Introduction
Both natural pigments obtained from dye plants and synthetic dyes can be used for dyeing purposes. The increasing interest in obtaining raw materials from renewable sources, the control of toxic effluents and the classification of many synthetic colourants as toxic when in contact with the skin are the main reasons for a refreshed interest in natural dyes by the dyestuff industry.
Woad (Isatis tinctoria L.) is among the plant-derived dyestuffs which produce the blue pigment indigo. Woad belongs to the Brassicaceae family and its centre of origin is Eurasia, but it was widely diffused throughout Europe (Spataro et al., Reference Spataro, Taviani and Negri2007).
Ancient Egyptians used woad to dye cloth (Barber, Reference Barber1991) and ancient Britons and Celts used it to colour their faces and bodies (Carr, Reference Carr2005). As the colour blue created was well suited to use with madder for cheaper purples and it was also used to dye fabric black, woad cultivation was widespread throughout the medieval Europe (Hofenk de Graaff et al., Reference Hofenk de Graaff, Roelofs and van Bommel2004; Cardon, Reference Cardon2007). The woad and dye production were highly profitable in Europe from the 12th to the 17th centuries (Oberthür et al., Reference Oberthür, Graf and Hamburger2004), but during the 16th century, its importance began to decrease with the import of blue dye derived from Indigofera tinctoria L. from the Far East, since it was cheaper (Galletti et al., Reference Galletti, Barillari, Jori and Venturi2006). From the 19th century onwards, woad almost disappeared from the European agriculture due to the development of synthetic indigo. The indigo molecule is not produced in significant amounts directly by woad. Instead, it is formed during the extraction process from the precursors accumulated in the leaves. During extraction, water-soluble isatan B (indoxyl-5-ketogluconate) is hydrolysed via fermentation to produce indoxyl, which further reacts with another indoxyl molecule, producing indigo (Gilbert et al., Reference Gilbert, Maule, Rudolph, Lewis, Vandenburg, Sales, Tozzi and Cooke2004).
The indole-derived compounds produced in the leaves, roots and seeds have anti-bacterial, anti-fever and anti-swelling properties and the essential oils produced in the seeds can be used in soap and body creams (Fréchard et al., Reference Fréchard, Fabre, Pean, Montant, Fauvel, Rollin and Fourasté2001; Chen et al., Reference Chen, Howard, Yang, Wang, Oppenheim and Krakaner2002; Oberthür et al., Reference Oberthür, Jäggi and Hamburger2005; Galletti et al., Reference Galletti, Barillari, Jori and Venturi2006).
While the studies on the assessment of morphological diversity revealed a great variation among the Isatis species (Kizil, Reference Kizil2006) and among plants and accessions (Spataro and Negri, Reference Spataro and Negri2008), quantitative analysis show that isatin B, indican and indigo amounts vary significantly throughout the year, being higher in spring and summer. The concentration also varies between young and older leaves. The younger leaves have a higher concentration of precursors (Maugard et al., Reference Maugard, Enaud, Choisy and Legoy2001).
The renewed interest showed by the dyestuff industry in natural dyes, the potential cultivations of woad in marginal lands and the use by the medicine and cosmetic industry shows that this can be an interesting crop in the near future.
The aims of this study were to evaluate the agronomic characteristics and to quantify the indigo precursors in a collection of woad accessions from different countries.
Material and methods
Ten woad accessions from nine countries (UK, Spain (SP), Belgium (BL), Germany (GR), Austria (AU), Italy (IT), Poland (PL), Kazakhstan (KZ) and Portugal (Portugal-Madeira (PT-M) and Portugal-Coimbra (PT-C)) were evaluated in Vila Real (41°17′N; 7°44′W; 460 m above the sea level), Portugal. The seeds were sown in jiffy pots in August and transplanted to the field in September 2008. From each accession, ten plants were characterized according to 5 qualitative and 15 quantitative traits.
The pigment content was also evaluated in the plants, by HPLC-DAD, regarding each main woad precursor. The identification of these precursors was done in comparison with Maugard et al., (Reference Maugard, Enaud, Choisy and Legoy2001) chromatograms. An analysis of variance (ANOVA) of quantitative data and a principal component analysis (PCA) were performed.
Results and discussion
ANOVA showed significant differences (P < 0.05) among accessions for all traits. The rosette leaves accessions from PT-M and UK had the longest (14.00 and 14.06 cm) and widest (4.06 and 3.50 cm) rosette leaves, but had the lower number (28.8 and 21.2, respectively).
The tallest height of the plants at the beginning of flowering (68.5 cm) as well as at the end of flowering (84.9 and 87.0 cm, respectively) was obtained in the accessions from PT-C and KZ.
The Portuguese accession from Madeira was the earliest to flower, while the accession from SP was the latest, with a 23 d difference. The longest period of flowering was registered in the accession from AU (23 d), while the shortest one was observed in the accession from SP (9 d).
The accession from SP had the highest average number of seeds/plant (3423), but a low total seed weight (14.75 g) as well as a low 1000 seeds weight (4.54 g), while the accession of PT-M had a high average number of seeds/plant (2837), the highest total weight (21.00 g) and the highest 1000 seeds weight (7.53 g).
High variability for morphological traits was also observed among woad accessions by Spataro and Negri (Reference Spataro and Negri2008) and between wild and cultivated woad species by Kizil (Reference Kizil2006). In other dye species like weld and madder, high diversity in morphological and agronomic traits were also observed (Angelini et al., Reference Angelini, Pistelli, Belloni, Bertoli and Panconesi1997; Angelini et al., Reference Angelini, Bertoli, Rolamdelli and Pistelli2003; Gaspar et al., Reference Gaspar, Moiteiro, Tukman, Coutinho and Carnide2009; Baghalian et al., Reference Baghalian, Maghsodi and Naghvi2010).
The first three main components of the PCA separated the accessions into four groups. One group includes the four accessions from South Europe (PT-M, PT-C, SP and IT); the second group contains the accessions from Central and Eastern European countries (GR, PL and KZ); the accessions from UK, BL and AU form two different groups (Fig. 1).
PCA for ten accessions estimated with morphologic traits.
A good correlation between the clusters and the geographic origin of the accessions was also observed by Spataro and Negri (Reference Spataro and Negri2008) for woad accessions from different European and Asian countries.
The first three components of the PCA accounted for 41.84, 22.74 and 12.18% of the total variation, respectively. The eigenvectors for the 14 morphological traits are shown in Fig. 1. PC1 was highly correlated with the length of rosette leaves, plant height, number of seeds/plant, total seed weight and 1000 seeds weight; PC2 was highly correlated with the number of day onset of flowering, number of rosette leaves and silique length and PC3 was highly correlated with the width of rosette leaves and number of days from seed germination to onset of scape.
Chemical analysis (Table 1) showed that the amount of isatan B present in the leaves of woad is higher in accessions from GR, BL and PT-C, ranging from 3.8 to 5.1 g/kg fresh weight. Reciprocal interactions between indoxyl, isatan B and indican precursors and fresh leaf weight were compared and significant differences (P < 0.05) were observed. Despite previous observations of Angelini et al. (Reference Angelini, Tozzi and Di Nasso2007) of a indican:isatan B ratio of 1:5 in woad, in our case, this ratio was 1:2 in the same species. The presence of indoxyl, isoindigo, cis-indirrubin and indigo is an indication of the physiological state of the leaf. While the levels of indoxyl and indigo are lower than those of indican and isatan B, their presence is due to the oxidation of their indigo precursors.
Amount of indigo and indigo precursors in ten populations of Isatis tinctoria
Natural dyes are important in modern dyeing procedures as they can contribute to an increasing sustainable textile dyeing, in what concerns, for example, water, chemicals and energy consumption.
In conclusion, we can say that this study revealed the high variability present in woad accessions, concerning the morphological and indigo precursors contents. This variability shows that there is a great potential for the improvement of woad, regarding both agronomic and quality characteristics.
Acknowledgements
The authors thank the Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; the Royal Botanic Garden of Madrid; the Botanic Garden of Coimbra; the National Botanic Garden of BL; the Germplasm Bank of Hungary and Dr. David Hill of University of Bristol for providing the woad material. This work was supported by FCT project POCTI/AGR/56087/2004.
