Genetic Transformation System Research Articles

Establishment of a Virus-Induced Gene Silencing System in Abelmoschus manihot L.

Abelmoschus manihot L. (Jinhuakui, JHK) is widely cultivated for its pharmacological properties owing to its high flavonoid content and is commonly used as a garden landscape plant. However, the absence of an efficient genetic transformation system poses significant challenges for functional gene studies in this species. Virus-induced gene silencing (VIGS) is a well-established technique for exploring plant gene functions; however, this technique has not been applied to JHK. Here, a tobacco rattle virus (TRV)-VIGS system was successfully developed for the first time in JHK using the gene encoding phytoene desaturase (AmPDS) as a marker gene. This study investigated the impact of various Agrobacterium infection methods on the efficiency of AmPDS silencing. The results demonstrated that administering two injections-the first on the day of complete cotyledon expansion and the second 14 days later-using pTRV1 and pTRV2-AmPDS cultures resuspended to an OD600 of 1.0 and via the backside of the blade-led to significant photobleaching in the cotyledons 2 days after the second injection. Subsequent analyses revealed a marked reduction in both chlorophyll content and AmPDS expression. These findings suggest that a VIGS system was successfully developed in JHK, thus providing a rapid and effective method for studying gene function in this species and facilitating future research in JHK genetics.

CRISPR/cas9 Allows for the Quick Improvement of Tomato Firmness Breeding.

Fruit firmness is crucial for storability, making cultivating varieties with higher firmness a key target in tomato breeding. In recent years, tomato varieties primarily rely on hybridizing ripening mutants to produce F1 hybrids to enhance firmness. However, the undesirable traits introduced by these mutants often lead to a decline in the quality of the varieties. CRISPR/Cas9 has emerged as a crucial tool in accelerating plant breeding and improving specific target traits as technology iterates. In this study, we used a CRISPR/Cas9 system to simultaneously knock out two genes, FIS1 and PL, which negatively regulate firmness in tomato. We generated single and double gene knockout mutants utilizing the tomato genetic transformation system. The fruit firmness of all knockout mutants exhibited a significant enhancement, with the most pronounced improvement observed in the double mutant. Furthermore, we assessed other quality-related traits of the mutants; our results indicated that the fruit quality characteristics of the gene-edited lines remained statistically comparable to those of the wild type. This approach enabled us to create transgenic-free mutants with diverse genotypes across fewer generations, facilitating rapid improvements in tomato firmness. This study offers significant insights into molecular design breeding strategies for tomato.

Construction and infection characteristics of a GFP‐labelled Botryosphaeria dothidea causing kiwifruit soft rot disease

AbstractSoft rot disease caused by Botryosphaeria dothidea is one of the most important fungal diseases affecting kiwifruit production. The symptoms of the disease generally appear during the fruit storage period, resulting in difficult disease management and serious economic losses. Therefore, the characterization of the infection mechanism needs to be explored to deepen our understanding of the pathogen and the disease. In this study, a highly pathogenic strain JY‐1, causing kiwifruit soft rot, was isolated and identified as B. dothidea. A polyethylene glycol‐mediated genetic transformation system for B. dothidea JY‐1 was established, enabling the successful acquisition of a green fluorescent protein (GFP)‐labelled strain with stable inheritance. The GFP‐labelled transformant Bd13 was generated and used in the research, and the infection process of kiwifruit tissues was studied through wound inoculation essays and fluorescence labelling microscopy. The results of the present study indicate that the transformant Bd13 not only damaged the kiwifruit fruits but also effectively infected and colonized the leaves and shoots of kiwifruit plants, causing shoot blight and leaf spot. These findings contribute to a deeper understanding of the pathogenic mechanisms underlying kiwifruit soft rot and lay a foundation for further investigations into the interaction between the soft rot pathogen B. dothidea and kiwifruit.

Highly Efficient Agrobacterium tumefaciens Mediated Transformation of Oil Palm Using an EPSPS-Glyphosate Selection System.

Oil palm (Elaeis guineensis Jacq.) is the most efficient oil-producing crop globally, yet progress in its research has been hampered by the lack of effective genetic transformation systems. The EPSPS gene, encoding 5-enolpyruvylshikimate-3-phosphate synthase, has been used as a transgenic selection marker in various crops, including rice and soybean. This study evaluated the EPSPS/glyphosate selection system for oil palm transformation. We constructed a binary expression vector, pCGlyDESCLI-C, containing the TIPS-EiEPSPS selection marker from goosegrass and the mScarlet-I red fluorescent reporter gene. This vector was introduced into oil palm embryonic callus (EC) via Agrobacterium-mediated transformation. After optimizing the transformation steps, positive calli were obtained, and integration of the foreign gene into the oil palm genome was confirmed through molecular analysis. Notably, the selection efficiency of the EPSPS/glyphosate selection system exceeded that of the traditional hpt/hygromycin selection system, demonstrating its advantages. Our findings support the effectiveness of the TIPS-EiEPSPS/glyphosate selection system for oil palm genetic transformation, marking its first application in this species and offering a valuable tool for advancing research on this economically significant crop.

Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD600 = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar ’Xiangling.’ This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system’s effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.

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.

Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis

Background Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.ResultsHighly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.ConclusionsHere, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

Establishment of Agrobacterium-Mediated Transient Transformation System in Desert Legume Eremosparton songoricum (Litv.) Vass.

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD600 values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD600 of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant's reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes.

An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava.

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Progress on the mining of functional genes of Lonicera japonica.

Lonicera japonica Thunb. is a semi-evergreen climbing shrub belonging to the Caprifoliaceae family, whose dried flower buds or flowers on the verge of blooming are known as Jin Yin Hua in traditional Chinese medicine. This plant is not only a high-value and widely used medicinal material but also possesses characteristics that make it suitable for both medicinal and culinary purposes. Currently, there is a robust market demand for Jin Yin Hua, yet the breeding technology for new varieties of Lonicera japonica lags behind, necessitating the integration of modern breeding techniques. With the advancement of genomics in Lonicera japonica, an increasing number of functional genes have been identified, amassing a rich reservoir of genetic resources for molecular breeding of this species. In this review, we summarize the progress in Lonicera japonica genomics, functional gene mining, and the establishment of genetic transformation systems. In light of the existing challenges and deficiencies in the research of functional genes and quality breeding of Lonicera japonica, it is imperative to establish a germplasm resource bank, a mutant library, and an efficient genetic transformation system for this plant. Intensive research into the mining and identification of functional genes should be conducted, and molecular markers closely linked to the functional genes of Lonicera japonica should be developed. This will lay a foundational basis for the identification and cultivation of breakthrough varieties with superior qualities in Lonicera japonica.

Advancements in plant transformation: from traditional methods to cutting-edge techniques and emerging model species.

The ability to efficiently genetically modify plant species is crucial, driving the need for innovative technologies in plant biotechnology. Existing plant genetic transformation systems include Agrobacterium-mediated transformation, biolistics, protoplast-based methods, and nanoparticle techniques. Despite these diverse methods, many species exhibit resistance to transformation, limiting the applicability of most published methods to specific species or genotypes. Tissue culture remains a significant barrier for most species, although other barriers exist. These include the infection and regeneration stages in Agrobacterium, cell death and genomic instability in biolistics, the creation and regeneration of protoplasts for protoplast-based methods, and the difficulty of achieving stable transformation with nanoparticles. To develop species-independent transformation methods, it is essential to address these transformation bottlenecks. This review examines recent advancements in plant biotechnology, highlighting both new and existing techniques that have improved the success rates of plant transformations. Additionally, several newly emerged plant model systems that have benefited from these technological advancements are also discussed.

Establishment of genetic transformation system in Lilium pumilum and functional analysis of LpNAC6 on abiotic stress

Lilium pumilum is widely distributed in northeast Asia. It exhibits strong resistance and possesses high ornamental value. However, it currently lacks an efficient and stable transformation system. Therefore, we aimed to establish an effective genetic transformation system using the Agrobacterium-mediated method for L. pumilum, enabling gene transfer into the plant for gene function research and genetic engineering breeding. Our genetic transformation system achieved a transformation efficiency of 7.25 % under specific conditions: a kanamycin (Kana) concentration of 120 mg/L, 3 days of pre-cultivation, an A. tumefaciens concentration of 0.7 OD600, an acetosyringone (AS) concentration of 20 mg/L, and a 15-minute infection period. We investigated the function of the LpNAC6 from L. pumilum by observing phenotypic and physiological changes under stresses induced by salt, alkali, and drought. Furthermore, overexpression of LpNAC6 resulted in enhanced stress tolerance as evidenced by increased levels of SOD, POD, CAT enzymes, improved photosynthetic indices, and elevated chlorophyll contents; as well as reduced levels of MDA and reactive oxygen species (ROS). These findings demonstrate that we have successfully established a transgenic transformation method for L. pumilum while also providing essential information for cultivating stress-tolerant Lilium species and advancing our understanding of the functions of LpNAC6 in plants.

Regeneration and Genetic Transformation in Eucalyptus Species, Current Research and Future Perspectives

Eucalyptus is an important plantation tree with a high economic value in China. The tree contributes significantly to China’s timber production. The stable and efficient Eucalyptus regeneration system and genetic transformation system are of great significance for exploring the regulatory function and possible genetic breeding capacity of important genes in the species. However, as a woody plant, Eucalyptus has problems, such as a long generation cycle, strong specificity of the regeneration system, and a low genetic conversion rate, which seriously limit the rapid development of Eucalyptus genetics and breeding programs. The present review summarizes the status of research on Eucalyptus regeneration and genetic transformation, with a focus on the effects of explants, media, plant growth regulators (PGRs), and concentrations in the Eucalyptus regeneration process. In addition, the effects of genotype, Agrobacterium, antibiotics, preculture, and co-culture on the genetic transformation efficiency of Eucalyptus are discussed. Furthermore, the study also summarizes the problems encountered in Eucalyptus regeneration and genetic transformation, with reference to previous studies, and it outlines future developments and prospects. The aim was to provide a reference for solving the problems of genetic instability and the low transformation efficiency of eucalyptus, and to establish an efficient and stable eucalyptus regeneration and transformation system to accelerate the process of its genetic improvement.

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.

Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii.

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.

Establishment of clary sage (Salvia sclarea L.) regeneration system and development of glyphosate-tolerant clary sage plants through Agrobacterium-mediated genetic transformation

Clary sage (Salvia sclarea L.) is well known as a highly aromatic herbaceous plant and is commonly used in aromatic products and medicine. The biggest problem in the cultivation of clary sage is weeds, which not only entails a great cost of labor for weeding, but also leads to a great fluctuation in the yield and quality of its products. Therefore, there is a need to breed herbicide-resistant clary sage using genetic engineering. However, the lack of suitable regeneration and genetic transformation systems for clary sage has limited the process of molecular breeding. In this study, we firstly reported the regeneration system of clary sage, where explants from hypocotyls of sage induced adventitious shoots at a rate of 22.22% when cultured on MS medium supplemented with 2.5mg·L-1 6-BA, 0.15mg·L-1 IAA, and 0.25mg·L-1 VC. Rooting of adventitious shoots was achieved at a rate of 94.44% on 1/2 MS medium containing 0.125mg·L-1 NAA, 0.5mg·L-1 IAA, and 0.1mg·L-1 IBA. Secondly, based on the regeneration system, an efficient and stable Agrobacterium-mediated genetic transformation system was established in this paper, and the rate of genetic transformation was determined to be in the range of 3%-5% by transforming the hypocotyls of clary sage with pCAMBIA1301 vector and pCAMBIA1301-GR79-GAT vector. Finally, glyphosate tolerance was obtained from GR79 and GAT co-expressed clary sage plants in this study. This study not only lays the foundation of genetic transformation for the functional gene research and in-depth investigation of various biological problems of clary sage, but also provides a feasible genetic engineering solution for its industry to solve the weed problem, as well as a promotional role for precise molecular breeding of clary sage.

Establishment of transient and stable gene transformation systems in medicinal woody plant Acanthopanax senticosus

BackgroundTransient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plants in vitro.ResultsEmbryogenic callus protoplast isolation and transient transformation system were successfully established. The highest yield of protoplasts was approximately 1.88 × 106 cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% macerozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng μL−1, and 25 min heat shock at 45 °C. In addition, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions.ConclusionThis study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research on A. senticosus.Graphical

De novo assembly of Idesia polycarpa transcriptome and unsaturated fatty acid biosynthesis candidate genes Mining and functional Identification

Unsaturated fatty acids (UFA) in lipids are the key to nutraceutical oil applications, with various potential applications in nutraceutical functional foods and pharmaceutical industries. In Idesia polycarpa (Salicaceae), more than 80 % of UFA have been found in the fruits; yet, the underlying genetic mechanism remains poorly understood. Due to the lack of theoretical research on the genes related to lipid biosynthesis and the complete genetic transformation system of I. polycarpa fruit, the selection and breeding of I. polycarpa, an excellent oil tree, has been severely restricted. In-depth understanding of the molecular mechanism and gene function of lipid biosynthesis of I. polycarpa fruit is therefore of great significance for the development of I. polycarpa resources. This is not only conducive to the genetic improvement of I. polycarpa by molecular breeding technologies but can also provide a reference for the study of the gene functions of other oil plants. In this study, the FA accumulation patterns of I. polycarpa fruits during 8 growth periods were analysed. Fruit from two developmental periods with different UFA levels were analysed for RNA sequencing by an Illumina NovaSeq 6000 HiSeq platform. De novo transcriptome assembly presented 115,350 unigenes and 4382 differentially expressed genes (DEGs). Functional annotation in the KEGG pathway and combined with DEG data revealed candidate genes potentially involved in UFA biosynthesis. Expression analysis of q-PCR of IpDGAT2, IpGPAT, IpKASII, IpSAD, IpFAD2, IpFAD3 and IpFAD8 suggested that these genes are highly involved in UFA biosynthesis. Full-length candidate genes were cloned and analysed by bioinformatic tools, and function analysis of IpSAD and IpFAD3 showed that these genes regulated the products of linoleic acid metabolism. This study provides a foundation for UFA biosynthesis in Idesia polycarpa, facilitating its genetic breeding in the future.

Integration of induction, system optimization and genetic transformation in Veratrum californicum var. vitro cultures to enhance the production of cyclopamine and veratramine

Cyclopamine, a compound found in wild Veratrum has shown promising potential as a lead anti-cancer drug by effectively blocking cancer signaling pathways. However, its complex chemical structure poses challenges for artificial synthesis, thus limiting its supply and downstream drug production. This study comprehensively utilizes induction, system optimization, and transgenic technologies to establish an efficient suspension culture system for the high-yield production of cyclopamine and its precursor, veratramine. Experimental results demonstrate that methyl jasmonate (MeJA) effectively promotes the content of veratramine and cyclopamine in Veratrum californicum var. callus tissue, while yeast extract (YE) addition significantly increases cell biomass. The total content of veratramine and cyclopamine reached 0.0638 mg after synergistic treatment of suspension system with these two elicitors. And the content of the two substances was further increased to 0.0827 mg after the optimization by response surface methodology. Subsequently, a genetic transformation system for V. californicum callus was established and a crucial enzyme gene VnOSC1, involved in the steroidal alkaloid biosynthesis pathway, was screened and identified for genetic transformation. Combined suspension culture and synergistic induction system, the total content of the two substances in transgenic suspension system was further increased to 0.1228 mg, representing a 276.69% improvement compared to the initial culture system. This study proposes a complete and effective genetic transformation and cultivation scheme for V. californicum tissue cells, achieving milligram-level production of the anticancer agent cyclopamine and its direct precursor veratramine for the first time. It provides a theoretical basis for the industrial-scale production of these substances.

Insights into the bZIP gene family in Osmanthus fragrans and the role of OfbZIP98 in the callus and flower

Osmanthus fragrans has important ornamental and economic value. As the callus re-differentiation process is difficult, a genetic transformation system has not been successfully established in O. fragrans. The basic leucine zipper (bZIP) transcription factors play an important role in plant growth and development. A total of 116 OfbZIP genes were identified in O. fragrans using bioinformatics methods. According to the evolutionary relationships, these genes were divided into 12 subfamilies and were found to be unevenly distributed on 23 chromosomes of O. fragrans. The quantitative real-time PCR results showed that the expression trend of OfbZIP98 was consistent with the proliferation of O. fragrans callus. The subcellular localization and yeast self-activation results showed that OfbZIP98 was localized in the nucleus and had self-activation activity. Further, phenotypic observation of over-expressed tobacco showed that compared with the wild type (WT), the transgenic strain formed callus 4 d earlier, and callus re-differentiation began on day 15. Analysis of the callus differentiation rate showed that after 20 d of culture, the differentiation rate of the leaf callus of the transgenic strain was about twice that of the WT plants. In addition, measurements of corolla diameter, corolla circumference, corolla area, and corolla tube diameter indicated that the petals of the transgenic strain were significantly larger than the WT plants. Our results showed that OfbZIP98 has the important function of promoting callus growth, re-differentiation, and flower organ enlargement. These findings provide new insights into the potential role of OfbZIP98 in the growth and development of O. fragrans.