Summary: Regeneration represents a fundamental biological process wherein an organism's tissues or organs repair and replace themselves following damage or environmental stress. In plant systems, injured tree branches can regenerate adventitious buds and develop new crowns through propagation techniques like cuttings and canopy pruning, while transgenic plants emerge via tissue culture in genetic engineering processes intimately connected to plant regeneration mechanisms. The advancement of plant regeneration technology is critical for addressing complex and dynamic climate challenges, ultimately ensuring global agricultural sustainability. This review comprehensively synthesizes the latest genetic transformation technologies, including transformation systems across woody, herbaceous and algal species, organellar genetic modifications, crucial regeneration factors facilitating Agrobacterium‐mediated transformations, the intricate hormonal networks regulating plant regeneration, comparative analyses of transient transformation approaches and marker gene dynamics throughout transformation processes. Ultimately, the review offers novel perspectives on current transformation bottlenecks and proposes future research trajectories.

Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs—19-nt duplex with 2-nt 3′ overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)—into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

As an ecofriendly biocontrol agent, antagonistic bacteria are a crucial class of highly efficient fungicides in the field against Verticillium dahliae, the most virulent pathogen for cotton and other crops. Toward identifying urgently needed bacterial candidates, we screened bacteria isolated from the cotton rhizosphere soil for antagonisitic activity against V. dahliae in an artificially infested nursery. In preliminary tests of antagonistic candidates to characterize the mechanism of action of on culture medium, 88 strains that mainly belonged to Bacillus strongly inhibited the colony diameter of V. dahliae, with inhibiting efficacy up to 50% in 9 strains. Among the most-effective bacterial strains, Bacillus sp. ABLF-18, and ABLF-50 and Paenibacillus sp. ABLF-90 significantly reduced the disease index and fungal biomass of cotton to 40–70% that of the control. In further tests to elucidate the biocontrol mechanism (s), the strains secreted extracellular enzymes cellulase, glucanase, and protease, which can degrade the mycelium, and antimicrobial lipopeptides such as surfactin and iturin homologues. The expression of PAL, MAPK and PR10, genes related to disease resistance, was also elicited in cotton plants. Our results clearly show that three candidate bacterial strains can enhance cotton defense responses against V. dahliae; the secretion of fungal cell-wall-degrading enzymes, synthesis of nonribosomal antimicrobial peptides and induction of systemic resistance shows that the strains have great potential as biocontrol fungicides.

The soil-borne ascomycete fungus Verticillium dahliae causes vascular wilt disease and can seriously diminish the yield and quality of important crops. Functional analysis of growth- and pathogenicity-related genes is essential for revealing the pathogenic molecular mechanism of V. dahliae. Phospholipase is an important virulence factor in fungi that hydrolyzes phospholipids into fatty acid and other lipophilic substances and is involved in hyphal development. Thus far, only a few V. dahliae phospholipases have been identified, and their involvement in V. dahliae development and pathogenicity remains unknown. In this study, the function of the patatin-like phospholipase gene in V. dahliae (VdPLP, VDAG_00942) is characterized by generating gene knockout and complementary mutants. Vegetative growth and conidiation of VdPLP deletion mutants (ΔVdPLP) were significantly reduced compared with wild type and complementary strains, but more microsclerotia formed. The ΔVdPLP mutants were very sensitive to the cell-wall-perturbing agents: calcofluor white (CFW) and Congo red (CR). The transcriptional level of genes related to the cell wall integrity (CWI) pathway and chitin synthesis were downregulated, suggesting that VdPLP has a pivotal role in the CWI pathway and chitin synthesis in V. dahliae. ΔVdPLP strains were distinctly impaired in in their virulence and ability to colonize Nicotiana benthamiana roots. Our results demonstrate that VdPLP regulates hyphal growth and conidial production and is involved in stabilizing the cell wall, thus mediating the pathogenicity of V. dahliae.