Micropropagation of an Eucalyptus hybrid (Eucalyptus benthamii x Eucalyptus dunnii)

This study was designed to micropropagate E. benthamii x E. dunnii, by testing chlorine concentrations for explant asepsis, the optimal concentrations of benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) for bud proliferation, and the ratio between BAP and gibberellic acid (GA3) in two nutrient media for shoot elongation. Nodal segments from H12, H19 and H20 clones were disinfected with 0.5, 1.0, 1.5 and 2.0% (v v) of chlorine. Explants were grown on 1⁄2MS medium supplemented with BAP (0, 0.25, 0.50, 0.75 and 1.00 mg L) and NAA (0, 0.025, 0.050, 0.075 and 0.100 mg L) for bud production. They were elongated on MS and 1⁄2MS media supplemented with BAP (0, 0.05 and 0.10 mg L) and GA3 (0, 0.1, 0.2 and 0.3 mg L). The 0.50 mg L BAP and 0.050 mg L NAA combination was optimal for bud proliferation for H12 and H20. GA3 concentrations of 0.10 and 0.20 mg L -1 combined with 0.10 mg L BAP on 1⁄2MS resulted in the longest shoots, for H12 and H20, respectively. Regardless of clone, the rooting rate was low, with an average of 12.0% and 14.4% of plants having roots for in vitro and ex vitro conditions, respectively.


Introduction
Economically important Eucalyptus species or varieties adapted to severe climate conditions are limited, but E. benthamii and E. dunnii stand out among these species, offering the possibility of climate adaptation and quality wood production. In regions where severe frosts compromise the establishment and growth of Eucalyptus, resistance to cold conditions is a crucial feature that may be introduced by interbreeding (BORÉM, 2007).
The natural hybridization between Eucalyptus species is common (BUTCHER et al., 2005). Recently, it was shown that the occurrence of spontaneous hybrid formation between E. benthamii and E. dunnii at Embrapa Forests in Colombo, Paraná State, might present frost-tolerant varieties. E. benthamii and E. dunnii hybrids are an alternative for future forest plantations, particularly due to frost tolerance and low temperature resistance (HIGA et al., 2000;JOVANOVIC et al., 2000). However, there are limitations regarding seed production, mainly because of the high price and low availability of hybrid seeds. Moreover, hybrid clones have low rooting rates when propagated by macropropagation techniques, such as mini-cutting (BRONDANI et al., 2008;BRONDANI et al., 2010a and b). Therefore, before the commercial use of these hybrids can be completed, studies are required to identify methods for obtaining seeds or plants from those hybrids.
One method for plant production is in vitro plant propagation (KOMATSU et al., 2011;MERKLE;NAIRN, 2005;NEHRA et al., 2005;VILLA et al., 2006;WATT et al., 2003), with micropropagation as the most widespread technique, with applications in wood production (BORÉM, 2007). According to Joshi et al. (2003), this technique is linked to genetic improvement programs that aim to maximize or maintain genetic material. Borém (2007) suggests that micropropagation from organ segments, with direct bud induction (direct organogenesis) or preexistent buds stimulus through apical dominance suspension, is the most recommended method for plant propagation and results in lower genetic variation than other methods.
Due to the absence of a protocol for E. benthamii x E. dunnii hybrids using in vitro cultivation, the present study was designed to: (i) to determinate the best chlorine concentration for the asepsis of nodal segments; (ii) to verify the best benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) ratio for bud proliferation and (iii) to verify the best BAP and gibberellic acid (GA 3 ) ratio and culture medium conditions for shoot elongation. A protocol was developed for optimal rooting and survival of explants ex vitro.

Plant material
The material used to obtain the mini-cuttings came from 1-year-old plants, located in Guarapuava, Paraná State, Brazil. The three best E. benthamii x E. dunnii hybrids (H12, H19 and H20) were propagated by conventional cutting techniques. Cuttings 15 cm long and 90 days old were planted in gutters containing sand and grown in a hydroponic system. After 20 days, the first pruning was performed at 5 cm high from the base of each rooted cutting. Plants received daily nutrient solution by dripping at a flow of 5 L m -2 (BRONDANI et al., 2009(BRONDANI et al., , 2010a.

Explant sampling and preparation
One week before shoots were removed for propagation, the mini-stumps were sprayed with Kumulus DF ® fungicide at 3 g L -1 (p v -1 ). Shoots from the 13 th collection were transported in polyethylene bags with 1% of ascorbic acid solution (p v -1 ). In the laboratory, leaves were removed and the vegetative material was washed with deionized water, for removal of dust and particles.

Culture medium preparation and in vitro conditions
The pH value was adjusted to 5.8 using HCl (0.1 M) and NaOH (0.1 M) and 250 mg L -1 PVP 40, BAP and NAA were added to the culture medium. After this, 6 g L -1 agar was added and the culture medium was autoclaved at 121°C (≈ 1.0 kgf cm -2 ) during 20 minutes. After autoclaving, GA 3 was added to the culture medium into a laminar flow chamber using a disposable filter Millex-GP ® , with a 0.22 μm sterile membrane. Explants were cultivated in an incubation room, at 25°C (± 2°C), 16-hour photoperiod and 40 μmol m -2 s -1 of luminosity.

Plant establishment
Nodal segments from the medium portion of shoots, with one pair of axillary buds, with leaves removed and a mean size of 1.5 cm were immersed in 70% solution of alcohol (v/v) during 15 seconds, washed in deionized and autoclaved water, and then submitted to 0.5, 1.0, 1.5 and 2.0% (v v -1 ) chlorine (NaOCl) with Tween 20 ® (0.05% v v -1 ) during 10 minutes. At the end, nodal segments were washed three times in deionized and autoclaved water, and inoculated in 7 x 3 cm test tubes containing 10 mL of MS medium, supplemented with 30 g L -1 sucrose (Merck ® ).
After 21 days, fungal and bacterial contamination and oxidation were evaluated. The number of leaves and shoots and total shoot length were quantified from the established explants. The experiment was performed in a randomized block design (3 x 4), with three clones (H12, H19 and H20), four chloride concentrations (0.5, 1.0, 1.5 and 2.0%) and five replications. Each replication was composed of five explants. Shoots with three to five buds were inoculated in 10 x 2 cm test tubes with 10 mL of ½MS medium supplemented with 0.25, 0.50, 0.75 and 1.0 mg L -1 BAP (Merck ® ) and 0.025, 0.050, 0.075 and 0.100 mg L -1 NAA (Merck ® ). Sucrose at 15 g L -1 was added to the medium. After 30 days, explants were transferred to a new culture medium maintaining the same treatments.
The number of developed buds per explant was quantified 60 days after the inoculation. The experiment was performed in a randomized block design (2 x 5 x 5), testing two clones (H12 and H20), five concentrations of BAP combined with five concentrations of NAA, and five replications. Each replication was composed of five explants.

Shoot elongation
Explants with 15 to 20 buds were grown in 7 x 6 cm recipients during 21 days in 30 mL of ½MS medium, which were supplemented with 250 mg L -1 PVP 40 (Merck ® ), 15 g L -1 sucrose, 6 g L -1 agar (Merck ® ) and pH adjusted to 5.8. After this, explants were inoculated in the same size recipients containing 30 mL of MS medium with 30 g L -1 sucrose or ½MS with 15 g L -1 sucrose, both supplemented with 0.05 and 0.10 mg L -1 BAP and 0.1, 0.2 and 0.3 mg L -1 GA 3 (Merck ® ). The culture medium was supplemented with 0.5 mg L -1 NAA.
The number of elongated shoots and the length of shoot mean per explant were quantified 30 days after inoculation. The experiment was performed in a randomized block design (2 x 2 x 3 x 4), testing two clones (H12 and H20), two culture media (MS and ½MS), three concentrations of BAP combined with four concentrations of GA 3 and five replications. Each replication was composed of five explants.

In vitro rooting
Elongated shoots of H12 and H20 clones with 1.5 cm long and one pair of buds were inoculated in 10 x 2 cm recipients with 10 mL of ½MS medium with 15 g L -1 sucrose. 0.1 g L -1 calcium pantotenate (Merck ® ), 0.1 mg L -1 Biotin (Merck ® ), 0.1 g L -1 Mio innosytol (Merck ® ) and 2 mg L -1 IBA (indole-3butyric acid) (Merck ® ) were added to the culture medium (LE ROUX; VAN STADEN, 1991). Rooting percentage was defined as the percentage of plants with roots and was quantified after 30 days.
Shoots remained inside the greenhouse for 40 days, with relative air humidity (RH ≥ 80%) and air temperature (T < 30°C) automatically controlled. Afterwards, shoots were transferred to a shade house (50% of light reduction) for acclimatization for 22 days. The micro aspersion system was controlled by timers and consisted of 12 micro aspersion nibs with 2.0 kg cm -2 of pressure, and flow of 144 L hour -1 (2.4 L minute -1 ), which were turned on during two minutes every two hours. After that, shoots were transferred to outdoor conditions during 28 days, for plant hardening and growth. The aspersion system worked with net water pressure (2.0 kg cm -2 ). The system consisted of 12 micro aspersion nibs of 97 L hour -1 , which were turned on three times a day during 20 minutes.
After 90 days, the rooting percentage was quantified. The experiment was performed in a randomized block design (2 x 2), testing two clones (H12 and H20), with presence and absence of IBA. To that end, six replications composed by five explants were used.

Statistical analysis
Data were analyzed using the Lilliefors test (p < 0.05), and then, with analysis of variance (ANOVA) (p < 0.01 and p < 0.05). Qualitative data were compared by Tukey test (p < 0.05) and quantitative data by polynomial regression (p < 0.01 and p < 0.05).

Results and discussion Establishment
There was no significant interaction between the clone and the concentration of chlorine on plant asepsis. However, there was a significant effect among clones regarding the percentage of plants with bacterial contamination (p < 0.01), oxidation (p < 0.05) and plants that were established (p < 0.01). H19 had 9% of the plants with bacterial contamination, much greater than H12 and H20, which had 1%, or the absence of bacterial contamination, respectively. The H20 clone did not exhibit oxidation and statistically differed from H19, which had 6% of oxidized explants. The H12 clone did not differ statistically from the others, presenting on average 3% of oxidation (Table 1)  In the columns, the mean values followed by the same letter do not significantly differ by Tukey test (p < 0.05). a data presented as: mean ± standard deviation.
H12 and H19 did not differ regarding the success of explant establishment, with 45 and 46% of the explants surviving after final acclimation, respectively. H20 had the greatest percentage of established explants, with 66% of the plants surviving (Table 1). From established explants, the number of leaves, number of shoots and mean shoot length did not statistically vary among clones, with a mean value of approximately 5 leaves per explant and 2 shoots of about 0.7 cm in length per explant.

Shoot Multiplication
Despite of its satisfactory establishment, the H19 clone proved to be highly recalcitrant in the following stages, with a high oxidation index during bud proliferation, which limited its in vitro cultivation under tested conditions.
The concentration of NAA and BAP significantly affected the number of bud induction for H12 and H20 clones (p < 0.01; Figure 1). The greatest number of buds for H12 occurred with 0.51 mg L -1 BAP combined with 0.057 mg L -1 NAA. Under these concentrations, there were 8.8 buds per explant (Figure 1a). For H20, 0.45 mg L -1 BAP and 0.040 mg L -1 NAA resulted in the most buds, with about 13 buds per explant after 60 days of inoculation ( Figure 1b).
Concentrations above 0.75 mg L -1 of BAP did not result in increased bud proliferation.
Furthermore, under concentrations of 1 mg L -1 BAP combined with 0.1 mg L -1 NAA, vitrification, explant oxidation and mortality were verified.

Shoot elongation
The medium and growth regulator concentrations significantly affected the number of shoots per explant (Figure 2). In ½MS medium, H12 had the greatest number of elongated shoots with medium supplemented with 0.11 mg L -1 BAP and 0.13 mg L -1 GA 3 with 10.6 shoots per explant (Figure 2a). H20 had the most shoots under nutrient conditions of 0.08 mg L -1 BAP with 0.22 mg L -1 GA 3 , with 5.82 elongated shoots per explant (Figure 2b).
Despite the fact that the estimation of BAP critical point for H12 was mathematically greater than the highest concentration tested on ½MS medium (Figure 2a), it can be noticed that the concentration of 0.1 mg L -1 BAP positively influenced the number of shoots, regardless of clone. Nevertheless, on MS medium, the H12 clone had, on average, 2.58 elongated shoots per explant under 0.07 mg L -1 BAP and 0.11 mg L -1 GA 3 (Figure 2c). The H20 clone, under 0.06 mg L -1 BAP and 0.18 mg L -1 GA 3 had 2.91 shoots (Figure 2d). When grown on MS medium, the clones had 3 elongated shoots per explant with 0.1 mg L -1 BAP concentration.
The clones had distinct behaviors with regard to GA 3 . Regardless of medium, the concentration of 0.1 mg L -1 and 0.2 mg L -1 GA 3 produced the most shoots for H12 and H20, respectively. Furthermore, H12 had approximately twice the number of shoots compared to H20, when it grew on ½MS medium (Figures 2a and b).  When it was grown on MS medium, H12 had a reduced percentage of shoots per explant (75% lower) compared to plants from ½MS medium. Similar results were found for the H20 clone, which had a 50% reduction in the number of shoots per explant when grown on MS medium. Furthermore, explants cultivated on MS medium showed oxidation symptoms, which did not occur when the culture medium had a 50% reduction in its saline composition (½MS).
H12 had the longest shoot length with medium supplemented with 0.07 mg L -1 BAP and 0.22 mg L -1 GA 3 , with shoots on average of 1.52 cm in length when grown on ½MS (Figure 3a). H20 had the longest shoot length, at 1.5 cm, with medium supplemented with 0.08 mg L -1 BAP and 0.21 mg L -1 GA 3 (Figure 3b).
When full-strength MS was used, H12 clones were 1.13 cm long with medium containing 0.08 mg L -1 BAP and 0.16 mg L -1 GA 3 (Figure 3c). For H20, the longest shoots were, on average, 1.43 cm long and occurred with medium containing 0.12 mg L -1 BAP and 0.30 mg L -1 GA 3 (Figure 3d).
In general, the longest shoots were found with medium supplemented with 0.10 mg L -1 BAP and 0.20 mg L -1 GA 3 , regardless of clone and MS concentration. Exceptions occurred for H20 cultivated on full-strength MS medium, where the GA 3 concentration that provided the longest shoots was at 0.30 mg L -1 .

Rooting
Both H12 and H20 had a rooting percentage of 12% of plants with roots in vitro (data not shown), 30 days after inoculation on ½MS medium. For ex vitro rooting, genetic materials did not differ in relation to IBA treatment, regarding survival after greenhouse (41.0%), after shade house (22.2%) and rooting under outdoor conditions (14.4%) ( Table 2).
The rooted material was vigorous and had excellent aboveground development (Figure 2d). Although significant differences were not found, it was observed that the H12 clone tended to have the highest mean values (    Regardless of the clone, there were a high percentage of plants with fungal contamination (41.33%, on average). The identification of contaminant sources (e.g. fungal or bacterial) during establishment becomes essential. Further studies on fungal and bacterial contamination are required, utilizing methods that reduce contamination with products application to source plants before shoots are collected, collaborating to the maximization of hybrid micropropagation processes.
The percentage of established explants were similar for our results compared to those found by Joshi et al. (2003) for 30-year-old E. tereticornis x E. grandis trees, both at approximately 50% survival after acclimation. Here, 50% of plants survived when treated with 30% concentration (v v -1 ) of NaOCl 2 for 20 minutes. A similar treatment was performed by Bennett et al. (1994), with sodium hypochlorite at 1% (v v -1 ) during 20 minutes for asepsis of 4-to 5-year-old nodal segments of E. globulus trees. George et al. (2008) emphasize that the physiological condition of stock plants, from which the explants are obtained, has a high influence on the later behavior of cultures. This fact may have influenced the clone response, because the clonal mini-garden management system provided homogeneity in the phytosanitary conditions. Due to the non-significant effect among tested sodium hypochlorite concentrations, 0.5% chlorite concentration is recommended for the asepsis of explants of hybrid material when it comes from a clonal mini-garden cultivated in sand.
Eucalyptus micropropagation responses are variable according to concentration of plant growth Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 655-663, 2011 regulators and depend on work conditions. Gomes and Canhoto (2003) verified that 0.9 μM BAP (≈ 0.20 mg L -1 ) combined with 0.05 μM NAA (≈ 0.01 mg L -1 ) on ½MS medium was the optimum concentration for E. nitens bud proliferation. Despite the low rate of bud proliferation (2.25 per explant), their results were promising because of the high recalcitrance of this species. Joshi et al. (2003) observed that E. tereticornis x E. grandis produced 20 to 25 buds per explant after 150 days on MS medium supplemented with 1 mg L -1 BAP and 1 mg L -1 NAA. Similar results were obtained by Bisht et al. (1999) for E. tereticornis x E. camaldulensis, where the highest multiplication, regardless of clone, occurred after 120 days on MS medium supplemented with 1 mg L -1 BAP and 1 mg L -1 NAA. For E. tereticornis, Sharma and Ramamurthy (2000) obtained the greatest number of buds (20 to 30 per explant) on MS medium supplemented with 1.0 mg L -1 BAP and 0.1 mg L -1 NAA. George et al. (2008) emphasize that excess cytokinin on the culture medium may be toxic to the explant, causing severe problems in the following stages. Bisht et al. (1999) verified for E. tereticornis x E. camaldulensis that the highest BAP concentrations (over 1 mg L -1 ) reduced the number of buds per explant, regardless of the studied clone. Bennett et al. (1994) recorded that BAP concentrations above 2.5 μM (≈ 0.56 mg L -1 ) on MS medium reduced the average number of buds per explant of E. globulus, which was similar to the results found in the present study, where concentrations over 0.5 mg L -1 BAP reduced the number of buds per explant (Figure 1). This fact supports that concentrations above 0.5 mg L -1 BAP are not recommended during the bud multiplication stage for the studied hybrid.
In general, H12 and H20 clones produced similar number of buds in relation to regulator concentrations. Furthermore, the combination of 0.50 mg L -1 BAP with 0.05 mg L -1 NAA was the most promising, and similar to the best values estimated by mathematical models, which increased the number of buds per explant (Figure 4a).
Several reports used different cytokinin and gibberellin combinations to induce shoot elongation, mainly for genetic materials of difficult in vitro propagation. Joshi et al. (2003) observed multiplication followed by shoot elongation of 30year-old E. tereticornis x E. grandis trees. The mean length of elongated shoots varied from 2.5 to 3 cm in length during 30 days when grown on MS medium supplemented with 1 mg L -1 BAP and 0.04 mg L -1 GA 3 . Similar effects were verified by Bisht et al. (1999) for E. tereticornis x E. camaldulensis, who reported multiplication followed by elongation after 120 days on MS medium supplemented with BAP and NAA. After 180 days on MS medium, all BAP and NAA treatments had elongation, producing shoots of 2.0 cm to 3.5 cm in length. Barrueto Cid et al. (1999) elongated buds regenerated from cotyledon leaves on SP medium (modified MS) supplemented with 1.0 μM BAP (≈ 0.24 mg L -1 ), 0.5 μM NAA (≈ 0.09 mg L -1 ) and 2.0 μM GA 3 (≈ 0.7 mg L -1 ) and, after 20 to 30 days of growth, produced vigorous shoots with mean length of 1.5 cm. In general, explants cultivated on ½MS supplemented with 0.10 mg L -1 BAP and 0.10 mg L -1 GA 3 for the H12 clone and 0.10 mg L -1 BAP and 0.20 mg L -1 GA 3 for the H20 clone had either the best standard development of number of elongated shoots and shoots length, then shoots presented a good aspect and vigor, with mean size around 1.5 cm in length (Figure 4b), 30 days after inoculation. The thinnest and the most fragile elongated shoots were produced in culture medium supplemented with GA 3 and without BAP; thus, its application is not recommended in the genetic material studied. H12 and H20 clones had similar rooting responses, in both in vitro and ex vitro conditions. Joshi et al. (2003) verified the highest rooting rates on ½MS culture medium for in vitro conditions. However, the maximum rooting (75% of rooting) occurred with 1 mg L -1 IBA, as concentrations above Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 655-663, 2011 1 mg L -1 IBA promoted callus induction. Bisht et al. (1999) had the same response where the maximum rooting was verified only after adding 1 mg L -1 IBA to MS medium for in vitro conditions. It is relevant to emphasize for the studied hybrid material, that 2 mg L -1 IBA concentration induced callus formation followed by rooting (Figure 4c), an effect similar to that verified by Joshi et al. (2003) and Bisht et al. (1999). Lower IBA concentrations might increase rooting rates without callus induction (Figure 4d). It was verified that the genotype had low rooting rates, even when cultivated in vitro, similar to the rates observed for ex vitro conditions. These results show that E. benthamii x E. dunnii hybrid material can be directly rooted ex vitro, thus maximizing micropropagation process tested. Despite low rooting rates through micropropagation, the results are promising due to rooting recalcitrance had by the hybrid at macropropagation (BRONDANI et al., 2008(BRONDANI et al., , 2010a. Further studies regarding this type of research will be complementary and necessary to develop a more efficient micropropagation protocol for the genotypes.

Conclusion
For nodal segments sterility, the use of 0.5% chlorine (NaOCl) is recommended.
The combination of 0.50 mg L -1 BAP and 0.05 mg L -1 NAA on ½MS basic medium provided the greatest number of buds for H12 and H20 clones. The concentrations of 0.10 mg L -1 GA 3 for the H12 clone and 0.20 mg L -1 GA 3 for the H20 clone, both combined with 0.10 mg L -1 BAP on ½MS basic medium, produced the longest shoots.
The GA 3 effect on shoot elongation depended on the basic composition of culture medium, where the ½MS provided the best results for the characteristics analyzed. Both H12 and H20 clones had low rooting rates, with an average of 12% of plants having roots for in vitro conditions and only 14.4% of plants having roots for ex vitro conditions, regardless of clone.