Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

Enhanced pollen grain embryogenesis and plant regeneration in anther cultures of Brassica juncea cv. PR-45

Summary Pollen grain embryogenesis in anther cultures of Brassica juncea cv. PR-45 was considerably enhanced by treating the donor plants with 4 mll^-1 (v/v) of ethrel or delayed sowing of the donor plants, the latter treatment being superior. The anthers derived from plants sown about two months after the normal sowing period showed 18% androgenesis as compared to 3.5% in the control.Pollen grain embryos normally showed very poor germination (10%) on B₅ or B₅ containing GA₃. However, ABA or cold treatment promoted normal germination of these embryos. Exposure of the embryos to 4°C for 6 days, which proved to be the best treatment, induced 66% germination of the embryos.     

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.     

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.     

The effect of cold and heat treatments on the anther culture response of diverse rye genotypes

The reduction of saturated fats in canola oil has recently been promoted as a goal for breeders for commercial and human health benefits. Currently, saturated fatty acids in Canadian produced canola oil are above the 7% level, and the objective of this study was to generate canola lines with reduced major saturates (palmitic and stearic), by several percent. Mutant embryos generated from direct ultraviolet radiation mutagenesis of microspores in vitro were subjected to heat during the maturation stage. Heat artificially elevated the saturate levels in developing mutant embryos, allowing efficient identification of those with reduced saturates within the expanded range using HPLC fatty acid analysis of the embryo cotyledons. Mutagenesis produced embryos with fatty acids altered in both directions. Major saturate levels in the cotyledons of heat-treated mutant embryos ranged from 3.3 to 16.4% (heated control ca. 6–9%) and 1.3–10% (heated control ca. 2–4%) for palmitic and stearic fatty acids, respectively. Doubled haploid seed derived from embryos grown at normal temperatures confirmed the reduction of major saturates. HPLC fatty acid analysis of DH seed identified saturate levels ranging from 3.9 to 6.5% (control ca. 5.5%) and 0.9–2.7% (control ca. 1.7%) for palmitic and stearic fatty acids, respectively. Various doubled haploids were identified with major saturate levels below 5.5%. Concomitant positive changes in the unsaturated fatty acids (18:1, 18:2, 18:3) among the mutant lines are also discussed.     

Mutagenic treatment of microspore-derived embryos of turnip rape (Brassica rapa) to increase oleic acid content

The article presents the results of a study on obtaining mutant doubled haploids of turnip rape. The culture medium for producing embryos in isolated microspore culture was optimized. The optimal medium formula was NLN + 13% S + 0.05 mg/L NAA + 0.05 mg/L BA. Embryos derived from isolated microspore culture were treated with EMS mutagen in three concentrations (4 mM, 8 mM, and 12 mM). Mutant doubled haploid plants and their seeds were obtained. Agronomic evaluation and analysis of fatty acid composition in mutant lines seeds showed that they had valuable traits in terms of seed oil quality and yield. Analysis of the fatty acid composition of the seeds of the obtained mutant doubled haploids showed an increase in the percentage of oleic acid (~11%–12%) in comparison with donor cultivars. Our results showed that mutagenesis of embryos from a culture of isolated microspores has potential for improving the qualitative traits of turnip rape.     

Selection of Semidwarf, Early Maturing and Blast Resistant Mutants after Mutagenic Seed Treatment in Two Locally Adapted Indian Rice Cultivars

The objective of this study was to induce semidwarf, early maturing and blast resistant mutants in two adapted Indian rice cultivars. Seeds of the rice cultivars ‘Madhu Malti’ and ‘Phul Patas 72’ were treated with gamma rays and ethyl methane sulfonate (EMS) as well as a combination of gamma rays and EMS. A total of 96 dwarf (< 75 cm) and semidwarf (< 100 cm) and 104 early flowering/maturing M₂ plants were identified among the 18,060 M₂ plants observed. Sixty-eight blast resistant M₂ plants were also identified. The true mutant status of these plants has yet to be determined in progeny tests. The induction of desired mutant types in locally adapted cultivars could prove to be superior over their introduction from non-adapted foreign materials via backcrossing in that the important agronomic characteristics of adaptation to the local environment are retained in the mutants.     

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.     

EMS离体诱变和抗大花萱草叶枯病突变体的筛选

利用甲基磺酸乙酯(EMS)诱变大花萱草‘红运’愈伤组织和分化芽,目的是获得抗叶枯病愈伤组织和分化芽突变体。结果表明：0.50%~0.75% EMS处理愈伤组织60 min;0.75%~1.00% EMS处理分化芽60min,分别获得“半致死剂量”效应。对存活的愈伤组织进行毒素浓度递增的多步筛选(10% 30 d—20% 30 d);分化芽80%毒素21d的一步筛选。经定向筛选,初步获得抗叶枯病分化芽突变体103株,有待进一步进行抗病鉴定。愈伤组织未能获得抗病突变体再生植株。     

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.     

EMS诱变谷子‘长农35号’M1代成熟期株型突变体的鉴定与分析

为建立谷子突变体库,以便在谷子分子生物学研究中应用,以生产上主要推广种植的谷子品种‘长农35号’干种子为实验材料,采用0.8%和1.0%甲磺酸乙酯(EMS)进行了诱变处理,试验共收获株型相关M1代突变体材料282份,其中,0.8% EMS诱变‘长农35号’获得株型突变体材料100个单株,可分为10个组;1.0% EMS诱变‘长农35号’获得株型突变体材料182个单株,可分为17个组。M1代成熟期株型相关性状突变体分析结果表明：1.0% EMS处理‘长农35号’所获突变体,在株高、穗下节粗、穗下第一节间粗和茎节数4个性状与对照有显著差异,而0.8%处理与对照差异不显著,因此,对于‘长农35号’来讲,采用1.0% EMS进行诱变处理更有利于多类型大量突变单株的获得。     

水稻“9311”突变体的筛选和突变体库的构建

利用γ射线和EMS溶液诱变处理籼稻“9311”种子,经过M₂筛选和M₃重复鉴定,分别获得465份和210份,共675份叶、茎、穗和根等性状变异的突变体,突变频率为5.62%。γ射线诱变群体的变异范围要大于EMS诱变群体,突变频率也较高,但紫色叶鞘和叶片类病斑等少数突变类型只在EMS诱变群体中出现。新构建的突变体库将有助于进一步开展水稻功能基因的研究。     

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.     

Genetic improvement of fenugreek (<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> L.) through EMS induced mutation breeding for higher seed yield under western Canada prairie conditions

Most fenugreek, Trigonella foenum-graecum L. suitable for western Canada exhibit an indeterminate growth habit and take about 120 days to produce a good proportion of high quality seed. Late maturity is a major inconvenience for seed producers in temperate climates prevalent in this region where plants must mature within a much shorter growing season. The objective of this study was to develop early maturing fenugreek mutants with a determinate growth habit to ensure uniform maturity within the 100 frost free days available on the Canadian prairies. Seeds from Tristar Fenugreek, a forage cultivar developed for production in western Canada, were treated with 10–300 mM ethyl methane sulfonate (EMS) for 2–24 h and plants were selected for determinate growth habit, early maturity and high seed yield. This mutation breeding approach has detected new breeding material exhibiting early seed maturity coupled with high seed yield, seed quality and determinate growth habit. Successful development of useful mutants in fenugreek was reported before, but this is the first report on successful use of mutation breeding for improvement in quantitative traits in this crop.     

Induction of microspore embryos in a CMS line of Brassica juncea and formation of the androgenic plantlets

Anther culture of two wide hybrids (Diplotaxis erucoides × Brassica campestris) × B. juncea and (D. berthautii × B. campestris) × B. juncea, their CMS lines and the parent species elicited a range of responses highlighting the importance of the genotype. Androgenesis was expressed in cultured anthers of CMS (D. erucoides) B. juncea (22.8%), in restored pollen fertile plants of this CMS line (1.66%), and in the parent, B. juncea cv Pusa Bold (13.02%). AgNO₃ was essential for androgenic response in the CMS lines, and it markedly increased the frequency of androgenesis in the cultivated species. Multiple crops of microspore embryos were obtained from responsive anthers of CMS plants in anther recultures. As high as92% microspore embryos of the CMS line germinated on basal B₅medium and formed normal plantlets. This revised version was published online in July 2006 with corrections to the Cover Date.     

绿豆EMS诱变突变体库的构建及表型分析

为探索适于绿豆突变体库构建的最佳甲基磺酸乙酯(EMS)浓度和处理时间,以‘皖科绿3号’种子为研究材料,设置4种EMS浓度（0.6%,1.0%,1.4%和1.8%）和4种时间（6 h,10 h,14 h和18h）共16个组合,并以半致死剂量为突变体库构建的条件。结果表明,‘皖科绿3号’的最佳诱变条件为1.0%/10 h。用该条件处理6000粒成熟的‘皖科绿3号’种子,获得M₁代植株998份。对M₂代群体中植株叶色、叶形、株高、花器官、开花期等性状进行鉴定。共发现表型变异材料324株,变异频率为2.97%。突变体中叶色变异和矮秆是主要的变异类型,分别占植株总数的1.10%和0.46%。利用基因组重测序技术对4份突变材料的突变位点进行鉴定,测序结果表明碱基突变频率为1/37.78 kb。实验确定‘皖科绿3号’的最佳诱变条件为1.0%/10 h。研究结果也表明利用EMS诱变的方法可获得丰富的表型变异材料,可用于绿豆品种遗传改良和功能基因组学研究。     

Production of mutants with high cold tolerance in spring canola (<Emphasis Type="Italic">Brassica napus</Emphasis>)

A major factor affecting spring canola (Brassica napus) production in Canada is killing frosts during seedling development in the spring and seed maturation in the fall. The objective of this study was to explore the possibility of producing spring canola lines with mutations that have altered biochemical pathways that increase cold tolerance. The approach was to generate UV point mutations in cultured microspores followed by chemical in vitro selection of individual mutant microspores or embryos resulting in measurable alterations to various biochemical pathways with elevated levels of key defense signaling molecules such as, salicylic acid (SA), p-Fluoro-d,l-Phenyl Alanine (FPA), and jasmonic acid (JA). In addition, since proline (Pro) is known to protect plant tissues in the cold-induced osmotic stress pathway, mutants that overproduce Pro were selected in vitro by using three Pro analogues: hydroxyproline (HP), azetidine-2-carboxylate (A2C); and, 3,4-dehydro-d,l-proline (DP). Of the 329 in vitro selected mutant embryos produced, 74 were identified with significant cold tolerance compared to their donor parents through indoor freezer tests at −6°C, and 19 had better winter field survival than winter canola checks. All chemically selected mutant doubled haploids with increased cold tolerance compared well with parent lines for all seed quality and agronomic parameters. Development of increased frost tolerant cultivars should allow for spring canola to be produced in western Canada without compromising seed quality.     

Establishing Cytoplasmic Male Sterility in Brassica napus by Mitochondrial Recombination with B. tournefortii

Fusion experiments between B. napus and X-ray-treated B. tournefortii protoplasts were carried out to develop cytoplasmic male sterility (ems) in B. napus. From the regenerants, six lines containing male sterile plants were selected; five lines segregated for male sterility, but one line (25–143) was completely male-sterile from the beginning. Molecular analyses of mitochondrial (mt) and chloroplast (cp) DNA of B. napus, B. tournefortii, B. juncea and cms juncea indicated that the original cytoplasmic donor of the cms juncea-system in B. napus was a B. tournefortii form, while the B. napus genotype used for the fusion experiments had a B. campestris cytoplasm. By analysis (it regenerated plants, line 25–143 was identified as possessing mt-DNA recombined between B. campestris and B. tournefortii. with the major part derived from B. campestris. No differences were detected between epDNAs from H. campestris and from line 25—143. The other five lines were similar to B. campestris with all the probes used. The low frequency of sterile lines from the fusion experiments and the inheritance of the cms in segregating progenies are both discussed.     

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.