Plant Cell Reports

Plant Cell Reports (1996) 15:924-928

9 Springer-Verlag1996

Transient and stable electrotransformations of intact black Mexican sweet maize cells are obtained after preplasmolysis N. Sabri 1, B. Pelissier 2, and J. Teissie 1 x Laboratoire de Pharmacologie et Toxicologie Fondamentale du CNRS, D~partement III 'Glycoconjugu~s et Biomembranes', t18, route de Narbonnc, F-3t062 Toulouse, France 2 D6partement de Biotechnologies, Rh6ne-Poulenc Agrochimie, Lyon, France Received 10 October 1995/Revised version received 1 February 1996 - Communicated by A. M. Boudet

Summary. When interested in plant cell transformation, the cell wall is often considered as a barrier to DNA transfer, which is only overcome by wounding or wall degrading enzymes. In this work, we demonstrate that cell plasmolysis before electropulsation is an efficient approach to DNA delivery into intact plant cells. Using such a method, transient expression (13-glucuronidase and chloramphenicol acetyltransferase) and stable expression (phosphinotricin acetyltransferase) of exogenous genes are obtained in intact black Mexican sweet maize cells. Abbreviations: BMS cells: Black Mexican Sweet cells, GUS: 13-glucuronidase, CAT: chloramphenicol acetyltransferase, PAT: Phosphinotricin acetyltransferase, MS: Murashige and Skoog, PCV: packed cell volume, 4MU and 4-MUG: 4-methylumbelliferone and 4methylumbelliferyl-glucuronide, BSA: bovine serum albumin, TTC: triphenyl tetrazolium chloride Introduction The incorporation of foreign gene sequences into plant cell genomes has become an important tool in the study of basic plant processes and in crop improvement. Due to the development of direct DNA delivery methods, cereal crop plant transformation is progressing rapidly. Ten years ago, maize transformation research focused on direct DNA delivery to protoplasts followed by cell regeneration (Fromm et al. 1986; Rhodes et al. 1988). Given the problems associated with regenerating plants from protoplasts (Potrykus 1990), other strategies have been developed with intact cells. Positive results were obtained with two different biophysical approaches. Direct DNA delivery by microprojectile bombardment was successful for the transformation of maize (Fromm et al. 1990; Gordon-Kamm et al. 1990; Waiters et al. 1992; Aves et al. 1992; Koziel et al. 1993). At the same time, positive results were reported using electrical methods. Dekeyzer et al. (1990) achieved transient expression of a reporter gene in electropulsed leaf base pieces of several monocot species in.eluding maize. Correspondence to: J. Teissie

Transient and stable transformations of maize via electropulsation after wall enzymatic treatment or a long incubation were described (D'Hallium et al. 1992; Songstad et al. 1993; Laursen et al. 1994; Pescitelli and Sukhapinda 1995). Recently, transgenic sugarcane plants were obtained (Arencibia et al. 1995) using the transformation procedure described by Dekeyser et al. (1990). All these reports indicate that in order to obtain plant cell transformation, it was always necessary to overcome the plant cell wall barrier by various pretreatments. This is a time consuming step during which cell viability can be affected. Various spaces and pores are indeed present in the plant cell wall. The size of these pores has been studied using hypertonic solutions of polyethyleneglycol or dextran (Carpita et al. 1979). They were found to be specific to species and tissues, nevertheless allowing 3-3.7 kDa PEG4000 and up to 6.56 kDa dextran molecules to pass through. Another study (Baron-Epel et al. 1988) suggested that the soybean cell wall allows dextran molecules as large as 41.0 kDa to pass through. More recently Wu and Cahoun (1995) reported that the passage of large macromolecules through the cell wall of onion, petunia and rice was greatly facilated by plasmolysis. It turns out that the cell wall, through which metabolites must pass in order to gain access to the cell cytoplasm, is not an impervious barrier. In the present work, plasmids were transfered into intact black Mexican sweet maize cells (BMS) simply by plasmolysis followed by electropulsation. 3 different plasmids: pRP-RD109 (6.32 kbp) encoding for ~glucuronidase activity (GUS), pBL345a (4.7 kbp) encoding for chloramphenicol acetyltransferase activity (CAT) and pJIT82 (4 kbp) encoding for phosphinotricin acetyltransferase activity (PAT) are expressed in intact BMS cells using this two step protocol. Materials and methods

Chemicals. MS culture medium, asparagine, 2,4-D, X-Glue, mannitol,

glucose, K+-aspartate, K+-glutamatc, Ca2+ glueonate, 4-MUG, MU, were purchased from Sigma (USA). Sucrose was purchased from

925 Fluka (Switzerland). All other chemicals and reagents were of analytical grade. Plant material. Black Mexican sweet (BMS) cell suspension culture was used for transformation. It was maintained in Murashige and Skoog culture medium: MS (Murashige and Skoog 1962) supplemented with 200 mg/l asparagine, 2 mg/l 2,4-dichlorophenoxyacetic acid and 30 g/l sucrose pH 5.8 at 25~ in the dark and shaked at 150 rpm (KS 501D, IKA-Labortechnik). BMS cells were subcultured every week in fresh MS medium. Plasmids used for transformation. 3 plasmids were used for transformation: pRP-RD109 and pBL345a for transient expression and pJIT82 for stable transformation. All p l a s m i d s contained the cauliflower mosaic virus 35S promoter, pJ'IT82 contained bar gene encoding for PAT activity, pRP-RD109 contained GUS gone and pBL345a contained CAT gone. These plasmids were given by RhrnePoulenc Agrochimie. Plasmids were purified on Qiagen columns. The plasmid DNA concentration and purity were determined spectrophotometrieaUy and checked by agarose gel electrophoresis. DNA electrotransfer into BMS cells. Plasmid DNA was isopropanol precipitated and resuspended at 1 mg/ml in sterile water. Plasmid DNAs were added to 100 p.1 aliquots of cell suspension at 25% PCV (packed cell volume) in electropulsation buffer. The DNA/cells were incubated at room temperature, The mixture was then electropulsed between two flat stainless-steel parallel electrodes (d= 4 ram) which were placed in contact with the bottom of a sterile Petri dish. Sterile conditions were obtained by working under a laminar flow hood (ESI, France). The electropulsator (Jouan, France) delivered a square wave pulse, the shape of which was controlled on line with an oscilloscope (Enertec, France). The field strength and the pulse duration could be adjusted independently, whatever the buffer conditions. Effect of osmotic pressure on cell transformation. BMS cells were incubated 30 min at room temperature in 10 mM Hepes pH 7.2, containing the osmoticum and plasmid DNA at 100 ].tg/ml. The products used as osmotic agent were mannitol at 0.2 M (M3) and 0.4 M (PM3) corresponding to 0.234 and 0.455 Osmol/kg respectively and NaC1 at 0.l M (NA) and 0.2 M (PNA) corresponding to 0.221 and 0.407 Osmol/kg respectively. Osmotic pressure was read on a cryoscopic osmometer (Osmomat 030, Gonotec, Germany). After incubation, cells were washed and then electropulsed by one 15 ms pulse at 750 V/cm in PM3 medium containing 100 l.tghnl DNA. A post pulse 15 rain incubation at room temperature followed before adding culture medium. Determination of GUS activity. GUS activity was assayed essentially as described by Jefferson (1987). Fluorimetric and colorimetric assays were used. Determination of CAT activity. After electropulsation, the cell suspension was diluted in fresh MS culture medium. 24 h after electropulsation, cells were frozen in liquid N2, thawed at 37~ for 5 min and vortexed, 3 times. Ceils were then centrifuged at 12000g for 15 rain at 4~ CAT activity was assayed in the supernatant according to the manufacturer's instructions(Biotechnology systems, NEN Research products) using 1 4 C acetyl coenzyme A and chloramphenicol as substrates. CAT activity is expressed as Acpm/mg protein/h. The protein concentration in crude extracts was determined by the Bradford assay using BSA as a standard (Biorad). Viability assay. Cell survival was measured by a triphenyltetrazolinm chloride (TTC) respiratory assay (Dixon 1985). This procedure was used with slight modifications. Briefly, 100 ~tl of electropulsed cell suspension were incubated with 100 lal of 2% TTC in 50 mM sodium phosphate buffer pH 7.2 for 24 h in the dark. Then the formazan was extracted with 1 ml 95% (v/v) ethanol and estimated spectrophotometrically at 495 nm. Selection of transformant callus After electropulsation, cells were dispensed onto filters (Whatman no 4) overlaying solid MS medium. After ten days, calluses on supporting filters were transferred to callus selection medium containing 3 mg/l bialaphos. Two weeks later, slowly growing tissues were removed from the filter and transferred as dumps to selective medium. Callus sectors, which continued to grow, were transferred to fresh selection medium every ten days. Determination of PAT activity. PAT activity was determined by an in vitro assay (Spenser et al. 1990). 14C acetylated phosphinotricin was visualized by an automatic TLC-linear analyser (Berthold, LB 2832) Southern blot analysis. Genomic DNA isolation was performed as described by Warren(1994). Approximately 20 p.g genomic DNA was digested with the restriction enzymes EcoRI and Hind III, separated by electrophoresis on a 1% agarose gel, transferred to a nyIon HybondN + membrane (Amersham) and hybridized with a 32p radioactive probe (the 0.6 kbp SmaI fragment from pJIT82 containing the bar gene), which was labelled with the Megaprime kit (Amersham).

Results Transient expression in intact BMS cells

Plasmolysis effect on electrotransformation: The effect of incubation of cells in plasmolysis medium prior to electropulsation was determined. BMS cells at 25% PCV were incubated with 100 l.tg/ml pRP-RD109 plasmid DNA in media at varying osmolarity levels and then electropulsed in PM3 buffer (one 750 V/cm pulse for 15 ms). Such a long pulse duration is known to be efficient for gene transfer. PM3 buffer was chosen because it gives high cell viability for electropulsed cells (25-40%). Results obtained indicate that when osmolarity is equal to 0.2 Osmol/kg, such as with M3 and NA media, no transformant was obtained. No GUS activity was detected when cells were incubated in MS culture medium, Blue spots indicating GUS activiy were obtained when cells were plasmolysed in PM3 and PNA media and then electropulsed in PM3 medium. The number of cell clusters that expressed GUS activity in PNA medium was approximately two times lower than in PM3 medium (about 6 blue spots per pulse). These media had the same osmolarity but cell viability was 4 times less in PNA medium than in PM 3 medium. PM3 electropulsation buffer was then used for subsequent transformation experiments. Electrotransformation as a function of pulsing conditions. GUS activity: The effect of elec~c field strength on GUS activity was also analysed (fig. 1A). There is no detectable GUS activity in cell extracts derived from cells incubated with DNA without pulses. The lowest GUS activity was detected at 350V/cm (one pulse lasting 15ms) and GUS activity increased with electrical field intensity. The effect of plasmid concentration on the level of GUS expression was determined using a fluorimetric GUS assay. Cells were electropulsed with a single 15 ms pulse at 750V/cm. Extractable activity increased with increasing concentrations up to approximately 100 ~tg/ml in cell suspension at 25% PCV (data not shown). At higher plasmid concentration, no further increase in GUS activity was observed. In all subsequent experiments, the plasmid DNA was added at 100 I.tg/ ml. While maximum activity was obtained 48h after electropulsation, GUS activity was detected up to five days after cell electropulsation (fig.2). The level of GUS activity was back to the background level seven days after the pulse. CAT activity: Cells were electropulsed in another plasmolysing buffer (Tada et al. 1990) in the presence of pBL345a DNA plasmid at 100 Ixg/ml. Viability of electropulsed cells in this medium was lower than in PM3 medium (data not shown). Results indicated that high CAT activity was associated with strong amplitude pulses (fig. 1B). No increase in CAT activity was detected when cells were pulsed at 350 V/cm. Background activity may be due to non-specific acetylases. Stable transformation Selection of stably transformed callus: Figure 3 shows calluses, derived from cells electropulsed with pJIT82 plasmid DNA, after 6 weeks in selective medium. Cells electropulsed without DNA did not divide and could not proliferate into callus on medium containing 3 mg/1

926 bialaphos. Cells electropulsed in the presence of pJIT82 plasmid showed some colonies that continued to proliferate one week after transfer to selective medium. These calluses were maintained in selective medium and then were subjected to molecular analysis.

herbicide revealed the same patterns (lanes 1-8). This indicated that transformed cells were from a single transformation event. Banding patterns indicated multiple insertions of the bar gene.

500

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20000

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NP 350 550 750 Electric field (V/cm) .

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Exp.3

Fig.2. GUS activity expressed in cells as a function of time after electric field treatment. Cells were electropulsed with one pulse of 15 ms at 750 V/era in the presence of pRP-RD109 plasmid (100 I.tg/ml). They were subcultured in MS medium for 2 days (black bars), 5 days (dashed bars) or 7 days (white bars) before GUS activity determination. 3 independent experiments were done. The control assay was conducted for cells electropulsed without DNA.

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Electric field (Were) Fig.1 (A): Expression of GUS activity as a function of electric treatment. MU production is measured by fluorimetric assay as described in Material and Methods. Each datum represents the mean of 3 replicates. (B): Expression of CAT activity in cell extracts The radioctivity of acetylated chloramphenicol is measured in extracts from electropulsed ceils with various electric field strengths. Controls were extracts from cells incubated in the presence of the plasmid but not electropulsed (NP) and extracts from ceils electropulsed without DNA (P).

Enzyme activity: Bialaphos-resistant colonies were checked for phosphinotricin acetyl-transferase (PAT) activity. PAT activity was found only in electrotransformed cells (fig.4). There was almost complete conversion of available acetyl-coenzyme A to Nacetyl-PPT. Molecular analysis: The presence of the bar gene was checked by Southern blot analysis. Results are shown in figure 5. There was no detectable hybridation of the radioactively labelled Sma I fragment in the case of control cells incubated with DNA without pulse (lane C). Analysis of eight phosphinotricin resistant calluses derived from an initial callus, which was resistant to the

Fig.3. Calluses phenotypically resistant to the Bialaphos. BMS cells were electropulsed with (I) or without pJIT82 plasmid 0I) containing the bar gene. After one week on non selective MS solid medium, calluses were transferred in MS medium containing bialaphos at 3mg,tl

Discussion In this study, electrotransformation of intact maize cell suspension without any pretreatment by wall degrading enzymes or wounding is reported. These results were obtained only when cells were plasmolysed before electropulsation. When plasmolysing PM3 medium was used and cell viability was not slrongly affected. While electroinduced cell permeability was not dependent on pulsing buffer, viability of cells was affected by the composition and the pH of the pulsing medium (data not shown). Positive results were obtained whatever the nature of the plasmolysing buffer or the plasmid DNA. This indicates that the DNA electrotransfer phenomenon is

927 accomplished if the passage through the cell wall is facilited (plasmolysis) and if the cell membrane is permeabilized (electropulsation). This does not depend on the nature of the plasmolysing medium and plasmid construction.

Control cells

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Fig.5. Southern blot analysis. Analysis was conducted on eight bialaphos resistant calluses derived from an initial resistant colony (lane 1-8). Lane C is obtained for DNA extracted from control cells. The arrow point to the location of the g d where a 0.6 kbp fragment containing the bar gene would migrate.

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q~ansformed cells

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_

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Fig.4. Analysis of PAT activity. Cell extracts were derived from electropulsed cells with (transformed cells) or without pJIT82 plasmid (control cells). PAT activity was detected as described in material and methods. Radioactivity is expressed in arbitrary units.

It was demonslrated that osmotic changes which create a void between the cell wall and plasmalemma, allowed macromolecules to pass through the cell wall, maximizing the contact of macromolecules with plasmalemma (Wu and Cahoun 1994). We suggest that hindrance to direct gene transfer in intact cells is not caused by the presence of the cell wall but by the low transfer of DNA molecules to the plasmalemma. The effect of osmotic pressure on DNA delivery has already been observed in other walled systems such as bacteria (Eynard et al. 1992). In this case, cells responded by a decrease in cytoplasmic volume and deformation of the membrane when submitted to an increase in external osmolarity. It was presumed that the osmolarity of pulsing buffer may affect the flow across the cell wall and, as a consequence, the transformation yield.

The present study describes a very fast protocol for transforming intact plant cells. Working on intact plant cells prevents all the problems described in the case of protoplasts (Fleck et al. 1979; Ingelbrecht et al. 1989; Loveys and Robinson 1987; Potrykus 1990; Vernet et al. 1982). Wounding cells or tissues induces the generation of defence mechanisms in the cell and negative effects on plasmid integrity may take place. Wall digestion requires careful control of timing and a tedious washing step. The two previously described approaches with intact cells were time consuming whereas the present protocol is run in 30 minutes. Positive results are obtained without special preparation of plasmids such as linearizing or coating of particles (biolistic). Gene electrotransfer to intact cells in plasmolysing medium appears as a promising approach for genetic plant transformation. Acknowledgements. "Fneauthors would like to gratefully acknowledge Dr J. Grima-Pettenati for helping to perform the southern blot analysis and Dr B. Bugler for her help in the CAT assay. We also thank Dr B. Gabriel for performing the Southern scan, B.Lagagne for technical assistance and Mr J. Robb who checked the english. This study was conducted under the Bioavenir programme funded by Rhtne-Poulenc, the Minist~re de la Recherche et de !'Espace and the Minist~re de l'Industrie et du Commerce Exttrieur.

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