Transgenic Calli Research Articles

PfGSTF2 endows resistance to quizalofop-p-ethyl in Polypogon fugax by GSH conjugation.

Populations of Polypogon fugax have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC-MS analysis of in vitro herbicide metabolism by Escherichia coli-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.

Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.

Identification and characterisation of 'No apical meristem; Arabidopsis transcription activation factor; Cup-shape cotyledon' (NAC) family transcription factors involved in sugar accumulation and abscisic acid signalling in grape (Vitis vinifera).

The 'No apical meristem; Arabidopsis transcription activation factor; Cup-shape cotyledon' (NAC) transcription factors are pivotal in plant development and stress response. Sucrose-non-fermenting-related protein kinase 1.2 (SnRK1) is a key enzyme in glucose metabolism and ABA signalling. In this study, we used grape (Vitis vinifera ) calli to explore NAC's roles in sugar and ABA pathways and its relationship with VvSnRK1.2 . We identified 19 VvNACs highly expressed at 90days after blooming, coinciding with grape maturity and high sugar accumulation, and 11 VvNACs randomly selected from 19 were demonstrated in response to sugar and ABA treatments. VvNAC26 showed significant response to sugar and ABA treatments, and its protein, as a nucleus protein, had transcriptional activation in yeast. We obtained the overexpression (OE-VvNAC26 ) and RNA-inhibition (RNAi-VvNAC26 ) of VvNAC26 in transgenic calli by Agrobacterium tumefaciens -mediated transformation. We found that VvNAC26 negatively influenced fructose content. Under sugar and ABA treatments, VvNAC26 negatively influenced the expression of most sugar-related genes, while positively influencing the expression of most ABA pathway-related genes. Dual-luciferase reporter experiments demonstrated that VvNAC26 significantly upregulates VvSnRK1.2 promoter expression in tobacco (Nicotiana benthamiana ) leaves, although this process in grape calli requires ABA. The levels of sugar content, sugar-related genes, and ABA-related genes fluctuated significantly in OE-VvNAC26 +RNAi-VvSnRK1.2 and OE-VvSnRK1.2 +RNAi-VvNAC26 transgenic calli. These findings indicated that VvNAC26 regulates sugar metabolism and ABA pathway, displaying synergistic interactions with VvSnRK1.2 .

VvRF2b interacts with VvTOR and influences VvTOR-regulated sugar metabolism in grape

The production of top-quality wines is closely related to the quality of the wine grapes. In wine grapes (Vitis vinifera L., Vv), sugar is a crucial determinant of berry quality, regulated by an interplay of various transcription factors and key kinases. Many transcription factors involved in sugar metabolism remain unexplored. Target of Rapamycin (TOR) is an important protein kinase in plants, recently found to regulate sugar metabolism in grapes. However, transcription factors or other factors involved in this process are rarely reported. Here, we utilized transgenic callus tissues from 'Cabernet Sauvignon' grape fruit engineered via gene overexpression (oe) and CRISPR/Cas9-based gene knockout (ko), and discovered a bZIP transcription factor, VvRF2b, whose knockout resulted in increased accumulation of fructose and sucrose, indicating that VvRF2b is a negative regulator of sugar accumulation. Subcellular localization and transcriptional activation tests showed that VvRF2b is an activator of transcription located both in the nucleus and cell membrane. Analysis of VvRF2b and VvTOR gene levels and sugar contents (glucose, fructose, and sucrose) in 'Cabernet Sauvignon' grape fruits at 30, 70, and 90 days after bloom (DAB) revealed that VvRF2b is expressed more highly during fruit development, while VvTOR is expressed more during the sugar accumulation phase, furthermore, VvTOR gene levels in koVvRF2b transgenic calli increased significantly, suggesting a strong relationship between the knockout of VvRF2b and the overexpression of VvTOR. Additionally, bimolecular fluorescence complementation and luciferase complementation assays demonstrated the interaction between VvRF2b and VvTOR proteins. After knocking out the VvRF2b gene in oeVvTOR calli, it was found that the knockout of VvRF2b promotes VvTOR-regulated sucrose accumulation and enhances the expression of sugar metabolism-related genes regulated by VvTOR. In summary, our results suggest that VvRF2b interacts with VvTOR protein and influences VvTOR-regulated sugar metabolism.

Elucidation of AsANS controlling pigment biosynthesis in Angelica sinensis through hormonal and transcriptomic analysis.

Angelica sinensis, a traditional Chinese medicinal plant, has been primarily reported due to its nutritional value. Pigmentation in this plant is an important appearance trait that directly affects its commercial value. To understand the mechanism controlling purpleness in A. sinensis, hormonal and transcriptomic analyses were performed in three different tissues (leave, root and stem), using two cultivars with contrasting colors. The two-dimensional data set provides dynamic hormonal and gene expression networks underpinning purpleness in A. sinensis. We found abscisic acid as a crucial hormone modulating anthocyanin biosynthesis in A. sinensis. We further identified and validated 7 key genes involved in the anthocyanin biosynthesis pathway and found a specific module containing ANS as a hub gene in WGCNA. Overexpression of a candidate pigment regulatory gene, AsANS (AS08G02092), in transgenic calli of A. sinensis resulted in increased anthocyanin production and caused purpleness. Together, these analyses provide an important understanding of the molecular networks underlying A. sinensis anthocyanin production and its correlation with plant hormones, which can provide an important source for breeding.

The effector SJP3 interferes with pistil development by sustaining SHORT VEGETATIVE PHASE 3 expression in jujube.

Jujube witches' broom (JWB) is a phytoplasma disease that causes severe damage to jujube (Ziziphus jujuba) crops worldwide. Diseased jujube plants show enhanced vegetative growth after floral reversion, including leafy flower structures (phyllody) and the fourth whorl converting into a vegetative shoot. In previous research, secreted JWB protein 3 (SJP3) was identified as an inducer of phyllody. However, the molecular mechanisms of SJP3-mediated pistil reversion remain unknown. Here, the effector SJP3 was found to interact with the MADS-box protein SHORT VEGETATIVE PHASE 3 (ZjSVP3). ZjSVP3 was expressed in young leaves and during the initial flower bud differentiation of healthy jujube-bearing shoots but was constitutively expressed in JWB phytoplasma-infected flowers until the later stage of floral development. The SJP3 effector showed the same expression pattern in the diseased buds and promoted ZjSVP3 accumulation in SJP3 transgenic jujube calli. The N-terminal domains of ZjSVP3 contributed to its escape from protein degradation in the presence of SJP3. Heterologous expression of ZjSVP3 in Nicotiana benthamiana produced typical pistil abnormalities, including trichome-enriched style and stem-like structures within the leaf-like ovary, which were consistent with those in the mildly malformed lines overexpressing SJP3. Furthermore, ectopic expression of ZjSVP3 directly bound to the zinc finger protein 8 (ZjZFP8) and MADS-box gene SHATTERPROOF 1 (ZjSHP1) promoters to regulate their expression, resulting in abnormal pistil development. Overall, effector SJP3-mediated derepression of ZjSVP3 sustained its expression to interfere with pistil development, providing insight into the mechanisms of pistil reversion caused by JWB phytoplasma in specific perennial woody plant species.

Construction and optimization of a genetic transformation system for efficient expression of human insulin-GFP fusion gene in flax

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins.Graphical

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

Overexpression of a transcription factor MdWRKY126 altered soluble sugar accumulation in apple and tomato fruit

The compositions and contents of soluble sugars highly determine the flavor and quality of flshy fruits. In the present study, we found that the overexpression of transcription factor MdWRKY126 localized on the nucleus enhanced sucrose concentration while decreased fructose and glucose concentration in transgenic apple calli and ripening tomato fruits. To comprehensively understand the effects of the MdWRKY126 on the content of various soluble sugars in apple and tomato fruits, enzyme activities and related essential genes associated with the sugar metabolism and transportation pathway in MdWRKY126-overrexpressed apple and tomato lines were analyzed. The results indicated that the overexpression of MdWRKY126 upregulated sucrose phosphate synthase (SPS) activity and the gene expression levels of SPS and sucrose transporter SUT, which was conducive to a large accumulation of sucrose in fruit cells. Meanwhile, MdWRKY126 overexpression downregulated the activity of enzymes involved in sucrose decomposition including cell wall invertase (CWINV), sucrose synthase (SUSY) and the corresponding gene expressions, as well as inhibited the expression levels of hexose transporter (HTs) and tonoplast sugar transporter (TSTs) that transport hexose into vacuoles, resulting in a reduced hexose level in apple calli and tomato fruit. These findings enrich our understanding of the metabolism and regulation of soluble sugars in apple fruits.

Brassinosteroids biosynthetic gene MdBR6OX2 regulates salt stress tolerance in both apple and Arabidopsis

Salt stress is a critical limiting factor for fruit yield and quality of apples. Brassinosteroids (BRs) play an important role in response to abiotic stresses. In the present study, application of 2,4- Epicastasterone on seedlings of Malus ‘M9T337’ and Malus domestica ‘Gala3’ alleviated the physiological effects, such as growth inhibition and leaf yellowing, induced by salt stress. Further analysis revealed that treatment with NaCl induced expression of genes involved in BR biosynthesis in ‘M9T337’ and ‘Gala3’. Among which, the expression of BR biosynthetic gene MdBR6OX2 showed a three-fold upregulation upon salt treatment, suggesting its potential role in response to salt stress in apple. MdBR6OX2, belonging to the CYP450 family, contains a signal peptide region and a P450 domain. Expression patterns analysis showed that the expression of MdBR6OX2 can be significantly induced by different abiotic stresses. Overexpressing MdBR6OX2 enhanced the tolerance of apple callis to salt stress, and the contents of endogenous BR-related compounds, such as Typhastero (TY), Castasterone (CS) and Brassinolide (BL) were significantly increased in transgenic calli compared with that of wild-type. Extopic expression of MdBR6OX2 enhanced tolerance to salt stress in Arabidopsis. Genes associated with salt stress were significantly up-regulated, and the contents of BR-related compounds were significantly elevated under salt stress. Our data revealed that BR-biosynthetic gene MdBR6OX2 positively regulates salt stress tolerance in both apple calli and Arabidopsis.

Transcriptional factor MdESE3 controls fruit acidity by activating genes regulating malic acid content in apple.

Acidity is a key factor controlling fruit flavor and quality. In a previous study, combined transcriptome and methylation analyses identified a P3A-type ATPase from apple (Malus domestica), MdMa11, which regulates vacuolar pH when expressed in Nicotiana benthamiana leaves. In this study, the role of MdMa11 in controlling fruit acidity was verified in apple calli, fruits, and plantlets. In addition, we isolated an APETALA2 domain-containing transcription factor, designated MdESE3, based on yeast one-hybrid (Y1H) screening using the MdMa11 promoter as bait. A subcellular localization assay indicated that MdESE3 localized to the nucleus. Analyses of transgenic apple calli, fruits, and plantlets, as well as tomatoes, demonstrated that MdESE3 enhances fruit acidity and organic acid accumulation. Meanwhile, chromatin immunoprecipitation quantitative PCR, luciferase (LUC) transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the ethylene-responsive element (ERE; 5'-TTTAAAAT-3') upstream of the MdMa11 transcription start site, thereby activating its expression. Furthermore, MdtDT, MdDTC2, and MdMDH12 expression increased in apple fruits and plantlets overexpressing MdESE3 and decreased in apple fruits and plantlets where MdESE3 was silenced. The ERE was found in MdtDT and MdMDH12 promoters, but not in the MdDTC2 promoter. The Y1H, LUC transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the MdtDT and MdMDH12 promoters and activate their expression. Our findings provide valuable functional validation of MdESE3 and its role in the transcriptional regulation of MdMa11, MdtDT, and MdMDH12 and malic acid accumulation in apple.

Unraveling the role of PlARF2 in regulating deed formancy in Paeonia lactiflora.

PlARF2 can positively regulate the seed dormancy in Paeonia lactiflora Pall. and bind the RY cis-element. Auxin, a significant phytohormone influencing seed dormancy, has been demonstrated to be regulated by auxin response factors (ARFs), key transcriptional modulators in the auxin signaling pathway. However, the role of this class of transcription factors (TFs) in perennials with complex seed dormancy mechanisms remains largely unexplored. Here, we cloned and characterized an ARF gene from Paeonia lactiflora, named PlARF2, which exhibited differential expression levels in the seeds during the process of seed dormancy release. The deduced amino acid sequence of PlARF2 had high homology with those of other plants and contained typical conserved Auxin_resp domain of the ARF family. Phylogenetic analysis revealed that PlARF2 was closely related to VvARF3 in Vitis vinifera. The subcellular localization and transcriptional activation assay showed that PlARF2 is a nuclear protein possessing transcriptional activation activity. The expression levels of dormancy-related genes in transgenic callus indicated that PlARF2 was positively correlated with the contents of PlABI3 and PlDOG1. The germination assay showed that PlARF2 promoted seed dormancy. Moreover, TF Centered Yeast one-hybrid assay (TF-Centered Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay analysis (Dual-Luciferase) provided evidence that PlARF2 can bind to the 'CATGCATG' motif. Collectively, our findings suggest that PlARF2, as TF, could be involved in the regulation of seed dormancy and may act as a repressor of germination.

VvERF117 positively regulates grape cold tolerance through direct regulation of the antioxidative gene BAS1

Cold stress significantly threatens grape quality, yield, and geographical distribution. Although ethylene-responsive factors (ERFs) are recognized for their pivotal roles in cold stress, the regulatory mechanisms of many ERFs contributing to tolerance remain unclear. In this study, we identified the cold-responsive gene VvERF117 and elucidated its positive regulatory function in cold tolerance. VvERF117 exhibits transcriptional activity and localizes to the nucleus. VvERF117 overexpression improved cold tolerance in transgenic Arabidopsis, grape calli, and grape leaves, whereas VvERF117 silencing increased cold sensitivity in grape calli and leaves. Furthermore, VvERF117 overexpression remarkably upregulated the expression of several stress-related genes. Importantly, BAS1, encoding a 2-Cys peroxidase (POD), was confirmed as a direct target gene of VvERF117. Meanwhile, compared to the wild-type, POD activity and H2O2 content were remarkably increased and decreased in VvERF117-overexpressing grape calli and leaves, respectively. Conversely, VvERF117 silencing displayed the opposite trend in grape calli and leaves under cold stress. These findings indicate that VvERF117 plays a positive role in cold resistance by, at least in part, enhancing antioxidant capacity through regulating the POD-encoding gene VvBAS1, leading to effective mitigation of reactive oxygen species.

MdWRKY45 contributes to resistance against Botryosphaeria dothidea in apple

Malus domestica “Fuji” is the predominant apple cultivar grown around the world, but it is highly susceptible to apple ring rot caused by Botryosphaeria dothidea. WRKY transcription factors (TFs) play key roles in multiple physiological responses to plant defense. We have previously reported the involvement of several MdWRKY TFs in plant defense against. In this study, we identified a new MdWRKY TF, MdWRKY45, which is induced by B. dothidea infection. MdWRKY45 was localized in the nucleus and exhibited transcriptional activity. Over-expression of MdWRKY45 enhanced the resistance of transgenic apple calli and fruits to B. dothidea infection. Conversely, suppression of MdWRKY45 decreased the disease resistance of apple calli and fruits. Further transcriptome profiling and expression analysis showed that various genes regulating the release of salicylic acid (SA), jasmonic acid (JA), and reactive oxygen species (ROS) were either up-regulated or down-regulated in MdWRKY45-RNAi transgenic apple calli compared with wild-type (WT). During B. dothidea infection, MdWRKY45 modulated the levels of SA and JA, as well as ROS burst. Moreover, MdWRKY45 could bind to the W-box cis-element in the promoters of MdPR1, MdJAZ1, and MdRbohF genes, thereby regulating their expression. In conclusion, MdWRKY45 primarily confers resistance to B. dothidea infection through pathways involving SA, JA, and ROS burst defense.

Calmodulin-like protein MdCML15 interacts with MdBT2 to modulate iron homeostasis in apple.

BTB and TAZ domain proteins (BTs) function as specialized adaptors facilitating substrate recognition of the CUL3-RING ubiquitin ligase (CRL3) complex that targets proteins for ubiquitination in reaction to diverse pressures. Nonetheless, knowledge of the molecular mechanisms by which the apple scaffold protein MdBT2 responds to external and internal signals is limited. Here we demonstrate that a putative Ca 2+ sensor, calmodulin-like 15 (MdCML15), acts as an upstream regulator of MdBT2 to negatively modulate its functions in plasma membrane H+-ATPase regulation and iron deficiency tolerance. MdCML15 was identified to be substantially linked to MdBT2, and to result in the ubiquitination and degradation of the MdBT2 target protein MdbHLH104. Consequently, MdCML15 repressed the MdbHLH104 target, MdAHA8's expression, reducing levels of a specific membrane H+-ATPase. Finally, the phenotype of transgenic apple plantlets and calli demonstrated that MdCML15 modulates membrane H+-ATPase-produced rhizosphere pH lowering alongside iron homeostasis through an MdCML15-MdBT2-MdbHLH104-MdAHA8 pathway. Our results provide new insights into the relationship between Ca2+ signaling and iron homeostasis.

Function of FT in Flowering Induction in Two Camellia Species.

FLOWERING LOCUS T (FT), belonging to the FT/TFL1 gene family, is an important gene regulating the flowering transition and inflorescence architecture during plant development. Given its importance to plant adaptation and crop improvement, FT has been extensively studied in related plant research; however, the specific role and underlying molecular mechanisms of FT in the continuous flowering of perennial plants remains elusive. Here, we isolated and characterized homologous FT genes from two Camellia species with different flowering-period phenotypes: CaFT was isolated from Camellia azalea, a precious species blooming in summer and flowering throughout the year, and CjFT was isolated from C. japonica, which blooms in winter and spring. The major difference in the genes between the two species was an additional five-amino acid repeat sequence in C. japonica. FT showed high expression levels in the leaves in both species from January to August, especially in April for C. japonica and in May for C. azalea. CaFT was expressed throughout the year in C. azalea, whereas CjFT was not expressed from September to December in C. japonica. The expression levels of FT in the floral buds were generally higher than those in the leaves. Overexpression of CaFT and CjFT in Arabidopsis indicated that both genes can activate downstream genes to promote flowering. Transgenic callus tissue was obtained by introducing the two genes into C. azalea through Agrobacterium-mediated transformation. Transcriptome and quantitative real-time polymerase chain reaction analyses indicated that both florigen FT genes promoted the expression of downstream genes such as AP1, FUL, and SEP3, and slightly up-regulated the expression of upstream genes such as CO and GI. The above results indicated that CaFT and CjFT played a role in promoting flowering in both camellia species. The expression pattern of CaFT in leaves suggested that, compared to CjFT, CaFT may be related to the annual flowering of C. azalea.

Development of an Agrobacterium tumefaciens-mediate transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis.1

Litchi holds paramount economic significance as a global fruit crop. However, the advancement of litchi functional genomics encounters substantial obstacles due to its recalcitrance to stable transformation. Here, we present an efficacious A. tumefaciens-mediated transformation system in somatic embryo of ‘Heiye’ litchi. This system was developed through meticulous optimization of variables encompassing explant selection, A. tumefaciens strain delineation, bacterium concentration, infection duration, and infection methodology. The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1, resulting in the generation of transgenic calli. However, it was discerned that the differentiation of transgenic calli into somatic embryos encountered substantial challenges. To delve into the intricate molecular underpinnings of LcMYB1’s inhibitory role in somatic embryo induction, a comprehensive transcriptome analysis was conducted encompassing embryogenic calli (C), globular embryos (G), and transgenic calli (TC). A total of 1166 common differentially expressed genes (DEGs) were identified between C-vs-G and C-vs-TC. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these common DEGs were most related to plant hormone signal transduction pathways. Furthermore, RT-qPCR corroborated pronounced down-regulation of numerous genes intricately associated with somatic embryos induction within the transgenic calli. The development of this transformation system has provided valuable support for functional genomics research in litchi.

Overexpression of AtMYB12 transcription factor simultaneously enhances quercetin-dependent metabolites in radish callus

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g−1 dry weight), flavonoid content (463 mg g−1 dry weight), and quercetin content (48.24 mg g−1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.

Overexpressing ATP Sulfurylase Improves Fe-Deficiency Tolerance in Apple Calli and Tobacco

Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions due to decreased availability of Fe in alkaline soils. The ATP sulfurylase (ATPS) gene has been reported to participate in regulating various abiotic stresses. Transcriptome data and qRT-PCR analysis revealed that the ATP sulfurylase gene MhATPS1 was notably induced by Fe-deficiency stress. Consequently, MhATPS1 (103410737) was isolated from Malus halliana, and transgenic tobacco and transgenic apple calli were successfully obtained by genetic transformation. Compared with the wild type (WT), transgenic MhATPS1 lines (transgenic tobacco and transgenic apple calli) displayed stronger resistance to Fe-deficiency treatment. To be specific, transgenic plants exhibited better growth, accumulated more Fe2+ content, had higher ferric chelate reductase (FCR) activity, and a greater active oxygen scavenging capacity. Furthermore, transgenic MhATPS1 lines up-regulated the expression of Fe uptake genes under Fe-deficit stress. Additionally, MhATPS1 transgenic lines secreted more H+ content compared to the WT. In summary, these findings indicate that the MhATPS1 gene may play a positive role in Fe-deficiency stress in both tobacco and apple calli.

The trihelix transcription factor MdSIP1-2 interacts with MdNIR1 promoter to regulate nitrate utilization in apple

Nitrate is not only the main nitrogen (N) source for plant growth and development, but also an important signaling molecule for plants to cope with the changing environment. To adapt to N fluctuations in the environment, plants have established sophisticated regulatory networks. Although some nitrate signaling regulatory genes have been determined, whether the trihelix transcription factors (TFs) regulate N metabolism remains unclear. Here, an apple trihelix TF-encoding gene 6B-INTERACTING PROTEIN1–2 (SIP1–2) was identified, which is inducible upon nitrate treatment. Under N deficiency condition, the MdSIP1–2-OE transgenic apple calli and Arabidopsis repressed the expression of genes involved in N transport and metabolism, and repressed the activity of both nitrate reductase and nitrite reductase, leading to decreased N content and biomass. These results indicated that MdSIP1–2 inhibited the utilization of nitrate, and MdSIP1–2 may play a negative regulatory role in nitrate signal transduction in plant. In vivo and in vitro experiments showed that MdSIP1–2 directly bound to the GT1 motif of the MdNIR1 promoter to repress its transcription. Taken together, these results revealed that the trihelix TF family gene MdSIP1–2 plays a crucial role in regulating nitrate transport and metabolism, and providing new insights for plants to adapt to N fluctuation in environment.