Plant Biosystems, Vol. 142, No. 2, July 2008, pp. 275 – 286 Effects of environmental factors on seed germination of Anthyllis barba-jovis L. MASSIMILIANO MORBIDONI1, ELENA ESTRELLES2, PILAR SORIANO2, ISABEL MARTI´NEZ-SOLI´S3, & EDOARDO BIONDI2 1Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Universita` Politecnica delle Marche, Ancona, Italy, 2ICBiBE - Jardı´ Bota`nic, Universitat de Vale`ncia, Espan˜a and 3Universidad CEU – Cardenal Herrera, Departamento de Fisiologı´a, Farmacologı´a y Toxicologı´a, Valencia, Espan˜a Abstract The influence of the main environmental factors on seed germination of Anthyllis barba-jovis L. were analysed. This work is part of a broader investigation aimed at the reintroduction of this species on Mount Conero, Ancona (central Italy), where it is at present extinct. The seeds were collected from the Gargano headland (southern Adriatic coast). Experimental analyses were carried out to determine: (i) dormancy levels of seeds collected in successive years, and also collected from the soil seed bank; (ii) effects of usual pre-treatments for overriding the physical dormancy of the seeds; (iii) optimal temperature range for maximum germination; (iv) effects of fire on seed germination; and (v) effects of NaCl on germination and on early stages of seedling development. Our results confirm that A. barba-jovis seeds have a physical dormancy due to their teguments, which are water-impermeable. This barrier persists in naked seeds that remain in the soil. Regularly water- drenched seeds show a high germinative ability. The optimal seed germination temperature is 208C, with germination decreasing progressively at lower temperatures, and falling drastically over 208C. Fire and high temperatures positively affected germination. The seeds were shown to be strongly resistant to salt stress, thus enabling the plants to colonize a habitat suitable for halophytes. Key words: Anthyllis barba-jovis, ecosystem restoration, fire species, germination, salt tolerance, seed dormancy affected by anthropic modifications, due to their Introduction inaccessibility, there have been quite recent cases Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 Anthyllis barba-jovis L. is an evergreen shrub that is where A. barba-jovis has disappeared from its natural found in different habitats along the rocky cliffs of habitats (Brilli-Cattarini 1965; Biondi 1986; Danton the western–central Mediterranean basin; in France & Baffray 1995; Paradis 1997; Benedi 1998). At (Var, Bouches du Rhoˆne, He´rault, Corsica), Italy present, it has to be considered as extinct from the (Liguria, Tyrrhenian coast, Sardinia, Sicily, Adriatic Mount Conero promontory, Ancona (central Italy; coast of Gargano, Tremiti Islands), Croatia, Algeria see Figure 2) (Brilli-Cattarini 1965; Biondi 1986), and Tunisia (Cullen 1968; Greuter et al. 1989; although this territory was, for a long time, considered Pignatti 1992; Trinajstic 1994; Biondi et al. 1997; to be its most northern known natural location along Paradis 1997) (Figure 1). the Adriatic coast of the Italian peninsula (Biondi Anthyllis barba-jovis is not included in the World et al. 2002). Its presence in the past is testified by a Conservation Union (IUCN) Red List, although it is herbarium record, as it was collected in 1808 by the a protected species at a national level in France naturalist Paolo Spadoni. In his work entitled (Danton & Baffray 1995) and Croatia (Trinajstic Xylologia Picena applicata alle arti, he claimed to have 1994). In Italy, it is not listed in the Protected Flora collected it along the coast between Ancona and (Conti et al. 1992), but it is considered to be in a risk Sirolo, including the coastal rocky marl and limestone category in seven of the nine regions where it is of Mount Conero (Spadoni 1826). found (Conti et al. 1997). Although the areas The present study is the first part of a broader inhabited by this species are not generally directly programme focused on acquiring further knowledge Correspondence: Edoardo Biondi, Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Universita` Politecnica delle Marche, via Brecce Bianche s.n., 60131 Ancona, Italy. E-mail: [email protected] ISSN 1126-3504 print/ISSN 1724-5575 online ª 2008 Societa` Botanica Italiana DOI: 10.1080/11263500802150514 276 M. Morbidoni et al. Figure 1. Present-day distribution of Anthyllis barba-jovis. Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 Figure 2. Geographic location of Mount Conero and the seed harvesting zone in the Gargano headland. of the autoecology of A. barba-jovis, its ex-situ (i) the dormancy level in seeds collected in suc- conservation, and its reintroduction in the Regional cessive years and taken from the soil seed bank; Natural Park of Mount Conero. Restoration pro- (ii) the effects of standard seed pre-treatments to grammes are a priority in the management of natural override their physical dormancy, with verifica- populations, and thus our study is aimed at providing tion of the results at a histological level; protocols that cover the first steps of plant culture. (iii) the optimal temperature range for maximum Present results relate to various aspects of the germination; germination physiology of A. barba-jovis seeds. In (iv) the effects of fire temperatures on seed particular, the following issues were addressed: germination; and Germination in Anthyllis barba-jovis 277 (v) the effects of NaCl on the germination phase, chemical, with 96% sulphuric acid for 5, 10 or the first stages of seedling development, and 15 min. For the pre-heating treatments, two different the relationships between light conditions, the types of heat were used: dry heat and humid heat, thermal optimum and salt concentrations. with a factorial experimental design. The dry heat was applied using a Selecta thermo-block: the seeds were exposed to at 808C or 1008C for 5 or 10 min. Materials and methods The seeds were subjected to humid heat by immer- sion in water at 808C for 5 s, 10 s and 5 min. The Plant material freeze/thaw protocol was for 20 min at –1968Cin Anthyllis barba-jovis seeds were collected from San liquid nitrogen, followed by 10 min thawing in a Menaio, in the Gargano headland territory of the waterbath at 408C, for 10, 20 and 30 successive Italian peninsula (southern Adriatic basin; Figure 2) cycles. in September 2003 and July 2004. In this region, the All of the basic germination tests for the dormancy A. barba-jovis population is large, and although seed of seeds were carried out in the dark at 208C. The harvesting was abundant, it was not enough to experiments were carried out in December 2003, damage the natural rate of reproduction. The with seeds gathered in September 2003. monosperm legumes are generally retained inside the dry flower remains; they were collected at Electron microscopy maturity directly from the plants and desiccated under laboratory conditions. The fruits were cleaned Observations were carried out on the morphology of by rubbing them between two rubber sheets, and the seed coat of three different seed samples: (i) non- then separating them through differently sized treated seeds collected from the plant; (ii) seeds metallic sieves; the small pods were then opened treated with 96% sulphuric acid for 15 min; and (iii) using a scalpel. The best procedure was to cut the tip non-treated seeds collected from the soil seed bank. and then open the pod longitudinally with a blunt The seeds, are, in general, found in a dehydrated blade. This had to be done with special care to avoid condition, so that they were analysed directly without damage to the seed coat, which could destroy seed the need for any dehydration process. The samples impermeability. The cleaned seeds were kept in for the ultramorphological analysis were mounted on airtight bags at room temperature until used. an aluminium base, and kept in place using double- sided carbon tape (STR tape, 8 mm, Sinto Paint Co. Ltd). They were then metallized with SC 500 Sputter Germination protocol Coater (Bio-Rad) for a cover of gold–palladium of For germination, the seeds were placed on 55 mm *200 A˚ , and examined by scanning electron diameter Petri dishes containing 0.6% agar, which microscopy (FE HITACHI 4100), which included were kept in climate-controlled rooms under various a system for the collection of digital images. The temperature and light conditions, as specified below. voltages used were 5 kV and 10 kV. Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 The observation period was generally one month, and for each treatment 4 replicates of 25 seeds were Germination temperature used. A seed was considered to have germinated when the radicle was longer than 1 mm. The optimum germination temperature was estab- lished by testing a range of temperatures from 58Cto 358C, at intervals of 58C. A second test was carried Mechanical and chemical scarification out to determine the percentages of seeds that The study of physical dormancy was performed germinated under conditions of alternating tempera- through the application of different treatments, ture, using 108C and 188C, with a period of 10 h and including mechanical and chemical scarification, 14 h, respectively. In all cases, the seeds were pre- pre-heating and the alternation of freeze/thaw tem- treated with chemical scarification using 96% sul- peratures using liquid nitrogen. Since similar experi- phuric acid for 15 min, followed by profuse washing ments have not been performed previously on A. with sterilized water; they were kept in the dark for barba-jovis, the various parameters were selected germination. The experiments were carried out in following the studies on Anthyllis cytisoides and October 2004 with seeds collected in September Anthyllis lagascana (Ibanez & Passera 1997; Prieto 2003. et al. 2004). These species are considered to be phylogenetically and ecologically similar to A. barba- Dormant seed collection from the seed bank jovis (Nanni et al.