Plant Physiology and Biochemistry 84 (2014) 197e202
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Research article
Endogenous hormones response to cytokinins with regard to organogenesis in explants of peach (Prunus persica L. Batsch) cultivars and rootstocks (P. persica Prunus dulcis) rez-Jime nez a, *, Elena Cantero-Navarro b, Francisco Pe rez-Alfocea b, Margarita Pe a Cos-Terrer Jose n y Desarrollo Agrario y Alimentario (IMIDA), C/ Mayor s/n, 30150 La Alberca, Departamento de Hortofruticultura, Instituto Murciano de Investigacio Murcia, Spain n Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Científicas (CSIC), Departamento de Nutricio Campus Universitario de Espinardo, 30100 Murcia, Spain a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Accepted 22 September 2014 Available online 29 September 2014
Organogenesis in peach (Prunus persica L. Batsch) and peach rootstocks (P. persica Prunus dulcis) has been achieved and the action of the regeneration medium on 7 phytohormones, zeatin (Z), zeatin riboside (ZR), indole-3-acetic acid (IAA), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA), has been studied using High performance liquid chromatography e mass spectrometry (HPLC-MS/MS). Three scion peach cultivars, ‘UFO-3’, ‘Flariba’ and ‘Alice Bigi’, and the peach almond rootstocks ‘Garnem’ and ‘GF677’ were cultured in two different media, Murashige and Skoog supplemented with plant growth regulators (PGRs) (regeneration medium) and without PGRs (control medium), in order to study the effects of the media and/or genotypes in the endogenous hormones content and their role in organogenesis. The highest regeneration rate was obtained with the peach almond rootstocks and showed a lower content of Z, IAA, ABA, ACC and JA. Only Z, ZR and IAA were affected by the action of the culture media. This study shows which hormones are external PGRs-dependent and what is the weight of the genotype and hormones in peach organogenesis that provide an avenue to manipulate in vitro organogenesis in peach. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Morphogenesis Abscisic acid Indole-3-acetic acid Zeatin Zeatin riboside 1-Aminocyclopropane-1-carboxylic acid
1. Introduction Somatic plant regeneration is a fundamental step in the way of obtaining transgenic plants, and is still lacking in certain genotypes defined as recalcitrant. Plant growth regulators (PGRs) are key factors in tissue culture. Actually, plant growth, rooting and multiplication in vitro are stimulated by externally applied PGRs. Exogenous PGRs added to culture media induce changes in cells and in the production of their own hormones, endogenous phytohormones, which play a major role in the regulation of morphogenesis (Huang et al., 2012). Furthermore, the interaction between the exogenous and endogenous PGRs influences the intrinsic natural process of cytokinins biosynthesis/accumulation (Aremu et al., 2014), phytohormones of importance in the organogenesis
* Corresponding author. rez-Jime nez). E-mail address: [email protected] (M. Pe http://dx.doi.org/10.1016/j.plaphy.2014.09.014 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
technology. Notwithstanding, cytokinins are not the only PGRs involved in organogenesis. It is acknowledged that a proper equilibrium between cytokinins and auxins has been used as a physiological index in plant regeneration (George, 1993) and ABA has been reported as a regulator of the organ induction process (Rai et al., 2011). There are also other hormones affecting organogenesis due to aspects related to tissue culture, as ethylene accumulation in the closed vessels or stress associated to the in vitro conditions. Although the addition of PGRs to the culture medium is the nez, preferred way to induce in vitro morphogenic responses (Jime 2005), carbon sources and genotypes are also well known to affect shoot regeneration in cultured cells (Lee and Huang, 2013). The genotype is determinant in the differences found among species, and in the least responsive, such as woody plants, it is common to find genotypes that react more readily than others to a particular nez, 2005). Peach has been set of inductive conditions (Jime
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M. Perez-Jimenez et al. / Plant Physiology and Biochemistry 84 (2014) 197e202
reported as one of the most recalcitrant species with regard to adventitious shoot regeneration (Bhansali et al., 1990; Padilla et al., 2006). However, a few shoot regeneration and transformation systems have been developed in adult tissues of peach but the regeneration frequency is commonly low and is cultivar-dependent rez-Jime nez et al., 2012). Although imma(Gentile et al., 2002; Pe ture tissues are more likely to be regenerated and transformed, each genotype is unique and not a clone of the parent (Abbott et al., 2008) and all their agronomic features would be unknown. In previous research a reproducible adventitious shoot regeneration protocol was developed through the induction of organogenic callus in the bottom of the proliferation clusters, as well as a first attempt to figure out the role of the endogenous hormones in rez-Jime nez et al., 2012, 2014). Nevertheless, a morphogenesis (Pe study regarding the response of different genotypes in terms of organogenesis-related hormones concentrations when they are cultured in an organogenic-inducing media was still lacking. In the present study, endogenous hormones are quantified in shoots cultured in a media with no PGRs and when they are exposed to the organogenic media. Our attempt is to establish a relationship between endogenous hormonal levels and in vitro morphogenic responses and to study the importance of the effect of the culture media and genotype in peach regeneration. To date, a few reports have been published concerning somatic regeneration and endogenous hormones in woody plants. Nevertheless, to our knowledge there are no studies about the culture media effect in the endogenous hormone content and/or genotype, and analyzing if the differences between the regenerative and the nonregenerative genotypes are due to the nature of the genotype, to its response to the tissue culture media or both. This study would be the first one in this sense. 2. Materials and methods 2.1. Plant material Plant material was obtained from 4-year-old peach trees grown at the Torreblanca experimental field station of the Instituto Mur n y Desarrollo Agrario y Alimentario (IMIDA), ciano de Investigacio located in Cartagena, Spain. Nodal segments of the scion cultivars ‘UFO-3’, ‘Flariba’, and ‘Alice Bigi’, and of the peach almond rootstocks ‘Garnem’ and ‘GF677’, were collected and transferred to the tissue culture laboratory. 2.2. In vitro establishment The nodal segments were surface sterilized by agitating in a solution of sodium troclosene dihydrate (CTX-200/GR®; CTX S.A.U., Barcelona, Spain) at a concentration of 3.5 g l 1, containing 0.1% (v/ v) Tween-20 for 2 h in a laminar flow hood. Shoot cultures were established in vitro and subcultured monthly on culture medium for 3 months. The medium (T0) was composed of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), 3% (w/v) sucrose and 0.7% (w/v) Plant Propagation Agar (Pronadisa®) in 300 ml culture vessels, each containing 100 ml culture medium. The pH was adjusted to 5.7 using 0.1 N KOH prior to autoclaving at 122 C (1.1 kg cm 2) for 16 min. Shoots were cultured in climatic chamber at 25 ± 1 C and with a 16 h light period (45 mmol m 2 s 1; Sylvania Gro-lux fluorescent tubes). 2.3. Culture treatments Half of the shoots were maintained on T0 and the other half were cultured on T0 supplemented with 0.1 mg l 1 of indolebutyric acid (IBA) and 1 mg l 1 of 6-benzylaminopurine (BA) (Duchefa®) (T1).
After 6 in vitro subcultures (30 days each) on the indicated media, the calli obtained from the base of the proliferation clusters were excised. Leaves were also excised, and the whole stem and all the leaves were weighted and analyzed in the HPLC-MS. 2.4. Callus induction and regeneration The calli were isolated and sliced prior to culturing in the organogenic media, composed of MS salts supplemented with 1 mg l 1 of a-naphthalene acetic acid and 2 mg l 1 of BA in Petri rez-Jime nez et al., 2012). After two in vitro subcultures on dishes (Pe the indicated media the organogenic rate (regenerated shoots/ callus) was recorded. 2.5. Hormone extraction and analysis The ABA, cytokinins [Z and ZR], IAA, JA, SA and ACC were extracted and purified according to the method described by Dobrev and Kaminek (2002), and were analyzed as described previously by Albacete et al. (2008). In summary, sampling material (1 g FW) was homogenized in liquid nitrogen and dropped in 2.5 ml of cold ( 20 C) extraction solution of methanol/water (80/20, v/v). Extracts were centrifuged at 20,000 g for 15 min at 4 C, and the pellets were re-extracted for 30 min in an additional 2.5 ml of the same extraction solution. Supernatants were collected and filtered through Sep-Pak Plus C18 (Waters, Milford, MA, USA) to remove interfering lipids and pigments, and evaporated at 40 C under vacuum. Residues were dissolved in 1 ml methanol/water (20/80, v/ v) solution using an ultrasonic bath. The dissolved samples were filtered through 13 mm-diameter nylon membrane Millex filters (Ø 0.22 mm) (Millipore, Bedford, MA, USA) and placed into tubes, adjusting the volume to 1.5 ml with the extraction solution. Analyses were carried out with an HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler connected to an Agilent Ion Trap XCT Plus mass spectrometer (©Agilent Technologies) using an electrospray interface. Before injection, 100 ml of each fraction were filtered again through Millex filters (Ø 0.22 mm). Each sample (8 ml) previously dissolved in mobile phase A was injected onto a Zorbax SB-C18 HPLC column (5 mm, 150 0.5 mm, Agilent Technologies) at 40 C and eluted at a flow rate of 10 ml min 1. Mobile phase A [water/acetonitrile/formic acid (94.9/5/0.1, v/v/v)] and mobile phase B [water/acetonitrile/formic acid (10/89.9/0.1, v/v/v)] were used for the chromatographic separation. The elution consisted of maintaining 100% A for a period of 5 min, and then using a 10 min linear gradient from 0 to 6% B, followed by another 5 min linear gradient from 6 to 100% B, and finally keeping 100% B for another 5 min. The column was equilibrated with the starting composition of the mobile phase for 30 min before each analysis. The UV chromatograms were recorded at 280 nm with the diode array detector module (Agilent Technologies). Different control samples with known concentrations of each component (0.001, 0.01, 0.05, 0.1, 0.2 and 0.5 mg l 1) were also analyzed under the same conditions. The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V and a scan speed of 22,000 (m/z)/s from 50 to 500 m/z. The nebulizer gas (He) pressure was set to 30 psi, while the drying gas was set to a flow of 6 l min 1 at 350 C. Mass spectra were obtained using the Data Analysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany). For quantification of ABA and JA, calibration curves were constructed for each component analyzed using internal standards: [2H6]cis, trans-abscisic acid and [2H5](±)-JA (Olchemin Ltd, Olomouc) (0.001, 0.01, 0.05, 0.1, 0.2 and 0.5 mg l 1) and corrected for 0.1 mg l 1. The ACC and SA were quantified by the external standard method, using the same concentration of the product
M. Perez-Jimenez et al. / Plant Physiology and Biochemistry 84 (2014) 197e202
(SigmaeAldrich Inc., St Louis, MO, USA). Recoveries ranged between 92 and 95%. Three biological replicas were quantified per sample. 2.6. Experiment design and data collection Three replications with 9 samples of stems and leaves from each of the cultivars per replication were used. The stem and leaves weight and the number of regenerated shoots per callus (regeneration rate) were recorded. Significance was determined by ANOVA and the significance (P 0.05) of differences between mean values was tested by Duncan's New Multiple Range Test. 3. Results and discussion Changes in the endogenous hormonal content produced by a rez-Jime nez et al., 2012, 2014) in proved-organogenic media (Pe plants of three peach cultivars and two peach almond hybrids have been studied. Furthermore, the differences between endogenous hormonal content and somatic organogenesis of the cited genotypes in a media without PGRs and in the organogenic media with cytokinins were also evaluated. In this study, weight of stems and leaves were recorded in plants cultured in T0 and T1 in order to study the relationship between the development of the shoot and its organogenic response. As it is shown in Table 1, stems and leaves weight were markedly lower in those shoots cultured in T0 than in those cultured in T1 due to PGRs action. Nonetheless, a lower leave weights was found in genotypes with the higher organogenesis capacity, the peach almond hybrids. Although the weight of leaves was higher in the cultivars than in the rootstocks, their number was observed to be lower, but with a longer size. This results are supported by the reports that relate inbreeding depression showed by highly inbreed cultivars (Charlesworth and Charlesworth, 1987), such as peach cultivars, and hybridity with rez-Jime nez et al., 2012). On the vigor and/or organogenesis (Pe other hand, regeneration rates obtained in this work are according rez-Jime nez et al., 2012, 2014), since with previous research (Pe shoot regeneration from peach rootstock calli were significantly higher (P 0.05) than the rates obtained in the studied peach cultivars (Table 1). The quantification of endogenous hormone content was carried out in stem and leaves of peach cultivars and rootstocks. Whilst stem results showed (Fig. 1) a relation with plant organogenesis, the hormone content in leaves could not have been related with organogenesis in any of the studied phytohormones (data not shown). The organogenic calli are induced from the bottom part of the stem; thus, the quantification of the organogenesis-causing hormones may be more accurate in this proximal organ than in leaves when studying the regeneration capacity of this baseformed callus. Results concerning the two endogenous cytokinins measured from the stem of different peach cultivars and rootstocks are
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presented in Fig. 1A, B. Levels of Z increased in the shoots cultured in T1 in every genotype when compared with shoots cultured in T0. Notwithstanding, the content in ZR decreased after the treatment with cytokinins except in the rootstocks, where the amount of ZR markedly increased. Z concentrations were higher in the stem of the peach cultivars than in the stem of the rootstocks. On the contrary, ZR content was markedly higher in hybrids than in cultivars. Other studies conduced in woody plants support higher s levels of Z in organogenic explants (Centeno et al., 1996; Valde et al., 2005; Pe rez-Jime nez et al., 2014) and et al., 2001; Mala lower levels of ZR (Centeno et al., 1996) when cultured in media s et al. (2001) and Pe rezwith BA. However, other authors as Valde nez et al. (2014) reported an increment of ZR in organogenic Jime explants. Furthermore, whilst endogenous concentrations of the studied cytokinins did not show any relation with organogenesis in explants cultured in T0, the differences between the most responsive genotypes were noted after being cultured in T1. Same results were obtained for ZR. Results showed in this study manifest a different response to the treatment depending on the genotype. A higher organogenesis rate does not seem to be related with natural endogenous cytokinin content but with the response to PGRs signals, since explant tissues consist of cells with distinct capacity to nez, 2005). respond to an induction treatment (Jime The genotypes showed the same pattern regarding the analysis resulting from the quantification of IAA (Fig. 1C). No differences among genotypes were found between the hybrids and the cultivars in T0. Nevertheless, results pointed out to a lower concentration of IAA in those genotypes that presented a higher organogenesis rate after T1, showing a clear response to the treatment. This is contrary to previous research developed in peach calli, where no relation between IAA and organogenesis was found rez-Jime nez et al., 2014). (Pe The results obtained in the analysis of the stems showed no differences in the ABA content between shoots grown in T0 and T1 (Fig. 1D). Only Flariba presents a slight difference between shoots cultured in different media. This finding indicates no influence of the media in the amount of the endogenous ABA; in this study, the ABA concentration depends on the genotype. However the effect of ABA on organogenesis is still not clarified. Lee and Huang (2013) reported a higher amount of ABA in organogenic genotypes of rice when compared with the non-organogenic ones. On the contrary, other studies suggest that a high level of ABA does not inrez-Jime nez crease shoot organogenic rate (Huang et al., 2012; Pe et al., 2014). This is according with our results, where ‘Garnem’ and ‘GF677’ were the genotypes with the lower ABA content and higher organogenic rate. The stress-related phytohormone JA did not show to be affected by the culture media, being levels of JA as high in T0 as in T1 (Fig. 1E). In both treatments the lower levels of JA were coincident with the genotypes with highest regeneration rates, this is contrary to previous results where no differences between hybrids and cultivar rez-Jime nez et al., 2014). Since high levels of JA have were found (Pe
Table 1 Adventitious shoot regeneration from callus explants, stem and leaves weight of three peach cultivars and two peach rootstocks. Cultivars
Flariba
Treatment without plant growth regulator Stem weight (g) 0.015 ± 0.004c Leaves weight (g) 0.161 ± 0.067c Treatment with plant growth regulator Regeneration rate 2.222 ± 0.830c Stem weight (g) 0.282 ± 0.069b Leaves weight (g) 1.146 ± 0.226b
Alice Bigi
UFO-3
0.042 ± 0.005a 0.120 ± 0.045c
0.037 ± 0.005 ab 0.235 ± 0.029b
3.889 ± 0.790b 1.147 ± 0.185a 1.940 ± 0.129a
2.667 ± 0.624c 0.343 ± 0.060b 1.304 ± 0.224b
Data represent average ± SD values. Different letters indicate a significant difference by LSD test (p 0.05).
GF677
Garnem
0.030 ± 0.004b 0.134 ± 0.020c
0.041 ± 0.003a 0.465 ± 0.027a
12.222 ± 1.832a 0.321 ± 0.059b 0.885 ± 0.129c
13.889 ± 2.330a 0.341 ± 0.051b 0.672 ± 0.088c
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Fig. 1. Endogenous content in (A) zeatin (Z), (B) zeatin riboside (ZR), (C) indole-3-acetic acid (IAA), (D) abscisic acid (ABA), (E) jasmonic acid (JA), (F) 1-aminocyclopropane-1carboxylic-acid (ACC), and (G) salicylic acid (SA) in stem of peach (cv. Flariba, cv. Alice Bigi, cv. UFO-3) and peach rootstocks (cv. GF677, cv. Garnem). Data represent average ± SD values. Bars with different letters indicate a significant difference by LSD test (p 0.05) between cultivars. * denotes significant differences between T0 and T1.
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Fig. 2. (A) Ratio between indole-3-acetic acid (IAA) and abscisic acid (ABA) in callus of peach (cv. Flariba, cv. Alice Bigi, cv. UFO-3) and peach rootstocks (cv. GF677, cv. Garnem) (B) Ratio between zeatin (Z) and indole-3-acetic acid (IAA) in organogenic and non-organogenic callus of peach. Data represent average ± SD values. Bars with different letters indicate a significant difference by LSD test (p 0.05). * denotes significant differences between T0 and T1.
been identified as stress indicators in tissue culture (Rudus et al., 2009), the peach cultivars seem to be more sensitive to the tissue culture techniques than the hybrids. Regardless of the results, the JA concentration seems to be more a consequence of the tissue culture conditions (Rudus et al., 2009) than an inductor of any regeneration process. This is supported by our previous findings in which no differences were found between the amount of JA and rez-Jime nez et al., 2014). somatic organogenesis (Pe The results of the quantification reflect lower levels of ACC in peach almond hybrids than in peach cultivars (Fig. 1F). The role played by ethylene in plant regeneration has been described as controversial and genotype-dependent (Yasmin et al., 2014). On one hand, some authors pointed out to a stimulating effect of ACC (ethylene precursor) in woody plants regeneration (Predieri et al., lez et al., 1997); on the other hand, ethylene has 1993; Gonza been linked with organogenesis and growth inhibition by Arigita et al. (2003). According to the data obtained in the current and in previous studies, ACC may be an inhibitor of the morphogenesis in peach, since its lower amount is found in those genotypes with the highest regeneration rates. The SA levels (Fig. 1G) found in calli of the studied genotypes remained constant in media with PGRs and in the control for all the genotypes. On the other hand, no relationship was found according to higher records of regenerated shoots. Although SA has been reported as an embryogenesis enhancer (Ahmadi et al., 2014), there are no records that conclude that SA has a clear effect on morphogenesis in woody plants. Huang et al. (2012) reported that ABA and IAA together may be involved in inducing shoot organogenesis on callus. In our study, a significant effect was observed in the case of the IAA/ABA ratio (Fig. 2A) where the lower levels of this ratio were found in the hybrids. Changes between T0 and T1 in this ratio were detected in every genotype but ‘Garnem’, in which they are the same. On the contrary, the Z/IAA ratio (Fig. 2B) could not be associated to organogenesis. The relation between Z and AA content varied in all the studied genotypes depending on the media where the genotypes were cultured but differences between the hybrids and the cultivars only were found in T0, where no organogenesis response were detected. In this study, the results showed an important influence of the genotype in organogenesis. Notwithstanding, this influence is not always given by the basal endogenous hormone naturally contained in each genotype but sometimes by the response of these studied genotypes to the culture media. It is well known that cytokinins and auxins at certain concentrations may be inducers of somatic morphogenesis. However, the basal concentrations of
these hormones in a media without PGRs is not related to organogenesis before being exposed to a media with PGRs, actually the concentrations of these hormones were very different in the most responsive genotypes. Only after the exposition to an organogenic callus inducing media, adventitious regeneration occur in these genotypes. On the other hand, hormones such as ABA, ACC, JA and SA seemed not to be affected by the culture media. A few articles showing the relationship between somatic regeneration and endogenous hormones content, genotype and/or culture media in woody plants have been published but to our knowledge this is the first study showing clearly the weight of each one of these elements in the regeneration process. Contributors rez-Jime nez. Planning of the experiment, lab work, Margarita Pe results analysis and writing. Elena Cantero-Navarro. Collaboration in endogenous hormones measurements. rez-Alfocea. Collaboration in results concerning Francisco Pe endogenous hormones measurements. Cos-Terrer. Planning of the experiment and results analysis. Jose Acknowledgments s Paredes Jime nez for the EnThe authors want to thank Andre glish revision of the manuscript. This report was supported by the Instituto Nacional de Investigaciones Agrarias (INIA) (RTA2008rez00121-00-00) and by a fellowship provided to Margarita Pe nez also by INIA. Jime References Abbott, A.G., Arús, P., Scorza, R., 2008. Genetic engineering and genomics. In: Layne, D.R., Bassi, D. (Eds.), The Peach: Botany Production and Uses. CABI, Wallingford, United Kingdom, pp. 85e105. Ahmadi, B., Shariatpanahi, M.E., da Silva, J.A.T., 2014. Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult. 116 (3), 343e351. , L., Bairu, M.W., Nova k, O., Plíhalova , L., Dolez Aremu, A.O., Pla ckova al, K., Finnie, J.F., Van Staden, J., 2014. How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea? Plant Cell Tissue Organ Cult. 118 (2), 245e256. Albacete, A., Ghanem, M.E., Martínez-Andújar, C., Acosta, M., S anchez-Bravo, J., rez-Alfocea, F., 2008. Hormonal changes in Martínez, V., Lutts, S., Dodd, I.C., Pe relation to biomass partitioning and shoot growth impairment in salinised tomato (Solanum lycopersicum L.) plants. J. Exp. Bot. 59, 4119e4131. s, R., Gonza lez, A., 2003. 1-methylcyclopropene and Arigita, L., Sanchez Tame ethylene as regulators of in vitro organogenesis in kiwi explants. Plant Growth Regul. 40, 59e64.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Endogenous hormones response to cytokinins with regard to organogenesis in explants of peach (Prunus persica L. Batsch) cultivars and rootstocks (P. persica Prunus dulcis) rez-Jime nez a, *, Elena Cantero-Navarro b, Francisco Pe rez-Alfocea b, Margarita Pe a Cos-Terrer Jose n y Desarrollo Agrario y Alimentario (IMIDA), C/ Mayor s/n, 30150 La Alberca, Departamento de Hortofruticultura, Instituto Murciano de Investigacio Murcia, Spain n Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Científicas (CSIC), Departamento de Nutricio Campus Universitario de Espinardo, 30100 Murcia, Spain a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Accepted 22 September 2014 Available online 29 September 2014
Organogenesis in peach (Prunus persica L. Batsch) and peach rootstocks (P. persica Prunus dulcis) has been achieved and the action of the regeneration medium on 7 phytohormones, zeatin (Z), zeatin riboside (ZR), indole-3-acetic acid (IAA), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA), has been studied using High performance liquid chromatography e mass spectrometry (HPLC-MS/MS). Three scion peach cultivars, ‘UFO-3’, ‘Flariba’ and ‘Alice Bigi’, and the peach almond rootstocks ‘Garnem’ and ‘GF677’ were cultured in two different media, Murashige and Skoog supplemented with plant growth regulators (PGRs) (regeneration medium) and without PGRs (control medium), in order to study the effects of the media and/or genotypes in the endogenous hormones content and their role in organogenesis. The highest regeneration rate was obtained with the peach almond rootstocks and showed a lower content of Z, IAA, ABA, ACC and JA. Only Z, ZR and IAA were affected by the action of the culture media. This study shows which hormones are external PGRs-dependent and what is the weight of the genotype and hormones in peach organogenesis that provide an avenue to manipulate in vitro organogenesis in peach. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Morphogenesis Abscisic acid Indole-3-acetic acid Zeatin Zeatin riboside 1-Aminocyclopropane-1-carboxylic acid
1. Introduction Somatic plant regeneration is a fundamental step in the way of obtaining transgenic plants, and is still lacking in certain genotypes defined as recalcitrant. Plant growth regulators (PGRs) are key factors in tissue culture. Actually, plant growth, rooting and multiplication in vitro are stimulated by externally applied PGRs. Exogenous PGRs added to culture media induce changes in cells and in the production of their own hormones, endogenous phytohormones, which play a major role in the regulation of morphogenesis (Huang et al., 2012). Furthermore, the interaction between the exogenous and endogenous PGRs influences the intrinsic natural process of cytokinins biosynthesis/accumulation (Aremu et al., 2014), phytohormones of importance in the organogenesis
* Corresponding author. rez-Jime nez). E-mail address: [email protected] (M. Pe http://dx.doi.org/10.1016/j.plaphy.2014.09.014 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
technology. Notwithstanding, cytokinins are not the only PGRs involved in organogenesis. It is acknowledged that a proper equilibrium between cytokinins and auxins has been used as a physiological index in plant regeneration (George, 1993) and ABA has been reported as a regulator of the organ induction process (Rai et al., 2011). There are also other hormones affecting organogenesis due to aspects related to tissue culture, as ethylene accumulation in the closed vessels or stress associated to the in vitro conditions. Although the addition of PGRs to the culture medium is the nez, preferred way to induce in vitro morphogenic responses (Jime 2005), carbon sources and genotypes are also well known to affect shoot regeneration in cultured cells (Lee and Huang, 2013). The genotype is determinant in the differences found among species, and in the least responsive, such as woody plants, it is common to find genotypes that react more readily than others to a particular nez, 2005). Peach has been set of inductive conditions (Jime
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reported as one of the most recalcitrant species with regard to adventitious shoot regeneration (Bhansali et al., 1990; Padilla et al., 2006). However, a few shoot regeneration and transformation systems have been developed in adult tissues of peach but the regeneration frequency is commonly low and is cultivar-dependent rez-Jime nez et al., 2012). Although imma(Gentile et al., 2002; Pe ture tissues are more likely to be regenerated and transformed, each genotype is unique and not a clone of the parent (Abbott et al., 2008) and all their agronomic features would be unknown. In previous research a reproducible adventitious shoot regeneration protocol was developed through the induction of organogenic callus in the bottom of the proliferation clusters, as well as a first attempt to figure out the role of the endogenous hormones in rez-Jime nez et al., 2012, 2014). Nevertheless, a morphogenesis (Pe study regarding the response of different genotypes in terms of organogenesis-related hormones concentrations when they are cultured in an organogenic-inducing media was still lacking. In the present study, endogenous hormones are quantified in shoots cultured in a media with no PGRs and when they are exposed to the organogenic media. Our attempt is to establish a relationship between endogenous hormonal levels and in vitro morphogenic responses and to study the importance of the effect of the culture media and genotype in peach regeneration. To date, a few reports have been published concerning somatic regeneration and endogenous hormones in woody plants. Nevertheless, to our knowledge there are no studies about the culture media effect in the endogenous hormone content and/or genotype, and analyzing if the differences between the regenerative and the nonregenerative genotypes are due to the nature of the genotype, to its response to the tissue culture media or both. This study would be the first one in this sense. 2. Materials and methods 2.1. Plant material Plant material was obtained from 4-year-old peach trees grown at the Torreblanca experimental field station of the Instituto Mur n y Desarrollo Agrario y Alimentario (IMIDA), ciano de Investigacio located in Cartagena, Spain. Nodal segments of the scion cultivars ‘UFO-3’, ‘Flariba’, and ‘Alice Bigi’, and of the peach almond rootstocks ‘Garnem’ and ‘GF677’, were collected and transferred to the tissue culture laboratory. 2.2. In vitro establishment The nodal segments were surface sterilized by agitating in a solution of sodium troclosene dihydrate (CTX-200/GR®; CTX S.A.U., Barcelona, Spain) at a concentration of 3.5 g l 1, containing 0.1% (v/ v) Tween-20 for 2 h in a laminar flow hood. Shoot cultures were established in vitro and subcultured monthly on culture medium for 3 months. The medium (T0) was composed of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), 3% (w/v) sucrose and 0.7% (w/v) Plant Propagation Agar (Pronadisa®) in 300 ml culture vessels, each containing 100 ml culture medium. The pH was adjusted to 5.7 using 0.1 N KOH prior to autoclaving at 122 C (1.1 kg cm 2) for 16 min. Shoots were cultured in climatic chamber at 25 ± 1 C and with a 16 h light period (45 mmol m 2 s 1; Sylvania Gro-lux fluorescent tubes). 2.3. Culture treatments Half of the shoots were maintained on T0 and the other half were cultured on T0 supplemented with 0.1 mg l 1 of indolebutyric acid (IBA) and 1 mg l 1 of 6-benzylaminopurine (BA) (Duchefa®) (T1).
After 6 in vitro subcultures (30 days each) on the indicated media, the calli obtained from the base of the proliferation clusters were excised. Leaves were also excised, and the whole stem and all the leaves were weighted and analyzed in the HPLC-MS. 2.4. Callus induction and regeneration The calli were isolated and sliced prior to culturing in the organogenic media, composed of MS salts supplemented with 1 mg l 1 of a-naphthalene acetic acid and 2 mg l 1 of BA in Petri rez-Jime nez et al., 2012). After two in vitro subcultures on dishes (Pe the indicated media the organogenic rate (regenerated shoots/ callus) was recorded. 2.5. Hormone extraction and analysis The ABA, cytokinins [Z and ZR], IAA, JA, SA and ACC were extracted and purified according to the method described by Dobrev and Kaminek (2002), and were analyzed as described previously by Albacete et al. (2008). In summary, sampling material (1 g FW) was homogenized in liquid nitrogen and dropped in 2.5 ml of cold ( 20 C) extraction solution of methanol/water (80/20, v/v). Extracts were centrifuged at 20,000 g for 15 min at 4 C, and the pellets were re-extracted for 30 min in an additional 2.5 ml of the same extraction solution. Supernatants were collected and filtered through Sep-Pak Plus C18 (Waters, Milford, MA, USA) to remove interfering lipids and pigments, and evaporated at 40 C under vacuum. Residues were dissolved in 1 ml methanol/water (20/80, v/ v) solution using an ultrasonic bath. The dissolved samples were filtered through 13 mm-diameter nylon membrane Millex filters (Ø 0.22 mm) (Millipore, Bedford, MA, USA) and placed into tubes, adjusting the volume to 1.5 ml with the extraction solution. Analyses were carried out with an HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler connected to an Agilent Ion Trap XCT Plus mass spectrometer (©Agilent Technologies) using an electrospray interface. Before injection, 100 ml of each fraction were filtered again through Millex filters (Ø 0.22 mm). Each sample (8 ml) previously dissolved in mobile phase A was injected onto a Zorbax SB-C18 HPLC column (5 mm, 150 0.5 mm, Agilent Technologies) at 40 C and eluted at a flow rate of 10 ml min 1. Mobile phase A [water/acetonitrile/formic acid (94.9/5/0.1, v/v/v)] and mobile phase B [water/acetonitrile/formic acid (10/89.9/0.1, v/v/v)] were used for the chromatographic separation. The elution consisted of maintaining 100% A for a period of 5 min, and then using a 10 min linear gradient from 0 to 6% B, followed by another 5 min linear gradient from 6 to 100% B, and finally keeping 100% B for another 5 min. The column was equilibrated with the starting composition of the mobile phase for 30 min before each analysis. The UV chromatograms were recorded at 280 nm with the diode array detector module (Agilent Technologies). Different control samples with known concentrations of each component (0.001, 0.01, 0.05, 0.1, 0.2 and 0.5 mg l 1) were also analyzed under the same conditions. The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V and a scan speed of 22,000 (m/z)/s from 50 to 500 m/z. The nebulizer gas (He) pressure was set to 30 psi, while the drying gas was set to a flow of 6 l min 1 at 350 C. Mass spectra were obtained using the Data Analysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany). For quantification of ABA and JA, calibration curves were constructed for each component analyzed using internal standards: [2H6]cis, trans-abscisic acid and [2H5](±)-JA (Olchemin Ltd, Olomouc) (0.001, 0.01, 0.05, 0.1, 0.2 and 0.5 mg l 1) and corrected for 0.1 mg l 1. The ACC and SA were quantified by the external standard method, using the same concentration of the product
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(SigmaeAldrich Inc., St Louis, MO, USA). Recoveries ranged between 92 and 95%. Three biological replicas were quantified per sample. 2.6. Experiment design and data collection Three replications with 9 samples of stems and leaves from each of the cultivars per replication were used. The stem and leaves weight and the number of regenerated shoots per callus (regeneration rate) were recorded. Significance was determined by ANOVA and the significance (P 0.05) of differences between mean values was tested by Duncan's New Multiple Range Test. 3. Results and discussion Changes in the endogenous hormonal content produced by a rez-Jime nez et al., 2012, 2014) in proved-organogenic media (Pe plants of three peach cultivars and two peach almond hybrids have been studied. Furthermore, the differences between endogenous hormonal content and somatic organogenesis of the cited genotypes in a media without PGRs and in the organogenic media with cytokinins were also evaluated. In this study, weight of stems and leaves were recorded in plants cultured in T0 and T1 in order to study the relationship between the development of the shoot and its organogenic response. As it is shown in Table 1, stems and leaves weight were markedly lower in those shoots cultured in T0 than in those cultured in T1 due to PGRs action. Nonetheless, a lower leave weights was found in genotypes with the higher organogenesis capacity, the peach almond hybrids. Although the weight of leaves was higher in the cultivars than in the rootstocks, their number was observed to be lower, but with a longer size. This results are supported by the reports that relate inbreeding depression showed by highly inbreed cultivars (Charlesworth and Charlesworth, 1987), such as peach cultivars, and hybridity with rez-Jime nez et al., 2012). On the vigor and/or organogenesis (Pe other hand, regeneration rates obtained in this work are according rez-Jime nez et al., 2012, 2014), since with previous research (Pe shoot regeneration from peach rootstock calli were significantly higher (P 0.05) than the rates obtained in the studied peach cultivars (Table 1). The quantification of endogenous hormone content was carried out in stem and leaves of peach cultivars and rootstocks. Whilst stem results showed (Fig. 1) a relation with plant organogenesis, the hormone content in leaves could not have been related with organogenesis in any of the studied phytohormones (data not shown). The organogenic calli are induced from the bottom part of the stem; thus, the quantification of the organogenesis-causing hormones may be more accurate in this proximal organ than in leaves when studying the regeneration capacity of this baseformed callus. Results concerning the two endogenous cytokinins measured from the stem of different peach cultivars and rootstocks are
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presented in Fig. 1A, B. Levels of Z increased in the shoots cultured in T1 in every genotype when compared with shoots cultured in T0. Notwithstanding, the content in ZR decreased after the treatment with cytokinins except in the rootstocks, where the amount of ZR markedly increased. Z concentrations were higher in the stem of the peach cultivars than in the stem of the rootstocks. On the contrary, ZR content was markedly higher in hybrids than in cultivars. Other studies conduced in woody plants support higher s levels of Z in organogenic explants (Centeno et al., 1996; Valde et al., 2005; Pe rez-Jime nez et al., 2014) and et al., 2001; Mala lower levels of ZR (Centeno et al., 1996) when cultured in media s et al. (2001) and Pe rezwith BA. However, other authors as Valde nez et al. (2014) reported an increment of ZR in organogenic Jime explants. Furthermore, whilst endogenous concentrations of the studied cytokinins did not show any relation with organogenesis in explants cultured in T0, the differences between the most responsive genotypes were noted after being cultured in T1. Same results were obtained for ZR. Results showed in this study manifest a different response to the treatment depending on the genotype. A higher organogenesis rate does not seem to be related with natural endogenous cytokinin content but with the response to PGRs signals, since explant tissues consist of cells with distinct capacity to nez, 2005). respond to an induction treatment (Jime The genotypes showed the same pattern regarding the analysis resulting from the quantification of IAA (Fig. 1C). No differences among genotypes were found between the hybrids and the cultivars in T0. Nevertheless, results pointed out to a lower concentration of IAA in those genotypes that presented a higher organogenesis rate after T1, showing a clear response to the treatment. This is contrary to previous research developed in peach calli, where no relation between IAA and organogenesis was found rez-Jime nez et al., 2014). (Pe The results obtained in the analysis of the stems showed no differences in the ABA content between shoots grown in T0 and T1 (Fig. 1D). Only Flariba presents a slight difference between shoots cultured in different media. This finding indicates no influence of the media in the amount of the endogenous ABA; in this study, the ABA concentration depends on the genotype. However the effect of ABA on organogenesis is still not clarified. Lee and Huang (2013) reported a higher amount of ABA in organogenic genotypes of rice when compared with the non-organogenic ones. On the contrary, other studies suggest that a high level of ABA does not inrez-Jime nez crease shoot organogenic rate (Huang et al., 2012; Pe et al., 2014). This is according with our results, where ‘Garnem’ and ‘GF677’ were the genotypes with the lower ABA content and higher organogenic rate. The stress-related phytohormone JA did not show to be affected by the culture media, being levels of JA as high in T0 as in T1 (Fig. 1E). In both treatments the lower levels of JA were coincident with the genotypes with highest regeneration rates, this is contrary to previous results where no differences between hybrids and cultivar rez-Jime nez et al., 2014). Since high levels of JA have were found (Pe
Table 1 Adventitious shoot regeneration from callus explants, stem and leaves weight of three peach cultivars and two peach rootstocks. Cultivars
Flariba
Treatment without plant growth regulator Stem weight (g) 0.015 ± 0.004c Leaves weight (g) 0.161 ± 0.067c Treatment with plant growth regulator Regeneration rate 2.222 ± 0.830c Stem weight (g) 0.282 ± 0.069b Leaves weight (g) 1.146 ± 0.226b
Alice Bigi
UFO-3
0.042 ± 0.005a 0.120 ± 0.045c
0.037 ± 0.005 ab 0.235 ± 0.029b
3.889 ± 0.790b 1.147 ± 0.185a 1.940 ± 0.129a
2.667 ± 0.624c 0.343 ± 0.060b 1.304 ± 0.224b
Data represent average ± SD values. Different letters indicate a significant difference by LSD test (p 0.05).
GF677
Garnem
0.030 ± 0.004b 0.134 ± 0.020c
0.041 ± 0.003a 0.465 ± 0.027a
12.222 ± 1.832a 0.321 ± 0.059b 0.885 ± 0.129c
13.889 ± 2.330a 0.341 ± 0.051b 0.672 ± 0.088c
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Fig. 1. Endogenous content in (A) zeatin (Z), (B) zeatin riboside (ZR), (C) indole-3-acetic acid (IAA), (D) abscisic acid (ABA), (E) jasmonic acid (JA), (F) 1-aminocyclopropane-1carboxylic-acid (ACC), and (G) salicylic acid (SA) in stem of peach (cv. Flariba, cv. Alice Bigi, cv. UFO-3) and peach rootstocks (cv. GF677, cv. Garnem). Data represent average ± SD values. Bars with different letters indicate a significant difference by LSD test (p 0.05) between cultivars. * denotes significant differences between T0 and T1.
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Fig. 2. (A) Ratio between indole-3-acetic acid (IAA) and abscisic acid (ABA) in callus of peach (cv. Flariba, cv. Alice Bigi, cv. UFO-3) and peach rootstocks (cv. GF677, cv. Garnem) (B) Ratio between zeatin (Z) and indole-3-acetic acid (IAA) in organogenic and non-organogenic callus of peach. Data represent average ± SD values. Bars with different letters indicate a significant difference by LSD test (p 0.05). * denotes significant differences between T0 and T1.
been identified as stress indicators in tissue culture (Rudus et al., 2009), the peach cultivars seem to be more sensitive to the tissue culture techniques than the hybrids. Regardless of the results, the JA concentration seems to be more a consequence of the tissue culture conditions (Rudus et al., 2009) than an inductor of any regeneration process. This is supported by our previous findings in which no differences were found between the amount of JA and rez-Jime nez et al., 2014). somatic organogenesis (Pe The results of the quantification reflect lower levels of ACC in peach almond hybrids than in peach cultivars (Fig. 1F). The role played by ethylene in plant regeneration has been described as controversial and genotype-dependent (Yasmin et al., 2014). On one hand, some authors pointed out to a stimulating effect of ACC (ethylene precursor) in woody plants regeneration (Predieri et al., lez et al., 1997); on the other hand, ethylene has 1993; Gonza been linked with organogenesis and growth inhibition by Arigita et al. (2003). According to the data obtained in the current and in previous studies, ACC may be an inhibitor of the morphogenesis in peach, since its lower amount is found in those genotypes with the highest regeneration rates. The SA levels (Fig. 1G) found in calli of the studied genotypes remained constant in media with PGRs and in the control for all the genotypes. On the other hand, no relationship was found according to higher records of regenerated shoots. Although SA has been reported as an embryogenesis enhancer (Ahmadi et al., 2014), there are no records that conclude that SA has a clear effect on morphogenesis in woody plants. Huang et al. (2012) reported that ABA and IAA together may be involved in inducing shoot organogenesis on callus. In our study, a significant effect was observed in the case of the IAA/ABA ratio (Fig. 2A) where the lower levels of this ratio were found in the hybrids. Changes between T0 and T1 in this ratio were detected in every genotype but ‘Garnem’, in which they are the same. On the contrary, the Z/IAA ratio (Fig. 2B) could not be associated to organogenesis. The relation between Z and AA content varied in all the studied genotypes depending on the media where the genotypes were cultured but differences between the hybrids and the cultivars only were found in T0, where no organogenesis response were detected. In this study, the results showed an important influence of the genotype in organogenesis. Notwithstanding, this influence is not always given by the basal endogenous hormone naturally contained in each genotype but sometimes by the response of these studied genotypes to the culture media. It is well known that cytokinins and auxins at certain concentrations may be inducers of somatic morphogenesis. However, the basal concentrations of
these hormones in a media without PGRs is not related to organogenesis before being exposed to a media with PGRs, actually the concentrations of these hormones were very different in the most responsive genotypes. Only after the exposition to an organogenic callus inducing media, adventitious regeneration occur in these genotypes. On the other hand, hormones such as ABA, ACC, JA and SA seemed not to be affected by the culture media. A few articles showing the relationship between somatic regeneration and endogenous hormones content, genotype and/or culture media in woody plants have been published but to our knowledge this is the first study showing clearly the weight of each one of these elements in the regeneration process. Contributors rez-Jime nez. Planning of the experiment, lab work, Margarita Pe results analysis and writing. Elena Cantero-Navarro. Collaboration in endogenous hormones measurements. rez-Alfocea. Collaboration in results concerning Francisco Pe endogenous hormones measurements. Cos-Terrer. Planning of the experiment and results analysis. Jose Acknowledgments s Paredes Jime nez for the EnThe authors want to thank Andre glish revision of the manuscript. This report was supported by the Instituto Nacional de Investigaciones Agrarias (INIA) (RTA2008rez00121-00-00) and by a fellowship provided to Margarita Pe nez also by INIA. Jime References Abbott, A.G., Arús, P., Scorza, R., 2008. Genetic engineering and genomics. In: Layne, D.R., Bassi, D. (Eds.), The Peach: Botany Production and Uses. CABI, Wallingford, United Kingdom, pp. 85e105. Ahmadi, B., Shariatpanahi, M.E., da Silva, J.A.T., 2014. Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult. 116 (3), 343e351. , L., Bairu, M.W., Nova k, O., Plíhalova , L., Dolez Aremu, A.O., Pla ckova al, K., Finnie, J.F., Van Staden, J., 2014. How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea? Plant Cell Tissue Organ Cult. 118 (2), 245e256. Albacete, A., Ghanem, M.E., Martínez-Andújar, C., Acosta, M., S anchez-Bravo, J., rez-Alfocea, F., 2008. Hormonal changes in Martínez, V., Lutts, S., Dodd, I.C., Pe relation to biomass partitioning and shoot growth impairment in salinised tomato (Solanum lycopersicum L.) plants. J. Exp. Bot. 59, 4119e4131. s, R., Gonza lez, A., 2003. 1-methylcyclopropene and Arigita, L., Sanchez Tame ethylene as regulators of in vitro organogenesis in kiwi explants. Plant Growth Regul. 40, 59e64.
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