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Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tplb20
SHAPE Strategies for ex situ conservation of Centaurea cineraria subsp. circae (Asteraceae), an endemic plant from Lazio (Italy) a
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Alessio Valletta , Anna Rita Santamaria , Giuseppe Fabrini , Noemi Tocci , Valdir b
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Cechinel Filho , Theodoro Wagner , Elisa Brasili & Gabriella Pasqua a
Dipartimento di Biologia Ambientale, Sapienza Università di Roma, Rome, Italy
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Nucleous of Chemical-Pharmaceutical Investigations (NIQFAR), University of Itajaí Valey, Itajaí, Brazil Accepted author version posted online: 19 Jun 2015.
To cite this article: Alessio Valletta, Anna Rita Santamaria, Giuseppe Fabrini, Noemi Tocci, Valdir Cechinel Filho, Theodoro Wagner, Elisa Brasili & Gabriella Pasqua (2015): SHAPE Strategies for ex situ conservation of Centaurea cineraria subsp. circae (Asteraceae), an endemic plant from Lazio (Italy), Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, DOI: 10.1080/11263504.2014.1001464 To link to this article: http://dx.doi.org/10.1080/11263504.2014.1001464
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Publisher: Taylor & Francis Journal: Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology DOI: http://dx.doi.org/10.1080/11263504.2014.1001464
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ALESSIO VALLETTA1, ANNA RITA SANTAMARIA1, GIUSEPPE FABRINI1, NOEMI TOCCI1, VALDIR CECHINEL FILHO2, THEODORO WAGNER2, ELISA BRASILI1 & GABRIELLA PASQUA1 Dipartimento di Biologia Ambientale, Sapienza Università di Roma, Rome, Italy and 2Nucleous of Chemical-Pharmaceutical Investigations (NIQFAR), University of Itajaí Valey, Itajaí, Brazil
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Corispondence: Gabriella Pasqua, Dipartimento di Biologia Ambientale, Sapienza Università di
Acknowledgements
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Email:
[email protected]
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Roma, Piazzale Aldo Moro 5, 00185, Roma, Italy. Tel: +39 06 49912414. Fax: +39 06 49912414.
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This work was supported by grants CNPq-Brazil and MIUR-Italy C26A11XLZ5.
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Strategies for ex situ conservation of Centaurea cineraria subsp. circae (Asteraceae), an endemic plant from Lazio (Italy)
Abstract Centaurea cineraria subsp. circae is an endemic plant with a distribution area limited to Circeo mountain (Lazio, Italy), whose population was estimated in a very low number of individuals. The
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aim of this work is to investigate ex situ conservation strategies such as achene collection and in
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the achenes demonstrated that only 5.5% of them were morphologically healthy. Seed germination tests showed that seeds do not display dormancy and that germination does not require pre-
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treatments. The higher germination rate (67.5%) was observed under a photoperiod of 12 h/12 h (light/dark) and temperature regime +20 °C/+10 °C. The in vitro studies demonstrated that
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micropropagation, acclimatization and the transfer outdoors of C. cineraria subsp. circae are not particularly difficult: 74% of shoot explants in MS medium added with 0.5 mg/L
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benzylaminopurine (BAP) and 2 mg/L kinetin (Kin) formed multiple shoots; 100% of shoots rooted in MS medium added with 0.5 mg/L IBA and over 90% survived the acclimatization phase. After
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been transferred outdoors the totality of in vitro propagated plants bloomed and appeared morphologically indistinguishable from wild plants. Preliminary chemical analyses showed a
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similar profile for in vitro propagated and wild plants.
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Keywords: Centaurea cineraria, ex situ conservation, seed germination, micropropagation, acclimatization Introduction
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vitro plant propagation, that will permit to carry out restoration programs. The test carried out on
The drastic reduction of global plant biodiversity that has occurred in recent decades (Sarasan et al.
2006), strongly imposes the implementation of efficient conservation strategies. There are two main strategies to preserve plant diversity: in situ conservation and ex situ conservation. In situ
conservation means “the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings”, while ex situ conservation
means “the conservation of components of biological diversity outside their natural habitats” (CBD definition, UNCED 1992). The importance of ex situ conservation has gained international recognition with its inclusion in Article 9 of the Convention on Biological Diversity (CBD, UNCED 1992) and in Target 8 of the Global Strategy for Plant Conservation (Sarasan et al. 2006).
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Ex situ conservation can be achieved through different methods, including seed collections and
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Seed storage in facilities, such as seed bank allows large numbers of viable seeds to be stored for extended periods (Walters et al. 2004), providing an effective and economically viable option to
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preserve genetic diversity of plant populations (Hong & Ellis, 1996). Long-term storage depends upon an understanding of desiccation tolerance and sub-zero temperature response. Plant seeds can
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be classified as orthodox, intermediate or recalcitrant. While orthodox seeds tolerate drying to 5% moisture content (10-15% relative humidity [RH]) and storage at −20 °C, recalcitrant seeds lose
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viability under drying or chilling exposure once they are shed from the parent plant. Intermediate seeds tolerate the removal of water to a moisture content equivalent to 20-50% RH, but do not
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tolerate storage below 0 °C (Tuckett et al. 2010). Low seed viability can also be caused by biotic factors such as microrganisms and insect attack. In Centaurea montis-borlae Soldano, it has been
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demonstrated that damages caused by Diptera belonging to Tephritidae family led to a strong
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reduction in seed viability (Boracchia et al. 2007). Tissue culture techniques have been widely used when traditional propagation methods have either failed or proved inadequate. Among other plant tissue culture techniques, micropropagation has
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tissue culture collections.
been successfully applied to ex situ conservation of numerous plant species (Piovan et al. 2011).
Through micropropagation, many endemic and/or endangered species can be quickly propagated and preserved from a minimum of plant material, and with low impact on wild populations. The in
vitro plants obtained through micropropagation can be used as germoplasm collection and in restoration projects.
Centaurea cineraria L. subsp. circae (Sommier) Cela Renz & Viegi (cliff cornflower, Figure 1 A and B) is an endangered endemic species belonging to the family Asteraceae. It is included in the Regional Red Lists of Italian Plants (Conti et al. 1997), LR Status in Italy and LR Status in Lazio. It has a very limited distribution area and it is present mostly in some rocky locations on Circeo
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mountain (San Felice Circeo, LT). In the stretch of coast between San Felice Circeo and Gaeta there
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cineraria subsp. circae is less than 5000 (unpublished results).
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cineraria (Cela Renzoni & Viegi 1982). It has been estimated that the number of individuals of C.
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In this study, we report: i) the results of germination tests and determination of the ecophysiological needs for seed germination of C. cineraria subsp. circae; ii) an efficient method for
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micropropagation of C. cineraria subsp. circae; iii) in vitro plant regeneration by indirect organogenesis from callus of C. cineraria subsp. circae; iv) an histological study to verify the
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vascular connection between the shoot and the root vascular system of in vitro propagated plants of C. cineraria subsp. circae; v) the preliminary results of GC/MS chemical analysis to investigate the
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presence of steroids and/or terpenes in aerial parts of C. cineraria subsp. circae plants harvested from the natural wild population, propagated and growing in vitro, or propagated in vitro and
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transferred in pots (ex vitro).
Materials and methods
Achene collection and storage
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are several intermediate forms between this subspecies and the most common C. cineraria L. subsp.
In August 2012, several hundreds of Centaurea cineraria subsp. circae flower heads were taken from plants growning in San Felice Circeo, Quarto Caldo (Latina, Italy), on limestone rocks and cliffs. The flower heads were collected from 150 plants, randomly and uniformly chosen within the entire population. The flower heads were stored at the Germplasm Bank of the Botanical Garden of Rome (Italy), in a dry environment for 7 days, then the achenes were isolated from the flower heads, and intact ones were separated from those that were empty or insect-damaged. Intact achenes
were separated in two groups, one group was selectred for the ex situ conservation. These achenes were placed in a desiccator containing silica gel for 60 days, before being stored in airtight boxes at a temperature of -20 °C. Humidity and temperature inside the dryer were held constant at 15% RH
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and 15 °C, respectively. The other group was used to carry out the seed germination tests.
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On a sample of 40 flower heads, taken randomly from 40 individuals of Centaurea cineraria subsp. circae natural population, the number of flowers and achenes was determined and the percentage of
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morphologically healthy achenes was calculated. Different tests were performed to investigate the ecophysiological needs for seed germination. The tests were performed on 600 achenes. The
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achenes were placed into 90 mm diameter Petri dishes in growth chambers under different temperature and light conditions. In each Petri dish, 25 achenes were placed on paper discs
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(Whathmann n. 2) soaked in distilled water and kept for 33 days The germination rate was monitored every three days. The dishes were maintained at the following temperatures +15 °C /+6
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°C, +20 °C/+10 °C, +25 °C /+15 °C (the first value is referred to 12 hours of light, while the second is referred to 12 hours of darkness). For each temperature regime, eight Petri dishes were used, four
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of which were exposed to a photoperiod 12 h/12 h (light/dark), while the other four were exposed to
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continuous darkness. The photoperiod was obtained using fluorescent tubes radiating a photon flux density of 80 µmol m-2 s-1. All tests were performed without the addition of hormones or physicochemical pretreatments. The temperature regimes above reported approximately reflect the seasonal
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Seed germination tests
temperature trend of C. cineraria subsp. circae growth station.
In vitro cultures The cultures were initiated from C. cineraria subsp. circae inflorescence stems harvested from natural wild population, at the flowering stage (early summer 2012), in the National Park of Circeo (Latina, Italy) (Figures 1 A and B). Inflorescence stem segments, 5 cm long, were thoroughly
washed with neutral soap and running tap water, then surface sterilized in 70% (v/v) ethanol for 1 min, followed by 20 min in 15% (v/v) commercial sodium hypochlorite (6% active chlorine) containing the wetting agent Tween-20 (one drop in 100 ml) and rinsed four times (5 min each) in sterile distilled water. Afterwards, the explants were cut into nodal segments, 2 cm in length, each
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bearing a single axillary bud.
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containing MS nutrient medium with vitamins (Murashige & Skoog, 1962), 3% (w/v) sucrose, and 0.8% (w/v) agar. The medium A was supplemented with 0.5 mg/L 6-benzylaminopurine (BAP) and
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2 mg/L kinetin (Kin), while the medium B was supplemented with 1 mg/L BAP and 0.2 mg/L 1naphthaleneacetic acid (NAA) (Table 1). The nodal explants were inoculated in 90 mm diameter
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Petri dishes sealed with Parafilm (five explants per plate), containing 30 mL of culture medium. After about 50 days (two subcultures), the newly formed shoots were separated from each other and
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transferred in hormone-free MS medium to stimulate shoot growth and development. The cultures
μmol m-2 s-1.
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were maintained in a growth chamber at 25±1 °C under a 16 h/8 h light/dark photoperiod at 70
Some nodal explants cultured on media A and B formed callus on the cut surfaces. Once formed,
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the callus was excised from the explants and inoculated in two different growth stimulating media
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(here referred to as C and D) both containing B5 nutrient medium with vitamins (Gamborg et al. 1968), 3% (w/v) sucrose, 0.8% (w/v) agar, and supplemented with 0.2 mg/L NAA and 1 or 0.1 mg/L Kin, respectively (Table 1). The callus cultures were maintained in a growth chamber at 25±1
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The nodal explants were cultured in two different culture media (here referred as A and B), both
°C under continuous darkness or under 16/8 h light/dark photoperiod at 70 μmol m−2 s−1 and subcultured every 25-30 days on fresh media with the same composition. To induce root formation, single shoots were inoculated in two media (here referred to as E and F), both containing half-strength MS medium with vitamins, 1.5% (w/v) sucrose, and 0.6% (w/v) agar and supplemented with 0.5 or 1 mg/L indole-3-butyric acid (IBA; Table 1). The cultures were
maintained in a growth chamber under 16/8 h light/dark photoperiod at 70 μmol m−2 s−1 for 25-30 days at 25±1 °C. The pH of all culture media was adjusted to 5.7 with NaOH 1N before adding sucrose and agar, and then sterilized in autoclave at 121 °C for 20 min.
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The media MS and B5, the sucrose and the agar were purchased from Duchefa (The Netherlands),
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Acclimatization of the in vitro regenerated plantlets
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(Italy).
The plantlets of C. cineraria subsp. circae obtained in vitro were collected and thoroughly washed
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with tap water to completely remove the agarized culture medium from the roots. Subsequently, they were placed in flasks containing 40 mL of tap water and hermetically sealed with Parafilm.
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After 3-4 days, 10 little holes in the Parafilm were made with sharp-tipped tweezers to allow gas exchange between the inner and outer atmosphere. After seven days the plantlets were transferred in
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pots containing approximately 400 mL of commercial soil and carbonate calcium gravel in a 1:1 ratio. Initially, the plantlets were covered with transparent film, to maintain a high degree of
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humidity. Seven days after, the film was removed and the plantlets were transferred in outdoors at
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the Botanical Garden of Sapienza University of Rome. The plants in pots were watered every day with about 20 mL of tap water.
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while the plant growth regulators, ethanol and Tween-20 were purchased from Sigma-Aldrich
Histological study of the in vitro rooted plantlets To verify the continuity between root and shoot vascular system, a histological analysis has been carried out on the in vitro rooted plantlets. Samples of about 1 cm in length, taken at the level of the transition zone between shoot and root, were embedded in 4% agar and then sectioned as described in Valletta et al. (2013). Fresh sections were stained with 0.1% toluidine blue (Sigma-Aldrich, Italy)
for 1 min, washed with distilled water and examined with a light microscope (Axioscop 2 Plus, Carl Zeiss Inc., Thornwood, NY, USA) fitted with a digital camera (Zeiss AxioCam MRc5).
Total phenol content
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Total phenol content in leaves of plants a) harvested from natural wild population, b) propagated
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the Folin-Ciocalteu reagent, as reported by Di Marco and coworkers (2014). Gallic acid was used as
Analysis of plant samples by GC-MS
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equivalents (GAE) in milligram per liter of extracts.
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standard to perform the calibration. The total phenolic content was expressed as gallic acid
Aerial parts of plants a) harvested from natural wild population, b) propagated and growing in vitro,
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c) propagated in vitro and transferred in pots (ex vitro), were extracted with dichloromethane and analyzed by GC-MS to investigate the presence of steroids and/or terpenes. Dichloromethane
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extracts were dissolved in chloroform and a 1-μL aliquot of the solution was injected onto the GCMS system. GC-MS analysis was performed using a Shimadzu GC-MS-QP2010S apparatus with a
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Shimadzu AOC-20i autosampler. A RTX-1 30 m x 0.25 mm, 0.10 μm, dimethylpolysiloxane
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analytical column (Agilent, Palo Alto, CA, USA) was employed for separation. Helium was used as the carrier gas at a flow rate of 0.8 mL/min. The injector (Splitless mode, 20:1 Split ratio) was maintained at 300 °C. The initial column temperature was set at 80°C and held for 2 min. It was
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and growing in vitro, c) propagated in vitro and transferred in pots (ex vitro), were determined with
then ramped by 30 °C/min up to 290 °C, then 25 °C/min up to 300 ºC and, finally, 20 °C/min up to 310 ºC, where it held for 15 min. Detector MS in a scan (m/z = 30 to 450 Da) and positive electron
impact ionization (EI) modes was used, and data were collected using single ion monitoring (SIM).
Results and Discussion Features of C. cineraria subsp. circae achenes
From an examination of 40 flower heads, it resulted that each head contained an average (± S.D.) of 36 ± 6 flowers and 20.3 ± 3.9 achenes, of which 1.1 ± 1.1 were morphologically healthy, corresponding to 5.5% of total achenes. The other achenes were empty or showed damages, frequently caused by insect larvae (Figure 1C). The achenes of many species of the genus
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Centaurea are parasitized by larvae of different insects, especially Diptera belonging to the family
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plant reproductive potential. Whereas on one hand this parasitic relationship may be advantageously exploited for biological control of invasive Centaurea species (Muller-Scharer et al. 1993; Turner et
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endangered plants, such as C. cineraria subsp. circae.
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al. 1996; Crowe & Bourchier 2006), on the other hand it may be a serious problem for rare and/or
Seed germination tests
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The results of germination tests (Figure 2) showed that C. cineraria subsp. circae seeds do not display particular types of deep dormancy and that germination does not require pre-treatments,
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such as scarification or stratification. The highest percentage of germination (67.5%) was observed in seeds subjected to photoperiod of 12 h/12 h (light/dark) and a temperature regime +20 °C/+10
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°C. Among the seeds subjected to continuous darkness, the highest germination percentage (47.5%)
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was obtained under the temperature regime +15 °C/+6 °C. These results suggest that the germination of C. cineraria subsp. circae seeds is positively influenced by light as demonstrated for many other species of Asteraceae (Grime et al. 1981;
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Tephritidae (Boracchia et al. 2007), which cause a great reduction in seeds viability, and then in
Baskin & Baskin 1998). These photoblastic seeds are typical of plant species that accumulate a persistent soil seed bank (Pons 1991). For plants that produce a large amount of seeds, it could be argued that the seeds represent the most suitable material for in vitro culture initiation to be used for the ex situ propagation, avoiding to damage wild plants. However, in C. cineraria subsp. circae, most of the seeds were found to be damaged, mainly by insect larvae. Moreover, when hybridization mechanisms is particularly
common, the seeds are not the most suitable material for ex situ conservation, and this is the case of several species of the genus Centaurea. For example, ibridization between C. paui Loscos ex Willk. with C. saguntina Mateo & M. B. Crespo and C. pinae Pau has been demonstrated (Aguilella et al. 1994); moreover, the existence of numerous intermediate forms between C. cineraria subsp.
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cineraria and C. cineraria subsp. circae have been reported by Renzoni & Viegi (1982). In these
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material by a specialist only when they have reached maturity. For these reasons we have decided to initiate in vitro cultures from axillary buds collected from inflorescence stems of identified adult
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plants. As stated by Cuenca and Amo-Marco (2000), the use of inflorescences as a source of explants avoids the destruction of the mother rosette plant, and this constitutes a significant
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advantage for conservation of rare and/or endangered species. In addition, this material result in
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lower microbial contamination than the explants obtained by the basal rosette at ground level.
Shoot multiplication from axillary buds
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To induce the formation of multiple shoots, nodal explants from inflorescence stems of C. cineraria subsp. circae plants collected on the Circeo mountain were grown on two media, named A and B
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(table 1). Axillary bud activation was already observed 20-30 days after explant inoculation (Figure
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2D) and multiple shoot formation was obtained after 2-3 subcultures in the same medium (Figure 2E). The medium B was found to be more effective in inducing multiple shoots formation, in terms of both percentage of responsive explants (74.33%) and number of shoots per explant (5.67) (Table
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cases, seeds could originate interspecific hybrids which can be distinguished from true-type plant
1). Since the medium B has proved very effective in promoting the activation of lateral buds (Figure 2E), other hormonal combinations were not tested. After transferring single shoots into hormonefree medium, within 20-30 days they reached a height of 3-5 cm (Figure 2F). Also in other species of the genus Centaurea, such as Centaurea paui Loscos ex Willk (Cuenca et al. 1999) and Centaurea ultreiae Silva Pando (Mallón et al. 2011), a high percentage of activated buds have been obtained under similar hormonal conditions.
Callus formation and indirect shoot regeneration On the cutting surfaces of some explants maintained in the media A and B, the formation of emerald-green friable callus was observed. The callus was separated from the explants, inoculated
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in the culture media C and D (Table 1) and grown under photoperiod or under continuous darkness.
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caulogenesis) was observed. In the callus grown under photoperiod, the newly formed shoots were numerous (about 50 per Petri dish containing 5-6 g fresh weight [FW] of callus) and light green,
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while in the callus grown under continuous darkness they were less numerous (five-six per Petri dish containing 5-6 g FW of callus) and colourless (etiolated). Although the shoots obtained from
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callus and maintained under photoperiod were morphologically similar to that obtained from axillary buds, they were not further investigated because, due to the high degree of somaclonal
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variation associated to indirect organogenesis (Larkin & Scowcroft 1981; Filipecki & Malepszy 2006), they would not be suitable for restoration programs (risk of genetic pollution). It should be
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stressed that the induction of somaclonal variation could be exploited for the selection of genotypes
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2013).
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useful for different application purposes, such as the obtainment of cold-tolerant crops (Liu et al.
In vitro rooting of shoots
The shoots transferred in the media added with 0.5 or 1 mg/L IBA (media E and F, respectively)
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In both growth conditions (photoperiod/darkness), the formation of vegetative buds (indirect
formed roots, however, in the medium E the roots were longer and more numerous (Figure 2G). Moreover, the highest IBA concentration caused callused roots. Among auxins, IBA is one of the most used to induce root formation (Ludwig-Müller et al. 2005), both from direct and indirect organogenesis (Casson & Lindsey 2003). The effective concentration of exogenous IBA is highly dependent on the plant species, the type of explant and the cultural conditions such as the pH of the medium (Harbage & Stimart 1996), so it must be determined empirically. If used in too high
concentrations, this growth regulator can interfere with the elongation and branching of newly formed roots, and induce callus formation (Tocci et al. 2011), which is an undesirable phenomenon in a micropropagation system.
Histology of the rooted shoots
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A hystological analysis was carried out on samples of C. cineraria subsp. circae taken at the level
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plants, the vascular system of the shoot was in continuity with the vascular system of the root. In
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micropropagated plantlets, the vascular connection between root and stem is of great importance for the functioning of the vascular system and the survival of plants after transplantation (De Oliveira et
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al. 2012-2013). In fact, the roots may originate either by direct or indirect organogenesis. Indirect rhizogenesis from small masses of callus at the base of the shoot could lead to an non functional
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root system (Monacelli et al. 1999; George et al. 2008; De Oliveira et al. 2012-2013). The success of the induction of adventitious rooting is due to many factors, which includes plant regulators. In
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Vismia guianensis we observed that direct adventitious root formation was favoured by IBA
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treatment, rather than other auxins (Monacelli et al. 1999).
Acclimatization of the vitro propagated plants
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The in vitro propagated plants of C. cineraria subsp. circae were subjected to acclimatization, transferred in pots outdoors in the botanical garden (in the same condition in which plants collected from natural wild population were grown) and observed for about one and a half years. A very high
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of the transition zone between shoot and root. As shown in Figures 2 I and L, in in vitro rooted
percentage of plants (> 90%) survived the acclimatization phase and the totality of these plants survived after moving them outdoor at the botanical garden. Already after 3-4 months, the ex vitro
plants were morphologically indistinguishable from the plants collected in the wild. After one year and a half all of these plants were still alive.
Even though many efforts have been directed to the in vitro propagation of plants, to date the process of acclimatization of micropropagated plants has been insufficiently studied (Hassan et al. 2014). Consequently, the transplantation stage continues to be a major bottleneck in the micropropagation of many species. The advantage of an in vitro propagation system may, however,
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be fully achieved only by the successful transfer of plantlets from the in vitro to the ex vitro
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results obtained in the present study, it seems that the acclimatization of Centaurea spp. is not particularly difficult. For example, the survival rate of plants after acclimatization was 70% in C.
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paui (Cuenca et al. 1999), 80% in C. spachii (Cuenca & Amo-Marco 2010), and 100% in C.
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ultreiae (Mallón et al. 2010). Chemical analysis
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Leaves of wild plants and of in vitro plants (propagated and growing in vitro or propagated in vitro and transferred in pots) were subjected to a preliminary analysis to evaluate if the in vitro conditions
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could affect their chemical profile.
Folin-Ciocalteu reagent revealed that plants propagated and cultivated in vitro contained a higher
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quantity of total phenols with respect to plants harvested from natural wild population, although the differences were not statistically significant (P>0.05). In particular, total phenols content, expressed
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as GAE (mg/L) was 102±7 in wild plants, 112±6 plants propagated in vitro and transferred in pots, and 122±11 in plantlets propagated and maintained in vitro. In other Centaurea species the values of total phenols, ranging from about 82 to 130 GAE (Aktumsek et al. 2011), were similar to that
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conditions (greenhouse or field) (Hazarika 2003). Based on the available literature and on the
observed in this work. It has been reported that the amount of total phenols can increase in plants under stress in order to protect cell vitality (Huseynova 2012). Surprisingly, Di Marco et al. (2014) in Iberis sempervirens leaves observed that tetracycline treatment, an environmental antibiotic contaminant, did not induce an increase of simple phenols.
Moreover chemical evaluation by GC/MS, carried out on dichloromethane extracts, evidenced that all the in vitro plants (still growing in vitro or transferred in pots) showed a similar profile to the plants harvested from natural wild population (Figure 3). Several components were present, five of which were identified as the alkanes octadecanal, n-
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hexadecanoic acid, heptacosane, octacosane, and nonacosane, the latter three being the most
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30%).
Senatore et al. (2003) published the first report on volatile components from aerial parts of C.
us
cineraria subsp. umbrosa and C. napifolia L., identifying 55 components, mostly sesquiterpenes and hydrocarbons. In previous studies, flavonoid, syringin glycosides and sesquiterpene lactones
an
and flavones have been found in C. cineraria L. subsp. umbrosa and sesquiterpene lactones in C. napifolia (Senatore et al. 2003 and references therein). Non polar compounds from the genus
M
Centaurea collected from different countries have been studied by other laboratories and the results demonstrated that terpenes (sesquiterpenoids), hydrocarbons and fatty acids are the main
ed
constituents (Senatore et al., 2006; Yayli et al., 2005; Formisano et al. 2008). However, in the present work, the expected steroids and/or terpenes were present only in low yield and additional
pt
studies are necessary to elucidate their molecular structures, since the peaks with very low intensity
ce
were not identified by the standard libraries. The components evidenced in the present study are apparently typical of propagated tissues and precursors of the terpenes, not yet formed in the in vitro plants. For example, cyclosativene (among several other terpenes), a bioactive tetracyclic
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represented compounds (about 55% of the total; Table 2). Octacosane gave the highest yield (ca.
sesquiterpene present in the essential oil of Centaurea cineraria, was not detected in none of the studied extracts (Senatore et al. 2003). Additional studies are in progress to elucidate the polar compounds, as flavonoids, acids, etc., which will be reported in the future.
References
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Phytochemistry 66: 1741-1745.
sc rip
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Tables
root formation, and main obtained results. Each data represent the mean of 5 replicates ± E.S. Sucrose (%)
Agar (%)
BAP
Kin
NAA
A
MS
3
0.8
0.5
2
-
-
B
MS
3
0.8
1
-
0.2
-
B5
3
Callus growth and indirect caulogenesis
-
B5
3
0.8
-
1
0.1
25±1 °C Photoperiod 16/8 25±1 °C Photoperiod 16/8
Responsive explants (%)
Shoots per explant
74.33 ± 6.22
5.67 ± 2.46
34.3 ± 9.97
2.78 ± 1.12
Shoots per plate (5-6 g callus) 0.2
0.2
-
-
pt
D
0.8
ed
C
Growth conditions
IBA
M
Shoot multiplication
Micro/macronutrients and vitamins
an u
Medium
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Table 1. Composition of culture media and hormone treatments used for C. cineraria subsp. circae in vitro shoot multiplication, callus growth, and
E
MS half strenght
1.5
0.6
-
-
-
0.5
F
MS half strenght
1.5
0.6
-
-
-
1
Shoot rooting
25±1 °C Photoperiod 16/8 25±1 °C Darkness 25±1 °C Photoperiod 16/8 25±1 °C Darkness
25±1 °C Photoperiod 16/8 25±1 °C Photoperiod 16/8
48.1 ± 6.32 5.3 ± 2.20 51.0 ± 5.54 4.9 ± 3.36 Rooted plantlets (%)
Roots per plantlet
100 ± 0.00
7.95 ± 1.02
100 ± 0.00
8.03 ± 2.32
Concentration (%)
octacosane
30.0
heptacosane
9.45
nonacosane
9.25
octadecanal
0.39
n-hexadecanoic acid
0.38
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pt
ed
M
an
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cr
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Compound
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Table 2: Main compounds of Centaurea cineraria subsp. circae identified by GC/MS.
Figure captions
Figure 1. (A) Centaurea cineraria subsp. circae plants on rocky soil in the promontory of Circeo (arrows); (B) a detail of developing (right) and fully developed (left) flower heads; (C) mature
t
achene damaged by an insect larva. (D) Nodal explant of C. cineraria subsp. circae at day 20 after
ip
cr
(arrow); (E) nodal explant at day 60 of culture (2 subcultures) in medium B, showing multiple shoot formation; (F) single shoots after been isolated from the explants and inoculated in MS hormone-
us
free medium to induce shoot growth and development; (G) rooted plantlets 25 days after the inoculation in medium E; (H) acclimatized plantlets. (I) Optical micrograph of a fresh longitudinal
an
section of an in vitro propagated C. cineraria subsp. circae plant, showing the vascular connection (white arrow) between the shoot (black arrows) and the root (yellow arrow) vascular system; (L)
ed
700 µm (I) and 1000 µm (L).
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detail of the vascular connection between shoot and root vascular system (arrow). Bars represents
Figure 2. Germination percentages of C. cineraria subsp. circae seeds incubated in different
pt
temperature regimes under photoperiod 12/12 h (ligt/dark) or continuous darkness. Each point
ce
represents the mean of four replicates ± SD. (□) +15/6 °C photoperiod; (■) +15/+6 °C continuous darkness; (▲) +20/+10 °C photoperiod; (▲) +20/+10 °C continuous darkness; (○) +25/15 °C photoperiod; (●) +25/+15 °C continuous darkness.
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the inoculation in medium B, in which it is possible to observe the activation of an axillary bud
Figure 3. Gas chromatographic profiles of aerial parts of Centaurea cineraria subsp. circae. (a)
plants collected in wild; (b) plants propagated and maintained in vitro; (c) plants propagated in vitro and transferred in pots (ex vitro).
