Biolistic Research Articles

Simple and efficient genetic transformation of sorghum using immature inflorescences

Sorghum is an important cereal crop and has been grown in a wide range of environments from tropic to warm-temperate zones. Genetic improvement of this crop has been based on those traditional breeding methods, but there is a limited application of genetic transformation for sorghum improvement due to its recalcitrance to genetic manipulation in vitro. The current study aimed at development of immature inflorescences as a genetic transformation system for sorghum. Immature inflorescences of two sorghum elite lines were chosen as the explants for developing a new method for genetic transformation. Sorghum immature inflorescences were collected from plants at the flowering stage and co-cultured with virulent Agrobacterium tumefaciens strain LBA4404 containing one of the special gene constructs. Once co-cultivation was completed, the treated immature inflorescences were moved onto regeneration medium to induce development of shoot and root from the resulted embryogenic callus. This system was also amenable for the microprojectile bombardment (biolistic particle delivery system) method. Putatively transgenic plants were confirmed for the presence of transgene in the plants based on the molecular analysis of the T1 plants. All resulted transgenic plants were normal in morphology and fully fertile. The transformation system developed with the immature inflorescence is a simple and efficient method for sorghum transformation, which can speed up the process of engineering new varieties.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Use of Sorghum bicolor leaf whorl explants to expedite regeneration and increase transformation throughput

Sorghum is considered a challenging species for transformation due to the limited choice in genotypes that can be transformed and the reliance on callus derived from immature embryos that need to be teased out of developing seeds. We have developed a more practical tissue culture procedure to generate embryogenic callus from leaf whorl explants obtained from 4 week-old seedlings. This procedure offers greater ease of obtaining explants, enables a more rapid turnover of growth chamber or greenhouse plants that can be used for explant collection, and eliminates the dependency on obtaining flowering plants with adequate numbers of seeds. Callus derived from these somatic embryos was amenable to biolistic transformation with DNA-coated gold particles and transformants were shown to express the Cas9 transgene. A comparison between genotypes RTx430 and P898012 indicated that the former was more suitable to tissue culture from leaf whorls based on greater callus induction and regeneration. Callus from both genotypes secreted colored compounds into the medium that we determined to be phenolic in nature, likely 3-deoxyanthocyanidins, based on UV–Vis absorbance profiles and thin layer chromatography. In order to reduce negative impacts of these compounds on regeneration, the addition of activated charcoal (AC) or polyvinylpyrrolidone (PVP) to the culture medium was examined. The addition of 0.5 g L−1 of AC after 4 weeks on callus induction medium accelerated callus regeneration, whereas culturing leaf whorl explants on medium containing 1 g L−1 PVP reduced the production of colored compounds and enhanced callus growth. The use of leaf whorl explants from developing sorghum plants serves as a practical alternative to immature embryos as a source of embryogenic callus and doesn’t require plants to flower.

Choice of Explant for Plant Genetic Transformation.

Particle bombardment or biolistic transformation is an efficient, versatile method. This method does not need any vector for the gene transfer and is not dependent on the cell type, species, and genotype. The success of any transformation technique depends on the starting experimental materials or the explants. Here, we describe the factors that have influenced the choice of explants in biolistic transformation. Many general factors in the selection of explants in the development of transgenic plants are presented here. Therefore, this chapter provides extensive guidelines regarding the choice of explants for researchers working on various plant genetic transformation techniques.

Biolistic DNA Delivery in Maize Immature Embryos.

One of the key factors for ensuring a successful genetic transformation is to effectively introduce genetic materials, such as plasmid DNA, into plant cells. A biolistic gun is one of the two best established and most popular tools for delivery of DNA into maize cells. It is the method that generated the first fertile transgenic maize plant. In this chapter, we describe steps involved in introducing single or paired plasmid DNAs into immature embryos of maize Hi II hybrid genotype, using Biolistic® PDS-1000/He particle delivery system. While we focus on the biolistic delivery process in the protocol presented here, we also provide step-by-step information required for successful regeneration of transgenic maize plants.

Biolistics-Mediated Gene Delivery in Sugarcane.

Biolistic transformation is one of two popular methods for introducing genes into sugarcane. However, unlike Agrobacterium-mediated transformation, the efficiency of gene transfer into sugarcane cells, using the biolistic method is very high. In addition to this, the biolistic transformation method is independent of the explant genotype or tissue. It also has the advantage that a minimum DNA sequence of linearized plasmid can be used, thus eliminating the introduction of undesirable plasmid derived genes, delivering low-copy transgenic events. In this chapter, we describe the method for efficient delivery of genes into sugarcane cells using a biolistic approach.

Biolistic DNA Delivery in Turfgrass Embryonic Callus Initiated from Mature Seeds.

We describe a protocol for the establishment and preparation of creeping bentgrass (Agrostis stolonifera L.) cultivar "Penn A-4" embryonic calli, biolistic transformation, selection, and regeneration of transgenic plants. The embryonic callus is initiated from mature seeds, maintained by visual selection under the dissecting microscope and subjected to bombardment with plasmid DNA containing a bialaphos-resistance (bar) gene. PCR, Southern, and Northern blot analyses are used to confirm the transgene integration and expression.

Agro-Infiltration and Microprojectile Bombardment Efficiency of Barley Stripe Mosaic Virus as a Virus-Induced Gene Silencing Vector in Brachypodium distachyon

Barley stripe mosaic virus (BSMV) works as a vector in the monocot plants, to deliver a small segment of gene for post‐transcriptional modification through the Virus-induced gene silencing (VIGS) mechanism. VIGS is derived from the plant defense mechanism as a tool to understand the gene function and the expected phenotype in a short time. Moreover, VIGS mechanism provides a large-scale analysis of screening phenotype with a low cost relatively compared to the stable transformation in the monocot plants. This study aimed to examine the microprojectile bombardment by biolistic particle bombardment device (gene gun) compared to the agro-Infiltration method with Agrobacterium tumefaciens to deliver the BSMV with genes to Brachypodium distachyon. The green fluorescent protein (GFP) and Phytoene desaturase gene (BdPDS) worked as a visual marker to confirm the conversion of pCass4-RZ-ND18 plasmid to the intact BSMV. The findings showed that the GFP signals appeared during the agro-Infiltration approach while the BSMV during the microprojectile bombardment approach failed to express the foreign GFP gene. Furthermore, the knockdown of BdPDS gene phenotype appears as a photo-belched strips on the B. distachyon leaves after inoculation of the BSMV with agro-Infiltration pathway. Conversely, B. distachyon leaves which injected through the microprojectile bombardment approach has no significant changes in the phenotype compared to the WT. This phenotype was confirmed by the qRT-PCR results which showed a highly significant decrease in the PDS expression level during the agro-Infiltration approach only. Eventually, the agro-Infiltration approach is much effective than the microprojectile bombardment approach although the former approach needs much time and many confirmation steps.

Nanobiolistics: An Emerging Genetic Transformation Approach.

Biolistic delivery of biomolecular cargoes to plants with micron-scale projectiles is a well-established technique in plant biotechnology. However, the relatively large micron-scale biolistic projectiles can result in tissue damage, low regeneration efficiency, and create difficulties for the biolistic transformation of isomorphic small cells or subcellular target organelles (i.e., mitochondria and plastids). As an alternative to micron-sized carriers, nanomaterials provide a promisingapproach for biomolecule delivery to plants. While most studies exploring nanoscale biolistic carriers have been carried out in animal cells and tissues, which lack a cell wall, we can nonetheless extrapolate their utility for nanobiolistic delivery of biomolecules in plant targets. Specifically, nanobiolistics has shown promising results for use in animal systems, in which nanoscale projectiles yield lower levels of cell and tissue damage while maintaining similar transformation efficiencies as their micron-scale counterparts. In this chapter, we specifically discuss biolistic delivery of nanoparticles for plant genetic transformation purposes and identify the figures of merit requiring optimization for broad-scale implementation of nanobiolistics in plant genetic transformations.

Use of Microspore-Derived Calli as Explants for Biolistic Transformation of Common Wheat.

There are specific advantages of using microspores as explants: (1) A small number of explant donors are required to obtain the desired number of pollen embryoids for genetic transformation and (2) microspores constitute a synchronous mass of haploid cells, which are transformable by various means and convertible to doubled haploids therefore allow production of homozygous genotypes in a single generation. Additionally, it has been demonstrated in wheat and other crops that microspores can be easily induced to produce embryoids and biolistic approach to produce a large number of transformants. In view of these listed advantages, we optimized the use of microspore-derived calli for biolistic transformation of wheat. The procedure takes about 6months to obtain the viable transformants in the spring wheat background. In the present communication, we demonstrated the use of this method to produce the reduced immunogenicity wheat genotypes.

Optimization of core phases of biolistic transformation of potato

We have established the optimal exposure time for internodes and callus plant material of Aksor potato variety on the OSS osmotic medium before and after biolistics and the type of plant explant that is most suitable for that. The optimal objects were explants from internodes of test plants. The best time for exposure of plant material to the osmotic medium before bombardment is 24 hours, and after biolistics – 48 hours. Callus tissue developed on the MS medium with zeatin two weeks later in internodes that were cultivated before bombarding on the OSS for 24 hours and we observed an increase in the callus mass by the end of fourth week. Not a single callus was obtained with rhizogenesis on the R4 medium from internodes and potato calli that were on the osmotic medium within 24 and 72 hours after biolistics. The regenerative capacity of internodes after cultivation on an osmotic medium for 48 hours after bombarding was higher than that of calli. We obtained 12 regenerant plants out of 200 internode explants and 4 regenerant plants out of 150 calli that were used in the experiment.

Genetic Transformation Methods for Crop Improvement- A Brief Review

Decades of documented successful pieces of evidence in agritech have shown us a clear picture of the importance of biotechnology in crop improvement. The production in agriculture should have steady growth and to achieve these objective conventional methods should go on parallel with biotechnological approaches. Genetic engineering which has revolutionized the path of crop improvement involves the identification and transfer of novel genes into the existing elite cultivars. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect method of gene transfer has its own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy, elite and eco-friendly method to transfer genes. The transformation method which is a base of genetic engineering is vital and Agrobacterium-mediated gene transfer and gene gun have shown to be doing well in recent years. As a whole the methodology involved, merits and demerits of different methods have been briefly discussed.

Gene transfer to German chamomile (L chamomilla M) using cationic carbon nanotubes

The use of nanoparticles to transfer genes to plant cells can solve some problems encountered in other gene transfer methods, including limited host range in the use of Agrobacterium, cell wall removal in the use of polyethylene glycol and electroporation, and high cell damage while using a gene gun. In this research, cationic carbon nanotubes (CNTs) were used to transfer ssDNA-FITC to German chamomile cells. The ability of nanoparticles to interact with and protect of DNA against enzymes and ultrasound damages was investigated using 0.8 % agarose gel. To investigate the morphology of CNTs loaded with DNA (Nanotube- Polyethyleneimine/DNA nanoparticles), scanning electron microscopy (SEM) was used. The biocompatibility effect of CNTs (Nanotube- Polyethyleneimine) on German chamomile cells was also determined by trypan blue staining. Agarose gel images showed that CNTs have a high ability to interact with DNA and can effectively protect it from damage by ultrasound and digestive enzymes. In addition, the SEM images of CNTs/DNA nanoparticles showed that these nanoparticles were rod-shaped with lengths around 100–200 nm. The fluorescence microscope results from German chamomile cells treated with CTNs/ssDNA-FITC nanoparticles showed the ability of these nanoparticles to transfer ssDNA-FITC to German chamomile cells. The results also revealed that the simultaneous use of ultrasound and CNTs significantly increased the transfer efficiency of ssDNA-FITC.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

N-terminal sequences affect expression of triterpene biosynthesis enzymes in Chlamydomonas chloroplasts

Metabolic engineering is an emerging technology to modify the biochemical properties of living cells. In microalgae, metabolic engineering has often been directed towards optimizing the production of desirable lipids or related bioproducts. Here we describe efforts to engineer the green alga Chlamydomonas reinhardtii for the production of botryococcene, a drop-in biofuel precursor. Genes encoding farnesyl diphosphate synthase (FPS) and squalene synthase-like (SSL)-1 and -3, were introduced into the chloroplast genome using biolistic transformation. Through a series of construct modifications, we identified intergenic sequences that promote expression of stable, discrete transcripts. We also found amino acids that dramatically increased the accumulation of SSL-3 when they were inserted at the N-terminal penultimate position, and similar manipulation of the N-terminal sequence of FPS appeared to improve its protein level as well. However, SSL-1 only accumulated to detectable levels when expressed as a chimera with SSL-3. In vitro assays showed that chloroplast-expressed SSL-3 was enzymatically active, but not SSL-1, although the SSL-1-SSL-3 chimeras were active when expressed in yeast. Taken together, our results suggest that the N-terminal sequence and other cellular factors are important when heterologous proteins are expressed in this model algal species.

Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection.

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aβ)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene.

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.

Biolistic co-transformation of rice using gold nanoparticles

Biolistic co-transformation of rice using gold nanoparticles

Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus

Miscanthus species, including M. sacchariflorus, are important bioenergy crops, used also as industry feedstocks and in environmental projects. M. sacchariflorus is also utilised as a donor in interspecific crossings to generate new forms of M. × giganteus. Research on Miscanthus species and their application potential may be considerably extended using methods of genetic modification. However, to date there are scare reports on miscanthus transformation. To improve transformation efficiency, new expression cassettes of a selectable marker gene were constructed and used for biolistic transformation of M. sacchariflorus. The sequence of the bacterial hpt gene encoding hygromycin phosphotransferase in the initial pCAMBIA vector was optimised according to codon usage characteristic of Zea mays, which is closely related to Miscanthus sp., and placed under control of 35S CaMV or the Zea mays ubiquitin promoter, resulting in the pCA35Sohpt and pCAUBQohpt vectors. Transformation rate using optimised vectors significantly increased in comparison to pCAMBIA, and reached 4.2–5.0% vs 2.5%. In respective transgenic plants, significantly higher transcription of optimised hpt was determined while the activity of the reporter GUS enzyme was similar. These results constitute basis for specific miscanthus transformation in a variety of research and practical applications.