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.

Gene transfer to plants by electroporation: methods and applications.

Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. The methods used for gene transfer fall into two main categories; natural and artificial transformation. The natural methods include the conjugation, transposition, bacterial transformation as well as phage and retroviral transductions, contain the physical methods whereas the artificial methods can physically alter and transfer genes from one to another organisms' cell using, for instance, biolistic transformation, micro- and macroinjection, and protoplast fusion etc. The artificial gene transformation can also be conducted through chemical methods which include calcium phosphate-mediated, polyethylene glycol-mediated, DEAE-Dextran, and liposome-mediated transfers. Electrical methods are also artificial ways to transfer genes that can be done by electroporation and electrofusion. Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. Electroporation is an electrical transformation method by which transient electropores are produced in the cell membranes. Based on the applications, process can be either reversible where electropores in membrane are resealable and cells preserve the vitality or irreversible where membrane is not able to reseal, and cell eventually dies. This problem can be minimized by developing numerical models to iteratively optimize the field homogeneity considering the cell size, shape, number, and electrode positions supplemented by real-time measurements. In modern biotechnology, numerical methods have been used in electrotransformation, electroporation-based inactivation, electroextraction, and electroporative biomass drying. Moreover, current applications of electroporation also point to some other uncovered potentials for various exploitations in future.

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 6 months 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.

Proteolistics: A Protein Delivery Method.

Intracellular protein delivery in plant tissues is becoming an important tool for addressing both basic and applied research questions by plant biologists, especially in the era of genome editing. The ability to deliver proteins or protein/RNA complexes into cells allows for producing gene-edited plants that are free of transgene integration in the genome. Here we describe a protocol for the delivery of a protein/gold particle mixture in plant cells through biolistics. The key for the delivery is the drying of the protein/gold suspension directly onto the gene-gun cartridge or macrocarrier. The intracellular protein delivery into plant cells is achieved through the bombardment using the Bio-Rad PDS-1000/He particle delivery device. We termed this methodology "proteolistics."

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Doubled Haploid Transgenic Wheat Lines by Microspore Transformation.

Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35-40 days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods. The genetic transformation methods of biolistic gene-delivery and electroporation gave rise to the chimeric plants, whereas the method involving cocultivation with Agrobacterium yielded homozygous transformants. These methods were tested on a large number of cultivars belonging to different market classes of wheat, and found to be fairly independent of the explant genotype. Other benefits of using microspores or derived tissues for transformation are: (1) a few explant donors are required to obtain desired transformants and (2) the time required for obtaining homozygous transformants is about 8 months in case of spring wheat genotypes and about a year in case of winter wheat genotypes.

Proteolistics: a biolistic method for intracellular delivery of proteins.

In this work, an intracellular protein delivery methodology termed "proteolistics" is described. This method utilizes a biolistic gun apparatus and involves a simple protein/projectile preparation step. The protein to be delivered is mixed with a gold particle microprojectile suspension and is placed onto a gene gun cartridge, where it is dehydrated using either lyophilization or room-temperature air-drying. Subsequent intracellular protein delivery is achieved in plant and mammalian tissues upon bombardment. Because the method does not require modification of delivery agents or cargo biomolecules and involves a simple physical deposition of the protein onto the microprojectiles, there is no restriction on protein type in terms of molecular weight, isoelectric point or tertiary structure. Because the method delivers protein through the widely used gene gun system, it can be readily applied to any tissue or organism amenable to biolistics. A variety of proteins with molecular weight ranging from 24 to 68 kDa and isoelectric point from 4.8 to 10.1 were tested in this work. It is anticipated that this simple and versatile technique will offer biologists a powerful tool for basic research in areas such as understanding of cell and gene functions and for biotechnological applications such as genome editing.

Plastid transformation for Rubisco engineering and protocols for assessing expression.

The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long-term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master-line and analyzing leaf Rubisco content.

Plastid transformation in potato: Solanum tuberosum.

Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts.

Plastid transformation in sugar beet: Beta vulgaris.

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of utmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories; conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

Biolistic-mediated production of transgenic oil palm.

The effectiveness of mannose (using phosphomannose isomerase [pmi] gene) as a positive selection agent to preferably allow the growth of transformed oil palm embryogenic calli was successfully evaluated. Using the above selection agent in combination with the previously optimized physical and biological parameters and the best constitutive promoter, oil palm embryogenic calli were transformed with pmi gene for producing transgenic plants. Bombarded embryogenic calli were exposed to embryogenic calli medium containing 30:0 g/L mannose to sucrose 3 weeks postbombardment. Selectively, proliferating embryogenic calli started to emerge around 6 months on the above selection medium. The proliferated embryogenic calli were individually isolated once they reached a specific size and regenerated to produce complete plantlets. The complete regenerated plantlets were evaluated for the presence of transgenes by PCR and Southern analyses.

Transformation of oil palm using Agrobacterium tumefaciens.

Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.

Parameters affecting the efficient delivery of mesoporous silica nanoparticle materials and gold nanorods into plant tissues by the biolistic method.

Applying nanotechnology to plant science requires efficient systems for the delivery of nanoparticles (NPs) to plant cells and tissues. The presence of a cell wall in plant cells makes it challenging to extend the NP delivery methods available for animal research. In this work, research is presented which establishes an efficient NP delivery system for plant tissues using the biolistic method. It is shown that the biolistic delivery of mesoporous silica nanoparticle (MSN) materials can be improved by increasing the density of MSNs through gold plating. Additionally, a DNA-coating protocol is used based on calcium chloride and spermidine for MSN and gold nanorods to enhance the NP-mediated DNA delivery. Furthermore, the drastic improvement of NP delivery is demonstrated when the particles are combined with 0.6 µm gold particles during bombardment. The methodology described provides a system for the efficient delivery of NPs into plant cells using the biolistic method.

Bimolecular fluorescence complementation.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

Bimolecular fluorescence complementation (BiFC) assay for protein-protein interaction in onion cells using the helios gene gun.

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay(1) allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)(2) and the visualization of their interactions mediated by BiFC in onion epidermal cells.

A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

Biolistic mediated production of transgenic oil palm.

Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun.

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis.

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

FW2.2 and cell cycle control in developing tomato fruit: a possible example of gene co-option in the evolution of a novel organ.

fw2.2 is one of the few QTLs thus far isolated from plants and the first one known to control fruit size. While it has been established that FW2.2 is a regulator (either directly or indirectly) of cell division, FW2.2 does not share sequence homology to any protein of known function (Frary et al. Science 289:85-88, 2000; Cong et al. Proc Natl Acad Sci USA 99:13606-13611, 2002; Liu et al. Plant Physiol 132:292-299, 2003). Thus, the mechanism by which FW2.2 mediates cell division in developing fruit is currently unknown. In an effort to remedy this situation, a combination of yeast two-hybrid screens, in vitro binding assays and cell bombardment studies were performed. The results provide strong evidence that FW2.2 physically interacts at or near the plasma membrane with the regulatory (beta) subunit of a CKII kinase. CKII kinases are well-studied in both yeast and animals where they form part of cell cycle related signaling pathway. Thus while FW2.2 is a plant-specific protein and regulates cell division in a specialized plant organ (fruit), it appears to participate in a cell-cycle control signal transduction pathway that predates the divergence of single- and multi-cellular organisms. These results thus provide a glimpse into how ancient and conserved regulatory processes can be co-opted in the evolution of novel organs such as fruit.