Introduction

Albizia lebbeck (Fabaceae) is an imperative multi-use perennial tree of the genus Albizia indigenously found in India, Bangladesh, Nepal and Pakistan etc. (Orwa et al. 2009). It owns several medicinal evidences such as anti-inflammatory, analgesic, antifertility and antimicrobial properties. In Indian traditional medicinal systems, the leaves, flowers, seeds, bark and roots are used to treat cough, boils, eye flu, gingivitis, piles, diarrhea, gonorrhea, hypoglycemia, abdominal tumors, lung problems, bronchial asthma and other allergic disorders (Rätsch and Christian 1998). Economically, it is used for fuel, timber, furniture, erosion control (Singh et al. 2004) and as a shade tree in tea, coffee and cardamom plantations (Saha and Ahmed 2009).

Unsystematic and illicit erosion, low natural rejuvenation, constricted habitat specificity and insufficient conventional methods resulted in the rigorous depletion of A. lebbeck population (Perveen et al. 2011). Therefore, keeping in views the outputs of the tissue culture techniques in woody trees, a number of efforts have been made for the in vitro propagation of A. lebbeck by using hypocotyl, root, cotyledon, leaflet, stem, petiole and seed explants (Gharyal and Maheshwari 1983; Mamun et al. 2004; Gharyal and Maheshwari 1990; Perveen et al. 2011, 2012, 2013b). For clonal plantlets production in vitro propagation through axillary shoots proliferation is a useful technique, while adventitious bud induction is a promising tool in order to obtained high frequency of plantlets at commercial level in a shorter period of time.

Moreover, the particular in vitro culture conditions i.e., high relative humidity, low CO2 concentration, low light intensity and massive of sugar etc., results in the formation of plantlets with anomalous physiology, morphology and anatomy (Ali et al. 2005). Transfer of these plantlets to ex vitro conditions having high light intensity, low relative humidity, high CO2 and temperature etc., might easily impaired them by hasty changes. Thus, the success of in vitro propagation depends on efficient transplantation protocols ensuring the establishment of in vitro plants under ex vitro environment (Hazarica 2006). Furthermore, study of changes in the physiological and biochemical parameters during acclimatization of the in vitro plants provide information on the effect of changed environmental conditions during acclimatization and helps in the standardization of acclimatization protocol (Van Huylenbroek et al. 2000; Kadlecek et al. 2001; Perveen et al. 2013a).

Hence, in vitro propagation of Albizia via adventitious bud induction followed by concerning the difficult stages of ex vitro acclimatization may prove highly efficient for the commercial protocol establishment. Therefore, the objectives of this study were (1) to develop a highly efficient in vitro protocol for routine plant regeneration in the A. lebbeck by using HP explants and (2) to assess the information on the changes in physiological and biochemical parameters during the acclimatization of in vitro raised plantlets under ex vitro conditions and its neutralization by biochemical characteristics.

Materials and methods

Seed germination and establishment of aseptic seedling

The mature fruits of A. lebbeck were collected from a 20 years old tree growing in the botanical garden of the Aligarh Muslim University, Aligarh, India. The healthy seeds, excised from the fruits were washed thoroughly in running tap water for 30 min. These were immersed in 1 % (w/v) solution of Bavistin, a fungicide, for 15 min to remove adherent particles and later treated with 5 % (v/v) Teepol solution for 20 min. Thereafter, the seeds were rinsed thrice with sterile distilled water followed by surface sterilization in 0.1 % (w/v) HgCl2 solution under laminar flow hood for 15 min. Finally, the seeds were washed thrice with sterile distilled water to remove the traces of HgCl2 solution. The disinfected seeds were inoculated on MS (Murashige and Skoog 1962) basal medium for seed germination. Hypocotyl segments (1.0–1.5 cm) (Fig. 1a) excised from 15-day-old seedlings were used as explants.

Fig. 1
figure 1

In vitro regeneration and plantlet establishment of A. lebbeck. a Hypocotyl explants (1.0–1.5 cm) obtained from 15 days old aseptic seedling. b Emergence of direct adventitious shoot buds from hypocotyl (HP) explants on MS + BA (7.5 μM) after 4 weeks of culture. c, d Histological section showing the direct induction of adventitious shoot buds (SB) in HP. e Cultures showing elongation and proliferation of adventitious shoots from HP on MS + BA (7.5 μM) + NAA (0.5 μM) after 8 weeks of incubation. f Acclimatized plantlets in sterile soilrite after 4 weeks. g Acclimatized plantlets in sterile soilrite + normal garden soil (1:1) after 10 weeks. h 4 month old plants acclimatized in garden soil under field conditions

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Culture media and culture condition

MS (Murashige and Skoog 1962) basal medium fortified with 3 % (w/v) sucrose, 0.8 % (w/v) agar (Qualigens, Mumbai, India) was used in all the experiments. The medium was adjusted to pH, 5.8 using 1N NaOH or HCl and sterilized by autoclaving at 121 °C and 1.06 kg cm−2 pressure for 20 min. Cultures were incubated at 25 ± 2 °C under 16/8 h (light/dark) photoperiod provided by cool white fluorescent tubes (Phillips, India) with a photon flux density of 50 μmol m−2 s−1 at 55–60 % relative humidity.

Adventitious shoot induction and multiplication

HP explants excised from 15-day-old seedlings were cultured onto MS medium augmented with various concentrations of BA, Kn, 2-iP (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) or TDZ (0.5, 1.0, 2.5, 5.0 and 7.5 μM) singly as well as in combination with different concentrations of NAA or IBA or IAA (0.1, 0.5, 1.0, 1.5 and 2.0 μM) for induction and multiplication of shoots.

Histological analysis

To confirm the regeneration of multiple shoot buds from the nodal explants, histological examination of explants was performed after 15 days. Tissues were fixed in formalin: glacial acetic acid: ethanol 4:6:90 (v/v) solution. Fixed tissues were dehydrated through an ethanol/xylol series and embedded in paraffin wax (60 °C). Serial sections 10 μM thickness were cut using a Spencer 820 microtome (American Optical Corp., Buffalo, NY, USA) and the resulting paraffin ribbons were passed through a series of deparaffinising solutions and stained in saffranin and fast green solutions. The sections were examined under an optical microscope (CH20i, Olympus, Tokyo, Japan).

Rooting and acclimatization

For rooting 4–5 cm shootlets were rooted and acclimatized in soilrite by utilizing Perveen et al. (2013b) method. After 4 weeks of soilrite acclimatization, plants were transferred to pots containing a mixture of garden soil [loam to clayey (Khan et al. 2012)] + soilrite (1:1) and maintained in the culture room conditions (25 ± 2 °C under 16/8 h light–dark conditions with a PPFD of 150 μmol m−2 s−1) up to the next 6 weeks, followed by green house transfer (garden soil) under normal day length conditions.

Fully developed leaves obtained at transplantation day (day 0, control) and after 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 days of acclimatization were used for the evaluation of different physiological and biochemical parameters (chl a/b, car content, PN, MDA, H2O2, SOD, CAT, APX and GR) as mentioned below.

Photosynthetic pigments estimation

The chl (a and b) and car contents were extracted using the method of MacKinney (1941). A total of 0.5 g of fresh leaf tissues from inter-venal areas was ground in 5 ml of 80 % acetone. The suspension was filtered with Whatman filter paper number 1. Absorbance of chlorophyll at 663, 645 nm and carotenoid at 480 and 510 nm was estimated on UV–Vis Spectrophotometer (UV-1700 Pharma Spec, Shimadzu, Kyoto, Japan).

Net photosynthetic rate (PN)

The net photosynthetic rate (PN) was measured on fully expanded leaves using a portable Infra-red Gas Analyzer (IRGA, LI-COR 6400, Lincoln, USA) on the basis of net exchange of CO2 between leaves and atmosphere by enclosing the leaves in a leaf chamber and monitoring the rate of CO2 concentration changed over short time intervals. The net photosynthetic rate was expressed as µmol CO2 m−2 s−1.

Estimation of MDA content

Lipid peroxidation was estimated according to malondialdhyde (MDA) production using the thiobarbituric acid (TBA) method (Heath and Packer 1968) by using a coefficient of absorbance of 155 mM−1 cm−1.

Estimation of H2O2 content

H2O2 content was measured after reaction with potassium iodide. Leaf tissue (0.5 g) was homogenized in 0.1 % (m/v) trichloroacetic acid (TCA) and centrifuged at 14,000×g and the homogenate was used for the determination of H2O2 content by the method of Alexieva et al. (2001). The supernatant (0.5 cm3) was mixed with 0.5 cm3 of 100 mM K-phosphate buffer (pH 7.8), and 2 cm3 of reagent (1 M KI in fresh double-distilled water). After 1 h in darkness, the absorbance was measured at 390 nm. The blank probe consisted of 0.1 % TCA in the absence of the leaf extract.

Antioxidant enzyme extraction and assay

To determine the activities of antioxidant enzymes, 0.5 g of fresh leaf tissue was homogenized in 2.0 cm3 of extraction buffer (pH 7.5) containing 1 % (m/v) PVP, 1 % (v/v) Triton X-100, and 0.11 g of ethylenediaminetetraacetic acid (EDTA) using a pre-chilled mortar and pestle. The homogenate was filtered through four layers of cheesecloth and centrifuged at 15,000×g for 20 min. The supernatant was used for enzyme assays. Extraction was carried out in dark at 4 °C.

SOD activity was assayed Dhindsa et al. (1981) by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). The reaction mixture consisting of 0.5 M phosphate buffer (pH 7.5), 0.1 mM EDTA, 13 mM methionine, 63 mM NBT, 1.3 mM riboflavin and 0.1 cm3 of enzyme extract in test tubes was incubated under 15 W fluorescent lamp (Philips, Kolkata, India) at 25 °C for 15 min. Absorbance was measured at 560 nm.

CAT activity was assayed from the rate of H2O2 decomposition as measured by the decrease of absorbance at 240 nm following the method of Aebi (1984). The assay mixture contained 50 mM phosphate buffer (pH 7.0) and 0.1 cm3 of enzyme extract in a total volume of 3 cm3 and the reaction began by adding 10 mM H2O2.

GR activity was measured using the protocol of Rao (1992) based on glutathione-dependent oxidation of NADPH at 340 nm. The assay mixture contained 50 mM phosphate buffer (pH 7.5), 1.0 mM EDTA, 0.2 mM NADPH and 0.5 mM glutathione disulfide (GSSG). The enzyme extract (0.1 cm3) was added to begin the reaction and the reaction was allowed to run at 25 °C for 5 min.

APX activity was measured by monitoring the decrease in absorbance at 290 nm within 1 min according to Nakano and Asada (1981). The reaction mixture included 50 mM phosphate buffer (pH 7.5), 0.5 mM ascorbate, 0.1 mM H2O2, 0.1 mM EDTA, and 0.1 cm3 of enzyme extract.

The protein content in enzymatic extracts was determined following the Bradford (1976) assay using bovine serum albumin as a standard.

Statistical analysis

The experiments were based on a completely randomized design with 20 explants per treatment and all the experiments were repeated thrice (=three replicates, 60 explants per treatment). The data on various parameters were subjected to one-way analysis of variance (ANOVA) using SPSS version 16 (SPSS Inc., Chicago, USA). The significance of differences among means was carried out using Duncan’s multiple range test (DMRT) at P = 0.05. The results were expressed as the mean ± SE.

Results

Effect of cytokinins on adventitious shoot proliferation

The ability of HP segments to produce shoots varied depending upon the PGRs supplied in the MS medium. The first visible change in the explants was a slight enlargement in size within the first week followed by multiple shoot bud induction (without an intervening callus formation) at the cut ends of the explants after 2 weeks of culture on MS + BA, Kn and 2-iP singly. After a period of 2 weeks, shoot buds started to stretch over the whole explant surface.

All the tested levels of BA supplied in MS medium were found more effective than Kn and 2-iP, for the regeneration of adventitious shoot buds from HP explants. Maximum shoot regeneration response 81 % with number of shoots (22.00 ± 1.10) was observed on MS + BA (7.5 µM) containing medium after 4 weeks of culture (Table 1; Fig. 1b). While, at the same level (7.5 µM), Kn and 2-iP produces maximum 68 and 59 % regeneration response respectively (Table 1).

Table 1 Effect of different cytokinins on adventitious shoot buds induction from hypocotyl explants after 4 weeks of culture
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Furthermore, the histological analysis confirms the direct organogenesis pathway showing shoot differentiation from HP explants and maintaining vascular connections from parent explants (Fig. 1c, d).

Synergistic effect of cytokinins and auxins on adventitious shoot proliferation

After 4 weeks, HP explants with regenerated shootbuds were transferred onto the shoot proliferation and elongation media containing MS + 7.5 μM BA or Kn or 2-iP with different concentrations of various auxins viz., NAA, IBA and IAA (0.1, 0.5, 1.0, 1.5, 2.0 μM) singly (Tables 2, 3, 4). Among the different combinations tested, BA (7.5 μM)+NAA (0.5 µM) was found to be the most effective, producing maximum regeneration response (88 %), number of shoots (34.00 ± 1.15) per explant and shoot length (6.30 ± 0.05 cm) after 8 weeks of culture (Table 2; Fig. 1f). While, BA combined with (0.5 µM) of IBA or IAA produced relatively low (78 and 65 %) shoot regeneration frequency (Table 2).

Table 2 Effect of different auxins amended with an optimal concentration of BA (7.5 µM) in MS medium on shoot proliferation and multiplication from hypocotyl explants after 8 weeks of culture
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Table 3 Effect of different auxins amended with an optimal concentration of Kn (7.5 µM) in MS medium on shoot proliferation and multiplication from hypocotyl explants after 8 weeks of culture
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Table 4 Effect of different auxins amended with an optimal concentration of 2-iP (7.5 µM) in MS medium on shoot proliferation and multiplication from hypocotyl explants after 8 weeks of culture
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Similarly, optimal level (7.5 µM) of Kn or 2-iP combined with NAA (0.5 µM) produced maximum regeneration frequency of 75 and 67 % respectively after 8 weeks of culture. While the combination of Kn (7.5 µM) + (0.5 µM) IBA or IAA and 2-iP (7.5 µM) + (0.5 µM) IBA or IAA (0.5 µM) proved comparatively less efficient (Tables 3, 4).

Effect of TDZ

The effect of various concentrations of TDZ was also evaluated on the direct adventitious shoot regeneration from HP explants. Among the various levels tested TDZ at (1.0 µM) showed maximum 12.60 ± 0.33 number of shoots in 70 % cultures after 4 weeks of culture (Table 5). While an increase in TDZ level beyond 1.0 μM resulted in callus formation. The TDZ exposed hypocotyl explants sub-cultured onto the same fresh medium showed vitrification and shoots did not elongate further. To overcome this problem, explants were subcultured onto the MS + BA (7.5 µM) + NAA (0.1, 0.5, 1.0, 1.5 and 2.0 µM) singly. The highest shoot regeneration response (77 %) with number of shoots (20.30 ± 0.32) and shoot length (5.15 ± 0.08 cm) was obtained on MS + BA (7.5 µM) + NAA (0.5 µM) after 8 weeks of culture (Table 6).

Table 5 Effect of different concentrations of TDZ on multiple adventitious shoot buds induction from hypocotyl explants after 4 weeks of culture
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Table 6 Effect of different concentrations of NAA supplied with 7.5 µM BA, on shoot multiplication from TDZ-exposed hypocotyl explants after 8 weeks of culture
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Rooting and acclimatization

The in vitro regenerated shootlets were rooted successfully by using previously standardized ex vitro rooting protocol and hardened off inside the culture room (in substrate soilrite) for 4 weeks (Perveen et al. 2013b). The primary hardened plants on soilrite for 4 weeks (Fig. 1f), were transferred to garden soil + soilrite mixture (1:1) showed survival rate of about 80 % after 6 weeks of transplantation (Fig. 1g, h). The plants grew well and did not show any variation in morphology and growth characteristics when compared with mother plant.

Estimation of physiological parameters during acclimatization

Photosynthetic pigments

Throughout the ex vitro acclimatization period of 10 weeks, in vitro raised plantlets showed a considerable increase in chl (a and b) and car content. However, there was a reduction of 14.2 and 36.3 % in Chl a and b pigments respectively after 14 days of transfer in soilrite as compared to day 0 plants but on subsequent days, the new leaves appeared and resulted in a significant increase of 67 and 118 % in both Chl a and b contents respectively at day 28. Again a significant decrease in photosynthetic pigments was observed after 28 days with the change in potting substrate [garden soil + soilrite mixture], but thereafter a significant increase in Chl a and b pigments was observed (Fig. 2). Similarly, with the increase in number of days of acclimatization (0–70 days), the car content was found to increase (62.8 %) up to the day 63 and get stabilized beyond 63 days (Fig. 2).

Fig. 2
figure 2

Changes in the levels of photosynthetic pigments (Chl a and b) and carotenoid content in the micropropagated plantlets acclimatized in soilrite (from 0 to 28 days) followed by garden soil + soilrite (1:1) transfer (from 28 to 70 days). Bars represent the mean ± SE. Bars denoted by the alphabets are significantly different (P = 0.05) using DMRT

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Net photosynthetic rate (PN)

The PN rate was found to decrease (26.6 %) during the first 2 weeks of acclimatization in the soilrite. While, after 2 weeks, it started to increase (30.2 %) up to 28 days. Transfer of the plants in changed substrate, again resulted in a significant decrease (09 %) in PN rate at day 35 compared to day 28, followed by steady increase (45 %) in the subsequent period (Fig. 3).

Fig. 3
figure 3

Change in net photosynthetic rate (µmol CO2 m−2 s−2) of micropropagated plantlets acclimatized in soilrite (from 0 to 28 days) followed by garden soil + soilrite (1:1) transfer (from 28 to 70 days). Bars represent the mean ± SE. Bars denoted by the alphabets are significantly different (P = 0.05) using DMRT

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Estimation of biochemical parameters during acclimatization

MDA and H2O2 content

An increase in the MDA and H2O2 content (3 and 2 %) respectively was observed in the plantlets in early 14 days of acclimatization, subsequently, a decreasing trend (23.1 and 30.3 %) respectively was observed up to the day 70, as compared to the day 0 plants (Fig. 4).

Fig. 4
figure 4

Changes in the levels of MDA and H2O2 content in micropropagated plantlets acclimatized in soilrite (from 0 to 28 days) followed by garden soil + soilrite (1:1) transfer (from 28 to 70 days). Bars represent the mean ± SE. Bars denoted by the alphabets are significantly different (P = 0.05) using DMRT

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Antioxidant enzymes

Plantlets exhibited a significant increase in SOD, CAT, GR and APX activity reaching a maximum on day 63 and thereafter got stabilized (Fig. 5). While, GR was found increasing up to the day 70 of acclimatization (Fig. 5).

Fig. 5
figure 5

Changes in the levels of SOD (Unit mg−1 protein), CAT, APX and GR (mmol min−1 mg−1 protein) content in micropropagated plantlets acclimatized in soilrite (from 0 to 28 days) followed by garden soil + soilrite (1:1) transfer (from 28 to 70 days). Bars represent the mean ± SE. Bars denoted by the alphabets are significantly different (P = 0.05) using DMRT

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Discussion

Successful genetic improvement of the plants for the useful trait expression (quality improvement in multipurpose trees) requires a suitable plant regeneration protocol. Plant production in vitro via adventitious shoot organogenesis on various juvenile explants (bearing high morphogenetic potential and low contamination rates) is the best system to obtain transgenic with ease (Yang et al. 1996). HP explants being juvenile and lacking pre-existing meristems can be suitably subjected to growth regulator manipulations to result in de novo plant regeneration as reported in number of plant species viz., Annona squamosa (Nagori and Purohit 2004), Euonymus japonicus (Shang et al. 2006) and Vigna subterranea (Mongomake et al. 2009).

In vitro organogenesis requires the moderation of cell differentiation by the application of appropriate plant growth regulators (Benson 2000). In the present study, BA followed by TDZ was found to be the most efficient cytokinin than Kn and 2-iP for maximum adventitious shoot buds production (without callus formation) from aseptic HP explants of A. lebbeck. The effectiveness of BA for adventitious organogenesis from HP explants has been demonstrated in number of woody tree species including Sesbania rostrata (Jha et al. 2002) and Fraxinus nigra (Beasley and Pijut 2013). While, in consistence with our finding, Parimalan et al. (2007) in Bixa orellana and Annapurna and Rathore (2010) in Embelia ribes found TDZ as effective cytokinin for in vitro adventitious shoot regeneration.

Direct shoot organogenesis without callus formation obtained from HP explants is beneficial as compared to indirect shoot organogenesis, as it generally results in a lower frequency of somaclonal variation (Annapurna and Rathore 2010). Hence, the structural analysis is an important step to corroborate adventitious shoot bud regeneration as it is an extremely useful approach in plant morphogenesis studies (Saha et al. 2012). The HP explants showed high mitotic division on cytokinin supplemented medium resulting in the proliferation of meristemetic zones nearby epidermis. As expected, vascular connections were observed to occur between meristemetic regions and mother explants as established by Neto et al. (2003) in Bixa orellana. Therefore, histological studies indicated that in vitro plant regeneration in A. lebbeck has developed through direct adventitious organogenesis.

The combination of various auxins with cytokinins enhances the shoot induction and multiplication rate from HP explants. Among all the combinations tested BA + NAA was found to be the most favorable producing maximum shoot proliferation and elongation, which is in accordance with the findings in other plant species, Artirrhinum majus (Cui et al. 2004) and Psoralea corylifolia (Baskaran and Jayabalan 2010; Tiwari and Pathak 2012). Gharyal and Maheshwari (1983) found NAA and IAA as best auxins with cytokinins for organogenesis from HP explants of A. lebbeck but the results (number of shoots) were comparatively less productive than our observations, it may be because of the supplementation of B5 basal medium, which was found less effective than MS medium for in vitro A. lebbeck production (Perveen et al. 2011).

The shoot buds induced in HP explants on TDZ-containing medium showed enhanced shoot induction and multiplication rate when cultured onto the secondary medium containing BA + NAA. The efficiency of BA + NAA on TDZ-exposed cultures for enhanced shoot induction and elongation was also confirmed by Khan and Anis (2012) in Salix tetrasperma and Ahmed and Anis (2012) in Vitex trifolia.

In vitro regenerated shootlets of A. lebbeck rooted by ex vitro rooting method gave the highest number of root regeneration response as compared to in vitro rooting (Perveen et al. 2011; Perveen et al. 2013b) and is less time consuming because of rooting and acclimatization took place simultaneously. The importance of ex vitro rhizogenesis over in vitro rooting have been reported by many researchers (Rogers and Smith 1992; Nas and Read 2004).

To safeguard in vitro grown plants against water stress and to encourage autotrophy, acclimatization under ex vitro conditions is one of the main processes to promote their field survival and physiological competence (Pospisilova et al. 1999; Hazarika 2003). For successful photoautotrophic acclimatization, a transitional environment is usually supplied for a duration ranging from one to several weeks using environmental controls, which has successfully improved the survival percentage of number of in vitro raised plant species under ex vitro environment (Xiao and Kozai 2004; Perveen et al. 2013a). In the same way, present study standardized an acclimatization period of 10 weeks for A. lebbeck. The primary hardened plantlets on soilrite for 4 weeks when transferred onto the mixture of soilrite + garden soil (1:1) showed good survival rate of 80 % after 10 weeks of acclimatization and did not show any visible defects in morphological and growth characteristics when compared with their respective donor plants. Similar findings have been reported by Balaraju et al. (2009) in Swertia chirata and Chabukswar and Deodhar (2005) in Garcinia indica, where sequencial hardening was found highly effective.

Furthermore, evaluation of physiological and biochemical characteristics during the hardening process may be helpful for the development of competent transplantation protocols for the in vitro raised plants and will help to make assessments on adjusting under ex vitro environmental conditions (Hazarica 2006). In the present study, during the course of acclimatization of regenerants initial decline in chl (a and b), car contents and PN rate (during 14 days) is in accordance with the findings of Kadlecek et al. (1998) in Nicotiana tabacum, wherein photomixotrophically grown plantlets exhibited decrease in chl a and b content during first week of ex vitro transplantation. Correspondingly, PN rate was found decreasing in Solanum tuberosum and Spathiphyllum floribundum regenerants in the initial days of transplantation and started increasing thereafter (Baroja et al. 1995; Van Huylenbroeck and Debergh 1996).

According to Borkowska (2001), during the ex vitro acclimatization of strawberry plants, newly formed leaves (during first week of acclimatization) exhibited low photochemical activity and with the progression of acclimation they become more active. Similarly in this study, newly formed leaves showed significant decline in chl (a and b) content at day 7 and 14 as compared to day 0 plants followed by an increase showing functional activeness of the newly originated leaves with the passage of acclimatization. While, a decline in photosynthetic parameters during initial 14 days of acclimatization, may be because of the low photochemical activity of the persistent leaves or newly formed leaves (Perveen et al. 2013a). These results are in accordance with the reports on Calathea louisae (Van Huylenbroek et al. 2000) and Tylophora indica (Faisal and Anis 2010). Likewise, in Calathea louisae and Spathiphyllum floribundum substantial increase in PN was measured with the fully developed new leafs (Van Huylenbroeck et al. 1998, 2000).

Light intensities has a crucial role for autotrophic plant development, as transfer of in vitro (50 PPFD) plants to ex vitro at increased light intensities (150 PPFD) favoured leaf expansion with autotrophic plant development mainly with regard to chlorophyll content as reported by Carvalho et al. (2002) in Vitis vinifera. Likewise, transfer of A. lebbeck plantlets under threefold (150 PPFD) light intensity than in vitro condition (50 PPFD) favours proper acclimatization of the plantlets as proved by the enhanced levels of photosynthetic parameters. Similar results have been reported in Gardenia jasmiinoides by Serret et al. (2001) and Ulmus minor by Dias et al. (2013). While, an increase in carotenoids level particularly signifies that plants sustained the light stress as play a key role in protecting of chlorophyll pigments under stress conditions (Ali et al. 2005).

Generally, water stress and photoinhibition promotes the production of reactive oxygen species (ROS) and in consequence of oxidative stress during the acclimatization of plants to ex vitro conditions (Pinto et al. 2011). ROS being inevitable byproducts of aerobic metabolism cause lipid peroxidation, protein degradation, damage of DNA; therefore, their production and removal must be controlled (Batkova et al. 2008). The extent of the damaging effects of ROS depends on the effectiveness of the antioxidative systems which include antioxidative enzymes viz., Superoxide dismutase (SOD), Catalase (CAT), Ascorbate peroxidase (APX) and Glutathione reductase (GR) (Perveen et al. 2013a). Therefore, an increase in SOD and CAT activities in the process of acclimatization suggests an up-regulation of the plant protective mechanism against oxidative stress. These results are in consistent with the earlier findings of Faisal and Anis (2010) in Tylophora indica, Varshney and Anis (2012) in Tecomella undulata and Perveen et al. (2013a) in Abrus precatorius. Similarly, elevation in activities of both APX and GR suggests chloroplast-based detoxification of ROS (Perveen et al. 2013a). Hence, plants with higher content of antioxidants are usually better adapted to stress (Mitrovic et al. 2012).

MDA is a product of lipid peroxidation and an indicator of tissue damage (Perveen et al. 2013a). Besides, H2O2 functions as a signaling molecule for antioxidant responses (Giampaoli et al. 2012). Hence a considerable decrease in MDA and H2O2 content and an augment in antioxidative enzymes (SOD, CAT, APX and GR) after 14 days of acclimatization clearly indicated the stress tolerance in A. lebbeck regenerants. Analogous decrease in MDA and H2O2 content has been observed by Chakrabarty and Datta (2008) in Gerbera jamesonii.

Therefore, reduction in photosynthetic pigments level and increase in antioxidative enzymes and stress indicator during the initial period (0–14 days) of acclimatization reflects the production of oxidative stress and its neutralization by the higher activities of antioxidative enzymes. While, enhancement in photosynthetic pigments and antioxidative pools from day 21–70 suggest that the micropropagated plants developed functional photosynthetic machinery to reduce oxidative stress during the acclimatization period. Hence, results prove that by using steady hardening processes plants can gradually and successfully establish in autotrophic environmental conditions from the heterotrophic environment by slowly neutralizing oxidative stresses and improving photosynthetic systems.

Our study successfully overcomes the drawback of tissue culture technique in tree species related with the establishment of in vitro plants under natural environmental conditions via gradual acclimatization process standardization. However there are several reports on Albizia tissue culture but none of these studies reports efficient results in comparison to our study in terms of large scale plant production as well as their autotrophic establishment. So, this study for the first time reported a highly efficient commercially usable protocol with successful autotrophic plant development as confirmed by several physiological and biochemical parameters also.