Agrobacterium-mediated Gene Transformation Research Articles

Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation.

Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method's robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology. Key features • Uses liquid nitrogen as a proxy for freezing. • Plasmid DNA from competent bacterial cells is extracted using a user-friendly high-copy isolation kit. • A maximum of five consecutive days is sufficient to complete the procedures.

A callus-derived regeneration and Agrobacterium-mediated gene transformation developed for bilberry, Vaccinium myrtillus

A callus-derived regeneration and Agrobacterium-mediated gene transformation developed for bilberry, Vaccinium myrtillus

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Visible gland constantly traces virus-induced gene silencing in cotton.

A virus-induced gene silencing (VIGS) system was established to induce endogenous target gene silencing by post-transcriptional gene silencing (PTGS), which is a powerful tool for gene function analysis in plants. Compared with stable transgenic plant via Agrobacterium-mediated gene transformation, phenotypes after gene knockdown can be obtained rapidly, effectively, and high-throughput through VIGS system. This approach has been successfully applied to explore unknown gene functions involved in plant growth and development, physiological metabolism, and biotic and abiotic stresses in various plants. In this system, GhCLA1 was used as a general control, however, silencing of this gene leads to leaf albino, wilting, and plant death ultimately. As such, it cannot indicate the efficiency of target gene silencing throughout the whole plant growth period. To address this question, in this study, we developed a novel marker gene, Gossypium PIGMENT GLAND FORMATION GENE (GoPGF), as the control to trace the efficiency of gene silencing in the infected tissues. GoPGF has been proved a key gene in gland forming. Suppression of GoPGF does not affect the normal growth and development of cotton. The number of gland altered related to the expression level of GoPGF gene. So it is a good marker that be used to trace the whole growth stages of plant. Moreover, we further developed a method of friction inoculation to enhance and extend the efficiency of VIGS, which facilitates the analysis of gene function in both the vegetative stage and reproductive stage. This improved VIGS technology will be a powerful tool for the rapid functional identification of unknown genes in genomes.

A Review: Agrobacterium-mediated Gene Transformation to Increase Plant Productivity

In genetics and molecular biology, Gene transformation is a gene alteration technique that involves the introduction and expression of a foreign gene into the host organism. There are many gene transformation methods like particle bombardment, electroporation, micro-injection, PEG for different biotechnological experiments. But Plant gene transformation is a widely used procedure for obtaining transgenic plants and plant models to understand gene functions. Agrobacterium tumefaciens is a natural genetic engineer which is rod-shaped, gram-negative soil-born bacteria. Initially, Agrobacterium was utilized to transform only dicot plants but over the year’s modification in plant transformation protocol it was now utilized in monocot plants as well as in fruits plants too. Agrobacterium tumefaciens inserts its DNA (Transfer DNA-T-DNA) into the host plant. The transmitted DNA is randomly integrated into the host cell's genetic material inside the infected plant cell nucleus. Alternatively, bacterial DNA can transiently remain in the nucleus without integrating into the genome, but it still replicates alongside the plant genome, using its machinery and expressing its genes to make separate gene products. Besides the traditional method, new research has also been done to transform the plants through agrobacterium. Various methods have been developed to transform monocotyledonous plants such as wheat, maize, rice, and fruity plants. Generally, dicotyledonous plants can be transformed by the traditional method of agrobacterium but various methods have also been developed for dicots for various applications. Here, we have taken an example of a tobacco plant (nicotiana tabacum) transformed with different methods.

Modeling Agrobacterium-Mediated Gene Transformation of Tobacco (Nicotiana tabacum)-A Model Plant for Gene Transformation Studies.

The multilayer perceptron (MLP) topology of an artificial neural network (ANN) was applied to create two predictor models in Agrobacterium-mediated gene transformation of tobacco. Agrobacterium-mediated transformation parameters, including Agrobacterium strain, Agrobacterium cell density, acetosyringone concentration, and inoculation duration, were assigned as inputs for ANN–MLP, and their effects on the percentage of putative and PCR-verified transgenic plants were investigated. The best ANN models for predicting the percentage of putative and PCR-verified transgenic plants were selected based on basic network quality statistics. Ex-post error calculations of the relative approximation error (RAE), the mean absolute error (MAE), the root mean square error (RMS), and the mean absolute percentage error (MAPE) demonstrated the prediction quality of the developed models when compared to stepwise multiple regression. Moreover, significant correlations between the ANN-predicted and the actual values of the percentage of putative transgenes (R2 = 0.956) and the percentage of PCR-verified transgenic plants (R2 = 0.671) indicate the superiority of the established ANN models over the classical stepwise multiple regression in predicting the percentage of putative (R2 = 0.313) and PCR-verified (R2= 0.213) transgenic plants. The best combination of the multiple inputs analyzed in this investigation, to achieve maximum actual and predicted transgenic plants, was at OD600 = 0.8 for the LB4404 strain of Agrobacterium × 300 μmol/L acetosyringone × 20 min immersion time. According to the sensitivity analysis of ANN models, the Agrobacterium strain was the most important influential parameter in Agrobacterium-mediated transformation of tobacco. The prediction efficiency of the developed model was confirmed by the data series of Agrobacterium-mediated transformation of an important medicinal plant with low transformation efficiency. The results of this study are pivotal to model and predict the transformation of other important Agrobacterium-recalcitrant plant genotypes and to increase the transformation efficiency by identifying critical parameters. This approach can substantially reduce the time and cost required to optimize multi-factorial Agrobacterium-mediated transformation strategies.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis.

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.

Forecasting and optimizing Agrobacterium-mediated genetic transformation via ensemble model- fruit fly optimization algorithm: A data mining approach using chrysanthemum databases.

Optimizing the gene transformation factors can be considered as the first and foremost step in successful genetic engineering and genome editing studies. However, it is usually difficult to achieve an optimized gene transformation protocol due to the cost and time-consuming as well as the complexity of this process. Therefore, it is necessary to use a novel computational approach such as machine learning models for analyzing gene transformation data. In the current study, three individual machine learning models including Multi-Layer Perceptron (MLP), Adaptive Neuro-Fuzzy Inference System (ANFIS), and Radial Basis Function (RBF) were developed for forecasting Agrobacterium-mediated gene transformation in chrysanthemum based on eleven input variables including Agrobacterium strain, optical density (OD), co-culture period (CCP), and different antibiotics including kanamycin (K), vancomycin (VA), cefotaxime (CF), hygromycin (H), carbenicillin (CA), geneticin (G), ticarcillin (TI), and paromomycin (P). Consequently, best-obtained results were used in the fusion process by bagging method. Results showed that ensemble model with the highest R2 (0.83) had superb performance in comparison with all other individual models (MLP:063, RBF:0.69, and ANFIS: 0.74) in the validation set. Also, ensemble model was linked to Fruit fly optimization algorithm (FOA) for optimizing gene transformation, and the results showed that the maximum gene transformation efficiency (37.54%) can be achieved from EHA105 strain with 0.9 OD600, for 3.8 days CCP, 46.43 mg/l P, 9.54 mg/l K, 18.62 mg/l H, and 4.79 mg/l G as selection antibiotics and 109.74 μg/ml VA, 287.63 μg/ml CF, 334.07 μg/ml CA and 87.36 μg/ml TI as antibiotics in the selection medium. Moreover, sensitivity analysis demonstrated that input variables have a different degree of importance in gene transformation system in the order of Agrobacterium strain > CCP > K > CF > VA > P > OD > CA > H > TI > G. Generally, the developed hybrid model in this study (ensemble model-FOA) can be employed as an accurate and reliable approach in future genetic engineering and genome editing studies.

Cloning and characterisation of an endosperm specific glutelin B1 promoter from Sri Lankan rice (Oryza sativa L. ssp. indica) variety Bg 250

Glutelins are the primary source of energy storage in the endosperm of rice grains. Among the glutelin promoters, Glutelin B-1 (GluB-1) is widely studied and used in transgenic rice plants to express recombinant proteins in the endosperm. In this study, three regions: 350 bp, 1308 bp and 2300 bp of the GluB-1 promoter were PCR amplified from the genomic DNA of Bg 250 rice variety. The amplified fragments were cloned into pGEM®-T Easy vector for characterisation of GluB-1 promoter. Each region of GluB-1 promoter was separately cloned into the promoterless binary vector pCAMBIA1391Z harbouring the β-glucuronidase (GUS) reporter gene. Putative transgenic plants were generated by Agrobacterium-mediated gene transformation and confirmed by PCR using nopaline synthase terminator primers. All GluB-1 promoter constructs showed expression of the GUS gene in the endosperm of T0 transgenic plant seeds. The 1308 bp GluB-1 promoter revealed the highest expression as determined by the GUS assay. This indicates the potential of this promoter for expression of recombinant proteins in rice endosperm.

Agrobacterium-mediated genetic transformation of ajowan (Trachyspermum ammi (L.) Sprague): an important industrial medicinal plant

Biotechnology-based breeding methods can improve useful industrial medicinal plants in a faster way. Tissue culture-based and in planta Agrobacterium-mediated gene transformation methods were applied to enhance drought and salinity tolerance in ajowan medicinal plant using betaine aldehyde dehydrogenase (BADH) gene. The pBI121 binary vector harboring kanamycin resistance gene (nptII) and BADH gene in its T-DNA region was applied to transfer BADH gene. Different concentrations of the selective antibiotic were tested on 15 day-old hypocotyl segments and 75 mg/L kanamycin was identified as the minimum inhibitory concentration to screen for transformed cells of ajowan. The different parameters of gene transformation including Agrobacterium optical density (OD), Agrobacterium strain, Agrobacterium killing antibiotic, acetosyringone concentration, and inoculation duration were tested during tissue culture-based Agrobacterium-mediated transformation. The OD600 = 0.6-0.8 of LB4404 strain of Agrobacterium × 160 mg/L timentin × 250 μmol/L acetosyringone × 30 min inoculation duration were identified as the best gene transformation parameters. Successful integration and expression of transferred gene were confirmed in T0 plants using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) methods. Five transgenic lines with 2.94% transformation efficiency were achieved by regular tissue culture-based Agrobacterium-mediated gene transformation method, whereas the dip flora in planta transformation method has led to one transgenic line with 1.42% transformation efficiency. Bioassay analysis revealed enhanced drought and salinity tolerance in transformed plants in comparison with non-transformed plants. The morphological characteristics of plant height and seedling fresh weight of BADH-transformed plants were improved under salinity stress. Under drought stress, the proline content and relative water content of transgenic plants were more than non-transformants. The results of gas chromatography mass spectrometry (GC–MS) revealed significant increase in thymol content of BADH-transformed plants (55.07%) compared with non-transformed plants (39.20%) under drought stress condition. The obtained transgenic plants are cultivable in unfavorable environments to give an acceptable level of useful secondary metabolites. The optimized protocol is applicable for gene transformation of other medicinal plants of Apiacea family for different purposes.

Application of genetics and biotechnology for improving medicinal plants.

Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.

Tissue culture-based Agrobacterium-mediated and in planta transformation methods

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much quicker than regular tissue culture-based Agrobacterium-mediated gene transformation and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular tissue culture-based Agrobacterium-mediated transformation methods and then recently successful transformations using these methods are presented.

Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation.

Metabolic engineering of Rhodosporidium toruloides, a robust lipid and caroteinoid producer, is of great importance for oleochemicals and carotenoids production. However, the Agrobacterium-mediated gene transformation is tedious and time consuming. Here, we described a fast and efficient genetic transformation of R. toruloides using electroporation with linear DNA fragments, and the process was optimized. The results showed that 2 × 103 transformants can be obtained at 0.7 kV/μg linear DNA by using hygromycin and bleomycin as selection markers after the competent cells pretreated with 25 mM DTT and 100 mM LiAc. Our results would facilitate mutant library construction and metabolic engineering of R. toruloides for production of oleochemicals and carotenoids. We further demonstrated that all transformants arose due to illegitimate integration of transforming DNA fragments by colony PCR.

Identification and Characterization of Multiple Intermediate Alleles of the Key Genes Regulating Brassinosteroid Biosynthesis Pathways

Most of the early identified brassinosteroid signaling and biosynthetic mutants are null mutants, exhibiting extremely dwarfed phenotypes and male sterility. These null mutants are usually unable to be directly transformed via a routinely used Agrobacterium-mediated gene transformation system and therefore are less useful for genetic characterization of the brassinosteroid (BR)-related pathways. Identification of intermediate signaling mutants such as bri1–5 and bri1–9 has contributed drastically to the elucidation of BR signaling pathway using both genetic and biochemical approaches. However, intermediate mutants of key genes regulating BR biosynthesis have seldom been reported. Here we report identification of several intermediate BR biosynthesis mutants mainly resulted from leaky transcriptions due to the insertions of T-DNAs in the introns. These mutants are semi-dwarfed and fertile and capable to be transformed. These intermediate mutants could be useful tools for future discovery and analyses of novel components regulating BR biosynthesis and catabolism via genetic modifier screen.

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. Protocorm-like bodies (PLBs) were cocultivated with the Agrobacterium tumefaciens strain AGL1 harboring the plant expression vector pGWB5-AcF3H, which contained the hygromycin phosphotransferase (hpt) gene as a selectable marker. The highest transformation efficiency (10.13%) was achieved when PLBs were cocultivated with Agrobacterium cells for 15 min. Three months after the transformation, the plantlets were regenerated, and the transgenic plants were confirmed by polymerase chain reaction (PCR) analysis using specific primers for the hpt gene and 35S promoter region. PCR products of approximately 400 and 500 bp, corresponding to the hpt gene and the 35S promoter, respectively, were detected in the transgenic plants, while no such product was observed in nontransgenic plants, indicating that the AcF3H gene was integrated into the genome of Dendrobium 5N white orchid plants. The transient expression of the AcF3H gene in Dendrobium 5N white and Dendrobium Anna petals was performed using the agroinfiltration technique, and the results demonstrated that no cyanidin content was detected in the Dendrobium 5N white petals after AcF3H infiltration. In contrast, the cyanidin content was increased by approximately 6% in the Dendrobium Anna petals, suggesting that the AcF3H gene was transiently expressed in this orchid.

Plant-Based Vaccines: Production and Challenges

Plant-based vaccine technologies involve the integration of the desired genes encoding the antigen protein for specific disease into the genome of plant tissues by various methods. Agrobacterium-mediated gene transfer and transformation via genetically modified plant virus are the common methods that have been used to produce effective vaccines. Nevertheless, with the advancement of science and technology, new approaches have been developed to increase the efficiency of former methods such as biolistic, electroporation, agroinfiltration, sonication, and polyethylene glycol treatment. Even though plant-based vaccines provide many benefits to the vaccine industry, there are still challenges that limit the rate of successful production of these third-generation vaccines. Even with all the limitations, continuous efforts are still ongoing in order to produce efficient vaccine for many human and animals related diseases owing to its great potentials. This paper reviews the existing conventional methods as well as the development efforts by researchers in order to improve the production of plant-based vaccines. Several challenges encountered during and after the production process were also discussed.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton.

Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.