Easy and Rapid Detection of Grain Iron Content in Fingermillet [ Eleusine Coracana (L.) Gaertn] Germplasm

In this study, a preliminary evaluation of grain iron content in fingermillet using Perls’ Prussian blue reagent, a stain for Fe 3+ was established. Prussian blue solution of two per cent concentration was used in identifying grain iron content of genotypes based on the development of blue colour intensity. The rank correlation between measured grain Fe content and the colour intensity score was highly significant and positive (r = 0.62; P<0.01), indicating that higher the Fe content in the grain, more will be the intensity of blue colour developed. Pearls’ Prussian blue method could be effectively used as an initial method of screening and genotypes can be scored for high grain Fe content. The grain Fe content of the same genotypes was quantified using Atomic Absorption Spectrophotometer method. Wide variation was observed in fingermillet genotypes for grain Fe content. It ranged from 3.46 (TNEc 0921) to 8.72 (TNEc 0601) mg per 100g of grain. The accessions namely TNEc 0308, TNEc 0407, TNEc 0601, TNEc 0788 and TNEc 0910 were rich in grain Fe content coupled with high grain yield per plant. Therefore, these accessions could be employed in the genetic improvement of fingermillet through hybridization or selection. This simple staining procedure could be used to screen genotypes with high Fe content in large number of germplasm accessions.

Fingermillet [Eleusine coracana (L.) Gaertn] 2n = 4x = 36, belongs to the tribe Chloridae of the family Poaceae. Fingermillet is third in importance among millets in the country in area and production after sorghum and pearlmillet. Fingermillet is the most important small millets in the tropics (12% of global millet area) and is cultivated in more than 25 countries in Africa (eastern and southern) and Asia (from Near East to Far East), predominantly as a staple food grain. Fingermillet is an important cereal because of its excellent storage properties and the nutritive value of the grains. It is also a good source of micronutrients like Calcium, Iron, Phosphorus, Zinc and Potassium. Fingermillet, being a promising source of micronutrients and protein (Malleshi and Klopfenstein, 1998) besides energy, can make a contribution to alleviate micronutrient and protein malnutrition, also called 'hidden-hunger', affecting more than half of the world's population, especially women and preschool children in most countries of Africa and South-East Asia (Underwood, 2000).
Increasing grain iron content is one of the effective way to increase iron intake and reducing the incidence of Fe-deficiency anemia (Welch and Graham et al., 2004). Intake of diet, poor in iron (Fe), zinc (Zn) and protein is the major cause for micronutrient and protein malnutrition. Iron deficiency leads to anemia; about 79 per cent of the preschool children between 6 and 35 months of age and 56 per cent of women between 15 and 49 years of age are anemic in India (Krishnaswamy, 2009). The most cost effective approach for mitigating micronutrient and protein malnutrition is to introduce fingermillet varieties selected and/or bred for increased Fe, Zn and protein contents through plant breeding. Attempts to breed fingermillet for enhanced grain micronutrient and yield are still in its infancy. Exploitation of existing variability among germplasm accessions is the first step and short term strategy for developing fingermillet cultivars to address the micronutrient malnutrition in the target population (Upadhyaya et al., 2011). Previous studies have shown that grain Fe content can vary widely among fingermillet genotypes. Most of the commonly cultivated fingermillet genotypes contain only about 3.9 mg of Fe per 100g of grain, but genotypes with 8 mg or more have also been found in germplasm collections. Hence, Selection and breeding for fingermillet with high grain Fe content is possible. However, in the past, Fe content of fingermillet grain could only be measured by chemical analysis. This poses a problem in screening when dealing with large numbers of germplasm and limited quantity of seed samples available in progenies of crosses. A procedure based on Prussian blue stain proposed for rapid screening of grain Fe content in rice (Promu-thai et al., 2003;Krishnan et al., 2003) and pearlmillet (Velu et al., 2006), which involves scoring of colour intensity development is for the first time used in this study to screen the fingermillet genotypes for high grain Fe content. The main objective of this study was to simplify the estimation and effective screening for grain Fe content in fingermillet and its variability.

Materials and Methods
Twenty five fingermillet genotypes with two check varieties were evaluated at Millet Breeding Station, Tamil Nadu Agricultural University, Coimbatore during rabi, 2011-2012. The field experiment was laid out in randomized complete block design with three replications. Each genotype was grown in single row of 3 metre length with a spacing of 30 cm x 10 cm.

Prussian blue staining method
Prussian blue solution of 2 per cent concentration was used in identifying high grain iron genotypes (Prom-u-thai et al., 2003). A quantity of 10.0 g of potassium ferrocyanide was mixed with distilled water, and the volume was made up to 500 ml. A volume of 10.0 ml concentrated hydrochloric acid (HCl) was mixed with distilled water to make the volume to 500 ml. This solution was prepared by mixing equal volumes of 2 per cent HCl and 2 per cent ferrocyanide solutions. Dry fingermillet grain samples were ground to flour with a pestle and mortar and 0.5 g of flour sample was placed in Borosilicate glass test tubes or Petri-dish. The Prussian blue solution (10 ml) was poured onto the flour in each test tube or Petri-dish. Colour development was recorded after 10 minutes and the color intensity was visually scored on a 1-4 scale, where score 1 represents formation of no colour; 2 for less intense blue colour; 3 for medium blue colour and 4 for more intense blue colour. Frequency distribution was studied for grain Fe content with colour intensity scores.
Simple correlation coefficients between the grain Fe content, 1000 grain weight and grain yield per plant were estimated to examine association among them (Snedecor and Cochran, 1994).

Results and Discussion
Perls' Prussian blue staining has been first reported by Baker (1958) in animal tissue for locating Fe 3+ because it is fast, reproducible and the reagent penetrates bulky tissue to give a distinctive blue reaction product. In plants, this technique had been first report by Krishnan et. al. (2001) in rice. In fingermillet, this technique was used for the first time to score the germplasm accessions based on the grain Fe content. These qualitative scoring based on the intensity of the colour development was validated by quantification of grain Fe content using Atomic Absorption Spectrophotometer (AAS) method (Table 1 In this study, frequency distribution was worked out for 27 genotypes based on grain Fe content with colour scoring and the results are given in Fig. 1. blue color served as a reliable qualitative selection criterion for grain Fe in fingermillet (Fig. 2). This method is efficient in classifying the genotypes with high grain Fe content. When a large number of germplasm accessions or progenies or breeding lines are to be screened for Fe content, this method will be highly efficient in discarding accessions with low Fe content or vice versa (Velu et al., 2006;Promu-thai et al., 2003;Krishnan et al., 2003).

Fig 1. Frequency distribution based on the Iron colour intensity
The frequency distribution showed that 48.15 per cent of the genotypes recorded a high score of 4 with high Fe content, while 29.63 per cent of the

Fig 2. Correlation between grain Fe content and colour intensity
genotypes recorded a score 3. Each of the less intense blue colour (score 2) and no colour (score 1) was show in 11.11 per cent genotypes.

Fig 3. Correlation between grain Fe content and 1000 grain weight
The rank correlation between measured grain Fe content using AAS method and the color intensity score using Perls' Prussian blue method was highly significant and positive, indicating that higher the Fe content in the grain, the more will be the intensity of blue colour developed. In general, the intensity of

Fig 4. Correlation between grain Fe content and grain yield per plant
The simple correlation between measured grain Fe content and the 1000 grain weight were non significant, indicating that the grain Fe content and 1000 grain weight are not associated (Fig. 3).  Similarly, there is no significant difference between grain Fe content and the grain yield per plant (Fig. 4). In general, grain Fe content did not significantly influence either the 1000 grain weight or the grain yield per plant. Hence, it can be inferred that genetic enhancement of grain iron content, 1000 grain weight and grain yield per plant are independent and does not influence each other.

Conclusion
The success of genetic improvement in any character depends on the nature of variability present for that character. Hence, an insight into the magnitude of variability present in the gene pool of a crop is of utmost important to a plant breeder for starting judicious plant breeding program. Although breeding for high yield is the primary objective of breeders, the improvement of the nutritional quality of these cereal crops should also be an important consideration to be given due to prevalence of malnutrition world-wide. Pearls' Prussian blue method can be effectively used as an initial method of screening, and genotypes could be identified for high grain Fe content. This saves the cost, time and labour involved in quantitative estimation of grain Fe content. Highly significant and positive correlation between grain Fe and colour intensity was observed in fingermillet genotypes. Entries having high grain Fe content combined with grain yield per plant can be used in crop improvement programmes.