**2.2. Morphological and anatomical analysis of the testa by scanning electron microscope (SEM)**

Seeds were selected according to their water permeability level; for this, seeds were classified by the hydration rate into rapid, slow, and very slow hydration [16, 17]. Seeds that imbibed water within the first 15 min were discarded due to possible damage in the seed coat. Seeds that hydrated after 2 h of being immersed in water were classified as of rapid hydration; seeds that hydrated between 4 and 14 h of water imbibition were classified as of slow hydration; and seeds with dormancy (hard seeds) were those did not imbibe water after 22 h of immersion and were determined as of very low hydration [18]. Testas of hard seeds that were immersed in water and failed to hydrate between 3000 h (125 days) and 7488 h (312 days) were observed. For each permeability level, five testas per sample were analyzed by SEM (2004 and 2006 harvests). The testa was observed and photographed on surface and lateral views under different magnifications. In micrographs, the testa topography and cuticle appearance were observed and compared among seeds; cell layers, cell size, and cuticle thickness were determined in lateral sections. Observations were made near and around the central point [19] and in the lens area. In addition, the cell wall of macrosclereids was observed and compared among testas of different permeability levels. SEM observations of the exomorphology and anatomy of the testa were made at CRIBABB (Centro Regional de Investigaciones Básicas y Aplicadas de Bahía Blanca) and CERIDE (Centro Regional de Investigación y Desarrollo de Santa Fe), using the method mentioned in [20, 21].

as testa, which is characterized by having several layers. Outermost is the epidermis, which is uniseriate and consists of palisade macrosclereids with uneven thickened walls and interior lumen, closely packed and containing different chemical substances (quinones); the hilar region consists of two palisade layers. Below the epidermis is the hypodermis or columnar cells (osteosclereids), followed by the lacunose parenchyma, which is composed of several layers of flattened cells, the aleurone layer and the endosperm, and then the embryo [2–4].

In some leguminous species, the cuticle wax present in the hard coat plays an important role in water permeability. Some studies suggested that the osteosclereids pose the main barrier to water entry, since most seeds start to imbibe water only after those cells are perforated [3, 5]. Studies of the structure and chemical components of the seed coat of some species indicated the presence of ions, such as K, Ca, and Mg, and some phenolic compounds with a role in hardening and protection [1, 6]. The anatomy of the seed coat does not vary between hard and a non-hard seeds [7, 8]; however, some differences in content of different components, porosity, and "linea lucida" or light line were found in soybean [9]. [10, 11] related lignin content to resistance to mechanical damage in soybean cultivars; however, they exhibit hydrophobic traits and can be related to impermeability [12]. Reports on extended seed longevity are mainly related to Fabaceae and Malvaceae taxa containing Malpighian cells or osteosclereids [2, 5, 8, 13, 14]. [15] assume that dormancy breakage in these seeds (except for mechanical scarification) is due to the formation of an opening in the specialized anatomical structure of the seed coat (or of the fruit) through which water enters

Seed coat of white clover (*Trifolium repens*) was found to have different water permeability levels, with the corresponding different dormancy levels, which influence seed physiological quality [16]. The factors hindering water entry are not clearly defined and may be more than one. A comparative study of testas of seeds with different water permeability levels may help to explain the different dormancy levels and to determine the necessary techniques useful for breaking dormancy in seed lots. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of white clover cv. NK Churrinche relative to water permeability levels.

Seeds of white clover cv. NK Churrinche (2004 and 2006 harvests) obtained from Criadero Barenbrug Palaversich S.A., Argentina, were used for the study. Seeds were checked under magnifying glass (10×) for purity, and those with visible physical damage were discarded.

Seeds were selected according to their water permeability level; for this, seeds were classified by the hydration rate into rapid, slow, and very slow hydration [16, 17]. Seeds that imbibed water within the first 15 min were discarded due to possible damage in the seed coat. Seeds

**2.2. Morphological and anatomical analysis of the testa by scanning electron** 

and hydrates the embryo.

188 Advances in Seed Biology

**2. Materials and methods**

**2.1. Seeds**

**microscope (SEM)**
