The Effect of Electric Field on Callus Induction with Rape Hypocotyls Maria Fileka,*, Magdalena Hołdaa, Ivana Macha´cˇkova´ b, and Jan Krekuleb a Institute of Plant Physiology, Polish Academy of Sciences, 30-239 Krako´ w, Niezapominajek 21, Poland. Fax: +48124253320. E-mail: [email protected] b Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova´ 135, 160502 Praha 6, Czech Republic *Author for correspondence and reprint requests Z. Naturforsch. 60c, 876Ð882 (2005); received April 11/May 19, 2005 The influence of electric field treatment on dedifferentiation and calli formation on rape hypocotyls was investigated. Segments, 10 mm long, of the upper part of rape (Brassica napus L., cv. Go´ rcza∆ski) hypocotyls were stimulated by different combinations of voltage/time (1.5 V/120 h, 3 V/3 h, 10 V/15 min and 30 V/30 s) under in vitro conditions. With all electric field treatments, segments oriented with their apical part towards the cathode produced more calli as compared to control (non-treated with electric field). Under opposite orientation slight inhibition of callus growth was observed. As the strongest effect on callus growth was observed after treatment with 30 V/30 s, this electric field treatment was selected for follow- ing analyses: the incorporation of [14C]-2,4-D (2,4-dichlorophenoxyacetic acid) and [14C]- BAP (benzylaminopurine) from the culture medium, changes in ACC (1-aminocyclopropane- 1-carboxylic acid) level and the redox activity in apical and bottom parts of hypocotyls during 18 d of culture. In contrast to changes in fresh weight, electric field treatment (30 V/30 s) stimulated a higher accumulation of 2,4-D and BAP in basal parts of hypocotyls than in apical ones. Moreover, orienting the apical part towards the cathode resulted in lower uptake of hor- mones as compared with the opposite orientation. The ACC concentration increased, espe- cially in the basal parts of hypocotyls, independently on electric field application. However, the highest level was observed after electric field treatment with orientation of the apical part towards the anode. The distribution of oxidative substances (measured as the amount of ferric ions) between the apical and bottom part of hypocotyls was not changed when the apical parts were oriented towards the cathode. Under these conditions a decrease in apical and an increase in basal parts was observed during culture. Opposite orientation influenced the redistribution of oxidative substances from the first day of electric field treatment. Based on these results we suggest that electric field action can be connected with its influence on specific concentration of oxidative substances and hormone distribution in cells. Key words: Electric Field, Rape, Growth Regulators Introduction critical value, mechanical instability and electro- poration (pore formation) of membranes occur The physical properties of lipids and proteins, (Wothers et al., 2001). the main compounds of biomembranes, predeter- It was observed in animal cells that exogenous mine the membranes to originate an electric field electric fields induce a variety of cellular re- (Chermomordic et al., 1987; Tsong and Astumian, sponses. It can influence cell surface charge redis- 1988). Endogenous currents occur in the form of tribution (Cho et al., 1994), cytoskeletal reorgani- transcellular potentials (Mycielska and Djamgoz, zation (Cho et al., 1996) and changes in the 2004). These electric fields are believed to play a intracellular calcium ion concentration (Wallec- significant role in biological processes (Le Saux zek, 1992). Because Ca2+ ions regulate numerous et al., 2001). Long-standing interest in the effect of biological processes including signal transduction electric fields was concentrated on animal systems cascades, cell orientation and migration and cell (Robinson, 1985). However, analysis of its influ- differentiation and proliferation, changes in Ca2+ ence on biological molecules raises several prob- concentration have been hypothesized to mediate lems. The main experimental problem is to apply cellular effects induced by an exogenous electric a transmembrane electric potential of comparable field (Cho et al., 1999). magnitude as the one existing in physiological In our earlier studies we observed the interac- conditions. When the applied potential exceeds a tion of an external electric field with generative 0939Ð5075/2005/1100Ð0876 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D M. Filek et al. ·The Effect of Electric Field on Callus Induction 877 development in plants (Macha´cˇkova´ et al., 1990; Materials and Methods Macha´cˇkova´ and Krekule, 1991; Filek et al., 2002, Twenty seeds of the winter rape (Brassica napus 2003). The final effect in the number of flowering L.) cultivar “Go´ rczan´ ski” were sown in Magenta plants was dependent on voltage and time of expo- (Sigma) vessels on Murashige-Skoog (Murashige sition of tissues as well as on the electric field po- and Skoog, 1962) medium and grown under light larity. Inhibition of bud formation and flower in- condition 16 h day/8 h night (150 µmol mÐ2 sÐ1)at duction was caused by direct current treatment 25/18 ∞C. After 7 d of culture segments of 10 mm with the cathode connected to the leaf tips in Che- were cut off from the upper part of hypocotyls (ex- nopodium rubrum (Adamec et al., 1989) or to the plants). These explants were placed into Magenta apical stem part in winter wheat and rape (Filek (Sigma) vessels equipped with two parallel silver et al., 2002, 2003) (the anode was in the nutrient electrodes (0.5 mm diameter, 50 mm long, distance solution around the roots). It was also described between them: 50 mm) dipped in the medium. To that an applied electric field induced a gradient 2+ induce dedifferentiation and callus induction, the of Ca ions from apex to base in Chenopodium medium was supplemented with 1 mg/l BAP and rubrum plants (Macha´cˇkova´ et al., 1995). Experi- 0.5 mg/l 2,4-D. Explants were placed in parallel to ments performed in various systems implicated lines of force of the electric field with the apical calcium in the hormonal signal transduction chain part toward the cathode or anode. The electric po- (Hobbie et al., 1994; Staxe´n et al., 1999; Anil and tential stimulation was applied after a 24 h culture Rao, 2000; Yang and Poovaiah, 2000; Pandey et al., as one direct electric current impulse. At the be- 2004). Moreover, electric field-induced changes in ginning explants were subjected for varying membrane potential can mediate the activation of lengths of time to electric current of different volt- plasma membrane hormone receptors (Barbier- ages. Four different voltage/duration combinations Brygoo et al., 1991; Astumian et al., 1995). It was were applied: 1.5 V/120 h, 3 V/3 h, 10 V/15 min also documented that an increase in auxin concen- and 30 V/30 s (see Fig. 1). Plants not stimulated by tration stimulates the activity of plasma membrane + the electric field were used as control. Fresh H -ATPase, which results in cell acidification weight of callus initiated on apical or bottom parts (Hager et al., 1991). The details of plant hormone of hypocotyls was determined after 4 weeks of cul- action remain largely unknown, however, their ture. On the basis of this experiment the electric participation in diverse aspects of plant growth field of 30 V lasting 30 s was chosen for further and differentiation is well documented. Especially studies. in in vitro experiments, the specific hormone com- In the following studies fresh weights of apical position of the medium is needed to induce both and basal part of in vitro cultured segments of hy- callus production on plant explants and embryo- pocotyls (hypocotyls were cut to two equal seg- genesis. ments) were measured after 1, 4, 7, 11 and 18 d The aim of presented experiments was to study after electric field treatment (30 V/30 s). In some the influence of an electric field on tissue dediffer- experiments the Murashige-Skoog medium was entiation and hormone redistribution during callus additionally supplemented with [14C]-2,4-D or formation on the segments of rape hypocotyl. At [14C]-BAP (radioactivity of both hormones was first the effect of voltage and time of the applied 380 MBq mmolÐ1). The samples from about 20 electric field on calli induction/inhibition was segments were collected in 5 replicates to measure tested. In the conditions of specific electric field 14 fresh weight and hormone incorporation. action the incorporation of [ C]-2,4-D (2,4-dichlo- 14 14 14 The accumulation of [ C]-2,4-D and [ C]-BAP rophenoxyacetic acid) and [ C]-BAP (benzylami- in apical and basal parts of hypocotyls was meas- nopurine) was detected. Both hormones are usu- ured after washing the tissue (3 times) in 0.6 m ally added to medium during rape in vitro cultures. mannitol and carefully drying to remove the ad- Because an electric field as well as auxins can in- sorbed substances. Then the tissue was homoge- duce changes in membrane potential and ion nized in the Bray solution and the radioactivity channel activities, the redox activity was also de- was detected by a scintillation counter (Beckman tected. To assess the possible stress effect of the LS 5801). treatments with an electric field, the level of ACC For detection of redox activity, hypocotyl seg- (1-aminocyclopropane-1-carboxylic acid), precur- ments were cut to apical and basal parts and incu- sor of ethylene, was measured. bated for 15 min in 1 mm Tris-HCl [tris(hydoxy- 878 M. Filek et al. ·The Effect of Electric Field on Callus Induction methyl)aminomethane hydrochloride] (pH 7.2) containing 0.5 mm CaCl2 and 50 mm KCl. Redox reaction was initiated by introducing 1 mm K3Fe(CN)6 and followed as the decrease in ab- sorbance at λ = 420 nm (Federico and Giartosio, 1983).