EMS诱变甘蓝型油菜M_2代群体的表型突变研究

利用4种浓度EMS处理甘蓝型油菜NJ7982种子,选取其中诱变效果好的处理(0.4%EMS),研究其后代突变型和突变频率。对4.8万株的M2代群体鉴定结果表明,6种器官性状在群体中均出现了突变。子叶突变性状包括3子叶、子叶黄化等,占群体的0.22%;叶片突变性状包括黄化叶、白化叶、紫色叶、上卷叶、下卷叶等,占0.74%;花器突变性状包括紫色花蕾、死蕾、3花瓣、6花瓣、白色瓣、花瓣黄白镶嵌、花瓣皱缩、完全不育、部分可育等,占9.38%;株型突变性状包括矮秆、紫茎等,占4.98%;角果突变性状包括粗角、长角、紫角等,占2.79%;种皮黄色,占0.40%;总的表型突变频率为18.51%。这些遗传多样性的突变材料,为甘蓝型油菜种质创新及品种遗传改良提供基础性材料。     

Effects of caffeine or EDTA post-treatment on EMS mutagenesis in soybean

Seeds of soybean cultivar LD4 were mutagenically treated with EMS (0.3, 0.5, 0.6, 0.9, 0.5 and 1.8%) for 3 h only or plus caffeine (50 mM) or EDTA (1 mM) post-treatment for 5 h. The experimental results indicated that: (1) of the different concentrations of EMS treatment, the M2 mutation frequency induced with 0.6% EMS was the highest (9.7%). When the EMS concentration was over 0.9%, the mutation frequency decreased rapidly. (2) Of the EMS treatments plus caffeine or EDTA post-treatment, the mutagenic effect of 0.6% EMS was the best for inducing morphological variations. Caffeine post-treatment decreased notably the mutation frequency of EMS treatment; when concentrations of EMS were very high (1.5% and 1.8%), mutation frequencies of EDTA post-treatment were still 5.0% and 4.88%, but no mutants were found in EMS treatment or plus caffeine post-treatment. (3) In the M2 mutation spectrum, 11 kinds of mutant types were observed in EMS treatment or plus caffeine or EDTA post-treatment. Relative frequencies of some mutant types (growth period, plant height, grain size, leaf shape and sterility, etc.) were similat among the three treatments, but EDTA post-treatment could change the relative frequencies of yield characteristics (number of pods and grains, grain weight/plant) induced by EMS treatment only.     

裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

Plant regeneration from in vitro leaf tissues of <Emphasis Type="Italic">Viburnum dentatum</Emphasis> L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.     

Somaclonal variation in tomato: effect of explant source and a comparison with chemical mutagenesis

Summary Plants were regenerated from leaf, cotyledon, and hypocotyl explants of tomato cv Moneymaker. Various phenotypic alterations were observed among regenerated plants (R₁), but were not transmitted to the progenies, except for ploidy variation. Variation in ploidy level, mainly tetraploidy, occurred in R₁ plants and their R₂ progenies, and the frequency of polyploid plants depended on the explant source. More than 50% of the regenerants derived from hypocotyl explants were found to be polyploid. A correlation was observed between the percentage of polyploid cells present in the explant material in vivo and the frequency of polyploid plants. Several monogenic mutations were recovered in the R₂, four of which were shown to be allelic to known, recessive, single-gene mutants. No significant effect of explant source or duration of tissue culture period on mutant frequency or spectrum was found. For several mutant types that could be scored unambiguously, somaclonal variation was compared to variation induced by treatment of seeds with ethyl methane sulphonate (EMS). The results showed that the mutant frequencies were higher after EMS treatment than those generated through tissue culture. With respect to the mutant spectrum, no clear differences were observed between the spectra obtained after EMS treatment and those after tissue culture. However, tissue culture gave rise to polyploid plants, whereas no ploidy variants occurred after EMS treatment.     

苦荞突变体库构建与突变体中芦丁合成相关基因表达分析

苦荞是重要的小杂粮作物之一,营养物质丰富,是天然芦丁的重要来源。突破苦荞育种难题,创制苦荞新种质是目前研究的重要方面。本试验利用甲基磺酸乙酯(EMS)构建了黑丰1号苦荞突变体库,明确了当EMS浓度为1.2%时,诱变效果较好。通过对M1突变株表型观察统计,共获得叶色、叶型、株型、粒型变异单株102株,突变率为3.85%;高效液相色谱技术(HPLC)测定1000株M3材料,获得高芦丁含量突变株系2个和低芦丁突变体株系5个;qRT-PCR对芦丁含量突变体株系中芦丁代谢关键酶基因(CHS、F3H、4CL、FLS、UFGT)进行表达量分析,发现不同株系中上述基因的表达量与芦丁含量相关性不明显,但个别基因如FtFLS基因表达量在高芦丁含量突变体中达到对照的4.55倍。通过突变体的筛选丰富了苦荞基因资源,创新了苦荞新种质,也为苦荞芦丁代谢的分子基础研究提供了材料保证与技术支持。     

甘蓝型油菜理化诱变和突变体库的构建

利用&#61543;&#61472;射线与甲基磺酸乙酯（EMS）溶液诱变技术复合处理甘蓝型油菜高油605的成熟种子。经田间M2筛选和M3验证, 共筛选到152个叶色、叶形、株高、分枝数、分枝角度、茎径、茎色、花色、花瓣数、花瓣大小、花蕊形态、雄性不育、死蕾及开花期等性状发生变异的突变体, 占M2诱变群体总数的12.67%。利用水培法进行M3和M4株系的子叶和根系变异性状筛选和验证, 发现子叶和根系变异频率分别为12.78%和7.07%。现已构建了包括叶片、株型、花器、子叶、根系及部分生理性状变异类型的突变体库, 可为今后油菜遗传改良和功能基因组学研究提供更多的种质资源。     

Effect of foliar application of phosphorus on growth and mineral composition in peanut plants (Arachis hypogaea L.) under salt-stress

Summary Peanut (Arachis hypogaea L., Var. TMV-2) plants were raised in sand cultures under natural photoperiod. Salt treatment (0.4% NaCl on dry weight basis) was given 10 days after sowing. Nutrient solution was supplied 15 days after sowing to control and salinized plants. Phosphate (0.1% NaH₂PO₄ with 0.01% Tween-80) was sprayed to the plants to the drip point once daily from 20th to 25th day and from 30th to 35th day. The plants were harvested at 30th and 40th day for analysis. The plants grown under saline conditions showed a market reduction in growth. When phosphorus was supplied to saline plants by foliar application, there was an increase in dry weight, leaf area, stomatal frequency, and yield and the increase was more marked when the plants received phosphate for two periods. Phosphorus content decreased due to salinity which was restored by foliar spray of phosphate. With salinity, sodium accumulated while potassium and calcium were lowered. Phosphate spray decreased sodium and increased potassium and calcium in general.     

Induction,germination and shoot development of somatic embryos in cassava

Four Indonesian and two Latin-American cassava genotypes (Manihot esculenta Crantz), were evaluated for their ability to develop somatic embryos from young leaf lobes. All genotypes formed somatic embryos but they differed in the frequency of embryos induced. The best genotypes, M. Col 22 and Tjurug, produced germinating embryos (GE) on 81% (22.1 GE/initial leaf lobe) and 46% (4.3 GE/initial leaf lobe) of the cultured leaf lobes, respectively. Up to 57% of the germinating embryos of M. Col 22 and 12% of Tjurug produced either normal or malformed shoots. Most malformed shoots developed into shoots with normal morphology after prolonged culture. All shoots formed roots after transfer to medium without BAP. Roots of all normal and most malformed regenerants had the original ploidy level (2n=36). Regardless of whether the plants were multipliedin vitro (150 plants) or in the greenhouse (30 plants) there were no morphological differences compared to parent plants.     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.     

A mutational analysis of leaf morphogenesis in Arabidopsis thaliana.

As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M2 individuals, arising from 5770 M1 parental seeds exposed to EMS, we isolated 1926 M2 putative leaf mutants, 853 of which yielded viable M3 inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants.     

EMS诱变西瓜突变体库的构建及表型分析

采用1.0%诱变剂甲基磺酸乙酯(EMS)处理西瓜品系W1-17种子9h,然后对M1和M2代群体单株在叶、花、茎、育性、分支习性等方面进行表型变异观察,同时选取M2代典型变异株系,利用23对西瓜SSR引物进行分析鉴定,构建西瓜突变体库。结果表明:(1)EMS诱变使M1代幼苗形态呈现出叶畸形、叶褶皱、部分黄化、花畸形、雄花不散粉、卷须畸形、矮小、生长缓慢、不育等特异性状,获得由1 252个单株组成的西瓜突变体M1群体,群体总变异频率为18.33%。(2)M2代共筛选到205个突变植株,40种表型变异,表现在子叶性状(黄化、扭曲不对称、折叠等)、叶和茎性状(叶黄化、变小、裂刻变深,茎变细,节间变短,分支少等)、花性状(花变大,花色变浅,两性花,花瓣皱缩、部分退化、数目突变,柱头畸形,雄蕊不成熟等)和其他性状(生长缓慢、不育等)等方面,总的表型突变率达到了19.59%。(3)针对M2代10个典型变异植株,通过SSR引物分析发现有9份材料在DNA水平上有变异。本研究初步构建了含有120个M1代家系及1 051株M2代植株、40种表型变异的西瓜突变体库。     

Morphometric and Biochemical Changes in <i>Agave americana</i> L. Plantlets Induced By Ethyl Methanesulfonate

A. americana L. is a crop with very little genetic variability. In order to evaluate the effect of ethyl methanesulfonate (EMS) to induce variability in in vitro plantlets of A. americana, different explants (meristems, leaves and roots) were evaluated for the production of callus. MS medium supplemented with ANA (2.68 μM) and BAP (2.68 μM) was used. Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D (0.11 μM) and BAP (44 μM). The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained, with 60% of the plantlets presenting differences such as dwarfism and different leaf forms, without the presence of spines, as well as an increase in fructan content of 30% with respect to the control plantlets. PAL was increased and this activity is related with higher anthocyanins concentration in A. americana L. plantlets.     

Ethylmethanesulfonate saturation mutagenesis in Arabidopsis to determine frequency of herbicide resistance

Plant resistance to glyphosate has been reported far less frequently than resistance to sulfonylurea and imidazolinone herbicides. However, these studies tend to be anecdotal, without side by side comparisons for a single species or natural isolate. In this study, we tested the frequencies of resistance of three herbicides in a controlled ethylmethanesulfonate (EMS) saturation mutagenesis experiment, allowing a direct comparison of the frequencies at which resistant mutant plants arise. The 100% growth inhibition dose rates of glyphosate, chlorsulfuron (a sulfonylurea herbicide), and imazethapyr (an imidazolinone herbicide) were determined for Arabidopsis. Populations of EMS-mutagenized M(2) seedlings were sprayed with twice the 100% growth inhibition dose of glyphosate, chlorsulfuron, or imazethapyr, and herbicide-resistant mutants were identified. Although there were no glyphosate-resistant mutants among M(2) progeny of 125,000 Columbia and 125,000 Landsberg erecta M(1) lines, chlorsulfuron resistance and imazethapyr resistance each appeared at frequencies of 3.2 x 10(-5). Given the observed frequency of herbicide resistance mutations, we calculate that there are at least 700 mutations in each EMS-mutagenized Arabidopsis line and that fewer than 50,000 M(1) lines are needed to have a 95% chance of finding a mutation in any given G:C base pair in the genome. As part of this study, two previously unreported Arabidopsis mutations conferring resistance to imidazolinone herbicides, csr1-5 (Ala-122-Thr) and csr1-6 (Ala-205-Val), were discovered. Neither of these mutations caused enhanced resistance to chlorsulfuron in Arabidopsis.     

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 μM 6-benzylaminopurine (BAP) and 2.5 μM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4°C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 μM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.     

The Effect of VA Mycorrhizal Infection and Phosphorus Status on Sunflower Hydraulic and Stomatal Properties

Koide, R. 1985. The effect of VA mycorrhizal infection and phosphorusstatus on sunflower hydraulic and stomatal properties.—J. exp. Bot. 36: 1087–1098. Mycorrhizal (M) and non-mycorrhizal (NM) sunflower plants weregrown in a soil of low phosphorus availability (with and withoutphosphorus amendment) and in a soil of moderate phosphorus availability(without phosphorus amendment). Using the Ohm's law analogyand measured leaf water potentials, stem water potentials, andtranspiration rates, hydraulic resistances were calculated forthe whole plant, leaf, and below leaf components. Mycorrhizalinfection (as high as 89%) was shown to have no effect on theintrinsic hydraulic properties of the soil/plant system overa wide range of transpiration rates in either soil when M andNM plants of equivalent root length were compared. When grownin the soil of moderate phosphorus availability, calculatedhydraulic resistances under given environmental conditions werethe same for M and NM plants, as were stomatal resistances andtranspiration rates. When grown in the soil of low phosphorusavailability, calculated values of hydraulic resistance werelower for M plants than for NM plants under given sets of environmentalconditions. These differences in calculated hydraulic resistancewere not due to a difference in the intrinsic hydraulic propertiesof M and NM plants. The differences were evident because stomatalresistances were lower and transpiration rates higher for Mplants and because hydraulic resistance varied inversely withtranspiration rate. When plants of significantly greater rootlength were compared to plants of lesser root length, the calculatedhydraulic resistances under given environmental conditions weremuch lower for the plants of greater root length. This differencewas largely due to a difference in the intrinsic hydraulic propertiesbetween large and small plants, and not because of differencesin transpiration rate. The elevated transpiration rates exhibitedby M plants were attributed to an enhanced phosphorus status.Short term phosphorus amendments made to phosphorus-deficientNM plants improved transpiration; transpiration rates were similarfor M and NM plants before NM plants became phosphorus-deficient,and phosphorus-amended M and NM plants had similar transpirationrates. The data are discussed in relation to other reports ofmycorrhizal influence on hydraulic and stomatal resistances.Possible mechanisms for the influence of infection on stomatalresistance are also briefly discussed. Key words: Hydraulic resistance, stomatal resistance, mycorrhizas     

Observations and experiments on the use of an avirulent mutant strain of tobacco mosaic virus as a means of controlling tomato mosaic

In 1973 tobacco mosaic virus (TMV) strain M II-16 was successfully used by growers in the United Kingdom to protect commercial tomato crops against the severe effects of naturally occurring strains of TMV. However, plants in many crops had mosaic leaf symptoms which were occasionally severe, so possible reasons for symptom appearance were examined. The concentration of the mutant strain in commercially produced inocula (assessed by infectivity and spectrophotometry) ranged from 28 to 1220 μg virus/ml; nevertheless all samples contained sufficient virus to infect a high percentage of inoculated tomato seedlings. Increasing the distance between the plants and the spray gun used for inoculation from 5 to 15 cm resulted in a significant decrease in the number of tomato seedlings infected. When M II-16 infected tomato plants were subsequently inoculated with each of fifty-three different isolates of TMV, none showed severe symptoms of the challenging isolates within 4 wk, although some isolates of strain o induced atypically mild leaf symptoms. In a further experiment, M II-16 infected plants showed conspicuous leaf symptoms only 7 wk after inoculation with a virulent TMV isolate. M II-16 multiplied more slowly in tomato plants and had a lower specific infectivity than a naturally occurring strain of TMV. More than 50% of plants in crops inoculated with strain M II-16 which subsequently showed conspicuous leaf mosaic contained TMV strain 1 or a form intermediate between strains o and 1. It is suggested that the production of TMV symptoms in commercial crops previously inoculated with strain M II-16 may result from an initially low level of infection, due to inefficient inoculation, which allows subsequent infection of unprotected plants by virulent strains. Incomplete protection by strain M II-16 against all naturally occurring strains may also be an important factor.     

An efficient mutational method for photosynthetic bacteria

The pigment and auxotrophic mutants of Rhodobacter sphaeroides Y6 were obtained by treatment with ethyl methanesulfonate (EMS) followed by lithium chloride (LiCI). Treatment with 0.081 M EPS and subsequent treatment with 0.071 M LiCI resulted in 12% higher frequency of pigment mutations than application of 0.081 M EMS alone; the frequency of auxotrophic mutations increased 2.5-fold when treatment with lithium chloride was applied. A blue shift 10 nm was recorded in the absorption spectrum of carotenoids form YM5-3 green mutant; considerable accumulation of neurosporine was revealed by HPLC and mass spectrometry. The method is efficient for isolating mutants of photosynthetic bacteria.     

A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)

Two large-scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M(2) plants were individually harvested, freeze-dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mm EMS treatments and assembled into 1152 eight-plant pools. To facilitate PCR-based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post-cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE-HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12-16 M(3) progeny from each of the M(2) plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/distilling.html).     

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties $\text{436}\text{35}$ and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M₁ and chlorophyll mutants in M₂ approximately being halved as compared with the controls (EMS treatment alone).An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M₁ and of chlorophyll mutants in M₂, was approximately doubled as compared with the control.Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone.Based on these results some aspects of the acetate/chloride effect are briefly discussed.