Genetic dissection of Brassica napus seed vigor after aging.

KEY MESSAGE: Stable and novel QTLs that affect seed vigor under different storage durations were discovered, and BnaOLE4, located in the interval of cqSW-C2-3, increased seed vigor after aging. Seed vigor is an important trait in crop breeding; however, the underlying molecular regulatory mechanisms governing this trait in rapeseed remain largely unknown. In the present study, vigor-related traits were analyzed in seeds from a doubled haploid (DH) rapeseed (Brassica napus) population grown in 2 different environments using seeds stored for 7, 5, and 3 years under natural storage conditions. A total of 229 quantitative trait loci (QTLs) were identified and were found to explain 3.78%-17.22% of the phenotypic variance for seed vigor-related traits after aging. We further demonstrated that seed vigor-related traits were positively correlated with oil content (OC) but negatively correlated with unsaturated fatty acids (FAs). Some pleiotropic QTLs that collectively regulate OC, FAs, and seed vigor, such as uq.A8, uq.A3-2, uq.A9-2, and uq.C3-1, were identified. The transcriptomic results from extreme pools of DH lines with distinct seed vigor phenotypes during accelerated aging revealed that various biological pathways and metabolic processes (such as glutathione metabolism and reactive oxygen species) were involved in seed vigor. Through integration of QTL analysis and RNA-Seq, a regulatory network for the control of seed vigor was constructed. Importantly, a candidate (BnaOLE4) from cqSW-C2-3 was selected for functional analysis, and transgenic lines overexpressing BnaOLE4 showed increased seed vigor after artificial aging. Collectively, these results provide novel information on QTL and potential candidate genes for molecular breeding for improved seed storability.

Plant Functional Genomics Based on High-Throughput CRISPR Library Knockout Screening: A Perspective.

Plant biology studies in the post-genome era have been focused on annotating genome sequences' functions. The established plant mutant collections have greatly accelerated functional genomics research in the past few decades. However, most plant genome sequences' roles and the underlying regulatory networks remain substantially unknown. Clustered, regularly interspaced short palindromic repeat (CRISPR)-associated systems are robust, versatile tools for manipulating plant genomes with various targeted DNA perturbations, providing an excellent opportunity for high-throughput interrogation of DNA elements' roles. This study compares methods frequently used for plant functional genomics and then discusses different DNA multi-targeted strategies to overcome gene redundancy using the CRISPR-Cas9 system. Next, this work summarizes recent reports using CRISPR libraries for high-throughput gene knockout and function discoveries in plants. Finally, this work envisions the future perspective of optimizing and leveraging CRISPR library screening in plant genomes' other uncharacterized DNA sequences.

Multi-Omics Analysis Revealed the AGR-FC.C3 Locus of Brassica napus as a Novel Candidate for Controlling Petal Color.

Variations in the petal color of Brassica napus are crucial for ornamental value, but the controlled loci for breeding remain to be unraveled. Here, we report a candidate locus, AGR-FC.C3, having conducted a bulked segregant analysis on a segregating population with different petal colors. Our results showed that the locus covers 9.46 Mb of the genome, harboring 951 genes. BnaC03.MYB4, BnaC03.MYB85, BnaC03.MYB73, BnaC03.MYB98, and BnaC03.MYB102 belonging to MYB TFs families that might regulate the petal color were observed. Next, a bulk RNA sequencing of white and orange-yellow petals on three development stages was performed to further identify the possible governed genes. The results revealed a total of 51 genes by overlapping the transcriptome data and the bulked segregant analysis data, and it was found that the expression of BnaC03.CCD4 was significantly up-regulated in the white petals at three development stages. Then, several novel candidate genes such as BnaC03.ENDO3, BnaC03.T22F8.180, BnaC03.F15C21.8, BnaC03.Q8GSI6, BnaC03.LSD1, BnaC03.MAP1Da, BnaC03.MAP1Db, and BnaC03G0739700ZS putative to controlling the petal color were identified through deeper analysis. Furthermo re, we have developed two molecular markers for the reported functional gene BnaC03.CCD4 to discriminate the white and orange-yellow petal colors. Our results provided a novel locus for breeding rapeseed with multi-color petals.

3D genome structural variations play important roles in regulating seed oil content of Brassica napus.

Dissecting the complex regulatory mechanism of seed oil content (SOC) is one of the main research goals in Brassica napus. Increasing evidence suggests that genome architecture is linked to multiple biological functions. However, the effect of genome architecture on SOC regulation remains unclear. Here, we used high-throughput chromatin conformation capture to characterize differences in the three-dimensional (3D) landscape of genome architecture of seeds from two B. napus lines, N53-2 (with high SOC) and Ken-C8 (with low SOC). Bioinformatics analysis demonstrated that differentially accessible regions and differentially expressed genes between N53-2 and Ken-C8 were preferentially enriched in regions with quantitative trait loci (QTLs)/associated genomic regions (AGRs) for SOC. A multi-omics analysis demonstrated that expression of SOC-related genes was tightly correlated with genome structural variations in QTLs/AGRs of B. napus. The candidate gene BnaA09g48250D, which showed structural variation in a QTL/AGR on chrA09, was identified by fine-mapping of a KN double-haploid population derived from hybridization of N53-2 and Ken-C8. Overexpression and knockout of BnaA09g48250D led to significant increases and decreases in SOC, respectively, in the transgenic lines. Taken together, our results reveal the 3D genome architecture of B. napus seeds and the roles of genome structural variations in SOC regulation, enriching our understanding of the molecular mechanisms of SOC regulation from the perspective of spatial chromatin structure.

Transcriptome Shock in Developing Embryos of a Brassica napus and Brassica rapa Hybrid.

Interspecific crosses that fuse the genomes of two different species may result in overall gene expression changes in the hybrid progeny, called 'transcriptome shock'. To better understand the expression pattern after genome merging during the early stages of allopolyploid formation, we performed RNA sequencing analysis on developing embryos of Brassica rapa, B. napus, and their synthesized allotriploid hybrids. Here, we show that the transcriptome shock occurs in the developing seeds of the hybrids. Of the homoeologous gene pairs, 17.1% exhibit expression bias, with an overall expression bias toward B. rapa. The expression level dominance also biases toward B. rapa, mainly induced by the expression change in homoeologous genes from B. napus. Functional enrichment analysis revealed significant differences in differentially expressed genes (DEGs) related to photosynthesis, hormone synthesis, and other pathways. Further study showed that significant changes in the expression levels of the key transcription factors (TFs) could regulate the overall interaction network in the developing embryo, which might be an essential cause of phenotype change. In conclusion, the present results have revealed the global changes in gene expression patterns in developing seeds of the hybrid between B. rapa and B. napus, and provided novel insights into the occurrence of transcriptome shock for harnessing heterosis.

Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library.

The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.

Dissecting the Meiotic Recombination Patterns in a Brassica napus Double Haploid Population Using 60K SNP Array.

Meiotic recombination not only maintains the stability of the chromosome structure but also creates genetic variations for adapting to changeable environments. A better understanding of the mechanism of crossover (CO) patterns at the population level is useful for crop improvement. However, there are limited cost-effective and universal methods to detect the recombination frequency at the population level in Brassica napus. Here, the Brassica 60K Illumina Infinium SNP array (Brassica 60K array) was used to systematically study the recombination landscape in a double haploid (DH) population of B. napus. It was found that COs were unevenly distributed across the whole genome, and a higher frequency of COs existed at the distal ends of each chromosome. A considerable number of genes (more than 30%) in the CO hot regions were associated with plant defense and regulation. In most tissues, the average gene expression level in the hot regions (CO frequency of greater than 2 cM/Mb) was significantly higher than that in the regions with a CO frequency of less than 1 cM/Mb. In addition, a bin map was constructed with 1995 recombination bins. For seed oil content, Bin 1131 to 1134, Bin 1308 to 1311, Bin 1864 to 1869, and Bin 2184 to 2230 were identified on chromosomes A08, A09, C03, and C06, respectively, which could explain 8.5%, 17.3%, 8.6%, and 3.9% of the phenotypic variation. These results could not only deepen our understanding of meiotic recombination in B. napus at the population level, and provide useful information for rapeseed breeding in the future, but also provided a reference for studying CO frequency in other species.

Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol.

Very long-chain fatty acids (VLCFAs) are important industrial raw materials and can be produced by genetically modified oil plants. For a long time, class A lysophosphatidic acid acyltransferase (LPAT) was considered unable to promote the accumulation of VLCFA in oil crops. The bottlenecks that the transgenic high VLCFA lines have an oil content penalty and the low amount of VLCFA in phosphatidylcholine remains intractable. In the present study, a class A LPAT2 from Camelina sativa (CsaLPAT2) promoting VLCFAs accumulation in phospholipid was found. Overexpression of CsaLPAT2 alone in Arabidopsis seeds significantly increased the VLCFA content in triacylglycerol, including C20:0, C20:2, C20:3, C22:0, and C22:1. The proportion of phosphatidic acid molecules containing VLCFAs in transgenic seeds reached up to 45%, which was 2.8-fold greater than that in wild type. The proportion of phosphatidylcholine and diacylglycerol molecules containing VLCFAs also increased significantly. Seed size in CsaLPAT2 transgenic lines showed a slight increase without an oil content penalty. The total phospholipid content in the seed of the CsaLPAT2 transgenic line was significantly increased. Furthermore, the function of class A LPAT in promoting the accumulation of VLCFAs is conserved in the representative oil crops of Brassicaceae, such as C. sativa, Arabidopsis thaliana, Brassica napus, Brassica rapa, and Brassica oleracea. The findings of this study provide a promising gene resource for the production of VLCFAs.

Lysophosphatidic acid acyltransferase 2 and 5 commonly, but differently, promote seed oil accumulation in Brassica napus.

BACKGROUND: Increasing seed oil content (SOC) of Brassica napus has become one of the main plant breeding goals over the past decades. Lysophosphatidic acid acyltransferase (LPAT) performs an important molecular function by regulating the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane and storage lipids. However, the mechanism underlying the effect of LPAT on the SOC of B. napus remains unclear. RESULTS: In the present study, significant elevation of SOC was achieved by overexpressing BnLPAT2 and BnLPAT5 in B. napus. RNAi and CRISPR-Cas9 were also successfully used to knock down and knock out these two genes in B. napus where SOC significantly decreased. Meanwhile, we found an accumulation of lipid droplets and oil bodies in seeds of BnLPAT2 and BnLPAT5 overexpression lines, whereas an increase of sugar and protein in Bnlpat2 and Bnlpat5 mutant seeds. Sequential transcriptome analysis was further performed on the developing seeds of the BnLPAT2 and BnLPAT5 overexpression, knockdown, and knockout rapeseed lines. Most differentially expressed genes (DEGs) that were expressed in the middle and late stages of seed development were enriched in photosynthesis and lipid metabolism, respectively. The DEGs involved in fatty acid and lipid biosynthesis were active in the overexpression lines but were relatively inactive in the knockdown and knockout lines. Further analysis revealed that the biological pathways related to fatty acid/lipid anabolism and carbohydrate metabolism were specifically enriched in the BnLPAT2 overexpression lines. CONCLUSIONS: BnLPAT2 and BnLPAT5 are essential for seed oil accumulation. BnLPAT2 preferentially promoted diacylglycerol synthesis to increase SOC, whereas BnLPAT5 tended to boost PA synthesis for membrane lipid generation. Taken together, BnLPAT2 and BnLPAT5 can jointly but differently promote seed oil accumulation in B. napus. This study provides new insights into the potential mechanisms governing the promotion of SOC by BnLPAT2 and BnLPAT5 in the seeds of B. napus.

The Transcriptome and Metabolome Reveal the Potential Mechanism of Lodging Resistance in Intergeneric Hybrids between Brassica napus and Capsella bursa-pastoris.

Lodging is one of the main reasons for the reduction in seed yield and is the limitation of mechanized harvesting in B. napus. The dissection of the regulatory mechanism of lodging resistance is an important goal in B. napus. In this study, the lodging resistant B. napus line, YG689, derived from the hybridization between B. napus cv. Zhongyou 821 (ZY821) and Capsella bursa-pastoris, was used to dissect the regulation mechanism of hard stem formation by integrating anatomical structure, transcriptome and metabolome analyses. It was shown that the lignocellulose content of YG689 is higher than that of ZY821, and some differentially expressed genes (DEGs) involved in the lignocellulose synthesis pathway were revealed by transcriptome analyses. Meanwhile, GC-TOF-MS and UPLC-QTOF-MS identified 40, 54, and 31 differential metabolites in the bolting stage, first flower stage, and the final flower stage. The differential accumulation of these metabolites might be associated with the lignocellulose biosynthesis in B. napus. Finally, some important genes that regulate the metabolic pathway of lignocellulose biosynthesis, such as BnaA02g18920D, BnaA10g15590D, BnaC05g48040D, and NewGene_216 were identified in B. napus through the combination of transcriptomics and metabolomics data. The present results explored the potential regulatory mechanism of lignocellulose biosynthesis, which provided a new clue for the breeding of B. napus with lodging resistance in the future.

Construction of a Quantitative Genomic Map, Identification and Expression Analysis of Candidate Genes for Agronomic and Disease-Related Traits in Brassica napus.

Rapeseed is the second most important oil crop in the world. Improving seed yield and seed oil content are the two main highlights of the research. Unfortunately, rapeseed development is frequently affected by different diseases. Extensive research has been made through many years to develop elite cultivars with high oil, high yield, and/or disease resistance. Quantitative trait locus (QTL) analysis has been one of the most important strategies in the genetic deciphering of agronomic characteristics. To comprehend the distribution of these QTLs and to uncover the key regions that could simultaneously control multiple traits, 4,555 QTLs that have been identified during the last 25 years were aligned in one unique map, and a quantitative genomic map which involved 128 traits from 79 populations developed in 12 countries was constructed. The present study revealed 517 regions of overlapping QTLs which harbored 2,744 candidate genes and might affect multiple traits, simultaneously. They could be selected to customize super-rapeseed cultivars. The gene ontology and the interaction network of those candidates revealed genes that highly interacted with the other genes and might have a strong influence on them. The expression and structure of these candidate genes were compared in eight rapeseed accessions and revealed genes of similar structures which were expressed differently. The present study enriches our knowledge of rapeseed genome characteristics and diversity, and it also provided indications for rapeseed molecular breeding improvement in the future.

The genome wide analysis of Tryptophan Aminotransferase Related gene family, and their relationship with related agronomic traits in Brassica napus.

Tryptophan Aminotransferase of Arabidopsis1/Tryptophan Aminotransferase-Related (TAA1/TAR) proteins are the enzymes that involved in auxin biosynthesis pathway. The TAA1/TAR gene family has been systematically characterized in several plants but has not been well reported in Brassica napus. In the present study, a total of 102 BnTAR genes with different number of introns were identified. It was revealed that these genes are distributed unevenly and occurred as clusters on different chromosomes except for A4, A5, A10 and C4 in B. napus. Most of the these BnTAR genes are conserved despite of existing of gene loss and gene gain. In addition, the segmental replication and whole-genome replication events were both play an important role in the BnTAR gene family formation. Expression profiles analysis indicated that the expression of BnTAR gene showed two patterns, part of them were mainly expressed in roots, stems and leaves of vegetative organs, and the others were mainly expressed in flowers and seeds of reproductive organs. Further analysis showed that many of BnTAR genes were located in QTL intervals of oil content or seed weight, for example BnAMI10 was located in cqOC-C5-4 and cqSW-A2-2, it indicated that some of the BnTAR genes might have relationship with these two characteristics. This study provides a multidimensional analysis of the TAA1/TAR gene family and a new insight into its biological function in B. napus.

Genome-wide unbalanced expression bias and expression level dominance toward Brassica oleracea in artificially synthesized intergeneric hybrids of Raphanobrassica.

Raphanobrassica (RrRrCrCr, 2n = 4x = 36), which is generated by distant hybridization between the maternal parent Raphanus sativus (RsRs, 2n = 2x = 18) and the paternal parent Brassica oleracea (C°C°, 2n = 2x = 18), displays intermediate silique phenotypes compared to diploid progenitors. However, the hybrid shares much more similarities in silique phenotypes with those of B. oleracea than those of R. sativus. Strikingly, the silique of Raphanobrassica is obviously split into two parts. To investigate the gene expression patterns behind these phenomena, transcriptome analysis was performed on the upper, middle, and lower sections of pods (RCsiu, RCsim, and RCsil), seeds in the upper and lower sections of siliques (RCseu and RCsel) from Raphanobrassica, whole pods (Rsi and Csi) and all seeds in the siliques (Rse and Cse) from R. sativus and B. oleracea. Transcriptome shock was observed in all five aforementioned tissues of Raphanobrassica. Genome-wide unbalanced biased expression and expression level dominance were also discovered, and both of them were toward B. oleracea in Raphanobrassica, which is consistent with the observed phenotypes. The present results reveal the global gene expression patterns of different sections of siliques of Raphanobrassica, pods, and seeds of B. oleracea and R. sativus, unraveling the tight correlation between global gene expression patterns and phenotypes of the hybrid and its parents.

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology.

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

The Expression Characteristics of NPF Genes and Their Response to Vernalization and Nitrogen Deficiency in Rapeseed.

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF) genes, initially characterized as nitrate or peptide transporters in plants, are involved in the transport of a large variety of substrates, including amino acids, nitrate, auxin (IAA), jasmonates (JAs), abscisic acid (ABA) and gibberellins (GAs) and glucosinolates. A total of 169 potential functional NPF genes were excavated in Brassica napus, and they showed diversified expression patterns in 90 different organs or tissues based on transcriptome profile data. The complex time-serial expression changes were found for most functional NPF genes in the development process of leaves, silique walls and seeds, which indicated that the expression of Brassica napus NPF (BnaNPF) genes may respond to altered phytohormone and secondary metabolite content through combining with promoter element enrichment analysis. Furthermore, many BnaNPF genes were detected to respond to vernalization with two different patterns, and 20 BnaNPF genes responded to nitrate deficiency. These results will provide useful information for further investigation of the biological function of BnaNPF genes for growth and development in rapeseed.

Influence of Operating Parameters on Chlorine Release and Pollutant Emission Characteristics of a 130 t/h BCFB Combustion System.

A 130 t/h biomass circulating fluidized bed (BCFB) boiler combustion system model, considering the chloride release and pollutant emissions during the biomass combustion, was established using the Modelica language. The effects of the biomass feed amount, limestone amount, excess air coefficients, and different ratios of primary and secondary air on the boiler furnace temperature and flue gas composition (O2, CO2, SO2, HCl, and KCl) were investigated. Upon the biomass feed amount step change, the variation ranges of NO and KCl concentrations were very large, which were 18.58 and 21.16% of the before step value, respectively. The step change of the limestone input had little effect on b ed temperature in the dense phase zone, but it could obviously reduce the SO2 concentration. The concentration of SO2 in flue gas decreased by 22.56% when the limestone input increased by 50%. The removal rate of SO2 gradually decreased with the increase of the limestone amount. The SO2 desulfurization rate decreased by 68.30% when the amount of limestone increased from 0.0275 to 0.0825 kg/s. More NO would be generated and KCl concentration would be significantly reduced with the increase of the excess air coefficient. When the ratio of primary and secondary air was 4:6, the NO concentration in flue gas was lower than 86.06 mg/Nm3.

Genome-wide identification of the restorer-of-fertility-like (RFL) gene family in Brassica napus and expression analysis in Shaan2A cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS) is very important in hybrid breeding. The restorer-of-fertility (Rf) nuclear genes rescue the sterile phenotype. Most of the Rf genes encode pentatricopeptide repeat (PPR) proteins. RESULTS: We investigated the restorer-of-fertility-like (RFL) gene family in Brassica napus. A total of 53 BnRFL genes were identified. While most of the BnRFL genes were distributed on 10 of the 19 chromosomes, gene clusters were identified on chromosomes A9 and C8. The number of PPR motifs in the BnRFL proteins varied from 2 to 19, and the majority of BnRFL proteins harbored more than 10 PPR motifs. An interaction network analysis was performed to predict the interacting partners of RFL proteins. Tissue-specific expression and RNA-seq analyses between the restorer line KC01 and the sterile line Shaan2A indicated that BnRFL1, BnRFL5, BnRFL6, BnRFL8, BnRFL11, BnRFL13 and BnRFL42 located in gene clusters on chromosomes A9 and C8 were highly expressed in KC01. CONCLUSIONS: In the present study, identification and gene expression analysis of RFL gene family in the CMS system were conducted, and seven BnRFL genes were identified as candidates for the restorer genes in Shaan2A CMS. Taken together, this method might provide new insight into the study of Rf genes in other CMS systems.

A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus.

BACKGROUND: Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. RESULTS: Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. CONCLUSION: A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.