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.     

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.     

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.     

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens

A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA. Transformation of vegetative cells of C. perfringens strain 13 with the 7.9-kb Escherichia coli-C. perfringens shuttle plasmid pHR 106 required pretreatment with lysostaphin (2 to 20 micrograms/ml) for 1 h at 37 degrees C. Cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases. The transformation frequency increased with the DNA concentration, to a saturating level at 5 to 10 micrograms DNA/ml. The transformation frequency was proportional to the field strength and time constant of the electroporation pulse; however, the field strength was a far more important parameter. A cell density between 1 x 10(8) and 5 x 10(8) cells/ml proved to be optimal for transformation. The procedure was capable of generating up to 3.0 x 10(5) transformants per micrograms DNA. The potential value of the method for the cloning of C. perfringens genes was demonstrated by the cloning of the clostridial tetracycline-resistance determinant, tetP, from the E. coli recombinant plasmid pJIR71, into C. perfringens strain 13.     

Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

We used a modified version of the method of Hanahan (D. Hanahan, J. Mol. Biol. 166:557-580, 1983) to transform Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica with the plasmids pBR322, pBR325, and pAT153. The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA. The nature of these transformants was confirmed by plasmid analysis. ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species.     

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.     

Efficient biolistic transformation of the moss Physcomitrella patens

High rates of homologous recombination (HR) in comparison to other plants make the moss Physcomitrella patens an attractive model organism for genetic studies as well as biotechnological applications. We describe a simple protocol for the efficient biolistic transformation of protonemal tissue with minimum tissue handling steps. The transformation efficiency depends on the biolistic conditions. The bombardment of tissue with 1 μm gold particles yielded between 20 and 40 stable transformants per 1 μg of DNA. Transformation with circular plasmids generates higher frequencies of random transgene integration, whereas linear plasmids are more efficient in generating gene-targeted insertions.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens.

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

Electro-stimulated transformation of E. coli cells pretreated by EDTA solution

The phenomenon of transformation of E. coli cells under electric treatment has been studied. The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension. The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA. The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

Transformation of bacteria with plasmid DNA by electroporation

The possibility of electric field-mediated transformation ("electroporation") of a gram-positive bacterium (Enterococcus faecalis) and two gram-negative bacteria (Escherichia coli and Pseudomonas putida) with plasmid DNA was investigated. E. faecalis protoplasts could be transformed by electroporation with a transformation frequency of 10(4) to 10(5) transformants/micrograms plasmid. Untreated--i.e., washed--cells of E. coli could be transformed with rates of 1 X 10(5) transformants/micrograms plasmid DNA. Transformation rates for P. putida cells were up to 3 X 10(4) if the method developed for E. coli was used. Detailed protocols for these systems, including the results of various optimization experiments, are given.     

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

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