Totipotency and pluripotency of plant cells serve as the theoretical basis for plant regeneration, underpinning various techniques such as plant tissue culture, genetic transformation, cutting, and grafting. In recent years, advances in understanding the molecular mechanisms of plant regeneration—particularly key transcription factors, hormonal signaling networks and epigenetic factors—have led to the development of a series of molecular tools capable of manipulating cell fate, offering new strategies to enhance regenerative capacity. This review summarizes the molecular and chemical tools that play central roles in four key regenerative processes: shoot regeneration, root regeneration, somatic embryogenesis, and grafting. It also discusses practical considerations such as delivery methods for these tools, selection of culture systems, and strategies for efficacy evaluation. Furthermore, the review explores future directions, including the rational combination of genetic and chemical tools to develop novel delivery systems and overcome epigenetic barriers, thereby providing theoretical and practical guidance for designing efficient regeneration protocols tailored to different species and regeneration objectives.

INTRODUCTION: Ficus hirta Vahl is a common Chinese herbal medicine and edible plant resource in the Lingnan area of China. It contains coumarins, flavonoids and other chemical compounds, which have antioxidant, anti-inflammatory, antibacterial, antiviral and antitumor effects. However, F. hirta exhibits significant morphological variation in leaf shapes due to its high genetic diversity. 

RATIONALE: To investigate the relationship between the leaf shape differences and the contents of major active compounds, herein, LC-MS/MS was performed to determine the contents of psoralen and bergapten in F. hirta roots from three different leaf shapes (entirc leaf, EL; lobed leaf, LL; Palmately deeply leaf, PDL), and transcriptome sequencing was further employed to explore the potential molecular mechanisms underlying these contents variations. 

RESULTS: Results showed that PDL exhibited significantly higher psoralen content compared to EL and LL, while EL with significantly higher bergapten content than that in LL, but no significant difference was observed between EL and PDL. A total of 60.9 Gb clean reads were obtained, and 46 194 unigenes were assembled. F. hirta had the highest homology with Morus notabilis, according to the homologous sequencing alignment. 2 355, 2 067 and 2 001 differentially expressed genes (DEGs) were screened from three comparison groups PDL_vs_EL, PDL_vs_LL, and LL_vs_EL, respectively. These DEGs were primarily enriched in the pathways such as phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant-pathogen interaction etc. 

CONCLUSION: There were differences in the contents of psoralen and bergapten among the three leaf shapes of F. hirta. The phenylpropanoid biosynthesis and plant-pathogen interaction pathway may play crucial roles in the biosynthesis and accumulation of psoralen and bergapten in F. hirta. This study preliminarily revealed the correlation between the leaf morphology and major active compounds of F. hirta, and extended its public transcriptome database, which will provide reference for further utilizing different leaf shape of F. hirta germplasm resources and quality breeding.

INTRODUCTION: Zizania latifolia is an important aquatic vegetable in China with both economic and nutritional values, but it is currently facing industrial challenges such as lack of crop rotation, declining quality, and insufficient supply, and is in urgent need of efficient means to increase production. As a novel small peptide hormone, phytosulfokine (PSK) has significant potential in plant growth regulation and agricultural applications, but its mechanism of regulating tillering in Z. latifolia has not yet been clarified. In this study, we used PSK-treated Z. latifolia as the material, combined with transcriptome sequencing (accession No.PRJNA1377146), fluorescence quantitative PCR validation and endogenous hormone determination, to investigate the molecular and physiological mechanisms of PSK in regulating the tillering of Z. latifolia, and to provide a theoretical basis and technological support for the high-yield and high-quality cultivation of Z. latifolia. 

RATIONALE: Z. latifolia is the second largest aquatic vegetable after lotus root in China, with remarkable economic and nutritional value, but the current cultivation is facing problems such as lack of crop rotation, strong seasonality, and declining soil fertility, which leads to quality degradation and insufficient supply, and there is an urgent need for efficient means of yield increase; PSK, as a small peptide hormone in plants, can regulate the physiological processes such as cell elongation and development of meristematic tissues, and it has demonstrated its value of promoting crop growth and delaying senescence in agriculture, but the molecular mechanism of PSK regulation has not been clearly defined. In agriculture, PSK has demonstrated its value in promoting crop growth, delaying aging and other applications. However, there are fewer studies on the regulation of plant growth and development, and the molecular mechanism has not yet been clarified. Therefore, it is of great significance to systematically analyze the changes in gene expression of Z. latifolia after PSK treatment by means of RNA-seq sequencing, combined with the determination of endogenous hormones and observation of the tillering phenotypes, to excavate key metabolic pathways and regulatory genes, and to elucidate the molecular and physiological mechanisms of its regulation of water bamboo tillering. 

RESULTS: After transcriptome sequencing, a total of 806 differentially expressed genes (365 up-regulated and 441 down-regulated) were screened out, and GO enrichment analysis showed that the differentially expressed genes were mainly involved in biological processes such as response to water deficit, cell wall organization, etc., and KEGG analysis showed that the differentially expressed genes were significantly enriched in the pathways of phenylpropane biosynthesis and tryptophan metabolism, etc. The results of fluorescence quantitative PCR of four differentially expressed genes were in line with the trend of expression of transcriptome, PSK treatment significantly promoted tillering in Z. latifolia, and the number of tillers at 14 days of treatment was 122.22% higher than that of the control group, which was the most significant effect. Two-way ANOVA confirmed that the treatment, time and the interaction effect were all highly significant, and there was a time-dependent “window period”. As for endogenous hormones, the content of growth hormone, gibberellin and jasmonic acid increased significantly after PSK treatment (GA3 increased by 188.59%), and the content of abscisic acid and cytokinin decreased significantly, and the hormone ratio was significantly reconfigured. 

CONCLUSION: Exogenous PSK regulates the tillering and growth of Z. latifolia through multiple pathways: it significantly alters the endogenous hormone levels, promotes the increase of growth-related hormones and inhibits the decrease of hormone-like hormones; it regulates the key metabolic pathways such as phenylpropane biosynthesis and fatty acid degradation, and influences the synthesis of the cell wall and energy metabolism; and it promotes the synthesis of indoleacetic acid through the expression of the key genes in the tryptophan metabolic pathway, which provides the material and signal basis for tillering. The optimal concentration of PSK treatment is 1 μmol·L^–1, and the optimal application “window” is about 14 days after treatment. This study reveals the molecular mechanism of PSK regulation tillering of Z. latifolia, and provides feasible technical solutions and theoretical support for large-scale high-yield and high-quality cultivation of Z. latifolia.

INTRODUCTION: Cibotium barometz is a medicinal fern species with high economic value, Chinese people produce the traditional medicine herb “Gou-ji” using its robust rhizome. To balance the conservation of wild plant resources with the sustainable development of herbal medicine, much more effort is needed in scientific research on C. barometz, which might be crucial to mitigate conflicts between wild resource protection and the development of the traditional medicine industry. Real-time quantitative PCR (RT-qPCR) is a commonly used method for characterizing plant gene molecular function, and an appropriate reference gene is pivotal for this technique. Currently, limited literature focuses on the gene function studies for C. barometz, and little validated reference genes is available. 

RATIONALE: Previous studies have demonstrated that the expression stability of reference genes is influenced by multiple factors, including species, tissue type, developmental stage, stress treatment, and sequence specificity. Consequently, distinct reference genes are required for different research subjects. When employing RT-qPCR to analyze gene expression pattern in C. barometz tissues, it is essential to pre-validate the stability of reference genes within the novel experimental system. This validation process ensures the accuracy and reliability of experimental results by maintaining proper data normalization. 

RESULTS: In this study, 12 commonly used plant reference genes were identified as candidate genes from transcriptome data of C. barometz rhizome. The amplification efficiency of candidate reference genes was calculated by template gradient dilution method, and eight pairs of reference gene primers with high amplification efficiency were screened out. The Ct values of these eight reference genes in ten tissues of C. barometz were measured using RT-qPCR method; the software RefFinder and Normfinder determined that the best reference genes are CbUBC4 and CbEF1A, respectively. If studying the gene expression patterns of C. barometz at different developmental stages, it was suggested that CbEF1A and CbUBC4 should be used as optimal reference genes. The reference genes CbUBC4 and CbUBC28 were recommended under mechanical injury and waterlogging conditions. 

CONCLUSION: CbUBC4, CbEF1A and CbUBC28 were validated as stable reference genes in C. barometz across distinct experimental contexts, demonstrating their suitability for normalization in gene expression studies for RT-qPCR assay. From a methodological rigor perspective, we recommended that the stability of reference genes should be evaluated prior to conducting qRT-PCR experiments, particularly when analyzing samples from diverse tissues or distinct developmental stages of a specific tissue.

INTRODUCTION: As an important ornamental species, chrysanthemum (Chrysanthemum morifolium Ramat.) depends on precise flowering time regulation, which is a key factor affecting its economic value and market demand. The bud sport of cut chrysanthemum cultivar 'Jinshan' maintains vegetative growth under normal cultivation conditions and fails to initiate floral transition. However, the underlying regulatory mechanisms responsible for its flowering defect remain unclear.

RATIONALE: To explore the molecular basis of the flowering inhibition in the ‘Jinshan’ bud sport, we conducted a comparative analysis using both the wild-type and the bud sport lines. Sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate their genetic backgrounds. In addition, transcriptome sequencing (RNA-seq) was performed on leaf and shoot apical meristem (SAM) tissues to identify differentially expressed genes (DEGs) associated with floral transition.

RESULTS: SRAP analysis revealed a high degree of genomic similarity between the bud sport and wild-type lines, suggesting that large-scale genetic structural variations are unlikely to be the cause of the floral defect. Transcriptomic analysis identified a substantial number of DEGs between the two lines. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated significant enrichment of plant hormone signal transduction pathways in both leaf and SAM tissues. Further analysis of flowering-related regulatory networks revealed widespread transcriptional dysregulation. In the photoperiod pathway, abnormal expression of the CO-FT module may have impaired the perception and transmission of flowering signals. In the autonomous pathway, upregulation of MAF2 (a homolog of FLC) and downregulation of FLD likely led to the repression of downstream floral integrators. In the gibberellin (GA) signaling pathway, a significant downregulation of the gibberellin receptor GID1, as well as the downregulation of the GA biosynthesis enzyme GA3ox and the upregulation of the GA deactivating enzyme GA2ox, may result in a reduction in active gibberellin content. In the early regulation network of flower organ formation, the expression of SOC1, AP1, FUL, AFL, and SPLs was significantly downregulated, and LFY expression in the apical meristem was nearly absent. These changes may collectively block floral primordium formation and flower organ differentiation in the bud sport.

CONCLUSION: This study reveals that the abnormal expression of multiple key genes in the photoperiod, autonomous, and gibberellin signaling pathways contribute to the failure of floral transition in the bud sport. These findings provide preliminary insights into the potential mechanism underlying flowering inhibition in the 'Jinshan' bud sport at the transcriptomic level, and provide theoretical support and candidate genes for molecular breeding of flowering time in chrysanthemum. 

Disrupted regulatory network of flowering in the bud sport of chrysanthemum ‘Jinshan’.

INTRODUCTION: Submergence tolerance is one of the most crucial stress resistances among garden plants. Willow is a plant that has strong tolerance to submergence. The objective of this study is to clone the TTF family gene SmTTF59 of willow and construct overexpression and silencing vectors in order to explore its role in responding to submergence stress in willow. 

RATIONALE: Studies have demonstrated that TTF family transcription factors play critical regulatory roles in plant responses to abiotic stresses.?Salix matsudana, a species with strong submergence tolerance, serves as an ideal model for investigating submergence stress adaptation mechanisms. By silencing?SmTTF59 in Salix matsudana and conducting hypoxia stress experiments on?SmTTF59-transgenic Arabidopsis, this study provides theoretical foundations for understanding the complex regulatory mechanisms of Trihelix transcription factors and lays a groundwork for genetic breeding of submergence-tolerant?Salix matsudana. 

RESULTS: The results showed that the coding sequence (CDS) of the SmTTF59 gene in Salix matsudana is 834 bp in length, encoding 277 amino acid residues with a protein molecular weight of 33,044.40 Da. Through PCR experiments, the 834-bp CDS of SmTTF59 was successfully amplified and used to construct recombinant vectors pWM101 and pYL156. Hypoxic stress experiments on transgenic Arabidopsis demonstrated that SmTTF59-overexpressing lines exhibited enhanced hypoxia tolerance compared to wild-type plants, characterized by longer root lengths and higher fresh weights. Physiological analyses revealed higher levels of antioxidant enzymes, such as peroxidase (POD) and catalase (CAT), in transgenic lines. Virus-induced gene silencing (VIGS) experiments in willow plants further indicated that SmTTF59 knockdown lines displayed poorer growth under submergence stress compared to controls, including severe leaf decay and abscission. Quantitative measurements showed that silenced plants had lower root fresh weight, root dry weight, and POD activity, along with higher relative electrical conductivity, compared to control plants. These results collectively confirm that SmTTF59 knockdown significantly reduces willow tolerance to submergence stress. 

CONCLUSION: The better anoxic tolerance of SmTTF59 transgenic Arabidopsis thaliana proved that this gene might be related to the response of willow to submergence stress. Through the VIGS experiment on willow, it was verified that this gene played a positive regulatory role in submergence. The results of this study are of great significance for the subsequent breeding work of submergence-tolerant willow varieties.

Phenotypic differences between pre- and post-flooding and phenotypic comparison between WT and SmTTF59 transgenic Arabidopsis thaliana under normoxic and hypoxic conditions. Elucidated the function of the SmTTF59 gene in response to submergence stress.

INTRODUCTION: Thermospermine, as a structural isomer of spermine, is an important substrate involved in plant growth and development, as well as responses to biotic and abiotic stress. 

RATIONALE: At present, there are few reports on the relationships between thermospermine and low-temperature stress, for which a primary analysis of their relationships in wucai was conducted. Firstly, the optimal concentration of exogenous thermospermine alleviating the damage of wucai caused by low temperature was analyzed. Then, thermospermine synthesis inhibitor combined with the selected appropriate concentration of thermospermine was used under low temperature condition in order to further analyze whether thermospermine plays a role in the resistance to low-temperature stress.

RESULTS: At lower concentrations of thermospermine (0.05 and 0.1 mmol·L^–1), electrolyte leakage, hydrogen peroxide, superoxide anion and malondialdehyde contents decreased with different degrees, and the dry and fresh weight increased, while the effects weakened with the increase of thermospermine concentration. The ranking order of the average membership function values obtained decreased gradually with the increase of the concentration, and the score of 0.05 mmol·L^–1 thermospermine was the highest. The addition of thermospermine synthesis inhibitor under low temperature made the two materials with different low-temperature tolerance accumulate more hydrogen peroxide and superoxide anion, electrolyte leakage and malondialdehyde content further increased. In addition, application of thermospermine at the same time can alleviate the damage of low temperature stress aggravated by inhibitor treatment, which was associated with higher activities of SOD and POD.

CONCLUSION: 0.05 mmol·L^–1 thermospermine could effectively alleviate the damage of low temperature on wucai seedlings, and thermospermine synthesis is closely related to the low-temperature stress tolerance of wucai, which may improve low-temperature tolerance through maintaining higher antioxidant enzyme activities, especially SOD and POD.

Phenotype (A), (B), H₂O₂ (C) and •O₂⁻ (D) staining of two wucai materials with different low-temperature stress tolerance (‘W18’, low-temperature tolerant type, and ‘SW-1’, low-temperature sensitive type) under treatment of thermospermine synthesis inhibitor.

INTRODUCTION: Global climate change and soil degradation have posed new challenges to the breeding efficiency of wheat (Triticum aestivum). Traditional breeding cycles are lengthy and inefficient, making it difficult to meet the ever-growing demand for food. As a major high-quality wheat producing region in China, Xinjiang urgently needs to establish a rapid generation-advancement system and an efficient genetic transformation platform tailored to its local cultivars, in order to shorten breeding cycles and expand the range of transgenic recipient materials.

RATIONALE: Compared to conventional greenhouse conditions, all five materials showed a significant reduction in generation time under artificial climate chamber conditions. XinChun 37 exhibited the greatest reduction, shortening its lifecycle from 92 days to 59 days; In Agrobacterium-mediated embryo transformation assays, XinChun 37 achieved a transformation efficiency of 23.30%, outperforming the other three Xinjiang cultivars but remaining lower than the Fielder control. XinChun 9, XinChun 44, and HeChun 137 also demonstrated measurable transformation efficiencies.

RESULTS: Under artificial climate chamber conditions compared to conventional greenhouse conditions, all five materials showed a significant reduction in generation time. XinChun 37 exhibited the greatest reduction, shortening its lifecycle from 92 days to 59 days; In Agrobacterium-mediated embryo transformation assays, XinChun 37 achieved a transformation efficiency of 23.30%, outperforming the other three Xinjiang cultivars but remaining lower than the Fielder control. XinChun 9, XinChun 44, and HeChun 137 also demonstrated measurable transformation efficiencies.

CONCLUSION: This study successfully established a novel rapid generation-advancement system for the main wheat cultivars of Xinjiang and, for the first time, compared their embryonic genetic transformation efficiencies. These findings will greatly shorten breeding cycles and provide key technological support for expanding the selection of transgenic recipient materials, thereby contributing to germplasm innovation and food-security strategies in Xinjiang and beyond.

The effects of speed breeding on agronomic traits of different spring wheat varieties. Under artificial climate chamber rapid-generation conditions, wheat grew well with normal fertility, all lines showed significantly shortened growth cycles and reduced spike length, while the number of effective tillers increased markedly.

Seed endophytic fungi are a type of fungi that spread through vertical transmission and have a mutualistic symbiotic relationship with host plants. They can be classified into three types based on their colonization sites and output characteristics. Currently, research on endophytic fungi in seeds mainly focuses on the Poaceae and Fabaceae plants. Seed endophytic fungi exhibit species diversity and community diversity, which not only manifest in the interspecies of plants, but also in different tissues of plants. Seed endophytic fungi have diverse functions, which can affect soil properties, improve seed germination, assist in seedling establishment, promote plant growth, enhance plant stress resistance, and influence plant community structure. Detecting and identifying species is the foundation of research on endophytic fungi in seeds, and figure out the structure of seeds and the colonization sites of fungi are the preliminary and basic work for detecting endophytic fungi in seeds. Currently, the research on seed endophytic fungi mainly includes identifying its species and their diversity, functions and their diversity, roles in the host's life history, and development and utilization. In terms of research methods, scanning electron microscopy can be used to observe the structure, morphology, and colonization location of fungi; whole-genome sequencing (WGS) technology can obtain microbial genome data and reveal the molecular mechanisms of interaction between seed endophytic fungi and plants. In terms of research content, species diversity, community diversity, ecosystem diversity, and development and utilization of seed endophytic fungi have become hot issues. Looking to the future, research on seed endophytic fungi is a field with great development prospects.