties.