Establishing plant regeneration systems and efficient genetic transformation techniques plays a crucial role in plant functional genomics research and the development of new crop varieties. The inefficient methods of transformation and regeneration of recalcitrant species and the genetic dependence of the transformation process remain major obstacles. With the advancement of plant meristematic tissues and somatic embryogenesis research, several key regulatory genes, collectively known as developmental regulators, have been identified. In the field of plant genetic transformation, the application of developmental regulators has recently garnered significant interest. These regulators play important roles in plant growth and development, and when applied in plant genetic transformation, they can effectively enhance the induction and regeneration capabilities of plant meristematic tissues, thus providing important opportunities for improving genetic transformation efficiency. This review focuses on the introduction of several commonly used developmental regulators. By gaining an in-depth understanding of and applying these developmental regulators, it is possible to further enhance the efficiency and success rate of plant genetic transformation, providing strong support for plant breeding and genetic engineering research.

Ginkgo biloba, usually referred to as a “living fossil,” is widely planted in many countries because of its medicinal value and beautiful appearance. Owing to ginkgo’s high resistance to drought stress, ginkgo seedlings can even survive withholding water for several days without exhibiting leaf wilting and desiccation. To assess the physiological and transcriptomic mechanisms involved in the drought stress and re-watering responses of Ginkgo biloba, ginkgo seedlings were subjected to drought treatment for 15 d (D_15 d) and 22 d (D_22 d) until they had severely wilted, followed by re-watering for 3 d (D_Re3 d) to restore normal growth. Variations in physiological characteristics (relative water content, malondialdehyde (MDA) content, stomatal aperture, and antioxidant enzyme activity) during drought and re-watering were assessed. In total, 1692, 2031, and 1038 differentially expressed genes (DEGs) were upregulated, while 1691, 2820, and 1910 were downregulated in D_15 d, D_22 d, and D_Re3 d, respectively, relative to the control. Three pathways, namely, plant hormone signal transduction, plant–pathogen interaction, and the plant MAPK signaling pathway, were enriched during drought stress and re-watering. The DEGs involved in plant hormone signal transduction pathways (those of IAA, CTK, GA, ABA, ETH, BR, SA, and JA) and the major differentially expressed transcription factors (TFs; MYB, bHLH, AP2/ERF, NAC, WRKY, and bZIP) were identified. Quantitative real-time PCR revealed six TFs as positive or negative regulators of drought stress response. These phenotype-related physiological characteristics, DEGs, pathways, and TFs provide valuable insights into the drought stress and re-watering responses in G. biloba.

Anthocyanins are important secondary metabolites in plants, which contribute to fruit color and nutritional value. Anthocyanins can be regulated by environmental factors such as light, low temperature, water conditions, and nutrition limitations. Nitrogen (N) is an essential macroelement for plant development, its deficiency as a kind of nutrition limitation often induces anthocyanin accumulation in many plants. However, there is a lack of reports regarding the effect of nitrogen deficiency on anthocyanin biosynthesis in pears. In this study, we found that N deficiency resulted in anthocyanin accumulation in pear callus and upregulated the expression of anthocyanin biosynthesis pathway structural genes (PyPAL, PyCHS, PyCHI, PyF3H, PyDFR, PyANS, and PyUFGT) and key regulatory factors (PyMYB10, PyMYB114, and PybHLH3). Through analysis of transcriptome data of treated pear callus and RT-qPCR assay, a differentially expressed gene PyNAC42 was identified as significantly induced by the N deficiency condition. Overexpression of PyNAC42 promoted anthocyanin accumulation in “Zaosu” pear peels. Additionally, dual luciferase assay and yeast one-hybrid assay demonstrated that PyNAC42 could not directly activate the expression of PyDFR, PyANS, and PyUFGT. Furthermore, yeast two-hybrid and pull-down assays confirmed that PyNAC42 interacted with PyMYB10 both in vivo and in vitro. Co-expression of PyNAC42 and PyMYB10 significantly enhanced anthocyanin accumulation in “Zaosu” pear peels. Dual luciferase assay showed that PyNAC42 significantly enhanced the activation of PyDFR, PyANS, and PyUFGT promoters by interacting with PyMYB10, which suggests that PyNAC42 can form the PyNAC42-PyMYB10 complex to regulate anthocyanin biosynthesis in pear. Thus, the molecular mechanism underlying anthocyanin biosynthesis induced by N deficiency is preliminarily elucidated. Our finding has expanded the regulatory network of anthocyanin biosynthesis and enhanced our understanding of the mechanisms underlying nutrient deficiency modulates anthocyanin biosynthesis in pear.

B-box (BBX) transcription factors, which are specific to the plant kingdom, play a crucial role in regulating light-dependent growth, development, secondary metabolite biosynthesis, and the response to biotic and abiotic stresses. Despite their significance, there has been a lack of systematic investigation into the BBX gene family in Ginkgo biloba. In the present study, we identified nine BBX genes within the G. biloba reference genome, distributed across seven chromosomes, and classified them into four groups based on their phylogenetic relationships with the BBX gene families of Arabidopsis thaliana. Our analysis of gene structure, conserved domains, and motifs suggests that GbBBXs exhibit a high degree of conservation throughout evolutionary history. Additionally, synteny analysis revealed that dispersed duplication events have contributed to the expansion of the BBX gene family in G. biloba. An examination of cis-regulatory elements indicated that numerous GbBBX genes contain motifs associated with light, hormones, and stress, suggesting their potential roles in responding to these signals and environmental adaptation. Expression profiles obtained from RNA-Seq data and quantitative Real-Time PCR (qRT-PCR) analyses of GbBBX genes across various organs, hormone treatments, and leaves with differing flavonoid content, as well as during both short-term and long-term water stress, demonstrated their potential roles in flavonoid regulation and responses to hormones and water stress. Subcellular localization studies indicated that the proteins GbBBX5, GbBBX7, GbBBX8, and GbBBX9 are localized within the nucleus. This study is the first thorough analysis of the BBX gene family in G. biloba, providing a valuable foundation for further understanding their evolutionary context and functional roles in flavonoid regulation and responses to water stress.

SQUAMOSA promoter-binding protein-like (SPL) transcription factors specific to plants are vital for regulating growth, development, secondary metabolite biosynthesis, and responses to both biotic and abiotic stresses. Despite their importance, no systematic investigations or identifications of the SPL gene family in Ginkgo biloba have been conducted. In this study, we identified 13 SPL genes within the Ginkgo biloba reference genome, spanning seven chromosomes, and categorized these genes into six groups based on their phylogenetic relationships with Arabidopsis thaliana SPL gene families. Our analysis of gene structure, conserved domains, motifs, and miR156 target predictions indicates that GbSPLs are highly conserved across evolutionary timelines. Furthermore, synteny analysis highlighted that dispersed duplication events have expanded the SPL gene family in Ginkgo biloba. Examination of the cis-regulatory elements revealed that many GbSPL genes possess motifs associated with light, hormones, and stress, implying their involvement in flavonoid biosynthesis and adaptation to environmental conditions. RNA-Seq and qRT-PCR expression profiles of GbSPL genes across various tissues and low- and high-flavonoid leaves and during both short-term and long-term water stress illustrated their roles in flavonoid biosynthesis and responses to water stress. Subcellular localization experiments showed that GbSPL2 and GbSPL11 proteins are situated within the nucleus. Our research offers the first systematic characterization of the SPL gene family in Ginkgo biloba, establishing a valuable foundation for understanding their evolutionary background and functional roles in flavonoid biosynthesis and water stress response.

To assess the regulatory mechanisms involved in the transcriptomic response of Ginkgo biloba to water shock and rehydration, ginkgo seedlings were subjected to dehydration for 0, 3, 6, 12, and 24 h, followed by rehydration for 12 h (Re12 h). A total of 1388, 1802, 2267, 2667, and 3352 genes were upregulated, whereas 1604, 1839, 1934, 2435, and 3035 genes were downregulated, at 3, 6, 12, 24, and Re12 h, respectively, compared to 0 h. Two KEGG pathways—the plant pathogen interaction pathway and mitogen-activated protein kinase (MAPK) signaling pathway—were enriched under water shock but not under rehydration. Moreover, plant hormone signal transduction was enriched under both water shock and rehydration. Differentially expressed genes (DEGs) involved in the ABA signaling pathway (PYR/PYLs, PP2Cs, and SnRK2s) and major differentially expressed transcription factors (MYB, bHLH, AP2/ERF, NAC, WRKY, and bZIP TFs) were identified. qRT-PCR analysis further revealed GbWRKY3 as a negative regulator of the water shock response in G. biloba. The subcellular localization results revealed GbWRKY3 as a nuclear protein. These phenotype-related DEGs, pathways, and TFs provide valuable insight into the water shock and rehydration response in G. biloba.