Somatic Embryogenesis in Elm

Annals of Botany 89: 637±644, 2002 doi:10.1093/aob/mcf080, available online at www.aob.oupjournals.org

Somatic Embryogenesis in Elm E . C O R R E D O I R A , A . M. V I E I T E Z and A . B A L L E S T E R * Instituto de Investigaciones AgrobioloÂgicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain Received: 19 July 2001 Returned for revision: 7 January 2002 Accepted: 28 January 2002

We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0´1 mg l±1 BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely. ã 2002 Annals of Botany Company Key words: Elm, somatic embryogenesis, Ulmus minor, Ulmus glabra, zygotic embryogenesis.

INTRODUCTION

MATERIALS AND METHODS

Elm is a tree of the northern hemisphere that is greatly valued for its landscape, amenity and timber qualities. Elm populations have been devastated by Dutch elm disease (Brasier, 1991), initially caused by the vascular wilt fungus Ophiostoma ulmi and later by the distinctly different and highly aggressive O. novo-ulmi (Brasier, 1994). There have been various attempts to control the disease through international research programmes, none of which have yet succeeded in signi®cantly halting the spread of the disease or reducing its impact (Sticklen and Sherald, 1993; Fenning et al., 1996). Genetic engineering may prove useful for control of the disease. Micropropagation and regeneration of various elm species through axillary branching has been achieved not only with juvenile starting material (Fenning et al., 1993), but also with material from mature trees (Dorion et al., 1987; Corchete et al., 1993). Adventitious buds have been induced from protoplast culture (Dorion et al., 1994), on leaf explants (Bolyard et al., 1991; George and Tripepi, 1994) and on strips of stem (Ben Jouira et al., 1998). Elm internodes have been shown to be suitable explants for genetic modi®cation via Agrobacterium tumefaciens (Bolyard et al., 1991; Dorion et al., 1995; Gartland JS et al., 2000, 2001; Gartland KMA et al., 2000). Somatic embryogenesis, too, might not only allow the clonal propagation of valuable genotypes, but also facilitate genetic engineering. In spite of this, to the best of our knowledge there have been no studies of the capacity of elm tissue to form somatic embryos. Here we describe the induction of somatic embryogenesis from zygotic embryo tissues of Ulmus minor and U. glabra.

In a ®rst experiment samaras were collected from three 20year-old Ulmus minor Miller trees (designated E, C and PB) and from two 70-year-old Ulmus glabra Hudson trees (designated RC and AV) at various times in March and April 1998 (Table 1). All the trees were growing in the Rosalia de Castro park (Lugo, NW Spain, 42°59¢39¢N, 7°32¢35¢W). In a second experiment carried out in 1999, samaras from the U. glabra tree AV were collected at weekly intervals between March and April, i.e. approx. 1±8 weeks post anthesis (WPA) if anthesis is de®ned as the time at which approx. half of the tree is in bloom. Samaras were surface sterilized by immersion for 3 min in a 0´5 % solution of free chlorine (Milliporeâ chlorine tablets) plus 1 % Tweenâ 80, and were then rinsed three times with sterile distilled water. Following sterilization, intact ovules were aseptically excised from the samaras collected 1±4 WPA, and zygotic embryos were isolated from those collected 5±8 WPA and divided into two parts (axes and cotyledons). All explants were placed in individual 20 3 150 mm culture tubes containing 16 ml of induction medium. At each collection date during the second experiment, 25 ovules or zygotic embryos were taken for morphological analysis (size and weight).

* For correspondence. Fax 34 981 592504, e-mail aballester@ iiag.cesga.es

Culture of initial explants

Basal medium consisted of Murashige and Skoog (1962) mineral salts and vitamins supplemented with 1g l±1 glutamine, 50 mg l±1 arginine, 50 mg l±1 glycine, 100 mg l±1 m-inositol, 30 g l±1 sucrose and 8 g l±1 Difco bacto agar. The pH was adjusted to 5´7 before sterilization by autoclaving at 121 °C for 20 min. ã 2002 Annals of Botany Company

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Corredoira et al. Ð Somatic Embryogenesis in Elm

TA B L E 1. Effect of 2,4-D concentration, size of zygotic embryo and seed collection date on induction of somatic embryogenesis in explants from different trees of Ulmus minor and U. glabra

Species/ tree

Collection date (WPA)

U. minor E C PB PB U. glabra RC AV

Length of zygotic embryo (mm)

4 4 2 5

4´2 3´8 2´6 5´3

6 6 6 6

6 6

6´2 6 0´3 nd

Embryogenesis induction (%) 2,4-D (mg l±1)

Surviving explants with callus (%)*

0.2

0.5

1

2

13´5 44´3 30´2 100

± 4´2 8´4 12´6

± ± ± ±

± ± 4´2 4´2

± ± 4´2 ±

100 100

± 12´5

± ±

± ±

± ±

0´3 0´2 0´4 0´4

For each tree and collection date 96 explants were cultured. WPA, Weeks post-anthesis. nd, not determined; ±, no response. * After 4 weeks' culture.

In the ®rst set of experiments explants were initially cultured on induction medium consisting of basal medium containing 0´2 mg l±1 benzyladenine (BA) in combination with 0´2, 0´5, 1 or 2 mg l±1 2,4-dichlorophenoxyacetic acid (2,4-D). For each tree and collection date, 24 zygotic embryos were cultured for each 2,4-D concentration giving a total of 96 explants per tree source. In the second set of experiments explants from AV were cultured on basal medium or on induction medium consisting of basal medium plus 0´2 mg l±1 BA, with or without 2,4-D (0´2 or 1´0 mg l±1). For each combination of induction medium and collection date, 72 explants were used (three replications with 24 explants each), giving a total of 2304 explants. After 4 weeks of culture in induction medium, explants were transferred to 300 ml jars (four or ®ve explants per jar) containing 50 ml basal medium supplemented with 0´1 mg l±1 BA, with subsequent monthly transfer to fresh medium. The numbers of explants showing germination of zygotic embryos, callus formation and somatic embryogenesis were recorded every 4 weeks. Proliferation of embryogenic tissues

Isolated somatic embryos and embryogenic calluses were excised from the original explants and subcultured separately in multiplication medium (basal medium supplemented with 0´1 mg l±1 BA) for proliferation of the somatic embryos and maintenance of embryogenic competence. Various embryogenic cell lines were established, each from the embryogenic callus induced on one original explant; these lines were maintained by embryogenic callus subculture at 4-week intervals. Histological analysis

For histological examination, zygotic embryos (collected at weekly intervals from 1 to 8 WPA) and somatic embryos were ®xed in a mixture of formalin, glacial acetic acid and 50 % ethanol (1 : 1 : 18 v/v/v), dehydrated through a graded

n-butanol series (50, 70, 85, 95, 100 %) and embedded in paraf®n. Transverse and longitudinal 8±10 mm sections were cut and stained with safranin-fast green (Jensen, 1962) for general examination. Starch and other insoluble polysaccharides were stained by the periodic acid Schiff's (PAS) reaction, and total proteins by naphtholblue black, which detects the reserve protein bodies and the densely protein-rich nucleus and cytoplasmic content of the cells. Culture conditions

Initial explant cultures were maintained in darkness at 25 °C for 6 weeks, and were then subjected to a 16 h photoperiod (30 mmol m±2 s±1 provided by cool-white ¯uorescent lamps) with temperatures of 25 °C in the light and 20 °C in the dark. These latter conditions were applied in all subsequent embryo proliferation and germination experiments. R E S U LT S Initiation of embryogenic cultures

In the ®rst set of experiments, both elm species tested, U. glabra and U. minor, proved to be amenable to induction of somatic embryogenesis via a straightforward procedure of culturing immature zygotic embryos in a medium containing the auxin 2,4-D (Table 1). Samples collected from two of the three U. minor trees and one of the two U. glabra trees exhibited an embryogenic response, generally mediated by callus formation. Only occasionally (most often when the 2,4-D concentration was low) did zygotic embryos develop roots or whole seedlings. The proportion of surviving explants with soft translucent callus 4 weeks after establishment in vitro ranged from 13´5 to 100 % and appeared to depend mainly on the tree and the size of the zygotic embryo, determined in turn by the collection date. Transfer of cultures from induction medium to 0´1 mg l±1 BA medium accelerated callus growth, and upon incubation under light conditions (6 weeks after culture initiation) the


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F I G . 1. Somatic embryogenesis in elm. A, Induction of somatic embryos and embryogenic callus on a zygotic embryo explant from Ulmus glabra (tree AV) cultured on 0´2 mg l±1 2,4-D + 0´2 mg l±1 BA induction medium (37´7). B and C, Isolated somatic embryos obtained from the AV2 embryogenic line after 4 weeks of culture in multiplication medium. Precocious germination is evident in C (B 310´5; C 310). D, Different morphological types of somatic embryos from the AV1 embryogenic line (311). E and F, Embryogenic callus with low capacity (E; U. minor line PB5) and high capacity (F; U. glabra line AV2) of embryo proliferation after 4 weeks of culture in multiplication medium (E 35´5). G, Plantlet regenerated from a somatic embryo (line AV6) after 4 weeks of culture in germination medium without plant growth regulators (34).

callus acquired a yellowish green colour. As culture progressed, some callus differentiated yellowish white nodules, which ®nally produced embryogenic masses 3±6 months after initiation of culture (Fig. 1A). Somatic

embryos at different stages of development, from globular shaped to cotyledonary shaped, had arisen by this time. The frequency of embryogenic cultures was greatest (12´5 %) for explants collected from the U. minor tree PB and the U.

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glabra tree AV 5 and 6 WPA, and the number of somatic embryos per embryogenic culture ranged from ®ve to 50. Within each species embryogenic capacity depended on the tree, and for all trees was greatest when the concentration of 2,4-D was lowest (Table 1). There were no differences in the induction capacity in embryonic axes or cotyledon explants. The somatic embryos were bipolar structures exhibiting a ®lamentous morphology with an elongated hypocotyl root pole and greenish leafy cotyledons (Fig. 1B and C). Many cotyledonary stage embryos failed to differentiate a wellstructured shoot meristem or had a ¯attened apical meristem. Anomalous morphologies, such as the presence of more than two cotyledons or fused embryos, were also observed (Fig. 1D). In¯uence of the developmental stage of the explant

A second set of experiments was carried out in 1999 to identify the `developmental window' during which somatic embryogenesis induction is possible in elm. Between 1 and 8 WPA, samples were collected at weekly intervals from the U. glabra tree AV and were cultured on four different induction media. At each collection date the developmental stage of the zygotic embryos was ascertained by means of morphological measurements (size and weight) and histological analysis. As in the ®rst set of experiments, the frequency of somatic embryogenesis was highest in induction medium supplemented with 0´2 mg l±1 2,4-D. Some embryogenesis (3±6 %) also took place in medium lacking plant growth regulators if samples were collected 5 or 6 WPA, but not in medium with BA but no 2,4-D. Figure 2, which is based on that reported for Cercis canadensis by Trigiano et al. (1999), shows the embryogenic response in induction medium containing 0´2 mg l±1 2,4-D as a function of collection time, together with data on the developmental stage of the zygotic embryos collected. Immature zygotic embryos collected 1 WPA showed no embryogenic response. Histological analysis showed them to be in the early division phase of the zygote giving rise to preglobular stage embryos, being composed of a small number of cells with densely staining cytoplasm, large nuclei and nucleoli, and no apparent vacuolation (Fig. 3A). The proembryos were surrounded by a developing endosperm of nuclear type. Embryos collected between the second and the fourth WPA had somatic embryogenesis rates of up to 3 %. During this period their length increased ®ve-fold and their fresh weight 35-fold (Fig. 2). By 2 WPA they had reached the globular stage, with distinct protoderm, radial symmetry and a multiseriate suspensor (Fig. 3B and C). The endosperm continued to develop by free nuclear division and subsequently, upon attainment of the heart and early torpedo stages 2±3 WPA, the formation of cell walls (Fig. 3C and D). Zygotic embryo development proceeded through the transitional stage from radial to bilateral symmetry over the next week, giving rise to the differentiation of heart-shaped and early torpedo-shaped embryos (3 WPA) (Fig. 3D). These embryos had organized root meristems and apical shoot meristems ¯anked by the developing cotyledons. The

F I G . 2. Embryogenic response (%) of elm zygotic embryos as a function of zygotic embryo explant age (weeks post-anthesis, WPA), with the length, fresh weight and developmental stage of the explants. For each collection date 288 explants were cultured.

central part of the embryo was formed by rather larger, more elongated cells destined to originate the procambium. By the torpedo stage, the embryo had a solid ®le of procambium extending the entire length of the axis and into the cotyledons. Early cotyledonary stage embryos collected at 4 WPA had well developed dome-shaped apical shoot meristems, root meristems, and closed vascular systems. Vacuolation of the cortex and pith of the embryonic axis was already evident (Fig. 3E). The cotyledons had epidermis, vacuolated parenchyma penetrated by vascular bundles and a small number of starch deposits, but no protein reserves (Fig. 3F). Mitotic activity continued in the axis and cotyledons. The embryo sac was full of cellular endosperm by this time. The frequency of somatic embryogenesis increased to 4 % in embryos collected 5 WPA, and to 14´4 % in those collected 6 WPA (Fig. 2). During weeks 5 and 6 there was further development of the hypocotyl root axis and cotyledons. Mitotic activity and further differentiation was still evident after 6 WPA (Fig. 3G): the root cap had become clearly distinguishable and on the adaxial side of the cotyledons the subepidermal cells had enlarged and were arranged as in the palisade cell layer of leaves. Starch grains had accumulated in cotyledons as well as in the cortex and pith of the embryonic axis. Protein bodies were evident in the cotyledons and cortex of mid-cotyledonary stage embryos (6 WPA), although they were not observed in the two apical meristems, procambial strands or epidermis. During week 6 the endosperm was almost totally depleted, while the fresh weight of the embryo almost tripled, increasing from 3´5 to 7´6 mg 5 WPA and 9´6 mg 6 WPA (Fig. 2).


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F I G . 3. Zygotic embryogenesis in Ulmus glabra. A, Proembryo inside the embryo sac in an ovule collected at 1 WPA (3202). B, Globular stage embryo (2 WPA) surrounded by endospermic nuclei (3304). C, Transitional stage from globular to heart-shaped embryo showing a distinct protoderm (p) and a multiseriate suspensor (s) (3150). D, Heart-stage embryo exhibiting bipolar organization with an integrated shoot±root axis surrounded by cellular endosperm (3 WPA, 375). E, Early cotyledonary stage embryo at 4 WPA exhibiting well developed shoot and root apical meristems (338). F, Section of a cotyledon with starch deposits (4 WPA, 3150). G, Further development of cotyledonary embryo (6 WPA) showing closed vascular system and characteristic cotyledonary notches (n) (320). H, Embryonic axis of a mature embryo (7 WPA) showing well elongated hypocotyl±root pole and differentiation of the root cap (340). I, Accumulation of storage products (starch and protein bodies) in cotyledons of an embryo collected at 7 WPA (351). J, Mature embryo collected at 8 WPA in which the embryonic axis and cotyledons are packed with protein bodies (332).

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In the seventh WPA, signi®cant hypocotyl elongation was re¯ected in a marked increase in the length of the zygotic embryo. In contrast, the embryo length hardly changed in week 8 (Fig. 2). At this time three-quarters of the embryonic axis was enclosed between the basal zones of the cotyledons (Fig. 3H). Storage substances continued to accumulate in the entire embryo: starch grains and protein bodies were abundant in the cotyledons (Fig. 3I), especially in the two or three palisade cell layers in the adaxial region. The root cap had become more mature, consisting of nine or ten layers of cells, the outermost of which contained starch deposits. The accumulation of storage reserves culminated 8 WPA in a morphologically mature cotyledonary stage zygotic embryo that was packed with protein bodies and partially desiccated in appearance (Fig. 3J). Since the size of the embryo hardly changed during the last week, the increase in fresh weight may be largely attributed to the intense accumulation of reserves that took place 6±8 WPA. The best time to collect zygotic embryos for induction of embryogenesis was 6 WPA, when they were approx. 85 % of their ®nal length and 66 % of their ®nal fresh weight. At this stage, induction of embryogenesis was even possible in medium with no plant growth regulators. Proliferation of embryogenic tissues and maintenance of embryogenic competence

The multiplication of embryogenic cultures was investigated by subculturing the somatic embryos and the embryogenic callus. When somatic embryos were isolated and cultured in the multiplication medium, they became desiccated and brown after 1 week without showing any signs of repetitive or secondary embryogenesis. However, subcultured embryogenic callus retained its embryogenic competence. At subculture the callus was formed by friable nodules consisting of embryogenic cells organized into proembryogenic cell masses (Fig. 4A and B), most of which later showed surface cell proliferation and differentiation of globular structures (Fig. 4C). Some of these structures were bordered by a distinct protoderm, an indication of the presence of globular or pre-globular embryos (Fig. 4D). The transition from the globular to the cotyledonary stage (Fig. 4E) was very rapid, as in the development of zygotic embryos. In some cases, somatic embryos differentiated in the embryogenic callus culture themselves began to produce further proembryogenic masses through rupture of the epidermis and proliferation of hypocotyl cortex cells (Fig. 4D). This can be considered as a form of repetitive embryogenesis response. The failure of isolated somatic embryos to behave likewise was probably due simply to their rapid desiccation and browning when cultured on semi-solid medium; culture of isolated somatic embryos in liquid medium is therefore required to clarify this issue. Production of embryogenic callus and embryos has now been maintained for over 3 years by subculturing the embryogenic masses every 4 weeks on multiplication medium supplemented with 0´1 mg l±1 BA. Nine U. minor lines and 24 U. glabra lines have been maintained in this way. Embryogenic capacity differs from line to line, some lines exhibiting a high capacity for embryo proliferation

while others mainly produce proembryogenic callus and only occasionally somatic embryos (Fig. 1E and F). Somatic embryos differentiated in proliferating cultures underwent precocious germination, showing elongation and development of the hypocotyl and root (Fig. 1C). There were also marked differences between zygotic embryos and cotyledonary somatic embryos (cf. Figs 3 and 4F), in which abnormal cytodifferentiation led to de®cient shoot meristem and cotyledon development, and the absence of storage products. These features and the precocious germination hindered completion of the maturation stage. In preliminary germination and conversion experiments, liquid MS medium with 30 g l ±1 sucrose and no growth regulators was used to avoid the rapid drying and browning of isolated embryos that had occurred previously on semisolid medium. Somatic embryos longer than 5 mm were placed on two ®lter paper discs (Whatman grade 181) in 90 3 15 mm Petri dishes containing 10 ml of liquid germination medium. In these conditions, root growth usually occurred ®rst, followed by enlargement and greening of hypocotyl and cotyledons, but shoot development was only rarely observed (Fig. 1G). Further research is therefore needed into the control of maturation and germination of somatic embryos. D I SC U S S IO N The production of embryogenic cultures by zygotic embryos of two elm species is reported here for the ®rst time. These ®ndings contrast with our unsuccessful attempts to induce somatic embryos on mature elm in vitro clonal material, namely leaf and internodal sections (results not shown). As in other woody species (Keinonen-MettaÈlaÈ et al., 1996; Wilhelm, 2000), genotype (tree source) clearly in¯uenced embryogenic capacity. The initiation of somatic embryogenesis was also most favoured during a particular developmental stage of the zygotic embryo. In this study, this stage occurred 5±6 WPA, although this may not necessarily be the case under other geographical and local climatic conditions. More reproducible parameters for selecting the appropriate developmental stage of the zygotic embryos are size, fresh weight or morphological development (Trigiano et al., 1999). In elm, embryogenic competence approximately coincided with the appearance of bilateral symmetry. This peaked during the mid-cotyledonary stage when the zygotic embryos had attained 85 % of their ®nal length and 66 % of their ®nal weight, and terminated with the appearance of signi®cant protein reserves. Endemann and Wilhelm (1999) have shown that the embryogenic response of immature zygotic embryos of Quercus robur fell with increasing embryo length. The zygotic embryos studied developed very rapidly, reaching maturity under ®eld conditions within 8±10 weeks of fertilization. This would explain why the `developmental window' during which somatic embryogenesis is higher is limited to 1 or 2 weeks. Histodifferentiation of the ontogenesis of zygotic embryos was observed during the ®rst 5±6 WPA, with subsequent maturation over the remaining weeks involving intense storage of protein and


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F I G . 4. Somatic embryo development from elm embryogenic callus. A, Friable embryogenic callus formed by proembryo cell masses (arrow) and non-embryogenic parenchymatic cells (arrow head) (358). B, Early phases of somatic embryo development from embryogenic masses (arrows) (3288). C, Mitotic divisions in cells of a proembryogenic nodule (3288). D, Section of the hypocotyl±root cortex of a somatic embryo showing embryogenic cell proliferation, rupture of the epidermis and dissociation of the subjacent layers. Note the presence of a young globular somatic embryo (386). E, Cotyledonary-stage embryo showing the root pole, a closed vascular system and a differentiating shoot meristem (386). F, Longitudinal section of a somatic embryo with elongated hypocotyl and small cotyledons. An anomalous shoot meristem structure is apparent (358).

starch deposits. As in other woody species, such as Picea glauca engelmanii (Roberts et al., 1989), Pinus sylvestris (Keinonen-MettaÈlaÈ et al., 1996), Cercis canadensis (Trigiano et al., 1999) and Tilia cordata (KaÈrkoÈnen, 2000), the zygotic embryos were embryogenically competent only prior to maturation; the appearance of signi®cant protein reserves signalled a decline in competence. Furthermore, Finch-Savage and Farrant (1997) found that

reduced moisture content and increased abscisic acid concentration (i.e. the ®nal stage of embryonic development) coincided with the decline of embryogenic response in zygotic embryos of Q. robur. In the present study, somatic embryogenesis was always indirect, via callus formation. According to the description of this process given by Evans et al. (1981), differentiated cells of the zygotic embryo were induced to undergo mitotic

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divisions that resulted in a callus composed partially of `induced embryogenic determined cells'. The induction of somatic embryos directly onto the explanted zygotic embryo was not observed in elm. The somatic embryos that have been obtained during the proliferation of embryogenic cultures in this study differ greatly both within and among cell lines. In particular, well developed somatic embryos similar to zygotic embryos have only rarely been produced. In general, the apical meristem of the somatic embryos obtained is underdeveloped or absent (probably the cause of conversion rates being very low); embryos with more than two cotyledons and fused embryos have also been observed. In a study of somatic embryogenesis by a grapevine rootstock, GoebelTourand et al. (1993) found that epicotyls only developed on embryos with normal cotyledon growth, and not on those with long hypocotyls and vestigial cotyledons. In conclusion, although the induction of somatic embryos on elm tissues has been achieved, further research is still required regarding their maturation, germination and development into plantlets. Failure to control these processes is currently the main obstacle to the use of somatic embryogenesis in elm biotechnology, including clonal propagation and genetic transformation. The integration of these approaches into elm improvement programmes could be highly bene®cial. A C K N O W L ED G EM EN T S We thank Y. Castro and C. G. Alvarez for technical assistance. This research was partially supported by CYCIT and Xunta de Galicia (Spain), through the projects AGF96± 0453 and XUGA 40001B98, respectively. L ITE R A T U R E C IT E D Ben Jouira H, Hassairi A, Bigot C, Dorion N. 1998. Adventitious shoot production from strips of stem in the Dutch elm hybrid `Commelin': plantlet regeneration and neomycin sensitivity. Plant Cell, Tissue Organ Culture 53: 153±160. Bolyard MG, Hajela RK, Sticklen C. 1991. Microprojectile and Agrobacterium-mediated transformation of pioneer elm. Journal Arboriculture 17: 34±37. Brasier CM. 1991. Ophiostoma novo-ulmi sp. novi, causative agent of Dutch elm disease pandemic. Mycopathologia 115: 151±161. Brasier CM. 1994. Missing link in tree disease. Nature 372: 227±228. Corchete MP, Diez JJ, Valle T. 1993. Micropropagation of Ulmus pumilla L. from mature trees. Plant Cell Reports 12: 534±536. Dorion N, Danthu P, Bigot C. 1987. Multiplication veÂgeÂtative in vitro de quelques espeÁces d'ormes. Annals Sciences ForestieÁres 44: 103±118. Dorion N, Ben Jouira H, Danthu P, Bigot C. 1994. Regeneration of plants from protoplasts of Ulmus species (elms). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry, Vol. 29. Plant protoplasts and genetic engineering V. Berlin: Springer-Verlag, 172±189.

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