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A procedure for shoot organogenesis in vitro from leaves and nodes of an elite Eucalyptus gunnii clone: comparative histology
Plant Science 161 (2001) 645– 653 www.elsevier.com/locate/plantsci
A procedure for shoot organogenesis in vitro from leaves and nodes of an elite Eucalyptus gunnii clone: comparative histology Philippe Herve´ a, Alain Jauneau b, Marc Paˆques c, Jean-Noe¨l Marien d, Alain Michel Boudet a, Chantal Teulie`res a,* a b
UMR CNRS/UPS 5546, Poˆle de biotechnologie 6e´ge´tale, 24 Chemin de Borde Rouge, BP 17, 31326 Castanet-Tolosan, France Institut Fe´de´ratif de Recherches Signalisation cellulaire et biotechnologie 6e´ge´tale (FR 40), 24 Chemin de Borde Rouge, BP 17, 31326 Castanet-Tolosan, France c Association Fore´t Cellulose (Afocel), Domaine de l’Etanc¸on, 77370 Nangis, France d Association Fore´t Cellulose (Afocel), Domaine de Saint-Cle´ment, 34980 St Cle´ment de Ri6ie´re, France Received 2 March 2001; accepted 14 May 2001
Abstract In Eucalyptus, shoot regeneration has mainly been achieved from juvenile material (i.e. seedlings). Here, we present a procedure for shoot regeneration from different organs (leaves, internodes, nodes) of micropropagated E. gunnii clones derived from selected trees using a basal regeneration medium that included 0.04 mM picloram and 2.25 mM N6-benzyladenine (BA). About 10% of the leaves or internodes regenerated an average of four buds after 10 – 12 weeks of culture and more than 50% of the nodes regenerated an average of 20 buds after 6–8 weeks of culture. Explant browning was reduced by increasing the BA concentration in a two-step procedure. The ability to regenerate buds was strictly correlated with the formation of protuberances of chlorophyll-containing cells from the different organs tested. Node-derived protuberances originated from pre-existing meristematic areas, whereas leaf-derived protuberance formation involved the leafs vascular system. Histological studies revealed that adventitious buds originated from the peripheral layers of both leaf-derived and node-derived protuberances. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Eucalyptus; Tissue culture; Regeneration; Histology; Woody plant
1. Introduction Among hardwood forest-tree species, eucalypts (Myrtaceae) are fast-growing and widely cultivated in productive forest short rotations. Some of the 600 Eucalyptus species are commercially important for timber, essential oil and pulp production. Only a few temperate species have been introduced into Europe for pulp production (covering more than 1 million ha). In France, E. gunnii, which was selected by the Association Foreˆt Cellulose through conventional breeding on Abbre6iations: BA, N6-benzyladenine; CPPU, N-(2-chloro-4pyridyl)-N%-phenylurea; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS, Murashige and Skoog medium; NAA, naphthaleneacetic acid; Picloram, 4-amino-3,5,6-trichloropicolinic acid; TDZ, thidiazuron. * Corresponding author. fax: + 33-5-62-193502. E-mail address: [email protected] (C. Teulie`res).
the basis of frost tolerance and growth is the major Eucalyptus species for industrial plantations. In order to extend the plantation area, the improvement of traits such as frost tolerance is still under investigation. Recently, new key forest biotechnology targets have been identified. These include enhanced growth, increased yield, uniformity of feed stock and customised feedstock [1]. In particular, genetic engineering of lignin profiles is a challenge for the qualitative improvement of forest trees [2]. However, plant regeneration from elite clones is still a limiting step in the application of genetic engineering to Eucalyptus species [3]. Shoot organogenesis and somatic embryogenesis have been reported for several commercially important tropical eucalypts, especially from young material (i.e. hypocotyls, cotyledons), and a few have been studied in-depth by histological studies [4]. Shoot regeneration from organs excised from micropropagated clones has
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been reported in E. citriodora [5], E. tereticornis [6], E. grandis [7,8] and E. camaldulensisi [9]. In contrast, only a limited number of reproducible regeneration methods have been published for temperate species, and again most of the successful results were obtained from juvenile organs such as excised hypocotyls or cotyledons for E. globulus [10 – 12] and E. gunnii [13]. However, only preliminary results were reported from organs excised from micropropagated clones of E. gunnii, and, it remains necessary to improve the regeneration procedure before Agrobacterium vectors can be used as an efficient mean of gene transfer. In this paper, we describe regeneration through indirect organogenesis from different organs of an elite E. gunnii clone that was selected for commercial pulp production. In addition, through a histological study, we show the organogenic pathways of shoot regeneration from leaves and nodes cultured in vitro.
2. Materials and methods
2.1. Source of plant material Two elite clones of E. gunnii Hook. provided by the Association Foreˆ t Cellulose (Nangis, France) were established from tissues excised from superior trees that were selected on the basis of frost tolerance and growth. Microcuttings grown in vitro were subcultured in Petri dishes every 2 weeks on a multiplication medium consisting of Murashige and Skoog medium (MS) salts [14] supplemented with 3% (w/v) sucrose, 100 mg l − 1 myoinositol, 0.1 mg l − 1 nicotinic acid, 0.1 mg l − 1 pyridoxine– HCl, 0.5 mg l − 1 thiamine –HCl and 0.45 mM BA, adjusted to pH 5.5 and solidified by Table 1 Conditions leading to shoot regeneration from one elite clone of E. gunnii leaves and internodes excised from microcuttings (45 explants for each condition, each condition being replicated at least twice) Auxin (mM)
None NAA O.1 NAA O.5 NAA NAA 2.7 NAA 4 2,4-D 0.04 2,4-D 4.5 Picloram 0.02 Picloram 0.04 Picloram 0.41 Picloram 4.1
Cytokinin (BA) (mM) 1
2.25
4.5
Nt Nt Nt Nt Nt C C C S C C
C S C C S C C C C S C C
C C C C S C C C C S C C
S, shoot regeneration; C, callus formation; Nt, not tested.
0.7% (w/v) Bacto Agar. All media were autoclaved at 110 °C for 30 min. At the beginning of the experiments, microcuttings had been subcultured bi-weekly for 2 years on multiplication medium at 25 °C under a 16-h photoperiod. Irradiance was 60 mmol m − 2 s − 1 from Sylvania Standard Daylight 36 W fluorescent lamps.
2.2. Explant preparation and tissue culture Leaves, internodes and nodes were tested for regeneration capacity. All explants were excised from shoot cultures in vitro under a stereo microscope, and in an antioxidant solution consisting of 1 mM ascorbic acid, 0.1 mM citric acid and 1 g l − 1 polyvinylpyrrolidone 40, pH 5. Leaves (three to four younger pairs) were excised from the upper part of proliferating shoots grown in the environmental conditions described for multiplication, and placed with the abaxial face in contact with the regeneration medium. Stem internodal and nodal segments (4–6 mm) were removed from etiolated shoots from microcuttings grown in the dark for 4 weeks and transferred either vertically or horizontally to the regeneration medium. All the explants were grown in 60 mm Petri dishes on regeneration media consisting of MS salts, 3% (w/v) sucrose, 100 mg l − 1 myoinositol, 2 mg l − 1 glycine, 1 mg l − 1 nicotinic acid, 1 mg l − 1 pyridoxine–HCl, 1 mg l − 1 thiamine–HCl. The media were adjusted to pH 5.5 before adding the gelling agent 0.25% (w/v) Gelrite, and then autoclaved at 110 °C for 30 min. In the first experiments, the morphogenic effects of various growth regulator combinations were tested. Naphthaleneacetic (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4amino-3,5,6-trichloropicolinic acid (picloram), BA, N(2-chloro-4-pyridyl)-N%-phenylurea (CPPU) and thidiazuron (TDZ) were thus combined in different concentrations (see Table 1). All the cultures were incubated at 25 °C in the dark for 1 week, then transferred to fresh medium and grown at 25 °C under a 16-h photoperiod (45 mmol m − 2 s − 1). They were subcultured every 2 weeks.
2.3. Experimental design and data analysis The morphogenic responses (% explants with protuberances, % explants regenerating buds and number of buds per protuberance) were observed every 2 weeks. Per experiment, 3–15 Petri dishes, each containing 15– 20 explants, were considered for each treatment. The data (% and S.D.) correspond to the average percentage of at least three independent replicates. Analysis of variance (ANOVA) was applied after the data were submitted to arcsin(x) or ln(x) transformation. Means were compared by the Tukey test with a critical value of P= 0.05.
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2.4. Histological studies At different regeneration steps, the harvested tissues were fixed in 0.5% (v/v) glutaraldehyde plus 2% (v/v) paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.2 for 3 h under vacuum, then washed and postfixed in 1% (v/v) osmium tetroxide solution in the same buffer for 1 h. After several washes in 0.1 M sodium phosphate buffer pH 7.2, the samples were dehydrated in a graded ethanol series and collected in acetone. Then, the samples were gradually embedded in Spurr’s resin (acetone/Spurr 2:1, 1:1, 1:2 (v/v) and pure Spurr’s resin). For polymerisation, samples were kept at 65 °C for 48 h. Six-mm-thick sections were obtained with an Ultracut Reichert ultramicrotome and stained with 0.5% toluidine blue in 2.5% sodium carbonate.
3. Results
3.1. Morphology of shoot organogenesis from different organs 3.1.1. Lea6es Fully expanded leaves (three to four upper pairs) were excised from the upper part of proliferating shoots grown in light/dark conditions and placed with the abaxial face in contact with a regeneration medium (Fig. 1A and B). Protuberances of densely-packed, chlorophyll-containing cells first appeared and developed at the leaf proximal end after 2– 6 weeks of culture (Fig. 1C and D). Most often, the protuberances were surrounded by callus, the cells of which were whitish to yellowish. Bud initiation occurred from the surface of the protuberances after 10– 12 weeks of culture (Fig. 1E). Leaves were totally incompetent for bud regeneration when the proximal part of the leaf was removed, whatever the culture conditions tested (data not shown). Protuberances excised from leaves and transferred to the regeneration medium gave rise to buds (Fig. 1F). When shoot initiation has been achieved, shoot proliferation was obtained by growing the adventitious shoots on multiplication medium. 3.1.2. Internodes Internodes were excised from etiolated microcuttings grown in the dark (Fig. 1G). Dense, chlorophyll-containing protuberances of tissue developed at the cut ends of the internodes after 4– 6 weeks of culture (Fig. 1H), and were surrounded by wound callus. Bud initiation occurred from the protuberances. Orientation of the explant on subsequent callus and bud formation was important, as the caulogenic protuberances were only observed on internodal segments that were placed horizontally on the medium. When the primary ex-
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plants were placed vertically, callus formation was greatly reduced and browning of both the primary explant and callus occurred.
3.1.3. Nodes Nodes excised from microcuttings grown in the dark were used as primary explants and grown on the same regeneration medium as for leaves and internodes (Fig. 2A). Dense, chlorophyll-containing protuberances of tissue first appeared at the nodal position after 2–3 weeks of culture (Fig. 2B). They differentiated into groups of buds and formed multiple shoots within 4–6 weeks on regeneration medium (Fig. 2C and D). In some cases, axillary shoots developed from bud primordia in the leaf axil. However, axillary shoot apices developed abnormally on regeneration medium (Fig. 2E), while normal shoots were obtained on multiplication medium. Shoot regeneration was associated with variable callus formation, at the cut end of the nodal segment and especially when the segments were grown in a horizontal position. A vertical orientation of the nodal segments reduced callus formation, which tended to be limited to the cut end of the explant positioned in the medium. To obtain normal shoot development, groups of buds were transferred to multiplication medium. Excised 3-week-old protuberances without differentiated buds could be induced to regenerate shoots (data not shown). 3.2. Design and efficiency of the shoot regeneration procedure We wished to develop a regeneration medium that could promote organogenesis from different organs of two elite E. gunnii clones. In the first set of experiments, different ratios of growth regulators were tested for the morphogenic responses of leaves and internodes that would lead to callus formation or root or shoot organogenesis. Shoot organogenesis was achieved by combinations of NAA/BA and picloram/BA for the two genotypes and the two explant types tested. The observations for the most reactive clone are presented in Table 1. In contrast to BA, bud regeneration was never obtained with the cytokinins TDZ (0.1, 1 and 2.25 mM) or CPPU (0.5, 1 and 2 mM) (data not shown). Further experiments were only carried out with the most reactive elite clone using the basal regeneration medium containing 0.04 mM picloram and 2.25 mM BA. The BA concentration in the medium during the first subculture influenced the amount, density and colour of the callus produced from the primary explants. To optimise the regeneration procedure, the effect of BA on shoot organogenesis during the first week of culture was evaluated. The primary explants were first grown on different BA concentrations combined with 0.04 mM picloram, and then after one week transferred to the
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Fig. 1. Shoot organogenesis from leaves and internodes. (A) Upper part of a single shoot of a microcutting of elite E. gunnii clone grown in vitro. Bar, 1.5 mm. (B) and (C) Leaf from the upper part of the shoot. The open arrow indicates the area at the proximal part of the leaf which produced (C) dense, chlorophyll-containing protuberances of tissue after 2 – 4 weeks of culture. Bars, 700 mm (B) and 250 mm (C). (D) 4– 6-week-old protuberance surrounded by wound translucent callus (arrowhead). Bar, 250 mm. (E) General view of a regenerating explant after 10 – 12 weeks of culture with buds at the surface of the protuberance (open arrow). Bar, 1.5 mm. (F) Isolated protuberance grown on culture medium with wound callus (arrowhead) and shoot primordia (arrow). Bar, l mm. (G) Internode from etiolated shoot grown horizontally on the culture medium. Bar, 1 mm. (H) 4– 6-week-old chlorophyll-containing protuberance at the cut end of the internode (open arrow). Bar, 1.2 mm.
basal regeneration medium. For leaf explants, an increase of BA concentration during the first subculture strongly decreased bud regeneration and induced browning of both primary explant and callus (Table 2). Therefore, a two-step procedure was used for shoot regeneration, whereby the explants were first grown on
modified MS medium supplemented with 0.04 mM picloram and 1 mM BA, and then after 1 week transferred to a regeneration medium consisting of 0.04 mM picloram and 2.25 mM BA. The best culture condition led to more than 35% of the leaves producing protuberances and about 9% pro-
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ducing buds, after 10–12 weeks of culture with an average of four buds per explant (Table 3). Under these conditions about 30% of the internodes produced protuberances and approximately, 10% produced buds after 10–12 weeks of culture. Although analysis of data by the Tukey test indicated that efficiency of shoot organogenesis was not significantly different between leaves and internodes, values were greater for nodes. More than 50% of the nodes produced protuberances with approximately, 20 buds per protuberance after 8 weeks of culture.
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3.3. Histology of shoot organogenesis from lea6es and nodes 3.3.1. Lea6es For the histological investigation, the samples were collected at different regeneration steps (day 0, 14, 28, 63, 77) and were excised at the proximal part of the leaves, which was found to be the reactive zone for shoot organogenesis. In longitudinal section, the proximal part of the leaf consisted of the leaf’s vascular system surrounded by parenchyma limited on both
Fig. 2. Shoot organogenesis from nodes. (A) Node from etiolated shoot grown vertically on the culture medium. Bar, 1 mm. (B) General view of 3-week-old protuberances (open arrow) developing at the nodal position and callus formation around the cut end of the primary explant (arrowheads). Bar, l mm. (C) and (D). Multiple buds originating from the protuberances (arrows). Bars, 800 mm (C) and 250 mm (D). (E) View of buds (arrows) formed at the distal part of a shoot developed from the axillary meristem of the primary explant. Bar, 250 mm. Table 2 Effect of BA concentration during the first subculture on protuberance formation from leaves, bud regeneration and primary explant browning percent primary explants were grown on various BA concentrations in combination with 0.04 mM picloram during the first week and then transferred to the regeneration medium (0.04 mM picloram, 2.25 mM BA) BA (mM)
% Explants with proluberancesa
% Explants regenerating budsb
% Explant broening
1 2.25 4.5
31.1a ( 9 27.7) 20.6a ( 9 21.5) 5.9a ( 9 5.2)
8.la ( 9 5.l) 0.7b ( 92.5) 0.7b ( 9 2.5)
5.2a ( 96.8) 17.8a ( 911.7) 4l.5b ( 9 13.0)
c
Data obtained from three independent experiments (45 explants per experiment). Means ( 92 S.D.) followed by the same letter, for each morphogenic response, are not significantly different by Tukey test (P= 0.05). a Determined 6 weeks after culture initiation. b Determined 10–12 weeks after culture initiation. c Determined 4 weeks after culture initiation.
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Table 3 Shoot organogenesis from leaves, internodes and nodes on modified MS medium supplemented with 0.04 mM picloram and 2.25 mM BA Explant
% Explants with protuberancesa
% Explants regenerating budsb
Number of buds per protuberancec
Leaf Internode Node
36.5a ( 9 8.8) 28.3a ( 9 15.8) 56.lb ( 9 24.8)
8.8a ( 94.7) 9.9a ( 94.9) 53.5b ( 921.7)
4.0a ( 9 4.2) 3.4a ( 9 3.3) 20.0b ( 912.2)
Data obtained from five independent experiments. Means ( 925 S.D.) followed by the same letter, for each morphogenic response, are not significantly different by Tukey test P= 0.05). a Determined 4–6 weeks after culture initiation. b Determined 8–12 weeks after culture initiation. c Determined from 50 node-derived protuberances and by combining independent experiments for leaves (41 protuberances) and internodes (14 protuberances).
sides by an epidermis (Fig. 3A). After 2 weeks of culture on the regeneration medium, a protuberance was produced at the proximal part of the leaf (Fig. 3B), and which appeared to be connected to the leaf’s vascular system. Wound callus was produced at the adaxial face of the leaf. The central part of the protuberance consisted mainly of parenchyma, active cambium and tracheary elements (Fig. 3C). At the connection zone between the leaf explant and the protuberance, an increasingly greater number of dividing cells were visible from the leaf vein to the distal part (Fig. 3D). These dividing cells were close to the tracheary elements of the vascular system (Fig. 3E). In transverse section, numerous dividing cells were visible in the leaf’s vascular system close to the zone connecting the leaf vein and the protuberance (Fig. 3F). After 8 –12 weeks of culture, bud regeneration occurred only from the protuberance in which the epidermal and subepidermal layers consisted of dividing cells deeply stained with toluidine blue (Fig. 3G). Anticlinal and periclinal divisions were observed. Adventitious buds originating from the peripheral cell layers were well developed after 10– 12 weeks of culture (Fig. 3H).
3.3.2. Nodes Tissue samples were excised from the upper part of the node from day 0 until day 42 of culture. In longitudinal section, an axillary meristematic area was visible at the leaf base and consisted of small, densely-stained and -cytoplasmic cells (Fig. 4A). After 4 days of culture, cell division resulted in the enlargement of the axillary meristem and its emergence (Fig. 4B). After 11 days of culture, protuberances could be identified at the axillary position, while cell proliferation occurred at the upper part of the node, resulting in wound callus (Fig. 4C). The central parts of the protuberance consisted of parenchyma with large intercellular spaces whereas the peripheral parts were composed of small and cytoplasmically-dense cells. After 21 days of culture, the protuberance exhibited 2– 3 densely stained peripheral cell layers, which resulted from both anticlinal and periclinal divisions in epidermal and subepidermal cells
(Fig. 4D). A cambial zone was visible. Finally, at 28 days the protuberance comprised an epidermal and subepidermal layer deeply stained with toluidine blue, oil glands, parenchyma with large, vacuolated cells and intercellular spaces, vascular tissue and cambial cells (Fig. 4E). New developing protuberance was visible in some cases from a well-structured protuberance. Adventitious buds originated from the peripheral cell layers after 4–6 weeks of culture (Fig. 4F).
4. Discussion In this study, morphogenic responses obtained from organs in vitro of elite E. gunnii clones of known genetic value are of interest regarding the potential of biotechnology and tissue culture for accelerating foresttree breeding programs. Procedures that could be applied to micropropagated selected clones would reduce the time involved in improving woody plants through genetic engineering. Furthermore, production of genetically improved Eucalyptus trees from clonal material is considered a more attractive prospect than the production of genetically engineered trees from seedlings whose the characteristics are unknown as they have not been field-tested. Our results may be of interest regarding the general recalcitrance of Eucalyptus clones to regenerate. In our hands, shoot organogenesis was successful from different organs (leaves, internodes, nodes) using the same regeneration medium consisting of 0.04 mM picloram and 2.25 mM BA. A similar growth regulator combination was used to induce multiple shoot formation from the cotyledonary node of Cicer arietinum [15]. The use of TDZ or CPPU as cytokinin did not promote shoot organogenesis of E. gunnii clones although they have been successfully used in various Eucalyptus species [16,17]. To reduce browning and death of primary explants, we developed a two-step regeneration procedure by increasing the BA concentration. With the regeneration procedure described in this paper, about 10% of the leaves and internodes pro-
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duced adventitious buds. This is low compared with efficiencies reported for certain very responsive Eucalyptus species such as E. camaldulensis [9] and E. grandis [7] but is similar to earlier results obtained from juvenile E. gunnii. A procedure based on node culture gave rise to more than 50% of primary explants producing groups of buds. Whatever the primary explant tested, shoot organogenesis occurred through the same general pathway. First, a protuberance was formed and then the -initiation of buds occurred from the surface of the protuber-
Fig. 3. Successive stages of shoot organogenesis in leaf explants. (A) Longitudinal section of the proximal part of the leaf showing the excised part (arrow), the adaxial (ad) and abaxial (ab) part of the leaf, vascular system (vs), parenchyma (p) and epidermis (e). Bar, 100 mm. (B) General view of 2 –4-week-old protuberance produced at the proximal part of the explant (longitudinal section) with wound callus (wc) formation. Bar, 200 mm. (C) The central part of the protuberance consists mainly of parenchyma (p), cambial cells (c) and vascular elements (arrowhead). Bar, 100 mm. (D) and (E). Zone between the leaf and the protuberance showing cell divisions progressing from the vein (v) to the distal part (arrowheads). Bar, 100 mm. At a higher magnification (E), dividing cells (arrowhead) are close to tracheary elements (arrows) of the leaf’s vascular system. Bar, 50 mm. (F) Transverse section of the zone between the leaf and the protuberance as indicated by the dotted line in B showing numerous cells in division in the leaf vein. Bar, 100 mm. (G) Distal part of a protuberance with peripheral cell layers heavily stained with toluidine blue. Bar, 80 mm. (H) Well-developed bud originating from the peripheral cell layers of a protuberance. Bar, 100 mm. Sections were stained with 0.5% toluidine blue.
Fig. 4. Successive stages of shoot organogenesis in node explants. (A) Longitudinal section of a meristematic area located at the basis of the leaf (1) near the axil. Bar, 50 mm. (B) Axillary meristematic area after 4 days of culture. Bar, 50 mm. (C) Organisation of the node after 11 days of culture with cell proliferation occurring at the upper part of the node and resulting in wound callus (wc). Emergence of a protuberance in the axillary position (arrowhead). Bar, 150 mm. (D) Organisation of a 21-day-old protuberance. Peripheral 2 – 3 cell layers consisted of densely stained cells. The centre consists mainly of parenchyma cells (p). Note organisation of a cambial zone (open arrow) and active cells at the basis of the protuberance (arrow). Bar, 120 mm. (E) A 28-day-old protuberance exhibiting an epidermal and subepidermal layer (arrows), oil glands (arrowheads), parenchyma (p), vascular tissues (v) and cambial cells (open arrow). A newly developing protuberance is indicated by solid arrowheads. Bar, 120 mm. (F) Developing bud from the peripheral cell layers of the protuberance. Bar, 60 mm. Sections were stained with 0.5% toluidine blue.
ance. The protuberances rapidly exhibited a well-organised structure composed of different cell and tissue types. At least four cell types were easily identified, epidermal cells, parenchyma, vascular elements and meristematic cells. Similar observations have been earlier reported in Populus [18], Pinus [19] and different species of Eucalyptus [20,21]. These authors used the terms nodule or nodule-like structures to describe independent, spherical and cohesive units obtained under their culture conditions. We did not observe spherical structures and found the protuberances from the explant always to remain attached to the explant. To establish a culture of isolated protuberances, it was thus
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necessary to excise them from the primary explant. Meristematic agglomerates were also obtained from nodal cultures of E. robusta and E. camaldulensis [22]. Such a structure was described as an axillary bud aggregate resembling an organogenic callus. So we favour the term protuberance to describe the structures obtained from the different explants of the E. gunnii clone. Depending on the explant, the origin of the cells which composed the caulogenic protuberances differed. The node-derived protuberances originated from a preexisting meristematic area located at the leaf basis. The leaf-derived protuberances were found to be connected to the leaf vein and numerous divisions occurred from cells located in the vein and close to the tracheary elements. This strongly suggests that the protuberances originated from the cambial cells of the leaf’s vascular system, even if it can not be excluded that some cells originated from fascicular parenchyma. The involvement of the vascular system has been earlier reported for leaves of Cichorium intybus [23] and for internodes of Populus [24]. Interestingly, the greater shoot regeneration capacity from node may be associated with the meristematic origin of the caulogenic protuberance [17]. Irrespective of their origin, the protuberances always exhibited two to three peripheral layers of reactive cells. The histological study confirmed that the regenerated shoots had an adventitious origin, and emphasised the epidermal or subepidermal origin of the adventitious buds from leaf-derived and node-derived protuberances. The involvement of superficial tissues in bud initiation has been earlier reported in different species [25,26]. In particular, Azmi et al. [11] demonstrated for shoot regeneration of E. globulus that the reactive cells were always in the vicinity of the oil glands, which differentiated in subepidermal and epidermal locations. We described a reproducible regeneration procedure in vitro by indirect shoot organogenesis for an elite clone of E. gunnii. This is an important step in further studies of genetic transformation in elite Eucalyptus genotypes. Furthermore, the protuberances described in this paper should be an interesting target for genetic transformation because of the superficial origin of bud initiation.
Acknowledgements The authors thank A. Souvre´ (INP-ENSAT Toulouse, France), D. Clriqui (Universite´ Pierre et Marie Curie, Paris) for helpful discussions, C.H. Bornman (Lund University, Sweden) for critically reading the manuscript and E. Dumas and P. Nouvet for excellent technical assistance. This research was financially supported by a CIFRE convention between the Socie´ te´ d’Exploitation des Bois du Sud-Ouest and the Associa-
tion Nationale de la Recherche Technique (France), by CNRS (Centre National de la Recherche Scientifique), by the University Paul Sabatier (Toulouse), by the Midi Pyre´ ne´ es Regional Council and by the European Union.
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P. Her6e´ et al. / Plant Science 161 (2001) 645–653 [17] L.P. Barrueto Cid, A. Machado, S. Carvalheira, A.C.M. Brasileiro, Plant regeneration from seedling explants of Eucalyptus grandis ×E. urophylla, Plant Cell. Tissue Organ. Cult. 56 (1999) 17 – 23. [18] B.H. McCown, E.L. Zeldin, H.A. Pinkalla, R.R. Dedolph, Nodule culture: a developmental pathway with high potential for regeneration, automated micropropagation, and plant metabolite production from woody plants, in: J.W. Hanover, D.E. Keathley (Eds.), Genetic Manipulation of Woody Plants, Plenum Press, New York, 1988, pp. 149 –166. [19] J. Aitken-Christie, A.P. Singh, H. Davies, Multiplication of meristematic tissue: a new tissue culture system for radiata pine, in: J.W. Hanover, D.E. Keathley (Eds.), Genetic Manipulation of Woody Plants, Plenum Press, New York, 1988, pp. 413 – 432. [20] E. Warrag, M.S. Lesney, D.J. Rockwood, Nodule culture and regeneration of Eucalyptus grandis hybrids, Plant Cell. Rep. 9 (1991) 586 – 589. [21] K. Ito, K. Doi, Y. Tatemichi, M. Shibata, Plant regeneration
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from rotating nodule culture, Plant Cell. Rep. 16 (1996) 42 – 45. P. Boxus, J.M. Terzi, C. Lievens, M. Pylyser, P. Ngaboyamahina, K. Duhem, Improvement and perspectives of micropropagation techniques applied to some hot climate plants, Acta Hortic. 289 (1991) 55 – 64. S. Pieron, P. Boxus, D. Dekegel, Histological study of nodule morphogenesis from Cichorium intybus L. leaves cultivated in vitro, In Vitro Cell. Dev. Biol-Plan 34 (1998) 87 – 93. G.C. Douglas, Formation of adventitious buds in stem internodes of Populus spp. cultured in vitro on basal medium: influence of endogenous properties of explants, J. Plant Physiol. 116 (1984) 313 – 321. C.H. Bornman, Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro, Physiol. Plant 57 (1983) 5 – 16. G. Hicks, Shoot induction and organogenesis in vitro: a developmental perspective, In Vitro Cell. Dev. Biol-Plant 30 (1994) 10 – 15.
A procedure for shoot organogenesis in vitro from leaves and nodes of an elite Eucalyptus gunnii clone: comparative histology Philippe Herve´ a, Alain Jauneau b, Marc Paˆques c, Jean-Noe¨l Marien d, Alain Michel Boudet a, Chantal Teulie`res a,* a b
UMR CNRS/UPS 5546, Poˆle de biotechnologie 6e´ge´tale, 24 Chemin de Borde Rouge, BP 17, 31326 Castanet-Tolosan, France Institut Fe´de´ratif de Recherches Signalisation cellulaire et biotechnologie 6e´ge´tale (FR 40), 24 Chemin de Borde Rouge, BP 17, 31326 Castanet-Tolosan, France c Association Fore´t Cellulose (Afocel), Domaine de l’Etanc¸on, 77370 Nangis, France d Association Fore´t Cellulose (Afocel), Domaine de Saint-Cle´ment, 34980 St Cle´ment de Ri6ie´re, France Received 2 March 2001; accepted 14 May 2001
Abstract In Eucalyptus, shoot regeneration has mainly been achieved from juvenile material (i.e. seedlings). Here, we present a procedure for shoot regeneration from different organs (leaves, internodes, nodes) of micropropagated E. gunnii clones derived from selected trees using a basal regeneration medium that included 0.04 mM picloram and 2.25 mM N6-benzyladenine (BA). About 10% of the leaves or internodes regenerated an average of four buds after 10 – 12 weeks of culture and more than 50% of the nodes regenerated an average of 20 buds after 6–8 weeks of culture. Explant browning was reduced by increasing the BA concentration in a two-step procedure. The ability to regenerate buds was strictly correlated with the formation of protuberances of chlorophyll-containing cells from the different organs tested. Node-derived protuberances originated from pre-existing meristematic areas, whereas leaf-derived protuberance formation involved the leafs vascular system. Histological studies revealed that adventitious buds originated from the peripheral layers of both leaf-derived and node-derived protuberances. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Eucalyptus; Tissue culture; Regeneration; Histology; Woody plant
1. Introduction Among hardwood forest-tree species, eucalypts (Myrtaceae) are fast-growing and widely cultivated in productive forest short rotations. Some of the 600 Eucalyptus species are commercially important for timber, essential oil and pulp production. Only a few temperate species have been introduced into Europe for pulp production (covering more than 1 million ha). In France, E. gunnii, which was selected by the Association Foreˆt Cellulose through conventional breeding on Abbre6iations: BA, N6-benzyladenine; CPPU, N-(2-chloro-4pyridyl)-N%-phenylurea; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS, Murashige and Skoog medium; NAA, naphthaleneacetic acid; Picloram, 4-amino-3,5,6-trichloropicolinic acid; TDZ, thidiazuron. * Corresponding author. fax: + 33-5-62-193502. E-mail address: [email protected] (C. Teulie`res).
the basis of frost tolerance and growth is the major Eucalyptus species for industrial plantations. In order to extend the plantation area, the improvement of traits such as frost tolerance is still under investigation. Recently, new key forest biotechnology targets have been identified. These include enhanced growth, increased yield, uniformity of feed stock and customised feedstock [1]. In particular, genetic engineering of lignin profiles is a challenge for the qualitative improvement of forest trees [2]. However, plant regeneration from elite clones is still a limiting step in the application of genetic engineering to Eucalyptus species [3]. Shoot organogenesis and somatic embryogenesis have been reported for several commercially important tropical eucalypts, especially from young material (i.e. hypocotyls, cotyledons), and a few have been studied in-depth by histological studies [4]. Shoot regeneration from organs excised from micropropagated clones has
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been reported in E. citriodora [5], E. tereticornis [6], E. grandis [7,8] and E. camaldulensisi [9]. In contrast, only a limited number of reproducible regeneration methods have been published for temperate species, and again most of the successful results were obtained from juvenile organs such as excised hypocotyls or cotyledons for E. globulus [10 – 12] and E. gunnii [13]. However, only preliminary results were reported from organs excised from micropropagated clones of E. gunnii, and, it remains necessary to improve the regeneration procedure before Agrobacterium vectors can be used as an efficient mean of gene transfer. In this paper, we describe regeneration through indirect organogenesis from different organs of an elite E. gunnii clone that was selected for commercial pulp production. In addition, through a histological study, we show the organogenic pathways of shoot regeneration from leaves and nodes cultured in vitro.
2. Materials and methods
2.1. Source of plant material Two elite clones of E. gunnii Hook. provided by the Association Foreˆ t Cellulose (Nangis, France) were established from tissues excised from superior trees that were selected on the basis of frost tolerance and growth. Microcuttings grown in vitro were subcultured in Petri dishes every 2 weeks on a multiplication medium consisting of Murashige and Skoog medium (MS) salts [14] supplemented with 3% (w/v) sucrose, 100 mg l − 1 myoinositol, 0.1 mg l − 1 nicotinic acid, 0.1 mg l − 1 pyridoxine– HCl, 0.5 mg l − 1 thiamine –HCl and 0.45 mM BA, adjusted to pH 5.5 and solidified by Table 1 Conditions leading to shoot regeneration from one elite clone of E. gunnii leaves and internodes excised from microcuttings (45 explants for each condition, each condition being replicated at least twice) Auxin (mM)
None NAA O.1 NAA O.5 NAA NAA 2.7 NAA 4 2,4-D 0.04 2,4-D 4.5 Picloram 0.02 Picloram 0.04 Picloram 0.41 Picloram 4.1
Cytokinin (BA) (mM) 1
2.25
4.5
Nt Nt Nt Nt Nt C C C S C C
C S C C S C C C C S C C
C C C C S C C C C S C C
S, shoot regeneration; C, callus formation; Nt, not tested.
0.7% (w/v) Bacto Agar. All media were autoclaved at 110 °C for 30 min. At the beginning of the experiments, microcuttings had been subcultured bi-weekly for 2 years on multiplication medium at 25 °C under a 16-h photoperiod. Irradiance was 60 mmol m − 2 s − 1 from Sylvania Standard Daylight 36 W fluorescent lamps.
2.2. Explant preparation and tissue culture Leaves, internodes and nodes were tested for regeneration capacity. All explants were excised from shoot cultures in vitro under a stereo microscope, and in an antioxidant solution consisting of 1 mM ascorbic acid, 0.1 mM citric acid and 1 g l − 1 polyvinylpyrrolidone 40, pH 5. Leaves (three to four younger pairs) were excised from the upper part of proliferating shoots grown in the environmental conditions described for multiplication, and placed with the abaxial face in contact with the regeneration medium. Stem internodal and nodal segments (4–6 mm) were removed from etiolated shoots from microcuttings grown in the dark for 4 weeks and transferred either vertically or horizontally to the regeneration medium. All the explants were grown in 60 mm Petri dishes on regeneration media consisting of MS salts, 3% (w/v) sucrose, 100 mg l − 1 myoinositol, 2 mg l − 1 glycine, 1 mg l − 1 nicotinic acid, 1 mg l − 1 pyridoxine–HCl, 1 mg l − 1 thiamine–HCl. The media were adjusted to pH 5.5 before adding the gelling agent 0.25% (w/v) Gelrite, and then autoclaved at 110 °C for 30 min. In the first experiments, the morphogenic effects of various growth regulator combinations were tested. Naphthaleneacetic (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4amino-3,5,6-trichloropicolinic acid (picloram), BA, N(2-chloro-4-pyridyl)-N%-phenylurea (CPPU) and thidiazuron (TDZ) were thus combined in different concentrations (see Table 1). All the cultures were incubated at 25 °C in the dark for 1 week, then transferred to fresh medium and grown at 25 °C under a 16-h photoperiod (45 mmol m − 2 s − 1). They were subcultured every 2 weeks.
2.3. Experimental design and data analysis The morphogenic responses (% explants with protuberances, % explants regenerating buds and number of buds per protuberance) were observed every 2 weeks. Per experiment, 3–15 Petri dishes, each containing 15– 20 explants, were considered for each treatment. The data (% and S.D.) correspond to the average percentage of at least three independent replicates. Analysis of variance (ANOVA) was applied after the data were submitted to arcsin(x) or ln(x) transformation. Means were compared by the Tukey test with a critical value of P= 0.05.
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2.4. Histological studies At different regeneration steps, the harvested tissues were fixed in 0.5% (v/v) glutaraldehyde plus 2% (v/v) paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.2 for 3 h under vacuum, then washed and postfixed in 1% (v/v) osmium tetroxide solution in the same buffer for 1 h. After several washes in 0.1 M sodium phosphate buffer pH 7.2, the samples were dehydrated in a graded ethanol series and collected in acetone. Then, the samples were gradually embedded in Spurr’s resin (acetone/Spurr 2:1, 1:1, 1:2 (v/v) and pure Spurr’s resin). For polymerisation, samples were kept at 65 °C for 48 h. Six-mm-thick sections were obtained with an Ultracut Reichert ultramicrotome and stained with 0.5% toluidine blue in 2.5% sodium carbonate.
3. Results
3.1. Morphology of shoot organogenesis from different organs 3.1.1. Lea6es Fully expanded leaves (three to four upper pairs) were excised from the upper part of proliferating shoots grown in light/dark conditions and placed with the abaxial face in contact with a regeneration medium (Fig. 1A and B). Protuberances of densely-packed, chlorophyll-containing cells first appeared and developed at the leaf proximal end after 2– 6 weeks of culture (Fig. 1C and D). Most often, the protuberances were surrounded by callus, the cells of which were whitish to yellowish. Bud initiation occurred from the surface of the protuberances after 10– 12 weeks of culture (Fig. 1E). Leaves were totally incompetent for bud regeneration when the proximal part of the leaf was removed, whatever the culture conditions tested (data not shown). Protuberances excised from leaves and transferred to the regeneration medium gave rise to buds (Fig. 1F). When shoot initiation has been achieved, shoot proliferation was obtained by growing the adventitious shoots on multiplication medium. 3.1.2. Internodes Internodes were excised from etiolated microcuttings grown in the dark (Fig. 1G). Dense, chlorophyll-containing protuberances of tissue developed at the cut ends of the internodes after 4– 6 weeks of culture (Fig. 1H), and were surrounded by wound callus. Bud initiation occurred from the protuberances. Orientation of the explant on subsequent callus and bud formation was important, as the caulogenic protuberances were only observed on internodal segments that were placed horizontally on the medium. When the primary ex-
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plants were placed vertically, callus formation was greatly reduced and browning of both the primary explant and callus occurred.
3.1.3. Nodes Nodes excised from microcuttings grown in the dark were used as primary explants and grown on the same regeneration medium as for leaves and internodes (Fig. 2A). Dense, chlorophyll-containing protuberances of tissue first appeared at the nodal position after 2–3 weeks of culture (Fig. 2B). They differentiated into groups of buds and formed multiple shoots within 4–6 weeks on regeneration medium (Fig. 2C and D). In some cases, axillary shoots developed from bud primordia in the leaf axil. However, axillary shoot apices developed abnormally on regeneration medium (Fig. 2E), while normal shoots were obtained on multiplication medium. Shoot regeneration was associated with variable callus formation, at the cut end of the nodal segment and especially when the segments were grown in a horizontal position. A vertical orientation of the nodal segments reduced callus formation, which tended to be limited to the cut end of the explant positioned in the medium. To obtain normal shoot development, groups of buds were transferred to multiplication medium. Excised 3-week-old protuberances without differentiated buds could be induced to regenerate shoots (data not shown). 3.2. Design and efficiency of the shoot regeneration procedure We wished to develop a regeneration medium that could promote organogenesis from different organs of two elite E. gunnii clones. In the first set of experiments, different ratios of growth regulators were tested for the morphogenic responses of leaves and internodes that would lead to callus formation or root or shoot organogenesis. Shoot organogenesis was achieved by combinations of NAA/BA and picloram/BA for the two genotypes and the two explant types tested. The observations for the most reactive clone are presented in Table 1. In contrast to BA, bud regeneration was never obtained with the cytokinins TDZ (0.1, 1 and 2.25 mM) or CPPU (0.5, 1 and 2 mM) (data not shown). Further experiments were only carried out with the most reactive elite clone using the basal regeneration medium containing 0.04 mM picloram and 2.25 mM BA. The BA concentration in the medium during the first subculture influenced the amount, density and colour of the callus produced from the primary explants. To optimise the regeneration procedure, the effect of BA on shoot organogenesis during the first week of culture was evaluated. The primary explants were first grown on different BA concentrations combined with 0.04 mM picloram, and then after one week transferred to the
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Fig. 1. Shoot organogenesis from leaves and internodes. (A) Upper part of a single shoot of a microcutting of elite E. gunnii clone grown in vitro. Bar, 1.5 mm. (B) and (C) Leaf from the upper part of the shoot. The open arrow indicates the area at the proximal part of the leaf which produced (C) dense, chlorophyll-containing protuberances of tissue after 2 – 4 weeks of culture. Bars, 700 mm (B) and 250 mm (C). (D) 4– 6-week-old protuberance surrounded by wound translucent callus (arrowhead). Bar, 250 mm. (E) General view of a regenerating explant after 10 – 12 weeks of culture with buds at the surface of the protuberance (open arrow). Bar, 1.5 mm. (F) Isolated protuberance grown on culture medium with wound callus (arrowhead) and shoot primordia (arrow). Bar, l mm. (G) Internode from etiolated shoot grown horizontally on the culture medium. Bar, 1 mm. (H) 4– 6-week-old chlorophyll-containing protuberance at the cut end of the internode (open arrow). Bar, 1.2 mm.
basal regeneration medium. For leaf explants, an increase of BA concentration during the first subculture strongly decreased bud regeneration and induced browning of both primary explant and callus (Table 2). Therefore, a two-step procedure was used for shoot regeneration, whereby the explants were first grown on
modified MS medium supplemented with 0.04 mM picloram and 1 mM BA, and then after 1 week transferred to a regeneration medium consisting of 0.04 mM picloram and 2.25 mM BA. The best culture condition led to more than 35% of the leaves producing protuberances and about 9% pro-
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ducing buds, after 10–12 weeks of culture with an average of four buds per explant (Table 3). Under these conditions about 30% of the internodes produced protuberances and approximately, 10% produced buds after 10–12 weeks of culture. Although analysis of data by the Tukey test indicated that efficiency of shoot organogenesis was not significantly different between leaves and internodes, values were greater for nodes. More than 50% of the nodes produced protuberances with approximately, 20 buds per protuberance after 8 weeks of culture.
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3.3. Histology of shoot organogenesis from lea6es and nodes 3.3.1. Lea6es For the histological investigation, the samples were collected at different regeneration steps (day 0, 14, 28, 63, 77) and were excised at the proximal part of the leaves, which was found to be the reactive zone for shoot organogenesis. In longitudinal section, the proximal part of the leaf consisted of the leaf’s vascular system surrounded by parenchyma limited on both
Fig. 2. Shoot organogenesis from nodes. (A) Node from etiolated shoot grown vertically on the culture medium. Bar, 1 mm. (B) General view of 3-week-old protuberances (open arrow) developing at the nodal position and callus formation around the cut end of the primary explant (arrowheads). Bar, l mm. (C) and (D). Multiple buds originating from the protuberances (arrows). Bars, 800 mm (C) and 250 mm (D). (E) View of buds (arrows) formed at the distal part of a shoot developed from the axillary meristem of the primary explant. Bar, 250 mm. Table 2 Effect of BA concentration during the first subculture on protuberance formation from leaves, bud regeneration and primary explant browning percent primary explants were grown on various BA concentrations in combination with 0.04 mM picloram during the first week and then transferred to the regeneration medium (0.04 mM picloram, 2.25 mM BA) BA (mM)
% Explants with proluberancesa
% Explants regenerating budsb
% Explant broening
1 2.25 4.5
31.1a ( 9 27.7) 20.6a ( 9 21.5) 5.9a ( 9 5.2)
8.la ( 9 5.l) 0.7b ( 92.5) 0.7b ( 9 2.5)
5.2a ( 96.8) 17.8a ( 911.7) 4l.5b ( 9 13.0)
c
Data obtained from three independent experiments (45 explants per experiment). Means ( 92 S.D.) followed by the same letter, for each morphogenic response, are not significantly different by Tukey test (P= 0.05). a Determined 6 weeks after culture initiation. b Determined 10–12 weeks after culture initiation. c Determined 4 weeks after culture initiation.
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Table 3 Shoot organogenesis from leaves, internodes and nodes on modified MS medium supplemented with 0.04 mM picloram and 2.25 mM BA Explant
% Explants with protuberancesa
% Explants regenerating budsb
Number of buds per protuberancec
Leaf Internode Node
36.5a ( 9 8.8) 28.3a ( 9 15.8) 56.lb ( 9 24.8)
8.8a ( 94.7) 9.9a ( 94.9) 53.5b ( 921.7)
4.0a ( 9 4.2) 3.4a ( 9 3.3) 20.0b ( 912.2)
Data obtained from five independent experiments. Means ( 925 S.D.) followed by the same letter, for each morphogenic response, are not significantly different by Tukey test P= 0.05). a Determined 4–6 weeks after culture initiation. b Determined 8–12 weeks after culture initiation. c Determined from 50 node-derived protuberances and by combining independent experiments for leaves (41 protuberances) and internodes (14 protuberances).
sides by an epidermis (Fig. 3A). After 2 weeks of culture on the regeneration medium, a protuberance was produced at the proximal part of the leaf (Fig. 3B), and which appeared to be connected to the leaf’s vascular system. Wound callus was produced at the adaxial face of the leaf. The central part of the protuberance consisted mainly of parenchyma, active cambium and tracheary elements (Fig. 3C). At the connection zone between the leaf explant and the protuberance, an increasingly greater number of dividing cells were visible from the leaf vein to the distal part (Fig. 3D). These dividing cells were close to the tracheary elements of the vascular system (Fig. 3E). In transverse section, numerous dividing cells were visible in the leaf’s vascular system close to the zone connecting the leaf vein and the protuberance (Fig. 3F). After 8 –12 weeks of culture, bud regeneration occurred only from the protuberance in which the epidermal and subepidermal layers consisted of dividing cells deeply stained with toluidine blue (Fig. 3G). Anticlinal and periclinal divisions were observed. Adventitious buds originating from the peripheral cell layers were well developed after 10– 12 weeks of culture (Fig. 3H).
3.3.2. Nodes Tissue samples were excised from the upper part of the node from day 0 until day 42 of culture. In longitudinal section, an axillary meristematic area was visible at the leaf base and consisted of small, densely-stained and -cytoplasmic cells (Fig. 4A). After 4 days of culture, cell division resulted in the enlargement of the axillary meristem and its emergence (Fig. 4B). After 11 days of culture, protuberances could be identified at the axillary position, while cell proliferation occurred at the upper part of the node, resulting in wound callus (Fig. 4C). The central parts of the protuberance consisted of parenchyma with large intercellular spaces whereas the peripheral parts were composed of small and cytoplasmically-dense cells. After 21 days of culture, the protuberance exhibited 2– 3 densely stained peripheral cell layers, which resulted from both anticlinal and periclinal divisions in epidermal and subepidermal cells
(Fig. 4D). A cambial zone was visible. Finally, at 28 days the protuberance comprised an epidermal and subepidermal layer deeply stained with toluidine blue, oil glands, parenchyma with large, vacuolated cells and intercellular spaces, vascular tissue and cambial cells (Fig. 4E). New developing protuberance was visible in some cases from a well-structured protuberance. Adventitious buds originated from the peripheral cell layers after 4–6 weeks of culture (Fig. 4F).
4. Discussion In this study, morphogenic responses obtained from organs in vitro of elite E. gunnii clones of known genetic value are of interest regarding the potential of biotechnology and tissue culture for accelerating foresttree breeding programs. Procedures that could be applied to micropropagated selected clones would reduce the time involved in improving woody plants through genetic engineering. Furthermore, production of genetically improved Eucalyptus trees from clonal material is considered a more attractive prospect than the production of genetically engineered trees from seedlings whose the characteristics are unknown as they have not been field-tested. Our results may be of interest regarding the general recalcitrance of Eucalyptus clones to regenerate. In our hands, shoot organogenesis was successful from different organs (leaves, internodes, nodes) using the same regeneration medium consisting of 0.04 mM picloram and 2.25 mM BA. A similar growth regulator combination was used to induce multiple shoot formation from the cotyledonary node of Cicer arietinum [15]. The use of TDZ or CPPU as cytokinin did not promote shoot organogenesis of E. gunnii clones although they have been successfully used in various Eucalyptus species [16,17]. To reduce browning and death of primary explants, we developed a two-step regeneration procedure by increasing the BA concentration. With the regeneration procedure described in this paper, about 10% of the leaves and internodes pro-
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duced adventitious buds. This is low compared with efficiencies reported for certain very responsive Eucalyptus species such as E. camaldulensis [9] and E. grandis [7] but is similar to earlier results obtained from juvenile E. gunnii. A procedure based on node culture gave rise to more than 50% of primary explants producing groups of buds. Whatever the primary explant tested, shoot organogenesis occurred through the same general pathway. First, a protuberance was formed and then the -initiation of buds occurred from the surface of the protuber-
Fig. 3. Successive stages of shoot organogenesis in leaf explants. (A) Longitudinal section of the proximal part of the leaf showing the excised part (arrow), the adaxial (ad) and abaxial (ab) part of the leaf, vascular system (vs), parenchyma (p) and epidermis (e). Bar, 100 mm. (B) General view of 2 –4-week-old protuberance produced at the proximal part of the explant (longitudinal section) with wound callus (wc) formation. Bar, 200 mm. (C) The central part of the protuberance consists mainly of parenchyma (p), cambial cells (c) and vascular elements (arrowhead). Bar, 100 mm. (D) and (E). Zone between the leaf and the protuberance showing cell divisions progressing from the vein (v) to the distal part (arrowheads). Bar, 100 mm. At a higher magnification (E), dividing cells (arrowhead) are close to tracheary elements (arrows) of the leaf’s vascular system. Bar, 50 mm. (F) Transverse section of the zone between the leaf and the protuberance as indicated by the dotted line in B showing numerous cells in division in the leaf vein. Bar, 100 mm. (G) Distal part of a protuberance with peripheral cell layers heavily stained with toluidine blue. Bar, 80 mm. (H) Well-developed bud originating from the peripheral cell layers of a protuberance. Bar, 100 mm. Sections were stained with 0.5% toluidine blue.
Fig. 4. Successive stages of shoot organogenesis in node explants. (A) Longitudinal section of a meristematic area located at the basis of the leaf (1) near the axil. Bar, 50 mm. (B) Axillary meristematic area after 4 days of culture. Bar, 50 mm. (C) Organisation of the node after 11 days of culture with cell proliferation occurring at the upper part of the node and resulting in wound callus (wc). Emergence of a protuberance in the axillary position (arrowhead). Bar, 150 mm. (D) Organisation of a 21-day-old protuberance. Peripheral 2 – 3 cell layers consisted of densely stained cells. The centre consists mainly of parenchyma cells (p). Note organisation of a cambial zone (open arrow) and active cells at the basis of the protuberance (arrow). Bar, 120 mm. (E) A 28-day-old protuberance exhibiting an epidermal and subepidermal layer (arrows), oil glands (arrowheads), parenchyma (p), vascular tissues (v) and cambial cells (open arrow). A newly developing protuberance is indicated by solid arrowheads. Bar, 120 mm. (F) Developing bud from the peripheral cell layers of the protuberance. Bar, 60 mm. Sections were stained with 0.5% toluidine blue.
ance. The protuberances rapidly exhibited a well-organised structure composed of different cell and tissue types. At least four cell types were easily identified, epidermal cells, parenchyma, vascular elements and meristematic cells. Similar observations have been earlier reported in Populus [18], Pinus [19] and different species of Eucalyptus [20,21]. These authors used the terms nodule or nodule-like structures to describe independent, spherical and cohesive units obtained under their culture conditions. We did not observe spherical structures and found the protuberances from the explant always to remain attached to the explant. To establish a culture of isolated protuberances, it was thus
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necessary to excise them from the primary explant. Meristematic agglomerates were also obtained from nodal cultures of E. robusta and E. camaldulensis [22]. Such a structure was described as an axillary bud aggregate resembling an organogenic callus. So we favour the term protuberance to describe the structures obtained from the different explants of the E. gunnii clone. Depending on the explant, the origin of the cells which composed the caulogenic protuberances differed. The node-derived protuberances originated from a preexisting meristematic area located at the leaf basis. The leaf-derived protuberances were found to be connected to the leaf vein and numerous divisions occurred from cells located in the vein and close to the tracheary elements. This strongly suggests that the protuberances originated from the cambial cells of the leaf’s vascular system, even if it can not be excluded that some cells originated from fascicular parenchyma. The involvement of the vascular system has been earlier reported for leaves of Cichorium intybus [23] and for internodes of Populus [24]. Interestingly, the greater shoot regeneration capacity from node may be associated with the meristematic origin of the caulogenic protuberance [17]. Irrespective of their origin, the protuberances always exhibited two to three peripheral layers of reactive cells. The histological study confirmed that the regenerated shoots had an adventitious origin, and emphasised the epidermal or subepidermal origin of the adventitious buds from leaf-derived and node-derived protuberances. The involvement of superficial tissues in bud initiation has been earlier reported in different species [25,26]. In particular, Azmi et al. [11] demonstrated for shoot regeneration of E. globulus that the reactive cells were always in the vicinity of the oil glands, which differentiated in subepidermal and epidermal locations. We described a reproducible regeneration procedure in vitro by indirect shoot organogenesis for an elite clone of E. gunnii. This is an important step in further studies of genetic transformation in elite Eucalyptus genotypes. Furthermore, the protuberances described in this paper should be an interesting target for genetic transformation because of the superficial origin of bud initiation.
Acknowledgements The authors thank A. Souvre´ (INP-ENSAT Toulouse, France), D. Clriqui (Universite´ Pierre et Marie Curie, Paris) for helpful discussions, C.H. Bornman (Lund University, Sweden) for critically reading the manuscript and E. Dumas and P. Nouvet for excellent technical assistance. This research was financially supported by a CIFRE convention between the Socie´ te´ d’Exploitation des Bois du Sud-Ouest and the Associa-
tion Nationale de la Recherche Technique (France), by CNRS (Centre National de la Recherche Scientifique), by the University Paul Sabatier (Toulouse), by the Midi Pyre´ ne´ es Regional Council and by the European Union.
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