Seed colour assessment in Brassica napus using a Near Infrared Reflectance spectrometer adapted for visible light measurements

Summary Improved oil, protein and fibre contents are associated with light seed colour in rapeseed but the lack of reliable and efficient methods to measure seed colour has hindered breeding efforts for this trait. The feasibility of using light reflectance to assess seed colour in Brassica napus was examined using scanning light reflectance spectrophotometry and near infrared reflectance (NIR). Light reflectance by seed samples from 30 doubled haploid (DH) lines segregating for seed colour increased as the wavelength of the illuminating light in the scanning spectrophotometer increased between 550 and 650 nm. The largest reflectance values were measured for the yellow seed samples; the brown seed samples were intermediate and the black seed samples had the lowest reflectance values. The areas under the reflectance curves were used to transform the spectra to single values. Average light reflectance area values for the seed colour classes were significantly different from each other. The DHs and their corresponding light reflectance area values were also used to calibrate a NIR analyzer modified with 670 and 710 nm filters. The best calibration curve used three wavelengths (670, 2190 and 2208 nm) and had a multiple correlation coefficient of 0.987. Light reflectance area values determined with the calibrated NIR analyzer for 30 randomly selected breeding lines could be used to categorize the colour of the seed samples with no discrepancies between the visual and instrument classifications. The results indicate that NIR can be used to assess seed colour in rapeseed.     

Seed Color Inheritance in Brassica carinata A. Braun, Cultivar S-67

The inheritance of seed color was studied in the brown seeded Ethiopian mustard (Brassica carinata A. Braun), cultivar S-67. Seed color was controlled by a single gene pair. The heterozygous condition resulted in light yellow-brown set-d indicating incomplete dominance [semidominance] of brown over yellow. The homozygous recessive condition resulted in yellow seed. The significance of these findings in relation to seed color inheritance in other Brassica species is discussed.     

Maternal and embryonal control of seed colour by different Brassica alboglabra chromosomes

Most oilseed rape, Brassica napus, cultivars are black‐seeded. The progenitor species, Brassica rapa, has either yellow or black seeds, while known cultivars of the other progenitor species Brassica oleracea/alboglabra have black seeds. To determine which chromosomes of B. alboglabra are carriers of seed colour genes, B. rapaalboglabra monosomic addition lines were produced from a B. napus resynthesized from yellow‐seeded B. rapa and brown/black‐seeded B. alboglabra. Eight out of nine possible lines have been developed and transmission frequencies of the alien chromosomes were estimated. Three B. alboglabra chromosomes in three of these lines influenced seed colour. B. rapa plants carrying alien chromosome 1 exhibited a maternal control of seed colour and produced only brown seeds, which gave rise to plants with either yellow or brown seeds. However, B. rapa plants carrying alien chromosome 4 or another as yet unidentified alien chromosome exhibited an embryonal control of seed colour and produced a mixture of yellow and brown seeds. The yellow seeds gave rise to yellow‐seeded plants, while the brown seeds gave rise to plants that yielded a mixture of yellow and brown seeds, depending on the absence or presence, respectively, of the B. alboglabra chromosome. Consequently, both maternal and embryonal control of seed colour are expected to contribute to the black‐seeded phenotype of oilseed rape.     

Inheritance of siliqua locule number and seed coat colour in Brassica juncea

The inheritance of siliqua locule number and seed coat colour in Brassica juncea was investigated, using three lines each of tetralocular brown seeded and bilocular yellow seeded. Three crosses of tetralocular brown seeded × bilocular yellow seeded lines were attempted and their F₁, F₂ and backcross generations were examined for segregation of these two traits. Brown seed colour and bilocular siliqua characters were found to be dominant over yellow seed and tetralocular siliqua, respectively. Chi‐square tests indicated that each trait is controlled by different sets of duplicate pairs of genes. Bilocular siliquae or brown seeds can result from the presence of either of two dominant alleles, whereas tetralocular siliquae or yellow seeds are produced when alleles at both loci are recessive. A joint segregation analysis of F₂ data indicated that the genes governing siliqua locule number and seed colour were inherited independently.     

Identification and inheritance of a partially dominant gene for yellow seed colour in Brassica napus

A yellow‐seeded doubled haploid (DH) line no. 2127‐17, derived from a resynthesized Brassica napus L., was crossed with two black‐seeded Brassica cultivars ‘Quantum’ and ‘Sprint’ of spring type. The inheritance of seed colour was investigated in the F₂, and BC1 populations of the two crosses and also in the DH population derived from the F1 of the cross ‘Quantum’× no. 2127‐17. Seed colour analysis was performed with the colorimeter CR‐300 (Minolta, Japan) together with a visual classification system. The immediate F1 seeds of the reciprocals in the two crosses had the same colour as the self‐pollinated seeds of the respective black‐ and yellow‐seeded female parents, indicating the maternal control of seed colour. The F1 plants produced yellow‐brown seeds that were darker in colour than the seeds of no. 2127‐17, indicating the partial dominance of yellow seed over black. In the segregating BC1 progenies of the two crosses, the frequencies of the black‐ and yellow‐seeded plants fit well with a 1 : 1 ratio. In the cross with ‘Quantum’, the frequencies of yellow‐seeded and black‐seeded plants fit with a 13 : 3 ratio in the F₂ progeny, and with a 3 : 1 ratio in the DH progeny. However, a 49 : 15 segregation ratio was observed for the yellow‐seeded and black‐seeded plants in the F₂ progeny of the cross with ‘Sprint’. It was postulated from these results that seed colour was controlled by three pairs of genes. A dominant yellow‐seeded gene (Y) was identified in no. 2127‐17 that had epistatic effects on the two independent dominant black‐seeded genes (B and C), thereby inhibiting the biosynthesis of seed coat pigments.     

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.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation of yellow-seeded rapeseed lines (Brassica napus L.) from different genetic sources

In order to breed yellow-seeded rapeseed, 16 yellow-seeded lines of Brassica napus L. derived from eight genetic sources were used. The genetic variation of the seedcoat ratio, the cellulose content of the seedcoat, the oil content of the seedcoat and of the embryo, and also the correlations between these characters of the yellow- and brown-seeded plants from the same line, were analysed by variance analysis and path analysis. The results show that the seedcoat ratio and cellulose content of brown seeds are 4.2% and 17.74%, respectively, higher than that of yellow seeds and the oil content of the seedcoat of brown seeds is 3% lower than that of the yellow seeds, these differences all being highly significant. However, the differences between yellow and brown seeds in 1000-seed weight and oil content of the embryo were very small. Both characters are determined mainly by the genetic background and not by seed colour or seedcoat thickness. The correlation analysis revealed that the seedcoat thickness has a highly significant positive correlation with the cellulose content of the seedcoat and is highly significantly negatively correlated with the seedcoat oil content and the 1000-seed weight. The oil content of the embryo alone has a highly significant negative correlation with 1000-seed weight. In yellow seeds, the seedcoat thickness has a large and directly positive effect on the oil content of the embryo whereas the 1000-seed weight has a negative one; the opposite was found in brown seeds. Selection objectives in breeding yellow seeds in Brassica napus are also discussed.     

Inheritance of siliqua orientation and seed coat colour in Brassica tournefortii

The inheritance of siliqua orientation and seed coat colour in Brassica tournefortii was investigated using four genotypes varying in these two characters. The F₁, F₂ and backcross generations of two crosses were used for studying the segregation pattern of the traits. The plants were classified for seed colour as having brown or yellow seeds and for siliqua orientation as having upright, semi‐spread or spread siliqua. Seed colour was found to be under monogenic control with brown being dominant over yellow. Siliqua orientation was under digenic polymeric gene action: upright siliqua was produced by the presence of two dominant genes and spread siliqua by two recessive genes. The absence of even a single dominant gene resulted in a third type of siliqua orientation, semi‐spread siliqua.     

Increasing erucic acid content in Ethiopian mustard through mutation breeding

Ethylmethane sulphonate (EMS) was applied to seeds of the Ethiopian mustard (Brassica carinata A. Braun) line C-101. Bulk samples of M₃ seeds from 8331 M₂ plants were evaluated for the fatty acid composition of their oil by near-infrared reflectance spectroscopy (NIRS) and by further gas chromatography on selected samples. A putative mutant, N2-6230, showing very low oleic acid content (4.7% vs. average of 8.6% in C-101) and erucic acid content within the range of variation of the line C-101 (40-49.3%) was identified. The M₃ progeny of this mutant showed a wide segregation for erucic acid content (39.1-57.9% vs. 41.8-50.3% in C-101), and maintained levels of oleic acid lower than in line C-101. Selection for high erucic acid content in the M₃ and M₄ generations led to the fixation of this mutation in the M₅ generation (52.2-59.3% vs. 39.0-47.6% in C-101). This is the first high erucic acid line obtained in Brassica species through mutation breeding. Its utility in future programmes to develop very high erucic acid lines is discussed.     

Inheritance of very high glucosinolate content in Ethiopian mustard seeds

Seed meal amendments rich in glucosinolates are of interest for soil pest and disease control. The Ethiopian mustard ( Brassica carinata A. Braun) line N2-6215, with very high levels of seed glucosinolates (160 μmol/g), was developed from the line C-101 (116 μmol / g) following mutagenesis. The objective of this research was to study the inheritance of very high seed glucosinolate content. Plants of N2-6215 were reciprocally crossed with plants of the line C-101. The F₁, F₂, and BC₁F₁ plant generations were evaluated in two environments and seeds from individual plants were analysed for total glucosinolate content. The very high glucosinolate content in N2-6215 seeds was largely subject to maternal control. No cytoplasmic effects were detected. The trait was found to be oligogenic and determined by at least two or three genes. The estimates of broad-sense heritability were 0.45 and 0.58 in both environments, whereas the estimates of narrow-sense heritability were 0.35 and 0.50. The moderate heritability and oligogenic control of the trait suggest the feasibility of breeding for increased seed glucosinolate content in Ethiopian mustard.     

Development of Strains with Yellow Seedcoat in Indian Mustard (Brassies juncea Czern. & Coss.)

A mutant with yellow seedcoat colour was isolated by Nayar (1968) in the mustard variety‘Rai-5′. This mutant was crossed to the national check cultivar ‘Varuna’ in order to develop improved strains with yellow seedcoat. Four such strains with yellow seeds were evaluated for their seed yield, yield components and percent oil. Two strains TM-9 and TM-17 were more productive than ‘Varuna’ in seed yield. All the yellow seeded strains showed higher oil percentage as compared to ‘Varuna’. The seedcoat in the yellow seeded strains accounts for 14-15% of the seed weight as compared to 18% in the black seeded ‘Varuna’. The higher proportion of the cotyledons and embryo accounts for the increased oil percentage in the yellow seeded types.     

Inheritance of petal colour and its independent segregation from seed colour in Brassica rapa

The inheritance of petal (flower) colour and seed colour in Brassica rapa was investigated using two creamy‐white flowered, yellow‐seeded yellow sarson (an ecotype from Indian subcontinent) lines, two yellow‐flowered, partially yellow‐seeded Canadian cultivars and one yellow‐flowered, brown‐seeded rapid cycling accession, and their F₁, F₂, F₃ and backcross populations. A joint segregation of these two characters was examined in the F₂ population. Petal colour was found to be under monogenic control, where the yellow petal colour gene is dominant over the creamy‐white petal colour gene. The seed colour was found to be under digenic control and the yellow seed colour (due to a transparent coat) genes of yellow sarson are recessive to the brown/partially yellow seed colour genes of the Canadian B. rapa cvs.‘Candle’ and ‘Tobin’. The genes governing the petal colour and seed colour are inherited independently. A distorted segregation for petal colour was found in the backcross populations of yellow sarson × F₁ crosses, but not in the reciprocal backcrosses, i.e. F₁× yellow sarson. The possible reason is discussed in the light of genetic diversity of the parental genotypes.     

Inheritance of black testa colour in lentil (Lens culinaris Medik.)

The inheritance of testa (seed coat) colour and interaction of cotyledon and testa colours were studied in seven crosses of lentil (Lens culinaris Medik.) involving parents with black, brown, tan or green testa and with orange, yellow or dark green cotyledons. Analysis of F₂ and F₃ seed harvested from F₁ and F₂ plants, respectively, revealed that although black testa is dominant over nonblack testa, its penetrance is not complete since both F₁ plants and heterozygous F₂ plants produced varying proportions of seeds with either black or nonblack testa. The F₂ populations of the crosses between parents with brown and tan, as well as brown and green, testa segregated in the ratio of 3 brown : 1 tan and 3 brown : 1 green, respectively, indicating monogenic dominance of brown testa colour over tan or green. The expression of testa colour was influenced by cotyledon colour when parents with brown or green testa are crossed with those having orange or green cotyledons. Thus F₂ seeds from these crosses with a green testa always had green cotyledons and never orange cotyledons. F₂ seeds from these crosses with a brown testa always had orange cotyledons and never green cotyledons. These results suggest diffusion of a soluble pigment from the cotyledons to the testa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of yellow-seeded Brassica napus of double low quality

Two yellow‐seeded white‐petalled Brassica napus F₇ inbred lines, developed from interspecific crosses, containing 26–28% emcic acid and more than 40 μmol glucosinolates (GLS)/g seed were crossed with two black/dark brown seeded B. napus varieties of double low quality and 287 doubled haploid (DH) lines were produced. The segregation in the DH lines indicated that three to four gene loci are involved in the determination of seed colour, and yellow seeds are formed when all alleles in all loci are in the homozygous recessive state. A dominant gene governed white petal colour and is linked with an erucic acid allele that, in the homozygous condition, produces 26–28% erucic acid. Four gene loci are involved in the control of total GLS content where low GLS was due to the presence of recessive alleles in the homozygous condition in all loci. From the DH breeding population a yellow‐seeded, yellow‐petalled, zero erucic acid line was obtained. This line was further crossed with conventional B. napus varieties of double low quality and, following pedigree selection, a yellow seeded B. napus of double low quality was obtained. The yellow seeds had higher oil plus protein content and lower fibre content than black seeds. A reduction of the concentration of chromogenic substances was found in the transparent seed coat of the yellow‐seeded B. napus.     

The measurements of acid detergent fibre in rapeseed by visible and near-infrared spectroscopy

Visible and near‐infrared spectroscopy (NIRS) calibrations for acid detergent fibre (ADF) in intact rapeseed (Brassica napus L.) were performed for two different sample volumes (10 ml, 500 seeds approximately; and 1 ml, 50 seeds approximately). The inclusion of brown and yellow‐seeded cultivars in this work has allowed the whole range of ADF currently described in the literature for this character to be covered. Chemometric techniques have been used for developing calibration equations for both procedures when measuring the two different seed sample volumes. On the basis of the coefficient of determination in the cross‐validation (R²cv) obtained for the 10 and 1 ml assays (0.80 and 0.73), and SECV/SEL ratios (2.30 and 2.57), respectively, both equations showed an accuracy sufficient for screening purposes in an ADF range from 6.80 to 13.46% dry wt, which is presented in this work.     

Inheritance of reduced linolenic acid content in the Ethiopian mustard mutant N2-4961

The zero erucic acid Ethiopian mustard lines developed so far are characterized by an exceptionally high linolenic acid content in the seed oil. The mutant line N2‐4961, expressing low linolenic acid content in a high erucic acid background, was developed through chemical mutagenesis. The objective of this research was to study the inheritance of low linolenic acid content in this mutant. Line N2‐4961 was reciprocally crossed with its parent line C‐101 and the linolenic acid content of the reciprocal F₁, F₂ and BC₁ generations was studied. No maternal, cytoplasmic or dominance effects were detected in the analysis of F₁ seeds and F₁ plants from reciprocal crosses. Linolenic acid content segregated in 1: 2: 1 ratios in all the F₂ populations studied, suggesting monogenic inheritance. This was confirmed with the analysis of the reciprocal backcross generation. The simple inheritance of low linolenic acid content in N2‐4961 will facilitate the transference of this trait to zero erucic acid lines of Ethiopian mustard.     

Isozyme variation in Ethiopian tetraploid wheat (Triticum turgidum) landrace agrotypes of different seed color groups

Summary Ethiopian landraces of tetraploid wheat can be grouped according to their seed colors in three major groups: brown, purple and white seeded. Seeds with different colours are used for different purposes, and the three seed colour groups are treated separately in breeding programs. The genetic variation between and within these groups was studied by isozyme analyses at six highly polymorphic loci in sixty landrace agrotypes. The mean number of alleles per locus was 1.95. The mean allele frequencies showed significant variation both within and between the seed colour groups. The brown and white seeded types had very high genetic identity and the genetic identity values between the brown and purple and the purple and white groups were only slightly smaller. The average coefficient of gene differentiation between the seed colour types was very low. Only about 5% of the total genetic variation was due to differences between the seed colour groups. This indicates that agrotypes of different seed colours can not be treated as genetically separate and distinct groups.     

Isolation of induced mutants in Ethiopian mustard (Brassica carinata Braun) with low levels of erucic acid

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop for the rain-fed Mediterranean area. However, its usage is limited by the high erucic and high glucosinolate content of the oil and meal, respectively. In the course of a mutagenesis programme, an agronomically good line of Ethiopian mustard was treated with EMS in order to widen the natural variability of nutritional traits in this species. As a result of this programme several low erucic mutants were isolated; two of these mutants showed erucic acid values in the M4 generation in the range 5–10% of total fatty acids. Near-infrared reflectance spectroscopy (N1RS) was successfully applied as a rapid screening method for erucic acid in this breeding programme.     

Production of yellow-seeded Brassica napus through interspecific crosses

Yellow‐seeded Brassica napus was developed from interspecific crosses between yellow‐seeded Brassica rapa var.‘yellow sarson’ (AA), black‐seeded Brassica alboglabra (CC), yellow‐seeded Brassica carinata (Bbcc) and black‐seeded B. napus (AACC). Three different interspecific crossing approaches were undertaken. Approaches 1 and 2 were designed directly to develop yellow‐seeded B. napus while approach 3 was designed to produce a yellow‐seeded CC genome species. Approaches 1 and 2 differed in the steps taken after trigenomic interspecific hybrids (ABC) were generated from B. carinata×B. rapa crosses. The aim of approach 1 was to transfer the yellow seed colour genes from the A to the C genome as an intermediate step in developing yellow‐seeded B. napus. For this purpose, the ABC hybrids were crossed with black‐seeded B. napus and the three‐way interspecific hybrids were self‐pollinated for a number of generations. The F₇ generation resulted in the yellowish‐brown‐seeded B. napus line, No. 06. Crossing this line with the B. napus line No. 01, resynthesized from a black‐seeded B. alboglabra x B. rapa var.‘yellow sarson’ cross (containing the yellow seed colour genes in its AA genome), yielded yellow‐seeded B. napus. This result indicated that the yellow seed colour genes were transferred from the A to the C genome in the yellowish‐brown seed colour line No. 06. In approach 2, trigenomic diploids (AABBCC) were generated from the above‐mentioned trigenomic haploids (ABC). The seed colour of the trigenomic diploid was brown, in contrast to the yellow seed colour of the parental species. Trigenomic diploids were crossed with the resynthesized B. napus line No. 01 to eliminate the B genome chromosomes, and to develop yellow‐seeded B. napus with the AA genome of ‘yellow sarson’ and the CC genome of B. carinata with yellow seed colour genes. This interspecific cross failed to generate any yellow‐seeded B. napus. Approach 3 was to develop yellow‐seeded CC genome species from B. alboglabra×B. carinata crosses. It was possible to obtain a yellowish‐brown seeded B. alboglabra, but crossing this B. alboglabra with B. rapa var.‘yellow sarson’ failed to produce yellow seed in the resynthesized B. napus. The results of approaches 2 and 3 demonstrated that yellow‐seeded B. napus cannot be developed by combining the yellow seed colour genes of the CC genome of yellow‐seeded B. carinata and the AA genome of ‘yellow sarson’.