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Gene Delivery into Intact Plants Using theHelios™ Gene Gun

ELINA HELENIUS1,*,**, MARIA BOIJE1,***, VIOLA NIKLANDER-TEERI2,E. TAPIO PALVA1,3 and TEEMU H. TEERI3

1Department of Biosciences, Division of Genetics, P.O. BOX 56, FIN-00014 Universityof Helsinki; 2Department of Plant Biology, Division of Botany, P.O.BOX 27, FIN-00014University of Helsinki; 3Institute of Biotechnology, P.O. BOX 56, FIN-00014 Universityof Helsinki

Abstract. Particle bombardment is a physical method for cell transformation in whichhigh density, sub-cellular sized particles are accelerated to high velocity to carryDNA/RNA into living cells. It is a versatile technique that can be used for transient ex-pression studies as well as for creating stable transformants. The Helios™ gene gun usesDNA-coated gold particles that are precipitated on the inner wall of a plastic tube and ac-celerated by pressurized helium. It differs from previous particle bombardment methodssince it operates in ambient pressure and can be used e.g. in the field. The aim of thisstudy was to optimize parameters for transient expression of gene constructs intoarabidopsis, tobacco and birch with the Helios™ Gene Gun. In order to investigate tran-sient gene delivery, we used constructs containing the constitutively active promoter of theCaMV 35S transcript fused with reporter genes encoding luciferase (LUC) andβ-glucuronidase (GUS). Optimization was performed in a step-by-step manner. We foundthat the most critical parameters were helium pressure, the optimum of which varied be-tween plant species, and the amount of gold. Gold particles with a diameter of 0.6 µmwere best for all plant species studied. The optimization procedure helped to increase theexpression levels five- to ten-fold.

Key words: optimization, particle bombardment, plant biotechnology, transient expressionGene delivery by the Helios™ Gene Gun Helenius et al.

Introduction

Particle bombardment is a physical method of cell transformation in which highdensity, sub-cellular sized particles are accelerated to high velocity in order tocarry DNA/RNA into living cells. The technique was first described as a methodof gene transfer into plants (Klein et al., 1987). As a physical method, particlebombardment is readily applicable to a variety of biological systems and it alsoeffectively overcomes physical barriers to gene transfer, such as the cell wall ofplants. It is a versatile technique that can be used both for transient expressionstudies (e.g. promoter analysis) and for creating stable transformants (Christou,

Plant Molecular Biology Reporter 18: 287a–287l, 2000© 2000 International Society for Plant Molecular Biology. Printed in Canada.

*Author for correspondence. e-mail: [email protected]; fax: +358 9 191 59079;ph: +358 9 191 59085.

**Both first authors have contributed equally to this work.***Present address: SkogForsk, Ekebo, S-26890 Svalöv, Sweden.

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1994). Particle bombardment has also been used for wounding plants in order topromote Agrobacterium transformation (Bidney et al., 1992).

The Helios™ Gene Gun is the second instrument in Bio-Rad´s particle de-livery product line. It uses DNA-coated gold particles, precipitated on the innerwall of a plastic tube and accelerated by a flow of pressurized helium (Bio-RadLaboratories, 1996). The most significant difference compared with other particlebombardment equipment, e.g. Bio-Rad’s previous model, PDS-1000/He, is thatthe Helios™ Gene Gun requires no vacuum, removing limitations to the targetand its size. Moreover, the cartridges can be stored for several months and thebombardment procedure is much faster than with the PDS-1000/He instrument. Inpractice, these two particle delivery products complement each other, the vacuumchamber method providing a more controlled bombardment environment, and theHelios™ Gene Gun providing a much wider selection of target material.

The aim of this study was to optimize parameters for transient expression ofgene constructs in plant material with the Helios™ Gene Gun. As target plants,we used thale cress (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), bothgenerally used as model organisms in plant molecular biology and genetics, andsilver birch (Betula pendula) as a representative woody plant. In order to investi-gate the efficacy of transient gene delivery, we used constructs containing the con-stitutively active promoter of the CaMV 35S gene fused with reporter genesencoding luciferase (LUC) and β-glucuronidase (GUS).

Materials and Methods

Equipment and reagents

Helios™ Gene Gun System (Bio-Rad Laboratories, USA)Helios™ Gene GunTubing Prep StationTubing CutterCartridge Extractor Tool

0.6 µm gold particles (Bio-Rad Laboratories, USA)Gold-Coat tubing (Bio-Rad Laboratories, USA)nitrogen, grade 4.8 or higherhelium, grade 4.5 or higherpolyvinylpyrrolidone (PVP), MW 360.000dry ethanol50 mM spermidine1 M CaCl2

1 mM 5-bromo-4-chloro-indolyl-β-D-glucuronide (X-Gluc) in buffered solution[100 mM Na-phosphate buffer (pH 7.0), 10 mM EDTA, 0.5 mM K3Fe(CN)6, 0.5mM K4Fe(CN)6, 0.1% Triton® X-100] (Stomp, 1996)modified lux-buffer [50 mM Na-phosphate (pH 7.0), 4% soluble PVP (MW360.000), 2 mM EDTA, 20 mM DTT] (Herrera-Estrella et al., 1994)Luciferase Assay Reagent (Promega, USA)luminometer (model 1254-001, Bio-Orbit, Finland)

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Plant material

Arabidopsis thaliana (L.) Heynh. ecotype Landsberg erecta plants were grown inpeat at 20°C with an 8 h photoperiod. 4-5 week-old plants were bombarded be-fore they started to bolt. Nicotiana tabacum (L.) cv. Petit Havana SR1 plants weregrown in vermiculite at 23°C with a 16 h photoperiod and fertilized with a com-mercial fertilizer (Substral®, Thompson Siegel, Germany). The birch plants wereyoung greenhouse Betula pendula (Roth.) JR 1/4 clone plantlets grown in forestpeat-vermiculite (2:1 ratio) mixture (Finnpeat, Kekkilä, Finland) under a 16 hphotoperiod. The arabidopsis, tobacco and birch leaves used as target materialwere attached to the plants during bombardment and until the enzymatic assayprocedure. Fully expanded young leaves were used in all assays.

Plasmid constructs

The reporter gene constructs used were pANU21 (5.2 kb) containing the uidAgene encoding β-glucuronidase (GUS) from Escherichia coli and pHTT308(6.6 kb) containing the luc gene from firefly encoding luciferase (LUC). Bothplasmid constructs contain the reporter gene under the control of the constitutiveCaMV 35S promoter (regular promoter for uidA and 4x35S for luc). In bothplasmids the promoter is followed by the TMV leader Ω that functions as atranslational enhancer (Gallie et al., 1987). Particles used for control bombard-ments were coated with pUC19 (Yanisch-Perron et al., 1985), the vector used forthe reporter constructs.

Cartridge preparation

Cartridges were prepared according to the Helios™ Gene Gun System InstructionManual (Bio-Rad Laboratories, 1996). In the following procedure, the amount ofgold and DNA are those found optimal in experiments described further below.

Precipitation of DNA onto microcarriers

• Weigh out 6.25 mg of 0.6 µm gold particles for each 30 inch length of tubing.Add to the gold 100 µL of 50 mM spermidine and mix well. Add 50 µg DNAin Tris-EDTA (a molar 1:1 mixture of plasmids pANU21 and pHTT308 wasused), and mix again.

• While mixing, add 100 µL of 1 M CaCl2 to associate the DNA with the goldparticles. Allow the suspension to settle at room temperature for 10 min.

• Centrifuge the suspension for 30 s in a microcentrifuge to pellet the gold. Washthe pellet three times with dry ethanol.

• Resuspend the pellet in 3 mL of 0.05 mg/mL PVP in dry ethanol.

Cartridge preparation with the Tubing Prep Station

• Dry the Gold-Coat tubing by purging with nitrogen (flow 0.3-0.4 liters per min[LPM]) for 15 min. 30 inches of tubing are needed for each 3 mL of gold sus-pension.

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• After mixing thoroughly, draw the gold suspension into the Gold-Coat tubingwith a syringe. Slide the loaded tubing into the Tubing Prep Station and allowit to settle for 3 min.

• Remove the ethanol slowly with the syringe from the tubing, turn it 180° andlet it settle for a few s.

• Start rotating the Gold-Coat tubing to allow the gold particles to spread onto itsinner surface. After 20-30 s, open the nitrogen flow (0.3-0.4 LMP) and con-tinue to rotate the tube for 3-5 min to dry it.

• Discard any unevenly coated sections and cut the remaining tubing into0.5 inch pieces with the Tubing Cutter.

Particle delivery

• Connect the Helios™ Gene Gun to the helium source, insert an empty cartridgeholder into the gun and pressurize the system by a few “pre-shots”.

• Load cartridges into the cartridge holder and insert it into the gun. Turn the he-lium regulator to the selected pressure.

• Touch the target area with the spacer, activate the safety interlock switch andpress the trigger simultaneously to deliver the DNA/gold to the target tissue.(We used intact leaves still attached to greenhouse-grown arabidopsis, tobaccoand birch plants as target material. The leaves were held in place during thebombardment by flattening them against a fine mesh with a suction pipe at-tached to a vacuum cleaner (Figure 1). In some experiments, a diffusion screenwas used on the base of the Helios™ Gene Gun barrel to reduce tissue damagein the center of the shot.)

• Ratchet to the next cartridge by pulling in and releasing the cylinder advancelever.

• After all the cartridges have been discharged, remove them with the CartridgeExtractor Tool.

• Depressurize and disconnect the gun from the helium source.After bombardment, the plants were placed for 24 h in the greenhouse be-

fore assaying for enzyme activities. Each bombarded region was cut into twoequal halves, one for histochemical GUS assay and one for quantitative LUC as-say.

Enzymatic assays

Histochemical GUS assay:• One half of the bombarded leaf disk was submerged in 1 mM

5-bromo-4-chloro-indolyl-β-D-glucuronide (X-Gluc) in buffered solution andincubated in the dark for 16 h at 37°C (Stomp, 1992).

• After staining, the chlorophyll was removed with absolute ethanol in order tobetter visualize the blue spots, which were counted using a stereo microscope.

Luciferase assay:• The other half of the bombarded leaf area was homogenized in 200 µL ice-cold

modified lux-buffer. Cell debris was removed by 10 min centrifugation at maxi-mal speed.

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• 20 µL of plant extract was mixed with 100 µL of Luciferase Assay Reagent(Promega, USA) at room temperature and the emitted light was measured for aperiod of 10 s in a luminometer (model 1254-001, Bio-Orbit, Finland). For to-bacco, 10 µL of extract and 50 µL of Luciferase Assay Reagent were used afterthe first step of optimization.

Experimental design

Two separate coatings were made for each optimization step and kept separatethroughout the experiment. In each step, the results were obtained from at leasttwo independent experiments with 3-5 parallel shots.

Results and Discussion

Optimization was performed in a step-by-step manner, as suggested in theHelios™ Gene Gun System Instruction Manual and outlined in Figure 2. Theprinciple of this procedure is that one parameter is varied while the others arekept constant. However, in the first optimization step, the helium pressure and thesize of microcarriers were optimized together, and different pressures were usedfor each gold particle size. In the second step, polyvinylpyrrolidone (PVP) con-centration was varied. PVP serves as an adhesive during the cartridge preparationprocess. In the final steps, the amount of gold and DNA were optimized. Thetested options for each parameter and the results of the optimization are listed inTable 1. The most critical parameters were helium pressure, the optimum ofwhich varied between plant species (Figure 3) and amount of gold (Figure 4). Re-ducing the amount of particles led to a significant increase in transient expressionlevels, particularly in arabidopsis and tobacco. However, in birch the amount ofgold did not have an equally significant influence. The particle size also had someeffect in all plant material tested, the best particle size being 0.6 µm for all targets(Figure 3). The optimal amount of DNA was 1 µg in all plant materials, but sig-nificant transient expression was also observed using only 0.04 µg of DNA per

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Figure 1. Bombarding birch leaves with the Helios™ Gene Gun.



shot. The total amount of DNA in these experiments consisted of two plasmidconstructs mixed in a 1:1 molar ratio.

In this study, the transient expression levels of two reporter genes (codingfor LUC and GUS) were measured from one shot. Luciferase was measured quan-titatively in plant extracts, while GUS was assayed by counting the number oftransformation loci after histochemical staining. For arabidopsis and tobacco, theparallel assays for luciferase activity and GUS histochemistry (number of bluespots) correlated well and led to identical optimal parameters in most steps of

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Figure 2. Outline of the gene delivery optimization procedure.



optimization. If the two assays differed, we used the luciferase results. The num-ber of blue spots per shot was similar for all species studied. However, in tobaccothe amount of luciferase in the extracts was 10-100 times higher than inarabidopsis. In the birch samples, we could not recover reliable amounts of activeluciferase. This problem was probably caused by inhibitory compounds that areco-extracted (Loponen, 1998), since mixing of birch extract with tobacco extractled to dramatic loss of activity (not shown). The inhibition was specific to lucifer-ase, since quantitative GUS assay (using 4-methylumbelliferryl-β-glucuronide,(Jefferson, 1987)) of birch extracts worked well (not shown).

We also investigated the effect of cartridge batch (i.e. coatings) and devel-opmental stage of the plant material. No significant difference was detected be-tween different coatings (data not shown). However, there were significantdifferences between leaves of different age within single tobacco (Figure 5) andbirch plants.

As a final step of optimization, arabidopsis, tobacco and birch leaves werebombarded with three different pressures with and without a diffusion screen. Thediffusion screen did not increase expression significantly, but reduced the damagein the center of the shot and made the transformation loci more evenly distributed.It was also possible to use higher pressures when using the screen, which may beimportant since pressures lower than 50 psi are hard to control with the instru-ment. The effect of the diffusion screen on transformation of arabidopsis is illus-trated in Figure 6. The screen could be useful for achieving stable transformation,

Gene delivery by the Helios™ Gene Gun 287g

ARABIDOPSISParameter Tested Options Optimumpressure of helium 50, 75, 100, 125 and 150 psi 75 psisize of gold particles 0.6, 1.0 and 1.6 µm 0.6 µmconcentration of PVP 0, 0.05 and 0.1 mg/mL No significant difference,

0.05 mg/mL chosenamount of gold per shot 0.5, 0.25 and 0.125 mg 0.125 mgamount of DNA per shot 5, 1, 0.2 and 0.04 µg 1 µg

TOBACCOParameter Tested Options Optimumpressure of helium 50, 75, 100, 200 and 250 psi 200 psisize of gold particles 0.6, 1.0 and 1.6 µm 0.6 µmconcentration of PVP 0, 0.05 and 0.1 mg/mL No significant difference,


BIRCHParameter Tested Options Optimumpressure of helium 50, 75, 100, 200 and 250 psi 200 psisize of gold particles 0.6, 1.0 and 1.6 µm 0.6 µmconcentration of PVP 0, 0.05 and 0.1 mg/mL No significant difference,


Table 1. Optimization parameters and their optimum values for arabidopsis, tobacco and birch.



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Figure 3. The effect of helium pressure and gold particle size. Bars indicate the standard deviation.Asterisk shows the chosen combination for further optimization.



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Figure 4. The effect of the quantity of gold. Bars indicate the standard deviation. Asterisk shows thechosen combination for further optimization.



as optimal conditions for stable transformation tend to be gentler than for tran-sient expression. While the highest transient expression is generally obtained withrather violent treatments giving better particle penetration, these conditions mayimpair cell division or growth (Sanford et al., 1993).

According to our experience, the biggest problem with the Helios™ GeneGun system is the high variation in and between experiments. This, however, ischaracteristic for all particle bombardment systems. According to Christou(1992), the variation can be due to physical, environmental and biological

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Figure 5. The effect of the age of the leaf in tobacco. The conditions used: helium pressure 200 psi,amount of gold 0.125 mg and amount of DNA 1 µg per shot. Bars indicate the standard deviation.

Figure 6. Arabidopsis bombarded with (A) and without (B) a diffusion screen. Conditions used:helium pressure 75 psi, amount of gold 0.125 mg and amount of DNA 1 µg per shot.



parameters. In this study, only physical parameters were optimized. Little isknown, for example, about biological interactions between physical parametersand the target tissue, or about the fate of DNA from the time the particles enterthe cells. Environmental and biological variation are particularly hard to control.This is also reflected in our results that show a significant difference in the tran-sient expression levels between leaves of different developmental stage in thesame plant (Figure 5). Therefore, statistical comparison of the results is usuallyquite difficult (Ritala, 1995).

Conclusions

We have shown that the Helios™ Gene Gun system can be successfully used fortransient gene expression studies in arabidopsis, tobacco and birch. The Helios™system is fast and easy to use and its operation in ambient pressure extends themethod to targets that cannot be bombarded in a vacuum chamber, e.g. fullygrown trees or plants in the field. Nevertheless, it was necessary to optimize keyparameters for each target. During the optimization we were able to increase theexpression levels five- to ten-fold.

To achieve optimum results, we recommend using plants grown under de-fined growth conditions to obtain plant material as homogenous as possible. Fur-thermore, it is important that the bombarded leaves are as near the same age andcondition as possible. For each new target, helium pressure and amount of goldshould be optimized. At least five parallel shots per construct and an internal con-trol should be used while doing e.g. promoter deletion analysis in order to controlthe variation. We also highly recommend supporting the leaves during bombard-ment. Using suction to keep the leaves in place and to flatten them evenly is espe-cially important for arabidopsis leaves.

Acknowledgements

The authors wish to thank M.Sc. Satu Ruokolainen for providing the tobaccoplants and Ms. Helena Rintala (Bio-Rad Laboratories) for providing part of thebiolistic consumables.

References

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Gallie DR, Sleat DE, Watts JW, Turner PC and Wilson TMA (1987) The 5’-leader se-quence of tobacco mosaic virus RNA enhances the expression of foreign gene tran-scripts in vitro and in vivo. Nucleic Acids Res 15: 3257-3273.

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Bio-Rad Laboratories (1996) Helios™ Gene Gun System Instruction Manual, Rev B. Her-cules, CA.

Herrera-Estrella L, León P, Olsson O and Teeri T (1994) Reporter genes for plants. In:Gelvin SB and Schilperoort RA (eds), Plant Molecular Biology Manual C2, pp 1-32,Kluwer Academic Publishers, Belgium.

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Loponen J (1998) Isolation and identification of plant phenolic compounds in birch leaves.Ph.D. Dissertation, University of Turku, Turku, Finland

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Sanford JC. Smith FD and Russell JA (1993) Optimizing the biolistic process for differentbiological applications. Methods Enzymol 217: 483-509.

Stomp A (1992) Histochemical localization of β-glucuronidase. In: Gallagher SR (ed),GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, pp 103-113,Academic Press, Inc, USA.

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