Christian Möllers

Estimation of seed weight, oil content and fatty acid composition in intact single seeds of rapeseed ( Brassica napus} L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of seed weight, total oil content and its fatty acid composition in intact single seeds of rapeseed was studied. A calibration set of 530 single seeds was analysed by both NIRS and gas-liquid chromatography (GLC) and calibration equations for the major fatty acids were developed. External validation with a set of 75 seeds demonstrated a close relationship between NIRS and GLC data for oleic (r = 0.92) and erucic acid (r = 0.94), but not for linoleic (r = 0.75) and linolenic acid (r = 0.73). Calibration equations for seed weight and oil content were developed from a calibration set of 125 seeds. A gravimetric determination was used as reference method for oil content. External validation revealed a coefficient of correlation between NIRS and reference methods of 0.92 for both traits. The performance of the calibration equations for oleic and erucic acid was further studied by analysing two segregating F2 seed populations not represented in the calibration set. The results demonstrated that a reliable selection for both fatty acids in segregating populations can be made by using NIRS. We concluded that a reliable estimation of seed weight, oil content, oleic acid and erucic acid content in intact, single seeds of rapeseed is possible by using NIRS technique.

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

SummaryThe effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

The availability of a highly efficient and reliable microspore culture protocol for many Brassica species makes this system useful for studying basic and applied research questions. Microspores and microspore-derived embryos are ideal targets for modification by mutagenesis and transformation. Regenerated doubled haploid plants are widely used in breeding programs and in genetic studies. Furthermore, the Brassica microspore culture system allows the identification of genomic regions and genes involved in the microspore embryogenic response, spontaneous diploidization and direct embryo to plant conversion. This review summarizes current achievements and discusses future perspectives.

Increased embryogenesis after colchicine treatment of microspore cultures of Brassica napus L.

Summary Efforts to improve the induction of embryogenesis in microspore culture of B. napus have focussed to date on growth conditions of the donor plants and culture conditions of the microspores. For initiating haploid development, the first pollen mitosis proceeds in a symmetrical fashion in contrast to the asymmetrical division in normal gametogenesis. Earlier studies have shown that disruption of this asymmetrical division by adding colchicine into the culture medium (25 mgL -1 for 12 h) of microspores increased the number of regenerated embryoids. In the present experiments the effect of higher concentrations and longer colchicine treatments have been analyzed in B. napus . Of the 57 microspore culture experiments with four different genotypes, 69% of the experiments produced a positive effect on embryogenesis. The best results were obtained with the treatment of 100 mgL -1 colchicine for 24 h. This produced a 3 fold increase in the average number of regenerated embryoids. The second best result was obtained with the 10 mgL -1 treatment for 3 days, leading to a 1.8 fold increase in regeneration. The individual treatments showed a strong tendency for the 24- h and 72-h duration of treatments to be significantly better than their controls, while the 6-h treatment resulted in a low level of significance. Colchicine concentrations as low as as 10 mgL -1 were sufficient to improve embryogenesis and the results suggest that treatment durations longer than 24h (or even 72h) may positively affect embryogenesis. A negative effect of colchicine on the regeneration rate was observed in 10% of the experiments, indicating that the developmental stage of microspores at the time of colchicine treatment might be important. During the culture of microspores and the regeneration of embryoids no signs of toxicity, e.g. malformations of embryoids, were observed. The experiments confirm that disruption of cytoskeleton components at or before the first pollen mitosis in vitro contributes to an improved embryogenesis in B. napus .

Reduction of sinapate ester content in transgenic oilseed rape (Brassica napus) by dsRNAi-based suppression of BnSGT1 gene expression

Seeds of oilseed rape (Brassica napus) accumulate high amounts of antinutritive sinapate esters (SE) with sinapoylcholine (sinapine) as major component, accompanied by sinapoylglucose. These phenolic compounds compromise the use of the protein-rich valuable seed meal. Hence, a substantial reduction of the SE content is considered essential for establishing rape as a protein crop. The present work focuses on the suppression of sinapine synthesis in rape. Therefore, rape (spring cultivar Drakkar) was transformed with a dsRNAi construct designed to silence seed-specifically the BnSGT1 gene encoding UDP-glucose:sinapate glucosyltransferase (SGT1). This resulted in a substantial decrease of SE content in T2 seeds with a reduction reaching 61%. In T2 seeds a high and significant correlation between the contents of sinapoylglucose and all other sinapate esters has been observed. Among transgenic plants, no significant difference in other important agronomic traits, such as oil, protein, fatty acid and glucosinolate content in comparison to the control plants was observed. Maximal reduction of total SE content by 76% was observed in seeds of one homozygous T2 plant (T3 seeds) carrying the BnSGT1 suppression cassette as a single copy insert. In conclusion, this study is an initial proof of principle that suppression of sinapoylglucose formation leads to a strong reduction of SE in rape seeds and is thus a promising approach in establishing rape, currently an important oil crop, as a protein crop as well.

Seed yield, oil and phytate concentration in the seeds of two oilseed rape cultivars as affected by different phosphorus supply

A greenhouse pot experiment was conducted for studying seed and oil yield, P uptake and phytate concentration in the seed of two oilseed rape cultivars (Brassica napus L. var. Oleifera, cv. Bristol and cv. Lirajet), grown on a soil substrate at diVerent levels of plant available phosphorus (6, 19, 31 and 106 mg P-CAL kg’1 soil, resp.). All other nutrients were maintained at a high level. At maturity, seed yield and seed quality were investigated. An increase in the phosphorus soil supply resulted in a significant (P<0.05) increase in seed and oil yield, oil and P concentration of the seeds, and P transported to the seeds. The phytate-phosphorus concentration ranged from 0.5 to 6.9 g kg’1 in the seeds and from 0.9 to 12.8 g kg’1 in rapeseed meal. InsuYcient P supply resulted in a reduced concentration of phosphorus and phytate in the seeds. Significant interactions between the factors cultivar and P supply were found for the traits seed yield, oil yield, and P-harvest index.

Development of synthetic Brassica napus lines for the analysis of “fixed heterosis” in allopolyploid plants

SummaryAllopolyploids are widely spread in the plant kingdom. Their success might be explained by positive interactions between homoeologous genes on their different genomes, similar to the positive interactions between different alleles of one gene causing heterosis in heterozygous diploid genotypes. In allopolyploids, such interactions can also occur in homozygous genotypes, and may therefore be called “fixed heterosis”. As to our knowledge, no experimental data are available to support this hypothesis. We propose an experimental approach to quantify “fixed heterosis” in resynthesised Brassica napus and the detection of loci contributing to “fixed heterosis” via comparative QTL mapping in B. napus and its parental species B. rapa and B. oleracea. In order to develop a genetically balanced material, interspecific crosses between 21 Brassicarapa and 16 Brassicaoleracea doubled haploid or inbred lines were performed. In total 3485 vital embryos have been obtained from 9514 pollinated buds. The success of interspecific hybridisation was highly depending on the maternal genotype (B. rapa) and ranged from 0 to 1.18 embryos per pollinated bud. For the genetic characterisation of the B. rapa and B. oleracea lines, a dendrogram was constructed based on 273 RAPD markers. Thus a well-characterised material is now available, which is suitable to analyse the effects of “fixed heterosis” and the interactions between homoeologous genes in allopolyploid species.

Nondestructive assessment of protein content in single seeds of rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) to detectwithin-plant differences for seed protein content was investigated. Fourhundred and fifty-one single seeds were scanned by NIRS using a specialadapter. After non-destructive NIRS scanning, the seeds were analysed forprotein content by the Dumas combustion method and a calibrationequation was developed. A validation set of 117 additional seeds fromthree individual plants from the cultivars Bristol, Lirajet and Maplus wasanalysed for protein content both by NIRS and combustion. The coefficientof determination between NIRS and combustion values in the validation setwas 0.94, with a standard error of performance (SEP) of 0.77% and aratio of the SEP to the standard deviation (SD) of the validation set of0.28. The coefficient of variation (CV) for seed protein content inindividual plants, as determined by the combustion method, was 11.7%for Bristol, 8.9% for Lirajet, and 9.5% for Maplus. The comparison ofsuch variation with the standard error (SE) of NIRS analysis, defined as thecombination of the SE of the combustion method and the SEP of NIRScalibration equation, revealed that the maximum explainable variance withinindividual plants that can be detected using NIRS analysis of proteincontent in single seeds was 0.86 for Bristol, 0.83 for Lirajet, and 0.85 forMaplus. These results demonstrated that NIRS is a powerful tool fornon-destructive assessment of within-plant variation for seed protein contentin rapeseed.

A New Near-Infrared Reflectance Spectroscopy Method for High-Throughput Analysis of Oleic Acid and Linolenic Acid Content of Single Seeds in Oilseed Rape (Brassica napus L.)†

The development of oilseed rape cultivars with a high content of oleic acid (18:1) and a low content of linolenic acid (18:3) in the seed oil is an important breeding aim. Oil of this quality is increasingly being sought by the food and the oleochemical industry. Since the oil quality is determined by the genotype of the seed, a selection can be performed among single seeds of segregating populations. For this purpose a high-throughput Near-Infrared Reflectance Spectroscopy (NIRS) method using an automated sample presentation unit for single seeds of oilseed rape and a spectrometer equipped with a photodiode array detector was developed. Single-seed analyses have been accomplished with a throughput of up to 800 seeds per hour. Seeds from segregating populations of different origin were analyzed by NIRS and gas chromatography. Calibration equations were developed and validated applying the Modified Partial Least Square regression (MPLS) and LOCAL procedure. In three independent validations, standard errors of prediction corrected for bias between 2.7% and 3.7% for oleic acid and 1.2% and 1.8% for linolenic acid were determined using MPLS. Similar results were obtained applying the LOCAL procedure. The results show that the new high-throughput method can be applied to predict the oleic acid and linolenic acid content of single seeds of oilseed rape.

Glucosinolate profile and distribution among plant tissues and phenological stages of field-grown horseradish

Profile and distribution of glucosinolates (GLS) were detected in plant tissues of horseradish at different developmental stages: beginning of vegetative re-growth, flowering and silique formation. The GLS profile varied widely in the different tissues: we identified 17 GLS in roots and sprouts, one of which was not previously characterized in horseradish, i.e. the 2(S)-hydroxy-2-phenylethyl-GLS (glucobarbarin) and/or 2(R)-hydroxy-2-phenylethyl-GLS (epiglucobarbarin), 11 already found in the roots, including the putative 2-methylsulfonyl-oxo-ethyl-GLS, and 5 previously recognized only in the sprouts. Fifteen of those GLS were also identified in young and cauline leaves, 12 in the mature leaves and 13 in the inflorescences. No difference in GLS profile was observed in plant among the phenological stages. Differences in concentrations of GLS, quantified as desulfated, were found in plant. At the beginning of vegetative re-growth, sprouts while showing the same profile of the roots were much richer in GLS having the highest total GLS concentrations (117.5 and 7.7μmolg(-1) dry weight in sprouts and roots, respectively). During flowering and silique forming stages, the roots still maintained lower amount of total GLS (7.4μmolg(-1) of dry weight, on average) with respect to the epigeous tissues, in which mature and young leaves showed the highest total concentrations (70.5 and 73.8μmolg(-1) of dry weight on average, respectively). Regardless of the phenological stages, the aliphatic GLS were always predominant in all tissues (95%) followed by indolic (2.6%) and benzenic (2.4%) GLS. Sinigrin contributed more than 90% of the total GLS concentration. Aliphatic GLS concentrations were much higher in the epigeous tissues, particularly in the mature and young leaves, while benzenic and indolic GLS concentrations were higher in the roots. Through the phenological stages, GLS concentration increased in young and mature leaves and decreased in cauline leaves and inflorescences, while it remained constant over time in roots.