Re-evaluating TiCl4 and UV assays for detection of vicine and convicine in high-throughput screening of immature and mature seeds of faba bean

Abstract

Genomic resources have just started to focus on the faba bean; the genes for synthesis of vicine and convicine (V-C) have not been determined, and recently developed genetic markers for these antinutritionals have not been used to examine these traits in very large scale in faba bean. Simple, rapid and cost-effective technologies are crucial in crop breeding programs, especially in the developing world, and in some cases, traditional methodologies are used in combination with genetic markers to assess agronomic traits and the value of gene markers. Here, two methodologies (TiCl4 assay and 274 nm absorption) are re-evaluated for their application in detection of V-C in faba beans. In comparison with TiCl4 assay, the method of 274 nm UV absorption without an HPLC analysis offers more reliable analysis for detection of V-C in immature and mature seeds of faba bean. Its application in high throughput screening by 60 min agitation of immature seeds or mature seed flour in 2% trichloroacetic acid (TCA) allows quick screening of low V-C faba beans. The level of V-C was maximum when seed moisture was 80% and V-C level was measured as 0.92% in CDC Fatima flour. Though V-C from 2% TCA extract of mature and immature seeds of CDC Fatima was detected by 274 wavelength in the TiCl4 assay reaction, a Ti-aglycone complex was not clearly detectible at 480 nm as previously suggested.Key words: Vicine, convicine, TiCl4 assay, UV assay, high throughput, seed, faba bean. INTRODUCTIONFaba bean (Vicia faba L.) is the third most important feed grain legume after soybean and pea. The largest producer is China, followed by Australia, France, the Americas, Egypt, Sudan, Morocco and Ethiopia (FAO, 2013). In North America, faba bean has been grown on a limited basis on the Canadian prairies since the 1970s (Gade, 1994). Production has expanded since 1997 and the annual growth rate was 5.82% between 2003 in Canada (FAO, 2013.Faba bean is rich in protein (Burstin et al., 2011) and essential amino acids (Alghamdi, 2009;Mortuza et al., 2009; Sosulski and Hold, 1980). It has potential as a meat substitute in many parts of the world where there is demand for non-animal protein sources. Faba bean produces the highest percentage of protein (24-38%) per seed (Burridge, 1999;Burstin et al., 2011) and per unit *Corresponding author. E-mail: ygungor27@gmail.com. Tel: 306-2414668.Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License land area as compared to all other temperate and tropical pulse crops, including lentil (Lens culinaris), common bean (Phaseolus vulgaris), chickpea (Cicer arietinum) and pea (Pisum sativum) (Broad beans world yield, FAOSTAT 2013). Mature faba beans provide high-quality carbohydrates (58%), fiber (25%) (USDA Nutritional Database, http://www.nal.usda.gov/fnic/foodcomp/search), antioxidants, and nutrients, with a comparatively low glycemic index (GI) (40 and 48 GI per 150 g serving for baked bean and dark durum wheat pasta, respe...