Inheritance of morphological traits of periwinkle mutants with modified contents and yields of leaf and root alkaloids

Periwinkle Catharanthus roseus is a medicinally important plant producing anticancer and antihypertension alkaloids. Two mutants, one dwarf and one semidwarf and both, with a high alkaloid content in the roots and one mutant with a wavy leaf margin and a high alkaloid content in its leaves were obtained after induced chemical mutagenesis with ethyl methane sulphonate and N-nitroso-N-ethylurea in the variety ‘Nirmal’, which is resistant to dieback disease. These mutants were evaluated in the M₃ and M₄ generations. The dwarf and semidwarf mutants differed from the parental variety for many morphological characters, while the mutant with the wavy leaf margin differed mainly in leaf size and leaf thickness. Although both dwarf and semidwarf mutants showed a significantly higher alkaloid content in their roots in both generations, only the semidwarf mutant gave a significantly higher (23%) root alkaloid yield than the parental variety. The mutant with the wavy leaf margin showed a significantly higher alkaloid content in the leaves in both the M₃ and M₄ generations and also had a significantly higher (21%) leaf alkaloid yield than the parental variety. All three mutants were found to be controlled by monogenic recessive genes; the genes for ‘dwarfness’ and ‘semidwarfness’ were allelic to each other, with the allele for semidwarfness being dominant over the allele for dwarfness. The gene for the wavy leaf margin was inherited independently of the genes for dwarfness and semidwarfness.     

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.     

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.     

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.     

Screening of Chinese cabbage mutants produced by 60Co γ‐ray mutagenesis of isolated microspore cultures

Since the release of the Chinese cabbage genome sequence, increasing interest has focused on the functional analysis of unidentified genes in Chinese cabbage. Mutant analysis forms the basis of functional genomics research. To produce a variety of Chinese cabbage mutants in the same genetic background, buds containing late uninucleate spores from a doubled haploid line of the Chinese cabbage variety ‘Fukuda 50’ were irradiated with ⁶⁰Co γ‐rays at doses of 20, 40 and 60 Gy. Then, the treated microspores were isolated and cultured. A total of 492 putative M₀ mutants were isolated from 1483 regenerated plants. Of these, six M₁ mutants were verified; the mutant frequency was 0.41%. These mutants comprise a mutant library that includes one plant shape mutant, two flower mutants and three male sterile mutants. Pollen viability detection and DNA flow cytometry were used to determine the ploidy of the regenerated plants. Some of the mutants isolated in this study may be useful for Chinese cabbage breeding and functional genomics research.     

Genetic mapping of the barley lodging resistance locus Erectoides‐k

The barley (Hordeum vulgare L.) mutant erectoides‐k.32 (ert‐k.32) was isolated in 1947 from an X‐ray‐mutant population of cultivar ‘Bonus’. The mutant was released as a cultivar in 1958 with the name ‘Pallas’ – one of the first cereal crop cultivars developed from induced mutants. ‘Pallas’ is a semi‐dwarf barley cultivar known for its culm stability and resistance to lodging. In total, eight allelic ert‐k mutants are known that show different phenotypic strength concerning culm length and spike architecture. They represent alternatives to the widely used, but pleiotropic ‘Green Revolution’ alleles of the Sdw1 (semidwarf1/denso) and Uzu1 (semi‐brachytic1) genes in breeding of robust elite barley cultivars. In the present study, we locate Ert‐k to a 15.7‐cM region in the centromeric region of chromosome 6H. Although the interval is estimated to contain approximately 700 genes, the work provides a solid foundation for the identification of the underlying mutations causing the ert‐k lodging‐resistant phenotype. In addition, the linked markers could be used to follow the ert‐k mutant genotype in marker‐assisted selection of new lodging‐resistant barley cultivars.     

Variation in seed proteins from mutagen-treated cultivars and selected lines of Vigna unguiculata L. Walp

The storage protein profiles of the seeds of two IITA cowpea cultivars (‘IT84E–124’ and ‘Vita 7’) exposed to three mutagens—sodium azide (NaN₃), ethyl methane sulphonate (EMS) and ⁶⁰Co gamma rays—and those of 18 selected M₃ lines were investigated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). The total seed protein, globulin and albumin fractions showed differences in number and intensity of subunit bands, but the least differences were found in the albumin fraction. Both high and low molecular weight bands were observed, the highest being 94.5 kDa and the lowest 12.0 kDa.‘Vita T showed less variability compared with ‘IT84E–124’, as indicated by the relative similarity indices (S.I.) of the two cultivars. The lowest S.I. of 0.200 was found among ‘IT84E–124’ lines while the lowest S.I. among ‘Vita T lines was 0.4706. A number of lines with particular traits were found to be characterized by the presence of specific polypeptide bands. This study demonstrates that induced mutation could create additional variability to supplement existing germplasm and that SDS-PAGE is a useful tool for discriminating and estimating genetic similarities among selections.     

Identification of a major blast resistance gene in the rice cultivar 'Tetep'

Analysis of near‐isogenic lines (NILs) indicated the presence of a novel resistance gene in the indica rice cultivar ‘Tetep’ which was highly resistant to the rice blast fungus Magnaporthe grisea.‘Tetep’ was crossed to the widely used susceptible cultivar ‘CO39’ to generate the mapping population. A Mendelian segregation ratio of 3 : 1 for resistant to susceptible F₂ plants further confirmed the presence of a major dominant locus, in ‘Tetep’, conferring resistance to the blast fungal isolate B157, corresponding to the international race IC9. Simple sequence length polymorphism (SSLP) was used for molecular genetic analysis. The analysis revealed that the SSLP marker RM 246 was linked to a novel blast resistance gene designated Pi‐tp(t) in ‘Tetep’.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of non-narcotic (opiumless and alkaloid-free) opium poppy, Papaver somniferum

A mutation breeding programme using gamma rays and ethyl methane sulphonate (EMS) was carried out for genetic conversion of narcotic ‘opium poppy’ into non-narcotic ‘seed poppy’. Two families contained 31 latexless (opiumless) and 23 partial latex-bearing plants which were identified in the M² and confirmed in the M³ generations by the ‘ray-pluck’ method. Thin-layer chromatography (TLC)-densitometry of powdered capsule hulls (straw) from 22 representative plants also revealed that none of the five opium alkaloids (morphine, codeine, thebaine, papaverine and narcotine) was present in the straw. Six plants also possessed high seed yield (4.0–5.66 g/capsule vs. 3.39 g for control) and seven had a high seed oil content (50.7-53.5%). The best mutant genotype, LL-34 of the family C¹-Comb-113-2, with 5.66 g/capsule seeds containing 52.6% oil was designated as cv. ‘Sujata’. This, perhaps the world's first opiumless and alkaloid-free seed poppy cultivar, offers a cheap and permanent (fundamental) solution to the global problem of opium-linked social abuse. Simultaneously, it serves as a safe and potential food crop with protein-rich seeds and healthy unsaturated seed oil.     

Broad-few-leaflets and outwardly curved wings: two new mutants of chickpea

This study was aimed at the induction of morphological mutations for increasing genetic variability and making available additional genetic markers for linkage studies in chickpea (Cicer arietinum L.). A wilt‐resistant, well‐adapted chickpea cultivar of central India,‘JG 315’(Jawahar gram 315), was used for the induction of mutations. Seeds presoaked in distilled water for 2 h were treated with ethyl methane sulphonate (EMS) using six different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%) and two different durations (6 and 8 h). Several morphological mutants were identified in M₂. One of the mutants, isolated from a treatment of 0.3% EMS for 8 h, had five to nine large leaflets per leaf in comparison with 11‐17 normal‐sized leaflets per leaf observed in the parental cultivar ‘JG 315′. The mutant was designated broad‐few‐leaflets. Many leaves of this mutant showed a cluster of three to five overlapping leaflets at the terminal end. The other mutant, designated outwardly curved wings, was isolated from the 0.5% EMS treatment for 6 h. In this mutant, the wings were curved outwards, exposing the keel petal, while the wings in typical chickpea flowers are incurved and enclose the keel. The lines developed from the broad‐few‐leaflets and outwardly curved wings mutants were named JGM 4 (Jawahar gram mutant 4) and JGM 5, respectively. Inheritance studies indicated that each of these mutant traits is governed by a single recessive gene. The gene for broad‐few‐leaflets was designated bfl and the gene for outwardly curved wings was designated ocw. The locus bfl was found to be linked with the locus lg (light green foliage) with a map distance of 18.7 ± 6.3 cM.     

Induced Vine Mutant in Vigna mungo

Pure dry seeds of variety T₉ of Vigna mungo were irradiated by 10, 20 and 30 kR of gamma rays at 33 °C followed by post irradiation treatment with 0.25 % EMS in phosphate buffer (pH 7.0) at 30 ± 1 °C. Vine mutants were isolated from segregating families after 20 kR and 10kR ± EMS treatment. The genetic nature of the mutants was tested by crossing with the wild type. The segregation in F² proved the mutant to be monogenic recessive. The mutant showed slight improvement in protein content and marked improvement in yield of seeds.     

大豆“南农86-4”突变体筛选及突变体库的构建

应用⁶⁰Co γ射线和甲基磺酸乙酯(EMS)分别对“南农86-4”大豆种子进行诱变，并构建大豆突变体库。结果在M₃代分别获得40份和145份叶、茎、花、种子、子叶等性状变异的材料。在两种方式诱变的M₃代都获得蛋白质和油含量变化的材料，尤其在EMS诱变的M₃代中获得47份蛋白质含量比对照高5个百分点以上的材料，5份蛋白质和油总含量比对照高5个百分点以上的材料。这些突变体可以作为新的种质资源，构建的突变体库也有助于大豆功能基因组研究的开展。     

Variability among advanced gamma-irradiation induced large-seeded mutant breeding lines in the 'Georgia Browne' peanut cultivar

The objective of this study was to compare the variability among advanced large‐seeded ‘Georgia Browne’ mutant breeding lines induced by γ‐irradiation. Seeds of the small‐seeded, high‐yielding, disease‐resistant peanut cultivar ‘Georgia Browne’ were exposed to a 200 Gy dose of γ‐radiation. Several advanced (M_6:7–M_6:9)‘Georgia Browne’ mutant breeding lines were developed and evaluated at the University of Georgia, Coastal Plain Experiment Station over three consecutive years 1997‐99 for disease incidence, pod yield, total sound mature kernels (TSMK) grade, pod weight, seed weight and seed size distribution. Field performance tests showed significant differences among the advanced large‐seeded mutant breeding lines compared with ‘Georgia Browne’ for each of these variables. The results obtained demonstrate the beneficial use of mutation breeding for inducing and developing variable and desirable advanced mutant breeding lines within peanut cultivars.     

A novel environment-induced genic male sterile (EGMS) mutant in indica rice

Summary Male-sterile mutants were isolated from M₂ and M₃ generations of indica rice variety 26 Zhaizao, dry seeds of which had been exposed to ⁶⁰Co- rays at a dose of 290 Gy. The mutants were planted in early season and ratooned in late season for two successive years for identification of fertility conversion in different growing seasons. One of the mutant lines was further observed in a growth chamber and in the field. Results showed that daily average temperature might be the major factor conditioning the male fertility conversion at a moderate daylength. The critical temperature for the male fertility conversion of the mutant grown under 12.5 h and 14.0 h daylength is about 23°C, below which the plant becomes completely male sterile. Its male fertility conversion character differs from other EGMS lines so far developed. The performance of the hybrids between the mutant and some other indica varieties demonstrated its good combining ability and its potential value in hybrid rice production. The obtained mutant line still sheds KI-stainable pollen grains under male sterilizing conditions. Nevertheless, pollen grains shed from the male sterile plants were much more vulnerable than from normal plants. At sucrose concentration below 1.5 M, the pollen grains from the mutant grown under male sterilizing conditions almost completely broke down, while above 1.5 M they became plasmolysed and shrunken. This is indicative of poor development of the membrane and walls of the pollen grains from the male sterile mutant, causing the pollen grains to be unfunctional. NBT test also clarified the abortion of the pollen grains from the mutant, which were formed in the male sterilizing environment.     

高能重离子辐射对水稻的生物学效应

本文观察了数种高能重离子辐射处理水稻干种子的生物学效应，结果表明；与具低传能线密度的＾Ｃｏ６０－γ射线相比，这些具高ＬＥＴ的重离子能更有效地抑制受照射种子萌发生出的幼苗的生长，诱导根尖细胞和花粉母细胞的染色体畸变和微核形成，降低当代植株的结实率，并发后代出现形态性状及农艺性状的变异，在一定剂量范围内，辐射诱导的上述数种生物学效应与吸收剂量间呈线性关系，根据剂一－效应关系曲线，估算了重离子辐射的上述     

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.     

理化诱变小豆京农6号突变体的鉴定

选用小豆品种京农6号种子,分别采用甲基磺酸乙酯(EMS)(0.5%、0.9%和1.4%处理12h和24h)、电子束(100、300、600Gy)、60Co-γ(400Gy)诱变处理,将处理后的种子种于大田,鉴定后代植株性状的变异。观察表明,EMS诱变的变异类型最丰富、60Co-γ射线次之、电子束产生的变异类型较单一。EMS处理小豆以浓度0.5%和0.9%处理24h为宜;0.5%EMS处理的粒色和荚色变异突出,有鲜红、黄白、绿白粒色和黑荚、褐荚、黑褐荚变异;0.9%处理的叶形变异突出,有鸡爪叶、剑叶、肾形叶、小密叶等突变类型;电子束诱变后,M2变异率分别为4.09%、3.64%和2.22%。400Gy60Co-γ射线处理种子,后代变异率为7.23%。通过两年的鉴定筛选,获得937个EMS诱变M3代株系,934个60Co-γ射线和电子束诱变M2代株系,已得到株高、叶形、叶色、粒形、粒色、荚色、无分枝、多分枝、叶簇生、分枝簇生、光叶、蔓生、有限结荚习性、株型松散、育性、成熟特性等突变体材料1490份。本研究为小豆基因遗传分析、基因定位与克隆及其进一步的基因功能分析奠定了基础,为小豆育种提供了重要的材料。     

Gamma Ray-Induced Field Tolerance to Phytophthora Blight in Sesame

Due to the non-availability of sources of resistance in Sri Lanka to Phytopbthora blight in sesame (Sesamum indicum) caused by Phytopbthora nicotianae var. parasitica, a mutation breeding programme was initiated. Seeds of three genotypes were subjected to six doses of gamma rays from 100 Gy to 750 Gy from a ⁶⁰Co source. Seeds of M₂ bulks, sampled by variety and treatment from the first five capsules formed on M₁ plants, were grown in a field with a history of repeated incidence of the disease. The best 21 lines, having the highest survival and seed production, identified in the screening of M₃ and M₄ progeny rows of selected M₂ single plants were then tested in a replicated field trial with the recommended cultivar ‘MI 3’ as a control. The plant survival in selected lines averaged 43.3 percent as against 7.2 percent in the recommended variety. Eight selections recorded significantly higher seed yield than ‘MI 3’ at P < 0.01 and another three at P < 0.05. Gamma ray treatments of 450 Gy and 600 Gy produced more lines tolerant to the disease than the other doses used.     

The mutagenic effect of treatment with EMS at different temperatures in Pisum sativum

Dry peas were treated with solutions of 0.13% and 0.2% EMS for 24 hours at temperatures of 6°, 12°, 18° and 24°C, respectively. The effect of the temperature during treatment was studied in the M₁ and in the M₂ generation.The germination capacity of the treated seeds and the percentage surviving M₁ plants were unfavourably affected only after treatment at 24°C. Leaf spotting and growth inhibition in the M₁ plants increased with increasing temperature, while fertility decreased. After treatment at 24°C more than 75% of the harvested M₁ plants were completely sterile.With rising temperature the percentage mutants in the M₂ increased from 8 to 50%. However, the fertility of the mutants was considerably reduced. The sterility after treatment with EMS, therefore, must be at least partly due to genetic changes. This sterility greatly diminishes the mutagenic efficiency of EMS treatment at high temperatures.