Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly.

Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization.

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines.

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET­gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA.

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.

Biolistic Transformation of Chlamydomonas reinhardtii and Saccharomyces cerevisiae Mitochondria.

Chlamydomonas reinhardtii and Saccharomyces cerevisiae are currently the two micro-organisms in which genetic transformation of mitochondria is routinely performed. The generation of a large variety of defined alterations as well as the insertion of ectopic genes in the mitochondrial genome (mtDNA) are possible, especially in yeast. Biolistic transformation of mitochondria is achieved through the bombardment of microprojectiles coated with DNA, which can be incorporated into mtDNA thanks to the highly efficient homologous recombination machinery present in S. cerevisiae and C. reinhardtii organelles. Despite a low frequency of transformation, the isolation of transformants in yeast is relatively quick and easy, since several natural or artificial selectable markers are available, while the selection in C. reinhardtii remains long and awaits new markers. Here, we describe the materials and techniques used to perform biolistic transformation, in order to mutagenize endogenous mitochondrial genes or insert novel markers into mtDNA. Although alternative strategies to edit mtDNA are being set up, so far, insertion of ectopic genes relies on the biolistic transformation techniques.

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics.

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 µm gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 °C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 °C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.

The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.

Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells.

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5-7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

Biolistic Plastid Transformation in Lettuce (Lactuca sativa) for Oral Delivery of Biopharmaceuticals.

The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide and ideal platform for the production of biopharmaceuticals.

Resistance Marker- and Gene Gun-Mediated Transformation of Trichoderma reesei.

Transformation enables the transfer of DNA into fungal cells for subsequent integration into the genome. Due to its versatility in industrial application, transformation is of utmost importance in Trichoderma reesei and hence continuously optimized. As one of the most crucial obstacles in fungal transformation efforts, removal of the cell wall is required to efficiently target genome modification cassettes to the genome. Here we describe resistance marker-mediated gene gun (biolistic) transformation of fungal spores of T. reesei as an alternative to protoplast transformation.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

Particle bombardment technology and its applications in plants.

Particle bombardment, or biolistics, has emerged as an excellent alternative approach for plant genetic transformation which circumvents the limitations of Agrobacterium-mediated genetic transformation. The method has no biological constraints and can transform a wide range of plant species. Besides, it has been the most efficient way to achieve organelle transformation (for both chloroplasts and mitochondria) so far. Along with the recent advances in genome editing technologies, conventional gene delivery tools are now being repurposed to deliver targeted gene editing reagents into the plants. One of the key advantages is that the particle bombardment allows DNA-free gene editing of the genome. It enables the direct delivery of proteins, RNAs, and RNPs into plants. Owing to the versatility and wide-range applicability of the particle bombardment, it will likely remain one of the major genetic transformation methods in the future. This article provides an overview of the current status of particle bombardment technology and its applications in the field of plant research and biotechnology.

Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide.

The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.

Biolistic Approach for Transient Gene Expression Studies in Plants.

Since its inception in the late 1980s, the delivery of exogenous nucleic acids into living cells via high-velocity microprojectiles (biolistic, or microparticle bombardment) has been an invaluable tool for both agricultural and fundamental plant research. Here, we review the technical aspects and the major applications of the biolistic method for studies involving transient gene expression in plant cells. These studies cover multiple areas of plant research, including gene expression, protein subcellular localization and cell-to-cell movement, plant virology, silencing, and the more recently developed targeted genome editing via transient expression of customized endonucleases.

Biolistic Transformation of Japonica Rice Varieties.

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2 months after the initiation of seed germination for transformation.

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 promising approach 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.

Biolistic DNA Delivery and Its Applications in Sorghum bicolor.

Biolistic DNA delivery has been considered a universal tool for genetic manipulation to transfer exotic genes to cells or tissues due to its simplicity, versatility, and high efficiency. It has been a preferred method for investigating plant gene function in most monocot crops. The first transgenic sorghum plants were successfully regenerated through biolistic DNA delivery in 1993, with a relatively low transformation efficiency of 0.3%. Since then, tremendous progress has been made in recent years where the highest transformation efficiency was reported at 46.6%. Overall, the successful biolistic DNA delivery system is credited to three fundamental cornerstones: robust tissue culture system, effective gene expression in sorghum, and optimal parameters of DNA delivery. In this chapter, the history, application, and current development of biolistic DNA delivery in sorghum are reviewed, and the prospect of sorghum genetic engineering is discussed.