Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles

Summary We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.Abbreviations BMS Black Mexican Sweet - RPM revolutions per minute - uidA -glucuronidase gene - GUS -glucuronidase protein - LB Luria-Bertani broth - OD600 optical density at 600 nm - psi pounds per square inch - Ap^r ampicillin resistance - Kn^r kanamycinresistance     

A new approach for the biolistic method: Bombardment of living nitrogen-fixing bacteria into plant tissues

Summary A new utilization of the biolistic gun was developed for the direct introduction of nitrogen-fixing bacteria (Azotobacter vinelandii) into strawberry (Fragaria x ananassa) tissues. This was the first case of using living bacteria as microprojectiles for the bombardment of plant tissues. Bacterial cells, adhered to tungsten particles, were accelerated by a nitrogen-powered device, and delivered into the target leaves and regenerating shoot meristems. The presence of bacteria in the developing strawberry callus tissues and regenerating plants was detected by microscopy, acetylene reduction assay, and selective polymerase chain reaction. Practically, the elaborated method proved to be suitable for the establishment of artificial intereellular, associations between nitrogen-fixing bacteria and higher plants.     

Optimization of biolistic transformation of embryogenic grape cell suspensions

Embryogenic suspensions of Chancellor (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 M kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 m (M10) resulted in more transient gene expression than 0.771 m diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.Abbreviations AC activated charcoal - GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - IAA-L-alanine indole-3 acetic acid L-alanine - MS Murashige and Skoog - CH casein hydrolysate - Km kanamycin - NPTII neomycin phosphotransferase II     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants

Summary Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed β-glucuronidase (GUS). Cells of wildtype Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed.     

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biolistic transformation of a procaryote, Bacillus megaterium

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.     

Biolistic transformation of a procaryote, Bacillus megaterium.

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.     

A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol

 An Aspergillus nidulans strain, auxotrophic for pyrimidine, was transformed to prototrophy by means of microprojectile bombardment. The transformation frequency was somewhat lower than conventional polyethyleneglycol-mediated transformation of protoplasts. However, the percentage of stable transformants was considerably higher with the biolistic approach. Typically, integrations of several copies of the plasmid introduced into chromosomal DNA were observed. The effect of several parameters, like the concentration of conidia, chamber pressure during bombardment and size of microprojectiles, on transformation frequencies were investigated and compared to previously published data on microprojectile bombardment of fungal conidia. Optimum results (6 transformants/μg plasmid DNA) were obtained when 10⁸ conidia were bombarded with a helium pressure of 5.5–8.3 MPa (800–1200 lb/in²). M5, M10 and M17 tungsten particles were equally efficient. Received: 9 August 1995/Received revision: 27 September 1995/Accepted: 4 October 1995     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA

Summary Stable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.Abbreviations YAC yeast artificial chromosome - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-3-acetic acid - GUS ß-glucuronidase - nptII neomycin phosphotransferase II     

Biolistic plant transformation

The biolistic process represents a completely new approach to the problem of how to deliver DNA into intact cells and tissues. High velocity microprojectiles are used to carry DNA or other substances past cell walls and membranes. Because DNA is being 'shot' into cells, it represents a type of biological ballistics, hence the term "biolistics".
There are several fundamental advantages to the biolistic process over other plant transformation techniques. The biolistic process appears to be effective regardless of species or tissue type, it is a rapid and very simple procedure, and it should facilitate the direct transformation of totipotent tissues such as pollen, embryos, meristems and morphogenic cell cultures. In addition, the biolistic process appears to be uniquely suitable for organelle transformation.
The disadvantages of the biolistic process are that it requires special instrumentation, and is still in the early stages of its development. Consequently, delivery efficiencies are still not as high as can be achieved in highly optimized transformation systems such as electroporation or agrobacterial-infection of tobacco. Furthermore, potential users should be prepared to spend some time adapting existing protocols to their specific species or tissue of interest.     

Mesoporous TiO2 nanoparticles: A new material for biolistic bombardment

To date, only solid heavy metals such as gold or tungsten have been used as DNA carriers in biolistic bombardment of algae. In this study, we show that even a metal oxide of lower density can act as a DNA carrier. We investigated the potency of size‐controlled mesoporous titanium dioxide (TiO₂) particles. Among the six tested gas pressures, TiO₂ particles best facilitated transformation of the green alga Chlamydomonas reinhardtii at 1100 psi (approximately 7.6 MPa) and 2000 psi (approximately 14 MPa). Surprisingly, a mesoporous metal oxide with a density of approximately only one‐tenth that of gold or tungsten could be effective as a DNA carrier in biolistic bombardment of a rigid cell wall‐containing alga. In addition, we found two peaks of gas pressures in the transformation ratio irrespective of whether the particles were made of gold, tungsten, or TiO₂.     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (≤ 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (≤ 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane

The comparison of direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic plants with DSE (12?weeks from explant to plants in soil). Microprojectiles of 0.3???m diameter produced 5 times more transgenic lines than 1???m microprojectiles when used at the same weight basis per shot. A significant reduction of the number of hybridization signals in the Southern blot was also observed with 0.3???m microprojectiles when compared to 1.0???m microprojectiles. This suggests that the lower DNA carrying capacity and greater number of the smaller microprojectiles contributes to more transgenic lines with less complex transgene integration. When geneticin sulfate, was used for selection following DSE, significantly more (4.8 times) transgenic plants were produced than with paromomycin sulfate and an equal number of non-expressing plants were produced with both selection agents. In conclusion, optimization of two alternative morphogenic routes for regeneration (DSE and ISE), biolistic and selection parameters generated rooted, transgenic plants of a commercially important sugarcane cultivar with simple transgene integration and within 12 or 19?weeks of culture initiation, respectively. Reducing the complexity of the transgene integration and minimizing the time in tissue culture will likely enhance the performance of the transgenic events.     

Major improvements in biolistic transformation of suspension-cultured tobacco cells

Summary Suspension cultures of the NT1 line ofNicotiana tabacum L. were used as a model system to study plant biolistic transformation, because of their uniformity, rapid growth, and ease of handling. The β-glucuronidase gene and the neomycin phosphotransferase genes were used to assay transient and stable transformation. Numerous factors were studied and optimized, such that the frequency of transformation was increased roughly 60-fold for transient transformants and 20-fold for stable transformants. Both biological parameters (the promoter used to drive gene expression, osmotic preconditioning and posbombardment handling of the cells) and physical parameters of the bombardment process (particle acceleration device and accelerator parameters) were tested. The factors that increased transformation rates the most were promoter strength, use of a helium-driven particle accelerator, and osmotic preconditioning of the cells.     

Development of an airgun device for particle bombardment

Construction and operation of an airgun device for transient gene-expression studies in monocots is described. Compressed air in a cylinder of an airgun was used as the source of propulsion for DNA-coated gold or tungsten particles. Under a partial vacuum of 700 mm Hg, velocity of the macrocarrier was measured at 520 m sec^?1 and 432 m sec^?1 at atmospheric pressure. Optimum distance from the stopping plate to different target cells during bombardment ranged from 4 to 7 cm. Mean transformation efficiency of the GUS-gene marker was estimated at 350 transformants per 65 mg fresh weight of the maize cultured cells. Up to 200 GUS transformed cells were detected per 100 mg of embryogenic rice callus. Use of gold flakes or tungsten powder as microcarriers resulted in similar transformation rates. No transformation was observed in any cells when DNA constructs contained prokaryotic translation initiation sequences for the GUS gene. Based on transient GUS assays, further modification of the airgun device will likely be necessary to obtain high stable transformation rates.     

Biolistic transformation of animal tissue

Summary The biolistic technique transforms cells by bombardment with DNA-coated microprojectiles. It has been used to transform plants, microbes, and organelles. We adapted a standard Biolistic PDS-1000 device for use with animals and have successfully transformed tissues in live mice. The firefly luciferase gene was introduced into mouse skin and ear tissue. One day after transformation 344±74 and 1648±254 pg of luciferase were detected in skin and ear samples, respectively. Expression of the gene product was transient but detectable up to 7 days after bombardment. A further modification of the device allowed transient transformation of liver tissue in vivo. Liver contained 293±122 pg of luciferase 1 day postransformation. Expression of the gene in liver tissue was unchanged at Day 3 but declined to low levels by Day 5. This new device allowed a fourfold increase in gene expression in ear tissue extending a minimum of 14 days. This technology is applicable to a broad range of tissues and organs in situ and makes it possible to test numerous reporters and the tissue specificity of promoters. It may also be useful in protocols for somatic cell therapy. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system

Effective biolistic transformation of intact conidia from the filamentous fungus Trichoderma reesei was achieved using the Bio-Rad Hepta Adaptor system with seven barrels for particle launch. Transformation frequencies of up to 39 colonies per microg of circular DNA and 37 colonies per microg of linear DNA were obtained at an optimal target distance of 3 cm and a helium pressure of 1350 psi. These values are about 3.5- to 6-fold higher than transformant yields reported earlier for T. reesei using the hygromycin phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin B as a selectable marker in combination with the PDS-1000/He single barrel system. High mitotic stability of the transformants (98-100%) was demonstrated. The Hepta Adaptor device allowing bombardment of seven lots of conidia in a single plate offers clear advantage in terms of transformant numbers over the single barrel system where target cells are restricted to the center of the plate.