TRANSIENT GENE EXPRESSION IN ELECTROPORATED INTACT TISSUES OF Stylosanthes guianensis ( AUBL . ) SW . 1

Genetic transformation though protoplast electroporation has been established for commercially important plant species. In this work, explant sources, electric field strengths, electroporation buffers, DNA forms and osmotic pretreatment were assayed in order to optimize transient reporter gene expression in electroporated tissues of Stylosanthes guianensis, a tropical forage legume. Higher transformation rates were obtained employing cotyledonary explants and an electric field strength of 250 V cm. Linear plasmid DNA, chloride-free electroporation buffer and osmotic pretreatment with 1.6 mol L mannitol also improved transient transformation but non-significantly. Transgene specific PCR amplification was employed to prove the transformed status of the tissues.


INTRODUCTION
Genetic transformation through protoplast electroporation has been established for commercially important plant species, such as Brassica napus (Guerche et al., 1987), maize (Rhodes et al., 1988) and rice (Shimamoto et al., 1989), but requires (i) an effective regeneration protocol from protoplast to fertile nonchimerical transgenic plants; (ii) development of genotype-specific regeneration protocols; (iii) long periods of in vitro cultivation which may induce genetic and epigenetic alterations; (iv) intense and expertise work.
DNA introduction into organized tissues is a promising means to avoid the forementioned constraints of protoplast electroporation and easily obtain wholly transformed plants (Lin et al., 1997).
Genetic transformation of Stylosanthes guianensis (Aubl.)Sw., a tropical forage legume, has been achieved with Agrobacterium infection, but at low efficiency (Wordragen & Dons, 1992;Sarria et al., 1994).Although being considered one of the less recalcitrant genus of legume to in vitro culture (Vieira et al., 1990), direct gene transfer has never been employed in Stylosanthes.Calli can be obtained from various explant sources, and they can further be induced to plant regeneration via organogenesis (Dornelas et al., 1992), thus suggesting that direct tissue electroporation could be effective to generate transformants.
We have evaluated the factors affecting plasmid DNA introduction and expression into organized tissue of Stylosanthes guianensis (Aubl.)Sw. and provided discussions about the effects of electric pulses on intact plant cell walls.To our knowledge, this is the first report of direct gene transference to this species.

Plant material and tissue cultures
Seeds of Stylosanthes guianensis cv.Mineirão were in vitro germinated on half-strength MS medium (Murashige & Skoog, 1962) as described by Dornelas et al. (1992), at a density of 15 seeds per flask.Hypocotyls and fully expanded cotyledons were excised from 7 to 8 day old seedlings and completely developed leaves from 14 to 15 day old ones.Explants were cut into segments of approximately 2 mm 2 .
After electroporation, the explants were thoroughly washed with autoclaved water, dried on sterile filter paper and placed on MS3 (MS salts, 3% sucrose, 0.18% Phytagel) during two days for the β-glucuronidase (GUS) test (Jefferson et al., 1987) or 14 days for polymerase chain reaction (PCR) analysis.The longer period of in vitro culture of the explants destined to PCR analysis was employed in order to rule out the possibility of amplification of non-transformed, surface contaminant DNA.

Plasmid
Transformation vector pCambia 1301 (CAMBIA GPO Box 3200, Canberra ACT 2601, Australia) carrying β-glucuronidase (GUS) -uidA, driven by 35S promoter from cauliflower mosaic virus (CaMV) (Figure 1).The presence of a catalase intron into the coding sequence of uidA rules out the possibility of reporter gene expression in transformed endophytic microorganisms.The plasmid was transformed to E. coli DH5α (Hanahan, 1983) and isolated by alkaline lysis (Sambrook et al., 1989).Supercoiled or plasmid DNA linearized through digestion with EcoRI (GIBCO BRL) was employed in transformation experiments.

Pre-pulse treatments
Hypocotyls (H), cotyledonary (C) and leaf (L) sections were presoaked in 5 mL of electroporation buffer supplemented with 0, 0.2, 0.8 or 1.6 mol L -1 mannitol for 20 min at 0 o C. Prior to pulsing, 10 µg of supercoiled or linear plasmid DNA were added to each 800 µL mixture of electroporation buffer and explants.The cuvettes were kept on ice for 10 min before pulsing.
Sample resistances were 20 Ω (buffer I and III) and 50 Ω (buffer II) as determined by the device measurements.The time, expressed as pulse decay constant, established by internal settings of the apparatus was τ = RC.

PCR analysis
After 14 days of culture in MS3, genomic DNA was isolated as described by Edwards et al. (1991) from randomly chosen samples of electroporated explants and non-electroporated controls.
Each 25 µL reaction mixture contained 20ng of genomic DNA, 10 mM TRIS-HCl (pH 8.4), 50 mM KCl, 2.0 mM MgCl 2 , 160 µM of each dNTP, 1.0 U of Taq polymerase (GIBCO BRL) and 200nM of each uidA specific primer (gus 1 -CCT GTA GAA ACC CCA CAA CG and gus 2 c -TGC AGC GCT ACC TAA GGC CG) (Figure 1), which provide an amplification product of 795 bp.The mixture was overlaid with sterile mineral oil and submitted to denaturation (2 min at 95 o C) followed by 25 cycles of amplification (1 min at 94 o C, 1 min at 45 o C and 1.5 min at 72 o C) and a final extension (7 min at 72 o C) in thermal cycler (Perkin Elmer Cetus).

Statistical procedures and data analysis
A cuvette containing ten explants (approximately 2 mm 2 ) was considered a replicate, and three replicates were evaluated for both GUS and PCR analysis in a randomized design experiment.
GUS stained areas were visually evaluated and attributes were given from 0 to 5 according to their size, intensity and frequency per explant.Data are the mean number of three independent replicates (30 explants) ± se (standard error).Mean comparisons were performed employing the Tukey test (1% and 5% probability).

Effect of explant type on transgene expression
Cotyledons showed the highest levels of GUS expression (Figures 2A and 3A) independent of the buffer employed, DNA form, osmotic treatment and electric field strength, followed by hypocotyl-derived explants (Figures 2B and 3A).Leaves showed the lowest transformation rates (Figures 2C and 3A).Songstad et al. (1993) also reported low levels of expression in electroporated leaves of maize embryos.Qualitatively, as evaluated by the arbitrary expression scale, leaves also showed poor transformation rates (Figure 3B).
Pools of DNA from 20 randomly chosen explants of all transformed tissues showed PCR amplification of the transgene after 14 days of in vitro culture from two independent experiments (Figure 4), confirming their transformed status.

Optimizing field strength to foreign DNA deliver
Fixed discharge capacity was employed (C = 900 µF) in an RC circuit, thus the energy input was correlated to the applied voltage (E = ½ CV 2 ) and ranged from 180 kJ (at 50 V cm -1 ) to 8820 kJ (at 350 V cm -1 ).The strength of the electric field promotes pore formation in cellular membranes (Kinosita & Tsong 1977;Neumann et al., 1982), alters the viscosity of homogalacturan gel-like structure (Zablackis et al., 1995) and affects ionic interactions of positively-charged lysine and histidine residues from extensins to negatively-charged uronic acid from pectin (Showalter, 1993;Reiter, 1998) allowing macromolecular migration across these structures.
Highest transformation levels were observed employing electric fields ranging from 100 to 250 V cm 1 (Figure 5A).Discharges of 350 V cm -1 caused tissue injuries and reduced GUS expression levels, even when compared to low energy electric fields (50 V cm 1 ) (Figure 5A).
Cotyledon and hypocotyl-derived tissues showed approximately the same patterns of GUS expression, with the highest values ranging from 150 to 250 V cm -1 (Figure 5B), which are similar to those reported by Dillen et al. (1995) for bean embryonic axes.No GUS expression was observed with electric field intensities lower than 50 V cm -1 or higher than 350 V cm -1 .

Effect of electroporation buffer on DNA introduction
No statistical differences (P<0.05) were observed concerning explants electroporated with buffer I, II and III.Tada et al. (1990) reported increased efficiency employing electroporation buffer lacking chloride in the transformation of rice protoplasts, but it is very likely that the effect of reactive oxygen derived from chloride reaction is not so drastic in intact cell walls.However, qualitative analysis of GUS expression showed that chloride-free buffers II and III had greater number of explants included in expression level 4 and 5 of the arbitrary scale in electroporated Stylosanthes cotyledons (Figure 6).
The transient nature of membrane pores and structural alterations of the cell wall induced by electric fields imposes conformational restrictions to the macromolecules to be introduced (Neumann et al., 1982).

Influence of the osmoticum pretreatment on transient GUS expression
No statistically significant enhancement (P < 0.05) was observed in the frequency of gene expression (Figure 8A).However, 0.8 mol L -1 and 1.6 mol L -1 mannitol increased the number and intensity of blue spots, as evaluated by the arbitrary scale (Figure 8B).There was positive correlation (r 2 = 0.9129) between the osmotic concentration (x) and the number of explants expressing GUS (y), described as y = 8.82 x + 37.35.
Osmotic treatment of the target tissue has been reported to increase transformation rates from 7-to 10-fold in microorganisms (Armaleo et al., 1990;Shark et al., 1991) and plant cells (Russell et al., 1992;Vain et al., 1993).The increased distance between the cell wall and cytoplasm in plasmolyzed cells makes them less susceptible to organelle damage and cytoplasm loss when immersed in an electric field.

CONCLUSIONS
Intact tissues of Stylosanthes guianensis (Aubl.)Sw. can be transformed with plasmid DNA after electroporation.Transient reporter gene expression is dependent upon biological and physical parameters.The explant source and energy input are the most important.Higher rates of transformation were obtained employing cotyledonary explants and electric fields ranging from 100 to 250 V cm -1 .Electroporation buffer, DNA form and osmotic pretreatment were not critical to transient transformation efficiency.To our knowledge, this is the first report on direct gene transfer in Stylosanthes, demonstrating the applicability of intact tissue electroporation to genetic manipulation of the genus.
Due to its importance for Latin American countries, the development of Stylosanthes biotechnological procedures must respond to increasing demands in terms of sustainable use of plant genetic resources as basic inputs for the future agriculture (see Izquierdo & de la Riva, 2000).

Figure 3 -Figure 1 -Figure 4 -
Figure 3 -Effect of the explant source on GUS expression in electroporated tissues of S. guianensis.(A) Number of explants expressing GUS.(B) Classification of GUS positive explants from different sources according to the arbitrary scale.Electroporation conditions: 900 mF, 100 V cm -1 , 20 W, buffer I, 10 mg of supercoiled DNA, absence of osmotic treatment.Bars indicate se and small cap letters, significant differences (P<0.05).

Figure 6 -Figure 7 -
Figure6-Effect of electroporation buffer on GUS expression in electroporated tissues of S. guianensis.GUS positive explants electroporated with different buffers were classified according to the arbitrary scale.Electroporation conditions: cotyledons, 900 µF, 20 Ω (buffer I and III) and 50 Ω (buffer II), 100 V cm -1 , 10 mg of supercoiled DNA, absence of osmotic treatment.Bars indicate se.