Biolistic Transformation of Wheat.

Biolistic transformation of wheat is one of the most commonly used methods for gene function study and trait discovery. It has been widely adapted as a fundamental platform to generate wheat plants with new traits and has become a powerful tool for facilitating the crop improvement. In this chapter, we present a complete and straightforward protocol for wheat transformation via biolistic bombardment system. Although wheat is still one of the hardest plant species to transform, this protocol offers an optimized and efficient system to produce transgenic plants. To demonstrate the application of this protocol, in this chapter we describe an example of obtaining transgenic wheat by the co-bombardment of two plasmids, containing a green fluorescent protein gene and a glufosinate herbicide selection gene, respectively. In addition, procedures for the screening and testing of putative transgenic plants are described. This protocol has been successfully applied to generate stable transgenic bread wheat (Triticum aestivum) in both spring and winter varieties.

Poplar Transformation.

First publications of successful Agrobacterium-mediated transformation of tobacco were published more than 30 years ago. Protocols for Agrobacterium-based transformation as well as biolistic bombardment and PEG transformation of protoplasts are available for more than 150 plant species from various plant families. Also for many Populus species and hybrids, adapted transformation protocols have been published. The standard protocol for Agrobacterium-mediated transformation of different Populus genotypes is the leaf-disc method. Here, we first describe the transfer of genes into poplar by using the Agrobacterium-based leaf disc methods. In addition, alternative basic transformation methods, namely, biolistic bombardment and PEG transformation of protoplasts, are also described. Further, we present improved poplar transformation protocols by simplifying the transformation procedure and optimizing tissue preparation and plant regeneration.

Biolistic Transformation of Cotton Zygotic Embryo Meristem.

Biolistic transformation of cotton (Gossypium hirsutum L.) meristems, isolated from mature seed, is detailed in this report. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He ) was used to bombard gold particles coated with a marker gene (uidA or "GUS") into the shoot meristem. The penetration of gold particles was dependent on bombardment parameters, and it was mostly one- to two-cell layers deep. Stable transformation of epidermal L1 layer was consistently observed in approximately 5% of the seedlings. Germ line transformation was observed in up to 0.71% of bombarded meristems by several laboratories. Using this method identification of germ line transformation is laborious and time-consuming. However, the protocol described here represents a simple and efficient method for generating germ line transformation events. In addition, this procedure offers a quick method to evaluate gene constructs in cotton tissues (embryos, cotyledons, leaf) especially fibers which originate as single cells from the maternal epidermis layer.

Biolistic Transformation of Cotton Embryogenic Cell Suspension Cultures.

Genetic transformation of cotton (Gossypium hirsutum L.) is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependent. However, once embryogenic cell cultures are available, they can be effectively used for transformation by Agrobacterium or biolistic bombardment methods. Here I describe a detailed procedure to transform cotton embryogenic cell suspension cultures by biolistic bombardment. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with plasmid DNA (for visual identification of transformed cells and/or selection) into embryogenic cells. Stable transformation at a high frequency (up to 4% of the transiently expressing cells) is possible. Regeneration of fertile transgenic plants from embryogenic cells takes only about 2 months. Another advantage of the embryogenic cell suspension cultures is that they are amenable for cryopreservation and long-term storage. It is highly preferable to transform commercial varieties of choice than obsolete varieties to avoid genetic drug due to backcrossing.

Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum.

Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.

Nanoparticle-Mediated Delivery towards Advancing Plant Genetic Engineering.

Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Nanoparticle-Mediated Recombinase Delivery into Maize.

We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants. Using the experimental procedures and conditions described here, as high as 20% of bombarded embryos can produce regenerable recombinant callus events. This nanomaterial-mediated, DNA-free methodology has potential to become an effective tool for plant genome editing.

Genetic Transformation of Date Palm Via Microprojectile Bombardment.

Efficient protocols for date palm embryogenic callus and somatic embryo transformation with uidA gene are described in this chapter. The embryogenic callus transformation procedure is 1.6 µm gold particle size coated with 2.5 µg DNA (pAct1-D plasmid), 1100 psi helium pressure, 9 cm target distance, 26 inHg vacuum pressure, 3 mm distance between the rupture disk and macrocarrier, and osmotic pretreatment with 0.4 M mannitol followed by 60 min air desiccation. The somatic embryo transformation procedure is 0.6 µm gold particle size coated with 2.5 µg DNA (pAct1-D plasmid), 1350 psi helium pressure, 6 cm target distance, 28 inHg vacuum pressure, 3 mm distance between the rupture disk and macrocarrier, and osmotic pretreatment with 0.4 M mannitol followed by 60 min air desiccation. Protocols for analysis of the transgenic plantlets have also been described.

Microprojectile Bombardment Transformation of Date Palm Using the Insecticidal Cholesterol Oxidase (ChoA) Gene.

The overall objective of this work is to optimize the transformation system for date palm as a first step toward production of date palm clones resistant to noxious pests. A construct harboring the cholesterol oxidase (ChoA) gene, which renders plant resistance against insect attack, is introduced into embryogenic date palm callus using the PDS-1000/He particle bombardment system. The process involves the establishment of embryogenic callus cultures as well as immature embryo-derived microcalli that are used as target tissues for shooting and optimization of transformation conditions. This chapter in addition explains molecular and histochemical assays conducted to confirm gene integration and expression.

Labeling of neuronal morphology using custom diolistic techniques.

BACKGROUND: Diolistic labeling is increasingly utilized in neuroscience as an efficient, reproducible method for visualization of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery allows for non-toxic fluorescent labeling of multiple neurons in both living and fixed tissue. Since first described, this labeling method has been modified to fit a variety of research goals and laboratory settings. NEW METHOD: Diolistic labeling has traditionally relied on commercially available devices for the propulsion of coated micro-particles into tissue sections. Recently, laboratory built biolistic devices have been developed which allow for increased availability and customization. Here, we discuss a custom biolistic device and provide a detailed protocol for its use. RESULTS: Using custom diolistic labeling we have characterized alterations in neuronal morphology of the lateral/dentate nucleus of the rat cerebellum. Comparisons were made in developing rat pups exposed to abnormally high levels of 5-methyloxytryptamine (5-MT) pre-and postnatally. Using quantitative software; dendritic morphology, architecture, and synaptic connections, were analyzed. COMPARISON WITH EXISTING METHOD(S): The rapid nature of custom diolistics coupled with passive diffusion of dyes and compatibility with confocal microscopy, provides an unparalleled opportunity to examine features of neuronal cells at high spatial resolution in a three-dimensional tissue environment. CONCLUSIONS: While decreasing the associated costs, the laboratory-built device also overcomes many of the obstacles associated with traditional morphological labeling, to allow for reliable and reproducible neuronal labeling. The versatility of this method allows for its adaptation to a variety of laboratory settings and neuroscience related research goals.

Particle Bombardment of Ex Vivo Skin to Deliver DNA and Express Proteins.

Particle bombardment of gold microparticles coated with plasmids, which are accelerated to high velocity, is used for transfection of cells within tissue. Using this method, cDNA encoding proteins of interest introduced into ex vivo living human skin enables studying of proteins of interest in real time. Here, technical aspects of particle bombardment of ex vivo skin are described using green fluorescent protein (GFP) as readout for efficiency. This method can be applied on numerous tissues, including in living model animals.

Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, ß-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

Using a Handheld Gene Gun for Genetic Transformation of Tetrahymena thermophila.

This chapter describes protocols for using a handheld gene gun to deliver transformation vectors for overexpression of genes or gene replacement in the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

The tracking of intercellular small RNA movement.

RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. In plants, one of the most fascinating aspects of RNA silencing is its mobile nature, in other words its ability to spread from the cell where it has been initiated to neighboring cells, through movement of sRNA molecules. To study this process, a key step is to directly monitor the spread of these nucleic acid species. Here we describe how this can be achieved through biolistic delivery of fluorescently labeled siRNA.

Microneedle-assisted microparticle delivery by gene guns: experiments and modeling on the effects of particle characteristics.

Microneedles (MNs) have been shown to enhance the penetration depths of microparticles delivered by gene gun. This study aims to investigate the penetration of model microparticle materials, namely, tungsten (<1 µm diameter) and stainless steel (18 and 30 µm diameters) into a skin mimicking agarose gel to determine the effects of particle characteristics (mainly particle size). A number of experiments have been processed to analyze the passage percentage and the penetration depth of these microparticles in relation to the operating pressures and MN lengths. A comparison between the stainless steel and tungsten microparticles has been discussed, e.g. passage percentage, penetration depth. The passage percentage of tungsten microparticles is found to be less than the stainless steel. It is worth mentioning that the tungsten microparticles present unfavourable results which show that they cannot penetrate into the skin mimicking agarose gel without the help of MN due to insufficient momentum due to the smaller particle size. This condition does not occur for stainless steel microparticles. In order to further understand the penetration of the microparticles, a mathematical model has been built based on the experimental set up. The penetration depth of the microparticles is analyzed in relation to the size, operating pressure and MN length for conditions that cannot be obtained in the experiments. In addition, the penetration depth difference between stainless steel and tungsten microparticles is studied using the developed model to further understand the effect of an increased particle density and size on the penetration depth.

A cost-effective method for preparing, maintaining, and transfecting neurons in organotypic slices.

The cellular and molecular mechanisms that underlie brain function are challenging to study in the living brain. The development of organotypic slices has provided a welcomed addition to our arsenal of experimental brain preparations by allowing both genetic and prolonged pharmacological manipulations in a system that, much like the acute slice preparation, retains several core features of the cellular and network architecture found in situ. Neurons in organotypic slices can survive in culture for several weeks, can be molecularly manipulated by transfection procedures and their function can be interrogated by traditional cellular electrophysiological or imaging techniques. Here, we describe a cost-effective protocol for the preparation and maintenance of organotypic slices and also describe a protocol for biolistic transfection that can be used to introduce plasmids in a small subset of neurons living in an otherwise molecularly unperturbed network. The implementation of these techniques offers a flexible experimental paradigm that can be used to study a multitude of neuronal mechanisms.

Transient gene expression in tobacco using Gibson assembly and the Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work(11), and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

Biolistic delivery of voltage-sensitive dyes for fast recording of membrane potential changes in individual neurons in rat brain slices.

Optical recording of membrane potential changes with fast voltage-sensitive dyes (VSDs) in neurons is one of the very few available methods for studying the generation and propagation of electrical signals to the distant compartments of excitable cells. The more lipophilic is the VSD, the better signal-to-noise ratio of the optical signal can be achieved. At present there are no effective ways to deliver water-insoluble dyes into the membranes of live cells. Here, we report a possibility to stain individual live neurons with highly lipophilic VSDs in acute brain slices using biolistic delivery. We tested four ANEP-based VSDs with different lipophilic properties and showed their ability to stain single neurons in a slice area of up to 150 µm in diameter after being delivered by a biolistic apparatus. In the slices of neocortex and hippocampus, the two most lipophilic dyes, di-8-ANEPPS and di-12-ANEPPQ, showed cell-specific loading and Golgi-like staining patterns with minimal background fluorescence. Simultaneous patch-clamp and optical recording of biolistically stained neurons demonstrated a good match of optical and electrical signals both for spontaneous APs (action potentials) and stimulus-evoked events. Our results demonstrate the high efficiency of a fast and targeted method of biolistic delivery of lipophilic VSDs for optical signals recording from mammalian neurons in vitro.

Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines.