**3. Results and discussion**

## **3.1. Morphological and anatomical SEM analyses of the testa**

The testa of the rapid-hydration white clover seed (nondormant seeds) observed under SEM (**Figure 1**) was 30–40 μm thick, showing a thin cuticle (1μm) with slight cracks, depressions, and openings, as mentioned by [24] for the soybean testa surface. At the surface level, the ends of the macrosclereids were visible, giving an irregular and rugged appearance. Macrosclereids (**Table 1**) were approximately 30 μm long and were arranged in palisade, but not too compressed, with a visible light line. In cross section, an irregular polygonal contour was observed (three to seven sides) (**Figure 2A**). The long and short axes varied in length; cell wall width was irregular and smaller than in macrosclereids of slow-hydration testas. Osteosclereids were not visible in these seed testas, and parenchyma cells were present.

Seed coats of very slow hydration (dormant seeds) (**Figure 3**) were 45–50 μm thick and had a thick cuticle (3–5 μm), giving the surface a smooth appearance. Macrosclereids (**Table 1**) were about 40 μm long; they were arranged in a single, very compressed palisade layer and had a visible clear line. In cross section, they had an irregular, polygonal contour (three to seven sides, **Figure 2B**). The long and short axes varied in length, depending on the cell; cell wall thickness was also variable but was thicker than in permeable testa macrosclereids. Osteosclereids (**Figure 3B** and **C**) were visible, 15–18 μm in width and relatively short (5–8 μm); parenchyma cells were present. Anatomical characteristics of hard seed testas with different immersion time were similar. The anatomical characteristics of the hard seed testas of 3000 h were similar to those of 5000 h and 7488 h (data not shown). Hard seeds of this species have very deep physical dormancy [16], suggesting similar anatomical and chemical traits among seeds within that category.

## **3.2. Determination of structural polymeric substances, pectic substances, and polyphenols, tannins, and cutins**

The results show (**Table 2**) that cellulose, hemicellulose, lignins, and condensed tannins were present in higher amounts in impermeable testas, whereas in permeable ones, a higher amount of cell content was observed. These findings are related to the presence of macrosclereids of greater size and thicker cell wall found in dormant seeds than in rapid-hydration seeds, as well as to the presence of osteosclereids.

Cellulose is associated with hemicellulose, with both being the most important structural substances in the cell wall. The cellulose values found in very low-hydration testas (48.6%) Anatomical and Chemical Insights into the White Clover (*Trifolium repens* L.) Seed Coat... http://dx.doi.org/10.5772/intechopen.70313 191

cutin via the Van Soest method. Determinations were made in 0.5 g testa samples with two repetitions (approximately 3000 testas) per permeability level, using testas of both harvests [20, 21], at the Institute of Cellulose Technology, Faculty of Chemical Engineering, National

The testa of the rapid-hydration white clover seed (nondormant seeds) observed under SEM (**Figure 1**) was 30–40 μm thick, showing a thin cuticle (1μm) with slight cracks, depressions, and openings, as mentioned by [24] for the soybean testa surface. At the surface level, the ends of the macrosclereids were visible, giving an irregular and rugged appearance. Macrosclereids (**Table 1**) were approximately 30 μm long and were arranged in palisade, but not too compressed, with a visible light line. In cross section, an irregular polygonal contour was observed (three to seven sides) (**Figure 2A**). The long and short axes varied in length; cell wall width was irregular and smaller than in macrosclereids of slow-hydration testas. Osteosclereids were not visible in these seed testas, and parenchyma cells were present.

Seed coats of very slow hydration (dormant seeds) (**Figure 3**) were 45–50 μm thick and had a thick cuticle (3–5 μm), giving the surface a smooth appearance. Macrosclereids (**Table 1**) were about 40 μm long; they were arranged in a single, very compressed palisade layer and had a visible clear line. In cross section, they had an irregular, polygonal contour (three to seven sides, **Figure 2B**). The long and short axes varied in length, depending on the cell; cell wall thickness was also variable but was thicker than in permeable testa macrosclereids. Osteosclereids (**Figure 3B** and **C**) were visible, 15–18 μm in width and relatively short (5–8 μm); parenchyma cells were present. Anatomical characteristics of hard seed testas with different immersion time were similar. The anatomical characteristics of the hard seed testas of 3000 h were similar to those of 5000 h and 7488 h (data not shown). Hard seeds of this species have very deep physical dormancy [16], suggesting similar anatomical and

**3.2. Determination of structural polymeric substances, pectic substances, and** 

The results show (**Table 2**) that cellulose, hemicellulose, lignins, and condensed tannins were present in higher amounts in impermeable testas, whereas in permeable ones, a higher amount of cell content was observed. These findings are related to the presence of macrosclereids of greater size and thicker cell wall found in dormant seeds than in rapid-hydration

Cellulose is associated with hemicellulose, with both being the most important structural substances in the cell wall. The cellulose values found in very low-hydration testas (48.6%)

University of Litoral, Santa Fe, Argentina.

**3.1. Morphological and anatomical SEM analyses of the testa**

chemical traits among seeds within that category.

seeds, as well as to the presence of osteosclereids.

**polyphenols, tannins, and cutins**

**3. Results and discussion**

190 Advances in Seed Biology

**Figure 1.** Testa of *T. repens* cv. NK Churrinche seed (2004 harvest) permeable after 2 h of water imbibition. (A) Testa surface. (Cu) Cuticle and (O) opening. (B and C) 3D view of testa. (T) Testa, (Cu) cuticle and waxes, (M) macrosclereids, (O) osteosclereids, (P) parenchyma, (Fs) slight fissure among cells, and (Ll) light line ×3000 (bar, 10μm).


Mean values (μm) were obtained through SEM observations (×2700; ×6700 and ×10,000) of cross sections of nine cells of three teguments, 2006 harvest. (Min) minimum value; (Max) maximum value.

are close to those mentioned by [25, 26] for secondary wall (41 and 45%, respectively), with the amount of hemicellulose found (16.1%) being lower than that reported by those authors (30%). In alfalfa seeds, cellulose and hemicellulose in testas amounted to 39.9 and 20.7%, respectively [20, 21]. Although these components exhibit hydrophilic characteristics, in the cell wall of macrosclereids, lignification eventually occurs [2], forming hydrophobic secondary walls that provide rigidness [11, 27]. Lignin was found in greater amount in impermeable seed teguments than in permeable ones. This component is a highly insoluble polymer of

**Figure 2.** Macrosclereids of seed testa of *T. repens* cv. NK Churrinche (2004 and 2006 harvests) (A) rapid-hydration testa, permeable after 2 h of water imbibition. (B) Very low-hydration testa, impermeable after 3000 h water imbibition. (Cw) Cell wall, (Ml) middle lamella, and (L) cell lumen. (A) ×2700, 1 cm, 0.91μm; (B) ×6700, 1 cm, 1.31μm.

**Table 1.** Macrosclereid measurements in testa of seeds of white clover (*Trifolium repens*) cv. NK Churrinche as a function of testa permeability to water.

Anatomical and Chemical Insights into the White Clover (*Trifolium repens* L.) Seed Coat... http://dx.doi.org/10.5772/intechopen.70313 193

are close to those mentioned by [25, 26] for secondary wall (41 and 45%, respectively), with the amount of hemicellulose found (16.1%) being lower than that reported by those authors (30%). In alfalfa seeds, cellulose and hemicellulose in testas amounted to 39.9 and 20.7%, respectively [20, 21]. Although these components exhibit hydrophilic characteristics, in the cell wall of macrosclereids, lignification eventually occurs [2], forming hydrophobic secondary walls that provide rigidness [11, 27]. Lignin was found in greater amount in impermeable seed teguments than in permeable ones. This component is a highly insoluble polymer of

Mean values (μm) were obtained through SEM observations (×2700; ×6700 and ×10,000) of cross sections of nine cells of

**Table 1.** Macrosclereid measurements in testa of seeds of white clover (*Trifolium repens*) cv. NK Churrinche as a function

**Testa Length (μm) Axis (cell width) (μm) Cell wall thickness (μm) Long Short**

Rapid hydration 25-30 2.72 2.51 3.26 1.79 1.67 1.86 0.54 0.37 0.93 Very slow hydration 30-40 9.40 7.20 11.38 3.41 2.35 4.59 2.2 1.18 3.67

three teguments, 2006 harvest. (Min) minimum value; (Max) maximum value.

of testa permeability to water.

192 Advances in Seed Biology

**Mean Min Max Mean Min Max Mean Min Max**

**Figure 2.** Macrosclereids of seed testa of *T. repens* cv. NK Churrinche (2004 and 2006 harvests) (A) rapid-hydration testa, permeable after 2 h of water imbibition. (B) Very low-hydration testa, impermeable after 3000 h water imbibition. (Cw)

Cell wall, (Ml) middle lamella, and (L) cell lumen. (A) ×2700, 1 cm, 0.91μm; (B) ×6700, 1 cm, 1.31μm.

**Figure 3.** Testa of *T. repens* cv. NK Churrinche seed (2006 harvest) of very low hydration, impermeable after 3000 h of water imbibition. (A) Testa surface, (Cu) cuticle; (B and C) cross section of two testas, (Ll) light line, (M) macrosclereid, (O) osteosclereid, and (P) parenchyma. A and C: SEM ×3000 (A and B, bar 10 μm; C, bar 20 μm).


Tp, Total polyphenols; Ct, Condensed tannins; Cw, Cell wall, (hc) hemicellulose, gums, mucilages, cellulose, lignin, and cysteine; Cc, cell content, glucose, fructose, sucrose, galactose, starch, and fructans (protoplasts and pectic substances); ADF, Acid detergent fiber. (c) Cellulose, (l) lignin, and (cu) cutin.

\* It could not be determined by the extraction method; the presence was observed via SEM.

**Table 2.** Determination of organic compounds (%) in dry matter of seeds testa of *T. repens* cv. NK Churrinche with different permeability levels.

phenolic units that form a large network of crossed bonds. The lignin content (5.5%) found in impermeable testas is relatively high, compared to the 8% of cereals straw [28] and 16.85% of wheat straw [29]. In alfalfa, 3.34 and 7% of lignins (cell wall) were reported at leaf and preflowering stages, respectively [30, 31]. And 7.7% was integument of very slow-hydration seeds [21]. Condensed tannin is another component found in higher amount (0.29%) in hard seed testas than in permeable seeds (0.16%) and might provide the testa with astringent and feeding deterrent characteristics, as well as with defense from predators.

Regarding cell content determined in the testa (**Table 2**), a great difference was observed between rapid-hydration (67%) and very low-hydration (30.1 and 22.9%) seeds. That fraction includes diverse substances, such as pectic substances. Based on observations of the lumen in micrographs of macrosclereids, we assume that the amount of cell content determined in hard seed testas largely corresponds to pectins of the middle lamella of macrosclereids. Pectins act as cementing substances [32] and become lignified in older cells [2].

Cutin values found in rapid- and slow-hydration testas were 0.5 and 9.1%, respectively (**Table 2**). In the very slow-hydration teguments, cutin proportion could not be determined via the extraction method used; however, SEM observations showed a proportionately thicker cutin layer than in seeds of other degrees of permeability.

From an anatomical perspective, the highest level of seed physical dormancy in white clover seeds would be attributed to the combination of the effects of a thicker cuticle, thicker cell wall of macrosclereids, and greater length and width of macrosclereids than in seeds of the other permeability levels, as well as to the presence of osteosclereids.

Physiologically and chemically, the dormancy mechanism via hard coats is explained by an increased amount of hydrophobic and cementing substances, such as cutins, lignins, tannins, and pectins. Lignification of the thick cell wall would be one of the main components determining physical dormancy. White clover seeds with physical dormancy contain viable embryos and very low water content, as found in alfalfa [17]; once water permeability is produced and other appropriate factors are present, embryo imbibition occurs, originating normal seedlings [16]. The traits found in these seeds would provide physical dormancy with the capacity to preserve the embryo during the time when seeds are dormant.
