Brassicaceae is at the forefront of evolution because of its frequent hybridization. Hybridization is responsible for the induction of widespread genetic and phenotype changes, making it important in agricultural production. In this study, we obtained resynthesized allotetraploid Brassica napus by performing interspecific crossing of B. rapa × B. oleracea combined with embryo rescue. We applied light microscopy and electronic microscopy to analyze the microstructure and ultrastructure of seeds of diploid parents and their allotetraploid progeny. Results showed that pigments in the seed coat were mainly distributed in the palisade layer. B. rapa presented the highest amount of pigment followed by B. napus and B. oleracea. B. napus had the thickest palisade layer followed by B. rapa and B. oleracea. The seed coat microsculpturing in B. rapa and B. napus was characterized as reticulate or reticulate-foveate, whereas that in B. oleracea was observed to be rugose and sulcate. The area index of the protein body was higher in central meristematic cells than in parenchyma cells. By contrast, the area index of the oil body was the lowest in central meristematic cells. Protein bodies were found to be heterogeneous with crystal globoids in two diploid parents and resynthesized allotetraploid progenies. Oil bodies consisted of large and small oil bodies, the sizes of which differed between two parents and allotetraploid progenies. Small oil bodies were spheroid, whereas large oil bodies were ovoid in shape. The quantity of oil bodies indicated that oil bodies were spheroid in two parents, ranging in size from 0.12 to 1.18 μm. In comparison, the size of large oil bodies in allotetraploid progenies exceeds 2.0 μm. These findings suggest that the anatomy of resynthesized allotetraploid seeds remarkably differs from that of two diploid parents, and these differences definitely affect the nutritional components of rapeseeds.

Drought is a major constraint of agriculture development. The intergeneric somatic hybrids between Brassica napus and Sinapis alba were created by electrofusion to obtain materials with enhanced drought tolerance. The drought tolerance of B. napus cv. Yangyou 6 (Y6) and one offspring line (W146) of the somatic hybrids was evaluated by morphological observation. Physiological parameters were determined in this study. Moreover, the activities of a few antioxidant enzymes and the transcript level of the antioxidant enzyme encoding genes were analyzed by qPCR. W146 and Y6 showed apparent wilting after drought stress for 7 days. However, Y6 wilted more severely than W146. The result of the physiological analysis showed that the relative electronic conductivity and malondialdehyde content of Y6 were higher than that of W146. The relative water content, net photosynthesis rate, proline content, and the activities of superoxide dismutase (SOD) and peroxidase in W146 were higher than that in Y6 after drought stress for 12 days. The DNA and nitrotetrazolium blue chloride staining analysis revealed less accumulation of O2·− and H2O2 in W146 than that in Y6 after drought stress. Moreover, the transcript level of some antioxidant enzyme encoding genes, such as Cu/ZnSOD, MnSOD, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase in W146, was higher than that in Y6 under drought stress. Results revealed that line W146 showed more drought stress tolerance than Y6 because line W146 could reduce oxidative damage by efficient antioxidant systems.

DNA methylation is a crucial modification process implicated in the epigenetic regulation of gene expression, which may be responsible for agronomic trait variation. However, the epigenetic regulation of the yellow- and black-seeded characters of Brassica napus has yet to be reported. In this study, methylation-sensitive amplification polymorphism (MSAP) analysis was performed to investigate the target regions of seed coat variation in B. napus. The yellow-seeded rapeseed line derived from the somatic hybrids of B. napus–Sinapis alba and the black-seeded rapeseed line were used as experimental materials. Extensive methylation changes were observed between yellow- and black-seeded B. napus. In particular, 10 % of demethylation and 5 % of hypermethylation were detected in yellow rapeseeds compared with black rapeseeds. Nonetheless, this variation was barely identified in different developmental stages. The relative expression levels of polymorphic fragments in MSAP profiles were also analyzed. The gene expression of the demethylated fragments in the yellow rapeseeds was upregulated. For instance, bHLH, a transcription factor regulating flavonoid biosynthesis, was upregulated at 3–5 weeks after flowering of yellow seeds. Therefore, epigenetic changes among rapeseed lines with different seed colors help elucidate the formation of the yellow seed character.

Tissue culture, a traditional technique broadly used for the genetic transformation and functional verification of target genes, induces epigenetic variations in transgenic acceptors of plants. This study compared the DNA methylation patterns during the callus formation of Brassica napus induced by different concentrations of 6-BA and 2,4-D through methylation-sensitive amplification polymorphism. The highest induction rate (85%) was observed in the hypocotyls cultured with 0.1 mg/L 2,4-D and the lowest methylation rate (25.09%) was detected in the hypocotyls cultured with 1.0 mg/L 6-BA. The methylation rates of the callus cultured with 0.2 and 0.05 mg/L 2,4-D were 29.99 and 28.31%, respectively. The callus induction rates were reduced to 79 and 80%. The methylation rates of the callus induced by 2.0 and 0.5 mg/L 6-BA were 28.17 and 33.98%, respectively. The callus induction rates were reduced to 76 and 74%. The expression analysis of methyltransferase under different induction conditions agreed with methylation modifications; therefore, the effects of hormones on callus induction may be partially indicated by methylation changes in B. napus genome.

Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (decrease in DNA methylation 1 and DNA methyl transferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.

A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA–ESI(−)-MSn/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA–ESI(−)-MSn/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential.

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rapeseed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.

Protoplast fusion offers an opportunity for circumventing barriers to sexual reproduction and allows for gene transfer of nuclear and cytoplasmic genomes to enrich the gene pool of cultivated species. Moreover, protoplast fusion effectively generates novel germplasm for elite breeding of conventional crosses and promotes crop improvement in existing cultivars. Over the past few decades, protoplast fusion in China has considerably progressed not only for food crops but also for economic plants. In this review, we present and discuss the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China. For example, an elite salt- and drought-resistant line, Shangrong No. 3, has already been registered as a commercial wheat cultivar. Some other hybrids have been found to have disease resistance as well as modified fatty acids, high oil and protein contents, novel cytoplasmic male sterility, and numerous other desirable agronomic characteristics that are useful for further breeding.

Rapeseed (Brassica napus L.) is one of the most important economic crops worldwide, and Sclerotinia sclerotiorum is the most dangerous disease that affects its yield greatly. Lipid transfer protein (LTP) has broad-spectrum anti-bacterial and fungal activities. In this study, B. napus was transformed using Agrobacterium tumefaciens harboring the plasmid-containing LTP gene to study its possible capability of increasing plant’s resistance. First, we optimized the petiole genetic transformation system by adjusting the days of explants, bacterial concentrations, ratio of hormones, and cultivating condition. Second, we obtained 8 positive plants by PGR analysis of T0 generation. The PGR results of T1 generation were positive, indicating that the LTP gene had been integrated into B. napus. Third, T1 transgenic plants inoculated by detached leaves with mycelia of S. sclerotiorum showed better disease resistance than non-transformants. Oxalic acid belongs to secondary metabolites of S. sclerotiorum, and several studies have demonstrated that the resistance of rapeseed to oxalic acid is significantly consistent with its resistance to S. sclerotiorum. The result from the seed germination assay showed that when T1 seeds were exposed to oxalic acid stress, their germination rate was evidently higher than that of non-transformant seeds. In addition, we measured some physiological changes in T1 plants and control plants under oxalic acid stress. The results showed that T1 transgenic plants had lower malondialdehyde (MDA) content, higher super oxide dismutase (SOD), and peroxidase (POD) activities than non-transformants, whereas disease resistance was related to low MDA content and high SOD and POD activities.

Comparative proteomics of seed filling between yellow-seeded progeny from somatic hybrids Brassica napus-Sinapis alba and black-seeded parent (B. napus) were taken out using two-dimensional electrophoresis (2-DE). The process indicated distinct differences in 2, 3, 4, 5, 6 weeks after fertilization (WAF) and mature seed. A total of 8 out of the 27 discriminate proteins were identified by mass spectrum analysis and MASCOT comparison, including protein kinase, enolase, triosephosphate isomerase, and dioxygenase. PCR primers contrived for the putative genes were applied for further identification of progenies and both parents, which indicated that spot A3-5 might be the novel protein of intergeneric hybrid, i.e., A5-2 derived from S. alba. Applying these specific primers, this study demonstrates that the new yellow-seeded germplasm is different from the existing yellow seed materials of rapeseed.

Polymerase chain reaction and genomic in situ hybridization techniques were conducted to demonstrate the genomic introgressions in somatic hybrid progenies between Brassica napus and Sinapis alba. With minisatellite core sequence 33.6 as primer, an S. alba-specific band (288 bp) was amplified in yellow seed progenies. No hybridization signals were found using the genomic DNA of S. alba as probe. In addition, degenerate primers were used for the detection of related genes based on the genes related to flavonoid biosynthesis of the model plant Arabidopsis thaliana. Sequencing results show that flavonoid pathway genes are highly conserved in A. thaliana, S. alba and B. napus, and present subtle differences because of genetic evolution. A specific band (1,672 bp) consistent with S. alba was characterized in the yellow seed lines. This study demonstrates that the new yellow seed germplasms, derived from backcrossed and self-crossed progenies of B. napus–S. alba hybrids, are stable and homozygous introgression lines with yellow seed color, and differ from existing yellow seed materials.

Embryogenic callus of Phalaenopsis amabilis derived from leaf tissue was cocultivated with Agrobacteriumtumefaciens strain LBA4404 harboring a plant cloning vector. The vector carried the lipid transfer protein (LTP) encoding gene cloned from cold tolerant Brazilian upland rice cv. IAPAR 9. The highest transformation efficiency (12.16%) was obtained when 1–2 mm calli were infected and cocultivated with 0.4 (OD600) A. tumefaciens for 20 min. Transgene integration of kan-resistant plants was confirmed through polymerase chain reaction analysis and Southern hybridization. Four hundred seventy transgenic plants, each derived from an independent protocorm-like body, were obtained. The expression of rice cold-inducible LTP gene in transgenic P. amabilis improved its adaptive responses to cold stress. The examination of transgenic plants revealed that enhanced cold tolerance was most likely due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, antioxidant superoxide dismutase, decreased accumulation of malondialdehyde, and maintained electrolytes within the membrane compared with controls.

Polyploidy is a prominent process in higher plants and is often described as a genomic shock that may induce stress and defense responses. The Brassica napus allotetraploid model was chosen to investigate the proteomic modifications that occur during allopolyploid formation. Large-scale analysis of the proteome from the leaves of B. napus was performed and compared with the homozygous diploid progenitors, Brassica rapa and Brassica oleracea, and among the proteomic changes in B. napus in the early generations (F1–F4). The abundance of all these differentially expressed proteins in the F1 generation differed from that of the corresponding proteins expressed in its progenitors, some of which relatively deviated from mid-parent predictions, exhibiting somewhat non-additive expression repatterning. Proteomic changes in the resynthesized B. napus from the first to the fourth generations were detected, which indicated that gene silencing was a permanent phenomenon and it could be reactivated at any moment. Although leaf proteins were extensively modified in synthetic B. napus, the distribution of the “housekeeping” proteins was not disturbed. Moreover, no evidence of chaos or large disorder was observed after the merging of the two genomes. Instead, a novel order quickly developed, which might evolve in further generations of synthetic B. napus.

Salt stress is one of the major abiotic stresses in agricultural plants worldwide. We used proteomics to analyze the differential expression of proteins in transgenic OsNAS1 and non-transformant Brassica napus treated with 20 mmol/L Na2CO3. Total protein from the leaves was extracted and separated through a high-resolution and highly repetitive two-dimensional electrophoresis (2-DE) technology system. Twelve protein spots were reproducibly observed to be upregulated by more than 2-fold between transgenic and non-transformant B. napus. These 12 spots were digested in-gel with trypsin and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain the peptide mass fingerprints. Protein database searching revealed that 5 of these proteins are involved in salt tolerance: dehydrogenase, glutathione S-transferase, peroxidase, 20S proteasome beta subunit, and ribulose-1,5-bisphosphate carboxylase/oxygenase. The potential functions of these identified proteins in substance and energy metabolism, stress tolerance, protein degradation, and cell defense are discussed. The salt tolerance of the transgenic rapeseed was significantly improved by the introduction of the OsNAS1 gene from Brazilian upland rice of Oryza sativa (cv. IAPAR 9).

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for Brassica napus. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose-selective medium, was transferred to B. napus hypocotyl explants by Agrobacterium-mediated transformation. More than 350 transgenic plants from three rapeseed varieties were obtained with transformation frequencies up to 24.2% when a combination of 4.5 g l−1 mannose and 10 g l−1 sucrose was used in the selection medium. For early identification of transgenic plants, histochemical staining with 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc) was used. Stable integration of the transgene was confirmed by PCR and Southern blot analysis. Mannose can be used as selective agent to identify transgenic plants in progeny i.e. segregation analysis. These results indicate that the mannose-selection system can be successfully used for Agrobacterium-mediated transformation of rapeseed.

The diploid species Brassica rapa (genome AA) and B. oleracea (genome CC) were compared by full-scale proteome analyses of seedling. A total of 28.2% of the proteins was common to both species, indicating the existence of a basal or ubiquitous proteome. However, a number of discriminating proteins (32.0%) and specific proteins (39.8%) of the Brassica A and C genomes, respectively, were identified, which could represent potentially species-specific functions. Based on these A or C genome-specific proteins, a number of PCR-based markers to distinguish B. rapa and B. oleracea species were also developed.

DNA methylation, an important epigenetic modification, serves as a key function in the polyploidization of numerous crops. In this study, early generations of resynthesized Brassica napus (F1, S1–S3), ancestral parents B. rapa and B. oleracea were analyzed to characterize their DNA methylation status during polyploidization, applying DNA methylation-sensitive amplification polymorphism (MSAP) and high-performance liquid chromatography methods. In F1, 53.4% fragments were inherited from both A- and C-genomes. Besides, 5.04 and 8.87% fragments in F1 were inherited from A- and C- genome, respectively. 5.85 and 0.8% fragments were newly appeared and disappeared in resynthesized B. napus, respectively. 13.1% of these gene sites were identified with methylation changes in F1, namely, hypermethylation (7.86%) and hypomethylation (5.24%). The lowest methylation status was detected in F1 (38.7%) compared with in S1–S3. In S3, 40.32% genes were methylated according to MSAP analysis. Sequencing of methylated fragments indicated that genes involved in multiple biological processes were modified, including transcription factors, protein modification, and transporters. Expression ananlysis of DNA methyltransferase 1 and DNA methyltransferase chromomethylase 3 in different materials was consistent to the DNA methylation status. These results can generally facilitate dissection of how DNA methylation contributes to genetic stability and improvement of B. napus during polyploidization.

Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.