Plant Cell Tiss Organ Cult (2013) 115:385–393 DOI 10.1007/s11240-013-0370-7
ORIGINAL PAPER
Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L. L. Cantelmo • B. O. Soares • L. P. Rocha • J. A. Pettinelli • C. H. Callado • E. Mansur A. Castellar • R. F. Gagliardi
•
Received: 11 April 2013 / Accepted: 10 August 2013 / Published online: 21 August 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 lM PIC were inoculated in hormone-free full-or half-strength liquid MS
L. Cantelmo B. O. Soares L. P. Rocha J. A. Pettinelli E. Mansur R. F. Gagliardi (&) Nu´cleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Rua Sa˜o Francisco Xavier, 524, PHLC sala 602, Maracana˜, Rio de Janeiro, RJ 20550013, Brazil e-mail:
[email protected] C. H. Callado Laborato´rio de Anatomia Vegetal, Universidade do Estado do Rio de Janeiro, Rua Sa˜o Francisco Xavier, 524, PHLC sala 225, Maracana˜, Rio de Janeiro, RJ 20550013, Brazil A. Castellar Laborato´rio de Produtos Naturais e Alimentos, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco A, 2o andar, salas 4 e 10, Ilha do Funda˜o, Rio de Janeiro, RJ 21941590, Brazil
medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide. Keywords Phytolaccacea Embryogenic cell suspension Direct somatic embryogenesis Callus Dibenzyl trisulfide Picloram
Introduction Petiveria alliacea L. (Phytolaccacceae) is a shrub native to the Amazon rainforest, with a wide distribution in the tropical and subtropical regions of South America, Central America, and Africa (Kubec and Musah 2001; Okada et al. 2008). There are many reports of its popular use (Taylor 2005; Mitchell and Ahmad 2006) in different countries as a therapeutic agent, because of its several pharmacological properties. A wide variety of biologically active substances such as saponins, alkaloids, flavonoids, sulfides, tannins and coumarins has been found in different parts of the plant (Delle-Monache and Suarez 1992; Delle-Monache et al. 1996; Zoghbi et al. 2002). The main compounds obtained in this species are lipophilic polysulfides known as petiverins, which include dibenzyl trisulfide (DTS) (Ro¨sner et al. 2001; Williams et al. 2007; Uruen˜a et al. 2008). DTS
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has been implicated in the immunomodulatory, cytostatic and neurotoxic activities found in Petiveria roots (De Sousa et al. 1990; Ayedoun et al. 1998; Kubec and Musah 2001; Williams et al. 1997, 2003, 2004, 2012). Treatment of patients with cancer and leukemia in Cuba was reported by Chirinos (1992). Among the popular uses, P. alliacea is recommended for treatment of stomach tumors, breast lumps and leukemia (Ruffa et al. 2002). Its inhibitory effect on the proliferation of neuroblastoma cells in vitro has also been reported (Ro¨sner et al. 2001). The dibenzyl trisulfide cytotoxic effects were not found in the cell line of hepatic carcinoma (Ruffa et al. 2002) but were observed in differentiation events of HL-60 promyelocytic cells, which are used as a model system for testing new substances with potential for the suppression of carcinogenesis (MataGreenwood et al. 2001). Some trisulfide derivatives have the same mode of action as the drug Paclitaxel, inhibiting tumor cells by disturbing the tubulin-microtubule dynamic equilibrium, and thus are useful for cancer chemotherapy (An et al. 2006). Natural populations of P. alliacea may suffer genetic erosion due to overexploitation in countries where this plant is in high demand. In addition, the biological activities of field plants are influenced by seasonal variations and microclimates that affect secondary metabolism (Bourgaud et al. 2001). Tissue culture is a suitable alternative for plant production and selection of genotypes with a high content of bioactive substances. Studies on tissue culture of P. alliacea established protocols for micropropagation from axillary buds, callus and cell suspension cultures (Castellar et al. 2011). Embryogenic systems were also obtained, but without recovery of whole plants (Webster et al. 2004). The development of in vitro systems for P. alliacea allowed phytochemical studies that showed the production of DTS in embryogenic cultures (Webster et al. 2008). Qualitative and quantitative differences in volatile compounds and essential oils between in vitro and in vivo plants were also described (Castellar et al. 2013). Considering that in vitro systems are also useful tools for modulating the production of substances of pharmacological interest, the objective of this study was the establishment of embryogenic cultures that can be used for both mass production of plants and for future isolation and characterization of bioactive constituents of P. alliacea.
Materials and methods Plant material and culture conditions Petiveria alliacea L. plantlets were maintained in vitro for 5 years (lineage AL) through the culture of nodal segments
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from mother plants cultivated in a residential garden (22°550 55.2700 S 43°110 49.9000 W, 167 m a.s.l.). The species was identified by Dr. Alexandre Gabriel Christo (Rio de Janeiro Botanical Garden), and a voucher specimen has been deposited in the Herbarium of Rio de Janeiro State University (HRJ No. 10371). In vitro plantlets were subcultured at 2-month intervals and maintained at 30 ± 2 °C, under a 16-h photoperiod and mean light intensity of 46 lmol m-2 s-1, according to Castellar et al. (2011). Embryogenic induction Leaf segments (1.0 cm2) of plantlets grown in vitro were inoculated on MS (Murashige and Skoog 1962) medium added with different plant-growth regulators: thidiazuron (TDZ) (2.2, 4.5, 13.6 or 22.8 lM), cis-zeatin (2.2, 4.5, 13.6 or 22.8 lM), picloram (PIC) (2.0, 4.0, 12.4 or 20.0 lM) or 2,4-dichlorophenoxyacetic acid (2,4-D) (2.2, 4.5, 13.5 or 22.6 lM), all from Sigma-Aldrich. Media were adjusted to pH 5.8 prior to adding agar (8 g L-1, Merck), autoclaved (121 °C, 104 kPa) for 15 min, and dispensed into 6 9 8 cm flasks (30 mL per flask) closed with polypropylene caps. In preliminary experiments, leaf segments were placed with their abaxial surface in contact with the culture medium and incubated either in darkness or under light with the same photoperiod described above. Monthly subcultures were performed by transferring the material to medium of similar composition, and were evaluated for 6 months. Liquid cultures Friable non-embryogenic callus tissue obtained from leaf pieces in response to 20.0 lM PIC or 22.6 lM 2,4-D after 6 months of culture was transferred (1.0 g per 10 mL) to liquid MS medium either devoid of growth regulators, or added with 20.0 lM PIC or 22.6 lM 2,4-D. The embryogenic portions that were simultaneously formed at the surface of the explants in response to 20.0 lM PIC were inoculated in hormone-free full-or halfstrength liquid MS (20 somatic embryos at the globularstage in 30 mL of medium). Cultures were maintained on a rotating shaker at 100 rpm under the same light conditions previously described. Subcultures were performed monthly to media of similar composition every 40 days. Culture biomass was evaluated by measuring fresh weight after vacuum filtration. Embryos were subcultured and counted every 40 days. Cotyledonary-stage embryos originated in these cultures were transferred to hormone-free halfstrength MS medium solidified with 0.7 % agar (MS0). At the end of each period, the number of somatic embryos was recorded, and the rate of conversion was calculated by the ratio between the total number of secondary embryos
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observed in each subculture, and the initial number of embryos inoculated. These experiments were performed in triplicate, repeated twice, and recorded for 120 days.
Results
Histological analysis
Primary embryogenesis
Leaf fragments with globular structures were fixed in a solution of 2.5 % glutaraldehyde and 4.0 % formaldehyde buffered with 50 mM phosphate at pH 7.2 after 35 and 90 days of culture on MS medium supplemented with 20.0 lM PIC or 22.6 lM 2,4-D, and stored at 4 °C. Subsequently, the samples were dehydrated in an ascending series of ethanol solutions (Johansen 1940), and embedded in glycol methacrylate resin (Feder and O’Brien 1968) or in paraffin (Sass 1958). Serial sections 5–11 lm thick were obtained using a rotary microtome (Leica RM 2025) and stained in O-toluidine blue (Johansen 1940) or astra bluefuchsin (Roeser 1972). The images were obtained using the image capture system Image-Pro Express 6.0 for Windows and a Collor Q R3 video camera attached to an Olympus BX41-BF-I-20 microscope.
Leaf segments showed different responses according to the culture conditions. Explants cultured in the presence of zeatin became chlorotic irrespective of the light conditions, and did not show any morphogenic response up to 120 days of culture. Explants cultured on semi-solid MS medium containing 22.7 lM TDZ showed a low frequency of friable callus formation (Table 1; Fig. 1a). In addition to direct somatic embryogenesis, the cultures obtained in the presence of 13.5 lM and 22.6 lM 2,4-D developed callus and roots (Fig. 1b), but showed increased browning following the subcultures. On the other hand, explants maintained on medium containing 20.0 lM PIC displayed direct somatic embryogenesis, with the formation of healthy embryos over a period of 4 months. Interestingly, even oxidized leaf pieces continued to produce primary embryos after an additional period of 6 months (Fig. 1d). Embryo differentiation was asynchronous, as all the different stages of development (globular, cordiform and cotyledon) were identified (Fig. 1e, f). Exposure to light significantly improved both the frequency of embryogenesis and the number of embryos per explant on both media (Fig. 2).
Acclimatization Whole plantlets were acclimatized in a greenhouse covered with mesh (SombriteÒ) that produced 50 % reduction in sunlight, in pots containing 100 % garden soil (pH 5.5), and mean temperature of 25 °C. The roots were washed, and the plantlets were then subjected to the following treatments: (1) direct transfer to greenhouse conditions; (2) prior treatment in glass vials containing 30 ml of filtered water, sealed with plastic film and kept at 30 ± 2 °C in a growth chamber under 16 h photoperiod and mean light intensity of 46 lmol m-2 s-1 for 10 days; the plastic covers were gradually perforated until total removal; (3) transfer to pots containing the same garden soil as above, and covered with plastic bottles, which were gradually opened over a period of 10 days, and finally removed. To evaluate the adaptation response of plantlets to the three acclimatization treatments, the survival, plant height, number of nodes, as well as length and width of the leaves were measured after 30 days of ex vitro growth. The experiment was repeated three times with 11 plantlets per treatment. Statistical analysis The data pertaining to embryogenic induction, proliferation and conversion were analyzed separately using a one-way analysis of variance (ANOVA). The differences among means were compared by high-range statistics through the Tukey–Kramer comparisons test (P \ 0.05), The acclimatization process was evaluated by Student’s t test using the GraphPad Instat Software, San Diego, CA, USA.
Somatic embryogenesis on semi-solid media
Secondary embryogenesis Culture of primary embryos on half-strength MS medium gelled with 0.2 % phytagel resulted in highly repetitive embryogenesis that was maintained over four subcultures during 6 months (Fig. 4a). Plantlets were obtained from these secondary embryos at an estimated frequency of 5 % after 180 days of culture. Subsequently, the material displayed gradual oxidation, impairing the development of new embryos (data not shown). Histological analysis Leaf segments after 35 days of culture showed significant proliferation of meristematic cells close to the vascular system (Fig. 3a), especially associated with the phloem tissue. In some cases, dedifferentiation of cells on the lower face of the epidermis could be observed. The material analyzed after 90 days of incubation showed direct development of somatic embryos, which were characterized by the presence of protoderm, procambium and ground meristems (Fig. 3b). Meristematic centers were still present, being formed by meristematic cells at the periphery and cells elongating and differentiating in the center (Fig. 3c).
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388 Table 1 Major morphogenetic responses by leaf explants of P. alliacea L., after 120 days, under different culture conditions
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Plant growth regulator
TDZ
PIC
The data represent the qualitative response and the percentage of two replicates or six leaf pieces per treatment CC compact callus, FC friable callus, SE somatic embryos, ND not determined
2,4-D
Concentration (lM)
Light Morphogenesis
Dark Response (%)
Morphogenesis
Morphogenesis response (%)
2.2
Necrosis
0
CC
66
4.5
Necrosis
0
FC
33
13.6
CC
33
ND
ND
22.8
FC
33
CC
33
2.0
SE/CC
100
SE
4.0
SE/CC
100
SE
100
12.4 20.0
SE/CC SE/FC
100 100
SE FC
100 100 100
2.2
SE/FC
100
CC
4.5
SE/FC
100
SE
13.5
SE/FC
100
SE/CC
100
22.6
SE/FC
100
SE/FC
100
66
Liquid cultures
Acclimatization
Following the occurrence of direct somatic embryogenesis, leaf pieces showed the formation of friable non-embryogenic callus in different regions of the explants, after 120 days of culture in the presence of 22.6 lM 2,4-D or 20.0 lM PIC (Fig. 1). Isolated embryos and non-embryogenic regions were inoculated separately into liquid medium in order to establish suspension cultures. Somatic embryos inoculated into MS0 or MS medium showed high rates of secondary embryogenesis (Fig. 4b), resulting in the production of a mean of 35 embryos from each embryo inoculated at the initiation of the culture, which resulted in a significant increase in biomass (Fig. 4c). Embryos started the conversion process in the same medium (Fig. 1g), and originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar (Fig. 1h). The secondary somatic embryos produced in liquid culture, in the cotyledon stage and starting the conversion (Fig. 1g), were isolated (Fig. 1h) and transferred to MS0 medium solidified with 0.7 % agar, where they developed into complete plants. In addition to plantlet development, the process of repetitive embryogenesis was continued in MS0 medium solidified with 0.7 % agar (Fig. 1i). Although part of the embryos (around 1 %) developed deformed buds, whole plants without apparent morphological abnormalities were formed after transfer to MS0 (Fig. 1j). Non-embryogenic cultures obtained from callus formed in the presence of 20.0 lM PIC or 22.6 lM 2,4-D showed an inverse relationship between the rate of cell growth and the number of subcultures (Fig. 4d). The formation of cell agglomerates of varying sizes occurred despite continuous stirring. These cultures showed consistent foaming, indicating the probable excretion of saponins into the liquid medium.
Phenotypically normal plantlets converted from secondary somatic embryos formed on liquid or solidified MS medium were acclimatized using three treatments. Plantlets directly transferred to pots containing garden soil and maintained in a greenhouse did not survive the acclimatization process; plantlets that were initially covered with plastic film or plastic bottles showed better ex vitro adaptation (Table 2). The highest percentage (90 %) survival was achieved when plastic bottles were used rather than PVC film (60 %) (Table 2). Among the variables evaluated, only the leaf width showed a statistically significant difference (P \ 0.5) among the treatments; wider leaves were obtained from plantlets hardened using plastic bottles (Table 2).
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Discussion The growth regulators used in plant tissue culture to produce de novo growth may produce a variety of responses, including formation of callus, somatic embryos, shoots and/or roots (Morini et al. 2000; Jimenez 2005). The morphogenic response of in vitro leaf pieces of P. alliacea was clearly influenced by the type of growth regulator used. Although there are reports of somatic embryogenesis from in vitro (Pacheco et al. 2007) and in vivo plants (Webster et al. 2008) in response to cytokinins (GoebelTourand et al. 1993; Gill and Saxena 1993; Pacheco et al. 2007), in this study in vitro leaf explants inoculated on MS medium supplemented with zeatin became chlorotic and showed no morphogenic response. Typically, the induction of somatic embryos is achieved in response to high concentrations of auxin. A second
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b
a
389
c
rt cl
d
e
fc
gs
g
j
f
c t
g
h
i
j
Fig. 1 In vitro morphogenesis from leaf explants of Petiveria alliacea L. under different culture conditions: a Friable callus (arrow) formed in the presence of 22.7 lM TDZ, after 30 days of culture; b Direct root formation (rt arrow) and callus (cl arrow) formed in the presence of 13.5 lM of 2,4-D, after 60 days of culture; c Somatic embryo (arrow) developed in the presence of 20.0 lM PIC, after 10 months of culture. d Friable callus (fc arrow) and globular structures (gs arrow) originated in the presence of 20.0 lM PIC after 120 days of culture. e Secondary somatic embryos at globular (g
arrow), torpedo (t arrow) and cordiform stages (c arrow) (bar = 1,000 mm); f Secondary somatic embryos at the cotyledonary stage (bar = 1,500 mm); g Somatic embryos cultured in hormonefree MS liquid medium; h Somatic embryos obtained from the hormone-free MS liquid medium starting conversion into plants; i Clusters of somatic embryos formed in MS medium solidified with agar, yielding new somatic embryos and plants; j Whole plant obtained from somatic embryos formed in MS liquid medium after transfer to gelled MS medium. Bar = 1 cm
medium with a reduced concentration or devoid of auxin is required for the development of these structures (Arnold et al. 2002). However, the mode of action of auxins in the embryogenic process is not fully understood (Kaur and Kothari 2004; Pinto et al. 2011; Ooi et al. 2012). In the present study, MS medium supplemented with 2,4-D promoted the formation of somatic embryos from in vitro leaf explants of P. alliacea. In addition to the formation of embryos, callus and roots were produced from the explants, confirming that 2,4-D can induce different morphogenetic responses such as the formation of somatic embryos, shoots and roots in the same propagule (Morini et al. 2000). This could be explained by the action of 2,4D and also of other plant growth regulators in the processes
of transcription and methylation of genes, which encode proteins that influence specific molecular and physiological processes that can lead to different morphogenetic pathways (Fehe´r et al. 2003). Picloram is also a highly potent auxin, capable of inducing somatic embryogenesis in different species (Mendoza and Kaeppler 2002; Hankoua et al. 2006; Karami et al. 2007; Ahmed et al. 2011). The occurrence of direct somatic embryogenesis from leaf explants of P. alliacea was confirmed by histological studies. According to Yeung (1995), one of the evidences of early embryo histodifferentiation is the formation of a protoderm, the tissue lining of the embryo that was identified in this study, confirming the direct origin of the somatic embryos. The histological analysis of the
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Average SE no./explant
300
a
a 250 200
a
150 100
b b
50 0 PIC light
PIC dark
2,4D light
2,4D dark
Culture conditions
Fig. 2 Effect of light conditions on somatic embryogenesis induction from leaf explants of P. alliacea, after 120 days in the presence of 20 lM PIC or 22.6 lM 2,4-D. *Different letters denote statistical differences between treatments (P \ 0.05) according to Tukey’s multiple range test
regenerative process clearly showed that the somatic embryos observed at the surface of in vitro leaves were formed from cells near the vascular bundle, without an intervening callus phase. This contrasts with the first study on somatic embryogenesis of P. alliacea, which reported the induction of an indirect pathway in response to BAP and NAA (Webster et al. 2004, 2008). The highly repetitive secondary embryogenesis from the primary embryos observed in this study was probably due to the lack of some of the natural controls that exist in the intact plant (George et al. 2008). Secondary embryogenesis has been described in several agronomic, ornamental and medicinal species (Devi and Narmathabai 2011; You et al. 2011, 2012; Chen and Hong 2012). The high multiplication rates observed in the liquid cultures established here may have been favored by the absence of auxin in the medium (Arnold et al. 2002), and by the beneficial effect of aeration provided by continuous stirring (Vargas et al. 2005). The efficiency of plantlet recovery from the secondary embryos obtained in liquid cultures was very high. Another positive characteristic of this system is that initiation of conversion did not require any specific treatment. Although no morphological abnormalities were observed, the clonal fidelity of recovered plants will be assessed in further studies. Suspension cultures derived from non-embryogenic callus showed intense foaming in the medium, suggesting the excretion of saponins. In spite of the lack of increase in biomass, these cultures may still represent an interesting system for investigating the production and excretion of metabolites. According to some authors, cell growth and metabolite production in cultures in vitro can be inversely proportional, considering that many cultures start to produce metabolites only after cell multiplication slows (Lee and Chan 2004; Maharik et al. 2009). The successful recovery of whole plants from somatic embryos in the present study demonstrates the possibility
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b
pt
gm pc
c
cl mc
ec
Fig. 3 Histological aspects of leaf culture of Petiveria alliacea L.: a Cross section of leaf explants showing proliferation of meristematic cells (arrows) in area adjacent to the vascular bundle (circle), after 35 days of culture in the presence of 22.6 lM 2,4-D; b Somatic embryo at cordiform stage, showing protoderm (pt), procambium (pc) and ground meristem (gm), after 90 days of culture in the presence of 20.0 lM PIC; c Meristematic center forming callus, after 90 days of culture in medium with 20.0 lM PIC added (cl). Note the meristematic cells in the periphery (mc) surrounding the elongated and partially differentiated cells (ec). Bar = 200 lm
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Fig. 4 In vitro proliferation of Petiveria alliacea L.: a Rate of secondary embryogenesis in MS medium gelled with 0.2 % phytagel over 180 days of culture; b Secondary embryogenesis and conversion process from secondary SE subcultured in liquid MS
medium, after 120 days; c Growth kinetics and increase in biomass from somatic embryos kept in liquid hormone-free culture medium; d Growth kinetics of nonembryogenic cells in suspension culture. Data are mean ± SD from 4 repetitions consisting of 20 S
Table 2 Influence of different acclimatization treatments on the survival and growth of somatic-embryo-derived in vitro plantlets of P. alliacea 30 days after transfer from in vitro culture Treatment
Survival (%)
Number of nodes*
Shoot length (cm)*
Leaf length (cm)*
Leaf width (cm)*
Plastic bottle
90
22.8 ± 7a
7.2 ± 1.8a
3.8 ± 1.1a
2.3 ± 0.7a
PVC film
60
19.6 ± 7.1
a
7.7 ± 2.7
a
3.2 ± 1
a
1.8 ± 0.6b
Obs.: Each value represents the mean ± SD of three repetitions with 11 in vitro plants per treatment * Means followed by same letter in column do not differ significantly by Student’s t test (P [ 0.05)
of using this system for mass propagation of this species, as suggested by Webster et al. (2008). In addition to plant propagation, embryogenic systems can be used for the production of plant metabolites of interest, often produced in quantities that are greater than those found in intact plant materials of several species (Szypula et al. 2005; Shohael et al. 2006, 2007; Webster et al. 2008, Aslam et al. 2009). In the case of P. alliacea, the production of DTS by somatic embryos was
approximately 30-fold greater than that obtained from rhizogenic and embryogenic callus, in contrast to the low yields obtained from the plant material grown in the wild (Webster et al. 2008). The formation of adventitious roots directly on the surface of the leaf explants in response to 2,4-D opens another interesting perspective for the study of in vitro DTS production, taking into account that its roots are the main organ for DTS accumulation in this species (Kubec and Musah 2001; Okada et al. 2008).
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To our knowledge, this is the first report on successful vegetative multiplication of P. alliacea via somatic embryogenesis. The repetitive embryogenic system described here also provides a tool for the study of the regulation of embryo development and the modulation of the synthesis of pharmacological substances. Acknowledgments This work was sponsored by the Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro (FAPERJ), the National Council for Scientific and Technological Development (CNPq/Brazil), and the Foundation for the Coordination of Improvement of Higher Education Personnel (CAPES).
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