Accepted Article
Received Date: 16-Jan-2015 Revised Date: 23-Mar-2015 Accepted Date: 31-Mar-2015 Article Type : Short Research Paper Editor: R Bekker
Article: short research paper
Old Sleeping Sicilian Beauty: seed germination study in the palaeoendemic Petagnaea gussonei (Spreng.) Rauschert (Saniculoideae, Apiaceae). Olga De Castro1*, Lorenzo Gianguzzi2, Francesca Carucci3, Adriana De Luca4, Renato Gesuele5, Marco Guida5 1
Dip. Biologia, Università degli Studi di Napoli Federico II, Via Foria 223 - Orto Botanico, I-80139
Napoli, Italy; 2
Dip. Scienze Agrarie e Forestali, Università degli Studi di Palermo, Viale delle Scienze, Ed. 4, I-
90128 Palermo, Italy; 3
Dip. Agraria (PgB Lab), Università degli Studi di Napoli Federico II, Via Università 100 - Castello,
I-80055 Portici, Italy 4
Dip. Medicina Veterinaria e Produzioni Animali, Via Federico Delpino 1, I-80137 Napoli, Italy;
5
Dip. di Biologia, Università degli Studi di Napoli Federico II, Via Cinthia, I-80126 Napoli, Italy.
*For correspondence. E-mail [email protected]; [email protected]
Abstract Petagnaea gussonei (Apiaceae) is a perennial herbaceous species endemic to northeastern Sicily (Nebrodi mountains). It is considered as a remnant of the Sicilian Tertiary
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12333 This article is protected by copyright. All rights reserved.
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flora, and it is endangered (EN) according to the Red List. In terms of its biology, no information is available in the literature about the germinability of its seeds, even though seed production occurs. The aim of this study was to obtain data to better understand the seed germination of this species and its biological implications. Thus, several approaches were employed, such as the following: vitality analyses, gibberellic acid supply, germination test and soil microbial flora analyses via end-point and qPCR. The results suggest that seed germination occurs in Petagnaea after ca. a year and a half and with a rate of ca. 11%. Its seeds can be classified as physiologically dormant (PD) and prolonged cold stratification and the microbial community present in natural environments could be the key to the germination of Petagnaea seeds. Because seed germination occurs in this plant but with a low rate, we acknowledge that agamic reproduction represents an important mean for its conservation and survival. These results have important implications for P. gussonei survival and should be taken into account in possible reintroduction attempts aimed at restoring threatened populations of this species.
Key words: Apiaceae, germination, Petagnaea gussonei, ITS, real-time PCR, seed dormancy, Saniculoideae, soil microbial flora, 16S gene
INTRODUCTION To understand the germination phenomenon in the Apiaceae subfamily Saniculoideae, several studies have been performed on some genera [i.e., Sanicula L. in Baskin & Baskin (1988), Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea F.Delaroche in Mulaudzi et al. (2009); Eryngium L. in Ekpong & Sukprakarn (2006), Curle et al. (2007) and Necajeva & Ievinsh (2013)]. To date, subfamily Saniculoideae has undergone an interesting phylogenetic rearrangement involving the following nine genera: Actinolema Fenzl, Alepidea, Arctopus L., Astrantia L., Eryngium, Petagnaea Caruel, Polemanniopsis B.L.Burtt., Sanicula and Steganotaenia Hochst. (Calviño et al. 2007, 2008; Kadereit et al. 2008). One of the most interesting Saniculoideae may be a palaeoendemic plant present in north-eastern Sicily, the monospecific genus Petagnaea. The species P. gussonei (Spreng.) Rauschert has captured the attention of many botanists due to its ecology, biology, phylogeny, population genetics and rarity (De Castro et al. 2015 and references therein). Briefly, P. gussonei is perennial and rhizomatous plant with a small number of isolated populations in the Nebrodi Mountains with moderate genetic variability (De Castro et al. 2009, 2013); it is considered a
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prepared according to the procedure described in AOSA (2005). Subsequently, seeds were cut, and each mericarp was removed to permit evaluation by microscope.
Action of gibberellic acid (GA3) on dormancy break To determine the effects of GA3, the achenes (three replicates of 20 achenes) were incubated in petri dishes (10 cm diameter, Falcon) on filter paper (3MM, Whatman) moistened with 0, 10, 100, and 1000 mgL-1 GA3 (AppliChem) solution in a growth chamber (18-23°C, 8 h-16 h dark-light). At the beginning of the experiments, the achenes were moistened with the appropriate GA3 concentration. During the following weeks, the filter papers were moistened with distilled water and the appropriate GA3 concentration every 10 days for a total of four administrations. Soil pot germination and microbial flora analyses Soil germination tests were performed in (1) the natural environment (NE) and (2) laboratory conditions (LC). Initially, five achenes of P. gussonei were potted in 36 plastic pots filled with sterile potting soil in a portable greenhouse (Jiffy-7 Windowsill Greenhouse 36, Jiffy). The greenhouse was located in the mesophilic habitat of the Botanical Garden of Naples where natural populations of P. gussonei are present (NE) and subject to minimum temperatures close to zero in the winter and a maximum of 25 °C in the summer. The same experiment was also performed in a growth chamber (1823 °C, 8 h-16 h dark-light) in the laboratory (i.e. LC). The soil pots were checked weekly for epicotyl emergence. At the end of the experiments, pH, water content and microbial flora (bacteria and fungi) were analysed in the soil pots of the two locations (NE and LC). The water content (u) was calculated according to Sibilio et al. (2014). Microbial flora of soil pots was analysed and quantified using real-time quantitative PCR (qPCR) and endpoint PCR (e-PCR). Two biological replicates for each location were processed. See the Supplementary file for the details about DNA extraction and PCR assays (Appendix S1).
RESULTS and DISCUSSION The tetrazolium test, which determines the viability of seeds based on the activity of enzymes involved in respiration (i.e. dehydrogenases), was employed to obtain initial data on seed germination in this species. The tetrazolium test is not influenced by the dormancy level of the seeds, and it is very rapid, although false positives can occur (e.g. microbial contamination; Bradbeer 1988; Sawma & Mohler 2002). To minimise the possibility of false positives, very careful microscopic examination of each stained Petagnaea seed was performed (e.g. embryo separation from the rest of seed). The
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In fact, the presence of AMF is important because these fungi promote germination in many plant species (see, Öpik et al. 2006; Smith & Smith 2012). It is also important to highlight the effect of GA3 on Petagnaea seed germination. According to a classification system for seed dormancy (Baskin & Baskin 2004), Petagnaea seeds can be considered physiologically dormant (PD) because GA3 promotes germination. In addition, low temperatures are an important factor in breaking seed dormancy in Petagnaea, as observed for the soil pots placed in nature (NL), which were exposed to colder temperatures. The presence of immature embryos has been documented for many representative species of Saniculoideae (e.g., Vandelook & Van Assche 2008; Hawkins et al. 2010; Necajeva & Ievinsh 2013), and their seeds are classified as morphologically dormant (MD). We can only speculate whether MD Petagnaea seeds may exist because there are no complete studies on the presence of underdeveloped embryos in Petagnaea seeds. In light of our data, we can conclude that seed germination does occur in Petagnaea, although after ca. a year and a half (i.e. 19 months) and with a low rate if compared with other study about Saniculoideae taxa [i.e., Sanicula in Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea in Mulaudzi et al. (2009); Eryngium in Ekpong & Sukprakarn (2006) and Necajeva & Ievinsh (2013)]. Petagnaea seeds can be classified only as physiologically dormant (PD), in fact, P. gussonei is sensitive to GA3 treatment. Cold temperatures are required to break its seed dormancy and grow embryos in pots placed in nature (NE). Moreover, a very different pattern of microbial flora abundance and fungal typology (including AMF presence) was observed in NE in contrast to the pots in the laboratory (LC). Arguably, prolonged cold stratification and a specialised microbial community present in the natural habitat are key to the germination of Petagnaea seeds. In terms of conservation, even if a low rate of seed germination has been observed in this study, the agamic reproduction result be an important prerequisite for its survival as confirmed by De Castro et al. (2013). As a final remark on the techniques applied in this study, for the real-time PCR data, it is important to highlight that qPCR does not provide an absolute measure of the different microbial groups due to several limitations that are both biological and technical (e.g., variability nuclei/cell among fungal species, universality of the primers) (Fierer et al. 2006; Plassart et al. 2012); yet, as shown in this study, qPCR can quickly give a good overview of the relative abundance of the biota without using time- and moneyconsuming techniques such as sub-cloning, Temperature Gradient Gel Electrophoresis (DGGE) and sequencing of PCR products.
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results showed that the majority of the seeds did not have an embryo (48.3%), and staining was observed for only 33.33% of seeds; no staining was present for 18.3% of seeds (i.e. non-viable seed). Only high concentrations of GA3 had an effect on Petagnaea seed germination (GA3 = 1000 mgL-1). Germination occurred between the fourth and fifth weeks for a total of ca. 12% (± 1.2) of germinated seeds. No seeds germinated after the fifth week at any GA3 concentration. The germinated seeds were planted in sterile jars with 0.8% agar (AppliChem) to examine the phenology of the seedlings. Figure 2 shows several growth stages of the Petagnaea seedlings. Seed germination was also observed in the soil pots placed in the NE location (i.e. in the mesophilic habitat near the Botanical Garden) with the 11.11% success. The germination started in March 2013 (i.e. one year and seven months after the beginning of experiment) and it lasted for 10 weeks with a germination average of 2/week (± 0.58 SE). No germination was observed in the soil pots located in laboratory conditions (LC). For pot soil analyses, eight pots were chosen from each location, and the water content (u) and pH were analysed. NE soil pots had u = 0.66 (± 0.011 SE) and pH = 7.3 (± 0.022 SE); LC soil pots presented u = 0.58 (± 0.004 SE) and pH = 6.92 (± 0.045 SE). The amount of DNA recovered (estimated from crude DNA extracts) from the different locations was less in the LC soil pots than the NE soil pots (1.055 µg ± 0.015 SE and 1.184 µg ± 0.016 SE per 1.5 g soil, respectively). According to our results (Fig. 3), there are differences in the abundance of both of fungi and bacteria between the two locations (LC and NE), with a higher presence of fungi and bacteria in the soil pots in the natural environment (NE, i.e. mesophilic habitat of the Botanical Garden). The PCR efficiency for each primer pair showed acceptable values (i.e. 2.1 for fungi and 1.9 for bacteria primers). The melting curve profiles of each qPCR assay revealed that different patterns were present in each of the two locations, confirming the presence of different fungal and bacterial communities (i.e. different amplicons) (Fig. 4). To confirm the presence of possible arbuscular mycorrhizal fungi (AMF) in the soil pots placed in nature (NE), an end-point PCR was carried out using Glomeromycota-specific primers (see Appendix 1). Positive amplifications were detected only in NE soil DNA templates, and BLAST analyses of amplicon sequences confirmed the specificity of the primers described by Kruger et al. (2009). Our molecular data showed a higher abundance and different composition of fungi and bacteria in the soil pots where P. gussonei germinated (i.e. NE) compared with the soil pots in laboratory conditions (LC) (Figs. 3-4). It is likely that the qualitative/quantitative microflora differences detected in NE soil pots had a role in establishing appropriate edaphic conditions to promote and/or improve germination in P. gussonei (Garret 1963).
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Accepted Article
In fact, the presence of AMF is important because these fungi promote germination in many plant species (see, Öpik et al. 2006; Smith & Smith 2012). It is also important to highlight the effect of GA3 on Petagnaea seed germination. According to a classification system for seed dormancy (Baskin & Baskin 2004), Petagnaea seeds can be considered physiologically dormant (PD) because GA3 promotes germination. In addition, low temperatures are an important factor in breaking seed dormancy in Petagnaea, as observed for the soil pots placed in nature (NL), which were exposed to colder temperatures. The presence of immature embryos has been documented for many representative species of Saniculoideae (e.g., Vandelook & Van Assche 2008; Hawkins et al. 2010; Necajeva & Ievinsh 2013), and their seeds are classified as morphologically dormant (MD). We can only speculate whether MD Petagnaea seeds may exist because there are no complete studies on the presence of underdeveloped embryos in Petagnaea seeds. In light of our data, we can conclude that seed germination does occur in Petagnaea, although after ca. a year and a half (i.e. 19 months) and with a low rate if compared with other study about Saniculoideae taxa [i.e., Sanicula in Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea in Mulaudzi et al. (2009); Eryngium in Ekpong & Sukprakarn (2006) and Necajeva & Ievinsh (2013)]. Petagnaea seeds can be classified only as physiologically dormant (PD), in fact, P. gussonei is sensitive to GA3 treatment. Cold temperatures are required to break its seed dormancy and grow embryos in pots placed in nature (NE). Moreover, a very different pattern of microbial flora abundance and fungal typology (including AMF presence) was observed in NE in contrast to the pots in the laboratory (LC). Arguably, prolonged cold stratification and a specialised microbial community present in the natural habitat are key to the germination of Petagnaea seeds. In terms of conservation, even if a low rate of seed germination has been observed in this study, the agamic reproduction result be an important prerequisite for its survival as confirmed by De Castro et al. (2013). As a final remark on the techniques applied in this study, for the real-time PCR data, it is important to highlight that qPCR does not provide an absolute measure of the different microbial groups due to several limitations that are both biological and technical (e.g., variability nuclei/cell among fungal species, universality of the primers) (Fierer et al. 2006; Plassart et al. 2012); yet, as shown in this study, qPCR can quickly give a good overview of the relative abundance of the biota without using time- and moneyconsuming techniques such as sub-cloning, Temperature Gradient Gel Electrophoresis (DGGE) and sequencing of PCR products.
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Accepted Article
CONCLUSION •
Half of Petagnaea seeds are abortive.
•
GA3 affects seed germination.
•
Seed germination occurs in the natural environment after ca. a year and a half.
•
The percentage of germination is low.
•
Seeds are physiologically dormant (PD).
•
Asexual reproduction is an important mechanism of Petagnaea survival.
•
Real-time PCR is a good tool to analyse the abundance and typology of biota without using time/money-consuming techniques.
ACKNOWLEDGEMENTS The authors are also grateful to Dr. Luca Cepollaro and Paolo Sbragia for technical assistance in the laboratory. Special thanks to Nando Peretti Foundation for allowing us to study this plant in the previous years (Project 2007-15).
SUPPLEMENTARY MATERIAL Appendix S1. Molecular methods employed in the soil pots including soil pot DNA extraction, real-time PCR (qPCR), PCR amplification and sequence analyses.
REFERENCES AOSA (Association of Official Seed Analyst) (2005) Tetrazolium Testing Handbook. Contribution No.29. AOSA, Las Cruces, New Mexico: 96 pp. Accessed on 16 January 2015. Baskin C.C., Baskin J.M. (1988) Germination ecophysiology of herbaceous plant species in a temperate region. American Journal of Botany, 75, 286–305. Baskin J.M., Baskin C.C. (2004) A classification system for seed dormancy. Seed Science Research, 14, 1–16.
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Bradbeer, J. W. (1988) Seed Dormancy and Germination (2nd Edn). Blackie, Chapman & Hall, New York, USA: 146 pp. Calviño C.I., Downie S.R. (2007) Circumscription and phylogeny of Apiaceae subfamily Saniculoideae based on chloroplast DNA sequences. Molecular Phylogenetic and Evolution, 44, 175–191. Calviño C.I., Martıñez S.G., Downie S.R. (2008) Morphology and biogeography of Apiaceae subfamily Saniculoideae as inferred by phylogentic analysis of molecular data. American Journal of Botany, 95(2), 196–214. Curle C.M., Stabbetorp O.E., Nordal I. (2007) Ervngium maritimum, biology of a plant at its northernmost localities. Nordic Journal of Botany, 24, 617–628. De Castro O, Cennamo P, De Luca P (2009) Analysis of the genus Petagnaea Caruel (Apiaceae), using new molecular and literature data. Plant Systematics and Evolution, 278, 239–249. De Castro O, Colombo P, Gianguzzi L, Perrone R (2015) Flower and fruit structure of the endangered species Petagnaea gussonei (Sprengel) Rauschert (Saniculoideae, Apiaceae) and
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Gianguzzi L., La Mantia A. (2006) Petagnaea gussonei. The IUCN Red List of Threatened Species.
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evolutionary implications for dormancy break. Plant Species Biology, 25, 103–113. Kadereit J.W., Repplinger M., Schmalz N., Uhink C.H., Wörz A. (2008) The phylogeny and biogeography of Apiaceae subf. Saniculoideae tribe Saniculeae: from south to north and south again. Taxon, 57(2), 365–382. Krüger M., Stockinger H., Krüger C., Schüßler A. (2009) DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytologist, 183, 212–223. Lakon G. (1942a) Topographischer nachweis der keimfiihigkeit der getreidefriichte durch tetrazoliumsalze. Berichte der Deutschen Botanischen Gesellschaft, 60, 299–305. Lakon G. (1942b) Topographischer nachweis der keimfiihigkeit von mais durch tetrazoliumsalze. Berichte der Deutschen Botanischen Gesellschaft, 60, 434–444. Mulaudzi R.B., Kulkarni M.G., Finnie J.F., Van Staden J. (2009) Optimizing seed germination and seedling vigour of Alepidea amatymbica and Alepidea natalensis. Seed Science and Technology, 37, 527–533. Necajeva J., Ievinsh G. (2013) Seed dormancy and germination of an endangered coastal plant Eryngium maritimum (Apiaceae). Estonian Journal of Ecology, 62(2), 150– 161. Öpik M., Moora M., Liira J., Zobel M. (2006) Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe. Journal of Ecology, 94, 778–790. Plassart P., Terrat S., Thomson B., Griffiths R., Dequiedt S., Mélanie Lelievre M., Regnier T., Nowak V., Bailey M., Lemanceau P., Bispo A., Chabbi A., Maron P.-A., Mougel C., Ranjard L. (2012) Evaluation of the ISO Standard 11063 DNA Extraction Procedure for Assessing Soil Microbial Abundance and Community Structure. PLoS ONE, 7(9), e44279.
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Sawma J.T., Mohler C.L. (2002) Evaluating seed viability by an unimbibed seed crush test in comparison with the Tetrazolium Test. Weed Technology, 16(4), 781–786. Sibilio G., Russo A., Vallariello R., Menale B., De Luca P., De Castro O. (2014) The past, present and future of thermophilous Cyperus polystachyos Rottb. (Cyperaceae) on the island
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FIGURE LEGENDS Figure 1. Detail of the stolons of Petagnaea gussonei in mesophilic habitat near the Botanical Garden of Naples (Italy). Figure 2. Phenology of Petagnaea gussonei seedlings. Figure 3. Relative abundance of bacterial and fungi flora within DNA samples from soil pots collocated in the natural environment (NE) and laboratory conditions (LC) estimated using qPCR. Error bars are the standard errors of the mean for the three replicates. Figure 4. Example of melting curves from the 16S (bacteria target) and ITS1 (fungi target) gene assays in the soil DNA pots in the natural environment (NE) and laboratory conditions (LC) estimated using qPCR.
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Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
Received Date: 16-Jan-2015 Revised Date: 23-Mar-2015 Accepted Date: 31-Mar-2015 Article Type : Short Research Paper Editor: R Bekker
Article: short research paper
Old Sleeping Sicilian Beauty: seed germination study in the palaeoendemic Petagnaea gussonei (Spreng.) Rauschert (Saniculoideae, Apiaceae). Olga De Castro1*, Lorenzo Gianguzzi2, Francesca Carucci3, Adriana De Luca4, Renato Gesuele5, Marco Guida5 1
Dip. Biologia, Università degli Studi di Napoli Federico II, Via Foria 223 - Orto Botanico, I-80139
Napoli, Italy; 2
Dip. Scienze Agrarie e Forestali, Università degli Studi di Palermo, Viale delle Scienze, Ed. 4, I-
90128 Palermo, Italy; 3
Dip. Agraria (PgB Lab), Università degli Studi di Napoli Federico II, Via Università 100 - Castello,
I-80055 Portici, Italy 4
Dip. Medicina Veterinaria e Produzioni Animali, Via Federico Delpino 1, I-80137 Napoli, Italy;
5
Dip. di Biologia, Università degli Studi di Napoli Federico II, Via Cinthia, I-80126 Napoli, Italy.
*For correspondence. E-mail [email protected]; [email protected]
Abstract Petagnaea gussonei (Apiaceae) is a perennial herbaceous species endemic to northeastern Sicily (Nebrodi mountains). It is considered as a remnant of the Sicilian Tertiary
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12333 This article is protected by copyright. All rights reserved.
Accepted Article
flora, and it is endangered (EN) according to the Red List. In terms of its biology, no information is available in the literature about the germinability of its seeds, even though seed production occurs. The aim of this study was to obtain data to better understand the seed germination of this species and its biological implications. Thus, several approaches were employed, such as the following: vitality analyses, gibberellic acid supply, germination test and soil microbial flora analyses via end-point and qPCR. The results suggest that seed germination occurs in Petagnaea after ca. a year and a half and with a rate of ca. 11%. Its seeds can be classified as physiologically dormant (PD) and prolonged cold stratification and the microbial community present in natural environments could be the key to the germination of Petagnaea seeds. Because seed germination occurs in this plant but with a low rate, we acknowledge that agamic reproduction represents an important mean for its conservation and survival. These results have important implications for P. gussonei survival and should be taken into account in possible reintroduction attempts aimed at restoring threatened populations of this species.
Key words: Apiaceae, germination, Petagnaea gussonei, ITS, real-time PCR, seed dormancy, Saniculoideae, soil microbial flora, 16S gene
INTRODUCTION To understand the germination phenomenon in the Apiaceae subfamily Saniculoideae, several studies have been performed on some genera [i.e., Sanicula L. in Baskin & Baskin (1988), Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea F.Delaroche in Mulaudzi et al. (2009); Eryngium L. in Ekpong & Sukprakarn (2006), Curle et al. (2007) and Necajeva & Ievinsh (2013)]. To date, subfamily Saniculoideae has undergone an interesting phylogenetic rearrangement involving the following nine genera: Actinolema Fenzl, Alepidea, Arctopus L., Astrantia L., Eryngium, Petagnaea Caruel, Polemanniopsis B.L.Burtt., Sanicula and Steganotaenia Hochst. (Calviño et al. 2007, 2008; Kadereit et al. 2008). One of the most interesting Saniculoideae may be a palaeoendemic plant present in north-eastern Sicily, the monospecific genus Petagnaea. The species P. gussonei (Spreng.) Rauschert has captured the attention of many botanists due to its ecology, biology, phylogeny, population genetics and rarity (De Castro et al. 2015 and references therein). Briefly, P. gussonei is perennial and rhizomatous plant with a small number of isolated populations in the Nebrodi Mountains with moderate genetic variability (De Castro et al. 2009, 2013); it is considered a
This article is protected by copyright. All rights reserved.
Accepted Article
prepared according to the procedure described in AOSA (2005). Subsequently, seeds were cut, and each mericarp was removed to permit evaluation by microscope.
Action of gibberellic acid (GA3) on dormancy break To determine the effects of GA3, the achenes (three replicates of 20 achenes) were incubated in petri dishes (10 cm diameter, Falcon) on filter paper (3MM, Whatman) moistened with 0, 10, 100, and 1000 mgL-1 GA3 (AppliChem) solution in a growth chamber (18-23°C, 8 h-16 h dark-light). At the beginning of the experiments, the achenes were moistened with the appropriate GA3 concentration. During the following weeks, the filter papers were moistened with distilled water and the appropriate GA3 concentration every 10 days for a total of four administrations. Soil pot germination and microbial flora analyses Soil germination tests were performed in (1) the natural environment (NE) and (2) laboratory conditions (LC). Initially, five achenes of P. gussonei were potted in 36 plastic pots filled with sterile potting soil in a portable greenhouse (Jiffy-7 Windowsill Greenhouse 36, Jiffy). The greenhouse was located in the mesophilic habitat of the Botanical Garden of Naples where natural populations of P. gussonei are present (NE) and subject to minimum temperatures close to zero in the winter and a maximum of 25 °C in the summer. The same experiment was also performed in a growth chamber (1823 °C, 8 h-16 h dark-light) in the laboratory (i.e. LC). The soil pots were checked weekly for epicotyl emergence. At the end of the experiments, pH, water content and microbial flora (bacteria and fungi) were analysed in the soil pots of the two locations (NE and LC). The water content (u) was calculated according to Sibilio et al. (2014). Microbial flora of soil pots was analysed and quantified using real-time quantitative PCR (qPCR) and endpoint PCR (e-PCR). Two biological replicates for each location were processed. See the Supplementary file for the details about DNA extraction and PCR assays (Appendix S1).
RESULTS and DISCUSSION The tetrazolium test, which determines the viability of seeds based on the activity of enzymes involved in respiration (i.e. dehydrogenases), was employed to obtain initial data on seed germination in this species. The tetrazolium test is not influenced by the dormancy level of the seeds, and it is very rapid, although false positives can occur (e.g. microbial contamination; Bradbeer 1988; Sawma & Mohler 2002). To minimise the possibility of false positives, very careful microscopic examination of each stained Petagnaea seed was performed (e.g. embryo separation from the rest of seed). The
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In fact, the presence of AMF is important because these fungi promote germination in many plant species (see, Öpik et al. 2006; Smith & Smith 2012). It is also important to highlight the effect of GA3 on Petagnaea seed germination. According to a classification system for seed dormancy (Baskin & Baskin 2004), Petagnaea seeds can be considered physiologically dormant (PD) because GA3 promotes germination. In addition, low temperatures are an important factor in breaking seed dormancy in Petagnaea, as observed for the soil pots placed in nature (NL), which were exposed to colder temperatures. The presence of immature embryos has been documented for many representative species of Saniculoideae (e.g., Vandelook & Van Assche 2008; Hawkins et al. 2010; Necajeva & Ievinsh 2013), and their seeds are classified as morphologically dormant (MD). We can only speculate whether MD Petagnaea seeds may exist because there are no complete studies on the presence of underdeveloped embryos in Petagnaea seeds. In light of our data, we can conclude that seed germination does occur in Petagnaea, although after ca. a year and a half (i.e. 19 months) and with a low rate if compared with other study about Saniculoideae taxa [i.e., Sanicula in Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea in Mulaudzi et al. (2009); Eryngium in Ekpong & Sukprakarn (2006) and Necajeva & Ievinsh (2013)]. Petagnaea seeds can be classified only as physiologically dormant (PD), in fact, P. gussonei is sensitive to GA3 treatment. Cold temperatures are required to break its seed dormancy and grow embryos in pots placed in nature (NE). Moreover, a very different pattern of microbial flora abundance and fungal typology (including AMF presence) was observed in NE in contrast to the pots in the laboratory (LC). Arguably, prolonged cold stratification and a specialised microbial community present in the natural habitat are key to the germination of Petagnaea seeds. In terms of conservation, even if a low rate of seed germination has been observed in this study, the agamic reproduction result be an important prerequisite for its survival as confirmed by De Castro et al. (2013). As a final remark on the techniques applied in this study, for the real-time PCR data, it is important to highlight that qPCR does not provide an absolute measure of the different microbial groups due to several limitations that are both biological and technical (e.g., variability nuclei/cell among fungal species, universality of the primers) (Fierer et al. 2006; Plassart et al. 2012); yet, as shown in this study, qPCR can quickly give a good overview of the relative abundance of the biota without using time- and moneyconsuming techniques such as sub-cloning, Temperature Gradient Gel Electrophoresis (DGGE) and sequencing of PCR products.
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results showed that the majority of the seeds did not have an embryo (48.3%), and staining was observed for only 33.33% of seeds; no staining was present for 18.3% of seeds (i.e. non-viable seed). Only high concentrations of GA3 had an effect on Petagnaea seed germination (GA3 = 1000 mgL-1). Germination occurred between the fourth and fifth weeks for a total of ca. 12% (± 1.2) of germinated seeds. No seeds germinated after the fifth week at any GA3 concentration. The germinated seeds were planted in sterile jars with 0.8% agar (AppliChem) to examine the phenology of the seedlings. Figure 2 shows several growth stages of the Petagnaea seedlings. Seed germination was also observed in the soil pots placed in the NE location (i.e. in the mesophilic habitat near the Botanical Garden) with the 11.11% success. The germination started in March 2013 (i.e. one year and seven months after the beginning of experiment) and it lasted for 10 weeks with a germination average of 2/week (± 0.58 SE). No germination was observed in the soil pots located in laboratory conditions (LC). For pot soil analyses, eight pots were chosen from each location, and the water content (u) and pH were analysed. NE soil pots had u = 0.66 (± 0.011 SE) and pH = 7.3 (± 0.022 SE); LC soil pots presented u = 0.58 (± 0.004 SE) and pH = 6.92 (± 0.045 SE). The amount of DNA recovered (estimated from crude DNA extracts) from the different locations was less in the LC soil pots than the NE soil pots (1.055 µg ± 0.015 SE and 1.184 µg ± 0.016 SE per 1.5 g soil, respectively). According to our results (Fig. 3), there are differences in the abundance of both of fungi and bacteria between the two locations (LC and NE), with a higher presence of fungi and bacteria in the soil pots in the natural environment (NE, i.e. mesophilic habitat of the Botanical Garden). The PCR efficiency for each primer pair showed acceptable values (i.e. 2.1 for fungi and 1.9 for bacteria primers). The melting curve profiles of each qPCR assay revealed that different patterns were present in each of the two locations, confirming the presence of different fungal and bacterial communities (i.e. different amplicons) (Fig. 4). To confirm the presence of possible arbuscular mycorrhizal fungi (AMF) in the soil pots placed in nature (NE), an end-point PCR was carried out using Glomeromycota-specific primers (see Appendix 1). Positive amplifications were detected only in NE soil DNA templates, and BLAST analyses of amplicon sequences confirmed the specificity of the primers described by Kruger et al. (2009). Our molecular data showed a higher abundance and different composition of fungi and bacteria in the soil pots where P. gussonei germinated (i.e. NE) compared with the soil pots in laboratory conditions (LC) (Figs. 3-4). It is likely that the qualitative/quantitative microflora differences detected in NE soil pots had a role in establishing appropriate edaphic conditions to promote and/or improve germination in P. gussonei (Garret 1963).
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In fact, the presence of AMF is important because these fungi promote germination in many plant species (see, Öpik et al. 2006; Smith & Smith 2012). It is also important to highlight the effect of GA3 on Petagnaea seed germination. According to a classification system for seed dormancy (Baskin & Baskin 2004), Petagnaea seeds can be considered physiologically dormant (PD) because GA3 promotes germination. In addition, low temperatures are an important factor in breaking seed dormancy in Petagnaea, as observed for the soil pots placed in nature (NL), which were exposed to colder temperatures. The presence of immature embryos has been documented for many representative species of Saniculoideae (e.g., Vandelook & Van Assche 2008; Hawkins et al. 2010; Necajeva & Ievinsh 2013), and their seeds are classified as morphologically dormant (MD). We can only speculate whether MD Petagnaea seeds may exist because there are no complete studies on the presence of underdeveloped embryos in Petagnaea seeds. In light of our data, we can conclude that seed germination does occur in Petagnaea, although after ca. a year and a half (i.e. 19 months) and with a low rate if compared with other study about Saniculoideae taxa [i.e., Sanicula in Vandelook & Van Assche (2008) and Hawkins et al. (2010); Alepidea in Mulaudzi et al. (2009); Eryngium in Ekpong & Sukprakarn (2006) and Necajeva & Ievinsh (2013)]. Petagnaea seeds can be classified only as physiologically dormant (PD), in fact, P. gussonei is sensitive to GA3 treatment. Cold temperatures are required to break its seed dormancy and grow embryos in pots placed in nature (NE). Moreover, a very different pattern of microbial flora abundance and fungal typology (including AMF presence) was observed in NE in contrast to the pots in the laboratory (LC). Arguably, prolonged cold stratification and a specialised microbial community present in the natural habitat are key to the germination of Petagnaea seeds. In terms of conservation, even if a low rate of seed germination has been observed in this study, the agamic reproduction result be an important prerequisite for its survival as confirmed by De Castro et al. (2013). As a final remark on the techniques applied in this study, for the real-time PCR data, it is important to highlight that qPCR does not provide an absolute measure of the different microbial groups due to several limitations that are both biological and technical (e.g., variability nuclei/cell among fungal species, universality of the primers) (Fierer et al. 2006; Plassart et al. 2012); yet, as shown in this study, qPCR can quickly give a good overview of the relative abundance of the biota without using time- and moneyconsuming techniques such as sub-cloning, Temperature Gradient Gel Electrophoresis (DGGE) and sequencing of PCR products.
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CONCLUSION •
Half of Petagnaea seeds are abortive.
•
GA3 affects seed germination.
•
Seed germination occurs in the natural environment after ca. a year and a half.
•
The percentage of germination is low.
•
Seeds are physiologically dormant (PD).
•
Asexual reproduction is an important mechanism of Petagnaea survival.
•
Real-time PCR is a good tool to analyse the abundance and typology of biota without using time/money-consuming techniques.
ACKNOWLEDGEMENTS The authors are also grateful to Dr. Luca Cepollaro and Paolo Sbragia for technical assistance in the laboratory. Special thanks to Nando Peretti Foundation for allowing us to study this plant in the previous years (Project 2007-15).
SUPPLEMENTARY MATERIAL Appendix S1. Molecular methods employed in the soil pots including soil pot DNA extraction, real-time PCR (qPCR), PCR amplification and sequence analyses.
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FIGURE LEGENDS Figure 1. Detail of the stolons of Petagnaea gussonei in mesophilic habitat near the Botanical Garden of Naples (Italy). Figure 2. Phenology of Petagnaea gussonei seedlings. Figure 3. Relative abundance of bacterial and fungi flora within DNA samples from soil pots collocated in the natural environment (NE) and laboratory conditions (LC) estimated using qPCR. Error bars are the standard errors of the mean for the three replicates. Figure 4. Example of melting curves from the 16S (bacteria target) and ITS1 (fungi target) gene assays in the soil DNA pots in the natural environment (NE) and laboratory conditions (LC) estimated using qPCR.
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