Introduction

The genus Eucalyptus (Myrtaceace) comprises of nearly 700 species distributed throughout the world (Brooker 2000). Eucalyptus tereticornis is a versatile member of the genus; known as forest red gum in Australia and Mysore gum in India, where it is one of the most extensively cultivated species (Rao et al. 2002). Besides its many other uses, E. tereticornis is a key source of raw material for the pulp and paper industry. The average annual yield from ordinary seed-raised agro forestry plantations is in the range of 5–6 m3 ha−1 yr−1 by the third year, and 10–15 m3 ha−1 yr−1 by the seventh yr of plant growth (Lal 1993). Whereas selected high-yielding clones can obtain mean annual productivity ranging between 16–20 m3 ha−1 yr−1 and 20–25 m3 ha−1 yr−1 by the third and seventh yr of plantation, respectively (Lal et al. 1993). This highlights the need for vegetative propagation of such clones within a clonal forestry program, and the requirement to develop more efficient and reliable multiplication methods. Although Eucalyptus multiplication through conventional vegetative propagation can be achieved, it has several constraints such as a poor rooting ability of stem cuttings and graft incompatibility (MacRae and Van Staden 1993; Bennett et al. 1994; Vengadesan and Pijut 2009). Micropropagation is an attractive alternative to conventional methods of vegetative propagation with the advantage of enhancing the rate of multiplication of valuable clones (Beck and Dunlop 2001). Moreover, micropropagation of these clones may result in rapid genetic gains and higher returns from plantations due to increased productivity. There are several micropropagation techniques used for the multiplication of Eucalyptus species. Among these, somatic embryogenesis (Nugent et al. 2001a; Prakash and Gurumurthi 2005; Pinto et al. 2008), organogenesis (Nugent et al. 2001b; Dibax et al. 2005; Aggarwal et al. 2010), and axillary shoot proliferation (Chang et al. 1992; Sharma and Ramamurthy 2000) are important. However, the response is reported to vary from clone to clone (Mullins et al. 1997, Aggarwal et al. 2010). Micropropagation of Eucalyptus from selected elite clones is a difficult task due to poor explant response, rapid browning, and hyperhydricity (Jones and Van Staden 1997; Jain 2006). Therefore, clone-specific micropropagation protocols are required. Moreover, Eucalyptus plantations raised from micropropagated plants can result in increased biomass yields compared to seedling-derived plantations (Khuspe et al. 1987). Therefore, the present investigation focused on factors affecting the efficient micropropagation and subsequent acclimatization of elite clone of E. tereticornis. We propose an easy and efficient method for rapid micropropagation of selected elite clones of E. tereticornis using nodal segments taken from freshly coppiced trees. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were used for assurance of clonal fidelity and uniformity of the micropropagated plantlets.

Materials and Methods

Plant material, chemicals, and glassware.

Plants of an elite clone of E. tereticornis (self-pruning and higher biomass productivity) growing at Thapar University Campus, Patiala, India were selected for this study. Freshly coppiced shoots from selected 10-yr-old trees provided explant material for the establishment of in vitro cultures. All routine chemicals were purchased from HiMedia Laboratories (Mumbai, India), and plant growth regulators were purchased from Sigma Chemical Co. (St Louis, MO). Unless otherwise mentioned, all experiments were conducted in 300 ml glass culture bottles (Kasablanka, Mumbai) with 50 ml of Murashige and Skoog medium (Murashige and Skoog 1962) containing 58 mM sucrose, 0.7 % (w/v) agar (MS medium), and supplemented with 2.5 μM 6-benzyladenine (BA) and 0.5 μM α-naphthalene acetic acid (NAA). The pH of medium was adjusted to 5.8 before autoclaving at 121°C for 20 min.

Cultures were established using nodal explants following the procedure of Aggarwal et al. (2010). Unless otherwise mentioned, cultures were incubated at 25 ± 1°C under cool white fluorescent lights (Philips India Ltd, Mumbai) with light intensity of 42.0 μmol m−2 s−1 (inside culture vessels) with a 16 h light/8 h dark cycle. Initially, the cultures were subcultured on to fresh medium at 7-d intervals (three subculture cycles) and subsequently these were subcultured on the same medium at 4-wk intervals until the emergence of shoots from explants. The actively growing shoot cultures were maintained on MS medium supplemented with 2.5 μM BA and 0.5 μM NAA for at least six subculture cycles at 4-wk intervals. These shoot cultures were then subsequently used for experimentation.

Effect of cytokinins on shoot proliferation and elongation.

Actively growing shoot clumps of 15–20 shoots (clump size 0.3–0.5 cm, three shoot clumps/culture vessel) were cultured on MS medium variously supplemented with (0.0–5.0 μM) BA, kinetin (KIN), or thidizuron (TDZ) in combination with 0.5 μM NAA.

Effect of inoculum size on shoot proliferation and elongation.

The effect of inoculum size was studied using two different sizes of shoot clumps (15–20 shoots per clump and 4–5 shoots per clump). These shoot clumps were cultured on MS medium supplemented with BA (2.5 or 0.1 μM) in addition to 0.5 μM NAA. Three shoot clumps per culture bottle were used in case of larger shoot clumps (15–20 shoots per clump) and ten shoot clumps per bottle were cultured in case of smaller shoot clumps (4–5 shoots per clump). Total number of shoots cultured in each bottle was kept approximately the same in both the cases.

Effects of light source.

Shoot multiplication and growth was measured in response to two different light sources. Larger shoot clumps (15–20 shoots per clump) were cultured on MS medium supplemented with BA (2.5 μM or 0.1 μM) together with 0.5 μM NAA for shoot proliferation and shoot elongation, respectively, and incubated either under photosynthetically active radiation (PAR; Lichtfarbe 77 fluora, Germany) or cool white fluorescent lights (CFL; Philips India Ltd, Mumbai). The light intensity obtained under both conditions was 42 μmol m−2 s−1 inside the culture vessel. The cultures were scored for the average number of shoots that had proliferated, and/or elongated, and shoot length after 4 wk of incubation. The chlorophyll content and osmotic potential were also assessed at this time.

Determination of chlorophyll content and osmotic potential.

Tissue samples from actively growing cultures (200 mg) were ground with mortar and pestle in 8.0 ml chilled aqueous acetone (80 %, v/v) and centrifuged (10,000×g for 20 min). The pellets were re-extracted with 80 % (v/v) aqueous acetone and centrifuged (10,000×g for 20 min). Supernatants were combined, the volume was made up to 20 ml and absorbance was recorded at 663 nm and 645 nm using a UV–VIS spectrophotometer (U-2900, Hitachi, Tokyo, Japan). Total chlorophyll contents were calculated following the method of Arnon (1949).

Osmotic potential (Ψs) was measured with vapor pressure osmometer (Wescor 5500, Logan, UT) following the method of Hernandey-Sebastia et al. (1999). Briefly, fresh tissue (500 mg) was placed in a 5-ml syringe and immediately frozen at −20°C. Samples were thawed and about 50–60 μl of sap was collected by squeezing the tissue inside the syringe. The sap was centrifuged (10,000×g for 30 min at 4°C) and 10 μl was used for each measurement. The Ψs values obtained in molarity were transformed to MPa using van’t Hoff’s empirical relationship Ψs = −CiRT, where C is the concentration of the solution expressed in molarity (moles of solute per kg of water), i is an ionization constant which is assumed to be one, R is a gas constant (0.00831 kg MPa mol−1 K−1) and T is absolute temperature (K = °C + 273).

Rooting of microshoots and acclimatization of plantlets.

Elongated shoots (2–3 cm in length) were excised from clumps just below the node, leaves were removed from lower nodes and microshoots were cultured on MS medium (full strength, half-strength, or one fourth-strength) supplemented with different concentrations (0.0–5.0 μM) of either NAA, indoleacetic acid (IAA), or indolebutyric acid (IBA). Similarly, the effect of PAR and CFL was also evaluated on rooting efficiency of microshoots cultured on one fourth-strength MS medium supplemented with 5.0 μM IBA. Acclimatization of plantlets was carried out in a polyhouse with controlled temperature (25–28°C) and humidity (90–95 %). Plantlets were planted in a mixture of soil and agropeat (3:1, w/w) in polythene bags and kept in a polyhouse. During the initial periods, 90 % relative humidity was maintained and slowly reduced to 40 % over a period of 1 mo. For each treatment, 100 plantlets were transferred to soil, and the survival data were recorded after 6 wk of transfer.

Biological acclimatization.

Plantlets (one per bag) were planted in a mixture of autoclaved soil and agropeat (3:1, w/w) and inoculated with 1.0 ml of rapidly growing bacterial suspension containing between 104 to 105 cells ml−1 in polythene bags. A total of three treatments were prepared consisting of plants transferred to soil and inoculated with Bacillus subtilis, Pseudomonas corrugata, and control plants with only nutrient medium (peptone 15.0 g l−1, yeast extract 3.0 g l−1, sodium chloride 6.0 g l−1 and d-glucose 1.0 g l−1). These bacteria were originally isolated from temperate locations in the Indian Himalayan Region and have been previously reported for their plant growth promotion and biocontrol properties with respect to agricultural, forest, and tissue culture raised plants (Pandey et al. 2000, 2002; Trivedi and Pandey 2007). The plant growth and survival were recorded after 15 d of transfer of plants to soil.

Testing of clonal fidelity.

Genomic DNA was extracted from leaves of the mother plants and randomly selected micropropagated plants following a 12-wk acclimatization period using the CTAB method (Doyle and Doyle 1990). The quality of the DNA was checked on a 0.7 % (w/v) agarose gel and the concentration determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). PCR amplifications were performed in 20 μl reaction volumes using 20 RAPD decamer primers (OPD1–OPD20; Operon Technologies, Alameda, CA) and 20 ISSR (16–20 nucleotide) primers (Table 1). Each reaction mixture consisted of 40 ng of genomic DNA, 1.0 U Taq DNA polymerase (Larova, Teltow, Germany), 100 μm dNTPs mixture, 2.0 μl reaction buffer (10×), and 10 nmol primer. Amplifications were performed in thermal cycler (model Gene Amp 9700, Applied Biosystems, San Francisco, CA). Amplification conditions were initial denaturation of 5 min at 94°C followed by 41 cycles of 1 min at 94°C, 45 sec at 35°C (or 55°C in case of ISSR) and 1.5 min at 72°C and the final extension for 5 min at 72°C. The amplified products were separated on 1.5 % (w/v) agarose gels and viewed using a UV transilluminator (Vilber Loumart, France) following ethidium bromide staining.

Table 1. Sequences of ISSR primers and characteristics of amplified markers
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Statistical analysis.

Unless otherwise stated, all experiments were conducted using four replicates with three explants in each culture vessel and repeated four times. The data were recorded after 4 wk of subculture. Osmotic potential and chlorophyll contents were estimated three times for each vessel, and repeated three times. Data were analyzed by analysis of variance and the means were compared with Duncan’s multiple range test (Duncan 1955).

Results

Effect of cytokinins on shoot proliferation and shoot elongation.

The effect of different concentrations of BA, KIN, and TDZ in combination with 0.5 μM NAA was examined on shoot proliferation and elongation of microshoots. The maximum number of shoots per culture vessel (342) was observed on medium supplemented with 2.5 μM BA Fig. 1A , whereas the maximum number of elongated shoots per culture vessel 54, Fig. 1B along with maximum shoot length (4.17 cm) was observed on medium supplemented with 0.1 μM BA. From the three cytokinins tested, BA was found to be the best for both shoot proliferation and shoot elongation followed by KIN and TDZ, respectively (Table 2). Raising the concentration of all cytokinins increased shoot multiplication, but inhibited shoot elongation.

Figure 1.
figure 1

Micropropagation of selected elite clone of E. tereticornis and clonal fidelity of micropropagated plants using RAPD and ISSR markers (A) shoot multiplication on MS medium supplemented with 2.5 μM BA and 0.5 μM NAA; (B) shoot elongation on MS medium supplemented with 0.1 μM BA and 0.5 μM NAA; (C) microshoots rooted on one fourth-strength MS medium supplemented with 5.0 μM IBA; (D) acclimatized plantlets; (E–F) RAPD profile with primers OPD 6 and OPD 20, respectively; (G–H) ISSR profile with primers ISSR- 2 and ISSR 13, respectively, Lanes 1 Mother plant, 2–13 micropropagated plants, M 1 kb molecular weight markers.

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Table 2. The effect of different cytokinins on proliferation and elongation of microshoots of Eucalyptus tereticornis propagated on MS medium
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Effect of initial shoot clump size on shoot proliferation and elongation.

The number of shoots elongated per culture vessel were significantly higher (65 shoots per culture vessel) when smaller shoot clumps (4–5 shoots per clump) were cultured on MS medium containing 0.1 μM BA along with 0.5 μM NAA, than cultures inoculated with larger shoot clumps (15–20 shoots per clumps) that resulted in lower number of shoot elongated (54 shoots per culture vessel). Despite that similar number of shoots were used in each flask for the initial inoculation, the number of shoots proliferated per culture vessel were significantly higher (342 shoots per culture vessel) when larger shoot clumps (15–20 shoots per clump) were cultured than cultures raised from smaller shoot clumps (4–5 shoots per clump), which showed lower shoot proliferation (245 shoots per culture vessel).

Effect of auxins and medium strength on rooting of microshoots.

Examination of the effect of various auxins (NAA, IBA, and IAA) and nutrient strengths (MS, half-strength MS, and one fourth-strength MS) on rooting efficiency of microshoots, showed that the rooting of maximum number of shoots (80.7 %) was achieved on one fourth-strength MS medium supplemented with 5.0 μM IBA. The maximum numbers of roots (4.25) per rooted shoot was also observed on the same medium, whereas maximum average root length (2.90 cm) was recorded on full-strength MS medium supplemented with 5.0 μM IBA (Table 3); Fig. 1C .

Table 3. The effect of medium composition (full-strength MS, half-strength MS and one fourth-strength MS) and auxins on rooting of microshoots of E. tereticornis
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Effect of light source on shoot multiplication, growth and rooting.

Both the number of proliferated and elongated shoots were significantly higher in cultures incubated under PAR compared to those incubated under CFL. Similarly, the percentage rooted shoots, root length, and number of roots per shoot also increased significantly in cultures incubated under PAR. Furthermore, in cultures incubated under PAR, root emergence was recorded on the fifth d, whereas 8 d were required for root emergence in cultures incubated under CFL (Table 4).

Table 4. The effect of light source on growth and morphogenesis of microshoots of E. tereticornis
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Effect of light source on chlorophyll content and osmotic potential.

Both chlorophyll content and osmotic potential were higher in cultures incubated under PAR compared to CFL. The total chlorophyll content was 0.853 mg g−1 in case of cultures incubated under PAR whereas chlorophyll content was 0.786 mg g−1 in case of cultures incubated under CFL. Similarly, osmotic potential of sap was also found to be higher (−0.9071) in cultures incubated under PAR as compared to those incubated under CFL (−1.0119; Table 4).

Acclimatization of plantlets.

The percentage of plants that survived transplantation to pots was higher (74.6 %) for the plantlets that were incubated under PAR as compared to plantlets incubated under CFL (Table 4). The survival of plants inoculated with P. corrugata was even higher (80.8 %), but the best results for acclimatization of plantlets (84.0 %) was achieved with B. subtilis Fig. 1D . Furthermore, plantlets inoculated with B. subtilis also showed significantly faster growth (19.3 mm increment) than plantlets inoculated with P. corrugata (16.1 mm) and control plantlets (11.7 mm), within the same time period.

Clonal fidelity of micropropagated plants.

Out of the 20 RAPD primers, 12 primers resulted in 58 scorable bands ranging from 250 to 1,500 bp in size. The number of bands for each primer varied from minimum of two to maximum of seven (OPD 1 and 18, respectively) with an average of 4.8 bands per RAPD primer. Out of 20 ISSR primers screened, 12 primers produced clear, scorable bands (Table 1). Bands for each primer varied from 3 (ISSR-9) to 7 (ISSR-6), with an average of 4.9 bands per ISSR primer. The screening with 12 ISSR primers generated 64 scorable bands, ranging in size from 250 to 2,500 bp. A total of 122 bands were generated, which were monomorphic in nature Fig. 1E–H , indicating the clonal uniformity of the micropropagated plants.

Discussion

In the present study, factors affecting micropropagation and subsequent acclimatization of plantlets of an elite clone of E. tereticornis were investigated. The effect of different cytokinins, in combination with NAA, was tested for shoot multiplication and elongation. Of the cytokinins tested, BA was found to be more effective than either KIN or TDZ (Table 2). Higher BA concentrations (≥2.5 μM) promoted shoot multiplication whereas lower concentrations (≤1.0 μM) promoted shoot elongation (Table 2). The use of BA for shoot multiplication of E. tereticornis (Rao 1988) and E. nitens (Gomes and Canhoto 2003 ) has been reported previously. The beneficial effect of BA over the other cytokinins for shoot multiplication or organogenesis is well documented (Rout et al. 2008; Vengadesan and Pijut 2009; Aggarwal et al. 2010). Cytokinins, in general, are known to suppress apical dominance and thus stimulate shoot multiplication (George 1996). The requirement of NAA for shoot multiplication and elongation could be due to its reported role in elimination of phenolic substances by competing for the active sites of auxin oxidase enzyme involved in oxidation of phenols (Pérez-Tornero et al. 2000), thus helping BA in the generation of multiple shoots (Sugimura et al. 2005). Previously, the presence of NAA along with BA in the medium has been reported to improve shoot induction and multiplication in Curculigo orchioides (Thomas 2007) and Spilanthes acmella (Saritha and Naidu 2008).

Smaller shoot clumps (4–5 shoots per clump) led to rapid shoot elongation, while larger shoot clumps (15–20 shoots per clump) showed poor shoot elongation, but better shoot multiplication. The effect of initial inoculum size on shoot multiplication and elongation is thought to be related to the activity of enzymes involved in different metabolic pathways influencing plant growth (Contin et al. 1998). Plant cells require a critical minimum inoculum density for growth. However, the optimum inoculum size is highly variable among different plants species (Figueiredo et al. 2000). The significant influence of inoculum size on shoot multiplication of Eucalyptus found in the course of this study seems to be a novel observation.

Root induction was observed in all tested media combinations (Table 3); however, maximum rooting of shoots (80.7 %) was achieved on one fourth-strength MS medium supplemented with 5.0 μM IBA. Similar results were also reported for other eucalypt species, where lowering of the nutrient salt concentration in the medium increased rooting ability of microshoots (Sharma and Ramamurthy 2000; Bennett et al. 2003). Auxins are widely used for induction of roots in microshoots. IBA was successful for root induction of microshoots of Eucalyptus hybrid cultivar ‘Urrbrae Gem’ (Glocke et al. 2006), and has similarly proved to be efficient for rooting in other species (Wardipura et al. 1986; Prakash and Van Staden 2008; Vengadesan and Pijut 2009).

Light quality is a critical factor that influences plant growth and development (Lee et al. 2007). The effect of two light sources i.e., PAR and CFL on shoot multiplication, growth, and rooting was also investigated. PAR was found to be beneficial both for shoot multiplication and elongation (Table 4). Tanaka et al. (1998) reported that the mixture of red and blue light enhances plant growth and development by increasing the P N (rate of photosynthesis) as the spectral energy distribution of red and blue light coincides with that of chlorophyll absorption (Goins et al. 1997). The emission spectra of PAR used in this study produced two major peaks, i.e., at 410–450 nm and 650–680 nm, whereas emission was in a broad range from 380–660 nm in case of CFL (Kumar et al. 2003). Significant increase in chlorophyll content was also observed in cultures incubated under PAR compared to CFL (Table 4). These results are consistent with earlier findings of Kumar et al. (2003) and Lee et al. (2007). PAR was also found to be more effective for efficient rooting than CFL (Table 4). Increase in rooting efficiency under PAR light may be due to the involvement of blue light responding cryptochromes and red/far-red light responding phytochromes as reported by Lin (2002). Light quality has been shown to promote rooting efficiency in some plant species (Rossi et al. 1993; Kumar et al. 2003).

Cultures incubated under PAR had higher osmotic potential compared to cultures incubated under CFL (Table 4). Osmotic potential is an important physiological parameter, which is reported to influence culture growth and subsequent plant survival during acclimatization (Hernandey-Sebastia et al. 1999). Light is reported to control expression and post-translational regulation of the nitrate reductase gene which is known to influence water relations and the growth of plants (Sharma et al. 1999; Appenroth et al. 2000). Kumar et al. (2003) also reported the increase in osmotic potential of plants incubated under PAR.

A major constraint for micropropagation of Eucalyptus is the considerable mortality that occurs during transfer to soil, due to the failure of plants to harden or acclimatize, and thus was a focus of this study. It was observed that plants produced under PAR showed higher survival rates and subsequently more vigorous growth following transfer to soil, which was commensurate with higher chlorophyll contents (Table 4). Often plants produced under in vitro conditions are reported to develop poor photosynthetic competence and slow growth during initial propagation periods (Brainerd and Fuchigami 1982; Kozai 1991). Higher survival rates and growth of plants produced under PAR following transfer to greenhouse conditions is possibly due to higher chlorophyll content, especially considering that modulation of photosynthetic efficiency is thought to be a key property for acclimatization of plants (Jeon et al. 2005).

Inoculation of plantlets with bacterial isolates during acclimatization has been found to be beneficial for the survival and subsequent growth of plants (Pandey et al. 2000). In vitro raised plants do not possess sufficient resistance against the soil microbial communities. Such plants can be susceptible to microbial (especially fungal) attack following field transfer (Palni et al. 1998). To overcome such a problem, several antagonistic plant growth-promoting bacteria have been used for biological acclimatization of micropropagated plants to increase the chances of survival and to augment plant growth overall (Pandey et al. 2000, 2002). Both Bacillus subtilis and Pseudomonas corrugate have been used for biological acclimatization of micropropagated plantlets (Pandey et al. 2000; Trivedi and Pandey 2007). The present study confirms the usefulness of these bacterial isolates for biological acclimatization of micropropagated plants of E. tereticornis.

Amplification of RAPD and ISSR markers established that a high level of genetic uniformity exists amongst the micropropagated plants and with that of mother plant. All scorable markers amplified (both RAPD and ISSR) were monomorphic thus supporting clonal uniformity. The utility of these markers in assessing the clonal uniformity is well documented (Aggarwal et al. 2010; Kumar et al. 2010). Previously, RAPD and ISSR markers have also been successfully used for the genetic analysis of Eucalyptus (Rani and Raina 1998, Aggarwal et al. 2010). The high degree of genetic uniformity observed in the micropropagated plants could be due to stability of genome during aseptic manipulations and culture conditions, which could be critical for longer-term commercial propagation of elite clones.

In conclusion, an efficient micropropagation protocol for an elite clone of E. tereticornis has been established. Both PAR light and bacterial isolates were found to be beneficial for micropropagation and acclimatization of E. tereticornis. The micropropagated plants were found to be genetically uniform and no genetic differences were detected compared to the mother plants. This protocol can be successfully exploited to undertake the commercial mass multiplication of other elite clones of E. tereticornis.