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PubMed2025

Efficient and versatile rapeseed transformation for new breeding technologies.

Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.

Brassica napus↗

PubMed2025

Phylogeny, chromosomal mapping and expression analyses of wheat CLAVATA pathway components suggest differential selection on receptor-like kinases, CLEs and T3 WOXes.

Ensuring continuous global food security is a major challenge of the 21st century. Wheat contributes approximately 20% of the total calories consumed by humans, and an estimated 60% increase in production will be required by 2050 to meet forecast global demand. In cereals like wheat, inflorescence (ear) size and branching patterns determine the number of flowers (florets) and grains produced, and these aspects of plant architecture are regulated by the activity of stem cells in the growing shoot tips. CLAVATA peptide and receptor-like kinase signalling regulates angiosperm stem cell activity, and as changes in CLAVATA function can improve crop yields, CLAVATA is a key target for reverse engineering. Here, we identify components of the wheat CLAVATA pathway using genome searches against Triticum aestivum and its wild relatives Triticum turgidum ssp. durum, Triticum turgidum ssp. dicoccoides, Triticum urartu and Aegilops tauschii. Using phylogenetic and synteny analysis, we determine the relationship between homoeologues and infer patterns of gene family evolution. Whilst CLAVATA1, BARELY ANY MERISTEM, RECEPTOR-LIKE PROTEIN KINASE 2, CORYNE and CLAVATA2 receptor-like kinase homologues are mainly present as single genome copies as in other grasses, CLAVATA3-like but not TRACHEARY ELEMENT DIFFERENTIATION FACTOR (TDIF)-like peptide encoding genes and WUSCHEL-LIKE HOMEOBOX (WOX) genes have expanded copy numbers with many gene gains and losses during evolution. Our results highlight wheat CLAVATA pathway components for reverse genetic analysis and indicate potential differential selection on wheat receptor-like kinases, their peptide ligands and WOXes.

Triticum↗

PubMed2026

MdWRKY75 interacts with MdWOX11 to modulate root growth under salt stress in apple.

The root system is pivotal for plant development, enabling both vegetative growth and tolerance to abiotic stresses like salinity. However, the molecular mechanisms governing root adaptive development in response to salt stress remain poorly understood in apple (Malus domestica Borkh.). In this study, we identified the salt stress-responsive WRKY transcription factor MdWRKY75. Overexpression of MdWRKY75 in transgenic apple negatively regulates adventitious root (AR) formation and salt stress tolerance, whereas reducing MdWRKY75 expression yields the opposite phenotype. Moreover, MdWRKY75 directly binds to the promoter of MdSAUR15 (SMALL AUXIN UP RNA15) and transcriptionally represses the expression of MdSAUR15, which, when overexpressed, promotes AR formation and enhances salt stress tolerance. We further demonstrated that MdWRKY75 interacts with MdWOX11, a WUSCHEL-related homeobox (WOX) transcription factor, both in vitro and in vivo. MdWOX11 expression is upregulated and enhances AR formation under salt stress. Additionally, MdWOX11 reduces the binding of MdWRKY75 to the MdSAUR15 promoter, and alleviates the MdWRKY75-mediated inhibitory effect on MdSAUR15 expression. Collectively, our study provides a MdWOX11-MdWRKY75-MdSAUR15 module regulating root adaptation in response to salt stress in apple.

Malus↗

PubMed2026

Identification and fine mapping of a locus controlling multi-main-stem trait in Brassica napus.

BACKGROUND: The main stem is a crucial component determining individual plant yield in rapeseed (Brassica napus). However, the genetic and developmental basis underlying the multi-main-stem trait remains largely unclear. RESULTS: In this study, we identified a multi-main-stem mutant, mms1, which exhibited a significantly increased silique number per plant and abnormal shoot apical meristem (SAM) development. Genetic analysis demonstrated that the multi-main-stem trait was controlled by a recessive gene. Using bulked segregant analysis combined with a Brassica napus 50 K SNP array and map-based cloning, the locus was mapped to a 340-kb interval on chromosome A09 of the ZS11 reference genome and was designated BnaA09.MMS1. Candidate gene analysis revealed that BnaA09G0254500ZS, which harbors sequence variations in both the promoter and coding regions and shows significantly increased expression in the mutant, was the most likely candidate gene. In addition, phytohormone analysis revealed reduced auxin accumulation in mutant SAMs, together with transcriptomic changes in genes associated with the CLAVATA3 (CLV3)-WUSCHEL (WUS) feedback loop. CONCLUSIONS: These findings provide an important foundation for elucidating the genetic basis of the multi-main-stem trait and offer a valuable genetic resource for rapeseed improvement.

Brassica napus↗

PubMed2026

Genome-wide identification of WOX transcription factors and functional characterization of WOX4 and WOX13 involved in cold stress response in Malus baccata.

INTRODUCTION: Cold stress is a major abiotic threat to apple production. Malus baccata has exceptional cold hardiness and is widely used as a superior cold-resistant rootstock. The WUSCHEL-related homeobox (WOX) transcription factor family regulates plant growth, development and stress adaptation, whereas the functions of WOX genes in cold tolerance of M. baccata remain elusive. METHODS: In the present work, 19 MbWOX family members were identified and characterized at the genome-wide level. Evolutionary analysis, cis-element prediction, transcriptome profiling and real-time quantitative PCR (RT-qPCR) were performed to screen core cold-responsive genes. Overexpression vectors were constructed and transformed into Arabidopsis seedlings for functional verification. RESULTS: Evolutionary analysis revealed that segmental duplication drove the expansion of the MbWOX family, and these genes contained a variety of stress-responsive cis-elements. Combined transcriptome and RT-qPCR analyses confirmed that MbWOX4 and MbWOX13 were core cold-responsive genes with distinct expression patterns. The two genes participated in cold signal transduction by interacting with different transcription factor networks. Functional tests revealed that MbWOX4 and MbWOX13 isoforms differentially modulated seedling cold tolerance under low-temperature stress.

Malus baccata↗

PubMed2026

The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.

Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.

Zea mays↗