Introduction

Maca (Lepidium meyenii Walp.), a plant belonging to the family of Brassicaceae, is primarily domesticated in the puna agro-ecological zone of central Peruvian Andes, at altitudes around 4,000 m. Maca roots have been used as source of food and medicine for at least 2,000 years (Rea 1992). Recently, maca has been utilized for its aphrodisiac effects (Zheng et al. 2000a; Cicero et al. 2002; Gonzales et al. 2002), with its consumption gradually increasing. Reported research on maca has focused on its composition and pharmacology. In order to develop possible markets, producing plants commercial growing systems are required. Because the source of available maca seeds is restricted, plant tissue and organ culture appears appropriate to produce sufficient numbers of plantlets for cultivation.

During micropropagation, hyperhydricity occurs frequently when maca adventitious shoots were induced from callus. Hyperhydricity can cause problems of differentiation and survival, and severely influences the efficiency of micropropagation. In general, the leaves of hyperhydric shoots are thicker, elongated, wrinkled and/or curled, and can be brittle when compared to normal leaves. The stems are also generally thicker with the distance between internodes being shorter than those of normal stems (Kevers et al. 2004). Hyperhydricity is considered as a physiology disorder and many physiological and biochemical changes have been observed (Franck et al. 2001; Franck et al. 2004; Saher et al. 2005).

Rare earth elements (REE) have many biological effects on the plant growth and development, such as improving cell growth, enhancing secondary metabolite synthesis (Yuan et al. 2002), increasing tolerance to acid rain and other adverse environmental conditions (Peng and Pang 2002). In this study, La3+, Ce3+and Nd3+ (REE) were added to induction media to reduce the hyperhydricity of adventitious maca shoots. We hypothesis that these REE may stimulate growth through enhanced activities of several antioxidative enzymes which have been measured here, i.e. peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR).

Materials and methods

Plant material

Maca (Lepidium meyenii Walp.) seeds were sterilized with 70% (v/v) ethanol for 2 min followed by 2% (v/v) sodium hypochlorite solution for 20 min. After sterilization, maca seeds were rinsed three times with sterile distilled water. The shoots sprouted from sterilized seeds (S) were cultured on hormone-free MS medium (Murashige and Skoog 1962) in 150 ml conical flasks at 25 ± 2°C for 25 days. Maca calli were induced from the roots of seed shoots, and 3–5-month old calli were used to differentiate. The solid induction medium for maca callus differentiation was MS medium supplemented with 2-mg l−1 6-benzylaminopurine, 0.25-mg l−1 naphthalene acetic acid and 6-g l−1 agar. When the REE effect experiments were carried out, different concentrations of La(NO3)3, Ce(NO3)3, and NdCl3 (purchased from Shanghai Yuelong chemical plant, China) were added to induction medium respectively. All media were adjusted to pH 5.85–5.90 and autoclaved at 120°C for 20 min. Every five pieces of calli (about 0.7 cm3) were cultured under 16 h fluorescent lamps at day length with an intensity of 24 μmol m−2 s−1, at 25 ± 2°C for 25 days, in a 150 ml conical flask containing 50 ml of media with or without REE. The shoots induced from maca calli on induction media without REE were used as control (C). Induction rate of the shoots was calculated as follows: Induction rate of adventitious shoot (%) = (the pieces of calli induced adventitious shoots/total pieces of calli) × 100%.

After 25 days, some shoots induced from maca calli (with and without REE) were used to analyze water content, soluble protein and H2O2 concentration along with MDA and enzyme activities, the others were subcultured on induction medium without REE. Subculture conditions were the same as those used for induction. After the shoots were subcultured for around two weeks, hyperhydric shoots were produced and counted as they appeared. Hyperhydricity rate (%) = (hyperhydric shoot number / total shoot number) × 100%.

Water content

The shoots were dried in an oven at 50°C until constant dry weight. Water content was calculated as follows: Water content (%) (w/w) = [(fresh weight − dry weight)/fresh weight] × 100%.

H2O2 and MDA concentrations

H2O2 concentration was determined according to Velikova et al. (2000) with some modification. Fresh maca shoots (0.3 g) were homogenized with 3 ml 0.1% (w/v) trichloroacetic acid (TCA) in an ice bath. The homogenate was centrifuged at 10,000 × g for 10 min at 4°C. For H2O2 determination, 0.5 ml supernatant was mixed with 0.5 ml 10 mM potassium phosphate buffer (pH 7.0) and 1 ml 1 M KI. The absorbance of the mixture was measured at 390 nm. The concentration of H2O2 was obtained based on a standard curve. MDA concentration was determined by thiobarbituric acid (TBA) reaction (Li 2000). Fresh shoots (0.5 g) were homogenized with 5 ml 5% TCA in an ice bath, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant (2 ml) with 2 ml 0.67% (w/v) TBA was incubated in boiling water for 30 min, then the mixture was centrifuged at 10,000 × g for 5 min. The absorbance of supernatant was assayed at 450 nm, 532 nm and 600 nm. MDA concentration in extracting buffer was calculated as the following: MDA (μmol g−1) = [6.45 (A532 − A600) − 0.56A450] × V t/W. V t = 0.005 l; W = 0.5 g.

Enzyme extraction

Fresh maca shoots (0.3 g) with 3 ml ice-cold extracting buffer were homogenized in an ice bath. The homogenized slurry was centrifuged at 10,000 × g for 10 min at 4°C and the supernatant was collected. The extracting buffer was 0.05 M potassium phosphate buffer (pH 7.0) with 0.1 mM EDTA and 1% (w/v) PVP. The protein extract was stored at −20°C for analyzing soluble protein concentration and enzyme activity within one week.

Soluble protein concentration

Soluble protein concentration was analyzed using the method of coomassie brilliant blue G-250 staining (Li 2000).

Enzyme activity

POD (EC 1.11.1.7) and SOD (EC 1.15.1.1) activities were measured according to the methods of Pütter (1974) and Beyer et al. (1987). POD activity was measured in the oxidation reaction of guaiacol at 470 nm (extinction coefficient, 26.6 mM−1 cm−1) for 3 min. The reaction system of POD was: 0.05 M potassium phosphate buffer (pH 5.5) 2.9 ml, 2% H2O2 1.0 ml, 0.05 M guaiacol 1.0 ml and enzyme extracting solution 0.1 ml. The reaction system of SOD was: 0.05 M potassium phosphate buffer (pH 7.8) 1.5 ml, 130 mM methionine 0.3 ml, 750 μM nitrotetrazolium blue tetrazolium (NBT) 0.3 ml, 100 μM EDTA-Na2 0.3 ml, 20 μM riboflavin 0.3 ml, distilled water 0.25 ml and enzyme extracting solution 50 μl. One unit of SOD was defined as the amount of enzymes that inhibited the rate of NBT reduction by 50%.

CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) activities were assayed according to Türkan et al. (2005) with modifications. CAT activity was determined by following the consumption of H2O2 (extinction coefficient, 39.4 mM−1 cm−1) at 240 nm for 3 min. The reaction mixture (2 ml) contained 0.05 mM potassium phosphate buffer (pH 7.0), 27 mM H2O2 and enzyme extracting solution 50 μl. The assay for APX depended on the reduction in absorbance at 290 nm as ascorbate was oxidized (extinction coefficient, 2.8 mM−1 cm−1). The reaction mixture (2 ml) contained 0.05 M potassium phosphate buffer (pH 7.0), 0.1 mM ascorbate, 0.3 mM H2O2 and enzyme extracting solution 50 μl.

MDHAR (EC 1.6.5.4) activity was assayed at 340 nm due to nicotinamide adenine dinucleotide (β-NADH) oxidation (extinction coefficient, 6.22 mM−1 cm−1) (Hossain et al. 1984). The reaction mixture (2.0 ml) consisted of 0.09 M potassium phosphate buffer (pH 7.0), 0.0125% Triton X-100, 0.2 mM NADH, 2.5 2M l-ascorbic acid and 20 μl enzyme extracting solution.

GR (EC 1.6.4.2) activity was assayed according to Türkan et al. (2005) which depends on the reducing rate and a change in the absorbance of oxidized glutathione (GSSG) at 340 nm (extinction coefficient, 6.22 mM−1 cm−1). The reaction mixture (2.0 ml) contained 0.1-M potassium phosphate buffer (pH7.8), 1-mM GSSG, 0.1-mM NADPH and 50-μl enzyme extracting solution.

Antioxidative activity of REE

The antioxidative activities of REE were assessed by 1,1-diphenyl-2-picrylhydarazyl (DPPH) radical scavenging method (Lee et al. 2005). Methanol solution of DPPH (100 μM) 2 ml with a series of concentrations of La3+, Ce3+ and Nd3+ solutions 2 ml respectively was placed into a cuvette. The reduction in absorbance at 515 nm was determined after 15 min. The DPPH scavenging capacity was defined as the following: % inhibition = [(A control−A sample)/A control] × 100. A control was the absorbance at time equaling 0, and A sample was the absorbance at the15th minute.

Statistical analysis

Induction rates and hyperhydricity rates were analyzed by χ2-test respectively. The data of other experiments were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

Results

Induction rates of adventitious shoot

The effects of REE on maca callus differentiation are shown in Table 1. REE reduced the induction rate of adventitious shoots from maca calli, with the effects depended on the concentrations of REE added to the induction media. When the concentrations of REE were in the range from 0.04 mM to 0.1 mM, the induction rates only slightly reduced (P > 0.05).

Table 1 Induction rates (%) of adventitious shoots induced from Lepidium meyenii Walp. calli on the induction media without rare earth elements (control) and at different concentrations of La3+, Ce3+ or Nd3+
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Hyperhydricity rates of adventitious shoot

Hyperhydric shoots were identified based on their appearance after shoots had been subcultured for two weeks. At this time, it was observed that hyperhydric shoots were thick, elongated, wrinkled and light green. The normal and the hyperhydic shoots are shown in Fig. 1.

Fig. 1
figure 1

Hyperhydric and normal shoots induced from Lepidium meyenii Walp. calli after subcultured for about two weeks. (a) Hyperhydric shoot; (b) Normal shoot

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The effects of different concentrations of REE on hyperhydricity rates in shoots are shown in Table 2. After subculturing for two weeks, most shoots induced from maca calli, on the media without REE, were hyperhydric, with a hyperhydricity rate of 92%. With the addition of 0.04–0.15 mM La3+, Ce3+ and Nd3+ to the induction media, hyperhydricity rates were reduced. When the concentrations of La3+, Ce3+ and Nd3+ in induction media were 0.1 mM, the hyperhydricity rates were 30%, 32% and 30%, respectively, lower than the controls (P < 0.05), showing that the survival rate of adventitious shoots was enhanced by REE.

Table 2 Hyperhydricity rates (%) of adventitious shoots induced from Lepidium meyenii Walp. calli after subcultured for abound 2 weeks
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Water content and soluble protein concentration

Water content in normal shoots sprouted from maca seeds (92.3%) was lower than that in the controls (94.7%) (P < 0.05). Adding different concentrations of La3+, Ce3+ and Nd3+ to induction medium, reduced the water content in shoots as the concentration of REE increased (Fig. 2). Shoot soluble protein concentration in the controls was 1.46 mg g−1 FW which was lower than that in seed shoots (2.15 mg g−1 FW) (P < 0.05). Increasing the concentration of La3+, Ce3+ and Nd3+ in the medium increased soluble protein concentration (Fig. 3). When the concentration of La3+, Ce3+ and Nd3+, in the culture media, was low, i.e. 0.1 mM, water content and soluble protein concentration for adventitious shoots were similar to those apparent with seed shoots (P > 0.05).

Fig. 2
figure 2

Water content in the shoots sprouted from Lepidium meyenii Walp. seeds (S), and in the shoots induced from the calli on induction media without rare earth elements (C) and at three concentrations of rare earth elements (REE). Values are mean ± S.D. (n = 3). *P < 0.05 respect to the controls (C)

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Fig. 3
figure 3

Soluble protein concentration in the shoots sprouted from Lepidium meyenii Walp. seeds (S), and in the shoots induced from the calli on induction media without rare earth elements (C) and at three concentrations of rare earth elements (REE). Values are mean ± S.D. (n = 3). *P < 0.05 respect to the controls (C)

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H2O2 and MDA concentrations

H2O2 concentration of shoots, in controls (0.057 mg g−1 FW) was much higher than that of seed shoots (0.029 mg g−1 FW) (P < 0.05) (Fig. 4). MDA concentration in the former (1.95 μmol g−1 FW) was also higher than that in the latter (1.33 μmol g−1 FW) (P < 0.05) (Fig. 5). Adding La3+, Ce3+ and Nd3+ to the induction media, reduced H2O2 and MDA concentrations as the concentrations of REE increased (Fig. 4 and Fig. 5). When La3+, Ce3+ and Nd3+ were added to the culture media at 0.1 mM, H2O2 and MDA concentrations, in adventitious shoots, were similar to seed shoots (P > 0.05).

Fig. 4
figure 4

H2O2 concentration in the shoots sprouted from Lepidium meyenii Walp. seeds (S), and in the shoots induced from the calli on induction media without rare earth elements (C) and at three concentrations of rare earth elements (REE). Values are mean ± S.D. (n = 3). *P < 0.05 respect to the controls (C)

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Fig. 5
figure 5

MDA concentration in the shoots sprouted from Lepidium meyenii Walp. seeds (S), and in the shoots induced from the calli on induction media without rare earth elements (C) and at three concentrations of rare earth elements (REE). Values are mean ± S.D. (n = 3). *P < 0.05 respect to the controls (C)

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Antioxidative activities of REE

Antioxidative activities of La3+, Ce3+ and Nd3+ were dependent on their concentrations (Fig. 6). When the concentrations of La3+ and Nd3+ increased from 0.01 mM to 0.1 mM, their antioxidative activities increased, up to a concentration of 0.1 mM. Antioxidative activity of Ce3+ also increased when its concentrations was less than 0.05 mM, but changed little when greater than 0.05 mM. Among the REE used, La3+ had the highest antioxidative activity.

Fig. 6
figure 6

The antioxidative activity of rare earth elements in aqueous solution analyzed by 1,1-diphenyl-2-picrylhydarazyl radical scavenging method. Values are mean ± S.D. (n = 3)

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Activities of antioxidative enzymes

The activities of antioxidative enzymes, such as CAT, APX, POD, SOD, MDHAR and GR of shoots in the controls were much lower than those in seed shoots (P < 0.05), and were enhanced to different extents by La3+, Ce3+ and Nd3+ (Fig. 7). The activities of these antioxidative enzymes were enhanced with an increase in Ce3+ and Nd3+ concentrations. The concentration at which La3+ enhanced the activities of antioxidative enzymes most was generally around 0.04 mM except for MDHAR.

Fig. 7
figure 7

The activities of antioxidative enzymes in the shoots sprouted from Lepidium meyenii Walp. seeds (S), and in the shoots induced from the calli on induction media without rare earth elements (C) and at three concentrations of rare earth elements (REE). Values are mean ± S.D. (n = 6). *P < 0.05 respect to the controls (C)

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Discussion

Hyperhydricity is considered as a physiological response that can be induced by different stress conditions, such as a high osmotic potential in the culture medium, high relative humidity, and specific hormonal treatment (Kevers et al. 2004). These different stresses can induce a sustained burst of reactive oxygen species (ROS) which can cause lipid peroxidation, DNA mutation and plant cell death (Cassells and Curry 2001). Plants have an efficient antioxidative system which scavenges ROS, which includes antioxidative metabolites such as ascorbic acid and glutathione, and antioxidative enzyme systems such as SOD, CAT, APX, POD, DHAR, MDHAR and GR. It has been hypothesised that the accumulation of undetoxified H2O2 due to an absence of antioxidative enzymes leads to hyperhydricity (Dily et al. 1993). Many studies support this hypothesis. Olmos et al. (1997) found that POD in the mesophyll and xylem cell walls in hyperhydric leaves of regenerated carnation plant was significantly reduced compared to normal tissue controls. Chen and Ziv (2001) showed that the activities of APX and CAT were correlated with hyperhydricity of Narcissus shoots. These results suggest that antioxidative enzymes play an important role in preventing shoot hyperhydricity. In our study, the shoots sprouted from maca seeds were normal, but most of the shoots induced from maca calli on induction medium without REE were hyperhydric after subculturing. We also showed that there were higher concentrations of H2O2 and MDA (a marker of lipid peroxidation) in the shoots induced from maca calli than those in seed shoots (P < 0.05). Meanwhile, antioxidative enzyme activities in the former was lower than those in the latter (P < 0.05).

Adding 0.04–0.15-mM La3+, Ce3+ and Nd3+ to the induction media, induction rates and hyperhydricity rates of adventitious shoots were reduced. REE have a number of biological activities in plants, although the mechanisms of how REE influence the plant are not clear (Hu and Ye 1996). As heavy metals, REE at low concentrations can have positive effects, while at higher concentrations, they can be harmful (Wang et al. 2004). In our study, when the concentrations of La3+, Ce3+ and Nd3+ were greater than 0.1 mM, maca callus differentiation was inhibited. The effects of REE on maca shoot induction and hyperhydricity rates suggest optimal concentrations of La3+, Ce3+ and Nd3+ in the media at around 0.1 mM.

When 0.04–0.1-mM La3+, Ce3+ and Nd3+ were added to the media, H2O2, MDA concentrations and shoot water content declined, while soluble protein concentrations increased. When the concentrations of La3+, Ce3+ and Nd3+ were 0.1 mM, the concentrations of H2O2, MDA, soluble protein and water content in adventitious shoots were similar to those in seed shoots (P > 0.05), and hyperhydricity rates of the shoots were much lower than the controls (P < 0.05).

The antioxidative activities of La3+, Ce3+ and Nd3+ were verified using DPPH radical scavenging (Lee et al. 2005). It has been reported that Ce3+ and Ce4+ have the capacity to remove ROS as they have similar functions to SOD; this capacity increases with concentration (Wang et al. 1997). Ce3+ and Ce4+ at appropriate concentrations can protect chloroplast membranes from ROS (Guo et al. 2000). In our study, oxidant stress was alleviated partly by the antioxidative properties of La3+, Ce3+ and Nd3+. However, whether REE can cross the cell membranes is still debated (Zheng et al. 2000a, b).

In present study, adding different concentrations of La3+, Ce3+ and Nd3+ to the media, enhanced the key antioxidative enzymes, i.e. CAT, POD and APX. It has been reported that REE can enhance SOD activity when plants are stressed (Peng and Pang 2002). Here SOD activities were increased slightly when 0.04–0.1-mM La3+, Ce3+ and Nd3+ was used. MDAHR activities were also enhanced by the addition of 0.04–0.1-mM La3+, Ce3+ and Nd3+ to the media. The increased MDHAR activity limited the formation of dehydroascorbate (DHA), generated from monodehydroascorbate (MDHA) which is harmful. It has been reported that stress tolerance can be related to increased GR activity (Azevedo Neto et al. 2006). GR activity was increased by adding 0.04-0.1 mM La3+, Ce3+ and Nd3+ to the media. The increase of APX, MDHAR and GR activities, suggests that ascorbate–glutathione cycle and oxygen stress tolerance may be enhanced by La3+, Ce3+ and Nd3+ at specific concentrations.