Introduction

Approximately 16 million ha of lands in Iran are located in the desert marginal area. Eucalyptus spp. is found to be an adaptable species which can be utilized in stabilizing the eroding lands (Assareh 1998). Over 700 species, subspecies and natural hybrids from Eucalyptus genus have been identified (Watt et al. 1999; Pinto et al. 2002). Among those, Eucalyptus microtheca F. Muell is native to Australia and has been introduced to the southern subtropical regions of Iran and has been adapted to the ecological conditions of this area. The species is not desirable for wood industry; however, improvements are possible using breeding techniques (Assareh 1998). Maintaining elite genotypes in this species is very difficult due to open pollination. The in vitro tissue culture enables us to control the essential factors in differentiation, regeneration, and proliferation. Moreover, it can be a proper tool to manipulate the environmental factors and plant material homogeneity (Morabito et al. 1994).

Differentiation is a multi-stage procedure, at the end of which the regeneration is carried out in forms of direct and indirect shooting, as well as somatic embryogenesis. In vitro regeneration is necessary for asexual proliferation of elite genotypes and gene transformation (Tang and Newton 2005). Explants derived from direct regeneration are genetically more stable than the ones passing through callus formation stage (Tang and Newton 2005). Adventitious shoot in E. camaldulensis (Muralidharan and Mascarenhas 1987) and E. gunnii (Herve et al. 2001), direct shoot in E. tereticornis (Sankara Rao 1988) and somatic embryogenesis in E. dunnii and E. grandis (Watt et al. 1999), E. nitens (Bandyopadhyay and Hamill 2000), E. globulus (Pinto et al. 2002) and E. sargentii and E. camaldulensis (Assareh 1998) has been reported. Comparing these methods indicates that (a) Juvenile explants have great potential for regeneration in Eucalyptus, (b) Using NAA with a proper cytokinin, will increase regeneration, (c) Using casein and glutamine amino acids or Thidiazuron (TDZ) for direct shoot, adventitious shoot and somatic embryogenesis induction in Eucalyptus is common and (d) A post-treatment with low level of hormone seems necessary for the growth of embryos.

Peroxidase (POD) has been reported to be used as a marker for rhizogenesis, organogenesis and embryogenesis (Coppens and Dewitte 1990; Gaspar et al. 1992; Preetha et al. 1995). This enzyme has high activity as a marker during stressful conditions (Ezaki et al. 1996). POD activity will increase during callus production, it has been reported to produce specific bands in embryogenic calli (Xiaoli et al. 1992) and also in different stages of regeneration (Rout et al. 1999; Panigrahi et al. 2007). On the other hand, significant change of POD and catalase (CAT) activity as a marker in pine direct shoot by TDZ has been observed (Tang and Newton 2005). Also, the effect of abscisic acid (ABA) on POD promoter has been recognized (Klotz and Lagrimini 1996) but no hormone has been identified to show a special band profile in different levels.

Peroxidase is affected by environment, genotype and their interaction. In this study, the objective was to study the effect of TDZ on regeneration efficiency. In vitro culture was exploited to eliminate environmental effects, and then variation in POD profiles at different TDZ levels was investigated in different types of regeneration.

Materials and methods

Investigation of the regeneration potential

Leaf discs, leafstalks, and green twigs without wood tissue, were separated as explants from a seedling plant which was kept in greenhouse for 1 year. For surface sterilization, the explants were immersed in sterilized water containing some drops of Tween for 15 min. Then they were rinsed with distilled water and soaked in mercuric chloride solution (0.01%) for 30 s. The explants were finally rinsed three times with sterile distilled water (5 min each time under laminar air flow cabinet). Modified MS (Murashige and Skoog 1962) media, with KNO3 and NH4NO3 at half-strength level (autoclaved for 20 min in 120°C) were used for regeneration. NAA and Kin were used in four (0.5, 1.0, 2.0, and 4.0 mg l−1) and two concentrations (0.0, 0.5, and 1.0 mg l−1), respectively, as in Table 1. After surface sterilization, the explants were cut into 1–1.5 cm long segments, cultured in regeneration media (culture) for 4 weeks, maintained in photoperiod of 16 h of light (27°C) and 8 h of darkness (19°C). Four replications containing six explants were used for each treatment.

Table 1 Callus production in E. microtheca explants using combinations of NAA and Kin levels
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Optimizing the process of differentiation

Based on the results of previous experiments, the green twigs were found as the best explants and were separated from the same seedling plant for the rest of the study. “Materials and methods” described above were used for sterilizing the explants, preparing the culture medium and maintaining culture conditions. Four different concentrations of TDZ plant growth regulator (0.0, 0.01, 0.1, 0.5, and 1.0 mg l−1) were added to the culture media that had shown superior performance (Table 2). The leaves of plantlets were used as samples for the rest of the study 30 days after culture.

Table 2 Optimizing differentiation by TDZ in E. microtheca explants
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POD quality survey

Crude extraction was conducted using Ebermann and Stich method (1982). Polyacrylamide gel electrophoresis was carried out using the method of Ebermann and Stich (1982). 0.1 g of fresh leaf was homogenized in 0.3 ml extraction buffer (1.2 g l−1 Tris, 3.8 g l−1 Borax, 3.6 g l−1 Chloride sodium, 2 g l−1 Ascorbic acid, 50 g l−1 polyethylene glycol 2000, 2 g l−1 Na2EDTA, pH = 7.5) and was kept for 48 h in 4°C. The samples were centrifuged at 6,000g, for 15 min in 4°C. Supernatant was used for electrophoresis with 12% separating gel (120 g l−1 acrylamide, 2 g l−1 bisacrylamide, 45.6 g l−1 Tris, pH = 8.3), which was loaded in the following conditions 5% staking gel (50 g l−1 acrylamide, 8.33 g l−1 bisacrylamide, 15 g l−1 Tris and 8.4 g l−1 citric acid) and electrolyte buffer (7.2 g l−1 glycine 1.5 g l−1 Tris) with 300 V and 100 mA during 4 h. 50 μl from each sample was loaded into the wells. Staining was done by 1,800 μl carbazole solution, (0.04 g carbazole, 1,200 μl absolute ethanol and 600 μl acetic acid) with 750 μl 3% H2O2 and 100 ml acetate buffer (50 mM, pH = 4.5) for 30 min. Then the gel was rinsed by distilled water and image was taken using a Sony digital camera. POD profile changes were carried out using two independent gels inclusive two repeats for maternal plant and minimum three independent repeats for other treatments.

Results

Tissue culture

In the first experiments, 7 days after culture initiation, callus production appeared from the cutting edge of the explants. Low level of NAA produced small calli, which showed increase in leaf disc and leafstalk explants in media with higher NAA levels. No embryogenic callus was observed (Table 1). Friable and embryogenic calli were formed in H6 and H11 treatments on the green twig explants (Table 1). Twig explants in H6 treatment produced very little callus (light green) on which microshoots appeared (Fig. 1a). Thirty days after the differentiation, when different amounts of TDZ were added to H6 treatment, the regeneration rate was 10% in B1 and 74% in B2 (Table 2). In B2 treatment callus production was not observed (Fig. 1b) and regeneration was of direct shoot type (Fig. 1c). There was no regeneration in treatment containing 0.1 mg l−1 TDZ. In B4 treatment, callus production was derived from cambium in twig explants and pre-embryonic white cells were appeared on green calli. In treatment containing 0.5 mg l−1 TDZ pre-embryonic cells passed dedifferentiation course (Fig. 2a–d) and regeneration was of adventitious shoot type, regeneration in B4 was 14% (Fig. 2e, f). Using B5 treatment, the calli became compact and hard, and the rate of adventitious shoot was reduced to 2%. Yellow-colored embryogenic calli showing globular and heart stage embryos were observed in H11 treatment (Table 1) but there were no regeneration (Fig. 3a). When 0.01 mg l−1 TDZ was added to H11 treatment (4 mg l−1 NAA and 0.5 mg l−1 Kin), there was 19% somatic embryogenesis induction (Fig. 3b). The highest amount of somatic embryogenesis (81%) was in B8 treatment (Fig. 3c).

Fig. 1
figure 1

Trend of callus suppression and direct shoot induction in 1 year old explants of twig E. microtheca plant. a Axillary shooting in H6 treatment without using TDZ, callus is seen in small amount (bar 0.4 cm). b Callus suppression in direct shoot formation using B2 treatment (bar 0.1 cm). c Regeneration in form of direct shoot (bar 0.3 cm)

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Fig. 2
figure 2

Regeneration trend in twigs of E. microtheca plant using B4 treatment. a Pre-embryonic cells in globular stage (bar 0.05 cm). b Pre-embryonic cells in heart shape stage (bar 0.05 cm). c Pre-embryonic cells in while start torpedo stage (bar 0.05 cm). d Pre-embryonic cells in cotyledon leaves appearance stage and beginning of photosynthesis (bar 0.1 cm). e,f Regeneration in adventitious shoot formation (bar 0.4 cm)

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Fig. 3
figure 3

Somatic embryogenesis induction in 1 year old explants of twig E. microtheca plant. a Embryogenic friable callus in H11 treatment that had heart shape and globular stages but would not make regeneration (bar 0.4 cm). b Embryogenic calluses resulted from B7 treatment in which they had a few regeneration (bar 0.5 cm). c More regeneration in somatic embryogenesis formation using B8 treatment (bar 0.5 cm)

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POD qualitative tests

Mother plants as controls and plantlets of adventitious origin were compared in qualitative surveys (Fig. 4). In the maternal plant, there was a group of three allele dimers and a group of five allele tetramers (Fig. 4a). There were two new extra bands in the plantlets derived from tissue culture. The first band with lower activity appeared in light molecular area (band 10), using NAA and Kin in all samples (100%). When different amounts of TDZ were added to culture media, 87.5% of regenerated plantlets had one extra band in heavy molecular weight area (band 1). In B1 treatment, all alleles existed and three, four and nine alleles had more activity (Fig. 4b). In B2 and B7 treatments, in plantlets which were regenerated via direct shooting and somatic embryogenesis, allele three was eliminated and the activity of dimer and tetramer alleles locus reduced (Fig. 4c, d). Plantlets derived from somatic embryogenesis in B8 treatment, had an active tetramer locus, while the activity of dimer locus was similar to treatments B2 and B7, namely allele three was disappeared (Fig. 4e). All alleles existed in 50% of B4 treatment plantlets and the activity of tetramer locus had increased drastically (Fig. 4f). In the other 50% of plantlets, dimer locus had lost its stability and became inactive (Fig. 4g). POD alleles in plantlets derived from adventitious shoot in B5 treatment showed similar behavior as in B4 but both locus were highly active (Fig. 4h).

Fig. 4
figure 4

POD bands resulted from somaclonal plantlets of E. microtheca. Maternal plant has been used as a check plant for B1 treatment plantlets and from B1 treatment for evaluated other treatments containing TDZ hormone were used. a Maternal plant that containing two locus of dimer (alleles 2–4) and tetramer (alleles 5–9). b Alleles activity in axillary shooting plantlets of B1 treatment and show new lower epigenetic band (band 10) in light molecular weight area. c,d The alleles are resulted from plantlets of B2 and B7 treatments (direct shoot and somatic embryogenesis), respectively that was with eliminated of heterozygote allele (allele 3) from dimer locus along with appearance of new epigenetic band (band 1) in heavy molecules weight area. e Shows reactivation of tetramer locus and disappearance of heterozygote allele (allele 3) in plantlets resulted from somatic embryogenesis in B8 treatment. f,g Show stability and instability in dimer locus also hyperactivity of dimer and tetramer locus in plantlets resulted from treatment B4 (adventitious shoot). h Shows hyperactivity of both locus in plantlets of B5 treatment. + and − show light and heavy molecules weight area, respectively

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Discussion

In this study, the medium containing 1 mg l−1 NAA and 1 mg l−1 Kin proved to be the best treatment for axillary shoot regeneration of E. microtheca twig explants. The result of this research agrees with report of Sankara Rao (1988) in which uniformity concerning type and concentration of used auxin for direct shoot in E. tereticornis was demonstrated.

The twig explants of E. microtheca in treatment containing 4 mg l−1 NAA along with 0.5 mg l−1 Kin showed the most friable embryogenic calli. For induction of somatic embryogenesis in Eucalyptuses, 3–5 mg l−1 NAA has been used in previous studies (Muralidharan et al. 1989; Pinto et al. 2002).

Adding 0.01 mg l−1 TDZ to H6 treatment resulted in decrease in callus production and regeneration was in direct shoot form. In other words, TDZ in small amount is likely to have suppressive effect on callus production but stimulated regeneration (Shan et al. 2000; Tang and Newton 2005). Visser et al. (1992) showed that benzyl amino purine and TDZ are hormones that can stimulate shoot formation.

Thidiazuron increase in H6 treatment to 0.5 mg l−1 increased callus production and stimulated the formation of adventitious shoots. In this treatment, the existence of compact bulks of white pre-embryonic cells is a possible evidence for a balance between cytokinin and auxin ratio. The presence of auxins, stimulates reproduction of pre-embryonic cells in callus, but has a preventive effect on growth (Nomura and Komamine 1985; Vries et al. 1998; Filonova et al. 2000). So it can be postulated that the combination of cytokinin and auxin has been optimized by adding TDZ. TDZ can highly stimulate and balance endogenous auxin/cytokinin levels (Visser et al. 1992). In the joint of pre-embryonic cells to the callus, phenol accumulation could be observed at the basal of embryos. Phenol accumulation has been reported in different stages of embryogenesis and regeneration (stages) of various species of Eucalyptus (Muralidharan and Mascarenhas 1987). Phenol affects the cell wall structure and plant growth regulators have role on their accumulation (Fry 1979).

In B7 and B8 treatments, that contain 0.01 and 0.1 mg l−1 TDZ, respectively, the regeneration took place through somatic embryo formation. Embryogenic calli in these treatments, contrary to H11 treatment, have formed many roots.

Peroxidase bands appearance or disappearance, and their rate of activity are affected by type and amount of hormones, as well as regeneration type (Joersbo et al. 1988). Band 10 is correlated with NAA and Kin, in all samples except the maternal plant, with very low activity. This band can be a marker for in vitro tissue culture or the use of NAA and Kin hormones. Compared to previous samples, band 1 exists whenever TDZ is added to the culture medium. In other words, this band is a marker for the use of TDZ in culture media. Some studies have discussed the impact of auxins and cytokinins on POD profile (Krsnik-Rasol 1991; Hassanian 2004; Panigrahi et al. 2007).

In axillary shoots, POD activity in dimer and tetramer locus, increases remarkably. There was no visible difference between B2 and B7 treatments which cause direct shoot and somatic embryogenesis, respectively. POD activity was low in direct shoot of white pine explants using TDZ hormone (Tang and Newton 2005). In B2 and B7 treatments the activity of all alleles decreases and heterozygote allele 3 disappears in all plantlets. Allele 3 disappeared in B8 treatment as well. So it can be concluded that TDZ in small amounts (0.01 and 0.1 mg l−1) may cause inactivity in a heterozygote allele from a dimer locus. This may be due to the synthesis of an inhibitor for POD promoter (Klotz and Lagrimini 1996) or due to mutation (Larkin and Scowcroft 1981). Phytohormones can affect POD promoter (Klotz and Lagrimini 1996). Tetramer locus activity in somatic embryos increased and heterozygote allele from a dimer loci disappeared. POD bands activity pattern in barley embryogenic calli, has been introduced as biochemical marker (Coppens and Dewitte 1990).

There’s stability in dimer and tetramer locus in all regeneration types under treatments containing 0.01 and 0.1 mg l−1 TDZ. Adventitious shoots initiated when TDZ level exceeds 0.5 mg l−1. This is while Tian et al. (2003) have reported reduction of POD activity in organogenesis. Tang and Newton (2005) observed reduction of POD activity in adventitious shoots for five to 6 weeks and after that they observed POD increase again. This reduction activity can be due to TDZ hormone ability for deleting a POD specific allele, which create instability or prevent activity of one or a few POD locus during regeneration.