Non-aerated liquid culture promotes shoot organogenesis in Eucalyptus globulus Labill
Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.
KeywordsAdventitious shoots Callus Liquid medium Micropropagation Phenolic compounds Rooting
The genus Eucalyptus (Myrtaceae) is among the most important hardwood forest crops worldwide, and due to their fast growth are largely exploited as a source of pulpwood to produce high-quality paper, construction timber, fuel and medicinal compounds (Abril et al. 2011; Mabona and Van Vuuren 2013). Eucalyptus globulus Labill is among the most desirable eucalyptus species in pulp industries. It is relatively frost resistant and its wood is low in lignin; cellulose is easily extracted, bleaching ability and pulp yield are some of the main properties that make it a superior raw material (Schwambach et al. 2005). Even though E. globulus has a wide geographical distribution, it is restricted to limited environments (Resquin et al. 2006). Nevertheless, this species is well adapted to southern Brazil where winter frosts are common.
Studies on Eucalyptus have been carried out to generate resistance to abiotic stress (Matsunaga et al. 2012), to modify the biosynthesis of cellulose and hemicelluloses and to increase biomass accumulation (Quoirin and Quisen 2006). However, plant transformation systems are mainly based on efficient methods for in vitro regeneration. Organogenesis is one of the important techniques associated to genetic transformation, which contributes to the success of obtaining transgenic plants. Reports on efficient regeneration of Eucalyptus species through indirect organogenesis are not abundant probably due to the recalcitrance of these species to in vitro manipulation, although success has been reported in some commercially important eucalyptus species such as E. tereticornis (Aggarwal et al. 2010), E. grandis (Hajari et al. 2006), E. camaldulensis (Dibax et al. 2005) and E. saligna (Dibax et al. 2010).
As for many other species, several factors affect shoot regeneration; plant growth regulators and type and age of explants are the most studied. Various auxin and cytokinin combinations are effective in promoting plant regeneration steps through indirect organogenesis (Hajari et al. 2006; Aggarwal et al. 2010; Matsunaga et al. 2012; Oliveira et al. 2016). 6-Benzyladenine (BA), an important plant growth regulator for callogenesis, is the preferred cytokinin to use with 1-naphthaleneacetic acid (NAA) to induce callus and subsequent plant regeneration by some Eucalyptus species (Bandyopadhyay et al. 1999; Nugent et al. 2001; Dibax et al. 2005).
Regarding the type of explant, young plant material such as hypocotyls and cotyledons has been the choice of explants for callus induction and adventitious shoot formation for Eucalyptus. However, regeneration methods are not always reproducible and genotype can markedly affect the morphogenesis capacity. Callus induction is commonly close to 70% (Nugent et al. 2001; Dibax et al. 2010), and a few studies have reported shoot organogenesis frequency higher than 50% (Nugent et al. 2001; Dibax et al. 2005; Matsunaga et al. 2012). Moreover, the establishment of efficient protocols has been based on organogenesis evaluation at 30–60 days from the onset of experiments, and cultures older than 30 days often show low frequency of shoot formation (Dibax et al. 2005). Secretion of phenolic compounds also adversely affects callus culture and regeneration capability of several species (Babaei et al. 2013; Kumar et al. 2015), including Eucalyptus (Navroski et al. 2014), although phenolic production by plantlets may enhance hardening and acclimatization steps during micropropagation (Quiala et al. 2012).
In various species, organogenesis is enhanced by the use of liquid culture systems, which promote more rapid plant growth (Rathore et al. 2014; Marbun et al. 2015; Ramírez-Mosqueda and Iglesias-Andreu 2016; Cuenca et al. 2017). However, mechanical stress and hyperhydricity, which is typically stress-induced, are often disadvantages in these systems (Quiala et al. 2012). Nevertheless, liquid culture is an alternative method for shoot multiplication in some species (Quiala et al. 2012; Sávio et al. 2012; Marbun et al. 2015). Shoots from E. globulus were grown in a liquid medium using a temporary immersion system, although long immersion periods induced hyperhydricity in shoots (González et al. 2011).
Despite the several reports on micropropagation of Eucalyptus, reproducible regeneration of plantlets has been difficult to achieve, and regeneration systems suitable for genetic engineering are desirable. The aim of this work was to establish a plant regeneration protocol via indirect organogenesis and develop a novel, efficient proliferative culture system for adventitious Eucalyptus shoots using non-aerated liquid medium. To the best of our knowledge, a non-aerated liquid system for Eucalyptus shoot propagation has never been reported.
Materials and methods
Seeds of Eucalyptus globulus (larger than 1 mm) were first surface-sterilized in 70% (v/v) ethanol for 30 s, followed by immersion in fungicide solution (3 g L−1 Benlate, DuPont, Wilmington, DE, USA) for 20 min. Seeds were then immersed in 1% (v/v) sodium hypochlorite solution for 10 min and rinsed three times in sterile distilled water. Seeds were sown in 250 mL glass flasks containing 25 mL of germination medium composed of ¼ MS salts and vitamins (Murashige and Skoog 1962), 10 g L−1 sucrose and 6 g L−1 bacteriological agar. No growth regulators were added. Flasks were kept under light conditions for 60 days, until plantlets were used for explant excision.
For callus induction, hypocotyls and leaf segments (1 cm2) were excised from plantlet and used as initial explants cultivated on half-strength MS medium supplemented with 1 g L−1 hydrolyzed casein, 30 g L−1 sucrose, 5 g L−1 PVP and 6 g L−1 agar, hereafter called ½ MS basal medium (½ MSBM). Based on previous work, BA (4.44 µM) and NAA (16.1 µM) were used as growth regulators (previous experiment; data not shown). Survival rate of explants and callus formation frequency were evaluated. Treatments consisted of hypocotyls and leaf segments distributed in 10 flasks with five explants each, maintained under dark conditions for 60 days.
Shoot induction and proliferation
Induced calli were transferred to shoot induction medium, consisting of ½ MSBM supplemented with 4.44 µM BA and 0, 0.5 or 2.7 µM NAA. Cultures were maintained in the dark until the onset of organogenesis, and then shoots induced on callus were excised and transferred to the same medium in light. Subcultures were carried out every 15 days, and the adventitious shoots induced on organogenic callus were counted at 30, 60 and 90 days after transfer to shoot induction medium. Each flask contained three calli (1 cm diameter, approximate 0.250 mg each), and 15 flasks were used in each treatment. Shoot proliferation was further carried out on ½ MSBM supplemented with 4.44 µM BA and 0.5 µM NAA.
Shoot proliferation was also tested comparing ½ MSBM and liquid system (LiqMSBM). A layer of liquid medium (5 mL/150 mL flask) with 4.44 µM BA and 0.5 µM NAA was used in the system without aeration. The efficiency of the system (semisolid and liquid) was evaluated by the number of shoots per cluster every 15 days for 45 days. Initial clusters contained an average of 12 shoots each. Proliferating cultures were kept in the light.
Rooting and acclimatization
Rooting capacity was evaluated on adventitious shoots cultured on ½ MSBM medium, with sucrose concentration reduced to 10 g L−1. The plant regulators tested were NAA or IBA at either 5 or 16 µM. No growth regulators were added to the control treatment. Experiment consisted of five flasks with three clusters of five to seven shoots each. After rooting, plantlets were isolated, and the rooting frequency and the number and length of roots were recorded.
Acclimatization was achieved by transferring complete plants to vases (500 mL) with sterile vermiculite, with one-fourth strength of MS basal salts (Rathore et al. 2014) with a plastic cover. Plants were gradually acclimatized over 14 days after transfer to the substrate. Plant survival was determined as the percentage of plants acclimatized after 30 days.
All culture media were adjusted to pH 5.8 before autoclaving at 120°C for 20 min. Cultures were maintained in the growth room at 26 ± 2 °C. When light was required, cultures were kept under a 16-h photoperiod at photosynthetic flux of 32.6 µmol m−2 s−1, provided by cool daylight fluorescent lamps (OSRAM 32 W). Subcultures were carried out every 15 days, in all the culture steps. The antioxidant polyvinylpyrrolidone (PVP) was maintained in the medium composition throughout the protocol to avoid oxidation of the explants.
Determination of phenolic compounds in shoots
Samples from adventitious shoots (0.5 g of fresh tissue) were taken from each treatment for proliferation (1/2MSBM or LiqMSBM) at 15-day intervals for 45 days, blot-dried on sterile filter and ground in aqueous methanol solution (80%, v/v). Extracts were filtered and centrifuged at 1250×g for 15 min. Total phenolic compounds were analyzed in the supernatant by colorimetric method using Folin–Ciocalteu reagent (765 nm) as previously described (Salla et al. 2014). Total phenolic compounds were expressed as mg g−1 of fresh mass (FM).
Data from either medium composition or system of culture and time of culture were analyzed by Two-way analysis of variance (ANOVA). The isolated effect of period of culture was determined by regression analysis. Whenever two treatments were compared, independent-samples test was used. Data from rooting experiment was subjected to one-way ANOVA and Duncan’s test was used to compare means. Data were evaluated for variance homogeneity (Levene’s test) and when necessary, data were transformed using arcsine \( \sqrt x \) or log x. All analyses adopted α = 0.05 as the level of significance. The results were presented as mean ± standard error (SE). Regression and statistical analyses were performed with the SPSS Statistical Software Program (SPSS v.17; SPSS, Chicago, IL, USA).
Results and discussion
Survival of explants and callus formation in Eucalyptus globulus, cultured on ½ MSBM, supplemented with 4.44 µM BA and 16.1 µM NAA
Callus formation (%)
85.0 ± 6.2a
97.5 ± 2.5a
82.2 ± 7.7a
74.0 ± 10.3b
Few reports have shown that indirect organogenesis in E. tereticornis, E. globulus, E. nitens and E. calmadulensis may succeed if juvenile tissue is used as initial explant (Bandyopadhyay et al. 1999; Dibax et al. 2005; Aggarwal et al. 2010), since less differentiated cells of young explants are more responsive to the growth regulators and nutritious conditions of the culture medium. Leaf maturity was found to influence the organogenic response of leaf segments of E. tereticornis, and 14- to 16-day-old leaves from in vitro plantlets resulted in 40.5% of organogenic explants (Aggarwal et al. 2010). Generally, leaves are not the explants of choice, probably due to tissue differentiation and endogenous hormone balance. However, results suggest that leaves of E. globulus from in vitro 60-day-old plants are still juvenile for callus formation, and a suitable combination of plant growth regulators might trigger de-differentiation, resulting in callus formation and shoot induction. In our study, an optimized culture medium, including antioxidant compounds and appropriate type and concentration of growth regulators, as well as dark cultivation might have accounted for the success of shoot induction on E. globulus leaf explants. It is worth mentioning that older seedlings generated more explants per plant with less labor than the use of explants such as cotyledons or hypocotyls.
Production of phenolic compounds by tissues in culture is frequently reported in relation to browning of excised explants, which may result in lower rates of regeneration and recalcitrance of the species, mainly wood species (Ahmad et al. 2013; Babaei et al. 2013; Jones and Saxena 2013; Kumar et al. 2015). Browning of tissues is caused by oxidation of polyphenols by polyphenol oxidases (PPO) and peroxidases (POD). This common problem in tissue culture is often minimized by addition of different absorbents or antioxidants such as PVP, citric acid, ascorbic acid or carbohydrates to the medium (Krishna et al. 2008; Kumar et al. 2015), by the inhibition of phenylalanine ammonia lyase (Jones and Saxena 2013) and several other strategies (Ahmad et al. 2013). In eucalyptus cultures, browning is a common problem since explants are usually rich in phenolic compounds (Dibax et al. 2005; Navroski et al. 2014; Lopes da Silva et al. 2015); however, addition of PVP to the medium can overcome this problem, by reducing PPO activity. Excessive PVP, however, can hinder tissue proliferation (González et al. 2011). When the content of phenolic compounds was compared between semisolid and liquid medium cultures, shoots cultured on MSBM medium steadily accumulated phenolic compounds (Fig. 3b). On the other hand, the content of phenolic compounds significantly decreased with culture time in shoots proliferating in LiqMSMB medium (Fig. 3b). Thus, the liquid system seems less stressful for culturing E. globulus shoots while stimulating proliferation. Low production of phenolic compounds could result in less secretion and, consequently, low oxidation of shoots. The novelty of this work is that a thin layer of liquid medium, even with no aeration, was sufficient to increase shoot multiplication of E. globulus, and cultures remained stable for up to 45 days with low production of phenolic compounds.
Rooting of Eucalyptus globulus shoots on ½ MSBM medium supplemented with different concentrations of either NAA or IBA after 30 days
Number of roots
Length of roots (cm)
Length of main root (cm)
0 ± 0.0b
0 ± 0.0c
0 ± 0.0c
0 ± 0.0c
15.3 ± 5.0b
2.3 ± 0.7bc
0.4 ± 0.0c
0.5 ± 0.0c
75.0 ± 15.0a
2.5 ± 0.2bc
2.8 ± 0.4ab
3.6 ± 0.9ab
80.3 ± 10.1a
6.2 ± 0.3ab
2.1 ± 0.3b
3.0 ± 0.5b
100.0 ± 0.0a
8.6 ± 0.0a
3.7 ± 0.2a
7.8 ± 0.8a
Our results showed that leaves from 60-day-old in vitro plants are suitable for callus formation and shoot induction of E. globulus. Multiplication of adventitious shoots was efficient in liquid medium up to 45 days of culture, creating a less expensive and less laborious system. Eucalyptus plant regeneration has been indicated as the most critical step in developing genetic transformation. Most eucalyptus species are recalcitrant using in vitro methods, probably due to the high production of phenolic compounds and the maturity level of the explants. In the liquid medium used for shoot proliferation, the production of phenolic compounds was lower, and thus a more useful system for regenerating genetically engineered Eucalyptus plants.
This work was supported by the National Council for Scientific and Technological Development (CNPq)/Brazil, under Grant 477538/2013-4. The authors are grateful to Suzano Papel e Celulose (former RioCell, Brazil) for providing seeds of E. globulus and to Janaína Belquis da S. P. Langois for technical assistance.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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