Plant Growth Regulation 38: 45 49, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 45 Local anaesthetic lidocaine modulates epiphyllous bud differentiation in Kalanchoe pinnata N. Sawhney 1 and S. Sawhney 2, * 1 S.S.N. College, University of Delhi, Alipur, Delhi, India; 2 Department of Botany, University of Delhi, 110 007, Delhi, India; *Author for correspondence (e-mail: sudhirsawhney@rediffmail.com; phone: 91-11-766 7725 ext. 1435, 91-11-544 0850 (res.)) Received 3 January 2002; accepted in revised form 7 February 2002 Key words: Epiphyllous bud differentiation, Kalanchoe pinnata, Lidocaine, Local anaesthetic Abstract A single treatment with lidocaine (2-Diethylamino-N-[2,6-dimethylphenyl]-acetamide) a potent local anaesthetic (LA), caused significant and extensive inhibition of epiphyllous bud differentiation in Kalanchoe pinnata, in a long term irreversible manner. These effects were both concentration and treatment duration dependent, with either variant generating similar and additive effects. The growth responses studied had differential sensitivity towards the applied dosage of the anaesthetic. Lidocaine also appears to influence expression of the organogenic potential of buds since for all inhibitory doses a significant percentage produced shoots but not roots. Abbreviations: LA local anaesthetic Introduction Local anaesthetics (LAs) are drugs that block nerve conduction when dispensed locally to the nerve tissue, resulting in both sensory and motor paralysis. These are considered to do so by acting at the cell membrane level by decreasing/preventing the large transient increase in permeability to Na + produced by slight polarization of membranes (Strichartz and Ritchie 1987). Few attempts have been made to investigate the influence of LAs on plant systems, obviously due to an apparent lack of a parallel neural system. A notable exception has been in the case of tactile and thermal responses of the sensitive plant Mimosa pudica where the transmission of electrical signals through the petiole affects the regulation of folding/unfolding movements of its leaflets, a situation nearest to the operation of potentials in nerves (Fromm 1991; Sibaoka (1966, 1969)). An exposure to anaesthetics, both inhalational and local, reversibly inhibited the motor mechanism of this plant (Milne and Beamish 1999; Okazaki et al. 1993). Since all higher plants utilize action potentials in regulating a variety of their physiological responses (Fromm 1991; Pickard 1973; Shepherd 1999; Sibaoka (1966, 1969)) and a wide range of cellular processes, even in non-nerve tissues, are influenced by LAs acting as calmodulin antagonists (Volpi et al. (1981), and references cited therein), work on the effect of LAs on diverse plant systems, the nature of their influence and probable mode of action, is warranted. In the present study, the effect of lidocaine the most widely used LA (Courtney and Strichartz 1987; Ritchie and Greene 1991), on epiphyllous bud differentiation in Kalanchoe pinnata has been investigated. In this plant, epiphyllous bud primordia lodged in marginal leaf notches are morphologically mature but physiologically dormant and differentiation is initiated in response to leaf detachment (Choudhary 1997; Karpoff 1982; Naylor 1932; Ross 1970). One cm diameter marginal leaf discs, each with an epiphyllous bud primordium, has been standardized as a model system that responds well to external stimuli (Sawhney et al. 1994).
46 Materials and methods Plant material and preparation of leaf discs From healthy garden grown vegetative plants of Kalanchoe pinnata, 35.40 ± 2.40 cm tall with 30.70 ± 2.20 unfolded leaves, 3 rd and 4 th leaf pairs (from top) were harvested. These were washed with diluted Teepol, rinsed thoroughly under running tap water, surface sterilized with ethyl alcohol and finally rinsed with sterilized double distilled water. One cm diameter marginal leaf discs each harboring a dormant epiphyllous bud, were made following the method described earlier (Sawhney et al. 1994). Lidocaine treatment and culture Lidocaine was dispensed as single treatments. For this, 16 sets each with 20 leaf discs were initially floated on 1, 2, 3 and 4% aqueous solutions of lidocaine hydrochloride separately for 1, 2, 3, and 4 h in each concentration. Subsequently the discs were thoroughly washed with sterile double distilled water and cultured on 0.8% agar in sterilized petriplates. An additional set of 20 water-treated discs was directly cultured on agar to serve as control. During treatment and culture, leaf discs were placed abaxially on the medium. Cultures were maintained at 25 ± 1 C under continuous illumination for 21 days. The experiment was repeated under identical conditions. Observations and statistics Observations were recorded for percent buds rooted and sprouted (those producing a shoot), total number of roots differentiated, maximum root length, shoot length and number of leaves differentiated on the shoot, after 21 days. All data presented are the mean values of two experiments. Values are presented as mean ± standard error of the mean (SE). Results Percent buds rooted and sprouted All water-treated (control) buds differentiated roots as well as shoots. Treatment of buds with lidocaine brought about significant inhibition of organogenesis, the effect increasing with both, the concentration as Table 1. Effect of lidocaine (concentration vs duration of treatment) on percent epiphyllous buds rooted and sprouted * (within parentheses). Lidocaine Duration of treatment concentration 1 h 2 h 3 h 4 h 0 100 1% 100 100 75 25 (90) (80) 2% 100 40 25 0 (90) (70) (0) 3% 100 40 0 0 (70) (0) (0) 4% 65 30 0 0 (55) (0) (0) * those producing a shoot. well as duration of treatment, although the magnitude of the effect varied for the two organs. Treatment with the anaesthetic at 1% level caused lowering of percent buds rooted when given for at least 3 h, whereas at 2% and 3% levels when given for 2 h and at 4% level when given even for one h (Table 1). The trends of results for percent buds sprouting a shoot in response to lidocaine treatment, were similar to those for rooting although the inhibitory effect of increasing duration of treatment was relatively less pronounced with its rising concentration (Table 1). It was observed that all buds receiving the anaesthetic at 2% level for 4 h and those receiving at 3 and 4% levels for 3 h and longer, died (lethal dosage) and therefore no data for any of the growth responses studied could be recorded for these treatments. It was also observed that for all inhibitory doses of lidocaine, a significant percentage of sprouted buds (with a shoot) did not root. Number of roots differentiated Although 1% lidocaine only slightly reduced root number when given for 1 h, it became increasingly inhibitory when the duration of treatment extended to 2 h and longer. Similarly, an increase in concentration from 1 to 2% for respective durations of treatment upto 3 h significantly reduced the number of roots produced, whereas a further rise in concentration could only slightly strengthen the effect (Table 2).
47 Table 2. Effect of lidocaine (concentration vs duration of treatment) on rooting characteristics of epiphyllous bud differentiation in Kalanchoe pinnata. Figures in parentheses are values relative to control taken as 100. Lidocaine Duration of treatment concentration 1 h 2 h 3 h 4 h Number of roots differentiated 0 4.40 ± 0.02 1% 3.95 ± 0.06 2.80 ± 0.08 1.50 ± 0.06 0.30 ± 0.04 (89.78) (63.64) (34.09) (6.81) 2% 1.90 ± 0.03 0.80 ± 0.05 0.50 ± 0.04 (43.18) (18.18) (11.36) 3% 1.80 ± 0.07 0.60 ± 0.05 (40.91) (13.63) 4% 1.20 ± 0.05 0.53 ± 0.06 (27.27) (12.04) Maximum root length 0 2.80 ± 0.05 1% 0.72 ± 0.01 0.28 ± 0.01 0.08 ± 0.02 0.08 ± 0.02 (25.71) (9.99) (2.85) (2.85) 2% 0.26 ± 0.01 0.22 ± 0.02 0.01 ± 0 (9.28) (7.85) (0.35) 3% 0.26 ± 0.01 0.22 ± 0.02 (9.28) (7.85) 4% 0.26 ± 0.02 0.21 ± 0.01 (9.28) (7.49) Maximum root length Treatment of buds with 1% lidocaine significantly reduced root length when given for 1 h, the effect increasing with the duration of treatment up to 3 h. A similar reduction in root length was seen with a rise in concentration from 1 to 2% for respective durations of treatment, although a further rise in concentration did not enhance the effect (Table 2). Shoot length Treatment with lidocaine also reduced the shoot length. The inhibition could be seen with 1% lidocaine dispensed for 1 h, the effect strengthening further with the duration of its treatment. A similar reduction of shoot length was also observed with an increase in concentration from 1 to 2%, for respective durations of treatment. A further rise in concentration did not enhance the effect (Table 3). Number of leaves differentiated Treatment of buds with lidocaine reduced the number of leaves produced on the shoot, the effect increasing with both the duration as well as the concentration of the anaesthetic treatment (Table 3). Discussion The results obtained in the present investigation show that a single application of lidocaine a potent local anaesthetic aminoethylamide caused a strong inhibition of epiphyllous bud differentiation on leaf discs of Kalanchoe pinnata. In fact it caused inhibition of all studied aspects of bud differentiation that included the number of roots differentiated, maximum root length attained, shoot length and number of leaves differentiated. The lidocaine effects were both concentration and treatment duration dependent, thus getting severer with rise in its concentration from 1 to 4% as well as the period for which dispensed from 1 to 4 h. A pe-
48 Table 3. Effect of lidocaine (concentration vs duration of treatment) on sprouting characteristics of epiphyllous bud differentiation in Kalanchoe pinnata. Figures in parentheses are values relative to control taken as 100. Lidocaine Duration of treatment concentration 1 h 2 h 3 h 4 h Shoot length 0 0.30 ± 0.05 1% 0.20 ± 0.06 0.10 ± 0.01 0.05 ± 0 0.01 ± 0 (66.66) (33.33) (16.66) (3.33) 2% 0.10 ± 0 0.02 ± 0 0.01 ± 0 (33.33) (6.66) (3.33) 3% 0.10 ± 0 0.01 ± 0 (33.33) (3.33) 4% 0.10 ± 0.08 0.01 ± 0 (33.33) (3.33) Number of leaves differentiated 0 3.71 ± 0.36 1% 2.94 ± 0.61 2.30 ± 0.08 1.90 ± 0.60 0.01 ± 0 (79.23) (61.98) (51.20) (0.26) 2% 2.63 ± 0.67 1.50 ± 0.60 0.01 ± 0 (70.87) (40.42) (0.26) 3% 2.00 ± 0.79 0.40 ± 0.04 (53.90) (10.78) 4% 1.30 ± 0.04 (35.03) rusal of data also shows that the concentration vis a vis duration of treatment produced identical and additive effects. This is exhibited even for its lethality, thus a dosage of 3% for 3hor2%for4hwere fatal treatments. The results show that the various growth responses studied had differential sensitivity towards applied dosage of lidocaine. For example, by 1 h exposure of buds to the anaesthetic treatment at a concentration of 1 %, the root length got most affected, followed by other responses in order of decreasing sensitivity, as given below, root length shoot length number of leaves number of roots For these responses the relative percent inhibitions caused by lidocaine, if calculated as reciprocals of their relative values would be 74.29, 33.34, 20.77 and 10.22, respectively. Thus, the root lengthening was more than twice as sensitive as shoot lengthening to the applied anaesthetic, although both are manifestations of extension growth. However, it remains to be seen whether this growth retardation is due to reduced rate of cell divisional or cell enlargement activity or both. There is a differential sensitivity in the expression of organogenic potential of buds as for all inhibitory doses of lidocaine, a significant percentage of sprouted buds (those producing a shoot) did not root. The above effects are long-term, irreversible morphogenetic modulations involving altered gene expression induced by an anaesthetic agent, unlike the blockade of nerve conduction in animals (Courtney and Strichartz 1987; Ritchie and Greene 1991; Strichartz and Ritchie 1987) and motor mechanism of the sensitive plant Mimosa pudica (Milne and Beamish 1999; Okazaki et al. 1993) operating primarily through action potentials of the membranes. Nevertheless, the two mechanisms need not be mutually excluding since the elicitor-induced membrane perturbations can eventually through a sequel of signal transductional events lead to an altered gene expression with morphogenetic consequences. Experiments are in progress to ascertain these possibilities.
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