Plant Molecular Biology 17: 259-263, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.

Update section Short communication

Transient expression of/ -glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

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Motoaki Seki ~, Yoshibumi Komeda 2, Asako Iida 3, Yasuyuki Yamada 1 and Hiromichi Morikawa 4. Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606,

Japan; 2Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113, Japan; 3 Present address: Sumitomo Chemical Co., Ltd., Biotechnology Laboratory, Takarazuka Research Center, 4-2-1 Takatsukasa, Takarazuka, Hyogo 665, Japan; 4present address: Department of Biological Sciences, Faculty of Science, Hiroshima University, Hiroshima 730, Japan (*author for correspondence)

Received 26 September 1990; accepted in revised form 4 April 1991

Key words: Gene transfer, Arabidopsis thaliana, Brassica napus, transient expression, pneumatic particle gun

Abstract

Successful transient expression of fl-glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm z) and of preculture periods of plant tissues (0 to 6 days) on the efficiency ofgene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm 2 of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm z of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.

Introduction

Since the pioneering studies by Klein [10] and Christou [2], biolistic gene delivery has now become one of the most useful methods to deliver foreign genes into intact plant cells [6, 7, 8, 9, 11, 14].

In our previous papers, we reported successful transient gene expression in cultured plant cells and tissues [ 12] and stable transformation of sus- pension-cultured tobacco cells using a gas- pressure-driven particle gun [5]. More recently, we developed a pneumatic particle gun that is driven by controllable compressed air [4].

Arabidopsis thaliana has been extensively studied as an experimental model plant for gen- etic, biochemical, and molecular biological studies. Also, Brassica napus is an interesting plant species, as it is both a major crop and a highly manipulable laboratory organism. But so far as we are aware, only a few reports on gene delivery into A. thaliana seedlings [ 1] and no reports on that into B. napus cells by particle gun have been published. Here we report the delivery offl-glucuronidase (GUS) gene into intact cells of A. thaliana leaves and roots and B. napus stems by our pneumatic particle gun.

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Materials and methods

Plant materials

Sterilized seeds of Arabidopsis thaliana races Columbia and Landsberg erecta and of Brassica napus cv. Hakuran were cultured on Murashige and Skoog (MS) [13] agar (0 .8~) for 3 to 4 weeks under 16h light/8h dark at 26 °C. A. thaliana leaf sections (1 cm × 0.5 cm) were placed on a plastic Petri dish (5.2 cm internal diameter) containing callus regeneration medium (CRM), consisting of MS inorganic salts, B-5 [3] vitamins, 3 ~o sucrose, 1.0 mg/1 6-benzyladenine (BA), and 0.1 mg/l 1-naphthaleneacetic acid (NAA). They were cultured for 0 to 6 days, and then subjected to the microprojectile bombard- ment.

Root sections of A. thaliana (0.5-1.0 cm long) were placed on a filter paper (Advantec Toyo No 2, 5.5 cm diameter) on a plastic mat in a plas- tic container (Toyobo Co., Osaka, Japan, ccp-102) containing B-5 medium with 3~o sucrose, 0.5 mg/1 2,4-dichlorophenoxyacetic acid and 0.05 mg/1 kinetin (0.5/0.05 medium). They were cultured for 0 to 6 days and then subjected to the microprojectile bombardment.

Stem sections of B. napus (0.5-1.0 cm long) were placed on MS inorganic salts, B-5 vitamins, 3 ~o sucrose, and 5.0 #M M BA. They were cul- tured at 26 °C under continuous light for 2 days and then subjected to the microprojectile bom- bardment.

Plasmid DNA and coating gold particles with DNA

Chimeric plasmid DNA, pBI221 (Clontech, Palo Alto, CA), which has the/~-glucuronidase (GUS) gene under the control of the cauliflower mosaic virus 35 S promoter and nopaline synthetase poly- adenylation region, was used. The closed circular form of the plasmid DNA was purified, and coated on gold particles (1-3/~m in diameter, Alfa Chemical Co., Danvers, MA) by co-precipi- tation in ethanol at a DNA concentration of 4 #g DNA/mg particles as reported previously [12].

The ethanol suspension of the DNA-coated gold particles was placed on the surface of the projectile so that the concentration of the gold particles was 0.2 mg/projectile, after which the particles were dried and used for bombardment.

Gene delivery to the cells

The particle acceleration device and the methods for gene delivery to the cells using this device were essentially as reported previously [4]. The acceler- ating pressure was varied between 85 and 200 kg/cm 2. The distance between target tissues and the stopper was fixed at 10 cm. Throughout this study, a single bombardment was given to a tissue sample.

After bombardment, the filter papers with A. thaliana roots were transferred onto a plastic mat in the plastic container containing about 17 ml of 0.5/0.05 medium, whileA, thaliana leaves and B. napus stems were kept on the agar medium. These tissues were then cultured for 24 h in a 16 h light/8 h dark cycle at 26 ° C, after which they were assayed for transient GUS expression.

Assay for GUS expression

The GUS activity assay was carried out essenti- ally according to Klein et al. [7] ; the filter papers with A. thaliana roots were transferred to plastic Petri dishes, and A. thaliana leaves and B. napus stems were transferred to plastic well plates (Corning Co., Cell Wells). Each dish or well contained 1.5 ml of filter-sterilized GUS sub- strate mixture. The substrate mixture consisted of 0.5 mM potassium ferricyanide, 0.5 mM potas- sium ferrocyanide, 1.9 mM 5-bromo-4-chloro- 3-indolyl glucuronide (X-gluc, the substrate of GUS) (Sigma Chemicals), and 0.3 To (v/v) Triton X-100. The tissues were incubated for 24 h at 37 °C, and then 3 ml of 70~/o (v/v) ethanol was added to the celI-GUS substrate mixture in order to stop the reaction and to keep aseptic condi- tions. GUS-expressing cells were detected as blue-colored spots. The size of the blue spots

varied from 50 to 1000 #m (see below). Each spot, regardless of its size, was considered as one GU S-expression unit, and the number of the units was counted under a binocular microscope ( × 6.6, Nikon, SMZ-10).

Results and discussion

Figures 1A and 1B show typical GUS results of A. thaliana leaves. Blue spots which stretch along veins were sometimes observed (Fig. 1B). Also, many blue spots were observed with the A. thaliana race Landsberg erecta roots (Fig. 1C) and B. napus cv. Hakuran stems (Fig. 1D). No blue spots were detected after bombardment with uncoated gold particles (data not shown). These results indicate that successful delivery of the plasmid DNA and expression of the gene in A. thaliana leaves and roots and B. napus stems were obtained by our device.

The effects of the accelerating pressure and preculture period on the biolistic gene delivery efficiency were studied; the results are sum- marized in Tables 1 and 2. Interestingly, the pre- culture period markedly influenced gene delivery efficiency in both leaves and roots ofA. thaliana. In general, without preculture poor gene delivery efficiencies were observed. The efficiency in each series of the accelerating pressure peaked with the sections that had been precultured for 3 to 4 days

Table 1. Effects of preculture period and accelerating pres- sure on the number of GUS expression units in Arabidopsis thaliana leaves I.

Preculture Accelerating pressure (kg/cm 2) (days)

85 115 150 200

0 1 7 ± 4 4 2 ± 16 2 6 ± 7 33± 15 1 7 0 ± 2 8 170±29 112± 1 6 9 ± 15 2 3 2 ± 2 1 177±33 1 6 1 ± 3 2 9 2 ± 17 3 3 5 7 ± 2 513± 105 464± 104 182±42 4 101 ± 4 2 2 9 9 ± 4 0 417± 19 159±34 5 4 8 ± 1 3 2 8 ± 9 6 3 2 7 ± 5 2 5 1 ± 2 6 5 8 ± 3 3 1 3 7 ± 5 0 2 7 8 ± 2 4 103± 1

Average of two experiments + standard deviation.

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Table 2. Effects of preculture period and accelerating pres- sure on the number of GUS expression units in Arabidopsis thaliana roots 1.

Preculture (days)

Accelerating pressure (kg/cm 2)

115 150 200

0 76 + 41 105 + 4 80 + 3 1 71 + 3 130 + 21 128 + 46 2 79 + 22 138 + 49 176 + 69 3 152 + 1 186 + 4 191 + 23 4 8 5 + 4 9 90+ 1 9 3 + 3 1 5 27+ 1 5 4 + 9 5 8 + 5 6 1 4 + 5 3 2 + 4 33+ 1

i Average of two experiments + standard deviation.

(leaf sections) or for 2 to 3 days (root sections), and then decreased with the sections precultured for a longer period of time (5 to 6 days for leaves, 4 to 6 days for roots).

This 'transient' dependence of gene delivery efficiency on the preculture period observed in these two different types of tissues indicates that preculture period is a vital factor for biolistic transformation of tissue sections of plants. One explanation might be that the activation of cellular or nuclear machinery of the leaf and root cells in response to exogenous plant hormone increases transient expression efficiency of the introduced gene. It is conceivable that callus for- mation itself is inhibitory to biolistic gene delivery because its efficiency decreased with the sections after a longer period of preculture.

The optimum accelerating pressure in each series of the preculture periods was observed to be higher in roots than in leaves; an accelerating pressure of 150 or 200kg/cm 2 gave the best results with roots and a pressure of 115 or 150 kg/cm 2 for leaves (see Tables 1 and 2). This suggests that cell walls of roots are harder to penetrate than leaf cells. We have previously reported a similar dependence of biolistic trans- formation of cultured tobacco cells on accelerat- ing pressure where the best results were obtained with 150 kg/cm 2 [4]. These results show that adjustment of the accelerating pressure depend-

262

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ing on the type of the tissues or cells is a vital factor for biolistic t ransformat ion .

In A. thaliana race Landsberg erecta leaves, the highest number of G U S expression units 513 + 105 per ca. 360 mg leaf sections) was observed at an accelerating pressure of 115 kg /cm 2 and after 3 days of preculture (Table 1). Also, in A. thaliana race Columbia leaves, 621 G U S expression units were observed at an accelerating pressure of 115 kg /cm 2 and after 4 days ofprecu l tu re (unpublished results). In A. thaliana race Landsberg erecta roots , the high- est number of G U S expression units (191 + 23 per ca. 150 mg root sections) was observed at an accelerating pressure of 200 kg /cm 2 after 3 days of preculture (Table 2). Also, in B. napus stems, 384 G U S expression units were observed at an accelerating pressure of 190 kg /cm 2 after 2 days of preculture.

We are currently studying the product ion of transgenic plants ofA. thaliana f rom the roots and leaves that are biolistically delivered with the neo- mycin phospho t rans fe rase gene under the opt imal b o m b a r d m e n t condit ions determined in this

study.

Acknowledgments

We thank Dr Hossa in (Universi ty of Kyoto , J a p a n ) for his help in the exper iment with Brassica

napus. This work was suppor ted by a grant f rom the Y a m a d a Science Founda t ion and by Gran t s - in-Aid f rom the Minis try of Educat ion, Science and Culture of Japan .

References

1. Bruce WB, Christensen AH, Klein T, Fromm M, Quail PH: Photoregulation of a phytochrome gene promoter

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from oat transferred into rice by particle bombardment. Proc Natl Acad Sci USA 86:9692-9696 (1989).

2. Christou P, McCabe DE, Swain WF: Stable transfor- mation of soybean callus by DNA-coated gold particles. Plant Physiol 87:671-674 (1988).

3. Gamborg OL, Miller RA, Ojima K: Nutrient require- ments of suspension cultures of soybean root cells. Exp Cell Res 50:151-158 (1968).

4. Iida A, Seki M, Kamada M, Yamada Y, Morikawa H: Gene delivery into cultured plant cells by DNA-coated gold particles accelerated by a pneumatic particle gun. Theor Appl Genet 80:813-816 (1990).

5. Iida A, Yamada Y and Morikawa H: Stable transfor- mation of cultured tobacco cells by DNA-coated gold particles accelerated by gas-pressure-driven particle gun. Appl Microbiol Biotechnol 33:560-563 (1990).

6. Klein TM, Fromm M, Weissinger A, Tomes D, Schaaf S, Sletten M, Sanford JC: Transfer of foreign genes into intact maize cells with high-velocity microprojectiles. Proc Natl Acad Sci USA 85:4305-4309 (1988).

7. Klein TM, Gradziel T, Fromm ME, Sanford JC: Factors influencing gene delivery into Zea mays cells by high- velocity microprojectiles. Biotechnol 6:559-563 (1988).

8. Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P: Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci USA 85:8502-8505 (1988).

9. Klein TM, Kornstein L, Sanford JC, Fromm ME: Gen- etic transformation of maize cells by particle bombard- ment. Plant Physiol 91:440-444 (1989).

10. Klein TM, Wolf ED, Wu R, Sanford JC: High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70-73 (1987).

11. Mendel PR, Muller B, Schulze J, Kolesnikov V, Zelenin A: Delivery of foreign genes to intact barley cells by high-velocity microprojectiles. Theor Appl Genet 78: 31-34 (1989).

12. Morikawa H, Iida A, Yamada Y: Transient expression of foreign genes in plant cells and tissues obtained by a simple biolistic device (particle-gun). Appl Microbiol Biotechnol 31 : 320-322 (1989).

13. Murashige T, Skoog F: A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962).

14. Wang Y-C, Klein TM, Fromm M, Cao J, Sanford JC, Wu R: Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment. Plant Mol Biol 11:433-439 (1988).