Plant Transformation Technology Research Articles

Phoenix dactylifera in vitro culture and transformation of Thio-60 antifungal gene via chitosan nanoparticle

The date palm, or Phoenix dactylifera L., is one of the world’s oldest fruit trees. The entire date palm tree is used to produce a variety of goods, including food, clothing, fibre, and shelter. Tissue culture is one of the most recent methods used to multiply date palms and produce disease-free offspring. Plant tissue culture technology has advantages over conventional methods of propagation for the quick and extensive multiplication of significant plants in vitro, regardless of the season, and disease-free. This is because it preserves space and time. For this study, three different cultivars were used (Barhy, Sakkoti, and Shamia). For the three cultivars, ¾ MS medium supplemented with 1 mg/L indole butyric acid IBA and 0.25 mg/l activated charcoal (AC) provided the optimal in vitro culture conditions. Plant gene function research and cultivar development are now both possible thanks to the advancement of plant transformation technologies. Date palm plants were given the AT1G12660 “Thio-60” gene to make them resistant to fungus infection. Utilizing chitosan nanoparticles for genetic transformation, the gene was introduced into three cultivars of dates (Barhy, Sakkoti, and Shamia). Run a conventional PCR to verify genetic fusion into all three cultivars. The fungal infection with Fusarium oxysporum was used to determine the resistance of the transgenic cultivar lines after it was established that the thionin gene had been transferred into transgenic date palm cultivars. Date crop transgenic lines showed strong resistance and a decline in the percentage of fungal infection-induced inhibition.

Plant-made vaccines against viral diseases in humans and farm animals.

Plants provide not only food and feed, but also herbal medicines and various raw materials for industry. Moreover, plants can be green factories producing high value bioproducts such as biopharmaceuticals and vaccines. Advantages of plant-based production platforms include easy scale-up, cost effectiveness, and high safety as plants are not hosts for human and animal pathogens. Plant cells perform many post-translational modifications that are present in humans and animals and can be essential for biological activity of produced recombinant proteins. Stimulated by progress in plant transformation technologies, substantial efforts have been made in both the public and the private sectors to develop plant-based vaccine production platforms. Recent promising examples include plant-made vaccines against COVID-19 and Ebola. The COVIFENZ® COVID-19 vaccine produced in Nicotiana benthamiana has been approved in Canada, and several plant-made influenza vaccines have undergone clinical trials. In this review, we discuss the status of vaccine production in plants and the state of the art in downstream processing according to good manufacturing practice (GMP). We discuss different production approaches, including stable transgenic plants and transient expression technologies, and review selected applications in the area of human and veterinary vaccines. We also highlight specific challenges associated with viral vaccine production for different target organisms, including lower vertebrates (e.g., farmed fish), and discuss future perspectives for the field.

Construction of a fusion anti-caries DNA vaccine in transgenic tomato plants for PAcA gene and cholera toxin B subunit.

Chronic bacterial infections in the oral cavity influence the development of dental caries. Mutans streptococci are the major pathogenic cause of dental caries. The World Health Organization (WHO) ranks dental caries, cancer, and cardiovascular diseases as the three major global diseases that need urgent preventative and curative measures. However, substantial evidence suggests that traditional prevention and treatment strategies are inefficient in reducing the prevalence of dental caries. For protection against caries, it is important to develop effective vaccines that induce anticolonizing immunity against Streptococcus mutans infections. In the present investigation, we constructed a fusion anti-caries DNA vaccine (PAcA-ctxB) through fusing A region of cell surface protein PAc (PAcA) coding gene of mutans streptococci with cholera toxin B subunit coding gene (CTB). Afterward, the plasmids were integrated into tomato genomes through agrobacterium-mediated plant transformation technology. The presence of transgenes in the tomato genome was confirmed by PCR, β-glucuronidase gene (GUS), and western blot. The expression of genes was confirmed at transcription and protein level. Altogether, the results presented herein showed that transgenic tomatoes may provide a useful system for the production of human caries antigen.

Overview of the appropriability mechanisms used in plant biotechnology industry

This paper aims to discuss the main appropriability mechanisms used by biotech firms to avoid imitation and appropriate the economic benefits from crop innovations. To this end, we analyze the strategic links between the technological development of genetically modified organisms (GMO) and the ownership rights over plant transformation technologies. We also discuss a key case study - the legal battle between Monsanto and Syngenta by the control of the GMO crops derived from the GA21 gene. Our findings show that a system of blocking patents has been built basically through strategies relating to intellectual property mechanisms. As result, Monsanto's efforts to sue competitors for patent infringement have proved to be a successful barrier to entry in plant biotechnology industry.

FPX is a Novel Chemical Inducer that Promotes Callus Formation and Shoot Regeneration in Plants.

Auxin and cytokinin control callus formation from developed plant organs as well as shoot regeneration from callus. Dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation are considered to be caused by the reprogramming of callus cells, but this hypothesis is still argued to this day. Although an elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus formation. In this study, we identified fipexide (FPX) as a useful regulatory compound through a chemical biology-based screening. FPX was shown to act as a chemical inducer in callus formation, shoot regeneration and Agrobacterium infection. With regards to morphology, the cellular organization of FPX-induced calli differed from those produced under auxin/cytokinin conditions. Microarray analysis revealed that the expression of approximately 971 genes was up-regulated 2-fold after a 2 d FPX treatment compared with non-treated plants. Among these 971 genes, 598 genes were also induced by auxin/cytokinin, whereas 373 genes were specifically expressed upon FPX treatment only. FPX can promote callus formations in rice, poplar, soybean, tomato and cucumber, and thus can be considered a useful tool for revealing the mechanisms of plant development and for use in plant transformation technologies.

TADEA-PCR is a highly efficient method of amplifying unknown flanking fragments of T-DNA transformants.

Forward genetic analysis, widely used to find new gene functions, benefits from the availability of mutants. At present, based on Agrobacterium-mediated plant transformation technology, many transfer (T)-DNA transformants have been created. However, cloning their T-DNA insertion sites, which enables identification of the mutated genes, is still challenging. In this study, we improved adapter ligation-mediated polymerase chain reaction (A-PCR), which mainly utilizes the Thermal Asymmetric interlaced reaction and Degenerate sequence-recognizing restriction Endonucleases (TADE). Using the new method TADE-mediated A-PCR (TADEA-PCR), we successfully cloned 22 of all the 24 junction sites in 10 Arabidopsis thaliana L. transformants that contained 12 T-DNA insertions in total, giving a success rate of 91.7%. In most cases, the two junction sites resulting from a single T-DNA insertion were simultaneously cloned. In addition, TADEA-PCR was able to clone more than two junction sites present in one transformant containing several T-DNA insertions. Overall, TADEA-PCR is a powerful technique for cloning T-DNA insertion sites.

Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon.

Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells.

Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed 'Ensifer-mediated transformation' (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of crop species. In this protocol, we adapted a previously published account that describes the use of Arabidopsis thaliana roots to investigate the interaction of A. thaliana and Agrobacterium tumefaciens. In our laboratory, we routinely use A. thaliana root explants to examine the factors that enhance the utility of EMT. In addition, the E-ART protocol can be used to study the transcriptional response of E. adhaerens and host plant following exposure to explant tissue, the transformability of different Ensifer adhaerens strains/mutants as well as testing the susceptibility of A. thaliana mutant lines as a means to decipher the mechanisms underpinning EMT.

Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice.

Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g−1 in transgenic plants. The M. oryzae population was constant at 31, 48, and 72 h after inoculation in transgenic plants, while it was increased in the inoculated control plants. This study successfully clarified that over-expression of the Pikh gene in transgenic plants can improve their blast resistance against the M. oryzae pathotype P7.2.

Isolation of stress-inducible promoter Oshox24 from rice

Plant improvement based on plant transformation technology has become a popular trend all over the world. The isolation and characterization of a complete set of genes/promoters associated with abiotic stress and the cross-talk among salinity, drought or temperature stresses are required for success in generating abiotic stress-resistant varieties. Recently, several studies on model plants demonstrated that the stress-responsive genes under the control of rice promoter Oshox24 were expressed specifically under stress conditions, while not affecting plant growth under normal conditions. In this study, we isolated DNA segments carrying the Oshox24 promoter from an Indica rice variety by PCR using specific primers. The Oshox24 promoter was cloned into the pGEM-T vector and fully sequenced. The nucleotide sequence of the isolated promoter Oshox24 was 1612 bp in size and had similarity of 90% in comparison to the Japonica rice promoter Oshox24 (published in GenBank as ID AP004868.3). Results of sequence analysis showed that the Indica Oshox24 promoter contains 15 stress-responsive cis-acting elements including 5 important groups ABRE, MYBRS/MYCRS, DRE, NACRS and ZFHDRS, and a TATA box located at position 1482 to 1488, suggesting that Oshox24 promoter plays an important role on regulation of abiotic stress inducible genes. The Oshox24 promoter was inserted into the expression vector pCAMBIA1300 for generating abiotic stress resistant plants in the future.

Biotechnological strategies for studying actinorhizal symbiosis in Casuarinaceae: transgenesis and beyond

Since the recovery of the first transgenic plant in the early 1980s, plant transformation technologies have enabled advances in many aspects of basic plant science, including nitrogen-fixing root endosymbioses. Using the biological vectors Agrobacterium tumefaciens and A. rhizogenes, gene constructs have been successfully introduced in the actinorhizal tree species Casuarina glauca, thereby paving the way for functional analysis of the key genes involved in the symbiotic process with the actinobacterium Frankia. In recent years, not only studies of gene promoters in transgenic Casuarinaceae, but also the use of RNA interference to down-regulate genes of interest, have provided new insights into the early stages of the interaction between the root system and the actinobacterium. Opportunities offered by recent developments in genome editing technologies based on the engineered nucleases ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases) and RNA-guided CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein/Cas) will be briefly presented.

Novel Techniques for Gene Delivery into Plants and Its Applications for Disease Resistance in Crops

From the early past to the present, biotechnologies have produced the ability to genetically transform a wide variety of plant species. The plant transformation technologies have changed the face of agriculture and plant biology. Plant genetic transformation is one of the key technologies for crop improvement in addition to emerging approach for producing recombinant proteins in plants. Both plastid genomes and plant nuclear can be genetically modified. Until now, essential functional differences between the prokaryotic-like genome of the plastid and the eukaryotic genome of the plant cell nucleus will have an impact on characteristics of transgenic organism. Thus, the main goals are to generate transgenic plants with the traits of interest as well as minimizing the amount of transgenic DNA in plants while maximizing stability of gene expression and trait performance. In this review, two broad groups of gene delivery methods will be discussed namely, (bilogical and physical methods) and subsequently there applications for improving disease resistance will be discussed.

Recent Progress of Genetically-Modification in Floriculture

Plant transformation technology (or genetic modification), along with breeding techniques such as cross-breeding and mutation breeding, has been widely used for breeding new varieties and enhancing the characteristics of floricultural plants. In particular, genetically modified (GM) floriculture has been developed for modified characteristics such as floral color, pigmentation, stress tolerance, disease resistance, and fragrance. In South Korea, studies on GM floriculture have been under progress since the 2000s, and there have been various research projects involving GM floriculture. In the global flower market, a mauve-colored carnation called “MoondustTM” was first developed and commercialized by the Australian biotechnology company Florigene, and a blue rose called “APPLAUSETM” was developed and commercialized by the Japanese company SUNTORY. Furthermore, new varieties of GM floriculture have recently been developed, and international trade in ornamental plant materials has grown. Given the rapid increase of studies on GM floriculture and the growing global market for GM ornamental plants, biotechnology may be used to increase farm household income by developing new ornamental varieties that have superior characteristics and match consumer preferences. In t his review, we summarize t he r esearch on G M ornamental plants during the last four years. This information will help in establishing more successful strategies for developing GM floriculture. Moreover, this report will facilitate the breeding of new and improved varieties with modified growth rates and superior product quality, including changes in flower color, fragrance, and stress tolerance.

THE NECESSITY OF TRANSGENIC TECHNOLOGY IN SUSTAINABLE PRODUCTION

It has been more than half a century that plant geneticists and breeders have been trying to assemble a combinationof genes in crop plants, in order to make them as suitable and productive as possible. Plant transformation technology incrop plants was first undertakenin the 1980s based on the ability of foreign gene integration into host plant genome andregeneration of transformed plant cells into whole plants. Soon after, transgenic plants were to be grown by farmers.Statistics show that farmers have started to cultivate genetically modified plants (GMPs) commercially since 1996.Between 1996 and 2012, the total surface area of land cultivated with GM crops has increased from 2 million hectaresto more than 170 million hectares in 29 countries. To this extent, some concerns have been raised by ecologists andconsumer organizations in West European countries based on the possibilities of horizontal and vertical gene flow ofantibiotic or herbicide resistance from transgenic plants into human intestinal bacteria and some weeds via outcrossing,respectively. Due to consumer and ecologist concerns, different approaches have been developed to eliminate marker(and/or reporter) genes from the nuclear or chloroplast genome after selection. Some of these proposed methods are:1. Replacing selectable markers with screenable ones.2. Elimination of marker genes by co-transformartion followed by classic recombination and selection.3. Excision of marker gene by some site-specific recombinases.4. Separation of the transgene and selectable marker by transposable elements.5. Avoiding gene pollution by chloroplasts genetic engineering followed by elimination of selectable marker.

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes.

BackgroundRecently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies.ResultsThe 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome.ConclusionsThis study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).

Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides.

BackgroundCowpea (Vigna unguiculata L.) is an important grain and forage legume grown throughout sub-Saharan Africa primarily by subsistence farmers on poor, drought prone soils. Genetic improvement of the crop is being actively pursued and numerous functional genomics studies are underway aimed at characterizing gene controlling key agronomic characteristics for disease and pest resistances. Unfortunately, similar to other legumes, efficient plant transformation technology is a rate-limiting step in analysis of gene function in cowpea.ResultsHere we describe an optimized protocol for the rapid generation of transformed hairy roots on ex vitro composite plants of cowpea using Agrobacterium rhizogenes. We further demonstrate the applicability of cowpea composite plants to study gene expression involved in the resistance response of the plant roots to attack by the root parasitic weed, Striga gesnerioides. The utility of the new system and critical parameters of the method are described and discussed herein.ConclusionsCowpea composite plants offer a rapid alternative to methods requiring stable transformation and whole plant regeneration for studying gene expression in resistance or susceptibility responses to parasitic weeds. Their use can likely be readily adapted to look at the effects of both ectopic gene overexpression as well as gene knockdown of root associated defense responses and to the study of a broader range of root associated physiological and aphysiological processes including root growth and differentiation as well as interactions with other root pests, parasites, and symbionts.

Genetic modification; the development of transgenic ornamental plant varieties

Plant transformation technology (hereafter abbreviated to GM, or genetic modification) has been used to develop many varieties of crop plants, but only a few varieties of ornamental plants. This disparity in the rate and extent of commercialisation, which has been noted for more than a decade, is not because there are no useful traits that can be engineered into ornamentals, is not due to market potential and is not due to a lack of research and development activity. The GM ornamental varieties which have been released commercially have been accepted in the marketplace. In this article, progress in the development of transgenic ornamentals is reviewed and traits useful to both consumers and producers are identified. In considering possible factors limiting the release of genetically modified ornamental products it is concluded that the most significant barrier to market is the difficulty of managing, and the high cost of obtaining, regulatory approval.

Trafficking and Subcellular Localization of Multiwalled Carbon Nanotubes in Plant Cells

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.

Recent Patents on Plant Transgenic Technology

Plant transformation technology plays an ever-increasing role in improving crop plant traits and addressing research questions. In recent years, a number of important patents on plant transgenic technologies have been published.These patents represent significant new progresses, many representing novel approaches in the field of plant transgenic technology. This review highlights selected as well as the most significant advancements made mostly within the past 2 years in plant transgenic research. These advancements include the improvements of plant transformation systems, development of new plant selectable markers, engineering for plant stress tolerance, production of vaccines in transgenic plants, etc. Of these, the engineering for plant stress tolerance accounts for a high percentage of the transgenic field of inventions. Some results are drastic and unexpected, having a great potential in their applications towards a wider range of plant species. This review also tries to avoid terms and languages pertaining to the patent and legal documents to ease readings.

RETRACTED: The myth of plant transformation

Technology development is innovative to many aspects of basic and applied plant transgenic science. Plant genetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs. Development of procedures in cell biology to regenerate plants from single cells or organized tissue, and the discovery of novel techniques to transfer genes to plant cells provided the prerequisite for the practical use of genetic engineering in crop modification and improvement. Plant transformation technology has become an adaptable platform for cultivar improvement as well as for studying gene function in plants. This success represents the climax of years of efforts in tissue culture improvement, in transformation techniques and in genetic engineering. Plant transformation vectors and methodologies have been improved to increase the efficiency of transformation and to achieve stable expression of transgenes in plants. This review provides a comprehensive discussion of important issues related to plant transformation as well as advances made in transformation techniques during three decades.