Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

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1 DOI /s ORIGINAL PAPER Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.) Robert Konieczny Maria Pilarska Monika Tuleja Terezia Salaj Tomasz Ilnicki Received: 28 August 2009 / Accepted: 8 October 2009 Ó Springer Science+Business Media B.V Abstract This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at torpedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylaminopurine (kinetin) or N 6 -[2-isopentenyl]-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytokinin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of cotyledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed morphological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44 68% (somatic embryos) and % (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells. Keywords Auxin Clover Cytokinin Leguminosae Plant regeneration Somatic embryo Abbreviations 2,4-D 2,4-Dihydrophenoxyacetic acid 2iP N 6 -[2-isopentenyl]-adenine CsE Cotyledonary-stage zygotic embryo Kinetin 6-Furfurylaminopurine NAA 1-Naphthaleneacetic acid PGR Plant growth regulator SE Somatic embryogenesis TsE Torpedo-stage zygotic embryo R. Konieczny (&) M. Pilarska M. Tuleja T. Ilnicki Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, Kraków, Poland robert.konieczny@uj.edu.pl; r.konieczny@iphils.uj.edu.pl T. Salaj Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, Nitra, Slovak Republic Introduction Trifolium nigrescens (Viv.) is a self-incompatible diploid (2n =2x =16) native to the Mediterranean, the Middle East and the Caucasus (Gillet 1985). In its countries of origin and in the United States it is used as a forage legume for pasture and soil improvement (Hoveland and Evers

2 1995). The main advantages of this species over many other clovers are high seed production under hard grazing and resistance to southern root knot (Meloidogyne incognita) and clover cyst (Heterodera trifolii) nematodes (Mercer 1988; Pederson and Windham 1989). T. nigrescens is investigated for its potential as germplasm for the improvement of T. repens through interspecific hybridization. So far, hybrids between these two species have been developed to introduce resistance to H. trifolii and some reproductive traits into T. repens as a means of improving seed yield (Hussain et al. 1997; Marshall et al. 2002, 2008). The general requirements of T. nigrescens for growth and differentiation in tissue culture remain largely unknown. Such knowledge, especially the establishment of an efficient protocol for whole-plant regeneration, is needed for it to improve the species by the production of true-to-type somaclonal variants and/or genetic transformants of desired features. Somatic embryogenesis (SE) is usually favored over other methods of plant regeneration in vitro as it allows propagation to be scaled up using bioreactors and cryopreservation of somatic embryos and/ or whole embryogenic cultures, which in turn makes it possible to establish gene banks. Besides its practical value, SE is useful in basic research on totipotency and on the fundamental processes underlying plant morphogenesis. In the genus Trifolium, comprising about 250 species (Gillet 1985), numerous protocols for plant regeneration via SE have been developed. Somatic embryos have been induced from different explants of almost all economically important clovers, including T. repens (Maheswaran and Williams 1984; Parrot 1991), T. pratense (Phillips and Collins 1980; Maheswaran and Williams 1986), T. fragiferum (Rybczyński 1997), T. incarnatum (Pederson 1986), T. vesiculosum (Pederson 1986), T. ambiguum (Pederson 1986), T. rubens (Parrot and Collins 1983), T. medium (Choo 1988), T. respupinatum and T. subterraneum (Maheswaran and Williams 1986). Webb et al. (1987) were the first to report the production of embryo-like structures in callus cultures of T. nigrescens. Later, Konieczny (1995) presented a method for whole plant regeneration via SE from cotyledon- and hypocotyl-derived callus of young T. nigrescens seedlings. In that study, however, the frequencies of SE induction and plant recovery were relatively low. Little attention has been paid to the culture conditions affecting the induction and differentiation of somatic embryos. In this report we describe a protocol whereby T. nigrescens plants can be efficiently and reliably regenerated from immature zygotic embryos via SE. The quantitative data on the effect of different auxins and cytokinins on regeneration are supplemented with karyological analysis of the regenerated plants. Materials and methods Plant material Seeds of Trifolium nigrescens (Viv.) ssp. nigrescens were kindly provided by the Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany. The seeds were sown in plastic pots containing soil, sand and perlite (1:1:1, v/v/v) and incubated in a glasshouse at 20 C under natural light for 8 weeks. After this time the plantlets were transferred to soil in an experimental plot. Pods derived from open-pollinated flowers were surface-sterilized in 70% ethanol (v/v) for 60 s and a 25% (v/v) solution of Domestos Ò commercial bleach (Unilever, Poland) for 10 min, followed by 3 washes with sterile distilled water. Then immature zygotic embryos at the torpedo stage (TsE; about 1 mm in length) and cotyledonary stage (CsE; about 3 mm in length) were excised from pods (4 5 mm and 6 7 mm in length, respectively) and placed on culture media for induction of regeneration. Culture media and culture conditions In preliminary experiments, TsE and CsE of T. nigrescens displayed different nutritional requirements for growth in vitro: TsE failed to survive when maintained on Murashige and Skoog (1962) basal medium (MS) but developed normally when cultured on Maheswaran and Williams (1984) mineral salts and vitamins (EC6), whereas CsE behaved in the opposite way. In view of those results, in all experiments described below we used EC6 salts as the basal medium for culture of TsE, and MS medium for culture of CsE. The media were supplemented with 3% (w/v) sucrose solidified with agar (0.8% w/v, Difco Bacto) and adjusted to ph 5.7 after the addition of plant growth regulators (PGRs). For induction of regeneration, the zygotic embryos were initially cultured in the presence of a cytokinin, either 6-furfurylaminopurine (kinetin) or N 6 -[2-isopentenyl]-adenine (2iP) at concentrations of 0.05, 0.1, 0.5, 1 or 2 mg/l or else an auxin, either 2,4-dihydrophenoxyacetic acid (2.4-D) or 1-naphthaleneacetic acetic (NAA) at 0.5, 1, 2, 4 or 8 mg/l. Next we tested the effect of adding a cytokinin (kinetin or 2iP) at 2 mg/l to media containing 0.5, 4 or 8 mg/l 2.4-D or NAA. A total 50 zygotic embryos, 10 per Petri dish ( mm, containing about 20 ml medium) for each growth regulator combination, were explanted. For plant regeneration, somatic embryos reaching torpedo stage as well as first shoots with unifoliate leaves of developing somatic embryos were harvested from the original explants and placed on growth regulator-free MS medium (as above) in 200 ml Erlenmeyer flasks; 25 somatic embryos and 25 shoots (5 per flask) from each culture

3 were transferred. Regenerated plantlets with well developed roots were transplanted to pots with a mixture of soil, sand and perlite (1:2:1, v/v/v) and grown to maturity. All cultures were maintained at 25 ± 2 C under diffuse cool white fluorescent light (80 lmol m -2 s -1 ) with a 16 h photoperiod. Culture evaluation After 4 weeks of culture on induction medium the frequency (%) of somatic embryogenesis (number of explants producing somatic embryos/total number of explanted zygotic embryos 9 100) and the mean number of somatic embryos produced per explant (total number of embryoids/ number of explants showing somatic embryogenesis) were assessed. The frequency (%) of plant recovery was estimated by calculating the number of flowering plants/ number of somatic embryos subcultured on MS medium Qualitative data of somatic embryogenesis were analyzed by two-way ANOVA followed by Duncan s multiple range test (P B 0.05) using Statistica for Windows ver.8.0 (StatSoft, Inc. Tulsa, Oklahoma). Karyology Chromosome counts were made in seed-germinated plants and in plantlets regenerated in vitro 4 weeks after transplanting to pots. Twenty-five metaphase plates from 10 root tips of 3 different plants regenerated from media containing 0.5 mg/l NAA? 2 mg/l 2iP and 4 mg/l 2,4- D? 2 mg/l 2iP were studied. Procedures for fixation, chromosome staining and metaphase plate analysis were as described earlier (Konieczny et al. 2003). Results Culture of torpedo-stage embryos On media containing 0.5 and 1 mg/l 2,4-D the explants produced small amounts of non-regenerative callus; when cultured in the presence of 2,4-D at 2, 4 or 8 mg/l they formed somatic embryos from cotyledons and hypocotyls (Fig. 1a c). SE was direct and occurred just below the cotyledonary nodes on the swelled region of hypocotyl and at the margin of cotyledons (Fig. 1d, e). The SE percentages were similar on media containing 2 and 4 mg/l 2,4-D (58 and 64%, respectively), and about double the percentage for culture with 8 mg/l 2,4-D (24%). The 2,4-D concentration had no effect on the number of embryoids produced per explant (Table 1). After 14 days of culture on media with 4 and 8 mg/l 2,4-D and after 18 days on medium with 2 mg/l of 2,4-D, the original explants became necrotic and produced a few whitish somatic embryos which soon turned brown and died. Necrosis began from the embryo axis of TsE and expanded to the whole explant within 7 10 days. Occasionally the cotyledons of TsE maintained in the presence of 2 mg/l 2,4-D remained green and retained low embryogenic potential (Fig. 1f). Plant regeneration was achieved from green torpedo-shaped somatic embryos produced before the occurrence of necrosis. When transferred to fresh MS without PGRs, somatic embryos induced Fig. 1 Plant regeneration via somatic embryogenesis in in vitro culture of TsE of T. nigrescens. a Torpedo-stage zygotic embryo before culture. b Production of non-regenerative callus on medium containing 0.5 mg/l 2,4-D. c Induction of SE on medium with 4 mg/l 2,4-D. Arrowheads point to somatic embryos. d, e High magnification of (c) showing direct somatic embryo production from regular row on hypocotyl (d) and at the margin of cotyledon (e). f Necrosis of original embryo with concomitant production of whitish somatic embryos after 18 days on medium with 2 mg/l 2,4-D. g Isolated somatic embryo from medium with 4 mg/l 2,4-D just after subculture on PGR-free MS medium. h Plantlet originating from somatic embryo after 3 weeks on PGR-free MS medium. Bar represents 0.5 mm

4 Table 1 Effects of 2,4-D on SE and plant regeneration from torpedostage zygotic embryos of T. nigrescens 2,4-D (mg/l) SE (%) Embryoid/explant Recovery (%) 2 58a 10.1a 24a 4 64a 12.9a 28a 8 24b 12.8a 0 Values with the same letter within a column do not significantly differ by Duncan s multiple range test (P B 0.05) by 8 mg/l 2,4-D turned brown and died, while those obtained on media with 2 and 4 mg/l 2,4-D converted to plantlets at very low frequency: 24 and 28%, respectively (Fig. 1h, i; Table 1). The regenerated plants showed typical morphology and grew to maturity in soil. A cytokinin (kinetin or 2iP at mg/l) and NAA (0.5 8 mg/l) alone did not induce somatic embryos from TsE but produced fast-growing and non-morphogenic callus. Similarly, no embryoids were induced when 0.5, 4 or 8 mg/l 2,4-D or NAA were combined with 2 mg/l 2iP or kinetin. Culture of cotyledonary-stage embryos When added alone to the medium, neither the auxins (0.5 8 mg/l NAA or 2,4-D) nor the cytokinins (kinetin or 2iP at mg/l) induced somatic embryogenesis from CsE. The explants developed into seedlings (kinetin or 2iP at 0.05 and 0.1 mg/l) or showed inhibited growth followed by production of non-regenerative (0.5 8 mg/l 2,4-D; cytokinins at mg/l) or rhizogenic (NAA at mg/l) callus (Fig. 2a d). Application of 2 mg/l 2iP or kinetin along with 8 mg/l 2,4-D or NAA was ineffective for SE and promoted callus growth but it stimulated somatic embryo induction from CsE when combined with 0.5 and 4 mg/l 2,4-D or NAA (Table 2). In all successful cultures SE was always associated with inhibition of shoot and root growth of the original embryo and considerable thickening of its hypocotyl. The concentration but not the type of auxin had an obvious effect on the timing of SE induction: at 0.5 mg/l NAA or 2,4-D with 2 mg/l kinetin or 2iP the first somatic embryos appeared after 7 days of culture; the process took 4 days longer on media with kinetin or 2iP together with 4 mg/l auxin. Initially the embryoids arose directly from the upper part of the hypocotyl (Fig. 2e, f); within 3 4 days after their appearance, CsE started to produce fast-growing embryogenic callus which developed from the embryo axis (NAA-supplemented media) or from whole embryos (media with 2,4-D) (Fig. 2g, h). The rate of callus growth did not depend on the type or amount of auxin used, but callus was more abundant with kinetin than with 2iP. After days of culture almost all the CsEs maintained on media containing 2,4-D with kinetin or 2iP were covered by embryogenic callus (Fig. 2i). When combined with a cytokinin (2iP or kinetin), 2,4-D gave a higher SE percentage than NAA (Table 2). Applying kinetin instead of 2iP decreased the frequency of SE on media containing 2,4-D (28 52% in the presence of kinetin vs % on media with 2iP), but did not affect the number of somatic embryos produced by a single explant. The dependence was similar when different cytokinins were used along with NAA (Table 2). When maintained continuously on media containing 2,4-D together with kinetin or 2iP, somatic embryos did not develop past the torpedo stage. Most of them displayed severe aberrations in cotyledon differentiation, and were tube- or trumpetshaped (Fig. 2i). When harvested from the original explants, somatic embryos converted to plantlets on MS without PGRs, at similar frequencies of about 25% for the different 2,4-D/cytokinin treatments (Table 2). Unlike embryoids obtained on media with 2,4-D and a cytokinin, those induced in the presence of NAA together with kinetin or 2iP generally showed a normal morphology (Fig. 2j) and often germinated while still on the callus. After 21 days of culture the calluses obtained on media containing NAA were covered with numerous shoots; on media with high NAA content (4 mg/l) they were unifoliate and displayed various symptoms of degeneration (vitrification and/or senescence), but in culture with 0.5 mg/l NAA they were normal and vigorous with unifoliate or trifoliate leaves (Fig. 2k, l). When harvested from the original explants and transferred to MS free from PGRs, most of the shoots rooted well and regenerated plants. The frequency of plant regeneration was comparable for shoots obtained on media containing 0.5 or 4 mg/l NAA with 2 mg/l kinetin and 4 mg/l NAA with 2 mg/l 2iP (100%) and significantly higher than for shoots obtained in the presence of 0.5 mg/l NAA together with 2 mg/l 2iP (72%). Additionally, from 44 to 68% of the torpedo-stage somatic embryos induced on NAA-enriched media developed into plantlets in a way similar to zygotic embryogeny when subcultured on MS (Fig. 2m o, Table 2). The regenerants appeared identical to each other and to control plants obtained from seeds; they produced flowers and grew in soil to maturity (Fig. 2p, q). Plants regenerated from embryoids induced on media containing 4 mg/l 2,4-D? 2 mg/l 2iP and 0.5 mg/l NAA? 2 mg/l 2iP showed the chromosome complement typical for the species, 2n = 16, in root tip cells (Fig. 2r). Discussion In this report we described a protocol for plant regeneration of T. nigrescens via somatic embryogenesis (SE). The explants used in the experiments were torpedo- (TsE) and

5 Fig. 2 In vitro culture and plant regeneration via somatic embryogenesis from CsE of T. nigrescens. a Cotyledonary-stage zygotic embryo before culture. b Normal development of CsE on medium with 0.1 mg/l 2iP. c, d Production of non-regenerative (c) and rhizogenic callus (d) on media with 2 mg/l kinetin and 4 mg/l NAA, respectively. e Induction of somatic embryos in a regular row on hypocotyl on medium with 4 mg/l 2,4-D and 2 mg/l 2iP. f High magnification of (e) showing numerous globular and oblong embryoids. g Production of embryogenic callus by axis of CsE; cotyledons are non-responsive on medium with 0.5 mg/l NAA and 2 mg/l 2iP. h Highly embryogenic callus derived from cotyledons on medium with 4 mg/l 2,4-D and 2 mg/l 2iP. i Callus culture after 21 days of explanting on medium with 0.5 mg/l 2,4-D and 2 mg/l kinetin. Arrowheads point to somatic embryos of abnormal morphology. j Group of bipolar somatic embryos induced on medium with 4 mg/l NAA and 2 mg/l 2iP. Arrowheads point to well-differentiated cotyledons. k, l Callus cultures after 21 days of culture: vitrified shoots with unifoliate leaves after 21 days of culture on medium with 4 mg/l NAA and 2 mg/l 2iP (k) and shoots of normal morphology with unifoliate and trifoliate leaves on medium with 0.5 mg/l NAA and 2 mg/l 2iP (l). m, n, o, p Development of somatic embryo induced on medium with 0.5 mg/l NAA and 2 mg/l 2iP after subculture on PGR-free MS. q T. nigrescens plants regenerated in vitro growing in soil. r Root tip squash of regenerant induced on medium with 0.5 mg/l NAA and 2 mg/l 2iP with 16 chromosomes visible. Bar represents: 1 mm (a o), 5 lm (r) cotyledonary-stage (CsE) zygotic embryos, which, among the different plant tissues, have been identified as the most suitable sources of embryogenic cells (Gaj 2004). In the genus Trifolium, the use of TsE and TsE-derived explants for SE induction has been described in culture of T. repens, T. pretense, T. resupinatum and T. subeterraneum

6 Table 2 Effects of 2iP or kinetin (2 mg/l) combined with different concentrations of NAA or 2,4-D on SE and plant regeneration from cotyledonarystage zygotic embryos of T. nigrescens Values with the same letter within a column do not significantly differ by Duncan s multiple range test (P B 0.05) Auxin (mg/l) 2iP Kinetin SE (%) Embryoid/ explant Recovery (%) SE (%) Embryoid/ explant 2,4-D a 10.4a 24c 28b 9.8a 24c 4 100a 11.9a 28bc 52a 11.5a 24c NAA b 8.6a 68a 17b 7.7a 64a 4 30c 10.9a 44b 24b 7.3a 48b Recovery (%) (Maheswaran and Williams 1984, 1986; Parrot 1991). To our knowledge this is the first report on somatic embryo production from CsE in clover. Different legumes respond differently to exogenous auxins and cytokinins. For example, a cytokinin alone has been found to be highly effective for induction of SE from torpedo-stage embryos of T. repens and T. subterraneum (Maheswaran and Williams 1986) and regeneration of shoots from cotyledonary embryos of T. michaelianum (Konieczny 1996). In our study, both TsE and CsE of T. nigrescens were non-regenerative on media containing a cytokinin (kinetin or 2iP) but without an auxin. The need for an exogenous auxin and not a cytokinin for induction of SE from immature embryos has been reported in many legumes including Cercis canadensis (Trigiano et al. 1988), T. repens (Parrot 1991) and Dalbergia laitifolia (Rao and Lakshmisita 1996). We found the same in culture of TsE of T. nigrescens. As in the work of Ozias-Akins (1989) on immature embryos of Arachis hypogea, these explants differed in their requirements for auxin type, being non-embryogenic on media supplemented and with NAA and embryogenic with 2,4-D. The cultural requirements for SE differed significantly between TsE and CsE of T. nigrescens. The addition of a cytokinin combined with 2,4-D or NAA was needed to elicit the embryogenic response of CsE, whereas both kinetin and 2iP completely inhibited SE from TsE when combined with 2,4-D. The literature data on the interaction between auxins and cytokinins during SE induction are not uniform. Repression of auxin-dependent SE by a cytokinin is not an unexpected outcome in legumes, and has been reported in direct regeneration from immature embryos of Coronilla varia (Arcioni and Mariotti 1982), Glycine max (Lazzerii et al. 1987) and Pisum sativum (Kysely and Jacobsen 1990). In many other studies, however, it was necessary to apply a cytokinin along with the auxin for induction of somatic embryos and/or to enhance the yield of regeneration (e.g., Nagarajan et al. 1986; Trinh et al. 1998; Nanda and Rout 2003). For expression of embryogenic competence, the plant growth regulators supplied to the culture medium and the endogenous hormones in the tissue of the primary explant must be in a proper balance (Jimenez 2005). The available data indicate that the concentration of endogenous cytokinins in the zygotic embryo is species-specific and that it changes during seed development (Van Staden 1983). An excess of a cytokinin is known to inhibit SE induction, as has been shown in legumes by Murthy et al. (1995) and more recently by Pintos et al. (2002). In our study of T. nigrescens the differential response of TsE and CsE to exogenously applied cytokinins might be directly related to differences in the endogenous level of these hormones in the explants. More detailed studies on the hormonal status of zygotic and somatic embryos of T. nigrescens are needed to verify this suggestion. In assessing the effect of exogenous growth regulators on SE induction, the regulatory role of auxins and cytokinins on the expression of somatic embryogenesis receptor kinases (SERKs) also needs to be taken into account (Nolan et al. 2003). These enzymes are differentially expressed during zygotic embryo development, and this is believed to be directly related to the SE competence of particular embryo cells (Hecht et al. 2001). When combined with a cytokinin (2iP or kinetin), 2,4-D gave a higher SE frequency than NAA, but the auxin type had no effect on the mean number of somatic embryos produced per single CsE. T. nigrescens is a highly heterozygous cross-pollinated species (Gillet 1985), and therefore each immature embryo represents in fact a different genotype. Our results suggest that 2,4-D-induced SE might not be as sensitive to genotype specificity as NAAinduced SE, and/or that the cells conferring embryogenic competence in the 2,4-D treatments were more common among the explanted CsE than those responding to NAA. A similar dependence between auxin type and qualitative SE data has been observed in cultures of seedling-derived hypocotyls and cotyledons of T. nigrescens (Konieczny 1995). In that study, however, the frequency of SE was significantly lower than obtained here. Though the SE frequency from CsE on media containing 2,4-D was high a large number of those embryoids were malformed, whereas generally the somatic embryos obtained on media with NAA resembled zygotic embryos. The teratogenic effect of

7 2,4-D on somatic embryo development is well documented (Gaj 2004). In legumes, abnormal embryoids showed poor further development into plants (e.g., Lazzerii et al. 1987; Shoemaker et al. 1991; Özcan et al. 1993). Likewise in our study, development similar to zygotic embryogeny and conversion to plantlets were about 3 times lower in somatic embryos produced in the presence of 2,4-D than in those obtained on media with NAA. Unlike with 2,4-D, the use of NAA did not prevent the development of somatic embryos on induction media, and after 21 days of culture there were shoots/leaves on the surface of CsE-derived callus. Most of these leaves were unifoliate, indicating the development of somatic embryos via typical precocious germination (Maheswaran and Williams 1984). However, on medium containing 0.5 mg/l NAA and 2 mg/l cytokinin, trifoliate leaves were also observed. The formation of trifoliate leaves is a typical phenomenon following the production of the initial unifoliate leaf during germination of zygotic embryo of clovers, but it can also be a sign of de novo organogenesis (Choo 1988; Konieczny 1995). In P. sativum, Griga (2002) reported that SE may convert to organogenesis when the auxin signal is insufficient. Using medium of the same content of PGRs as in our work (0.5 mg/l NAA and 2 mg/l 2iP), shoot, leaf and somatic embryo regeneration was induced concomitantly from callus of T. nigrescens (Webb et al. 1987; Konieczny 1995). Thus, it cannot be ruled out that prolonged exposure of CsE to a high concentration of a cytokinin (2 mg/l 2iP or kinetin) and a low concentration of a relatively weak auxin (0.5 mg/l NAA) could favor induction of de novo organogenesis from callus along with ongoing SE. Detailed histological/anatomical work is needed to elucidate the developmental processes involved during continuous culture of CsE on media containing NAA and a cytokinin. The onset of regeneration from zygotic embryos of T. nigrescens was rapid, suggesting that the explants might already be SE-competent before explanting. Initially all the embryoids were formed directly, but with continuous culture we observed the formation of embryogenic callus from CsE maintained in the presence of auxin and 2iP or kinetin. When zygotic embryos are used as explants, the entire embryo may not be embryogenic, as in the case of T. repens and T. subterraneum (Maheswaran and Williams 1986) or P. sativum (Kysely and Jacobsen 1990), where the axis was found to be responsive but not the cotyledons. In our cultures we also noted a specific pattern of embryoid formation. The arrangement of somatic embryos in a narrow strip of tissue on the hypocotyl just below the cotyledonary nodes is reminiscent of that reported previously in Helianthus annuus (Bronner et al. 1994). This region of the explant was the site of direct shoot regeneration in the legume T. michaelianum (Konieczny 1996). In T. nigrescens, however, there were also somatic embryos at the margin of TsE cotyledons, whereas the cotyledons of more mature explants (i.e., CsE) remained non-regenerative throughout culture. This observation seems to confirm the general suggestion that the propensity of particular cells of the zygotic embryo to follow SE diminishes with embryo maturation (Gaj 2004). Our aim in this study was to determine the conditions for whole plant regeneration via somatic embryogenesis from immature zygotic embryos of T. nigrescens. The easily managed protocol we describe here seems suitable for propagation of this plant. Its main advantages over previously published methods (Konieczny 1995) are rapid onset of regeneration as well as a much higher frequency of SE induction and plant recovery. Additionally, the spatial limitation of direct SE to well-defined regions of the explanted embryos makes this system useful for some transformation techniques such as particle bombardment. Our results also point up some theoretical aspects of SE which need to be explored with the use of cytological and biochemical approaches. These involve the specific action of NAA and 2,4-D on somatic embryo formation and the role of the cytokinin and its interaction with an auxin during SE induction. Acknowledgments We thank the Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany for providing T. nigrescens seeds for this study. References Arcioni S, Mariotti D (1982) Tissue culture and plant regeneration in the forage legumes Onobrychis vicaefolia Scop., Coronilla varia and Lotus corniculatus L. 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