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Introduction: Acer truncatum Bunge, belonging to the Acer genus in the Aceraceae family, is a commonly planted afforestation species across China, Japan, Korea, Europe, and North America. Renowned for its vibrant fall colors, it holds significant ecological and ornamental value. Methods: In this study, Acer truncatum ' Lihong ' was used as the research object. Starting from the callus induction of explants, the embryogenic callus of Acer truncatum 'Lihong' was obtained by systematically optimizing the medium and culture conditions. Then, the candidate gene AtrGST894 screened by transcriptome sequencing was transformed into embryogenic callus by Agrobacterium-mediated transformation. The genetic transformation system of Acer truncatum 'Lihong' embryogenic callus was initially established by continuously adjusting the conditions of Agrobacterium tumefaciens infection receptor materials, thus laying a material foundation for the study of the molecular regulation mechanism of Acer truncatum 'Lihong' leaf color, and also preparing for the later molecular improvement breeding of Acer truncatum. Therefore, this study has important theoretical and practical significance. Results: The results showed that the best medium for callus induction of Acer truncatum was 1/2MS+2 mg/L 2,4-D+0.3 mg/L 6-BA+0.5 mg/L NAA; The embryogenic callus induction medium of Acer truncatum was 1/2MS+3.0mg/L 6-BA+2.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA3; The proliferation medium of embryogenic callus of Acer truncatum was WPM+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA3+3mg/L 6-BA+1.0mg/L KT; The infection experiment of Agrobacterium tumefaciens on the embryogenic callus of Acer truncatum showed that the best antibacterial medium was WPM+30g/L sucrose+8g/L agar+0.5g/L acid-hydrolyzed casein+0.2mg/L KT+1.0 mg/L TDZ+0.5 mg/L IBA+0.1 mg/L GA3+200mmol/L carboxybenzyl+200mg/L cephalosporin, and then WPM+30g/L sucrose+8g/L agar+0.5g/L acid-hydrolyzed casein+0.2mg/L KT+1.0 mg/L TDZ+0.5 mg/L IBA+0.1 mg/L GA3+300mmol/L carboxybenzyl+200mg/L cephalosporin+25mg/L hygromycin. Screening medium screening, The obtained embryogenic callus browning rate, pollution rate and mortality rate were the lowest, and maintained vigorous growth. Discussion: The embryogenic callus was used as the infection material to verify that we successfully transferred the target gene into the embryogenic callus, which means that the genetic transformation system of Acer truncatum embryogenic callus was partially completed, and the infection process could be effectively inhibited. Although there was partial browning, it could continue to proliferate. Therefore, in future experiments, the focus is still to continue to verify the optimal conditions for optimizing the genetic transformation of Acer truncatum embryogenic callus and to solve the problems of difficulty in embryonic callus germination.

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The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P<0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications.

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Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an effective genetic transformation method for purslane, various organ tissues were used as explants for callus induction and shoot regeneration. Leaf tissue exhibited the highest dedifferentiation and regeneration ability, making it the optimal explant for tissue culture. By culturing on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzyleaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA), somatic cells from leaf explants could be developed into calli, shoots, and roots. The shoot induction results of 27 different purslane accessions elucidated the impact of genotype on somatic-cell regeneration capacity and further confirmed the effectiveness of the culture medium in promoting shoot regeneration. On this basis, a total of 17 transgenic plants were obtained utilizing the genetic transformation method mediated by Agrobacterium. The assessment of GUS staining, hygromycin selection, and polymerase chain reaction (PCR) amplification of the transgenic plants as well as their progeny lines indicated that the method established could effectively introduce foreign DNA into the purslane nucleus genome, and that integration was found to be stably inherited by offspring plants. Overall, the present study demonstrates the feasibility and reliability of the Agrobacterium-mediated genetic transformation method for introducing and integrating foreign DNA into the purslane genome, paving the way for further research and applications in purslane genetic modification.

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The Agrobacterium-based transgenic technique is commonly used for gene function validation and molecular breeding. However, it is not suitable for plants with a low regeneration capacity or a low transformation rate, such as Panax notoginseng (Burk) F.H. Chen and Lilium regale Wilson. In this study, a novel Agrobacterium transformation method based on injection in the meristems was developed using P. notoginseng and L. regale as experimental models. PCR analysis confirmed the successful integration of the reporter gene DsRed2 (Discosoma striata red fluorescence protein 2) into the genome of two experimental models. QRT-PCR and Western blot analysis demonstrated the transcriptional and translational expression of DsRed2. Additionally, laser confocal microscopy confirmed the significant accumulation of the red fluorescent protein in the leaves, stems, and roots of transformed P. notoginseng and L. regale. Most importantly, in the second year after injection, the specific bright orange fluorescence from DsRed2 expression was observed in the transgenic P. notoginseng and L. regale plants. This study establishes a fast, efficient, and tissue-culture-independent transgenic technique suitable for plants with a low regeneration capacity or a low transformation rate. This technique may improve the functional genomics of important medicinal and ornamental plants such as P. notoginseng and L. regale, as well as their molecular breeding.

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The genus Selaginella holds a key phylogenetic position as a sister species to vascular plants, encompassing desiccation-tolerant members. Some Selaginella species thrive in extremely arid conditions, enduring significant water loss and recovering upon rehydration. Consequently, Selaginella has emerged as a model system for studying desiccation tolerance in plant science. However, the absence of an efficient genetic transformation system has limited the utility of Selaginella species as a model. To address this constraint, we developed a nanoparticle-mediated transformation tool utilizing arginine-functionalized nanohydroxyapatites. This biocompatible system enabled the transient expression of the GFP, GUS, and eYGFPuv reporter genes in Selaginella moellendorffii. Establishing a stable genetic transformation technique for S. moellendorffii holds promise for application to other Selaginella species. This tool could be instrumental in identifying genetic resources for crop improvement and understanding genome-level regulatory mechanisms governing desiccation tolerance in Selaginella species. Furthermore, this tool might aid in identifying key regulatory genes associated with desiccation tolerance, offering potential applications in enhancing drought-sensitive crops and ensuring sustainable food production.

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As a typical bulb flower, lily is widely cultivated worldwide because of its high ornamental, medicinal and edible value. Although breeding efforts evolved over the last 10000 years, there are still many problems in the face of increasing consumer demand. The approach of biotechnological methods would help to solve this problem and incorporate traits impossible by conventional breeding. Target traits are dormancy, development, color, floral fragrance and resistances against various biotic and abiotic stresses, so as to improve the quality of bulbs and cut flowers in planting, cultivation, postharvest, plant protection and marketing. Genetic transformation technology is an important method for varietal improvement and has become the foundation and core of plant functional genomics research, greatly assisting various plant improvement programs. However, achieving stable and efficient genetic transformation of lily has been difficult worldwide. Many gene function verification studies depend on the use of model plants, which greatly limits the pace of directed breeding and germplasm improvement in lily. Although significant progress has been made in the development and optimization of genetic transformation systems, shortcomings remain. Agrobacterium-mediated genetic transformation has been widely used in lily. However, severe genotypic dependence is the main bottleneck limiting the genetic transformation of lily. This review will summarizes the research progress in the genetic transformation of lily over the past 30 years to generate the material including a section how genome engineering using stable genetic transformation system, and give an overview about recent and future applications of lily transformation. The information provided in this paper includes ideas for optimizing and improving the efficiency of existing genetic transformation methods and for innovation, provides technical support for mining and identifying regulatory genes for key traits, and lays a foundation for genetic improvement and innovative germplasm development in lily.

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Agrobacterium-mediated transformation is a preferred method for genetic engineering and genome editing of plants due to its numerous advantages, although not all species exhibit transformability. Genetic engineering and plant genome editing methods are technically challenging in recalcitrant crop plants. Factors affecting the poor rate of transformation in such species include host genotype, Agrobacterium genotype, type of explant, physiological condition of the explant, vector, selectable marker, inoculation method, chemical additives, antioxidative compounds, transformation-enhancing compounds, medium formulation, optimization of culture conditions, and pre-treatments. This review provides novel insights into the key factors involved in gene transfer facilitated by Agrobacterium and proposes potential solutions to overcome existing barriers to transformation in recalcitrant species, thereby contributing to improvement programs for these species. This review introduces the key factors that impact the effectiveness of a molecular breeding program using Agrobacterium-mediated transformation, specifically focusing on recalcitrant plant species.

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Citrus yellow vein-clearing virus (CYVCV) is an increasing threat to citrus cultivation. Notably, the role of zinc finger proteins (ZFPs) in mediating viral resistance in citrus plants is unclear. In this study, we demonstrate that ZFPs ClSUP and ClDOF3.4 enhance citrus defense responses against CYVCV in Eureka lemon. ClSUP interacted with the coat protein (CP) of CYVCV to reduce CP accumulation and inhibit its silencing suppressor function. Overexpression of CISUP triggered reactive oxygen species (ROS) and salicylic acid (SA) pathways, and enhanced resistance to CYVCV infection. In contrast, ClSUP-silencing resulted in increased CP accumulation and down-regulated ROS and SA-related genes. ClDOF3.4 interacts with ClSUP to facilitate its interactions with CP. Furthermore, ClDOF3.4 synergistically regulated the accumulation of ROS and SA with ClSUP and accelerated the down-regulation of CP accumulation. Transgenic plants co-expressing ClSUP and ClDOF3.4 remarkedly decrease the CYVCV. These findings provide a new reference for understanding the interaction mechanism between the host and CYVCV.

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BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.

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Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid S. nigrum, and 7 weeks for diploid Solanum americanum. In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation (Dianthus caryophyllus L.). Furthermore, we applied the improved method to generate S. americanum plants expressing AcMYB110 from kiwifruit (Actinidia chinensis spp.). This method will contribute to speeding up gene functional analysis and trait improvement in S. nigrum and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture.

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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.

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In recent decades an extensive mortality and decline of Quercus suber populations mainly caused by Phytophthora cinnamomi has been observed. In the current study, a chestnut gene homologous to ginkbilobin-2 (Cast_Gnk2-like), which in Ginkgo biloba codifies an antifungal protein, was transferred into cork oak somatic embryos of three different embryogenic lines by Agrobacterium mediated transformation. The transformation efficiency varied on the genotype from 2.5 to 9.2%, and a total of 22 independent transformed lines were obtained. The presence of Cast_Gnk2-like gene in transgenic embryos was verified in all lines by PCR. The number of transgene copies was estimated by qPCR in embryogenic lines with high proliferation ability and it varied between 1 and 5. In addition, the expression levels of Cast_Gnk2-like gene were determined in the embryogenic lines, with higher levels in lines derived from the genotype ALM6-WT. Transgenic plants were obtained from all transgenic lines and evaluated after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In vitro tolerance tests made under controlled conditions and following zoospore treatment showed that plants overexpressing Cast_Gnk2-like gene improved tolerance against Pc when compared to wild type ones.

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Plants are often exposed to biotic or abiotic stress, which can seriously impede their growth and development. In recent years, researchers have focused especially on the study of plant responses to biotic and abiotic stress. As one of the most widely planted grapevine rootstocks, 'Beta' has been extensively proven to be highly resistant to stress. However, further research is needed to understand the mechanisms of abiotic stress in 'Beta' rootstocks. In this study, we isolated and cloned a novel WRKY transcription factor, VhWRKY44, from the 'Beta' rootstock. Subcellular localization analysis revealed that VhWRKY44 was a nuclear-localized protein. Tissue-specific expression analysis indicated that VhWRKY44 had higher expression levels in grape roots and mature leaves. Further research demonstrated that the expression level of VhWRKY44 in grape roots and mature leaves was highly induced by salt and cold treatment. Compared with the control, Arabidopsis plants overexpressing VhWRKY44 showed stronger resistance to salt and cold stress. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly increased, and the contents of proline, malondialdehyde (MDA) and chlorophyll were changed considerably. In addition, significantly higher levels of stress-related genes were detected in the transgenic lines. The results indicated that VhWRKY44 was an important transcription factor in 'Beta' with excellent salt and cold tolerance, providing a new foundation for abiotic stress research.

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Synthetic promoters are powerful tools to boost the biotechnological potential of microalgae as eco-sustainable industrial hosts. The increasing availability of transcriptome data on microalgae in a variety of environmental conditions allows to identify cis-regulatory elements (CREs) that are responsible for the transcriptional output. Furthermore, advanced cloning technologies, such as golden gate-based MoClo toolkits, enable the creation of modular constructs for testing multiple promoters and a range of reporter systems in a convenient manner. In this chapter, we will describe how to introduce in silico-identified CREs into promoter sequences, and how to clone the modified promoters into MoClo compatible vectors. We will then describe how these promoters can be evaluated and screened for transgene expression in an established microalgal model for genetic perturbation, i.e., Chlamydomonas reinhardtii.

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Panax ginseng is a perennial herb with the main active compounds of ginsenosides. Among the reported ginsenosides, ginsenoside Rg_1 not only has a wide range of medicinal functions and abundant content but also is one of the major ginsenoside for the quality evaluation of this herb in the Chinese Pharmacopoeia. The main biosynthesis pathway of ginsenoside Rg_1 in P. ginseng has been clarified, which lays a foundation for the comprehensive and in-depth analysis of the biosynthesis and regulatory mechanism of ginseno-side Rg_1. However, the biosynthesis of ginsenoside Rg_1 is associated with other complex processes involving a variety of regulatory genes and catalyzing enzyme genes, which remain to be studied comprehensively. With the transcriptome data of 344 root samples from 4-year-old P. ginseng plants and their corresponding ginsenoside Rg_1 content obtained in the previous study, this study screened out 217 differentially expressed genes(DEGs) with Rg_1 content changes by DEseq2 analysis in R language. Furthermore, the weighted gene co-expression network analysis(WGCNA) revealed 40 hub genes among the DEGs.Pearsoncorrelation analysis was further perforned to yield 20 candidate genes significantly correlated with ginsenoside Rg_1 content, and these genes were annotated to multiple metabolic processes including primary metabolism and secondary metabolism. Finally, the treatment of P. ginseng adventitious roots with methyl jasmonate indicated that 16 of these genes promoted the biosynthesis of ginsenoside Rg_1 in response to methyl jasmonate induction. Finally, one of the 16 genes was randomly selected to verify the function of the gene by genetic transformation and qRT-PCR and to confirm the rationality of the methodology of this study. The above results lay a foundation for studying the mechanism for regulation on the synthesis of ginsenoside Rg_1 and provide genetic resources for the industrial production of ginsenoside Rg_1.

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This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of ß-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

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Cordyceps chanhua is a well-known edible and medicinal mushroom with a long history of use in China, and it contains a variety of secondary metabolites with interesting bioactive ingredients. However, recent researches have mainly focused on cultivation conditions, secondary metabolite compositions and pharmacological activities of C. chanhua, the lack of an efficient and stable genetic transformation system has largely limited further research on the relationship between secondary metabolites and biosynthetic gene clusters in C. chanhua. In this study, single-factor experiments were used to compare the effects of different osmotic stabilizers, enzyme concentrations and enzyme digestion times on protoplast yield, and we found that the highest yield of 5.53 × 108 protoplasts/mL was obtained with 0.7 M mannitol, 6 mg/mL snail enzyme and 4 h of enzyme digestion time, and the regeneration rate of protoplasts was up to approximately 30% using 0.7 M mannitol as an osmotic stabilizer. On this basis, a PEG-mediated genetic transformation system of C. chanhua was successfully established for the first time, which lays the foundation for further genetic transformation of C. chanhua.

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KEY MESSAGE: CsDGAT1A and CsDGAT2D play a positive regulatory role in cucumber's response to low-temperature stress and positively regulate the synthesis of triacylglycerol (TAG). Triacylglycerol (TAG), a highly abundant and significant organic compound in plants, plays crucial roles in plant growth, development, and stress responses. The final acetylation step of TAG synthesis is catalyzed by diacylglycerol acyltransferases (DGATs). However, the involvement of DGATs in cucumber's low-temperature stress response remains unexplored. This study focused on two DGAT genes, CsDGAT1A and CsDGAT2D, investigating their function in enhancing cucumber's low-temperature stress tolerance. Our results revealed that both proteins were the members of the diacylglycerol acyltransferase family and were predominantly localized in the endoplasmic reticulum. Functional analysis demonstrated that transient silencing of CsDGAT1A and CsDGAT2D significantly compromised cucumber's low-temperature stress tolerance, whereas transient overexpression enhanced it. Furthermore, the TAG content quantification indicated that CsDGAT1A and CsDGAT2D promoted TAG accumulation. In conclusion, this study elucidates the lipid metabolism mechanism in cucumber's low-temperature stress response and offers valuable insights for the cultivation of cold-tolerant cucumber plants.

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Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

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