Improving ovary and embryo culture techniques for efficient resynthesis of <Emphasis Type="Italic">Brassica napus</Emphasis> from reciprocal crosses between yellow-seeded diploids <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

The primary aim of this study was to optimize in vitro culture protocols to establish an efficient reproducible culture system for different Brassica interspecific crosses, and to synthesize yellow-seeded Brassica napus (AACC) for breeding and genetical studies. Reciprocal crosses were carried out between three B. rapa L. ssp. oleifera varieties (AA) and five accessions of B. oleracea var. acephala (CC). All the parental lines were yellow-seeded except one accession of B. oleracea. Hybrids were obtained through either ovary culture from crosses B. rapa × B. oleracea, or embryo culture from crosses B. oleracea × B. rapa. A higher rate of hybrid production was recorded when ovaries were cultured at 4–7 days after pollination (DAP). Of different culture media, medium E (MS with half strength macronutrients) showed good response for ovaries from all the crosses, the highest rate of hybrid production reaching 45% in B. rapa (1151) × B. oleracea (T₂). In embryo culture, the hybrid rate was significantly enhanced at 16–18 DAP, up to 48.1% in B. oleracea (T₃) × B. rapa (JB₂). The combinations of optimal DAP for excision and media components increased recovery of hybrids for ovary and embryo culture, and constituted an improved technique for B. rapa × B. oleracea crosses. In addition, yellow seeds were obtained from progenies of two crosses, indicating the feasibility of developing yellow-seeded B. napus through the hybridization between yellow-seeded diploids B. rapa and B. oleracea var. acephala.     

Development of Yellow Seeded Brassica napus Through Interspecific Crosses

Yellow seeded Brassica napus was developed through interspecific crosses with the two mustard species, B. juncea and B. carinata. The objective of these two interspecific crosses was the introgression of genes for yellow seed colour from the A genome of B. juncea and C genome of B. carinata into the A and C genomes of B. napus, respectively. The interspecific F₁ generations were backcrossed to B. napus in an attempt to eliminate B genome chromosomes and to improve fertility. Backcross F₂ plants of the (B. napus×B. juncea) ×B. napus cross were then crossed with backcross F₂ plants of the (B. napus×B. carinata) ×B. napus cross. The objective of this intercrossing was to combine the A and C genome yellow seeded characteristics of the two backcross populations into one genotype. The F₂ generation of the backcross F₂ intercrosses was grown in the field, plants were individually harvested and visually rated for seed colour. Ninety-one yellow seeded plants were identified among the 4858 plants inspected. This result indicated that the interspecific crossing scheme was successful in developing yellow seeded B. napus.     

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 effects on biomass yield in interspecific hybrids between Brassica napus and B. rapa

This study was conducted to estimate the genetic effects on biomass yield in the interspecific hybrids between Brassica napus and B. rapa, and to evaluate the relationship between parental genetic diversity and its effect on biomass yield of interspecific hybrids. Six cultivars and lines of oilseed B. napus and 20 cultivars of oilseed B. rapa from different regions of the world were chosen to produce interspecific hybrids using NC design II. Obvious genetic differences between B. rapa and B. napus were detected by RFLP. In addition, Chinese B. rapa and European B. rapa were shown genetically differences. Plant biomass yield from these interspecific hybrids were measured at the end of flowering period. Significant differences were detected among general combining ability (GCA) effects over two years and specific combining ability (SCA) effects differences were detected in 2000. The ratios of mean squares, (σ² _GCA(f) + σ² _GCA(m)) / (σ² _GCA(f) + σ² _GCA(m) + σ² _SCA), were 89% and 88% in 1999 and 2000, respectively. This indicates that both additive effects and non-additive effects contributed to the biomass yield of interspecific hybrids and the former played more important role. Some European B. rapa had significant negative GCA effects while many of Chinese B. rapa had significant positive GCA effects, indicating that Chinese B. rapa may be a valuable source for transferring favorable genes of biomass yield to B. napus. Significant positive correlation between parental genetic distance and biomass yield of interspecific hybrids implies that larger genetic distance results in higher biomass yield for the interspecific hybrids. A way to utilize interspecific heterosis for seed yield was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different Behavior of Brassica juncea and B. carinata as Sources of Phoma lingam Resistance in Experiments of Interspecific Transfer to B. napus

With the aim to transfer Phoma lingam resistance into rape, successful interspecific crosses were made between three oilseed rape varieties (Brassica napus) and the resistant species B. carinata and B. carinata. Although both hybrid types B. napus×B. juncea and B. napus×B. carinata showed the same high level of resistance as the respective resistant parent, the resistance could be only transferred from juncea crosses. After three backcross generations, lines morphologically undistinguishable from rape, fertile, and with a high degree of resistance were obtained. The resistance of B. carinata was practically lost in the first backcross. A possible explanation of this different behavior could be a higher recombination between the genomes B and C (juncea crosses) than between B and A (carinata crosses). The: applied embryo culture increased the yield of hybrids and first backcross plants and reduced considerably the generation time.     

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.     

A study on disease variation in the populations of an interspecific cross of Brassica juncea L. x B. napus L.

Summary F₁ behaviour and F₂ variation in disease reaction were studied in the interspecific cross Brassica juncea x B. napus. Gene(s) for adult resistance to blackleg (Leptosphaeria maculans) were found to be present in the A genome of B. juncea and could be transferred to B. napus. Gene(s) for complete (seedling plus adult) resistance in B. juncea appeared to be located in the B genome. The chance of their transfer to the oilseed rapes (B. napus or B. campestris) would therefore seem to be remote.     

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)     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

Plant Regeneration from the Hybridization of Brassica juncea and B. napus Through Embryo Culture

Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l^?1 naphthylacetic acid (NAA) + 1.5 mg l^?1 6‐benzylaminopurine (BAP) and with 0.3 mg l^?1 NAA + 2.0 mg l^?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.     

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     

Long-term monitoring of feral genetically modified herbicide-tolerant Brassica napus populations around unloading Japanese ports

Genetically modified, herbicide-tolerant (GMHT) Brassica napus plants originating from seed spill have recently been found along roadsides leading from Japanese ports that unload oilseed rape. Such introductions have potential biodiversity effects (as defined by the Cartagena Protocol): these include replacement of native elements in the biota through competitive suppression or hybridization. We conducted surveys in the period 2006–2011 to assess such threats. We examined shifts in the population distribution and occurrence of GMHT plants in 1,029 volunteer introduced assemblages of B. napus, 1,169 of B. juncea, and 184 of B. rapa around 12 ports. GMHT B. napus was found around 10 of 12 ports, but its proportion in the populations varied greatly by year and location. Over the survey period, the distributions of a pure non-GMHT population around Tobata and a pure GMHT population around Hakata increased significantly. However, there was no common trend of population expansion or contraction around the 12 ports. Furthermore, we found no herbicide tolerant B. juncea and B. rapa plants derived from crosses with GMHT B. napus. Therefore, GMHT B. napus is not invading native vegetation surrounding its populations and not likely to cross with congeners in Japanese environment.     

Chromosomal structural changes in Capsicum annuum L. and C. chinense Jacq.

Summary Wide hybridizations between M. arvensis and Brassica amphidiploid species (B. napus and B. juncea) were carried out in order to incorporate desirable traits of M. arvensis into Brassica crops. Crossing barriers between them were present without the use of in vitro techniques. F₁ hybrids have been produced through ovary culture, when M. arvensis were used as a female parent. Higher hybrid embryo productivity (3.07 embryos per pollination) was obtained in the cross of M. arvensis x B. napus than in that of M. arvensis x B. juncea (0.79 embryos). The hybridity was confirmed by morphology, cytology, isozyme and Southern analyses. The first backcrossing progenies and open pollinated ones were produced.     

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.     

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.     

Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue

Black rot is a bacterial disease of Brassica oleracea caused by Xanthomonas campestris pv. campestris. Resistance to the major black rot races 1 or 4 has been identified in related Brassica species including B. carinata and B. napus. In this study, two B. juncea accessions (A 19182 and A 19183) that are resistant to races 1 and 4 of Xcc were used as maternal and paternal parents to generate interspecific hybrids with B. oleracea cultivars. Interspecific hybrids were recovered using the embryo rescue technique and confirmed through inheritance of paternal molecular markers. Twenty-six interspecific hybrid plants were obtained between A 19182 and B. oleracea cultivars, but no interspecific hybrids were obtained using A 19183. Although interspecific hybrid plants were male sterile, they were used successfully as maternal parents to generate backcross plants using embryo rescue. All hybrid and BC₁ plants were resistant to black rot races 1 and 4.     

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

Summary Allopolyploids 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 Brassica rapa and 16 Brassica oleracea 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.     

Possibilities of direct introgression from Brassica napus to B. juncea and indirect introgression from B. napus to related Brassicaceae through B. juncea

The impact of genetically modified canola (Brassica napus) on biodiversity has been examined since its initial stage of commercialization. Various research groups have extensively investigated crossability and introgression among species of Brassicaceae. B. rapa and B. juncea are ranked first and second as the recipients of cross-pollination and introgression from B. napus, respectively. Crossability between B. napus and B. rapa has been examined, specifically in terms of introgression from B. napus to B. rapa, which is mainly considered a weed in America and European countries. On the other hand, knowledge on introgression from B. napus to B. juncea is insufficient, although B. juncea is recognized as the main Brassicaceae weed species in Asia. It is therefore essential to gather information regarding the direct introgression of B. napus into B. juncea and indirect introgression of B. napus into other species of Brassicaceae through B. juncea to evaluate the influence of genetically modified canola on biodiversity. We review information on crossability and introgression between B. juncea and other related Brassicaseae in this report.     

Cytogenetics of Brassica juncea×Brassica rapa hybrids and patterns of variation in the hybrid derivatives

Interspecific hybridization is an important tool to elucidate intergenomic relationships, transfer characters across species and develop synthetic amphidiploids, and it has been widely applied for improving Brassicas. The objective of the present study was to create genetic variability in Brassica through interspecific hybridization. Crosses between Brassica juncea (AABB, 2n= 36), and Brassica rapa (AA, 2n = 20) vars toria, yellow sarson, and brown sarson were attempted, and the hybrid derivatives were advanced to the F4 generation. Hybrids were obtained from the crosses B. juncea× toria and B. juncea× yellow sarson. The F₁ plants were vigorous and intermediate to the parents in many morphological traits. The meiotic study of AAB hybrids showed 10 II + 8 I in the majority (71.8%) of cells analysed. A maximum of 12 and a minimum of seven bivalents were also observed in a few cells. The occurrence of multivalent associations (trivalents to pentavalents) at diakinesis/metaphase I and a bridge‐fragment configuration at anaphase I were attributed to homoeology between A and B genomes. A high percentage of plants resembling B. juncea was observed in the F₂ generation. Transgressive segregation in both directions was found for plant height, primary branches, main raceme length, siliquae on main raceme, siliqua intensity, seeds per siliqua and seed yield. There were significant differences for the 14 characters in the F₄ derivatives. Moderate to high estimates of phenotypic and genotypic coefficients of variation, broad‐sense heritability, and expected genetic advance were found for seed yield, 1000‐seed weight, siliquae per plant, seeds per siliqua and days to flowering. Intergenomic recombination, reflected as wide variation in the hybrid progenies, permitted the selection of some useful derivatives.