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.     

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.     

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。     

Induction of <Emphasis Type="Italic">Fusarium</Emphasis> wilt (<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis>) resistance in garden pea using induced mutagenesis and in vitro selection techniques

Wilt caused by Fusarium oxysporum f. sp. pisi is a serious production constraint for peas worldwide. An attempt was made to isolate wilt-resistant mutants in two susceptible pea genotypes, Arkel and Azad P-1, employing induced mutagenesis and in vitro selection techniques. Two thousand seeds of each genotype were mutagenized either with ethyl methane sulfonate (EMS, 0.2% and 0.3%) or gamma rays (5-22.5 kR) in ⁶⁰Co gamma cell for three consecutive years. Screening of different mutagenized populations under wilt-sick plots resulted in the isolation of 25 mutants exhibiting complete or enhanced wilt resistance compared to parental genotypes. Five of these wilt-resistant mutants also outperformed the susceptible background genotypes in terms of yield and other horticultural traits. Efforts were also made to isolate wilt-resistant regenerants from callus cultures exhibiting insensitivity to culture filtrate (CF) of F. oxysporum f. sp. pisi. A total of 250 regenerants (R ₀) were obtained from CF-insensitive calli selected from medium supplemented with 20% culture filtrate. When evaluated in artificially inoculated sick plots, only five R ₂ lines obtained from the regenerants exhibited enhanced wilt resistance compared to parental cultivars. However, the selected lines did not exhibit resistance levels equivalent to those shown by wilt-resistant lines isolated through in vivo mutagenesis. To conclude, induced mutagenesis through irradiation and EMS treatments exhibited superiority over in vitro selection for inducing wilt resistance in peas.     

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.     

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.     

Individual and combined effects of plant height reducing genes in periwinkle

Inheritance of an induced bushy mutant (EMS 24-5) of periwinkle (Catharanthus roseus) and its genetic relationship with an earlier reported dwarf mutant (NEU 6-15) was studied by crossing it with its parental variety, Nirmal and the dwarf mutant. The bushy mutant was found to be under the control of a monogenic recessive gene (by), which was inherited independently of the recessive gene (dw ₁ ) involved in the dwarf mutant. The double mutant (bydw ₁ ) recombinant, which was recovered from the F₂ generation of the cross (NEU 6-15) × (EMS 24-5), its parental mutants and variety, Nirmal were used for determining the individual and combined effects of plant height reducing genes dw ₁ and by. Individually, the recessive genes by and dw _1, reduced plant height by 27–30, 64–68%, respectively, while together they reduced plant height by 73–77%. The double mutant recombinant was found to be 63–68 and 25–29% shorter than its parental mutants (EMS 24-5) and (NEU 6-15), respectively. The effects of the recessive gene by on plant height were discernable only after the age of 4 months while those of dw ₁ and combined effects of dw ₁ and by became apparent one to 2 weeks after germination. The double mutant recombinant was found to have higher content of total alkaloids in the roots than parental mutants as well as the variety, Nirmal.     

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。     

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.     

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.     

Genetics of Seed Yield and its Components in Common Bean (Phaseolus vulgaris L.) of Middle-American Origin

Genetic variance, heritability, and expected response from selection arc useful in devising alternative methods and criteria of: selection. The objectives of this study were to estimate these for seed yield and its components from 200 F₂: populations involving 80 cultivars and lines of mostly small-seeded dry bush bean (Phaseolus vulgaris L.) of habits growth I, II, and III of Middle-American origin. All cultivars and lines were crossed in eight sets of ten parents each in a Design II mating system. The F₂ populations, without parents, were evaluated in the field in a replicates-in-sets design at two locations in Colombia in 1983. Estimates of additive genetic variance were significant for yield, pods/m², seeds/pod, and seed weight. Interaction with environments was also significant. Values for nonadditive genetic variance were not significant for either yield or yield components. The estimates of narrow sense heritability, based on the F₂ population mean and unbiased by genotype x environment interaction, were 0.21 ± 0.13 for yield. 20 ± 0.13 for pods/m², 0.57 ± 0.13 for seeds/pod, and 0.74 ± 0.15 for seed weight. The expected direct response from selection of the top 20 % of F₂ populations for yield per se would result in a 4.30 % increase in yield with a correlated response of 0.21 % in seed weight. In contrast, the expected gain from direct selection for seed weight would result in a 11.76 % increase in seed weight with a, correlated gain of 0.28 % for yield. Direct selection for pods/m² would decrease yield, seeds/pod and seed weight, while direct selection for seeds/pod would reduce pods/m² and seed weight but increase seed yield by 0.37 %. Data on yield from replicated trials in the early segregating generations could be utilized for identification and selection of promising crosses and families or lines with crosses for dry bean yield improvement.     

Genotypic differences in utilization of assimilate sources during maturation of wheat under chronic heat and heat shock stresses

Heat stress from chronic, prolonged exposure up to 32 ^°C or heat shock from brief exposure to 33 ^°C and above alters the source of assimilates for grain growth of wheat (Triticum asetivum L.). Our objectives were to identify genotypes that resist chronic heat stress and heat shock and to determine the relative contributions of photosynthesis and stem reserves to grain filling under both conditions. Twenty-eight genotypes were grown in controlled enviroments at 20/15 and 30/25 ^°C day/night in light and darkness during maturation in the first experiment, and six genotypes were grown in light at the same temperatures and at 40/35 ^°C followed by 20/15 or 30/25 ^°C in the second experimnet. Heat susceptibility indices (HSI) were calculated from grain yields of the genotypes in both experiments. The ratio of chlorophyll variable fluorescence to maximum fluorescence (Fv/Fm), a measure of the stability of photosynthesis, and carbohydrate reserves in the stems were measured in the second experiment. Photosynthesis provided 63 and 65% of assimilates in the grain at 20/15 and 30/25 ^°C, respectively, but both stable photosynthesis in some genotypes and high content of reserves in other genotypes were associated with low susceptibility to stress. The Fv/Fm ratio was decreased by heat shock and returned to normal values intolerant genotypes when the treatment was followed by 20/15 ^°C but not 30/25 ^°C. Grain yield was highly correlated among 20/15, 30/25, and 40/35 ^°C followed by 20/15 ^°C treatments, suggesting that similar plant traits were involved. We conclude that assimilates from either stable photosynthesis or high reserve levels provided for high grain yields during heat stress. Combining the two traits could improve heat tolerance of wheat but might not be feasible if other traits are impeded. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of Seed Dormancy in Lisianthus (Eustoma grandiflorum)

The genetics of chill-related seed dormancy, exhibited by a lisianthus (Eustoma grandiflorum) genotype native to North America, was investigated. Less than 1 % of the seeds germinated by direct hydration at 20°C, while full germination (98%) was attained by chilling imbibed seeds, for at least 11 days, at 3°C, before transferring them to 20°C. The inheritance of seed dormancy was analyzed in F₁, BC₁, and F₂, populations, derived from mating between a normal non-chill-requiring genotype (P₁) and the above chill-requiring genotype (P₂). F₁ seeds, of both reciprocals, and BC₁ (P₁) seeds were all non-dormant. The proportion of seed dormancy in F₂ and BC, (P₂) was 0.06 and 0.68, respectively. The inheritance model proposed includes six diallelic loci with cumulative effects, in which the presence of at least nine ‘dormancy-conferring’ alleles is necessary for inducing phenotypic seed dormancy. Possible implications of inherent seed dormancy on the utilization of native lisianthus germplasm for breeding are discussed.     

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.)     

Mutagenic Effects of Combined and Single Doses of Gamma Rays and EMS in Opium Poppy*

Mutagenic effects of gamma rays and EMS using both single and combined dosages were examined in opium poppy (Papaver somniferum L.). For inducing both macro- and micro-mutations in the present study, combined mutagenic doses, especially 5 kR + 0.4 % EMS appeared to be greatly superior to all single doses of the two mutagens: gamma rays and EMS. The lowest dose of combined mutagen (5 kR + 0.2 % EMS) was about twenty times more effective than the most effective dose (15 kR) of gamma rays and twice as effective as the most effective concentration (0.4 %) of EMS. The combined dose 5 kR + 0.4 % EMS enhanced morphine content (%) to 0.98 as against 0.64 in the control. In accordance with earlier findings (GAUL 1964) the study revealed a correlation between induced macro- and micro-mutations.     

Further Studies on the Restoration of Fertility in Triticum durum×T. timopheevi Hybrids

Twenty one F₁ hybrids involving three Triticum durum cultivars and eleven forms of T. timopheevi were backcrossed to their respective durum parents. Backcross fertility (BC₁ grain set) of these sterile hybrids improved with the rise in temperature at the time of pollination. A mean temperature of less than 20 °C results in poor seed set while higher mean temperatures around 24 °C result in increased seed set. It is suggested that a large number of backcross seeds can be produced by pollinating these hybrids late in the season, i.e., after 20th of March in northern India.     

Interspecific cross of Brassica oleracea var. alboglabra and B. napus : effects of growth condition and silique age on the efficiency of hybrid production, and inheritance of erucic acid in the self-pollinated backcross generation

Interspecific hybrids were produced from reciprocal crosses between Brassica napus (2n = 38, AACC) and B. oleracea var. alboglabra (2n = 18, CC) to introgress the zero-erucic acid alleles from B. napus into B. oleracea. The ovule culture embryo rescue technique was applied for production of F₁ plants. The effects of silique age, as measured by days after pollination (DAP), and growth condition (temperature) on the efficiency of this technique was investigated. The greatest numbers of hybrids per pollination were produced under 20°/15°C (day/night) at 16 DAP for B. oleracea (♀) × B. napus crosses, while under 15°/10°C at 14 DAP for B. napus (♀) × B. oleracea crosses. Application of the ovule culture technique also increased the efficiency of BC₁ (F₁ × B. oleracea) hybrid production by 10-fold over in vivo seed set. The segregation of erucic acid alleles in the self-pollinated backcross generation, i.e. in BC₁S₁ seeds, revealed that the gametes of the F₁ and BC₁ plants carrying a greater number of A-genome chromosomes were more viable. This resulted in a significantly greater number of intermediate and a smaller number of high-erucic acid BC₁S₁ seeds.     

Oxidative damage in forage rape (Brassica napus L.) seeds following heat stress during seed development

A short period (240°C hr; Tb = 25°C) of heat stress (30°C day/25°C night) during forage rape (Brassica napus L.) seed development or at seed physiological maturity can reduce seed vigour, but the extent of oxidative damage associated with this short heat stress was not known. Heat-stressed seeds were assessed for malondialdehyde (MDA) content, hydrogen peroxide (H₂O₂) accumulation, antioxidant enzyme activity, adenylate energy charge and seed ultrastructural integrity, and compared with that of non-heat-stressed seeds. Heat stress increased both MDA content and H₂O₂ accumulation by 35%–50%, reduced antioxidant enzyme activity by between 12% and 67%, and significantly reduced adenosine energy charge. Transmission electron microscope images showed clear evidence of seed deterioration in heat-stressed seeds, including ruptures in cell wall and plasma membranes, fused lipid bodies and damaged mitochondria. Heat stress at physiological maturity caused more oxidative damage than the same heat stress during seed development. Seed vigour decreased as H₂O₂ accumulation increased and antioxidant enzyme activity decreased, but no direct relationship between lipid peroxidation and seed vigour was established. The extent of damage resulting from even shorter periods of heat stress (<240°C hr) before or at seed physiological maturity requires investigation.     

Effects of day-length and temperature on floral structure and fertility restoration in a season-dependent male-sterile Solanum villosum Mill. mutant

Solanum villosum is an important African leafy vegetable whose yield is limited mainly by competition from early and excess fruit-set. Induced male-sterility is a potential tool to reduce this competition and enhance yields. This study was conducted to investigate the influence of photoperiod and temperature on the floral dynamics of a season-dependent male-sterile mutant. The mutant, named T-5, has flowers which are sepaloid, mostly stamenless, indeterminate and partially restored in winter, late-spring, summer and autumn, respectively. Floral organ restoration was found to be largely independent of photoperiod conditions. Day/night temperatures of 25/25 and 30/20°C were found to favour restoration of the floral organ but most flowers were stamenless and infertile. High night temperature favoured the formation of indeterminate flowers both in the growth chamber (30°C) and in the greenhouse (>25°C). On the other hand, low growth chamber (10°C) and greenhouse (<15°C) night temperature favoured the formation of sepaloid flowers. The optimum temperatures for floral structure and fertility restoration were between 20°C and 25°C (day) and 15–20°C (night). Propagation of T-5 mutant can thus be achieved by growing in regions or seasons with such temperature ranges. Under temperatures unfavourable for fruit-set, leaf productivity is expected to be high.     

Differences in Starch Characteristics of Rice Strains Having Different Sensitivities to Maturation Temperatures

The amylose synthesis in rice with Wx^b is affected by environmental temperature during seed development. We previously isolated a mutant candidate (coi) in which amylose synthesis is insensitive to a cool temperature. Here, the effects of maturation temperature (21 °C vs. 28 °C) on the starch characteristics of coi and its original strain 76‐3/T65 were studied. Amylose contents and gelatinization properties of 76‐3/T65 were affected by maturation temperatures, while coi was less affected. The amylopectin chain length distributions were almost the same between the two strains maturated at both temperatures. Gelatinization onset (T_o), peak (T_p), conclusion temperatures (T_c) and enthalpies (ΔH) decreased with environmental temperatures, and T_p, T_c and ΔH of coi at 21 °C were significantly higher than those of 76‐3/T65. Thus, the coi mutant, less affected by maturation temperature than that of 76‐3/T65, may be a useful strain in understanding biochemical and genetic starch biosynthesis response to slight changes in temperature.