Development of B-Genome Chromosome Addition Lines of B. napus Using Different Interspecific Brassica Hybrids

By interspecific hybridization within the genus Brassica, trigenomic haploids were produced and back-crossed four times with B. napus, variety ‘Andor’. From this material, monosomic B-genome chromosome addition lines were selected with the extra chromosome derived from three different B-genome sources, i.e., B. nigra (BB), B. carinata (BBCC), and B. juncea (AABB). After selfing and/or microspore culture, disomic addition lines were obtained. Meiotic behavior was studied of the trigenomic hybrids, the pentaploid BC₁ plants, and the monosomic addition lines. The addition lines were shown to possess cytological stability and good fertility.     

Alloplasmic effects of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis> on seed characteristics of <Emphasis Type="Italic">B. carinata</Emphasis>

Effects of Brassica napus (N) and B. juncea (J) cytoplasm on seed characteristics of B. carinata (C) were examined. Alloplasmic lines of B. carinata were produced from N × C and J × C hybrids by recurrent backcrossing to the BC₈ generation. Fourteen sets of reciprocal crosses were used. Compared with their euplasmic sibs, alloplasmic B. carinata line seeds with B. napus cytoplasm showed reduced dormancy, higher seed weight, lower germination rate at high temperatures, higher germination rate at low temperatures, and had lower erucic acid and higher linoleic acid contents. Alloplasmic B. carinata line seeds with B. juncea cytoplasm had higher seed weight but lower germination rate than their corresponding euplasmic sibs. These results showed a cytoplasmic effect on seed development, and an influence on seed weight, dormancy, and fatty acid composition. B. carinata was more deleteriously affected by cytoplasm from B. napus than by cytoplasm of B. juncea.     

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.     

Development and characterisation of a <Emphasis Type="Italic">Brassica carinata</Emphasis> inbred line incorporating genes for low glucosinolate content from <Emphasis Type="Italic">B. juncea</Emphasis>

The presence of high levels of sinigrin in the seeds represents a serious constraint for the commercial utilisation of Ethiopian mustard (Brassica carinata A. Braun) meal. The objective of this research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata. BC₁F₁ seed from crosses between double zero B. juncea line Heera and B. carinata line N2-142 was produced. Simultaneous selection for B. carinata phenotype and low glucosinolate content was conducted from BC₁F₂ to BC₁F₄ plant generations. Forty-three BC₁F₄ derived lines were selected and subject to a detailed phenotypic and molecular evaluation to identify lines with low glucosinolate content and genetic proximity to B. carinata. Sixteen phenotypic traits and 80 SSR markers were used. Eight BC₁F₄ derived lines were very close to N2-142 both at the phenotypic and molecular level. Three of them, with average glucosinolate contents from 52 to 61 micromoles g⁻¹, compared to 35 micromoles g⁻¹ for Heera and 86 micromoles g⁻¹ for N2-142, were selected and evaluated in two additional environments, resulting in average glucosinolate contents from 43 to 56 micromoles g⁻¹, compared to 29 micromoles g⁻¹ for Heera and 84 micromoles g⁻¹ for N2-142. The best line (BCH-1773), with a glucosinolate profile made up of sinigrin (>95%) and a chromosome number of 2n = 34, was further evaluated in two environments (field and pots in open-air conditions). Average glucosinolate contents over the four environments included in this research were 42, 31 and 74 micromoles g⁻¹ for BCH-1773, Heera and N2-142, respectively. These are the lowest stable levels of glucosinolates reported so far in B. carinata.     

Synthesis and cytogenetic characterization of intergeneric hybrids of Diplotaxis siifolia with Brassica rapa and B. juncea

Intergeneric hybrids involving a wild crucifer, Diplotaxis siifolia (2n = 20; D^sD^s), and two crop Brassica species, namely Brassica rapa (2n = 20; AA) and B. juncea (2n = 36; AABB), were developed through sequential ovary/ovule culture. Hybridization was successful only when D. siifolia was used as the female parent, indicating unilateral cross incompatibility. Hybrids were intermediate between the parents for morphological characteristics but had low male as well as female fertility. Meiotic studies of hybrids revealed partial homoeology between D^s and A/B genomes.     

Intra- and intergenomic relationships in interspecific hybrids between Brassica (B. rapa, B. napus) and a wild species B. maurorum as revealed by genomic in situ hybridization (GISH)

Interspecific hybridization plays a crucial role in plant genetics and breeding. The efficiency of interspecific crosses to a considerable extent depends on the genetic relatedness of genomes from parental species. Interspecific hybrids involving Brassica maurorum (2n = 16, MM) and two Brassica crop species, viz B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC), were produced and analyzed for their meiotic chromosome pairings in pollen mother cells (PMCs) by using genomic in situ hybridization (GISH) with the labeled DNA of B. maurorum (MM) as probe. In hybrids B. maurorum × B. rapa (2n = 18, MA), all chromosomes remained unpaired in 28% PMCs, and the maximum of autosyndetic bivalents was two and one among the chromosomes of A and M genomes, with the average per cell being 0.27 and 0.12, respectively. Up to two allosyndetic bivalents between A and M genomes appeared, averagely 0.48 per cell. In hybrids B. maurorum × B. napus (2n = 27, MAC), the maximum of autosyndetic bivalents in M genome was two and the average was 0.11, while the maximum of allosyndetic bivalents between M and A/C genomes was two and the average was 0.78. The 2–7 bivalents formed by A/C-genome chromosomes showed their high homology. The results were compared and discussed with the chromosome pairings in the hybrids of B. maurorum with B. juncea and B. carinata with respect to the genome relationships and the potential for chromosome recombination.     

The effect of the cytoplasms of Brassica napus and B. juncea on some characteristics of B. carinata, including flower morphology

This study was conducted to assess the cytoplasm effects of Brassica napus and B. juncea on the some characteristics of B. carinata, as well as the phylogenetic distances separating the three species. Alloplasmic lines of B. carinata were developed from B. napus × B. carinata and B. juncea × B. carinata hybrids by recurrent backcrossing to the BC₇ generation. Sixteen populations from three generations were compared for a number of characteristics. Plants with the cytoplasm of B. napus flowered later, had shorter filaments and longer pistils, lower pollen amount, lower seed set, lower petal length and width and different petal color; plants with the cytoplasm of B. juncea had shorter pistils and filaments, and lower petal length and width than their corresponding euplasmic sibs, respectively. The results suggest that the cytoplasm is involved in the development of flower organs. The natural species, B. carinata showed a balance between the nucleus and cytoplasm. The cytoplasm from B. napus showed a stronger disturbing effect than that of B. juncea, suggesting that B. carinata might be genetically closer to B. juncea than to B. napus. The significant difference in the alloplasmic effect of the cytoplasms of B. napus and B. juncea also suggests that in B. carinata the B genome may play a greater role than the C genome. An erratum to this article can be found at     

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’.     

Brassilexin accumulation and resistance to Leptosphaeria maculans in Brassica spp. and progeny of an interspecific cross B. juncea × B. napus

Summary Resistance to Leptosphaeria maculans was assessed in Brassica napus, B. juncea, B. carinata, B. nigra and progeny issuing from an interspecific cross B. napus × B. juncea, using a cotyledon-inoculation test. In these individual plants, brassilexin accumulation was determined following an abiotic, non-specific, elicitation. All the tested B. napus cultivars were highly susceptible to the parasite and weakly accumulated brassilexin. In contrast, B. juncea, B. carinata, and B. nigra usually displayed a hypersensitive response to the inoculation and accumulated more brassilexin than B. napus. The same correlation between resistance to L. maculans and phytoalexin accumulation was observed in the interspecific hybrid progeny. The cotyledon-inoculation test allowed the discrimination of plants displaying a hypersensitive response to the inoculation from those highly sensitive to the parasite, but intermediate disease severity classes were not usually representative of resistance or susceptibility. In this respect, brassilexin determination allowed differentiation, within a set of plants presenting an intermediate response to the pathogen, of plants with a high (B. juncea-like), and with a weak (B. napus-like) ability to accumulate brassilexin.Abbreviations IHP interspecific hybrid progeny - JR B. juncea-type complete resistance to blackleg (Roy, 1984) - W&D test cotyledon-inoculation test as described by Williams & Delwiche (1979)     

Glucosinolate content in interspecific crosses of Brassica carinata with B. juncea and B. napus

Brassica carinata A. Braun is a highly productive oilseed crop in the Ethiopian highlands, but the seed has a high 2-propenyl glucosinolate content, which is undesirable. The objective of this study was to introgress, through interspecific crosses, genes for low 2-propenyl glucosinolate content from the B genome of B. juncea and C genome of B. napus into the B. carinata B and C genomes and thus develop low glucosinolate B. carinata. The cross [(B. carinata×B. juncea) ×B. carinata] yielded plants that contained only ～ 20 μmoles of 2-propenyl glucosinolate, which was an 85% reduction compared with levels in B. carinata seed. Plants of the [(B. carinata×B. napus) ×B. carinata] cross had normal high concentrations of 2-propenyl glucosinolate. Backcross plants of both interspecific crosses also contained 3-butenyl and 2-hydroxy-3-butenyl glucosinolates. The results of these crosses suggested that genes for glucosinolate synthesis were located on B genome chromosomes of B. carinata because B. napus C genome introgressions did not result in reductions of total glucosinolate contents. The total alkenyl glucosinolate content of one F₃ family of the B. juncea backcross was similar to that of the B. juncea parent. It was concluded that through further selection in this family, B. carinata plants could be identified that would be basically free of 2-propenyl glucosinolate, and have a low total alkenyl glucosinolate content.     

Genetic effect on phytoaccumulation of arsenic in <Emphasis Type="Italic">Brassica juncea</Emphasis> L

The presence of excessive amounts of heavy metals such as arsenic in the environment is a problem in many parts of the world. Many countries e.g., Bangladesh are already severely affected by arsenic contamination of soil and groundwater. Phytoaccumulation is a method in which plants are used to uptake heavy metals from soil. The genus Brassica has many species that can efficiently accumulate arsenic. Despite many studies on phytoaccumulation, little is known about the genetics of phytoaccumulation of Brassica. This study investigated the genetic effect on phytoaccumulation of arsenic in Brassica juncea L. and attempted to identify any morphological trait(s) for phytoaccumulation. Two cultivars of B. juncea namely, B. juncea cv. Rai and B. juncea cv. BARI-11 were chosen as parents for the study. A set of their morphological characteristics were selected to evaluate their potential as marker(s) for phytoaccumulation. A single concentration, 15 ppm of arsenic was used to study the genetic effect on phytoaccumulation using quantitative genetics. The results revealed that phytoaccumulation of arsenic (by the root and shoot systems) is heritable in the broad sense and polygenic in nature. The stem diameter was also found to be heritable in the broad sense and path analysis indicated that it has a significant direct effect on the shoot uptake. It is concluded that phytoaccumulation of arsenic in B. juncea (cvs. Rai and BARI-11) is a genetic phenomenon and stem diameter may be a potential marker of phytoaccumulation of arsenic in B. juncea (cvs. Rai and BARI-11).     

Production of intergeneric hybrids between Brassica juncea and Diplotaxis virgata through ovary culture, and the cytology and crossability of their progenies

The cytogenetic study was investigated in the intergeneric F₁ hybrid, F₂and backcross progenies (BC₁). The plants used were Brassica juncea(2n=36) and Diplotaxis virgata(2n=18). Three intergeneric F₁ hybrids between two species were produced through ovary culture. They showed 36 chromosomes. It might consist one genome of B. juncea and two genomes of D. virgata. The morphology of the leaves resembled that of B. juncea. The color of the petals was yellow that was like in D. virgata. The size of the petal was similar to that of B. juncea. The mean pollen fertility was15.3% and the chromosome associations in the first meiotic division were(0–1)_IV+(0–2)_III+(8–12)_II+(12–20)_I. Many F₂ and BC₁seeds were harvested after open pollination and backcross of the F₁ hybrids withB. juncea, respectively. The F₂seedlings showed different chromosome constitutions and the range was from 28 to54 chromosomes. Most seedlings had 38chromosomes followed by 36, 40 and 54. The BC₁ seedlings also showed different chromosome constitutions and the range was from 29 to 62. Most seedlings had both 40and 54 chromosomes followed by 36, 46 and52. In the first meiotic division of F₂ and BC₁ plants, a high frequency of bivalent associations was observed in all the various kinds of somatic chromosomes. Many F₃ and BC₂ seeds were obtained by self-pollination and open pollination of both F₂ and BC₁ plants, and by backcrossing both F₂ and BC₁plants with B. juncea, respectively,especially, three type progeny with 36, 40or 54 chromosomes. The somatic chromosomes of the F₃ and BC₂ plants were further investigated. The bridge plants between B. juncea and D. virgata with 36 chromosomes may be utilized for breeding of other Brassica crops as well as B. juncea. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

Transgene introgression from Brassica napus to different varieties of Brassica juncea

Transgene introgression from transgenic rapeseed (Brassica napus) to different varieties of B. juncea was assessed in this study. Crossability between a transgenic rapeseed line Z7B10 (pollen donor) and 80 cultivars of 16 B. juncea varieties (including two wild accessions) was estimated by artificial pollination in a greenhouse. As a result, interspecific crossability between the transgenic Z7B10 line and the 80 B. juncea cultivars varied considerably, with seeds per flower from 0.00–10.67. Seed germination rates of the interspecific F₁ hybrids ranged from 49.0%–89.3%. The estimated frequencies of natural gene flow from the transgenic Z7B10 line to 10 B. juncea cultivars with different uses in the experiment field varied from 0.08% to 0.93%. The natural F₁ hybrids were highly sterile, with seeds per silique ranging from 0.27 to 1.03. In addition, seeds per flower of hybrid descendants varied from 0.02 to 0.22 when F₁ hybrids were self‐pollinated, and those ranged from 0.03 to 0.30 when F₁ hybrids were backcrossed with their corresponding B. juncea parents. Results of this study suggest a low level of transgene introgression from transgenic rapeseed to different B. juncea varieties, which provides a sound scientific basis for the safety management of coexisting transgenic B. napus and B. juncea varieties in China.     

Intergeneric hybridization of Diplotaxis erucoides with Brassica napus. I. cytogenetic analysis of F1 and BC1 progeny

Summary Intergeneric hybrids (F₁) Diplotaxis erucoides (DeDe) x Brassica napus (AACC) and the first backcross to B. napus (BC₁) have been obtained through in vitro culture of excised ovaries. The chromosome numbers of F₁ and BC₁ plants proved the occurrence of unreduced gametes. The study of metaphase I chromosome pairing showed that autosyndesis in De genome and allosyndesis between De and A/C genomes might exist. The male fertility of the F₁ plants was low. Some male-sterile plants were found in F₁ and BC₁ progeny. The possibilities of creating addition lines B. napus-D. erucoides and of obtaining a new cytoplasmic male sterility in B. napus are discussed.     

Mapping of Molecular Markers on Brassica B-Genome Chromosomes Added to Brassica napus

Using interspecific hybridization among various Brassica species, B-genome chromosomes from different sources of Brassica, i.e. B. nigra (BB, 2n = 18), B. carinata (BBCC), 2n = 34) and B. juncea (AABB, 2n = 36) were transferred into the Canadian variety ‘Andor’ of B. napus. Monosomic addition lines were selected (AACC + 1B, 2n = 39) by cytological control. For characterization of the alien chromosomes, series of isozymes, RFLPs and RAPD markers were employed. This permitted the identification of a total of 39 lines representing seven of the eight B-genome chromosomes.     

甘蓝型油菜亚基因组间杂种（ArAnCcCn）的细胞学和育性研究

以新型甘蓝型油菜（A^rA^rC^cC^c）与自然甘蓝型油菜品种湘油15（AⁿAⁿCⁿCⁿ）杂交，人工配制了A^rAⁿC^cCⁿ油菜亚基因组间杂种。亚基因组间杂种花粉母细胞减数分裂基本正常，中期I大多以19个二价体的构型存在，但偶尔也有单价体、三价体和四价体等异常情况出现。在减数分裂后期Ⅰ和后期Ⅱ，绝大多数细胞中的同源染色体和姊妹染色单体正常分离，仅在很小比例的花粉母细胞中观察到落后染色体。不同亚基因组间杂种的减数分裂正常程度存在一定的差别，其中Ar和An基因组之间的遗传分化可能是主要原因。产量分析表明亚基因组间杂种具有较强的杂种优势潜势，并且亚基因组间杂种的种子产量和正常花粉母细胞所占比例呈显著正相关。     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. II. Morphology,fertility and cytology of the F1 hybrid

Summary F₁ hybrids of triazine resistant Brassica napus and triazine susceptible B. oleracea were morphologically intermediate to the parent species. Of 49 hybrids examined, 44 had 28 chromosomes, two had 37, one had 38 and two had 56. The 38-chromosome plant was thought to be a matromorph, the others, A₁C₁C (28), A₁C₁CC (37) or A₁A₁C₁C₁CC (56) type hybrids. Pollen stainability averaged 9.0% in the sesquidiploid, 32.0% in the tetraploids and 89.5% in the hexaploids. All the interspecific hybrids were resistant to 1.0×10^-4 mol L^-1 atrazine. The sesquidiploid hybrids produced gametes with chromosome numbers ranging from 9 to 17 and the tetraploid hybrid gametes had chromosome numbers from 15 to 22. Most hybrids produced self-seed. The partial fertility of these hybrids may permit their backcrossing to one or both parents.     

Meiotic nondisjunction in resynthesized <Emphasis Type="Italic">Brassica napus</Emphasis> and generation of aneuploids through microspore culture and their characterization

Meiotic nondisjunction during microsporegenesis can lead to aneuploid gametes formation and reduced pollen fertility in plants. This paper reports the prevalence of meiosis I nondisjunction in a resynthesized Brassica napus (AACC, 2n = 38) and its use for aneuploid production. Meiosis in the amphidiploids was characterized by high frequencies of univalents and multivalents per PMC at diakinesis/metaphase I and notably unbalanced chromosome segregations at anaphase I (AI). In all the plants observed, 18.95–44.3% of PMCs exhibited a segregation of 18:20 (n − 1:n + 1) at AI which was caused by nondisjunction of one bivalent or the distribution of two homologous univalents to the same pole. Meiosis proceeded normally after AI then, thus led to the formation of viable n − 1 and n + 1 gametes and high pollen fertility of these plants. Microspore culture was subsequently carried out using these plants in an attempt to isolate Brassica nullisomics. Four nullisomics (2n = 36), two nullihaploids (2n = 18) and one tetrasomic haploid (2n = 20) were identified cytologically and characterized morphologicaly and physiologically. Amplified fragment length polymorphism (AFLP) survey suggested that of the six nullisomics/nullihaploids, one nullihaploids lost one A-genome chromosome and the other five lost C genome chromosome(s). Altogether, different C-genome chromosomes were thought to have been lost in the nullisomics/nullihaploids. The mechanisms underlying the meiotic abnormalities and the implications of these B. napus nullisomics are discussed.