2.1 Plant material and cultural conditions

Seeds of J. effusus cultivar Nonglin-4 were obtained from the University of Nottingham Ningbo, Ningbo, China. Upon receipt at the Zhejiang University, Hangzhou, China, these seeds were used for germinating plants and culturing callus. For the plant growth, glass-house facilities of Institute of Crop Science Zhejinag University were used, while laboratory-based experiments/analyses were performed at the College of Agriculture and Biotechnology, Zhejinag University. Healthy seeds were surface sterilized with 1% sodium hypochlorite (NaOCl) for 20 min, and then washed three times with distilled water (Xu et al., 2009). Nursery was developed on substrate by mixing perlite and vermiculite in 3:1 ratio (v/v). The uniform size (about 15 cm) seedlings were selected and shifted to plastic trays having half strength basal nutrient solution, and were allowed to grow in glass-house at 19 ± 2 °C temperature under 16/8 h light/dark regime.

For comparison, plantlets were raised from callus at the College of Agriculture and Biotechnology, Zhejiang University by the following method. Mat rush seeds were first surface-sterilized and placed in growth vessels containing hormone free MS media (Murashige and Skoog, 1962). Vessels were sealed and placed under fluorescent light (100 μmol m⁻² s⁻¹) with light/dark regime of 16/8 h and 19 ± 2 °C temperature to develop seedlings. These seedlings were used for callus induction and subsequent plant regeneration through the protocols developed by Xu et al. (2009). The basal culm segments (0.5–1.0 cm) of seedling explants were cultured in Petri dishes containing solidified MS callus induction medium supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA), and 2 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). From the callus, plant regeneration was achieved by shifting embryogenic calli on MS regeneration medium-I and II (containing 0.5–1.0 mg L⁻¹ BA, 1.0 mg L⁻¹ kinetin and 3.0 mg L⁻¹ indoleacetic acid). Plantlets, induced from the regenerative calli were transferred to half strength MS plant multiplication medium. At a certain height (approximately 15 cm), these plants were shifted to Hoagland’s modified nutrient solution for hardening (2 weeks), and then treated with Pb and EDTA.

2.2 Sample preparation and treatments (Pb and EDTA)

Two week old uniform size seedlings were transferred to the pots each containing 3.2 L Hoagland’s modified nutrient solution. Hydroponics solution had micro-molar (μM) concentration of nutrients as: 2000 KNO₃, 50 KCl, 500 Ca(NO₃)₂.4H₂O, 200 MgSO₄.7H₂O, 100 NH₄NO₃, 10 KH₂PO₄, 12 H₃BO₃, 2.0 MnSO₄.H₂O, 0.5 ZnSO₄.7H₂O, 0.2 CuSO₄.5H₂O, 0.1 Na₂MoO₄, 0.1 NiSO₄, 20 Fe-EDTA. Concentration of KH₂PO₄ was adjusted regularly at 10 μM to prevent Pb precipitation in the nutrient solution.

After establishment of the seedlings to Hoagland’s solution, two set of J. effusus seedlings, obtained from seed and regenerated from callus, were exposed to various concentrations of Pb [applied as Pb(NO₃)₂] and EDTA. There were seven treatments of Pb/EDTA in triplicates both for seed and callus grown plants. These were: control (without Pb and EDTA), Pb 0.5, Pb 1.0, Pb 0.5 + EDTA 2.5, Pb 1.0 + EDTA 2.5, Pb 0.5 + EDTA 5.0, Pb 1.0 + EDTA 5.0 (all in mM concentration). The two Pb concentrations as 0.5 and 1.0 mM were selected for treatment on the basis of previous results of Verma and Dubey (2003) who suggested these concentrations as moderate to high Pb respectively in the heavy metal polluted soils. EDTA application rate was selected on the basis of our previous studies, where 2.5 and 5.0 mM EDTA concentrations significantly enhanced the metal accumulation by J. effusus (Najeeb et al., 2009, 2011). Treatment pots were arranged with two factor factorial randomized complete design under glass-house conditions with 30/25 °C temperature and 70%/90% relative humidity (day/night). The pH of nutrient solution was maintained at 5.8 by adding 0.1 M NaOH or 0.1 M HCl, and it was continuously aerated. Since our previous results showed that J. effusus performs well under lower pH level, we used relatively low pH (5.8) for the growth media (Najeeb et al., 2009; Xu et al., 2009). The nutrient solution was regularly changed after every 4 days, followed by the repetition of respective Pb/EDTA treatments. On the 15th day of treatments, plants were collected from each pot, cleaned, weighed and then stored at −80 °C for further analysis.

2.3 Pb and chlorophyll determination

The Pb concentrations in plant tissues (root and shoot) were measured following the procedure of Hsu and Kao (2003). Topmost fully expended leaf samples were used for analyzing Pb contents, while for roots, whole root mass was dried at 65 °C for 24 h and ground, and 0.2 g of the prepared sample was used for elemental analysis. A sub-sample of the dried plant mass (root and shoot) was converted into ash in Muffle furnace at 550 °C for 12 h. The ash residues were digested with 31% HNO₃ and 17.5% H₂O₂ at 72 °C for 2 h and transferred to 50 mL flask to make the volume up to the mark by de-ionized water. The Pb contents were determined through atomic absorption spectrophotometer (AA-6800, Shimadzu, Japan). Standard reference material GBW10010 from the Institute of Geophysical and Geochemical of Earth, China, and internal reference materials were used for quality assurance and measuring Pb contents in plants. The analysis showed high Pb recovery rates (>90%), where Pb concentration in the standard reference material was very close to the given value, confirming the accuracy of the method. We extracted chlorophyll from fresh leaves at the top 3rd position using 800 mL L⁻¹ acetone, and leaf chlorophyll contents were measured by the method described by Lichtenthaler (1987).

2.4 Biochemical assays

Fresh plant root and shoot samples were ground in phosphate buffer (pH 7.8) using pre-chilled mortar and pestle. The extract was used for measuring activities of SOD, POD and malondialdehyde (MDA) contents following the method reported by Leu and Zhou (1999).

Catalase (CAT) activity of J. effusus roots and shoots was determined by the reduction rate of H₂O₂ in a specific period of time (Cakmak et al., 1993). Plant extracts were treated with 5 mL of 0.1 N H₂O₂ and kept at 20 °C for 5 min. Then 1 mL of a 20% KI solution was added with 3 drops of 10% (NH₄)₆Mo₇O₂₄ and 5 drops of 1% starch solution. A 0.02 N Na₂S₂O₃ reagent was used for titration of reaction solution until the disappearance of blue color.

2.5 Statistical analysis

Two-way analysis of variance (ANOVA) was used to analyze the data with SAS statistical package (version 9.0) for ANOVA, followed by Fisher’s protected LSD test to identify homogenous groups within the treatment means. If the F-test showed significant differences among the means, multiple range tests were performed at the level of 5% probability.

3.1 Plant growth attributes

Application of higher Pb (1.0 mM) concentrations in hydroponics culture inhibited the growth of J. effusus plants, while, control plants appeared healthy containing greater biomass and greenish in color. Despite no significant change in root and shoot lengths of the plants originating from different sources, the callus grown seedlings attained significantly greater dry biomass than seed grown seedlings under all the treatments. Development of relatively thicker roots and shoots not only helped callus grown plants to survive under metal toxicity but also to sequester higher Pb contents in their tissues. Lower Pb concentration (0.5 mM) had no effect on dry biomass of seed grown plants, however, it significantly reduced the shoot and non-significantly the root dry biomass of callus grown plants (Table 1). Higher dose of Pb (1.0 mM) significantly decreased the plant dry biomass (root and shoot) of the seedlings originating from both sources. EDTA amendments appreciably improved the shoot dry biomass of Pb-treated callus grown plant, while, no significant change was recorded in dry biomass of Pb-treated seed grown plants under EDTA amendments. The positive impact of EDTA on shoot growth was probably due to the restricted movement of Pb in roots and lower translocation.

Increasing Pb concentrations in the growth media inhibited the root and shoot length of two kinds of J. effusus plants (Table 2). No significant improvement in plant height was noticeable under lower EDTA (2.5 mM) concentration but the higher EDTA concentration (5.0 mM) recovered root length of Pb-stressed plants. In general, seed grown plants had longer roots than callus grown seedlings, while, callus grown plants possessed relatively longer shoots, although the difference was statistically non significant.

3.2 Lead accumulation

J. effusus plants experienced a consistent rise in Pb contents in their tissues (shoots and roots) under the growing Pb concentrations in nutrient media that was further enhanced by EDTA amendments (Table 3). The plants tended to store greater Pb contents in their roots compared to their aboveground parts as was evident from the low translocation factor (TF). Furthermore, the callus grown plants deposited more Pb contents in root tissues than seed grown plants under all the treatments, and the difference was significant. Contrastingly, seed grown plants contained significantly greater Pb contents in their shoots as compared to the callus grown plants. Increasing EDTA concentrations significantly enhanced the uptake and translocation of Pb in J. effusus plants.

3.3 Variations in chlorophyll contents

Incremental Pb concentrations in growth media significantly suppressed the chlorophyll (chlorophyll a and b) contents of seed and callus grown plants (Table 4). EDTA application improved chlorophyll contents of Pb-stressed plants. The plants developed from callus exhibited relatively better response toward EDTA application in terms of chlorophyll recovery, while in seed grown plants, improvement was significant in chlorophyll b contents only. Overall, treatment Pb 0.5 + EDTA 5.0 mM proved the best dose for chlorophyll contents of both type of plants. The Pb-induced stress variably affected the chlorophyll a and b contents of seed and callus grown plants. In seed grown plant, increasing Pb concentration in the growth media gradually increased the chlorophyll a/b value that was reduced by EDTA amendments. No significant change in chlorophyll a/b value of callus grown plants was observed under application of Pb alone, however, chlorophyll a/b value significantly increased under treatment of Pb 1.0 + EDTA 2.5 mM. On an average, the seed grown plants contained greater chlorophyll a contents, while relatively higher chlorophyll b contents were observed in callus grown seedling but the difference was non-significant.

3.4 Biochemical attributes

Application of Pb and ETDA considerably altered the activities of various biochemical attributes of J. effusus. Lipid peroxidation in Pb-treated plants of J. effusus was estimated in terms of MDA contents. Compared to the control plants, Pb (1.0 mM) treatment significantly raised the root and non-significantly the shoot MDA contents of seed and callus grown plants (Fig. 1a). EDTA (2.5, 5.0 mM) amendments non-significantly reduced root MDA contents of Pb-stressed callus and seed grown plants, while, shoots MDA contents increased under higher EDTA (5.0 mM) concentrations.

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Figure 1 (a) Alterations in malondialdehyde (MDA) contents (nmol g⁻¹ fresh weight) in the roots and shoots (Juncus effusus L.) as influenced by Pb and EDTA levels. (b) Changes in superoxide dismutase (SOD) activity (Ug g⁻¹ fresh weight) in the roots and shoots (Juncus effusus L.) under different levels of Pb and EDTA. (c) Peroxidase (POD) activity (OD470 g⁻¹ fresh weight min⁻¹) in the roots and shoots (Juncus effusus L.) as varied by Pb and EDTA levels. (d) Catalase (CAT) activity (μmol mg⁻¹ protein min⁻¹) in the roots and shoots (Juncus effusus L.) as influenced by Pb and EDTA additions. T1: control (without Pb and EDTA), T2: Pb 0.5 mM was added, T3: Pb concentration was increased to 1.0 mM, T4: Pb 0.5 + 2.5 mM EDTA, T5: Pb 0.5 + 5.0 mM EDTA, T6: Pb 1.0 + 2.5 mM EDTA and T7: Pb 1.0 + 5.0 mM EDTA. Fresh weights (FW) of both seed-grown (SG) and callus-grown (CG) plant parts were used for measuring the enzymatic activities.

Export to PPT

Modification in antioxidative enzyme activities of J. effusus was noted under different levels of Pb alone or in combination with EDTA (Fig. 1b–d). Under increasing Pb concentrations, both seed and callus grown plants experienced a consistent rise in the activity of tested antioxidative enzymes i.e. POD, CAT and SOD in roots as well as in shoots. J. effusus roots experienced a further increase in the activity of these enzymes under increasing EDTA levels. However, changes in antioxidative enzyme activities were variable in plant shoots under Pb + EDTA application. The SOD activity progressively reduced in shoots of Pb-stressed plants under increasing EDTA concentrations. Compared to Pb treatment alone, EDTA application significantly elevated POD activity in the shoots of Pb-stressed plants. The CAT activity of Pb-treated shoots showed no significant increase under EDTA application.