Introduction

In 1987, a new gaseous signaling molecule was introduced to the biological world when endothelium derived relaxation factor (EDRF) in mammals was identified as nitric oxide (NO), a gas until than considered obnoxious (Ignarro et al. 1987). NO is a small, highly diffusible and ubiquitous bioactive molecule. Its chemical properties make NO a versatile signaling molecule that functions through interactions with cellular targets via either redox or additive chemistry (Stamler et al. 1992). It plays important role in a number of biological processes in both plants and animals (Wilson et al. 2008). In mammals, it is involved in the central nervous, cardiovascular and immune systems; platelet inhibition, programmed cell death and host responses to infection (Crawford 2006). It has turned out to be an important regulatory molecule in growth and development of plants and their responses to biotic and abiotic stresses (Delledonne 2005; Arasimowicz and Floryszak-Wieczorek 2007). Its involvement has been shown in seed germination, hypocotyl elongation (Beligni and Lamatina 2000), stomatal movements (Desikan et al. 2004; Distéfano et al. 2008), flowering (He et al. 2004; Khurana et al. 2011), xylem differentiation (Gabaldón et al. 2005), senescence (Leshem and Haramaty 1996), and rhizogenesis (Pagnussat et al. 2002; Lanteri et al. 2008). Recently, we have reported the promotory effect of NO on in vitro caulogenesis in Linum usitatissimum, an herbaceous plant (Kalra and Babbar 2010). The present work reports stimulatory effect of NO on in vitro caulogenic response of Albizzia lebbeck, a leguminous tree. The hypocotyl explants of this plant have been well characterized for their morphogenic potential (Gharyal and Maheshwari 1981, 1982, 1990). Because of the ease to regenerate shoots on basal media, A. lebbeck hypocotyls explants have been used as experimental material for various in vitro morphogenic studies (Baweja et al. 1995; Khurana and Khurana 2000; Jain and Babbar 2002, 2006).

Materials and methods

The seeds of Albizzia lebbeck were procured from Pratap Nursery and Seed Stores, Dehradun, India. After surface sterilization with 0.1% (w/v) mercuric chloride for 7–8 min followed by thorough rinsing with sterile distilled water, these were implanted on to the B5 basal medium (BM; Gamborg et al. 1968), supplemented with 3% sucrose and gelled with 0.8% bacteriological grade agar (Qualigens Fine Chemicals, Mumbai, India). 1 cm long hypocotyl segments from 11-day-old in vitro seedlings, having 5.5–6.0 cm long hypocotyls, were used as the explants. BM was supplemented with different concentrations of freshly prepared NO donor, sodium nitroprusside (SNP, alone or along with NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB) at concentrations equimolar to the optimal concentration of SNP. Potassium ferrocyanide {K4[Fe(CN)6]}, sodium nitrite (NaNO2) or sodium nitrate (NaNO3) were supplemented to the BM individually at 4 μM. Thermolabile chemicals {SNP, cPTIO, K4[Fe(CN)6] and MB}were filter sterilized using Millipore filters of 0.45 μm pore size (Millipore, Bangalore, India) and added to the autoclaved medium. Subsequent to adjustment of pH to 5.8, autoclaving was done at 1.06 kg cm−2 at 121°C for 15 min. The media were dispensed in glass test tubes (25 mm × 150 mm, Borosil India), each containing 20 ml of medium. These were closed with cotton plugs (non-adsorbent cotton covered with two layers of cheese cloth) without any sealant. Two explants were cultured in each culture tube. All cultures were exposed to 16 h photoperiod of 18 μE m−2 s−1 provided by 40 W cool daylight fluorescent tubes, and incubated at 25 ± 2°C. After 45 days, the cultures were scored for the percentage of responding explants, number of shoots per responding explant and the average length of shoots. All experiments were repeated at least twice with 30 explants per treatment each time.

To find if any morphogenic gradient existed along the Albizzia hypocotyls, in the very first experiment, hypocotyl segments were segregated based on their position on the seedlings. These segments, designated as I to V based on their proximity to the root-shoot junction of the seedling, were cultured on BM. To arrive at the concentration of SNP optimal for shoot differentiation, effect of its three concentration ranges (0–40, 0–10 and 0–5 μM) was studied in three independent experiments. To know whether for its promotory effect, SNP is required during the entire culture duration (45 days) or only during specific period(s) and if latter is the situation, does it influence the response adversely for the remaining period, the explants cultured on BM or BM + SNP (4 μM) for different periods (5, 10, 15 and 20 days) were transferred to the other medium for rest of the culture period (45 days).

The data were subjected to one-way analysis of variance (ANOVA, p ≤ 0.05) using SPSS version 10 to test the significance of the observed differences and comparisons between the mean values of treatments were made by Post Hoc Tukey HSD (Honestly Significant Different) test at p ≤ 0.05.

Results and discussion

Before initiating the experiments to study the possible role of NO in in vitro morphogenesis, it was considered necessary to ascertain the absence of any inherent variability among the selected explants. Earlier, differential morphogenic responses by the segments of the same organ have been reported. For example, hypocotyl segments of Santalum album and Solanum melongena proximal to the root-shoot junction exhibited better caulogenic response than the distal ones (Bapat and Rao 1984; Sharma and Rajam 1995). Contrary to this, hypocotyl segments from different positions along the seedling of Liquidambar stryaciflua did not exhibit any significant difference in adventitious shoot differentiation (Kim et al. 1997). In the present study too, no significant difference in the caulogenic response of different hypocotyl segments, in terms of percentage of responding explants, average number of shoots per responding explant and growth of shoots as evidenced by their length was recorded (data not presented). This ruled out the existence of any morphogenic gradient in the hypocotyls of Albizzia. Therefore, for further experiments, hypocotyl segments were randomly mixed and distributed among different treatments.

Pharmacological studies using NO donors and scavengers have generally been used to gain insight in the role of NO in plant growth and development (Besson-Bard et al. 2008). NO donors, well suited to mimic a NO response (Floryszak-Wieczorek et al. 2006; Ederli et al. 2009), are routinely used for physiological studies as treatment of biological samples directly with NO gas is difficult due to its short half-life in vivo and difficulty in maintaining a steady-state concentration of exogenously supplied NO in an experimental system (Delledonne et al. 1998, 2001; Clarke et al. 2000; Neill et al. 2003; Miller and Megson 2007). Commonly used NO donors are SNP (sodium nitroprusside) GSNO (S-nitrosoglutathione), SNAP (S-nitroso-N-acetylpenicillamine), RBS (Roussin’s Black Salts), NOR-3 (+/−)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (Neill et al. 2003). In the present study, SNP was used as a NO donor. In the initial experiment conducted to study the effect of 0–40 μM SNP on the caulogenic response of the hypocotyl explants, the lowest concentration (5 μM), did not significantly affect the caulogenic response in terms of percentage of caulogenic explants, average number of shoots per responding explants and average shoot length. At 10 μM, average number of shoots per responding explant was significantly reduced, while the other two parameters were statistically comparable to the control. However, 20 and 40 μM SNP totally inhibited the response (data not shown). When a narrow range (0–10 μM) of SNP with increments of 2 μM was used, 4 μM SNP significantly promoted the average number of shoots per explant. Though, at the higher levels (6–10 μM), average number of shoots gradually declined, the values were not significantly different from that of the control cultures. The percentages of responding explants and the average shoot length among these treatments were not significantly different (data not presented). Further narrowing of the range of concentrations of SNP to 1–5 μM, with increment of 1 μM, revealed the stimulatory effect of 3 and 4 μM on the caulogenic response. The enhancement was only in terms of the number of shoots per responding explants, with 4 μM being the optimum (Table 1) as was also observed with explants of Linum usitatissimum (Kalra and Babbar 2010).

Table 1 Caulogenic response of hypocotyl explants of A. lebbeck cultured on BM supplemented with different concentrations of SNP (1–5 μM) for 45 days
Full size table

Although B5 basal medium, used in the present study, contains about 27 mM of nitrate in the form of ammonium and potassium nitrate, the observed stimulatory effect of NO donor could have been due to a negligible increase in the concentrations NO2 − and NO3 −, two stable metabolites of NO (Ullrich et al. 1997). This remote possibility was ruled out as neither of NaNO2 and NaNO3, provided individually at 4 μM, had any effect on the caulogenic response (Table 2).

Table 2 Caulogenic response of hypocotyl explants of A. lebbeck cultured on BM supplemented with 4 μM of SNP, NaNO2, NaNO3 or potassium ferrocyanide {K4[Fe(CN)6]} for 45 days
Full size table

SNP, along with NO, generates cyanide (Meeussen et al. 1992; Yamamoto and Bing 2000; Butler and Megson 2002; Murgia et al. 2004; Bethke et al. 2006a, b; Manjunatha et al. 2008) as the first volatile breakdown product. Cyanide, also a small gaseous molecule, has been extensively studied to reveal its role in plant growth, metabolism and development. It acts as a protective molecule against herbivores (Nahrstedt 1985; Zagrobelny et al. 2004). It plays regulatory role in seed germination, nitrate assimilation, sugar and lipid metabolism and in different plant responses to some environmental stimuli (Solomonson and Barber 1990; Bogatek et al. 1991, 1999; Bethke et al. 2006a, b). It also interplays with ethylene signaling (Grossmann 1996; Smith and Arteca 2000) and is mostly correlated with the inhibition of the terminal cytochrome oxidase in mitochondria (Siegień and Bogatek 2006). Therefore, to rule out the possibility of the observed effect of SNP due to cyanide, effect of 4 μM K4 [Fe(CN)6] (a structural analog of SNP that releases cyanide but does not possess NO moiety, was also studied. K4[Fe(CN)6] had no effect on the caulogenic response (Table 2), thus ruling out the role of CN− in observed promotion of caulogenic response due to SNP.

To further ascertain that the observed promotion in the caulogenic response was due to NO released by its donor, specific NO scavenger, cPTIO (Goldstein et al. 2003; Beligni and Lamattina 2000; París et al. 2007), was added alone or along with SNP. cPTIO reacts stoichiometrically with NO (Kasprowicz et al. 2009) and is known to be specific for NO scavenging (Cragon 1999). The NO scavenger added along with the donor brought down the average number of shoots per responding explant statistically equivalent to the control level (Table 3). cPTIO, when added alone, did not affect the caulogenic response. This observation seemingly suggests that there is no role of endogenous NO in in vitro caulogenesis of A. lebbeck, similar to what was observed with the caulogenic response of Linum (Kalra and Babbar 2010).

Table 3 Caulogenic response of hypocotyl explants of A. lebbeck cultured on BM supplemented with cPTIO (4 μM) alone or in combination with SNP (4 μM) for 45 days
Full size table

The results of the experiment involving transfer of the explants from BM to BM + SNP and vice-versa revealed that (a) SNP stimulated caulogenesis even if present only during the initial phase (5 days) of culture, (b) continued presence of SNP for period beyond 5 days neither promoted nor inhibited the response and (c) SNP if provided after initial 5 days of culture had no effect on the caulogenic response (Table 4). SNP is known to release NO for about 12 h (Floryszak-Wieczorek et al. 2006). Therefore, by extrapolation one can say that NO stimulated the caulogenesis by acting during the first 12 h of culture, during which determination and differentiation leading to shoot differentiation at cell level might have taken place. Moreover, no response of SNP beyond 5 days is quite expected as by this time SNP is completely spent as far its capability of releasing NO is concerned. As administration of SNP after 5 or more days of culture did not have affect caulogenic response, it can be concluded that NO influences only the initial stages of shoot differentiation and development.

Table 4 Caulogenic response of hypocotyl explants of A. lebbeck cultured on BM or BM + SNP (4 μM) for 45 days and those cultured on either of these media for 5, 10,.15, 20 days before being transferred to the other medium for rest of the culture period of 45 days
Full size table

To conclude, the present paper on A. lebbeck, a tree legume, affirms the role of NO in in vitro caulogenesis reported recently in Linum usitatissimum, an herbaceous plant (Kalra and Babbar 2010). This extends the versatility of NO molecule by confirming its effect on caulogenesis of two taxonomically divergent taxa. The ease with which A. lebbeck hypocotyls explants develop shoots on various basal media makes them a good experimental material for further biochemical and molecular analyses for the elucidation of signaling cascade downstream NO leading to its stimulatory effect on caulogenesis.