Biolistic Particle Delivery Research Articles

Optimization of Biolistic Transformation Parameters for <i>Nicotiana tabacum</i>

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species.

Routine and efficient in vitro regeneration system amenable to biolistic particle delivery in chickpea (Cicer arietinum L.)

Routine and efficient in vitro regeneration system amenable to biolistic particle delivery in chickpea (Cicer arietinum L.)

Biolistic Inoculation of Fruit Trees with Full-Length Infectious cDNA Clones of RNA Viruses.

Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectiousclones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system.

Enabling Transgenic Plant Cell–Derived Biomedicines with Nanotechnology

Transgenic plants are promising factories for manufacturing pharmaceutical small molecules or proteins safe for human consumption. Eukaryotic plant cells can synthesize proteins with precise posttranslational modifications, but microbes cannot. Conventional transformation methods (e.g., electroporation, Agrobacterium‐mediated transformation, and biolistic particle delivery) often suffer from inefficient transformation, damage to plant tissues and cells, or applicability to a narrow range of plant species. Notably, the cell wall poses a physical barrier that obstructs effective delivery of nucleic acids to plant cells. Nanoparticles (NPs) are emerging carriers of nucleic acids to plants because they are sufficiently small to diffuse through the cell wall, enter plant cells without the aid of external forces, and inflict limited damage to the plant cells in a broad variety of plants. Herein, three areas of the phytonanotechnology field that merit comprehensive investigations are outlined, namely, the design considerations of NPs for gene delivery to plant cells, homing of NPs to specific organelles (e.g., nucleus and chloroplast), and transformation of plant suspension cells. This perspective concludes with recent insights into scale‐up production of therapeutics by using bioreactors. NPs are poised to catalyze the transformation of plant cells for producing therapeutics in bioreactors at reduced costs, high purity, and improved scale.

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.

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

Background Capsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results In this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 μm of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant. Conclusions We have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants.

Plant genetics enters the nano age?

Plant transformation has for many years relied on agrobacterium infection or biolistic particle delivery. However, these two methods are limited to model plant systems or a small number of crop species. This commentary highlights recent developments in the nanoparticle-mediated transformation that have the potential to revolutionize how plants are transformed.

A new toolset for protein expression and subcellular localization studies in citrus and its application to citrus tristeza virus proteins

BackgroundTransient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium.ResultsHere we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of Xanthomonas citri effector AvrGF1. To further expand the available tools for subcellular localization studies in citrus, we also generated a new set of transgenic citrus plants that contain organelle markers labelling the nuclei, actin and endoplasmic reticulum. Using these new tools, we were able to show that the coat protein of citrus tristeza virus localizes to the cytoplasm and nuclei when expressed in epidermal cells fused to GFP.ConclusionWe have optimized a new method for transient expression in citrus leaves, to give highly reproducible and efficient transformation without producing a high level of injury or artifacts to the bombarded tissue. We also generated the first set organelle markers for use in citrus. These fluorescent protein markers label the nucleus and the actin. With these new resources, protein activity and subcellular localization can be studied in citrus rapidly and in high throughput. The handheld gene-gun device can also be used in the grove to deliver therapies for citrus diseases, such as canker and Huanglongbing, into trees.

Virus-Induced Gene Silencing in Maize with a Foxtail mosaic virus Vector.

Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation. This chapter provides a protocol for constructing FoMV VIGS clones and inoculating them into maize seedlings.

Therapeutic NKG2D Ligation Facilitates Improved Wound Healing

Abstract NKG2D is a C-type lectin-like receptor in the CD94/NKG2 family and is expressed by a variety of leukocyte populations including natural killer (NK) cells, γδ T cells, CD8+ T cells and certain innate lymphoid cell (ILC) subsets. NKG2D can bind proteins in two protein families – MIC and RAET1/ULBP. Expression of NKG2D ligands is low/absent under homeostatic conditions, but these proteins are upregulated in several contexts, including infection, cancer, physiologic stress, and senescence. NKG2D-driven activation of NK and CD8+ T cells has previously been shown to result in augmented control of infections and tumor growth. However, the role of NKG2D in the context of tissue injury and repair has not been established. Here we tested the effects of augmented NKG2D ligation in the context of a skin injury model. Biopsy punches were used to generate six 3- mm wounds on the dorsal surface of C57BL/6 mice. On days 0 and 1, NKG2D stimulating antibodies or isotype controls were injected into the peritoneal cavity and wound healing was assessed daily. Mice treated with NKG2D activating antibodies had more rapid wound repair than controls – wounds were healed by day 5 in NKG2D-treated mice as compared to day 7 in controls. Analogous results were observed in an ex vivo human skin model. Using a biolistic particle delivery system to induce upregulation of RAE (an NKG2D ligand), we observed more rapid wound repair compared to controls. Genetic ablation of NKG2D resulted in a 40% delay in wound healing. Moreover, depletion of NK1.1+ or CD8+ leukocytes blunted the benefit of NKG2D ligation, suggesting that T cells, NK cells, and/or ILCs may play a role in driving tissue repair. Thus, we demonstrate a potential therapeutic function of NKG2D engagement in tissue regeneration.

RNAi‐Mediated, Stable Resistance to Triticum mosaic virus in Wheat

Triticum mosaic virus (TriMV) was discovered in 2006 and affects wheat (Triticum spp.) production systems in the Great Plains of the United States. Genetic resistance is available in a few commercial varieties; however, it is not effective above 20°C. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA‐mini‐based hairpin expression vector was created from 272 bp of the TriMV coat protein (CP) sequence. Immature embryos of the wheat cultivar ‘Bobwhite’ were co‐transformed by biolistic particle delivery system with the pANDA::TriMVCP vector and pAHC20, containing the bar gene for glufosinate selection. T0 plants were analyzed by PCR for the presence of the RNAi TriMV CP fragment. Transgenic T1 lines were tested for transgene expression via reverse transcription PCR Virus resistance was evaluated by mechanical inoculation with TriMV and indirect enzyme‐linked immunosorbant assay (ELISA). Several lines were identified that were resistant, having little or no viral RNA. Resistant lines underwent single plant selection to the T6 generation. Real‐time PCR and ELISA showed that T6 lines had a high level of resistance when challenged with the virus. To test if the transgene would behave similarly in an adapted variety, homozygous transgenic T3 lines were crossed with the virus‐susceptible winter wheat, ‘Overley.’ Real‐time PCR results indicate decreased viral titer by as much as 20‐fold in the T6 transgenic lines, the F1 crosses, and the BC1F1 lines when compared with control plants, providing evidence that this RNAi‐silencing construct can provide stable resistance to TriMV and has great potential benefits to both breeders and producers.

Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens.

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.

A new LxxxA motif in the transmembrane Helix3 of maize aquaporins belonging to the plasma membrane intrinsic protein PIP2 group is required for their trafficking to the plasma membrane.

Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER.

Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l−1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.

Plastid transformation of tomato (Solanum lycopersicum L.) with HIV-1 p24 gene by biolistic particle delivery system

Plastid transformation in tomato (Solanum Lycopersicum L.) is carried out using vector carrying HIV-1 p24 gene and a selection of aadA gene, spectinomycin for resistance to antibiotics, and streptomycin. We used the hepta adaptor to replace the rupture disk retaining cap and microcarrier launch assembly. Three days after bombardment, the leaves and cotyledons were cut into small pieces each of 3 × 3 mm and placed on medium MS salts and B5 vitamins containing 2.0 mg/l Zeatin, 0.2 mg/l IAA, 50 mg/l spectinomycin for leaves, and medium MS salts and B5 vitamins containing 2.0 mg/l BAP, 1.0 mg/l IAA, 0.5mg/l Kinetin, 50 mg/l spectinomycin for cotyledons. Selected further time on the medium containing spectinomycin was up to 300 mg/l. PCR analysis of aadA and HIV-1 p24 genes of the tomato plastid transformation lines showed the presence of DNA amplification with the corresponding 250bp and 700bp. Southern blot analysis confirmed that the foreign genes were inserted into the tomato plastid genome. The result indicated the presence of HIV-1 p24 in the tomato plastid genome with the presence of DNA band of 5.8 kb. However, it is heteroplasmic transformant because it has a DNA band of 3.5 kb of wide type plant.

Ca2+ Induces Spontaneous Dephosphorylation of a Novel P5A-type ATPase

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.

Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method

AbstractThe synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au‐MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green fluorescent protein (eGFP) are investigated. As a proof‐of‐concept demonstration, Au‐MSN with large average pore diameters (10 nm) are shown to deliver and subsequently release proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non‐denatured proteins to plant cells. This advance represents the first example of biolistic‐mediated codelivery of proteins and plasmid DNA to plant cells via gold‐functionalized MSN and provides a powerful tool for both fundamental and applied research of plant sciences.

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii.

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.

Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel α1C Subunit in Chicken Cone Photoreceptors

MicroRNAs (miRNAs) modulate gene expression by degrading or inhibiting translation of messenger RNAs (mRNAs). Here, we demonstrated that chicken microRNA-26a (gga-mir-26a) is a key posttranscriptional regulator of photoreceptor L-type voltage-gated calcium channel alpha1C subunit (L-VGCCalpha1C) expression, and its own expression has a diurnal rhythm, thereby explaining the rhythmic nature of L-VGCCalpha1Cs. Circadian oscillators in retinal photoreceptors provide a mechanism that allows photoreceptors to anticipate daily illumination changes. In photoreceptors, L-VGCC activities are under circadian control, which are higher at night and lower during the day. Interestingly, the mRNA level of VGCCalpha1D oscillates, but those for VGCCalpha1C do not. However, the protein expression of both VGCCalpha1C and alpha1D are higher at night in cone photoreceptors. The underlying mechanism regulating L-VGCCalpha1C protein expression was not clear until now. In vitro targeting reporter assays verified that gga-mir-26a specifically targeted the L-VGCCalpha1C 3'-untranslated region, and gga-mir-26a expression in the retina peaked during the day. After transfection with gga-mir-26a, L-VGCCalpha1C protein expression and L-VGCC current density decreased. Therefore, the rhythmic expression of gga-mir-26a regulated the protein expression of the L-VGCCalpha1C subunit. Additionally, both CLOCK (circadian locomoter output cycles kaput) and CREB (cAMP-response element-binding protein-1) activated gga-mir-26a expression in vitro. This result implies that gga-mir-26a might be a downstream target of circadian oscillators. Our work has uncovered new functional roles for miRNAs in the regulation of circadian rhythms in cone photoreceptors. Circadian regulated miRNAs could serve as the link between the core oscillator and output signaling that further govern biological functions.

Simulations of transient shock motion within a biological contoured-shock-tube system

This study is motivated by the author’s interest in developing needle-free powdered vaccine/drug delivery systems. One system configuration is called the Contoured Shock Tube (CST). Of great importance is the behaviour of a transonic gas flow with a strongly nonlinear starting process, which accelerates powdered vaccines in micro-form to a sufficient momentum to penetrate the outer layer of human skin or mucosal tissue. In this paper, an established Modified Implicit Flux Vector Splitting (MIFVS) solver for the Navier–Stokes equations is extended to numerically study these transient transonic gas flows. A low Reynolds number k – ϵ turbulence model, with the compressibility effect considered, is integrated into the MIFVS solver to predict the turbulent structures and interactions with inherent shock systems. The MIFVS is first calibrated for NASA validation case, NPARC, and the resulting flow characteristic are compared with experimental date and simulations published. The MIFVS calculation with the modified k – ϵ model shows the best agreement. Subsequently, the MIFVS is applied to model the transient gas flow within a biolistic CST prototype. Comparison with experimental pressure traces shows the MIFVS captures gas flow mechanics with more accuracy than calculations with a commercial code (Fluent). This illustrates that the MIFVS is well-suited to model the strongly nonlinear fluid dynamics associated with the CST biolistic particle delivery system.