Genetic Transformation Method Research Articles

Knockout of BnaX.SGT.a caused significant sinapine reduction in transgene-free rapeseed mutants generated by protoplast-based CRISPR RNP editing.

Rapeseed (Brassica napus L.) is known for its high-quality seed oil and protein content. However, its use in animal feed is restricted due to antinutritional factors present in the seedcake, with sinapine being one of the main compounds that reduces palatability. Attempts to develop rapeseed germplasm with lower sinapine levels through traditional breeding methods have shown limited progress. Genetic transformation methods could create new genotypes with reduced sinapine levels by silencing key genes involved in sinapine biosynthesis, though these methods often result in transgenic or genetically modified plants. The recent development of CRISPR-Cas technology provides a precise and efficient approach to crop improvement, with the potential to generate transgene-free mutants. In this study, we targeted the BnaX.SGT.a genes for knockout using CRISPR-Cas editing. By utilizing our newly established protoplast regeneration and transfection protocol for rapeseed, we demonstrated that DNA-free CRISPR editing via protoplast-based ribonucleoprotein (RNP) delivery was highly effective. We achieved successful knockout of the BnaX.SGT.a paralogues, with an average mutation efficiency of over 30%. Sequencing results revealed a variety of mutation types, from 1 bp insertions to 10 bp deletions, with most mutants exhibiting frameshift mutations that led to premature stop codons. The mutants displayed no visible phenotypic differences in growth patterns or flowering compared to the wild type. Importantly, sinapine content was significantly reduced in all T2 generation mutants analysed, while seed weight remained comparable between mutants and the wild type.

Sonication-assisted Rhizobium radiobacter-mediated genetic transformation of Indian Lotus (Nelumbo nucifera Gaertn.).

This study aimed to develop a reliable and efficient genetic transformation method for the ornamental Indian Lotus (Nelumbo nucifera Gaertn.) using the sonication-assisted Rhizobium radiobacter-mediated transformation technique. To conduct the transformation, shoot apical meristem explants were infected with Rhizobium radiobacter (synonym Agrobacterium tumefaciens) strain LBA 4404 containing a binary vector pBI121 that harbours the GUS reporter gene (uidA) and kanamycin resistance gene nptII for plant selection. To improve the transformation efficiency, we optimized parameters such as bacterial cell density, sonication duration, infection time, co-cultivation duration, acetosyringone concentration, cefotaxime, and kanamycin concentrations. Sonication treatment at 42kHz for 90s recorded the highest transformation efficiency. The selection of regenerated plantlets was performed on a kanamycin-supplemented selection medium. The putative transformants showed GUS expression in the leaves and petioles. The presence of the GUS gene was also confirmed in the putative transformants through PCR, with the appearance of the expected amplicon size of 520bp. The presence of nptII was confirmed by PCR in the putatively transformed plants with an amplicon size of 530bp. The maximum regeneration frequency obtained was 72.66%, and the highest transformation efficiency achieved was 9.0% in the Indian Lotus.

Somatic Embryogenesis from the Leaf-Derived Calli of In Vitro Shoot-Regenerated Plantlets of Rosa hybrida 'Carola'.

Roses are one of the most important flowers applied to landscape, cut flowers, fragrance and food industries widely. As an effective method for plant reproduction, the regeneration via somatic embryos is the most promising method for breed improvement and genetic transformation of woody plants. However, lower somatic embryogenesis (SE) induction rates and genotypic constraints impede progress in genetic transformation in rose. This study describes a plant regeneration system for the famous red cut flower cultivar Rosa hybrida 'Carola'. The stems without petioles cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg·L-1 6-benzylaminopurine (6-BA), 0.05 mg·L-1 a-naphthalene acetic acid (NAA) and 30 g·L-1 sucrose showed the maximum proliferation coefficient of shoots with 3.41 for the micropropagation system. We evaluated the effects of different plant growth regulators (PGRs) on the induction, proliferation and conversion of somatic embryos. The induction rate of calli reached 100% on MS medium supplemented with 2.0 g·L-1 NAA and 30 g·L-1 glucose. The highest induction rate of somatic embryos achieved a frequency of 13.33% on MS medium supplemented with 2.0 mg·L-1 zeatin (ZT), 0.1 mg·L-1 NAA and 30 g·L-1 glucose. The most suitable carbohydrate with 60 g·L-1 glucose resulted in a proliferation rate of somatic embryos (4.02) on MS medium containing 1.5 mg·L-1 ZT, 0.2 mg·L-1 NAA and 0.1 mg·L-1 gibberellic acid (GA3). The highest somatic embryos germination rate (43.33%) was obtained from the MS medium supplemented with 1.0 mg·L-1 6-BA, 0.01 mg·L-1 IBA and 30 g·L-1 glucose. Finally, the germinated somatic embryos successfully rooted on 1/2 MS medium containing 1.0 mg·L-1 NAA, 30 g·L-1 sucrose, and the vigorous plantlets were obtained after hardening-off culture. This study provided a stable and efficient protocol for plant regeneration via somatic embryos in R. hybrida 'Carola', which will be beneficial to the further theoretical study and genetic improvement in roses.

Transgenic poplar for resistance against pest and pathogen attack in forests: an overview

Forests are potential habitats for immense terrestrial ecosystems and aquatic biodiversity, performing an essential role in ecological preservation and regulation of climate. The anthropogenic pressures on the forests lead to forest loss, fragmentation and degradation. Requirements for sustainable methodologies for forest protection are of utmost priority under the climate change regime. Among forest trees, poplar trees (Populus L.) have attracted attention in global forestry as a promising material for improving the quality and quantity of urban landscapes. These plants provide wood, which can be utilized as raw resources for the paper industry and as a potential source of biofuel. However, several biotic stresses, such as attacks by pests and pathogens, severely affect poplar production and productivity. The improvement of Populus trees through conventional tree breeding methods is restricted due to their long-life cycles and the lack of suitable donors with resistance genes. Populus has been utilized as a model plant for studying gene functions due to its highly efficient genetic transformation capabilities. The present review will provide a comprehensive overview of pest and pathogen attacks on poplar, focusing on their infection mechanisms, transmission routes, and control strategies. Additionally, it will examine the most widely used genetic transformation methods (gene gun-mediated, Agrobacterium tumefaciens-mediated, protoplast transformation, micro-RNA mediated and micro-RNA clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR-Cas) systems methods and RNA interference) for improving tolerance in poplar trees against pest and pathogens attack. Furthermore, it will delve into prospects, challenges, and recent advances in molecular biology tools and their safe application for genetic transformation to improve insect and pest resistance in poplar trees. Finally, the regeneration of transgenic poplar trees with enhanced resistance, developed through various genetic engineering techniques, is discussed.

The antisense CircRNAVvcircABH controls salt tolerance and the brassinosteroid signaling response by suppressing cognate mRNA splicing in grape.

Soil salinization is a major factor limiting the sustainable development of the grape industry. Circular RNAs (circRNAs) are more stable than linear mRNAs and are involved in stress responses. However, the biological functions and molecular mechanisms underlying antisense circRNAs in plants remain unclear. We identified the antisense circRNA VvcircABH through high-throughput sequencing. Using genetic transformation methods and molecular biological techniques, we analyzed the effects of VvcircABH on the response to salt stress and the mechanisms underlying its effects. VvcircABH was located in the nucleus and upregulated by salt stress, while the expression level of its cognate gene VvABH (alpha/beta-hydrolase) was downregulated. VvcircABH overexpression or VvABH silencing greatly enhanced grape salt tolerance. VvcircABH could bind to the overlapping region and inhibits VvABH pre-mRNA splicing, thereby decreasing the expression level of VvABH. Additionally, VvcircABH repressed the additive effect of VvABH on the interaction between VvBRI1 (brassinosteroid-insensitive 1) and VvBKI1 (BRI1 kinase inhibitor 1), thus influencing the plant's response to BR, which plays important roles in plant salt tolerance. We conclude that VvcircABH and VvABH play distinct roles in the salt tolerance and brassinosteroid signaling response, and VvcircABH could govern the expression of VvABH by inhibiting its splicing.

Establishment and Validation of an Efficient Agrobacterium Tumefaciens-Mediated Transient Transformation System for Salix Psammophila.

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD600 of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.

MaMPK19, a key gene enhancing cold resistance by activating the CBF pathway in banana

MPKs play an essential part role in the process of plant low temperature stress. In this study, the specific inhibitor SB203580 of MPK was used to spray banana leaves and MaMPK19 was overexpressed in N.benthamiana and banana to explore the effect of MaMPK19 on cold resistance and the regulation mode of downstream genes. Additionally, we optimized the method of genetic transformation of banana laying the foundation for the establishment of an efficient genetic transformation system. The results showed that 40 μmol L−1 SB203580 could significantly reduce the expression of MaMPK19 and MaCBFs, as well as weaken the cold resistance of banana at 4 °C. After agrobacterium tumefaciens infection, the regeneration rates of adventitious buds in ‘Tianbao’, ‘Brazilian’ and‘Indonesia’ (Musa spp. AAA Group, Cavendish) reached 10.43%, 15.81% and 14.23%, respectively. And the positive rates reached 10.71%, 2.25% and 6.94%, respectively. Overexpression of MaMPK19 enhanced the cold resistance of N.benthamiana and bananas. MaMPK19 promoted the expression of MaICE1, MaDREB1D and MaCOR413. Furthermore, MaMPK19 increased POD activity and the content of ABA and JA. Our study highlights the importance of MaMPK19 in improving the cold resistance of bananas and provides a reference for biological breeding.

A Rapid Method for Obtaining the Transgenic Roots of Crassulaceae Plants.

Crassulaceae plants are valued for their horticultural, ecological, and economic significance, but their genetic improvement is hindered by the absence of efficient and stable genetic transformation methods. Therefore, the development of a tailored genetic transformation method is crucial for enhancing the progress of the genetic improvement of Crassulaceae plants. The results indicate that, in the transformation experiments conducted on Kalanchoe tetraphylla, the K599 strain exhibited the highest transformation efficiency (76.67%), while C58C1 was least efficient (21.43%). An acetosyringone concentration of 100 μM was optimal for the hairy root transformation, and the immersion method yielded the highest efficiency. Additionally, the Silwet L-77 concentration significantly influenced the transformation efficiency, with 0.05% leading to a decrease. Upon four Crassulaceae species, notable differences were observed, with K. tetraphylla exhibiting the highest efficiency of 100%, and Sedum alfredii displaying the lowest efficiency of 5%. The RUBY reporter gene offers a more distinct advantage over GFP in observing the transformation effects. This study developed a simple, feasible, and cost-effective method for obtaining transgenic roots from leaves of Crassulaceae. The methodology provides technical support for the genetic improvement and gene function research of Crassulaceae plants.

Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia.

Descurainia sophia L. Webb ex Prantl is used in traditional medicine globally. However, the lack of an efficient and reliable genetic transformation system has seriously limited the investigation of gene function and further utilization of D. sophia. In this study, a highly efficient, time-saving, and cost-effective Agrobacterium tumefaciens-mediated genetic transformation system has been developed in D. sophia. In this method, the transformation was accomplished by simply dipping developing D. sophia inflorescences for 45 s into an Agrobacterium suspension (OD600 = 0.6) containing 5% sucrose and 0.03% (v/v) Silwet L-77. Treated plants were allowed to set seeds which were then plated on a selective medium with hygromycin B (HygB) to screen transformants. Additionally, the CRISPR/Cas9 genomic editing system was validated by targeting phytoene desaturase (PDS) gene using this floral dip method, and mutant plants with the expected albino phenotype could be obtained in 2.5 months. This genetic transformation and targeted editing system will be a valuable tool for routine investigation of gene function and further exploitation in D. sophia.

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Screening and Functional Evaluation of Four Larix kaempferi Promoters.

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation.

Map-based cloning of LPD, a major gene positively regulates leaf prickle development in eggplant.

A critical gene for leaf prickle development (LPD) in eggplant was mapped on chromosome E06 and was confirmed to be SmARF10B through RNA interference using a new genetic transformation technique called SACI developed in this study Prickles on eggplant pose challenges for agriculture and are undesirable in cultivated varieties. This study aimed to uncover the genetic mechanisms behind prickle formation in eggplant. Using the F2 and F2:3 populations derived from a cross between the prickly wild eggplant, YQ, and the prickle-free cultivated variety, YZQ, we identified a key genetic locus (LPD, leaf prickle development) on chromosome E06 associated with leaf prickle development through BSA-seq and QTL mapping. An auxin response factor gene, SmARF10B, was predicted as the candidate gene as it exhibited high expression in YQ's mature leaves, while being significantly low in YZQ. Downregulating SmARF10B in YQ through RNAi using a simple and efficient Agrobacterium-mediated genetic transformation method named Seedling Apical Cut Infection (SACI) developed in this study substantially reduced the size and density of leaf prickles, confirming the role of this gene in prickle development. Besides, an effective SNP was identified in SmARF10B, resulting in an amino acid change between YQ and YZQ. However, this SNP did not consistently correlate with prickle formation in eight other eggplant materials examined. This study sheds light on the pivotal role of SmARF10B in eggplant prickle development and introduces a new genetic transformation method for eggplant, paving the way for future research in this field.

Developmental regulators in promoting genetic transformation efficiency in maize and other plants

Given global agricultural challenges such as population growth, climate change, and limitations on resources and the environment, as well as increasing diversity in breeding goals, relying on traditional breeding methods is inadequate to provide food security requirements and promote sustainable development. Genetic transformation technology has become an effective tool for performing functional genomics research and molecular breeding. In this study, we conducted an in-depth analysis of 1669 literary works to investigate the potential of developmental regulators (DRs) in enhancing the efficiency of plant genetic transformation, with a concentration on their use in maize. Through multi-omics data analysis, we identified 12 homologous DRs from various species that are potentially applicable to maize. We identified a total of 41 possible disease resistances (DRs) for maize genetic transformation. Further experimental verification of ZmWIND1, a novel regulator belonging to the ERF/AP2 transcription factor (TF) family, showed that it significantly improved the efficiency of plant regeneration and transformation efficiency in maize. Specifically, compared to the control group, the callus induction rates for the pG3GB411-ZmWIND1 vector increased to 60.22 % and 47.85 % in Xiang249 and Zheng58, respectively. Transformation efficiency increased to 37.5 % in Xiang249 and 16.56 % in Zheng58, both significantly surpassing the control group. These findings have the potential to broaden the range of transformable maize varieties and lines, as well as introduce new genetic transformation methods in agricultural biotechnology, underscoring the immense potential to enhance genetic transformation efficiency through systematic exploration and application of DRs in maize.

An Effective Somatic-Cell Regeneration and Genetic Transformation Method Mediated by Agrobacterium tumefaciens for Portulaca oleracea L.

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.

A simple and efficient non-tissue culture method for genetic transformation of grape immature zygotic embryos via VvBBM overexpression

The process of genetic transformation of grapevine has long been hampered by the inefficiency and complexity of tissue culture-based methods, severely limiting cultivar improvement and functional genome research. In this study, we aimed to develop a novel, non-tissue culture-based method for the genetic transformation of grape immature zygotic embryos without relying on tissue culture. This approach involves the overexpression of the Vitis vinifera BABY BOOM (VvBBM) gene, a key member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor family, which is known to enhance plant regeneration and transformation efficiency. Using ‘Cabernet Sauvignon’ immature zygotic embryos, we achieved a transformation efficiency of 42.85 % under non-tissue culture conditions, significantly reducing the cycle duration to approximately two months compared to traditional genetic transformation methods. Phenotypic and molecular analyses of the transformed grapevine plants confirmed the integration and overexpression of the VvBBM gene, resulting in improved growth metrics, including increased plant height, root length, and total root number, in a genotype-independent manner. Our results highlight the potential of VvBBM overexpression to circumvent the limitations imposed by tissue culture dependence, providing a rapid, efficient, and scalable alternative to conventional methods. Our study not only provides a practical approach to overcoming the barriers to genetic transformation in grapevine, but also opens new avenues for the application of this technology in the breeding of other economically important crops.

Micropropagation and genetic transformation of Byblis liniflora

Byblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL−1) concentrations. Zeatin supplementation (1 mgL−1) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL−1 facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

Impact of activated charcoal on the progression of somatic embryogenesis in grape cells: Histological and transcriptomic perspectives

Somatic embryogenesis is a crucial genetic transformation method in plants. Despite numerous studies being conducted for acquiring embryogenic competence in plant cells, somatic embryo (SE) induction from embryogenic cells (ECs) has attracted relatively limited research attention. In grapes, somatic cell induction is impeded by unidentified inhibitors, which hinders the progression of globular embryo (GE) formation and subsequently embryo development. We found that addition of activated charcoal (AC) to the grape SE induction medium augmented the speed, consistency, and synchronization of embryogenesis. Cell samples cultured in the induction medium with and without AC were thoroughly evaluated through histological anatomy analysis, subcellular structure observation, and comparative transcriptomic analysis. The histological observation revealed the specific timeline of SE formation. An intermediate developmental state, referred to as the globular embryo precursor (PGE), was observed during the induction of GE formation from EC. Members of transcription factor families such as WRKY, MYB, MADS, AP2/ERF, HB-KNOX, HB-HD-ZIP, HB-WOX, NAC, and GRF were identified in the early and middle stages of GE formation. Additionally, we explored the key roles of reactive oxygen species (ROS)-, starch-, cell wall-, and hormone-related genes. The cell wall-related genes were highly enriched in differentially expressed gene sets. Several amyloplasts in EC disappeared, potentially because of hydrolysis, and numerous mitochondria were observed. In AC-added samples, starch was consumed more, and cellulose hydrolysis-related genes were downregulated, whereas cellulose synthesis-related genes had higher expression than AC-free samples. ROS response-related genes were induced when GE induction was initiated. When applied, an optimal concentration of external H2O2 promoted SE initiation in grapes, whereas a high concentration caused delayed GE formation. This study suggests that, by absorbing and releasing substances, the added AC can create a stable buffer environment for cell survival, protect against excessive ROS- or inhibitor-induced cell damage, and support uninterrupted progress of somatic embryogenesis.

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

Microalgae-made human vaccines and therapeutics: A decade of advances.

Microalgal emergence is a promising platform with two-decade historical background for producing vaccines and biopharmaceuticals. During that period, microalgal-based vaccines have reported successful production for various diseases. Thus, species selection is important for genetic transformation and delivery methods that have been developed. Although many vaccine prototypes have been produced for infectious and non-infectious diseases, fewer studies have reached immunological and immunoprotective evaluations. Microalgae-made vaccines for Staphylococcus aureus, malaria, influenza, human papilloma, and Zika viruses have been explored in their capacity to induce humoral or cellular immune responses and protective efficacies against experimental challenges. Therefore, specific pathogen antigens and immune system role are important and addressed in controlling these infections. Regarding non-communicable diseases, these vaccines have been investigated for breast cancer; microalgal-produced therapeutic molecules and microalgal-made interferon-α have been explored for hypertension and potential applications in treating viral infections and cancer, respectively. Thus, conducting immunological trials is emphasized, discussing the promising results observed in terms of immunogenicity, desired immune response for controlling affections, and challenges for achieving the desired protection levels. The potential advantages and hurdles associated with this innovative approach are highlighted, underlining the relevance of assessing immune responses in preclinical and clinical trials to validate the efficacy of these biopharmaceuticals. The promising future of this healthcare technology is also envisaged.

Application of CRISPR/Cas9 gene editing technology in edible fungi: a review

The CRISPR/Cas9 gene editing system is a versatile technology for modifying gene, playing a crucial role in the study of functional genes and genetic breeding of plants, animals, fungi, and microorganisms. This review provides a comprehensive analysis of the application of this technology in gene research and genetic breeding of edible fungi. The review covers various aspects, including the delivery and expression strategies of Cas9 and sgRNA, genetic transformation methods, mutant screening, and repair strategies for target sites following DNA double-strand breaks. Additionally, the review summarizes the main challenges and optimization strategies associated with the application of this technology in edible fungi. Lastly, the future application potential of this technology in edible fungi research is discussed, drawing from the authors' personal research background.