Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding.     

白菜型冬油菜温敏不育系PK3-12S育性转换的差异蛋白质组学分析

为揭示温敏不育系PK3-12S育性转换机制,本研究以白菜型冬油菜温敏不育系PK3-12花药为材料,采用2-DE和LC-MS/MS质谱鉴定等差异蛋白组学方法,分离鉴定了PK3-12在不育/可育条件下花药差异表达蛋白质,并对差异表达蛋白进行了生物信息学分析;进而采用RT-PCR检测了PK3-12在不育/可育条件下花蕾发育进程中差异蛋白编码基因表达量变化。结果表明,高温不育条件下, PK3-12花药形态瘪小,药室有少量败育花粉,育性转换受1对隐性基因控制,表达变化量在2倍以上差异蛋白质点31个,其中增量表达蛋白质点6个,减量表达蛋白质点11个,表达完全抑制蛋白点12个,不育花药特异表达蛋白点2个。质谱鉴定出15个差异蛋白质,参与信号转导通路、二羧基乙醛酸代谢、糖酵解代谢、次生合成代谢、氨基酸生物合成、分支酸生物合成、碳代谢途径等细胞过程。Rubisco亚基连接蛋白编码基因BrrbcL开放读码框(open reading frame, ORF)长度为1095 bp,编码364个氨基酸;与可育花蕾相比,发育进程中不育花蕾BrrbcL基因、膜联蛋白基因(ANN)、BetVI过敏原家族基因(BetV...     

The introgression of S alleles into forage rape,Brassica napus L. from turnip. Brassica campestris L. ssp. Rapifera

Summary Forage rape. B. napus, is self-compatible, the work described illustrates the introgression of functional incompatibility alleles into B. napus from turnips, B. campestris, in two generations. By grading seed, produced by backcrossing turnip/rape hybrids to rape, the frequency of 2n=38 semi articial B. napus recovered, amply justifies the cytological screening involved in such breeding programmes.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp. oleifera (DC.) Metzger

The influence of temperature/photoperiod treatment and gibberellic acid concentration (0, 0.1 or 1.0 mg/l) on direct conversion of microspore-derived embryos (MDEs) to plantlets of winter oilseed rape was investigated. Physiologically mature, 21-day-old MDEs were transferred to a solid B5 medium supplemented with gibberellic acid, and cultured at 24 ^°C, 4 ^°C or 1 ^°C for 14 days, and then at 24 ^°C for the next 21 days. Low temperature was linked with short photoperiod (8 h light/16 h dark), and high temperature was linked with long photoperiod (16 h light/8 h dark). The highest embryo conversion rate was at 1 ^°C with over 70%, compared to<20% at 4 ^°C. Two-way analysis of variance confirmed the significance of the effect of temperature/photoperiod treatment. By contrast, gibberellic acid concentration had no significant effect on stimulation of shoot development from apical meristems of MDEs. Roots developed from apical root meristems of MDEs very easily. The best obtained conversion rate of MDEs induced with cold treatment at1 ^°C for 14 days was 86.5%. Observations on morphological development of MDEs showed clear differences in reaction at various temperature/photoperiod treatments. This revised version was published online in August 2006 with corrections to the Cover Date.     

A comparison of traditional and haploid-derived breeding populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil

Summary Microspore embryogenesis technology allows plant breeders to efficiently generate homozygous micros-pore-derived breeding populations of oilseed rape (Brassica napus L.) without traditional generations of inbreeding. This study was conducted to compare the frequency distribution of microspore-derived population and single seed descent populations with respect to fatty acids of seed oil. Both microspore-derived populations and single seed descent populations were produced from each of three crosses made between selected parents containing contrasting amount of erucic, oleic, linoleic and linolenic acids. The fatty acid content of F₃ plants derived lines (F₅ seed) developed by single seed descent was compared to that of microspore-derived populations. The means, ranges and distribution pattern of seed fatty acid contents were similar in both populations for each fatty acid studied, although a few heterozygous lines were observed in the single seed descent populations. The results indicated that microspore-derived population form random, homozygous F₁ plant derived gametic arrays for all fatty acids evaluated. Selection for altered fatty acid composition in microspore-derived and single seed descent homozygous populations should be equally efficient, in the absence of linkage of traits investigated.     

Identification of molecular markers associated with oleic and linolenic acid in spring oilseed rape (Brassica napus)

The quality of the oil derived from oilseed rape is determined by its fatty acid composition. Breeding oilseed rape for enhanced oil quality includes the development of cultivars with high oleic and low linolenic acid. Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) techniques were investigated for the development of molecular markers for genes controlling oleic and/or linolenic acid. Markers that were identified were converted to sequence characterized amplified region (SCAR) markers for use in breeding. Molecular markers associated with these two fatty acids were identified in a doubled haploid population derived from a cross between the oilseed rape lines TO99‐5318‐20, very high oleic (>79%) and very low linolenic acid (<2%) × DH12075, high oleic (68%) and higher linolenic acid (>7%). Eight RAPD markers were associated with oleic and linolenic acid contents. The RAPD marker UBC 2₈₃₀ accounted for 43% and 13% of the genetic variation for oleic and linolenic acid levels, respectively. The RAPD marker UBC 153₅₅₀ accounted for 19% of the genetic variation for linolenic acid. The UBC 2₈₃₀ fragment was converted to a SCAR marker. The markers identified in this study should be useful tools for the early generation selection of high oleic and low linolenic acid genotypes in oilseed rape breeding programmes.     

Evaluation of microspore-derived embryos as models for studying lipid biosynthesis in seed of rapessed (Brassica napus L.)

Summary Microspore culture of rapeseed (Brassica napus L.) has provided a powerful tool not only for breeding but also in developmental studies. In this study, microspore-derived embryos (MDE) of B. napus were evaluated as a model in seed for studying accumulations of triacylglyceride (TAG) fatty acids in both a low and high erucic acid rapeseed line; and accumulations of TAG and free fatty acids (FFA) in a high erucic acid rapessed line. The accumulation patterns confirmed that MDE had a similar TAG fatty acid profile to seed during the embryo development within each genotype. The oil accumulation in MDE after 36 days in culture (DIC) approached levels similar to those in zygotic seed 25 days after flowering (DAF). Significant differences were detected in contents of both total free fatty acids and specific free fatty acids between MDE and seed. During the developmental period, total free fatty acids changed from 16% to 2.1% in MDE, but from 10.5% to 0.1% in seed. MDE had much higher percentage of free linolenic and erucic acids than seed, particularly during the late developmental stages. The current study indicated that MDE can be used as a model to study TAG and TAG fatty acids in seed but caution must be taken to study free fatty acid metabolism.     

The transfer of characters from Brassica campestris L. to Brassica napus L.: Production of clubroot-resistant oil-seed rape (B. napus ssp oleifera)

Summary Four oil-seed rape lines were crossed with a clubroot resistant Brassica campestris line from the European Clubroot Differential sct. The allotriploid hybrids were backcrossed to the rape lines to introgress clubroot resistance into oil-seed rape. Using a combination of screening for disease resistance and chromosome number, a high proportion of 38-chromosome, clubroot resistant selections were obtained.     

Emergence and performance of volunteer oilseed rape (Brassica napus) in different crops

Volunteer oilseed rape (OSR, Brassica napus L.) causes various agronomic problems in crop rotations and can contribute to gene dispersal by pollen and by seed admixture. A 4-year field experiment (2008–2011) was set up in south-west Germany to investigate the performance of volunteers derived from two OSR cultivars with different levels of seed dormancy. Volunteers of a high-dormancy (HD) and a low-dormancy (LD) OSR cultivar were deliberately generated by spreading 10,000 seeds m⁻² on a field in August 2008 and 2009. Four different crops were grown on that area in the first year following the seed rain: winter wheat (Triticum aestivum L.), winter turnip rape (Brassica rapa L.), spring barley (Hordeum vulgare L.) and field pea (Pisum sativum L.). In the second year, maize (Zea mays L.) was sown uniformly across all plots. Numbers of OSR seedlings emerging in early autumn shortly after seed rain were not connected with the size of the soil seed bank in early spring of the following year. The seeds of the HD-cultivar formed a much greater soil seed bank (up to 14% of the initially spread seed number) compared with the LD-cultivar (up to 1.3%) in the soil layer of 0 to 30 cm in early spring 2009 and 2010). Across all crops, considerably more volunteers of the HD-cultivar than of the LD-cultivar were present at several survey dates in the first year following seed rain. The highest number of volunteers originated from the HD-cultivar with up to 11 volunteers m⁻² in winter turnip rape compared with a maximum of 0.48 plants m⁻² in the other crops. Cultivar-specific differences in volunteer density were observed as well in maize two years after OSR seed rain. Flowering and seed setting volunteers were only present in 2010 and the flowering time was crucially overlapping with that of sown winter OSR. The reproductive ability (seeds produced m⁻²) of the LD-volunteers was five times lower in winter turnip rape than of the HD-volunteer; a similar trend was observed for the OSR volunteers in the other host crops.Strategies to definitely reduce unwanted effects of OSR volunteers, such as gene flow, should include the use of LD-cultivars with a low potential to form a soil seed bank, particularly if selective herbicides are not available, for instance in broad-leaved crops, or if the volunteers are herbicide-tolerant.     

Inheritance of progoitrin and total aliphatic glucosinolates in oilseed rape (Brassica napus L)

Inheritance of progoitrin and total aliphatic glucosinolate concentrations were investigated in oilseed rape, using parental, F₁, F₂ and first backcross generations, derived from a cross between resynthesized spring rape and a double-low spring rape cultivar. Progoitrin and total aliphatic glucosinolate concentrations were measured in mature seeds of single plants from these generations, using micellar electrokinetic capillary chromatography. For progoitrin, an additive/dominance model of gene action adequately explained the variation among the generation means, but for total aliphatic glucosinolate concentration, non-allelic interactions were also detected. Predictions based on estimates of the genetic parameters indicated that recombinant inbred lines, rather than second cycle hybrids, appeared to offer a better prospect of reducing glucosinolate concentrations in this material. Estimates of the minimum number of genes controlling these two characters were broadly in line with the number required for the known stages of their biosynthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of rape seed cultivars (Brassica napus) by starch gel electrophoresis of enzymes

Summary Isozyme patterns were used to characterize ten commercial rape seed (Brassica napus) cultivars. Extracts of cotyledons were made 4–7 days after germination and separated by electrophoresis on starch gels. A sample of more than 100 plants of each variety was analysed and stained for 4 polymorphic enzyme loci (Lap, Gpi, Acon, and Sdh). Therewith, it was possible to distinguish all ten rape varieties qualitatively by at least one typical enzyme pattern. Further, it could be demonstrated that all pairs of varieties showed clear qualitative differences in isozyme patterns, when only tow loci were screened (Acon, Sdh). Using ² test of homogeneity all pairs of varieties differ significantly in their frequencies of isozyme patterns for Acon and in all but one for Sdh.     

Doubled haploid lines of Brassica carinata with modified erucic acid content through mutagenesis by EMS treatment of isolated microspores

Brassica carinata is a potential oilseed crop for the Mediterranean area. Chemical mutagenesis has been applied to microspores of B. carinata with the purpose of identifying lines with altered erucic acid content. From a population of nearly 400 doubled haploid plants recovered, nine lines have been identified that exhibit promising useful changes in erucic acid concentration in the seed oil. Three lines showed erucic acid contents below 25%, with a minimum of 17.1%, and in six lines the level of this fatty acid was greater than 52%. Changes in other fatty acids are also described and discussed.     

Analysis of QTL responsible for grain iron and zinc content in doubled haploid lines of rice (Oryza sativa) derived from an intra-japonica cross

Quantitative trait loci (QTL) related to the grain iron and zinc contents of brown rice were mapped by using a doubled haploid population derived from an intra-japonica cross between 'Hwaseonchal' and 'Goami 2'. QTL–QTL, background–QTL, and background–background interactions and candidate genes that affect grain iron and zinc contents were preliminarily identified. Twenty-one iron- and zinc-related QTL were found. The major-effect QTL qFe7 and qZn7 provided the highest contribution to phenotypic variance for grain iron and zinc contents. The colocation of zinc- and iron-related QTL on chromosomes 1, 4, 7 and 11 may account for the strong correlation between iron and zinc contents. A region on chromosome 7 and epistatic interaction between loci on chromosomes 2 and 10 affected iron content. qZn7 and qZn11.3 exerted additive effects on zinc content. Eleven iron- and zinc-related candidate genes colocated with qFe7, qZn7 and the region on chromosome 7 with an additive effect on iron content. The major-effect QTL identified here may be useful for breeding biofortified rice.     

Genetic variation for saturated fatty acid content in a collection of European winter oilseed rape material (Brassica napus)

Oil quality is an important issue in oilseed rape breeding programmes. Recently, the seed oil content of saturated fatty acids has received substantial interest, because in a number of industrialized countries dietary recommendations not only limit total fat intake but also intake of saturated fatty acids. The aim of the present study was to analyse the genetic variation for saturated fatty acid content in two sets of modern winter oilseed rape cultivars ( Brassica napus L.) tested in field experiments under typical German growing conditions. The results showed highly significant genetic differences among the cultivars for total saturated fatty acid content, which ranged from 6.8% to 8.1%, with a mean of 7.4%. For both sets medium to high genotypic heritabilities were found for most fatty acids and for oil and protein content. Close negative correlations between palmitic acid, the predominant saturated fatty acid, and oleic acid as well as oil content were found.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

Identification of RAPD markers linked to linolenic acid genes in rapessed

Summary The inheritance of the low linolenic acid content (derivated from mutant lines) in rapeseed was investigated. Molecular techniques of gene mapping through RAPD markers were applied on a microspore-derived progeny from a high × low linolenic acid F₁ hybrid. Bulked segregant analysis made it possible to test rapidly number of RAPD primers. Two linkage groups of 6 markers (72.7 cM and 75.6 cM) were determined. Each corresponded to a major QTL which explained 24% and 30.7% of the total phenotypic variation of the linolenic acid content. It was confirmed that two independant mutations were implied in the low linonenic acid content.     

白菜型冬油菜叶片结构和光合特性对冬前低温的响应

为明确白菜型冬油菜在冬前低温下叶片结构特征、光合作用特性及其抗寒性,本研究在0℃和–7.6℃自然低温条件下,选用白菜型冬油菜品种陇油7号(超强抗寒)和天油4号(弱抗寒),测定并比较其叶片气孔性状、解剖结构和光合、荧光参数的日变化等指标。结果表明,随着冬前温度下降,2个白菜型冬油菜叶片气孔密度、气孔面积、气孔周长、栅栏组织和海绵组织厚度均变小,细胞间隙变大,叶片变薄;P_n日变化呈"单峰"型曲线,无光合"午休"现象;叶片的P_n、G_s、T_r和CE均降低,而C_i均升高,说明是非气孔限制引起P_n降低。白菜型冬油菜在冬前低温条件下发生了光抑制现象,表现为F_m和F_v/F_m下降,F_o上升。与超强抗寒品种陇油7号相比,弱抗寒品种天油4号叶片气孔密度和气孔面积均较大,气孔总周长较长,叶片较厚,P_n、F_m和F_v/F_m均较高,说明冬前低温条件下,天油4号光合能力较强,光抑制程度较弱。白菜型冬油菜在冬前低温条件下的叶片气孔密度越大、气孔面积越大、气孔周长越长、叶片及栅栏组织和海绵组织越厚,光合能力越强,地上部生长越旺盛,品种抗寒性越差。本研究为冬油菜抗寒种质创新和育种提供了部分理论支撑。     

Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.)

An essential quality improvement of rapeseed oil can be obtained by reduction of its linolenic acid (C18:3) content from about 10% to less than 3% of the total fatty acids. Genotypes low in C18:3 have been developed by mutagenesis. The initial summer rapeseed mutant had been low yielding and highly susceptible to various diseases. It has been debated whether the low C18:3 character can be successfully combined with high seed yield for physiological reasons. Therefore, the low linolenic character of mutant M48 was transferred into high-yielding genotypes by repeated backcrossing to well-adapted low erucic acid, low glucosinolate (00-) winter rapeseed cultivars. Lines with low C18:3 content were selected from BC3 and BC4 generations and examined in 1990–95. Positive selection response for seed yield was shown to continue over the years. Presently, the best lines are yielding as well as the control cultivars being equivalent also in oil and glucosinolate contents. In order to test the effect of a low C18:3 content on seed yield, plants with low and with high C18:3 content, respectively, were selected from 16 segregating BC5-F₂ populations and bulked to form 32 F₃ populations. These ‘isogenic’ bulk populations were tested for field performance at four locations in 1995. The results show that C18:3 content of the seed oil is not associated with seed yield, oil content, beginning of flowering, plant height and disease resistance. Means of relative seed yield for the high and the low linolenic F₃ bulk populations were not significantly different with 88.0% and 86.9% of the control cultivars, respectively. There was a significant interaction between genotypes with high or low C18:3 content and location. This shows that under specific environmental conditions a low C18:3 content may be either favourable or unfavourable. The results indicate that the low C18:3 character of the original mutants per se does not cause a decrease in seed yield, oil content or general field performance.