To establish a systematic approach for evaluating heat tolerance at the seedling stage in broccoli, we investigated 14 representative cultivars cultivated in China. Physiological indicators such as electrical conductivity, malondialdehyde, proline, and chlorophyll content were measured before and after heat stress, alongside phenotypic scoring of heat injury, to characterize the differential thermotolerance among genotypes. The results indicated that significant differences (p < 0.05) in heat tolerance at the seedling stage were observed among different broccoli cultivars after heat stress treatment at 40 °C. Among them, Yanxiu exhibited the strongest heat tolerance, followed by Meiqing, Naihanyouxiu, Meiao 7172, Feicui 5, Guowang 11, Zheqing 80, Zhongqing 15, Zhongqing 318, Zhongqing 319, and Qianghan. Lvxiong 90, Zhongqing 11, and Zhongqing 16 were the least heat tolerant. Pearson’s correlation analysis demonstrated that the seedling heat tolerance of different broccoli cultivars was significantly negatively correlated with electrical conductivity, with a correlation coefficient of 0.542 (p < 0.05). In this study, a rapid and robust method for determining the heat resistance response of broccoli was established, providing a scientific basis and technical support for the identification of the heat resistance of new broccoli cultivars and the selection and breeding of heat-tolerant cultivars in China.

To elucidate differences in nutritional components and glucosinolates between broccoli and lacinato kale leaves, and to promote consumption of broccoli and kale leaves for human nutrition, food development, and by-product utilisation, this study used UPLC-MS/MS and HPLC for both qualitative and quantitative analyses of their metabolome and glucosinolate profiles. In broccoli leaves, 11 glucosinolates were identified, notably including high concentrations of glucoraphanin, glucobrassicin, and neoglucobrassicin. In contrast, lacinato kale leaves contained seven glucosinolates, with glucoraphasatin being the most abundant. Moreover, our analysis revealed 644 metabolites, 87 of which were differentially expressed between these crucuiferous vegetables. These findings not only offer new insights into nutritional value but also highlight their potential as plant chassis for the biosynthesis of valuable products, thereby broadening food diversity and improving by-product utilisation.

Clubroot, caused by Plasmodiophora brassicae, poses a serious threat to cruciferous crop production worldwide. Breeding resistant varieties remains the most cost-effective strategy to mitigate yield losses, yet achieving durable, stable, and broad-spectrum resistance continues to be a formidable challenge. Recent advances in genetic and genomic technologies have improved the understanding of complex host-pathogen interactions, leading to the identification of key resistance loci, including dominant resistance genes such as CRa and Crr1, as well as quantitative trait loci. This review discusses the genetic mechanisms governing clubroot resistance and highlights applications in breeding, such as marker-assisted selection and CRISPR/Cas9-based genome editing, which are accelerating the development of resistant germplasm. Furthermore, integrated management strategies, encompassing resistant cultivars, crop rotation, biocontrol agents, and soil amendments, are emphasized as critical components for sustainable disease management. This review summarizes the major resistance genes against clubroot and discusses potential strategies to address the persistent threat posed by the disease.

To investigate role of ELONGATED HYPOCOTYL 5 (HY5) in the regulation of glucosinolate (GSL) metabolism, we conducted RNA-seq on BoHY5 overexpression line B42OE, knockout line B42CR, wild-type B42WT. BoHY5 plays a negative regulatory role in the transcription of MYB transcription factors (TFs) and GSL pathway genes. Furthermore, BoHY5 overexpression reduces the content of aliphatic GSLs (especially glucoraphanin), but increases the content of indolic GSLs (especially glucobrassicin). Knocking out BoHY5 can increase the content of aliphatic and aromatic GSLs and raise the total GSL content, but it can decrease the content of indolic GSLs. These results suggest that BoHY5 is a key regulatory factor in balancing the metabolic levels between aliphatic and indolic GSLs, which provides a theoretical basis for efficient biosynthesis of GSL in broccoli.

To elucidate the comprehensive mechanism by which nitric oxide (NO) enhances low-temperature tolerance in cucumber, we utilized two cucumber cultivars (Jinyan No. 4 and Jinyou No. 1) as experimental materials. By integrating transcriptomic analysis with physiological indicators, we investigated the physiological and molecular mechanisms underlying the NO-mediated improvement of cold tolerance. Both molecular and physiological data revealed that phytohormone signal transduction and alpha-linolenic acid metabolism were significantly affected by low-temperature stress alone and in combination with exogenous SNP treatment in both cultivars. Under low-temperature stress, most transcripts associated with abscisic acid (ABA) biosynthesis, ABA signal transduction, and flavonoid biosynthesis were coordinately downregulated in cucumber. In contrast, transcripts related to secondary metabolism, lipid metabolism, glutathione biosynthesis, and hormone signal transduction—including salicylic acid (SA), ethylene (ETH), gibberellin (GA), and jasmonic acid (JA) pathways—were coordinately upregulated. Additionally, exogenous SNP was found to regulate both phytohormone signal transduction and endogenous hormone levels. These results suggest that exogenous NO improves low-temperature tolerance in cucumber seedlings primarily by modulating phytohormone signaling and secondary metabolism.

Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.

This review provides a comprehensive summary of the latest international research on detection methods for glucosinolates in cruciferous plants. The article examines various analytical tech-niques, including high-performance liquid chromatography (HPLC), liquid chromatog-raphy-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA), while highlighting their respective advantages and limitations. Additionally, the review delves into recent advancements in sample preparation, extraction, and quantification methods, offering valuable insights into the accurate and efficient determination of glucosinolate content across diverse plant materials. Furthermore, it underscores the critical importance of standardization and validation of these methodologies to ensure reliable glucosinolate analysis in both scientific research and industrial applications.

This study aimed to develop a systematic method for assessing the cold tolerance of broccoli seedlings, which is helpful to evaluating the cold tolerance of broccoli cultivars. We selected 14 classical broccoli cultivars and evaluated their cold tolerance by examining physiological indicators including the levels of cold resistance, electrical conductivity, malondialdehyde (MDA), soluble sugar, and hydrogen peroxide (H2O2). The results showed that there were significant differences in detected characteristics in all cultivars after cold stress during the seedling stage (p < 0.05). Among them, Meiqing had the strongest cold tolerance, followed by King 11, Zhongqing 15, Meiao 7172, Zhongqing 318, Zheqing 80, Zhongqing 16, Zhongqing 319, and Lvxiong 90. However, Zhongqing 11, Yanxiu, Qianghan, and Feicui 5, showed the worst cold tolerance (all died). Pearson correlation analysis indicated that there was a significantly negative correlation between the cold tolerance and the electrical conductivity during broccoli seedling stage, with a correlation coefficient of −0.586 (p < 0.05). At the same time, we found that the electrical conductivity of all broccoli cultivars showed a positive correlation with the MDA and soluble sugar levels, with the correlation coefficients 0.650 and 0.573, respectively (p < 0.05). This study not only firstly provides a fundamental method for evaluating the cold tolerance in different broccoli genotypes and the other cruciferous vegetables, but also offers a scientific evidence explaining the cold tolerance of the Meiqing, King 11, and Zhongqing 15 cultivars widely cultivated in China.

Heterosis utilization in a large proportion of crops depends on the use of cytoplasmic male sterility (CMS) tools, requiring the development of homozygous fertile lines and CMS lines1. Although doubled haploid (DH) technology has been developed for several crops to rapidly generate fertile lines2,3, CMS lines are generally created by multiple rounds of backcrossing, which is time consuming and expensive4. Here we describe a method for generating both homozygous fertile and CMS lines through in vivo paternal haploid induction (HI). We generated in-frame deletion and restored frameshift mutants of BoCENH3 in Brassica oleracea using the CRISPR/Cas9 system. The mutants induced paternal haploids by outcrossing. We subsequently generated HI lines with CMS cytoplasm, which enabled the generation of homozygous CMS lines in one step. The BoCENH3-based HI system provides a new DH technology to accelerate breeding in Brassica and other crops.

Roots are fundamental to the growth, development, and survival of plants. Beyond anchoring the plant, roots absorb water and nutrients, supporting the plant’s ability to grow and function normally. Root systems, originating from the apical meristem, exhibit significant diversity depending on the plant species. ROS are byproducts of aerobic metabolism, present in both above- and below-ground plant tissues. While ROS were once considered merely harmful byproducts of oxygen metabolism, they are now recognized as critical signaling molecules that regulate plant growth and development. Under stress conditions, plants produce elevated levels of ROS, which can inhibit growth. However, moderate ROS levels act as signals that integrate various regulatory pathways, contributing to normal plant development. However, there is still a lack of comprehensive and systematic research on how ROS precisely regulate root growth and development. This review provides an overview of ROS production pathways and their regulatory mechanisms in plants, with a particular focus on their influence on root development.

A causal gene BoUGT76C2, conferring clubroot resistance in wild Brassica oleracea, was identified and functionally characterized. Clubroot is a devastating soil-borne disease caused by the obligate biotrophic pathogen Plasmodiophora brassica (P. brassicae), which poses a great threat to Brassica oleracea (B. oleracea) production. Although several QTLs associated with clubroot resistance (CR) have been mapped in cultivated B. oleracea, none have been cloned in B. oleracea. Previously, we found that the wild B. oleracea B2013 showed high resistance to clubroot. In this study, we constructed populations using B2013 and broccoli line 90196. CR in B2013 is quantitatively inherited, and a major QTL, BolC.Pb9.1, was identified on C09 using QTL-seq and linkage analysis. The BolC.Pb9.1 was finely mapped to a 56 kb genomic region using F2:3 populations. From the target region, the candidate BoUGT76C2 showed nucleotide variations between the parents, and was inducible in response to P. brassicae infection. We generated BoUGT76C2 overexpression lines in the 90196 background, which showed significantly enhanced resistance to P. brassicae compared to the WT line, suggesting that BoUGT76C2 corresponds to the resistance gene BolC.Pb.9.1. This is the first report on the CR gene map-based cloning and functional analysis from wild relatives, which provides a theoretical basis to the understanding of the molecular mechanism of CR, and lays a foundation to improve the CR of cultivated B. oleracea.

This review provides a comprehensive summary of the latest international research on detection methods for glucosinolates in cruciferous plants. This article examines various analytical techniques, including high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), and capillary electrophoresis (CE), while highlighting their respective advantages and limitations. Additionally, this review delves into recent advancements in sample preparation, extraction, and quantification methods, offering valuable insights into the accurate and efficient determination of glucosinolate content across diverse plant materials. Furthermore, it underscores the critical importance of the standardization and validation of these methodologies to ensure reliable glucosinolate analyses in both scientific research and industrial applications.

Male sterility has been used for crop hybrid breeding for a long time. It has contributed greatly to crop yield increase. However, the genetic basis of male sterility has not been fully elucidated. Here, we report map-based cloning of the cabbage (Brassica oleracea) dominant male-sterile gene Ms-cd1 and reveal that it encodes a PHD-finger motif transcription factor. A natural allele Ms-cd1PΔ-597, resulting from a 1-bp deletion in the promoter, confers dominant genic male sterility (DGMS), whereas loss-of-function ms-cd1 mutant shows recessive male sterility. We also show that the ethylene response factor BoERF1L represses the expression of Ms-cd1 by directly binding to its promoter; however, the 1-bp deletion in Ms-cd1PΔ-597 affects the binding. Furthermore, ectopic expression of Ms-cd1PΔ-597 confers DGMS in both dicotyledonous and monocotyledonous plant species. We thus propose that the DGMS system could be useful for breeding hybrids of multiple crop species.

Cabbage has significant heterosis and most commercial cultivars are hybrids. To explore genetic basis of cabbage heterosis and promote cabbage heterosis utilization, we constructed two populations by crossing 100 DH lines derived from a cabbage hybrid 01–20 × 96–100 with two female parents. Hybrids exhibited different extents of heterosis, the mean value of economic yield was 2.6 times bigger than parents. We identified 66 and 73 QTLs associated with mid-parent heterosis and transgressive heterosis of twelve yield-related traits, respectively. Some QTLs could be detected under the two-year experiment existed in two populations with different testers, showing relatively high phenotypic contribution rate (15.8%–20.0%). Heterosis QTLs exhibited clustered distribution in several cabbage chromosome regions. Two dominant genetic regions, mk300–316 and mk258–268, originated from the elite parent 01–20, exhibited significant genetic effects for yield-related heterosis, which were first identified. Three elite DH lines (D22, D46, D83) harboring these two dominant regions were selected as having strong heterosis in cabbage production. Candidate gene analysis revealed that some genes participating in biosynthetic processes of carbohydrates and some responses to auxin might affect cabbage yield heterosis. QTL identification and genetic dissection of yield-related traits provide new insights into the genetic effects of cabbage heterosis.

Introduction Agrobacterium-mediated genetic transformation has been widely used for the identification of functional genes and regulatory and developmental mechanisms in plants. However, there are still some problems of low genetic transformation efficiency and high genotype dependence in cruciferous crops. Methods In this study, broccoli, a worldwide Brassica crop, was used to investigate the effects of genotype, explant type, concentration of hygromycin B used during seedling selection, overexpression vector type, RNAi and CRISPR/cas9 on the genetic transformation efficiency. At the same time, two vectors, PHG-031350 and PHG-CRa, were used for subcellular localization of the glucoraphanin synthesis-related gene FMOGS-OX5 and clubroot resistance gene by a PEG-Ca2+-mediated transient transformation system for broccoli protoplasts. Finally, the Agrobacterium-mediated genetic transformation system of broccoli was optimized and improved. Results and Discussion This study showed that hypocotyl explants are more suitable for Agrobacterium-mediated transgene and CRISPR/Cas9 gene editing of broccoli. In contrast to previous studies, we found that 5 mg/L hygromycin B was more advantageous for the selection of resistant broccoli sprouts, and genotype 19B42 reached the highest transformation rate of 26.96%, which is higher than that in Brassica oleracea crops. In addition, the inbred line 19B42 successfully achieved high genetic transformation of overexpression, RNAi and CRISPR/Cas9 vectors; thus, it is powerful recipient material for the genetic transformation of broccoli. Subcellular localization proved that the glucoraphanin metabolism-related gene Bol031350 and clubroot resistance gene CRa were both expressed in the cytoplasm and nucleus, which provided a scientific basis for studying the regulation of glucosinolate metabolism and clubroot resistance in cruciferous crops. Therefore, these findings will provide new insight into the improvement of the genetic transformation and molecular breeding of Brassica oleracea crops.

CRISPR/Cas9 has been widely used for precise and efficient genome editing in a number of organisms, notably in major crops, for gene function characterization and trait improvement. However, the application of this genome editing tool is very limited in Brassica oleracea, especially in broccoli (Brassica oleracea var. italica). Here, we report that the genome of broccoli, a worldwide important Brassicaceae vegetable, can also be precisely edited by a CRISPR/Cas9 system with multiple single-guide RNA-expressing cassettes. The gene BoGL5, previously identified as a candidate for the glossy green mutant, was chosen as the target gene. We generated 14 T0 transgenic plants, among which 14.3% and 50% produced mutations at the expected position in targeted regions 1 and 2, respectively. Two of them with a 100% mutation rate showed a glossy green phenotype and dramatically reduced cuticular wax load. These results indicate that CRISPR/Cas9 can be used as a promising technique for gene functional verification and trait improvement in broccoli.

In order to breed broccoli and other Brassica materials to be highly resistant to clubroot disease, 41 Brassicaceae varieties were developed and identified between 2020 and 2021. Seven known clubroot genes were used for screening these materials. In addition, the resistant and susceptible broccoli cultivars were designed for observing their differences in the infection process with Plasmodiophora brassicae. The results showed that 90% of total materials had carried more than two clubroot resistance genes: one material carried two disease resistance genes, four materials carried seven genes for clubroot resistance, two materials carried six genes for clubroot resistance, and in total 32% of these materials carried five genes for clubroot resistance. As a result, several new genotypes of Brassicaceae germplasm were firstly created and obtained based on distant hybridization and identification of loci conferring resistance against Plasmodiophora brassicae in this study. We found and revealed that similar infection models of Plasmodiophora brassicae occurred in susceptible and resistant cultivars of broccoli, but differences in infection efficiency of Plasmodiophora brassicae also existed in both materials. For resistant broccoli plants, a small number of conidia formed in the root hair, and only a few spores could enter the cortex without forming sporangia while sporangia could form in susceptible plants. Our study could provide critical Brassica materials for breeding resistant varieties and new insight into understanding the mechanism of plant resistance.

The effects of a nanocarbon solution on the nutrients, glucosinolate metabolism and glucoraphanin pathway in broccoli were investigated. Significant positive linear relationships were observed between the nanocarbon solution and total protein yield, although effects on the soluble sugars, vitamin C and dry matter production were not observed. All nanocarbon solutions significantly increased the glucoraphanin content (p < 0.05), and the 18.75 L·ha-1 nanocarbon solution maximally increased the glucoraphanin content by 22.9 %. However, these treatments also significantly reduced the contents of glucobrassicin, 4-methoxyglucobrassicin, 4-hydroxyglucobrassicin and neoglucobrassicin. Further research demonstrated that the 18.75 L·ha-1 nanocarbon solution significantly upregulated the MAM1, IPMI2, CYP79F1, FMOgs-ox2, AOP2, and TGG1 expression levels, which directly resulted in the accumulation of glucoraphanin and glucoerucin. This study provides insights into the prospective nanotechnological approaches for developing efficient and environmentally friendly nanocarbon solution for use on crops.

To better serve breeding of broccoli, the electrophysiological, morphological and transcriptomic profiling of the isogenic Ogura-CMS, DGMS and their maintainer fertile lines, were carried out by scanning electron microscopy, investigation of agronomic traits and RNA-sequencing analysis. The agronomic traits of plant height, length of the largest leaf, plant spread angle, single head weight, head width and stem diameter showed stronger performance in Ogura-CMS broccoli than in DGMS line or maintainer fertile line. However, the Ogura-CMS broccoli was poorer in the seed yield and seed germination than in the DGMS line and maintainer fertile line. Additionally, the DGMS broccoli had longer maturation and flowering periods than the Ogura-CMS and maintainer fertile lines. There were obvious differences in the honey gland, happening in the male sterility and fertile lines of broccoli. Additionally, the mechanism regulating Ogura-CMS and DGMS in broccoli was investigated using florets transcriptome analyses of the Ogura-CMS, DGMS and maintainer fertile lines. As a result, a total of 2670 differentially expressed genes (DEGs) were detected, including 1054 up- and 1616 downregulated genes in the Ogura-CMS and DGMS lines compared to the maintainer fertile line. A number of functionally known genes involved in plant hormones (auxin, salicylic acid and brassinosteroid), five Mitochondrial Oxidative Phosphorylation (OXPHOS) genes of atp8, LOC106319879, LOC106324734, LOC106314622 and LOC106298585, and three upregulated genes (Lhcb1, Lhcb3 and Lhcb5) associated with the photosynthesis-antenna protein pathway, were obviously detected to be highly associated with reproductive development including flowering time, maturity and reproductive period in the Ogura-CMS and DGMS broccoli comparing to their maintainer fertile line. Our research would provide a comprehensive foundation for understanding the differences of electrophysiological, morphological and transcriptomic profiles in the Ogura-CMS, DGMS and maintainer broccoli, and as well as being beneficial to exploring the mechanism of male sterility in Brassica crops.