Tree Physiology Advance Access published April 15, 2015
Tree Physiology 00, 1–13 doi:10.1093/treephys/tpv028
Research paper
Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees
1Department 2ENCE,
of Plant Physiology, Instituto de Investigaciones Agrobiológicas de Galicia (CSIC), Apartado 122, 15705 Santiago de Compostela, Spain; 28046 Madrid, Spain; 3Corresponding author ([email protected])
Received January 18, 2015; accepted March 8, 2015; handling Editor Ron Sederoff
A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/ genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l−1 gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloramcontaining medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. Keywords: eucalypt, leaf explant, NAA, picloram, shoot apex explant, somatic embryo.
Introduction Forest trees cover ∼30% of the earth’s land surface, providing renewable fuel, wood, timber and fruits, and are also a source of medicinal products (R amawat et al. 2014). Several strategies have been adopted to increase the supply of wood in order to meet industrial and domestic demands. One strategy used was to introduce rapidly growing species in plantation forestry. Among these species, the most widely planted hardwood crop in the tropical and subtropical world is Eucalyptus (Grattapaglia and Kirst 2008), a fast growing evergreen tree of the Myrtaceae
family native to Australia, Tasmania and nearby islands. By the mid 1800s, eucalypts had been introduced to Southern Europe and Northern Africa (Penford and Willis 1961). Since then, the importance of eucalypts has increased over time until currently eucalypt plantations can be found in more than 90 countries, with the largest plantations in Brazil, India and China. The eucalypts are characterized by their fast growth rate and large biomass production, their ability to grow in a wide range of environments and soils, and their good wood quality for solid wood products and paper production. Of the 894 taxa including subspecies and
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E. Corredoira1,3, A. Ballester1, M. Ibarra2 and A.M. Vieitez1
2 Corredoira et al.
Tree Physiology Volume 00, 2015
2008a), but there are no reports on the induction of somatic embryos from tissues of mature trees. Somatic embryogenesis induction in explants derived from mature trees has only been mentioned in three species of the genus. Qin and Kirby (1990) described the initiation of embryo-like structures from leaves of cultures derived from adult E. grandis genotypes, whereas Termignoni et al. (1998) described SE induction from explants of mature trees of E. saligna Smith and E. dunnii, attaining in the latter a high frequency of plant regeneration (Patent No. PI 9801485-4 INPI). Naphthaleneacetic acid (NAA) has been commonly used for induction of SE in the majority of previous studies in eucalypt species (Pinto et al. 2013, Chauhan et al. 2014). Picloram (4-amino-3,5,6-trichloropicolinic acid), a compound known to have a stronger auxin effect, could be more effective than other auxins such as 2,4-d in inducing SE (Steinmacher et al. 2011). Although picloram has hardly been tested in Eucalyptus species, this compound has been used to stimulate the SE in material derived from adult trees (K arun et al. 2004, Stefanello et al. 2005, Steinmacher et al. 2007, Correia et al. 2011). The main objective of the present work was to develop a reliable protocol for the induction of SE in tissues of mature trees of E. globulus and the hybrid E. saligna × E. maidenii. The potential of both leaf and shoot apex explants excised from axillary shoots established from mature trees to initiate SE was investigated. The ability of NAA and picloram to induce an embryogenic response and the maintenance of embryogenic competence in the embryogenic lines was also studied.
Materials and methods Plant material and culture conditions Clonal axillary shoot cultures, previously established from axillary buds of crown branches of two E. globulus Labill. trees and one E. saligna × E. maidenii hybrid tree (all 12-year-old elite trees) by the private company Foresta Mantenimiento de Plantaciones, were used as sources of initial explants. The genotypes were designated as 41-1-AC and 22-6-RP for the E. globulus trees and Sal-May for the hybrid tree. Axillary shoots (1 cm long) were multiplied by subculture every 3–4 weeks on a basal shoot proliferation medium consisting of MS (Murashige and Skoog 1962) mineral salts and vitamins (M0222, Duchefa Biochemie, Amsterdam, The Netherlands) supplemented with 10 mg l−1 ascorbic acid, 10 mg l−1 citric acid, 1 mg l−1 folic acid, 0.054 µM NAA and 7 g l−1 agar (Vitroagar, Pronadisa, Spain). This basal medium was supplemented with 0.41 µM meta-topolin (mT) and 2% (w/v) glucose for the genotype 22-6-RP, whereas 0.44 µM 6-benzylaminopurine (BA) and 2% (w/v) sucrose were included in the shoot proliferation medium for genotypes 41-1-AC and Sal-May. All the culture media were brought to pH 5.6–5.7 before autoclaving at 121 °C for 20 min. Stock shoot cultures of the three genotypes were maintained by periodic
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natural hybrids that belong to the genus (Brooker et al. 2014), the predominant species cultivated on commercial plantations are E. grandis Hill ex Maiden, E. globulus Labill., E. camaldulensis Dehnh, E. urophylla Blake and their hybrids (Potts and Dungey 2004, Grattapaglia and Kirst 2008). Among these species, E. globulus (Tasmanian Blue Gum or Blue Gum) is one of the most important and commonly cultivated eucalypts and was the first species of the genus to become widely known outside Australia. The economic importance of this species owes to the fact that it combines fast growth under appropriate conditions with good tree form and excellent wood quality for pulp production, including a high pulp yield, resulting in pulps with excellent technical properties (Patt et al. 2006). Furthermore, E. globulus is the main source for global Eucalyptus oil production. Biotechnological approaches such as cloning and hybrid production, breeding, the use of molecular markers, application of tissue culture for micropropagation and genetic engineering have all been employed for the development and production of eucalypt trees with better quality wood for the pulp and paper industry, energy production, stress tolerance and disease resistance (Labate et al. 2008). For Eucalyptus species, conventional breeding is a difficult and time-consuming process since these species possess a genetic background with a high-level of heterozygosity. Despite the clear potential of genetic engineering, this technology remains costly, difficult or impossible for most eucalypts, mainly due to the absence of an effective in vitro regeneration system (Prakash and Gurumurthi 2010). Somatic embryogenesis (SE) is an important in vitro plant regeneration approach that has demonstrated significant benefits when applied to forest tree species, bringing important advantages including clonal mass propagation, cryostorage of valuable germplasm and genetic transformation (Corredoira et al. 2006). Somatic embryogenesis is the procedure of choice for transformation protocols, since plant regeneration from transformed tissues via organogenesis can often give rise to the production of chimeras, whereas regeneration via SE offers the advantage of a single cell origin that reduces this problem (Giri et al. 2004). In spite of its benefits for genetic transformation, SE in Eucalyptus species has low induction frequency and the somatic embryos germinate at a low frequency. Initiation of somatic embryos has been obtained in E. grandis (Lakshmi Sita et al. 1986, Watt et al. 1991, 1999), E. citriodora Hook (Muralidharan and Mascarenhas 1987, Muralidharan et al. 1989), E. gunnii Hook (Boulay 1987, Franclet and Boulay 1989), E. dunnii Maiden (Qin and Kirby 1990, Termignoni et al. 1996, Watt et al. 1999), E. nitens Deane et Maiden (Ruaud et al. 1997), E. tereticornis Smith (Prakash and Gurumurthi 2005) and E. camaldulensis (Prakash and Gurumurthi 2010). However, in most of these reports embryogenic cultures were initiated from zygotic embryos and/or young seedlings (1–4 weeks old), and, therefore, the material being propagated is of unknown genetic value. In E. globulus, SE has also been achieved from immature and mature zygotic embryos (Nugent et al. 2001, Pinto et al. 2002,
A reproducible procedure for induction of somatic embryogenesis 3 subculture for 2 years before being used in SE induction experiments. Stock cultures were incubated in a growth chamber with a 16-h photoperiod (provided by cool-white fluorescent lamps at a photon flux density of 50–60 µmol m−2 s−1) at 25 °C light/20 °C dark (i.e., standard culture conditions).
Induction of SE
Effect of NAA on the induction of SE In a preliminary experiment, leaf (1 and 2) and shoot apex explants excised from shoot cultures of the Sal-May and 41-1-AC were cultured on induction medium supplemented with different NAA concentrations (10.74, 16.11 and 21.48 µM). For each genotype, explant type and NAA treatment, 80–100 explants were used. Based on the results obtained in the above experiment, a second experiment was performed to analyse the embryogenic capacity of the three explant types (leaf 1, leaf 2 and shoot apex) of the three eucalypt genotypes using the induction medium supplemented with 16.11 µM NAA. Gum arabic
Effect of picloram on induction of SE Picloram was tested at 20, 30 and 40 µM in combination with 40 mg l−1 GA. According to the embryogenic capacity shown by leaf explants cultured in NAA-induction medium, in this experiment leaf 1 and shoot tips from 41-1-AC stock cultures were utilized as initial explants. For each picloram and explant type treatment, 60–100 explants were used (see Table 2). In a further experiment, induction medium containing 40 µM picloram and 40 mg l−1 GA was selected to test the embryogenic abilities of two explant types (leaf 1 and shoot apex) from each of the three eucalypt genotypes. For each explant type, at least 180 explants of each E. globulus genotype and 80 explants of the Sal-May genotype were subcultured on induction medium (see Figure 4). In all the SE induction experiments, the cultures were incubated in darkness at 25 °C for 8 weeks. After this period, the following data were recorded: the percentage of explants forming callus, the percentage of explants forming adventitious roots, the percentage of explants with an embryogenic response (Table 1 and Figure 4) and the relative rate of explants showing an embryogenic response (Table 2). The embryogenic response was defined as the presence of embryogenic structures and/or somatic embryos (torpedo-cotyledonary stage) in the initial
Table 1. Influence of explant type and Eucalyptus species/genotype on the percentages of root formation and embryogenic response, following culture for 8 weeks in medium supplemented with NAA (16.11 µM) and gum arabic (40 mg l−1). Influence of the genotype/species and explant type on the percentage of root formation and embryogenic response was evaluated by the χ2 test (P ≤ 0.05). Genotype/species
Explant type
Root formation (%)
Embryogenic response (%)
Leaf 1 Leaf 2 Apex
40.8 (98/240) 33.0 (33/100) 67.1 (215/320)
7.5 (18/240) 1.0 (1/100) 14.6 (47/320)
Leaf 1 Leaf 2 Apex
6.0 (6/100) 0.0 (0/100) 29.0 (58/200)
1.0 (1/100) 0.0 (0/100) 0.0 (0/200)
Leaf 1 Leaf 2 Apex
3.0 (3/100) 6.0 (6/100) 20.0 (20/100)
2.0 (2/100) 1.0 (1/100) 0.0 (0/100)
Sal-May/E. saligna × E. maidenii
41-1-AC/E. globulus
22-6-RP/E. globulus
Source of variation Genotype (A) Explant type (B) A × B
P ≤ 0.001 P ≤ 0.001 P ≤ 0.001
P ≤ 0.001 P ≤ 0.001 P ≤ 0.01
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The shoot apex (1.5–2 mm long) comprising the apical meristem and 2–3 leaf primordia and the two most apical expanding leaves collected from the first (leaf 1) and second (leaf 2) nodes in the apical region of the shoots in an active state of growth were excised from stock shoot cultures and used as initial explants. Leaf 1 and leaf 2 explants had mean lengths of 2.5 and 3 mm, respectively. Ten shoot apices (horizontally orientated) and 10 leaf explants (abaxial side down) were placed in 90 × 15 mm Petri dishes containing 25 ml of induction medium consisting of MS mineral salts and vitamins, 500 mg l−1 casein hydrolysate, 3% (w/v) sucrose and 6 g l−1 agar supplemented with plant growth regulators (PGRs) as stated in the following experiments.
(40 mg l−1) from acacia tree (GA; Sigma, St Louis, USA, G-9752), as a source of arabinogalactan proteins, was also added after filter sterilization to the autoclaved NAA-induction media. The use of 40 mg l−1 GA was based on its positive effect as reported in previous studies on somatic embryo induction in Quercus species (Corredoira et al. 2014). For each genotype and explant type, at least 100 explants were cultured on induction medium (see Table 1).
4 Corredoira et al. Table 2. Morphogenic response of leaf 1 and shoot apex explants excised from shoot cultures of E. globulus (41-1-AC) after culture for 8 weeks in medium supplemented with different picloram concentrations and gum arabic (40 mg l−1).
The interaction of both factors was analysed by a log-linear model (P ≤ 0.05). SPSS for Windows (version 19.0, SPSS, Chicago, IL, USA) was used for statistical analysis.
Picloram Watery callus (%) (µM)
Relative embryogenic response1
Results
Leaf 1 Shoot apex
Effect of NAA on induction of SE
Leaf 1 20 30 40
Shoot apex
98.3 (59/60) 100.0 (60/60) No Yes (+) 100.0 (100/100) 98.0 (98/100) Yes (+) Yes (++) 98.8 (79/80) 100.0 (100/100) Yes (+) Yes (+++)
1Characterization
of relative embryogenic response: No: no embryogenic response; Yes: presence of nodular embryogenic structures/somatic embryos: +, ++, +++: low, medium and high embryogenic response, respectively.
Maintenance of embryogenic competence For somatic embryo proliferation and maintenance of embryogenic competence, individual cotyledonary stage embryos or small clusters of globular and torpedo stage embryos were isolated and transferred to 90 × 15 mm Petri dishes containing 25 ml of embryo proliferation medium. This medium consisted of MS mineral salts and vitamins, 500 mg l−1 casein hydrolysate, 3% (w/v) sucrose, 6 g l−1 agar, 20 µM silver thiosulfate (STS) and 16.11 µM NAA. The STS was filter sterilized and added to the autoclaved medium. The embryogenic lines were maintained by secondary embryogenesis with subcultures at 4–5-week intervals, and were incubated in darkness at 25 °C.
Histological study Explants with visible somatic embryos and/or embryogenic structures were collected at different periods during the experiments for histological analysis. Samples were fixed in FAA solution (formalin, glacial acetic acid and 50% ethanol [1 : 1 : 18 (v/v/v)]), dehydrated through a graded n-butanol series and embedded in paraffin wax (Merck, Kenilworth, NJ, USA). Sections (8 μm) were cut on a Reichert-Jung rotary microtome (Leica, Heidelberg, Germany) and stained with periodic acid-Schiff (PAS)-naphthol blueblack (Merck) to detect starch and other insoluble polysaccharides and total proteins, respectively (Feder and O’Brien 1968). The stained sections were mounted with Euckit®, and photomicrographs were taken with an Olympus DP71 digital camera fitted to a Nikon-FXA microscope (Nikon, Tokyo, Japan).
Statistical analysis The influence of the main experimental factors (the explant type and genotype) on the percentage of root formation and the percentage of embryogenic response (Table 1 and Figure 4) was evaluated by the χ2 test (P ≤ 0.05) from a contingency table.
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explants. The explants were observed under a stereomicroscope (Olympus SZX9, Olympus, Tokyo, Japan) and photographed with an Olympus DP10 digital camera.
In a preliminary experiment, shoot apex and leaf explants from the Sal-May (E. saligna × E. maidenii) and 41-1-AC (E. globulus) were cultured in induction medium supplemented with three NAA concentrations. Most of the explants from the two genotypes responded by forming callus; differentiation of adventitious roots was also observed, especially in explants of the hybrid material, with the highest frequency of adventitious root formation produced on medium with 16.11 μM NAA (data not shown). The embryogenic response, defined as the presence of bipolar embryos and/or embryogenic structures, was observed in shoot apex and leaf explants from both genotypes (Figure 1a–d), with the best response obtained in shoot apex explants of Sal-May treated with 16.11 μM NAA (data not shown). Although embryogenic cultures were obtained in E. globulus with all three NAA concentrations (Figure 1c and d), their embryogenic ability was far lower than that observed in the hybrid genotype. Based on the results in the first experiment, explants of the three genotypes were cultured in induction medium supplemented with 16.11 μM NAA and 40 mg l−1 GA (Table 1). The root formation frequency was significantly affected by the species/genotype (P ≤ 0.001) and explant type (P ≤ 0.001), with a significant interaction (P ≤ 0.001) between these two factors (Table 1). Whereas leaf 2 showed the lowest rooting frequencies in Sal-May and 41-1-AC, the leaf at position 2 of 22-6-RP exhibited a higher rooting ability than leaf 1 explants (Table 2). In all three genotypes, shoot apex explants showed the highest rooting rates, with frequencies ranging between 20% (22-6-RP) and 67.1% (Sal-May). The embryogenic response was also significantly influenced by the species/genotype (P ≤ 0.001) and explant type (P ≤ 0.001), and a significant interaction (P ≤ 0.01) between both factors was also observed (Table 1). The interaction was due to the fact that the highest embryogenic induction frequency was achieved in shoot tip explants of the hybrid genotype (14.6%), while, in contrast, in E. globulus only leaf 1 explants generated somatic embryos. Leaf 1 showed a greater ability for SE than leaf 2, especially in the Sal-May genotype (Table 1). Generally, the embryogenic structures and/or somatic embryos were indirectly formed from callus tissue developed from the initial explants, although a direct origin was also observed (Figure 1c). The embryogenic structures were round or oblong and translucent in appearance (Figure 1d). Histological study confirmed that these structures were somatic embryos (Figure 1e). Embryo production ranged between 2 and 10 embryogenic structures and/or somatic embryos per embryogenic explant. Somatic embryos
A reproducible procedure for induction of somatic embryogenesis 5
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Figure 1. Induction of somatic embryogenesis after 8 weeks of culturing explants in NAA induction medium. Explants were excised from axillary shoot cultures of E. saligna × E. maidenii (Sal-May genotype) and E. globulus (41-1-AC genotype). (a and b) Somatic embryos initiated in leaf (a) and shoot apex explants (b) of the Sal-May genotype. (c) Somatic embryo generated directly from the adaxial surface of a leaf explant of the 41-1-AC genotype. (d) Cluster of embryogenic structures originated from a shoot apex explant of the 41-1-AC genotype. (e) Histological section of the same explant as in (d) showing somatic embryo differentiation. Note a heart-shaped embryo arising from a nodular structure (arrow). (f) Somatic embryos isolated from a shoot apex explant of the Sal-May genotype showing small cotyledons, a hypocotyl and root elongation. (g and h) Longitudinal sections of cotyledonary stage somatic embryos in which the vascular system (vs), root meristem (rm) and shoot apical meristem (sam) are evident. Note the presence of different sized cotyledons in (h). Scale bar: a–d = 1 mm.
could easily be detached from the initial explant and showed a clear bipolarity, evidenced by the development of root and shoot poles and two small cotyledons that were of a scale-leafy appearance (Figure 1c). A number of somatic embryos on initial
explants exhibited precocious germination and root growth (Figure 1f). At the histological level, bipolar organization of somatic embryos was demonstrated, with root and shoot apical meristems, and closed vascular tissue that bifurcated into small
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6 Corredoira et al. cotyledons (Figure 1g and h). The primary root pole was more developed than the shoot pole, which was made up of a few meristematic cell layers.
Effect of picloram on induction of SE
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Somatic embryo maintenance and plant regeneration To establish embryogenic lines of E. globulus and Sal-May, individual cotyledonary somatic embryos or small clusters of globular and torpedo embryos were transferred to embryo proliferation medium containing 16.11 µM NAA, where new somatic embryos were obtained by secondary embryogenesis (Figure 5a). The frequency of subcultured embryo clusters producing secondary SE ranged from 60 to 75%, with 6–8 new secondary embryos obtained per embryo cluster. Secondary embryo production was reduced when isolated cotyledonary stage embryos were used as subcultured explants. Secondary embryo development was asynchronous; they frequently formed in the cotyledonary region of the primary somatic embryos (Figure 5b). The histological
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Given the low embryogenic frequencies obtained in E. globulus explants treated on NAA induction medium, picloram was tested to determine whether it would improve this response. Leaf 1 and shoot apex explants of the 41-1-AC were cultured in induction medium supplemented with three concentrations of picloram (Table 2). In all the treatments, almost 100% of the initial explants proliferated and formed calli (Table 2), but adventitious roots were not generated in the presence of picloram. Both explant types developed a watery callus of friable consistency that initially appeared yellow. As the culture progressed, the callus began to turn necrotic, dark brown and disaggregated (Figure 2a and b). The embryogenic tissue appeared as nodular structures that arose from the disaggregating watery callus. These structures were white-translucent, shiny and varied in shape; most of them were nodular-ovoid but cup-shaped or somewhat curved shapes were also observed (Figure 2c–e), and they could be easily separated from the surrounding callus. Interestingly, each embryogenic structure seemed to be surrounded by a translucent and mucilaginous coating layer. Histological study showed that the mucilaginous layer appeared to be made of vacuolated stratified cells with a large accumulation of starch grains (Figure 3a–c). Shoot apex explants cultured on medium supplemented with the three picloram concentrations produced embryogenic structures, although the greatest response was obtained in medium with 40 µM picloram (Table 2) compared with those relatively fewer embryogenic tissues induced in leaf explants cultured on 30 and 40 µM picloram. There was no embryogenic response in leaves treated with 20 µM picloram. Based on these results, a further experiment was performed by selecting induction medium supplemented with 40 µM picloram to evaluate the embryogenic ability of leaf and shoot tip explants from the three genotypes (Figure 4). After 8 weeks of culture, embryogenic cultures were induced in leaf and shoot apex explants of all three genotypes with frequencies ranging between 13.8% (leaf 1 of 41-1-AC) to 68.8% (leaf 1 of SalMay). The frequency of explants developing embryogenic structures was significantly influenced by the species/genotype (P ≤ 0.001) and the explant type (P ≤ 0.001), with a significant interaction (P ≤ 0.001) between these two factors. The most responsive material was the Sal-May hybrid, in which both types of explant showed a similar embryogenic capacity (Figure 4). However, in the two E. globulus genotypes, the embryogenic ability of leaf explants was significantly lower than those of shoot tip explants. Nevertheless, in both explant types of the three genotypes, the embryogenic frequencies were considerably improved when picloram was included in the induction
medium in comparison with those obtained in induction medium supplemented with NAA (see Table 1). This was especially relevant for the two genotypes of E. globulus, in which induction of SE in picloram-containing medium was higher than 50% in shoot apex explants. The number of nodular embryogenic structures produced per embryogenic explant appeared to be influenced by the species/genotype, with production of few structures (2–6 per explant) in E. globulus genotypes, while more than 50 embryogenic structures were recorded in the hybrid genotype (Figure 2a–c). Histological analysis revealed that the nodular embryogenic structures generated on picloram medium were made up of cells with a dense protein-rich cytoplasm, small vacuoles, a high nucleocytoplasmic ratio and starch grains that were especially abundant in tightly packed cells of the external layers (Figure 3b and c). Small, undifferentiated and more vacuolated cells were also evident in the central area of the nodular structures (Figure 3a–c). In addition, somatic embryos at different developmental stages, including globular-, torpedo- and early cotyledonarystage embryos, were also apparent (Figure 3d–g). As occurred with somatic embryos generated in NAA induction medium, cotyledonary somatic embryos developed in the presence of picloram had a distinct primary root pole, while the apical shoot meristem appeared to be less developed, blocked or even absent (Figure 3g). Embryo abnormalities such as the presence of fused embryos, cotyledons of different size or fused cotyledons in a cup-like structure were also observed. After 8 weeks of culture in picloram-containing medium, the embryogenic structures were subcultured for 6 weeks onto secondary medium devoid of PGRs, where embryo histodifferentiation and the production of somatic embryos at the globular, torpedo and cotyledonary stages and even precociously germinated embryos were promoted (Figure 2f–h). When the secondary medium was supplemented with NAA (16.11 µM), with or without the addition of mT (0.41 µM), embryo differentiation was less pronounced, whereas the embryogenic structures became necrotic following culture on PGR-free medium containing 0.4% activated charcoal.
A reproducible procedure for induction of somatic embryogenesis 7
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Figure 2. Induction of somatic embryogenesis after 8 weeks of culturing explants in picloram induction medium. Explants were excised from axillary shoot cultures of E. saligna × E. maidenii (Sal-May genotype) and E. globulus (41-1-AC and 22-6-RP genotypes) trees. (a and b) Nodular embryogenic structures arising from a watery callus generated in a leaf explant of the 22-6-RP genotype (a) and a shoot apex explant of the 41-1-AC genotype (b). (c–e) Isolated embryogenic structures detached from the watery callus induced in explants of the Sal-May genotype (c) and the 41-1-AC genotype (d and e). Note the different shapes of the embryogenic structures. (f) Developing somatic embryos (globular-torpedo stage) obtained following subculture of embryogenic nodular structure on PGR-free medium. (g and h) Cotyledonary stage embryos (g) and a somatic embryo with precocious germination (h) after subculture of embryogenic nodular structures on PGR-free medium. Scale bar: a–h = 1 mm.
study of embryogenic lines showed the presence of distinct meristematic/embryogenic zones located in the epidermal and subepidermal cell layers of cotyledons, these embryogenic cells gave the origin to the secondary embryos (Figure 5c). Moreover, precociously germinating somatic embryos were also found on
proliferation medium. Embryogenic cultures were maintained for more than 3 years under these conditions. Alternating the cultures between 16.11 µM NAA medium and PGR-free medium or the addition of a cytokinin to the proliferation medium did not improve the multiplication rates.
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Figure 3. Histological analysis of nodular embryogenic structures and somatic embryos originated in picloram induction medium. (a) Section of the nodular structure shown in Figure 2b which exhibited the presence of a stratified coating layer (arrow) surrounding the nodular structure. (b) Nodular embryogenic structures of different sizes arising between the disaggregate callus tissue (arrows). (c) Higher magnification of the embryogenic structure in (b) (square) to show the meristematic cell surface layers and the high starch content of the vacuolated cell coating layer. (d) Globular stage somatic embryo with well-developed protodermis (pt). (e–g) Longitudinal sections of torpedo stage (e) and cotyledonary stage (f and g) embryos. A cotyledonary stage somatic embryo with developing root (r) and poor development of shoot apical meristem (sam) is presented in (g).
Well-developed cotyledonary somatic embryos with defined root and shoot poles were separated from secondary embryogenic cultures and transferred to semisolid germination medium consisting of MS medium supplemented with 3% sucrose, 6 g l−1 agar, 0.44 µM BA and 1.44 µM gibberellic acid, where drying and browning of the isolated embryos occurred rapidly.
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To prevent this disorder, the use of liquid germination medium was tested by placing cotyledonary embryos on two filter paper discs (Whatman grade 181) in Petri dishes (90 mm) containing 10 ml of liquid germination medium. Under these conditions, enlargement and greening of hypocotyl and cotyledons followed by root growth occurred in most of the somatic embryos;
A reproducible procedure for induction of somatic embryogenesis 9 owever, embryo conversion with both root and shoot developh ment (Figure 5d) was observed in only a few instances (
Tree Physiology 00, 1–13 doi:10.1093/treephys/tpv028
Research paper
Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees
1Department 2ENCE,
of Plant Physiology, Instituto de Investigaciones Agrobiológicas de Galicia (CSIC), Apartado 122, 15705 Santiago de Compostela, Spain; 28046 Madrid, Spain; 3Corresponding author ([email protected])
Received January 18, 2015; accepted March 8, 2015; handling Editor Ron Sederoff
A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/ genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l−1 gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloramcontaining medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. Keywords: eucalypt, leaf explant, NAA, picloram, shoot apex explant, somatic embryo.
Introduction Forest trees cover ∼30% of the earth’s land surface, providing renewable fuel, wood, timber and fruits, and are also a source of medicinal products (R amawat et al. 2014). Several strategies have been adopted to increase the supply of wood in order to meet industrial and domestic demands. One strategy used was to introduce rapidly growing species in plantation forestry. Among these species, the most widely planted hardwood crop in the tropical and subtropical world is Eucalyptus (Grattapaglia and Kirst 2008), a fast growing evergreen tree of the Myrtaceae
family native to Australia, Tasmania and nearby islands. By the mid 1800s, eucalypts had been introduced to Southern Europe and Northern Africa (Penford and Willis 1961). Since then, the importance of eucalypts has increased over time until currently eucalypt plantations can be found in more than 90 countries, with the largest plantations in Brazil, India and China. The eucalypts are characterized by their fast growth rate and large biomass production, their ability to grow in a wide range of environments and soils, and their good wood quality for solid wood products and paper production. Of the 894 taxa including subspecies and
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E. Corredoira1,3, A. Ballester1, M. Ibarra2 and A.M. Vieitez1
2 Corredoira et al.
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2008a), but there are no reports on the induction of somatic embryos from tissues of mature trees. Somatic embryogenesis induction in explants derived from mature trees has only been mentioned in three species of the genus. Qin and Kirby (1990) described the initiation of embryo-like structures from leaves of cultures derived from adult E. grandis genotypes, whereas Termignoni et al. (1998) described SE induction from explants of mature trees of E. saligna Smith and E. dunnii, attaining in the latter a high frequency of plant regeneration (Patent No. PI 9801485-4 INPI). Naphthaleneacetic acid (NAA) has been commonly used for induction of SE in the majority of previous studies in eucalypt species (Pinto et al. 2013, Chauhan et al. 2014). Picloram (4-amino-3,5,6-trichloropicolinic acid), a compound known to have a stronger auxin effect, could be more effective than other auxins such as 2,4-d in inducing SE (Steinmacher et al. 2011). Although picloram has hardly been tested in Eucalyptus species, this compound has been used to stimulate the SE in material derived from adult trees (K arun et al. 2004, Stefanello et al. 2005, Steinmacher et al. 2007, Correia et al. 2011). The main objective of the present work was to develop a reliable protocol for the induction of SE in tissues of mature trees of E. globulus and the hybrid E. saligna × E. maidenii. The potential of both leaf and shoot apex explants excised from axillary shoots established from mature trees to initiate SE was investigated. The ability of NAA and picloram to induce an embryogenic response and the maintenance of embryogenic competence in the embryogenic lines was also studied.
Materials and methods Plant material and culture conditions Clonal axillary shoot cultures, previously established from axillary buds of crown branches of two E. globulus Labill. trees and one E. saligna × E. maidenii hybrid tree (all 12-year-old elite trees) by the private company Foresta Mantenimiento de Plantaciones, were used as sources of initial explants. The genotypes were designated as 41-1-AC and 22-6-RP for the E. globulus trees and Sal-May for the hybrid tree. Axillary shoots (1 cm long) were multiplied by subculture every 3–4 weeks on a basal shoot proliferation medium consisting of MS (Murashige and Skoog 1962) mineral salts and vitamins (M0222, Duchefa Biochemie, Amsterdam, The Netherlands) supplemented with 10 mg l−1 ascorbic acid, 10 mg l−1 citric acid, 1 mg l−1 folic acid, 0.054 µM NAA and 7 g l−1 agar (Vitroagar, Pronadisa, Spain). This basal medium was supplemented with 0.41 µM meta-topolin (mT) and 2% (w/v) glucose for the genotype 22-6-RP, whereas 0.44 µM 6-benzylaminopurine (BA) and 2% (w/v) sucrose were included in the shoot proliferation medium for genotypes 41-1-AC and Sal-May. All the culture media were brought to pH 5.6–5.7 before autoclaving at 121 °C for 20 min. Stock shoot cultures of the three genotypes were maintained by periodic
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natural hybrids that belong to the genus (Brooker et al. 2014), the predominant species cultivated on commercial plantations are E. grandis Hill ex Maiden, E. globulus Labill., E. camaldulensis Dehnh, E. urophylla Blake and their hybrids (Potts and Dungey 2004, Grattapaglia and Kirst 2008). Among these species, E. globulus (Tasmanian Blue Gum or Blue Gum) is one of the most important and commonly cultivated eucalypts and was the first species of the genus to become widely known outside Australia. The economic importance of this species owes to the fact that it combines fast growth under appropriate conditions with good tree form and excellent wood quality for pulp production, including a high pulp yield, resulting in pulps with excellent technical properties (Patt et al. 2006). Furthermore, E. globulus is the main source for global Eucalyptus oil production. Biotechnological approaches such as cloning and hybrid production, breeding, the use of molecular markers, application of tissue culture for micropropagation and genetic engineering have all been employed for the development and production of eucalypt trees with better quality wood for the pulp and paper industry, energy production, stress tolerance and disease resistance (Labate et al. 2008). For Eucalyptus species, conventional breeding is a difficult and time-consuming process since these species possess a genetic background with a high-level of heterozygosity. Despite the clear potential of genetic engineering, this technology remains costly, difficult or impossible for most eucalypts, mainly due to the absence of an effective in vitro regeneration system (Prakash and Gurumurthi 2010). Somatic embryogenesis (SE) is an important in vitro plant regeneration approach that has demonstrated significant benefits when applied to forest tree species, bringing important advantages including clonal mass propagation, cryostorage of valuable germplasm and genetic transformation (Corredoira et al. 2006). Somatic embryogenesis is the procedure of choice for transformation protocols, since plant regeneration from transformed tissues via organogenesis can often give rise to the production of chimeras, whereas regeneration via SE offers the advantage of a single cell origin that reduces this problem (Giri et al. 2004). In spite of its benefits for genetic transformation, SE in Eucalyptus species has low induction frequency and the somatic embryos germinate at a low frequency. Initiation of somatic embryos has been obtained in E. grandis (Lakshmi Sita et al. 1986, Watt et al. 1991, 1999), E. citriodora Hook (Muralidharan and Mascarenhas 1987, Muralidharan et al. 1989), E. gunnii Hook (Boulay 1987, Franclet and Boulay 1989), E. dunnii Maiden (Qin and Kirby 1990, Termignoni et al. 1996, Watt et al. 1999), E. nitens Deane et Maiden (Ruaud et al. 1997), E. tereticornis Smith (Prakash and Gurumurthi 2005) and E. camaldulensis (Prakash and Gurumurthi 2010). However, in most of these reports embryogenic cultures were initiated from zygotic embryos and/or young seedlings (1–4 weeks old), and, therefore, the material being propagated is of unknown genetic value. In E. globulus, SE has also been achieved from immature and mature zygotic embryos (Nugent et al. 2001, Pinto et al. 2002,
A reproducible procedure for induction of somatic embryogenesis 3 subculture for 2 years before being used in SE induction experiments. Stock cultures were incubated in a growth chamber with a 16-h photoperiod (provided by cool-white fluorescent lamps at a photon flux density of 50–60 µmol m−2 s−1) at 25 °C light/20 °C dark (i.e., standard culture conditions).
Induction of SE
Effect of NAA on the induction of SE In a preliminary experiment, leaf (1 and 2) and shoot apex explants excised from shoot cultures of the Sal-May and 41-1-AC were cultured on induction medium supplemented with different NAA concentrations (10.74, 16.11 and 21.48 µM). For each genotype, explant type and NAA treatment, 80–100 explants were used. Based on the results obtained in the above experiment, a second experiment was performed to analyse the embryogenic capacity of the three explant types (leaf 1, leaf 2 and shoot apex) of the three eucalypt genotypes using the induction medium supplemented with 16.11 µM NAA. Gum arabic
Effect of picloram on induction of SE Picloram was tested at 20, 30 and 40 µM in combination with 40 mg l−1 GA. According to the embryogenic capacity shown by leaf explants cultured in NAA-induction medium, in this experiment leaf 1 and shoot tips from 41-1-AC stock cultures were utilized as initial explants. For each picloram and explant type treatment, 60–100 explants were used (see Table 2). In a further experiment, induction medium containing 40 µM picloram and 40 mg l−1 GA was selected to test the embryogenic abilities of two explant types (leaf 1 and shoot apex) from each of the three eucalypt genotypes. For each explant type, at least 180 explants of each E. globulus genotype and 80 explants of the Sal-May genotype were subcultured on induction medium (see Figure 4). In all the SE induction experiments, the cultures were incubated in darkness at 25 °C for 8 weeks. After this period, the following data were recorded: the percentage of explants forming callus, the percentage of explants forming adventitious roots, the percentage of explants with an embryogenic response (Table 1 and Figure 4) and the relative rate of explants showing an embryogenic response (Table 2). The embryogenic response was defined as the presence of embryogenic structures and/or somatic embryos (torpedo-cotyledonary stage) in the initial
Table 1. Influence of explant type and Eucalyptus species/genotype on the percentages of root formation and embryogenic response, following culture for 8 weeks in medium supplemented with NAA (16.11 µM) and gum arabic (40 mg l−1). Influence of the genotype/species and explant type on the percentage of root formation and embryogenic response was evaluated by the χ2 test (P ≤ 0.05). Genotype/species
Explant type
Root formation (%)
Embryogenic response (%)
Leaf 1 Leaf 2 Apex
40.8 (98/240) 33.0 (33/100) 67.1 (215/320)
7.5 (18/240) 1.0 (1/100) 14.6 (47/320)
Leaf 1 Leaf 2 Apex
6.0 (6/100) 0.0 (0/100) 29.0 (58/200)
1.0 (1/100) 0.0 (0/100) 0.0 (0/200)
Leaf 1 Leaf 2 Apex
3.0 (3/100) 6.0 (6/100) 20.0 (20/100)
2.0 (2/100) 1.0 (1/100) 0.0 (0/100)
Sal-May/E. saligna × E. maidenii
41-1-AC/E. globulus
22-6-RP/E. globulus
Source of variation Genotype (A) Explant type (B) A × B
P ≤ 0.001 P ≤ 0.001 P ≤ 0.001
P ≤ 0.001 P ≤ 0.001 P ≤ 0.01
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The shoot apex (1.5–2 mm long) comprising the apical meristem and 2–3 leaf primordia and the two most apical expanding leaves collected from the first (leaf 1) and second (leaf 2) nodes in the apical region of the shoots in an active state of growth were excised from stock shoot cultures and used as initial explants. Leaf 1 and leaf 2 explants had mean lengths of 2.5 and 3 mm, respectively. Ten shoot apices (horizontally orientated) and 10 leaf explants (abaxial side down) were placed in 90 × 15 mm Petri dishes containing 25 ml of induction medium consisting of MS mineral salts and vitamins, 500 mg l−1 casein hydrolysate, 3% (w/v) sucrose and 6 g l−1 agar supplemented with plant growth regulators (PGRs) as stated in the following experiments.
(40 mg l−1) from acacia tree (GA; Sigma, St Louis, USA, G-9752), as a source of arabinogalactan proteins, was also added after filter sterilization to the autoclaved NAA-induction media. The use of 40 mg l−1 GA was based on its positive effect as reported in previous studies on somatic embryo induction in Quercus species (Corredoira et al. 2014). For each genotype and explant type, at least 100 explants were cultured on induction medium (see Table 1).
4 Corredoira et al. Table 2. Morphogenic response of leaf 1 and shoot apex explants excised from shoot cultures of E. globulus (41-1-AC) after culture for 8 weeks in medium supplemented with different picloram concentrations and gum arabic (40 mg l−1).
The interaction of both factors was analysed by a log-linear model (P ≤ 0.05). SPSS for Windows (version 19.0, SPSS, Chicago, IL, USA) was used for statistical analysis.
Picloram Watery callus (%) (µM)
Relative embryogenic response1
Results
Leaf 1 Shoot apex
Effect of NAA on induction of SE
Leaf 1 20 30 40
Shoot apex
98.3 (59/60) 100.0 (60/60) No Yes (+) 100.0 (100/100) 98.0 (98/100) Yes (+) Yes (++) 98.8 (79/80) 100.0 (100/100) Yes (+) Yes (+++)
1Characterization
of relative embryogenic response: No: no embryogenic response; Yes: presence of nodular embryogenic structures/somatic embryos: +, ++, +++: low, medium and high embryogenic response, respectively.
Maintenance of embryogenic competence For somatic embryo proliferation and maintenance of embryogenic competence, individual cotyledonary stage embryos or small clusters of globular and torpedo stage embryos were isolated and transferred to 90 × 15 mm Petri dishes containing 25 ml of embryo proliferation medium. This medium consisted of MS mineral salts and vitamins, 500 mg l−1 casein hydrolysate, 3% (w/v) sucrose, 6 g l−1 agar, 20 µM silver thiosulfate (STS) and 16.11 µM NAA. The STS was filter sterilized and added to the autoclaved medium. The embryogenic lines were maintained by secondary embryogenesis with subcultures at 4–5-week intervals, and were incubated in darkness at 25 °C.
Histological study Explants with visible somatic embryos and/or embryogenic structures were collected at different periods during the experiments for histological analysis. Samples were fixed in FAA solution (formalin, glacial acetic acid and 50% ethanol [1 : 1 : 18 (v/v/v)]), dehydrated through a graded n-butanol series and embedded in paraffin wax (Merck, Kenilworth, NJ, USA). Sections (8 μm) were cut on a Reichert-Jung rotary microtome (Leica, Heidelberg, Germany) and stained with periodic acid-Schiff (PAS)-naphthol blueblack (Merck) to detect starch and other insoluble polysaccharides and total proteins, respectively (Feder and O’Brien 1968). The stained sections were mounted with Euckit®, and photomicrographs were taken with an Olympus DP71 digital camera fitted to a Nikon-FXA microscope (Nikon, Tokyo, Japan).
Statistical analysis The influence of the main experimental factors (the explant type and genotype) on the percentage of root formation and the percentage of embryogenic response (Table 1 and Figure 4) was evaluated by the χ2 test (P ≤ 0.05) from a contingency table.
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explants. The explants were observed under a stereomicroscope (Olympus SZX9, Olympus, Tokyo, Japan) and photographed with an Olympus DP10 digital camera.
In a preliminary experiment, shoot apex and leaf explants from the Sal-May (E. saligna × E. maidenii) and 41-1-AC (E. globulus) were cultured in induction medium supplemented with three NAA concentrations. Most of the explants from the two genotypes responded by forming callus; differentiation of adventitious roots was also observed, especially in explants of the hybrid material, with the highest frequency of adventitious root formation produced on medium with 16.11 μM NAA (data not shown). The embryogenic response, defined as the presence of bipolar embryos and/or embryogenic structures, was observed in shoot apex and leaf explants from both genotypes (Figure 1a–d), with the best response obtained in shoot apex explants of Sal-May treated with 16.11 μM NAA (data not shown). Although embryogenic cultures were obtained in E. globulus with all three NAA concentrations (Figure 1c and d), their embryogenic ability was far lower than that observed in the hybrid genotype. Based on the results in the first experiment, explants of the three genotypes were cultured in induction medium supplemented with 16.11 μM NAA and 40 mg l−1 GA (Table 1). The root formation frequency was significantly affected by the species/genotype (P ≤ 0.001) and explant type (P ≤ 0.001), with a significant interaction (P ≤ 0.001) between these two factors (Table 1). Whereas leaf 2 showed the lowest rooting frequencies in Sal-May and 41-1-AC, the leaf at position 2 of 22-6-RP exhibited a higher rooting ability than leaf 1 explants (Table 2). In all three genotypes, shoot apex explants showed the highest rooting rates, with frequencies ranging between 20% (22-6-RP) and 67.1% (Sal-May). The embryogenic response was also significantly influenced by the species/genotype (P ≤ 0.001) and explant type (P ≤ 0.001), and a significant interaction (P ≤ 0.01) between both factors was also observed (Table 1). The interaction was due to the fact that the highest embryogenic induction frequency was achieved in shoot tip explants of the hybrid genotype (14.6%), while, in contrast, in E. globulus only leaf 1 explants generated somatic embryos. Leaf 1 showed a greater ability for SE than leaf 2, especially in the Sal-May genotype (Table 1). Generally, the embryogenic structures and/or somatic embryos were indirectly formed from callus tissue developed from the initial explants, although a direct origin was also observed (Figure 1c). The embryogenic structures were round or oblong and translucent in appearance (Figure 1d). Histological study confirmed that these structures were somatic embryos (Figure 1e). Embryo production ranged between 2 and 10 embryogenic structures and/or somatic embryos per embryogenic explant. Somatic embryos
A reproducible procedure for induction of somatic embryogenesis 5
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Figure 1. Induction of somatic embryogenesis after 8 weeks of culturing explants in NAA induction medium. Explants were excised from axillary shoot cultures of E. saligna × E. maidenii (Sal-May genotype) and E. globulus (41-1-AC genotype). (a and b) Somatic embryos initiated in leaf (a) and shoot apex explants (b) of the Sal-May genotype. (c) Somatic embryo generated directly from the adaxial surface of a leaf explant of the 41-1-AC genotype. (d) Cluster of embryogenic structures originated from a shoot apex explant of the 41-1-AC genotype. (e) Histological section of the same explant as in (d) showing somatic embryo differentiation. Note a heart-shaped embryo arising from a nodular structure (arrow). (f) Somatic embryos isolated from a shoot apex explant of the Sal-May genotype showing small cotyledons, a hypocotyl and root elongation. (g and h) Longitudinal sections of cotyledonary stage somatic embryos in which the vascular system (vs), root meristem (rm) and shoot apical meristem (sam) are evident. Note the presence of different sized cotyledons in (h). Scale bar: a–d = 1 mm.
could easily be detached from the initial explant and showed a clear bipolarity, evidenced by the development of root and shoot poles and two small cotyledons that were of a scale-leafy appearance (Figure 1c). A number of somatic embryos on initial
explants exhibited precocious germination and root growth (Figure 1f). At the histological level, bipolar organization of somatic embryos was demonstrated, with root and shoot apical meristems, and closed vascular tissue that bifurcated into small
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6 Corredoira et al. cotyledons (Figure 1g and h). The primary root pole was more developed than the shoot pole, which was made up of a few meristematic cell layers.
Effect of picloram on induction of SE
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Somatic embryo maintenance and plant regeneration To establish embryogenic lines of E. globulus and Sal-May, individual cotyledonary somatic embryos or small clusters of globular and torpedo embryos were transferred to embryo proliferation medium containing 16.11 µM NAA, where new somatic embryos were obtained by secondary embryogenesis (Figure 5a). The frequency of subcultured embryo clusters producing secondary SE ranged from 60 to 75%, with 6–8 new secondary embryos obtained per embryo cluster. Secondary embryo production was reduced when isolated cotyledonary stage embryos were used as subcultured explants. Secondary embryo development was asynchronous; they frequently formed in the cotyledonary region of the primary somatic embryos (Figure 5b). The histological
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Given the low embryogenic frequencies obtained in E. globulus explants treated on NAA induction medium, picloram was tested to determine whether it would improve this response. Leaf 1 and shoot apex explants of the 41-1-AC were cultured in induction medium supplemented with three concentrations of picloram (Table 2). In all the treatments, almost 100% of the initial explants proliferated and formed calli (Table 2), but adventitious roots were not generated in the presence of picloram. Both explant types developed a watery callus of friable consistency that initially appeared yellow. As the culture progressed, the callus began to turn necrotic, dark brown and disaggregated (Figure 2a and b). The embryogenic tissue appeared as nodular structures that arose from the disaggregating watery callus. These structures were white-translucent, shiny and varied in shape; most of them were nodular-ovoid but cup-shaped or somewhat curved shapes were also observed (Figure 2c–e), and they could be easily separated from the surrounding callus. Interestingly, each embryogenic structure seemed to be surrounded by a translucent and mucilaginous coating layer. Histological study showed that the mucilaginous layer appeared to be made of vacuolated stratified cells with a large accumulation of starch grains (Figure 3a–c). Shoot apex explants cultured on medium supplemented with the three picloram concentrations produced embryogenic structures, although the greatest response was obtained in medium with 40 µM picloram (Table 2) compared with those relatively fewer embryogenic tissues induced in leaf explants cultured on 30 and 40 µM picloram. There was no embryogenic response in leaves treated with 20 µM picloram. Based on these results, a further experiment was performed by selecting induction medium supplemented with 40 µM picloram to evaluate the embryogenic ability of leaf and shoot tip explants from the three genotypes (Figure 4). After 8 weeks of culture, embryogenic cultures were induced in leaf and shoot apex explants of all three genotypes with frequencies ranging between 13.8% (leaf 1 of 41-1-AC) to 68.8% (leaf 1 of SalMay). The frequency of explants developing embryogenic structures was significantly influenced by the species/genotype (P ≤ 0.001) and the explant type (P ≤ 0.001), with a significant interaction (P ≤ 0.001) between these two factors. The most responsive material was the Sal-May hybrid, in which both types of explant showed a similar embryogenic capacity (Figure 4). However, in the two E. globulus genotypes, the embryogenic ability of leaf explants was significantly lower than those of shoot tip explants. Nevertheless, in both explant types of the three genotypes, the embryogenic frequencies were considerably improved when picloram was included in the induction
medium in comparison with those obtained in induction medium supplemented with NAA (see Table 1). This was especially relevant for the two genotypes of E. globulus, in which induction of SE in picloram-containing medium was higher than 50% in shoot apex explants. The number of nodular embryogenic structures produced per embryogenic explant appeared to be influenced by the species/genotype, with production of few structures (2–6 per explant) in E. globulus genotypes, while more than 50 embryogenic structures were recorded in the hybrid genotype (Figure 2a–c). Histological analysis revealed that the nodular embryogenic structures generated on picloram medium were made up of cells with a dense protein-rich cytoplasm, small vacuoles, a high nucleocytoplasmic ratio and starch grains that were especially abundant in tightly packed cells of the external layers (Figure 3b and c). Small, undifferentiated and more vacuolated cells were also evident in the central area of the nodular structures (Figure 3a–c). In addition, somatic embryos at different developmental stages, including globular-, torpedo- and early cotyledonarystage embryos, were also apparent (Figure 3d–g). As occurred with somatic embryos generated in NAA induction medium, cotyledonary somatic embryos developed in the presence of picloram had a distinct primary root pole, while the apical shoot meristem appeared to be less developed, blocked or even absent (Figure 3g). Embryo abnormalities such as the presence of fused embryos, cotyledons of different size or fused cotyledons in a cup-like structure were also observed. After 8 weeks of culture in picloram-containing medium, the embryogenic structures were subcultured for 6 weeks onto secondary medium devoid of PGRs, where embryo histodifferentiation and the production of somatic embryos at the globular, torpedo and cotyledonary stages and even precociously germinated embryos were promoted (Figure 2f–h). When the secondary medium was supplemented with NAA (16.11 µM), with or without the addition of mT (0.41 µM), embryo differentiation was less pronounced, whereas the embryogenic structures became necrotic following culture on PGR-free medium containing 0.4% activated charcoal.
A reproducible procedure for induction of somatic embryogenesis 7
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Figure 2. Induction of somatic embryogenesis after 8 weeks of culturing explants in picloram induction medium. Explants were excised from axillary shoot cultures of E. saligna × E. maidenii (Sal-May genotype) and E. globulus (41-1-AC and 22-6-RP genotypes) trees. (a and b) Nodular embryogenic structures arising from a watery callus generated in a leaf explant of the 22-6-RP genotype (a) and a shoot apex explant of the 41-1-AC genotype (b). (c–e) Isolated embryogenic structures detached from the watery callus induced in explants of the Sal-May genotype (c) and the 41-1-AC genotype (d and e). Note the different shapes of the embryogenic structures. (f) Developing somatic embryos (globular-torpedo stage) obtained following subculture of embryogenic nodular structure on PGR-free medium. (g and h) Cotyledonary stage embryos (g) and a somatic embryo with precocious germination (h) after subculture of embryogenic nodular structures on PGR-free medium. Scale bar: a–h = 1 mm.
study of embryogenic lines showed the presence of distinct meristematic/embryogenic zones located in the epidermal and subepidermal cell layers of cotyledons, these embryogenic cells gave the origin to the secondary embryos (Figure 5c). Moreover, precociously germinating somatic embryos were also found on
proliferation medium. Embryogenic cultures were maintained for more than 3 years under these conditions. Alternating the cultures between 16.11 µM NAA medium and PGR-free medium or the addition of a cytokinin to the proliferation medium did not improve the multiplication rates.
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Figure 3. Histological analysis of nodular embryogenic structures and somatic embryos originated in picloram induction medium. (a) Section of the nodular structure shown in Figure 2b which exhibited the presence of a stratified coating layer (arrow) surrounding the nodular structure. (b) Nodular embryogenic structures of different sizes arising between the disaggregate callus tissue (arrows). (c) Higher magnification of the embryogenic structure in (b) (square) to show the meristematic cell surface layers and the high starch content of the vacuolated cell coating layer. (d) Globular stage somatic embryo with well-developed protodermis (pt). (e–g) Longitudinal sections of torpedo stage (e) and cotyledonary stage (f and g) embryos. A cotyledonary stage somatic embryo with developing root (r) and poor development of shoot apical meristem (sam) is presented in (g).
Well-developed cotyledonary somatic embryos with defined root and shoot poles were separated from secondary embryogenic cultures and transferred to semisolid germination medium consisting of MS medium supplemented with 3% sucrose, 6 g l−1 agar, 0.44 µM BA and 1.44 µM gibberellic acid, where drying and browning of the isolated embryos occurred rapidly.
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To prevent this disorder, the use of liquid germination medium was tested by placing cotyledonary embryos on two filter paper discs (Whatman grade 181) in Petri dishes (90 mm) containing 10 ml of liquid germination medium. Under these conditions, enlargement and greening of hypocotyl and cotyledons followed by root growth occurred in most of the somatic embryos;
A reproducible procedure for induction of somatic embryogenesis 9 owever, embryo conversion with both root and shoot developh ment (Figure 5d) was observed in only a few instances (
