Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

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1 DOI /s ORIGINAL PAPER Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants L. Sáenz A. Azpeitia C. Oropeza L. H. Jones K. Fuchsova L. Spichal M. Strnad Received: 25 March 2010 / Revised: 13 July 2010 / Accepted: 22 July 2010 Ó Springer-Verlag 2010 Abstract Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was Communicated by M. Jordan. L. Sáenz (&) C. Oropeza Centro de Investigación Científica de Yucatán, A.C., Calle 43 No. 130, Col. Chuburna de Hidalgo, C.P., Mérida, Yucatán, Mexico vyca@cicy.mx A. Azpeitia Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuaria, Campo Experimental Huimanguillo Km 1 Carr., Apdo. Postal No. 17, C.P Huimanguillo, Tabasco, Mexico L. H. Jones 17 Marriotts Close, Felmersham, Bedford MK43 7HD, UK K. Fuchsova L. Spichal M. Strnad Laboratory of Growth Regulators, Institute of Experimental Botany ASCR and Palacký University, Slechtitelu 11, Olomouc, Czech Republic observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents. Keywords Coconut palm Somatic embryogenesis Endogenous cytokinins Abbreviations 2,4-D 2,4-Dichlorophenoxiacetic acid BAP 6-Benzylaminopurine BAP9G 6-Benzylaminopurine-9-glucoside BAPR 6-Benzylaminopurine riboside BAPR5 0 P 6-Benzylaminopurine ribotide DHZ Dihydrozeatin DHZR Dihydrozeatin riboside DHZ9G Dihydrozeatin-9-glucoside DHZR5 0 P Dihydrozeatin ribotide ip Isopentenyladenine ipr Isopentenyladenine riboside ip9g Isopentenyladenine-9-glucoside ipr5 0 P Isopentenyladenine ribotide mt Meta-Topolin 6-(3- hydroxybenzylamino)purine mt9g Meta-Topolin-9-glucoside mtr Meta-Topolin-riboside ot Ortho-Topolin 6-(2- hydroxybenzylamino)purine ot9g Ortho-Topolin 9-glucoside

2 otr Z ZR Z9G ZR5 0 P ZOG ZROG FW SE Introduction Ortho-Topolin riboside Zeatin Zeatin riboside Zeatin-9-glucoside Zeatin ribotide Zeatin-O-glucoside Zeatin riboside-o-glucoside Fresh weight Somatic embryogenesis Cytokinins are purine derivatives with phytohormone activity that can influence several plant processes such as growth of lateral buds, leaf expansion and leaf senescence (see Davies 1995). They can also influence (in combination with auxins) cell division and morphogenesis in in vitro plant tissue cultures (see Krikorian 1995). For instance, small amounts of exogenously provided cytokinin inhibited embryogenesis in Dactylis glomerata explants of embryogenic genotypes (Wenck et al. 1988). Further, the addition of anticytokinins promoted somatic embryo formation in low embryogenic genotypes (Somleva et al. 1995). When the endogenous cytokinin contents of different genotypes of D. glomerata were analysed, it was found that they were inversely related to their somatic embryogenic potential (Wenck et al. 1988). On the other hand, exogenous cytokinins can favour embryogenesis in different species (see Gaj 2004). With respect to endogenous cytokinin Centeno et al. (1997) found that embryogenic potential was directly related to the amount of Z-type and inversely related to the ip-type in Corylus avellana. In the case of coconut previous data shows a correlation between the endogenous content of zeatin and isopentenyladenine and the formation of somatic embryos from foliar explants cultured in vitro (Verdeil and Hocher 1997). In plumular explants the synthetic auxin 2,4-D induces in vitro somatic embryogenesis through callus formation (Chan et al. 1998). In this system, embryogenic calli and non-embryogenic calli can be formed from the initial calli (Chan et al. 1998). Whether the difference in embryogenic capacity between embryogenic callus and non-embryogenic callus is correlated to differences in endogenous cytokinins is not known. This paper reports on the relationship of endogenous cytokinins in initial calli, nonembryogenic calli and embryogenic calli obtained from coconut plumular explants and the effect of exogenous cytokinins and anticytokinin (8-azaadenine), (see George 1993), on the morphogenetic responses of plumule explants cultured in vitro. Materials and methods Plant material Zygotic embryos of mature coconut fruit (12 14 months after pollination) of 15-year-old Malayan dwarf palms were collected. The plumules were extracted and cultivated as in Chan et al. (1998). Three different types of in vitro cultured tissues derived from plumule explants were analysed: initial callus formed after 1 month of culture; embryogenic callus formed after 3 months of culture that shows globular structures (embryogenic structures); and non-embryogenic callus formed after 3 months of culture which lacks embryogenic structures. These cultures were weighed, deep frozen in liquid nitrogen and stored in a freezer at -70 C and, subsequently lyophilised. Culture media and conditions Media preparation and culture conditions were done according to Chan et al. (1998). Media I and II, each prepared using Y3 medium (Eeuwens 1976) were supplemented with 3 g l -1 Gelrite and 2.5 g l -1 charcoal (acidwashed, plant cell culture tested). Medium I contained 0.55 mm 2,4-D, while Medium II contained 6 lm 2,4-D and 300 lm BAP. All chemicals were reagent grade (Sigma, St. Louis MO, USA). Medium ph was adjusted to 5.75 before autoclaving for 20 min at 120 C. Each explant was cultured in 35 ml vessels containing 10 ml of medium I and was incubated in the dark for 3 months at 27 ± 2 C without subculturing and then transferred to medium II under illumination (45 60 lmol m -2 s -1 PPFD) at 27 ± 2 C, subculturing every 2 months. Experiments with exogenous cytokinins and 8-azaadenine Filter sterilized zeatin, isopentenyladenine, 6-benzylaminopurine (Sigma, St. Louis MO, USA) or 8-azaadenine (Fluka, Buchs, Switzerland)) were added to an autoclaved medium prior to solidification to give finals concentrations of 1, 5, 25, 100 lm for the cytokinins or 0, 4.5, 9 and 18 lm for the 8-azaadenine. These compounds were maintained in medium I throughout the in vitro culture period. None of these compounds were added to medium II. Chemicals and reagents Cytokinins ip, ipr, Z, ZR, DHZ, DHZR, BAP, BAPR were purchased from Sigma. ot, otr, ot9g, mt, mtr and mt9g were synthesized as described by Holub et al. (1998). Before use all cytokinins were purified by HPLC as described by Strnad (1996). Bovine serum albumin,

3 ovalbumin and 4-nitrophenylphosphate were purchased from Fluka; DEAE cellulose and acid phosphatase from Sigma, Sep-Pak C18 cartridges from Waters Assoc., and methanol and acetonitrile for chromatography from Merck (Germany). Tritium-labelled cytokinins, immunogens and alkaline phosphatase tracer syntheses as well as immunization schedule and isolation of immunoglobulins were performed as described by Strnad (1996). Cytokinin analyses Cytokinins from each plant part studied were extracted and purified by the methods of Kraigher et al. (1991) and Faiss et al. (1997). The extracts were purified using combined DEAE Sephadex-Sep Pack C18 columns. Cytokinin bases, ribosides and glucosides retained on the reverse-phase cartridge were eluted in 5 ml 80% methanol (v/v) and after drying fraction B (basic cytokinins) was obtained. After washing with 10 ml distilled water the DEAE Sephadex column was coupled to another Sep Pack and cytokinin nucleotides were eluted with 10 ml 6 M HCOOH. The nucleotides retained on the C18 cartridge were eluted in 5 ml 80% methanol, dried and dephosphorylated (fraction NT). The conversion of cytokinin nucleotides to their dephosphorylated forms was carried out using acid phosphatase for 30 min in the dark (25 C, 0.05 U ml -1, Sigma P-3627, EC ) in 40 mm ammonium acetate buffer (ph 6.5), as described by MacDonald et al. (1981). Both fractions were immunopurified on a cytokinin monoclonal antibody column (Faiss et al. 1997). Cytokinin-O-glucosides (fraction OG) occurring in PBS eluates from the immunoaffinity columns were treated with b-glucosidase (Sigma, St. Louis MO, USA) and repurified on the same monoclonal column. All three immunopurified fractions were separated by reversed-phase HPLC and assayed by ELISA following the methods described in Jones et al. (1995). The aromatic cytokinins were analysed by the method of Strnad (1996). The following modifications of the methods have been used for cytokinin analyses: (1) HPLC Alliance 2690 Separations Module (Waters, Milford, MA, USA) separation was realised on Microsorb C1 column (Rainin, mm, 3 lm particle size), (2) the HPLC elution was performed with a methanolic gradient in (A): 10% methanol in 40 mm acetic acid (AcA) adjusted to ph 3.4 with triethylamine, and (B): 80% methanol in 40 mm AcA. The following gradient sequences were used: 0 min, 90% A? 10% B, 10 min, 60% A? 40% B, 14 min, 65% A? 35% B, 18 min, 50% A? 50% B, 24 min, 50% A? 50% B, 26 min, 100% B, 30 min, 100% B, 31 min, 90% A? 10% B, the flow rate was 0.6 ml min -1 ; (3) aliquots of 50 ll were used in different group specific ELISA; (4) the resulting immunohistograms were quantified on the basis of cross-reactivity and losses of tritiated cytokinin recovery markers; (5) the endogenous content was also calculated from the integration of HPLC peaks when the cytokinin levels were higher than 5 pmol g -1 FW. Recoveries were usually greater than 60%. Total cytokinin content was calculated as the sum of the individual cytokinins analysed. The pool of active cytokinins was obtained by the sum of the ribosides, free bases, nucleotides and O-glucosides. Identification of cytokinins Cytokinin identity was confirmed by photodiode-array 996 (Waters, Milford, MA, USA) HPLC detection and by gas chromatography mass spectrometry (Jones et al. 1996). HPLC fractions containing appropriate cytokinin were evaporated in l ml hypovials (Pierce, Chester, UK) and permethylated using methyl iodide in dimethylsulphonyl carbanion. The samples were dried in a stream of N 2. The permethylated cytokinins were dissolved in 10 ll of methanol and 3 ll injected onto a 30 m mm PTE-5, (Supelco, INC, Bellefonte, PA, USA) 0.25 lm i.d. capillary column: He carrier gas, flow rate 2 ml min -1, 2 min at 40 C, from 40 C at10 C min -1 to 300 and 300 C for 10 min. The mass spectrometer was an electron impact HP 6890/HP5073 operating at electron energy of 70 ev with an autosampler HP 7673 (Hewlett Packard) in MSD regime: TIC, amu, and 2.91 scan s -1. Statistics The data presented are means values ± the standard deviation (SD) of two independent batches from in vitro tissue culture for the analysis of endogenous cytokinins. It was subjected to analysis of variance (ANOVA). Significant differences were determined by the Newman Keuls s test at p = For the study of the response of plumules explants cultured in vitro, a completely randomised block design with three or four replication depending of experiment as indicated in the text. Data were subjected to analysis of variance (ANOVA) followed by means separation using the Fischer s least significant difference (LSD) test at p = Results Morphogenic development The initial explants were plumule explants from coconut zygotic embryos as described by Chan et al. (1998) (Fig. 1a). After 30 days of culture initial callus was formed. This callus was beige in colour and 2 3 mm in diameter (Fig. 1b). At 90 days, the callus measured

4 Fig. 1 Embryogenic callus formation during in vitro culture: initial explant (a). Initial callus formed at 30 days (b) and embryogenic callus at 90 days of culture (c). Nonembryogenic callus at 90 days (d). ESt embryogenic structures, p plumule, ze zygotic embryo % formation of embryogenic callus Concentration of exogenous cytokinins (µm) 6 9 mm diameter and showed clear embryogenic structures (Fig. 1c). During the development of explants, calli that did not show embryogenic structures or somatic embryo formation were referred to as non-embryogenic calli (Fig. 1d). Effect of exogenous cytokinins a b bcd cd BAP Z ip The capacity of plumule explants to form initial calli and embryogenic calli was tested in media containing different concentrations (0, 1, 5, 25, 100 lm) of the cytokinins BAP, ip or Z. The percentage of explants forming initial calli (nearly 100%) was not affected by the addition of any of these cytokinins within the concentration range tested (data not shown). On the other hand, the percentage of explants forming embryogenic calli with these cytokinins was reduced and the extent of this reduction increased with increasing concentrations of cytokinin (Fig. 2). At 1 lm embryogenic calli formation was reduced from 60 to 39, 32 and 28% with BAP, ip and Z, respectively. With the highest concentration of cytokinin tested, 100 lm, embryogenic calli formation was further reduced to 17, 16 and 8% with BAP, ip and Z, respectively. bc de ef dede ef ef fg g Fig. 2 Effect of different exogenously added cytokinins (6-benzylaminopurine, zeatin and isopentenyladenine) on the formation of embryogenic callus. Each replicate consisted of 20 individual plumule explants (n = 3). Different letters represents significant differences at p [ 0.05 Fig. 3 Effect of different concentrations of 8-azaadenine on the formation of callus and embryogenic callus in explants of plumule cultured in vitro either with or without 1 lm of BAP. Each replicate consisted in 10 individual plumule explants (n = 4). Different letters represent significant differences at p [ IC initial callus, EC embryogenic callus Effect of 8-azaadenine application The results shown above suggested that there was an inverse correlation between cytokinin levels and the formation of embryogenic calli. In order to further validate this negative effect on the somatic embryogenesis process, the effect of the addition of 8-azaadenine in plumule explants cultured in vitro was tested. The results showed that the addition of 9 lm 8-azaadenine produced a slight but significant increase in the formation of initial callus, from 90 to 100% and in the formation of embryogenic calli from 52 to 65% (Fig. 3). Moreover, the addition of 8-azaadenine at 18 lm could restore the percentage of formation of embryogenic calli in plumule explants cultured in vitro with 1 lm BAP with values similar to control (without 8-azaadenine and BAP). The formation of somatic embryos was further evaluated in medium II as outlined in Materials and methods. It was observed that the explants cultured with 4.5 lm of 8-azaadenine in medium I showed 5.14 somatic embryos/embryogenic calli at 30 days and 8.80 at 60 days and were significantly higher than all the other treatments tested including the control forming 3 and 5.7 somatic embryos/embryogenic calli at 30 and 60 days, respectively (Fig. 4). The effect of 8-azaadenine on

5 Fig. 4 Effect of different concentrations of 8-azaadenine on the formation of somatic embryos from embryogenic callus in explants of plumule cultured in vitro. Each replicate consists in 10 individual plumule explants (n = 4). Different letters represent significant differences at p [ SE somatic embryos embryo formation was almost twice that of the control. However, higher concentrations (18 lm) of this compound lowered the embryogenic response showing that an optimal level of cytokinin is necessary to increase the formation of somatic embryos. Endogenous isoprenoid cytokinins The concentrations of naturally occurring cytokinins were measured in the three types of calli that form during the induction of in vitro coconut somatic embryogenesis from plumule explants: initial calli, embryogenic calli and nonembryogenic calli. The highest concentration of total isoprenoid cytokinins was found in non-embryogenic calli with 222 pmol g -1 FW and in initial calli with 189 pmol g -1 FW; and the lowest in embryogenic calli with, 105 pmol g -1 FW (Fig. 5); and the difference was significant. When the Fig. 5 Comparison of endogenous concentrations of different types of cytokinins in different stages of plumule explants cultured in vitro. Figures presented are means ± SD (n = 2). Different letters represent significant differences at p [ 0.05 contents of the different types of isoprenoid cytokinins were calculated, the ip-type were the ones with the largest concentrations, followed by the Z-type and then by the DHZtype, in each of the three types of callus analysed. For each type of callus differences were observed in the contents of each type of isoprenoid cytokinin. In the case of the ip-type the highest concentrations were found in non-embryogenic calli with pmol g -1 FW and in initial calli with pmol g -1 FW; and the lowest in embryogenic calli with pmol g -1 FW; and the difference was significant (Fig. 5). Similarly for the Z-type, the highest concentrations were found in non-embryogenic calli with pmol g -1 FW and in initial calli with pmol g -1 FW; the lowest in embryogenic calli with 6.68 pmol g -1 FW; and the difference was significant (Fig. 5). In the case of the DHZ-type, the highest concentration was found in initial calli with 5.3 pmol g -1 FW; the lowest in embryogenic calli with 1.67 pmol g -1 FW; and an intermediate value in nonembryogenic calli with 3.51 pmol g -1 FW (Table 1); and the differences were significant. With respect to individual isoprenoid cytokinins, all 14 analysed were detected in each of the three types of calli studied. The concentration of each of the isoprenoid cytokinins was always significantly lower in embryogenic Table 1 Endogenous concentrations of individuals cytokinins (pmol g -1 FW) in explants of plumule cultured in vitro of Cocos nucifera L. Cytokinins Initial callus Embryogenic callus Non-embryogenic callus Z9G ± 2.93 b 2.74 ± 0.49 c ± 2.54 a Z 3.66 ± 0.22 a 1.20 ± 0.25 c 2.16 ± b ZR 7.93 ± 0.46 a 1.34 ± 0.17 b ± 3.82 a ZR5 0 P 1.63 ± 0.12 ab 0.16 ± 0.09 b 1.76 ± 0.74 a ZOG 0.27 ± a 0.07 ± 0.01 a 0.23 ± 0.05 a ZROG 2.84 ± 0.96 a 1.16 ± 1.0 a 1.92 ± 0.57 a IP9G ± 16 a ± 8 b 167 ± 1.06 a IP 0.58 ± 0.19 a 0.33 ± 0.08 a 0.43 ± 0.46 a IPR 4.41 ± 0.15 a 1.18 ± 0.20 b 3.35 ± 0.63 a IPR5 0 P 0.34 ± 0.09 a 0.16 ± 0.15 a 0.42 ± 0.23 a DHZ9G 1.17 ± 0.25 a 0.29 ± 0.05 b 0.71 ± 0.22 ab DHZ 1.17 ± 0.25 a 0.29 ± 0.05 b 0.71 ± 0.22 ab DHZR 2.51 ± 0.20 a 0.48 ± 0.1 c 1.56 ± 0.41 b DHZR5 0 P 0.44 ± 0.14 a 0.6 ± 0.03 a 0.52 ± 0.13 a BAP9G 4.13 ± 0.22 b 3.91 ± 0.86 b 6.20 ± 0.18 a BAP 2.01 ± 1.01 a 0.25 ± 0.05 a 0.32 ± 0.13 a BAPR 4.73 ± 0.56 a 1.86 ± 0.99 b 1.60 ± 0.05 b BAPR5 0 P 0.27 ± 0.11 a 0.55 ± 0.09 a 0.48 ± 0.19 a Active CKs ± 2.09 a 9.66 ± 1.17 b ± 7.22 a Values presented are means ± SD (n = 2). Different letters are significantly different (p [ 0.05)

6 calli than in the other two types of calli, with the exception of ZOG, ZROG, ip, IPR5 0 P and DHZR5 0 P (Table 1). The concentrations of Z, DHZ9G, DHZ and DHZR were significantly higher in initial calli, and the concentrations of Z9G and ZR5 0 P were significantly higher in non-embryogenic calli (Table 1). The most abundant ones were: ip9g (167 pmol g -1 FW), Z9G (30.29 pmol g -1 FW), ZR (11 pmol g -1 FW), and Z (2.16 pmol g -1 FW) in nonembryogenic calli (Table 1); ip9g (145 pmol g -1 FW), Z9G (17.72 pmol g -1 FW), ZR (7.93 pmol g -1 FW), and Z (3.66 pmol g -1 FW) in initial calli (Table 1); and ip9g (95.44 pmol g -1 FW), Z9G (2.74 pmol g -1 FW), ZR (1.34 pmol g -1 FW), and Z (1.20 pmol g -1 FW) in embryogenic calli (Table 1). Endogenous aromatic cytokinins The results also showed the occurrence of aromatic cytokinins in each of the different types of callus. The significantly highest concentration of total aromatic cytokinins was present in initial calli with pmol g -1 FW; the lowest in embryogenic calli with 6.58 pmol g -1 FW; and an intermediate value in non-embryogenic calli with 8.61 pmol g -1 FW (Fig. 5); and the differences were significant. The aromatic cytokinins were, depending on the type of callus, from 26- to 16-fold less abundant than the isoprenoid ones (Fig. 5). With respect to individual aromatic cytokinins only BAP types were present, and all four metabolites analysed were detected in each of the calli studied. BAPR (4.73 pmol g -1 FW) was significantly higher in initial calli, BAP9G (6.20 pmol g -1 FW) was significantly higher in non-embryogenic calli. In the case of BAP and BAPR5 0 P no differences were found between different types of calli (Table 1). The concentrations of individual cytokinins were summed to calculate the total concentration of cytokinin. The calli with the highest concentrations of total cytokinins were non-embryogenic calli with 230 pmol g -1 FW and initial calli with 201 pmol g -1 FW, and the lowest concentration was in embryogenic calli with 112 pmol g -1 FW (Fig. 5); and this difference was significant. Active cytokinins Contents of these cytokinins were calculated by the sum of the ribosides, free bases, nucleotides and O-glucosides. The highest concentrations were found in initial calli with 32.8 pmol g -1 FW and non-embryogenic calli with 26.6 in pmol g -1 FW, and the lowest in embryogenic calli with 9.7 pmol g -1 FW (Table 1); and this difference was significant. Discussion Effect of exogenous cytokinins The present results show that addition of the aromatic cytokinin BAP to the culture medium reduced the formation of embryogenic calli promoted by auxin, in coconut plumule cultures, and that the extent of this reduction was dependent on the concentration. Similarly, this effect was observed with other cytokinins of the isoprenoid type, Z and ip. Therefore, this effect is not associated with only one type of cytokinin. Wenck et al. (1988) reported that exogenous Z inhibits somatic embryogenesis in cultures of a D. glomerata embryogenic genotype. Our results further show that none of the cytokinins affected the yield of initial calli. These observations prompted the question of whether the concentrations of endogenous cytokinins were different between initial calli and embryogenic calli, and later forming embryogenic and non-embryogenic calli. Therefore analysis of both the isoprenoid and the aromatic cytokinins was performed in the three different types of calli. Effect of 8-azaadenine Previous studies have shown that exogenous cytokinins suppressed somatic embryogenesis in leaf explants of D. glomerata (Wenck et al. 1988) while anticytokinins applied during the whole culture period stimulated somatic embryo formation in this species (Somleva et al. 1995). Therefore the addition of 8-azaadenine was tested. The results showed that there was an increase in the formation of both embryogenic calli and somatic embryos. Further when the explants were initially cultured with BAP, the addition of 8-azaadenine restored the percentage of embryogenic calli formation. Our results suggest that there is a negative correlation between the action of endogenous cytokinins and the formation of embryogenic calli in plumule cultured coconut palm explants. Analysis of endogenous cytokinins The analysis showed that all isoprenoid and aromatic cytokinins studied were present in each type of callus, and that the isoprenoid cytokinins were from 17- to 26-fold (depending on the type of callus) more abundant than the aromatic ones, coinciding with what has been found in different plant parts of the coconut palm (Sáenz et al. 2003). Comparison of the total cytokinin concentrations in the different calli showed that it was two times lower in embryogenic calli than in initial calli or non-embryogenic calli, and basically the same pattern was observed for total isoprenoid cytokinin concentrations. However, in the case of total aromatic cytokinin concentrations the pattern was

7 different. This time the concentration in embryogenic calli was no different than that in non-embryogenic calli, but the concentration in initial calli was more than twofold higher than in the other two calli. Isoprenoid cytokinins Within the isoprenoid cytokinins, the most abundant ones for each of the calli studied were the ip-type, the Z-type and the DHZ-type in decreasing order. In in vitro culture of oil palm (Jones 1990) and tobacco (Gaudinová et al. 1995) the ip type cytokinin were the most abundant. This result is in contrast with what was observed for the coconut palm and other species where the Z-type was more abundant than the ip-type. For instance the Z-type cytokinins comprised 90% of the total cytokinins in Urtica dioica (Wagner and Beck 1993) 80 90% in Rosa hybrida (Dieleman et al. 1997) and 43% in Pistacia vera seedlings (Ahmadi and Baker 2000). In a hormone-autotrophic genetic tumour line of tobacco the Z types were the dominant endogenous cytokinins (Nandi et al. 1990). Regarding individual isoprenoid cytokinins, in each of the calli studied the most abundant one was ip9g. This contrasts with what was observed in the coconut palm and plants in general (for instance: U. dioica, (Wagner and Beck 1993); R. hybrida (Dieleman et al. 1997); and P. vera (Ahmadi and Baker 2000) where ZR was the most abundant cytokinin. Aromatic cytokinins The present study shows the occurrence of BAP and the metabolites BAP9G, BAPR and BAP5 0 P in each of the three types of calli studied, as has been previously reported for the coconut palm (Sáenz et al. 2003). As in the present case, these types of cytokinins have been always reported in lower concentrations than the isoprenoid ones (Jones et al. 1995). No particular pattern was observed. One of the first reports about the detection of endogenous aromatic cytokinins came from cell suspensions of anise, where BAPR was detected (Ernst et al. 1983). Other reports from in vitro culture have shown the presence of aromatic cytokinins as reported by Danin et al. (1993) in Apium graveolens, embryogenic cultures, Jones et al. (1995) in Elaeis guineensis, and Centeno et al. (1997) incorylus avellana. Correlation between endogenous cytokinins and morphogenetic response The results above showed that there were indeed differences in cytokinin concentration between different types of calli, particularly for the concentrations of the isoprenoid cytokinins, which comprised most of the cytokinins in the tissues. In the embryogenic calli these were always lower than in either initial calli or non-embryogenic calli, thereby demonstrating an inverse correlation between isoprenoid cytokinin concentrations and embryogenic capacity of the calli. This suggested an inhibitory effect of cytokinins on the development of embryogenic calli, which is consistent with the fact that addition of exogenous cytokinins decreased the formation of embryogenic calli in this system. Other authors have found an inverse relationship between the endogenous cytokinin content and the embryogenic capacity of plant tissues (Rajasekaran et al. 1987; Ivanova et al. 1994; Centeno et al. 1997). On the contrary, Jones et al. (1995) reported higher levels of endogenous cytokinins in the embryogenic callus of oil palm (30 1,500 pmol g -1 FW) than in initial callus ( pmol g -1 FW). There are observations that coconut tissue cultures behave differently to oil palm culture as reported by Jones and Hughes (1989). Oil palm forms somatic embryos reluctantly and produces a callus of large vacuolated cells that cease division and die. In coconut Verdeil and Hocher (1997) found higher levels of Z and ip in the coconut calli derived from foliar explants oriented towards embryogenesis than in the part remaining at the multiplication stage; however, in this study only Z, ip and their respective ribosides were analysed. It is probable that the initial source of endogenous cytokinins affects the response of the in vitro culture. Sáenz et al. (2003) have shown that zygotic embryos had higher endogenous cytokinin content than the foliar tissues; even the pattern of individual cytokinins was different. The information presented here is useful, not only to understand more about the role of cytokinins in in vitro coconut tissue cultures capable of forming somatic embryos, but also for practical purposes. We know that we should avoid adding cytokinins to these cultures during callogenesis, and the addition of anticytokinin compound can increase the embryogenic response. Further increases may be obtained with testing other compounds that block cytokinin action. Acknowledgments We are grateful to H. Martínková for excellent technical assistance. L. Sáenz would like to acknowledge the continuing support from the Centro de Investigación Científica de Yucatán and CONACyT (88207). A. Azpeitia would like to acknowledge continuing support from Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias and CONACyT (119335). Research in the Czech Republic was supported by Ministry of Education Grant No. MSM and Academy of Science Grant No. IBS References Ahmadi M, Baker DA (2000) Identification and quantification of the major endogenous cytokinins in pistachio seedlings. Plant Growth Regul 32: Centeno ML, Rodríguez R, Berros B, Rodríguez A (1997) Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons. Plant Cell Rep 17:

8 Chan JL, Sáenz L, Talavera C, Hornung R, Robert M, Oropeza C (1998) Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis. Plant Cell Rep 17: Danin M, Upfold SJ, Levin N, Nadel BL, Altman A, van Staden J (1993) Polyamines and cytokinins in celery embryogenic cell cultures. Plant Growth Regul 12: Davies PJ (1995) The plant hormones: their nature, occurrence and functions. In: Davies PJ (ed) Plant hormones: physiology, biochemistry and molecular biology. Kluwer, Dordrecht, pp 1 38 Dieleman JA, Verstappen FWA, Perik PRJ, Kuiper D (1997) Quantification of the export cytokinins from root to shoots of Rosa hybrida and their degradation rate in the shoot. Physiol Plant 101: Eeuwens CJ (1976) Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms (Cocos nucifera) and cultured in vitro. Physiol Plant 36:23 28 Ernst D, Schaffer W, Oesterhelt D (1983) Isolation and identification of a new, naturally occurring cytokinin (6-benzylaminopurine riboside) from anise cell culture (Pimpinella anisum L.). Planta 159: Faiss M, Zalubilová J, Strnad M, Schmulling T (1997) Conditional expression of the ipt gene indicates a function for cytokinins in paracrine signalling in whole tobacco plants. Plant J 12: Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27 47 Gaudinová A, Süssenbeková H, Vojtechová M, Kamínek M, Eder J, Kohout L (1995) Different effects of two brassinosteroids on growth, auxin and cytokinin content in tobacco callus tissue. Plant Growth Regul 17: George EF (1993) Plant propagation by tissue culture, Part 1, 2nd edn. Exegetics, England, p 444 Holub J, Hanuš J, Hanke DE, Strnad M (1998) Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul 26: Ivanova A, Velcheva M, Denchev P, Atanassov A, Onckelen A (1994) Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata. Physiol Plant 92:85 89 Jones LH (1990) Endogenous cytokinins in oil palm (Elaeis guineensis L.) callus, embryoids and regenerant plants measured by radioimmunoassay. Plant Cell Tiss Org Cult 20: Jones LH, Hughes WA (1989) Oil palm (Elaeis guineensis Jacq). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry 5. Trees II. Springer, Berlin, pp Jones LH, Hanke DE, Eeuwens CJ (1995) An evaluation of the role of cytokinins in the development of abnormal inflorescences in oil palm cultures (Elaeis guineensis Jacq.) regenerated from tissue culture. J Plant Growth Regul 14: Jones LH, Martínková H, Strnad M, Hanke DE (1996) Occurrence of aromatic cytokinins in oil palm (Elaeis guineensis Jacq.). J Plant Growth Regul 15:39 49 Kraigher H, Grayling A, Wang T, Hanke DE (1991) Cytokinin production by two ectomycorrhizal fungi in liquid culture. Phytochemistry 30: Krikorian D (1995) Hormones in tissue culture and micropropagation. In: Davies PJ (ed) Plant hormones: physiology, biochemistry and molecular biology. Kluwer, London, pp MacDonald EMS, Akioshi DE, Morris RO (1981) Combined high performance liquid chromatography radioimmunoassay for cytokinins. J Chromatogr 214: Nandi SK, Palni LM, Parker CW (1990) Dynamics of endogenous cytokinins during the growth cycle of hormone-autotrophic genetic tumor line of tobacco. Plant Physiol 94: Rajasekaran K, Hein MB, Davis GC, Carnes MG, Vasil IK (1987) Endogenous growth regulators in leaves and tissue cultures of Pennisetum purpureum Schum. J Plant Physiol 130:13 25 Sáenz L, Jones LH, Oropeza C, Vlácil D, Strnad M (2003) Cytokinins in Cocos nucifera L. Plant Growth Regul 39: Somleva MM, Kapchina V, Alexieva V, Golovinsky E (1995) Anticytokinin effects on in vitro response of embryogenic and non embryogenic genotypes of Dactylis glomerata L. Plant Growth Regul 16: Strnad M (1996) Enzyme immunoassays of N 6 -benzyladenine and N 6 -(meta-hydroxybenzyl)adenine cytokinins. J Plant Growth Regul 15: Verdeil J-L, Hocher V (1997) Coconut: development of methods for the clonal propagation of elite, disease resistant palms by somatic embryogenesis. Third Annual Report. EC STD3 Wagner BM, Beck E (1993) Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status. Planta 190: Wenck AR, Conger BV, Trigiano RN, Sams CE (1988) Inhibition of somatic embryogenesis in Orchardgrass by endogenous cytokinins. Plant Physiol 88: