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

Posted on: Saturday, 22 November 2003, 06:00 CST

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 intercellular associations between nitrogen-fixing bacteria and higher plants.

Key words: Azotobacter; Fragaria; biolistic; bombardment; in vitro culture; nitrogen fixation; symbiosis.

INTRODUCTION

Particle bombardment (Sanford et al., 1987) is very effective for the delivery of DNA into intact cells and tissues. This method is especially beneficial for those plants which cannot be transformed by Agrobacterium-based gene transfer (Cocking, 1990). The biolistic process has found application in the transformation of diverse organisms including animal cells and tissues (Zelenin et al., 1989; Fitzpatrick-McElligot, 1992), monocot (Klein et al., 1989; Weeks et al., 1993; Wan and Lemaux, 1994) and dicot (Klein et al., 1988; McCabe et al., 1988; Finer and McMullen, 1990) plants, yeasts, and other fungi (Armaleo et al., 1990), algae (Kindle et al., 1989), and bacteria.

Several cultured plant cells and explants have been targeted by microprojectiles for DNA transfer. Finer and McMullen (1991) used cell suspension, whereas Vasil et al. (1992) and Weeks (1995) transformed callus tissues. Gene delivery directly into the explants offers several advantages. Immature zygotic embryos, especially those of monocolyledonous plants, were frequently used as recipients (Kartha et al., 1989; Sautter et al., 1991; Christou, 1992; Dunder et al., 1995). Gene transfer to shoot apical meristems by the biolistic gun is an attractive approach although meristems are small and sensitive targets and are therefore difficult to handle. However, successful meristem bombardment has been reported (McCabe et al., 1988; Bilang et al., 1993; Gambley et al., 1993; Sautter, 1993). The method was developed further for biolistic microtargeting of inflorescences and flower meristems (Leduc et al., 1994) as well as pollen grains (Twell et al., 1989, 1991). Stable transformants were recovered after the bombardment of whole leaves and leaf strips (Klein et al., 1988; Tomes et al., 1990). In addition to nuclear transformation, particle bombardment also delivered genes into plastids (Boynton et al., 1988; Daniell et al., 1990; Svab et al., 1990; Boynton and Gillham, 1993; Daniell, 1993; Klein and Bogorad, 1995) and mitochondria (Johnston et al., 1988).

Until now, only Rasmussen et al. (1994) have used bacteria as microprojectiles for the transformation of plant cells, but they utilized dead Escherichia coli and Agrobacterhim tumefaciens cells, pretreated with phenol, to deliver DNA into cultured tobacco and maize cells. The bacterial cultures were combined with tungsten particles. Their work demonstrated that these biological particles can be used for the delivery of high molecular weight DNA and the transformation process is simpler and faster without the time- consuming isolation and purification of DNA. However, transformation frequencies were much lower than those obtained by the original DNA delivery method.

This paper reports on a novel use of biolistic bombardment. In our experiments living nitrogen-fixing Azotobacter vinelandii cells were delivered into strawberry tissues to create working artificial associations between the two organisms. Our previous works cover other in vitro methods for establishing nitrogen-fixing associations (Varga et al., 1994; Gyurjan et al., 1995; Preininger et al., 1997). According to the results, this method can be used for the extension of nitrogen-fixing ability to originally asymbiotic plant species with the use of azotobacters which can fix atmospheric nitrogen in photosynthesizing plant organs.

MATERIALS AND METHODS

Bacterial strains. The CCM 289 and DSM 2289 strains of aerobic, free-living, Gram-negative, poly-[beta]-hydroxybutyrate-forming Azotobacter vinelandii were maintained in nitrogen-free liquid and solid culture (Newton el al., 1953) at 30[degrees]C in the dark.

Plant material and maintenance of shoot culture. Shoot cultures of strawberry (Fragaria x ananassa 'Fertodi F5') were initiated from virus-free shoot tips and maintained on MS (Murashige and Skoog, 1962) basal medium (30) supplemented with 2.5 [mu]M N^sup 6^- benzyladenine (BA), 0.3 [mu]M gibberellic acid (GA^sub 3^), 2.2 [mu]M indole-3-butyric acid (IBA), and 3% (w/v) sucrose. The pH of the medium was adjusted to 5.7 before adding 0.7% (w/v) agar and autoclaving for 20 min at 121[degrees]C and 108 kPa. Cultures were incubated at 25[degrees]C, at a photosynthetic photon flux of 40 [mu]mol m^sup -2^s^sup -1^, and under a 16 h photoperiod. The shoot culture was subcultured every 6 wk.

Preparation of target plant material for bombardment. Young expanding leaves of the micropropagated strawberries were excised from the 1-mo.-old shoots and the petioles were removed. Leaves were placed on callusinducing medium [MS basal medium supplemented with 4.4 [mu]M BA, 0.5 [mu]M IBA, 0.3 [mu]M [alpha]-naphthaleneacetic acid (NAA), 0.5 [mu]M 2,4-dichlorophenoxyacetic acid (2,4-D), 3% lactose, and 0.6% agar] with both abaxial and adaxial sides touching the medium for 3 d prior to bombardment. The pH was adjusted to 7.0. After biolistic treatment, the leaves were left in their original Petri dishes until callus formation, and then subcultured with the same medium for plant regeneration.

Alternatively, regenerating callus tissues with a great number of developing 1-3-mm long shoot primordia were placed on the same medium as described above. In both cases, the target plant tissues were arranged in a circle in the center of a 9 cm Petri dish.

Preparation of bacterial microprojectiles and biolistic bombardment Tung- sten particles of an average size of 2-3 [mu]m were suspended at 60 mg ml^sup -1^ in absolute ethanol and stored at -20[degrees]C. For bombardments, 25 [mu]l of the stock suspension was placed into a microcentrifuge tube and washed thoroughly. The supernatant was discarded and the particles were resuspended in 175 [mu]l bacterial suspension (about 10^sup 9^ cells ml^sup -1^). Five [mu]l of 0.1 M spermidine was added and, after centrifugation, 130 [mu]l of supernatant were removed. The mixture was briefly vortexed and a 5 [mu]l aliquot was pipetted onto the plastic macroprojectiles which were accelerated by a nitrogen-powered biolislic gun al 30 bar. The shooting distance was 13 cm.

Re-isolation of bacteria. Callus pieces and regenerated plants were aseptically homogenized with a mortar and pestle and placed onto nitrogen-free medium used for the maintenance of bacteria. Petri dishes were kept downwards at 30[degrees]C in the dark. Colonies were observed by an Olympus BH-2 light microscope and the single bacterial cells by a Hitachi 7100 election microscope after the same procedure as described above.

Microscopy. For light and transmission electron microscopy, plant tissues were fixed in 2% (v/v) glutaraldehyde for 2 h and postfixed in 1% (w/v) OsO^sub 4^ for 2 h in 70 mM K-Na phosphate (pH 7.2). Samples were embedded in Durcupan ACM epoxy resin (Fluka Chemie AG). Semi-thin sections were stained with toluidine blue (1%) and observed with an Olympus BH-2 light microscope. Ultrathin sections were resectioned from the selecled 5 [mu]m thick light microscopy sections (Kristof, 1997) and stained with uranyl acetate and lead citrate and observed with a Hitachi 7100 electron microscope.

For scanning electron microscopy, small parts of leaves and shoot apexes were fixed in 2% (v/v) glutaraldehyde for 3 h and postfixed in 1% (w/v) OsO^sub 4^ for 3 h. After dehydration with increasing concentrations of ethanol, samples were placed in amyl acetate. The intermediate fluid was removed from the samples by critical point drying. Before investigation, specimens were coated with coal and gold evaporation. Observation was made using a Hitachi S-2360 N scanning microscope at 15 and 25 kV accelerating voltages.

Molecular detection of A. vinelandii in strawberry tissues by PCR. A. vinelandii and strawberry genomic DNA was prepared using the Qiagen DNeasy Plant Mini Kit (Qiagen, Germany). To detect the nitrogenase 3 (anfH) gene of A. vinelandii from plant DNA using PCR, the primers ANF3FOR (5'-GGCCTATTTCCACGACAAGA-3') and ANF3REV (5'- ATCATCTTGGTACCGATGGC-3' were used. DNA amplification was achieved in 30 cycles of denaturation at 94[degrees]C (1 min), annealing at 55[degrees]C (1 min), and elongation at 72[degrees]C (2 min) using a Perkin Elmer Thermal Cycler. The PCR products were separated on 1.0% agarose gels by electrophoresis.

Measurement of acetylene reduction activity (ARA). The nitrogen- fixing activity of reisolated bacteria and bacteria in regenerated plantlets after the formation of the first leaves \was detected by ARA. Plants were maintained on MS medium with one-sixth of the normal nitrogen content for 4 wk prior to measurement. The assay was performed as described previously (Preininger et al., 1997).

Surface-sterilization of plant tissues. To eliminate outer bacterial cells on the plant tissues, plant pieces were surface- sterilized with 0.5% HgCl^sub 2^ for 1 min. This procedure was made prior to different detection methods: reisolation of bacteria, DNA isolation, and measurement of acetylene reduction activity.

RESULTS

Biolistic treatment of leaf inocules. Three days alter bombardment, brownish spots were observed on the leaves using a light microscope (Fig. 1A). Scanning electron micrographs showed clearly the hit sites of the microprojectiles on the surface. There were holes several cells in diameter scattered on the leaves which were probably caused by the tungsten aggregates (Fig. 1B) and contained the delivered bacteria (Fig. 1C). A larger magnification revealed smaller penetration sites as well on the cell surfaces torn by solitary microprojectiles (Fig. ID).

Yellow primary calluses appeared 14-18 d after bombardment and scanning electron microscopy detected the early stages of callus development (Fig. 2A). There were no differences between the abaxial and adaxial sides, neither in frequency of penetration gaps nor in that of callus induction. Bombarded leaves developed calluses at the penetration sites and bacteria were surrounded by dividing callus cells. Figure 2B demonstrates the presence of azotobacters in the intercellular spaces of the strawberry callus and dividing bacteria were also detected (Fig. 2C). Plants were regenerated from bacterium- containing callus tissues on the same medium. Bombarded leaves regenerated plants at high frequency 3-4 wk after callus formation (Fig. 2D). The bacteria gained access into the regenerating plants during organogenesis.

Bombardment of shoot apexes with bacterial microprojectiles. If regenerating shoot tips were treated, first the in vitro phases of callus formation and plant regeneration were induced and then the sprouting tiny green shoot apexes were bombarded with the bacterial microprojectiles (Fig. 3A). This enhanced the efficiency of bacterium delivery and shortened the time after the biolistic treatment. The elimination of different steps of tissue culture after the particle transfer minimized the loss of introduced bacteria. Continuous division of the meristematic region ensured the spread of bacteria in the whole plant.

The effect of bombardment was detected by scanning electron microscopy shortly after the treatment. Injuries of different sizes were seen on the surface of the shoot apices and leaf primordia. Some of them were located at only a restricted part of the tunica cells (Fig. 3B), while tungsten aggregates caused more extensive damage. The injuries showed different appearances in terms of their depth. Some injuries were rather shallow and the delivered bacteria were seen near to the surface (Fig. 3C). Others appeared as deeper holes caused by the aggregates penetrating several cell layers (Fig. 3D).

A light micrograph of the bombarded strawberry meristem (Fig. 4A) shows that bacteria were delivered into the outer cell layers of the corpus, but they were also observed in the lower and more differentiated ground tissue region (Fig. 4B). Electron micrographs (Fig. 4C, D) demonstrated the situation 2 h after the biolistic treatment. A sufficient number of bacterial cells landed in this part of the shoot apex. Tungsten particles caused injuries in the internal meristem tissues, which, despite this, were able to regenerate and develop into vigorous plantlets. Azotobacters survived the treatment and dividing bacteria were also observed (Fig. 4D).

FIG. 1. Injuries on the leaf surface after bombardment. Leaves were derived from micropropagated strawberry shoot culture, and examined 3 d after the biolistic treatment. A, Stereomicroscopic picture of a leaf with black spots caused by tungsten aggregates. B, SEM micrograph of a penetration site of tungsten aggregate in size comparable with several epidermal cells (bar = 50 [mu]m). C, SEM micrograph of a deeper hit site among the epidermal cells with the delivered bacteria (bar = 10 [mu]m). D, SEM micrograph of small holes caused by single tungsten particles and bacteria on the leaf surface (bar = 10 [mu]m).

FIG. 2. Callus formation and plant regeneration from bombarded leaves of strawberry and detection of bacteria. A, Early stage of callus development on the abaxial surface of the leaf at the injuries caused by the tungsten microprojectiles 14-18 d after biolistic treatment (bar = 100 [mu]m). B, Electron micrograph of bacteria among callus cells developed from the bombarded leaves (bar = 2 [mu]m). C, Dividing bacterium in the intercellular space of strawberry callus initiated at the penetration site of the microprojectiles (bar = 1 [mu]m). D, Plant regeneration from callus tissues developed from the leaves after biolistic treatment (bar = 2 mm).

FIG. 3. Bacterium bombardment of shoot apexes regenerating from strawberry callus. Samples were taken and fixed 3 h after the treatment. A, Tiny shoot tips representing the developmental stage of the biolistic treatment (bar = 100 [mu]m). B, Small hole with bacteria on a tunica cell (bar = 10 [mu]m). C, Shallow injury on the shoot apex surface, full of bacteria (bar = 10 [mu]m). D, Deeper hole with the delivered azotobacters on the shoot tip (bar = 5 [mu]m).

FIG. 4. Light and electron microscopical detection of the delivered bacteria in the shoot apices of strawberry 3 h after biolistic treatment. A, A group of bacteria is present in the external part of the corpus (arrow) (bar = 20 [mu]m). B, Intercellularly placed bacteria in the deeper region of the shoot tip. Single and dividing bacteria can be seen among differentiated parenchymatic cells with high starch content (arrows) (bar = 10 [mu]m). C, D, TEM micrographs of azotobacters (arrows) among shoot apex cells destroyed by the microprojectiles (C, bar = 10 [mu]m; D, bar = 2 [mu]m).

Re-isolation of bacteria from the strawberry tissues. Re- isolation of the introduced bacteria is a quick and simple method to check the bacterium content of the treated calluses and plants. Re- isolation was successful in about 60% of the randomly selected tissues. Colonies grew slowly on nitrogen-free medium at the beginning but multiplied with normal vigor after subculturing twice. Electron microscopical examinations verified that the reisolated diazotrophic bacteria were Azotobacter vinelandii cells. In the case of 3-4-mo.-old regenerated plants, poly-[beta]-hydroxybutyrate accumulation was observed in the bacteria.

Molecular detection of A. vinelandii in plant tissues. In order to detect A. vinelandii in bombarded plant material, oligonucleotide primers were designed to verify the presence of the nitrogenase 3 (anfH) gene (Joerger et al., 1989). Nitrogenase 3 was chosen because it is an alternative nitrogenase encoded by the anfHDGK structural genes found only in Azotobacter vinelandii. The primers amplified a 546-bp fragment in DNA isolated from A. vinelandii or bombarded strawberry tissues, while PCR product was not detected in DNA isolated from untreated plant tissues (Fig. 5).

Acetylene reduction activity. The nitrogen-fixing ability of bacteria in the regenerated plants was checked by ARA. Since inorganic nitrogen compounds repress nitrogenase activity, the nitrogen content in the MS medium was restricted to a one-sixth part (0.14 g l^sup -1^ N) for in vitro plants. This ensures normal growth and regeneration of the strawberries as well as detection of the bacterial enzyme activity. Azotobacter-containing plants showed the ARA in 15% of the plants (Fig. 6). Only qualitative and not quantitative analysis of the forming ethylene was attempted, as offered by Urquiaga et al. (1989) and Peoples and Craswell (1992).

DISCUSSION

The developed biolistic method made possible the delivery of living bacteria into plant tissues. Formerly, only Rasmussen et al. (1994) used bacterial cells as microprojectiles, but they used phenol-treated, dead bacteria as vectors for DNA delivery. Applying the method of DNA transfer, bacterium cells adhered to tungsten particles were shot into the leaves and shoot tip meristems. The diameter of tungsten particles (2-3 [mu]m) was chosen according to the size of bacteria and was twice as much as generally used for normal gene delivery. It appears that these particles did not cause significant and irreversible damage in the plant target tissues.

The pressure of the accelerating nitrogen gas (30 bar) ensured penetration of the bacterium cells into the deeper plant tissue layers. Bacterial cells could tolerate the arising energy and survived the treatment. Dividing bacteria were also observed, demonstrating that conditions inside the plant tissues are acceptable for their growth and reproduction. The injured tissues were able to regenerate which was proven by the spread of azotobacters in the developing strawberry organs and tissues. The presence of bacteria was detected by re-isolation, light and electron microscopy as well as acetylene reduction assay. The detection of a 546-bp fragment of the anfH gene verified the identity of azotobacters present in the artificial associations. Surface-sterilization of plant tissues prior to all detection methods ensured that data concerned only the introduced, inner bacteria.

FIG. 5. Detection of Azotobacter vinelandii anfH gene in strawberry tissues after plant regeneration by PCR. M, 500 hp ladder; lane 1, A. vinelandii genomic DNA; lane 2, genomic DNA from leaves of non-treated strawberry; lane 3, genomic DNA from strawberry leaves bombarded with A. vinelandii; lane 4, genomic DNA from strawberry callus bombarded with A. vinelandii; lane 5, control.

The elaborated technique made possible, for the first time, the intro\duction of living bacteria into plant tissues. Particle bombardment seems to be an efficient alternative method for establishing artificial nitrogen-fixing associations because it can ensure the delivery of diazotrophic bacteria in sufficiently high numbers into the target tissues. Theoretically, any higher plant can be inoculated by nitrogen-fixing bacteria using this method. Bacteria can gain access to the newly forming tissues and organs and this has great importance for vegetatively propagated plants, because the transfer of bacteria into new generations is ensured. Biolistic bombardment utilizing living bacteria is a more direct and effective procedure of establishing nitrogen-fixing plant-microbe associations as compared to those mixing the plant cells or tissues with bacteria suspensions (Varga et al., 1994; Gyurjan et al., 1995; Preininger et al., 1997).

FIG. 6. Acetylene reduction assay from a regenerated control plant (a) and an artificial association (b) of strawberry at the same age. Peaks: 1, ethylene; 2, acetylene.

Another advantage of biolistic delivery is the possibility of random incorporation of some bacteria into the plant cells. This can lead to the establishment of intracellular symbioses between nitrogen-fixing bacteria and higher plants. In the 1970s, several papers reported on the results of polyethylene glycol-induced uptake of diazotrophic cyanobacteria into isolated higher plant protoplasts (Davey and Cocking, 1972; Davey and Power, 1975; Burgoon and Bottino, 1976; Meeks et al., 1978; Bradley and Leith, 1979), but bacteria could be detected in the protoplasts only for some hours. Whether the biolistic method can establish an artificial intracellular nitrogen-fixing association remains to be tested.

ACKNOWLEDGMENT

This work was supported by OMFB 96-97-46-1032, OTKA T 021068, and OTKA T 34875 grants.

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EVA PREININCER1*, ISTVAN GYURJAN1, KAROLY BOKA1, TAMAS PONYI2, JOZSEF ZATYKO3, AND PAL KORANYI1

1 Department of Plant Anatomy, Eotvos Lorand University, Budapest 1117, Hungary

2 Agricultural Biotechnology Center, Godollo 2101, Hungary

3 Institute for Fruitgrowing and Ornamentals, Fertod 9431, Hungary

(Received 25 April 2002; accepted 20 February 2003; editor L. Herrera-Estrella)

* Author to whom correspondence should be addressed: Email preininger@ludens.elte.hu

Copyright Society for In Vitro Biology Sep/Oct 2003

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