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Variation in grain iron between seed lots of some upland rice varieties

Chanakan Prom-u-thai, Saicome Pintasen, Sansanee Jamjod, Benjavan Rerkasem

Agronomy Department, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand Email g4268101@cm.edu

Abstract

High levels of grain Fe concentration were found in many local Thai rice varieties. These varieties were upland rice from hill-tribe villages. Seed lots of these local rice varieties from individual farmers have also been found to be genetically diverse. We have developed an easy and rapid method for assessing Fe content of individual rice grains by staining with Perl’s Prussian blue. This experiment set out to detect the source of high grain Fe in individual seed lots of some varieties of upland rice by staining individual grains with Perl’s Prussian blue. A total of 66 accessions of rice seeds with 17 variety names were collected from Tee Cha village, Sob Moei District, Mae Hong Son. Included as checks were one variety with high grain Fe (IR68144) and one with low grain Fe (KDML105). After the husk was removed by hand, 20 seeds of brown rice from each accession were stained with Perls’ Prussian blue, and reaction rated as intense (+++), medium (++), low (+) and none (0). All of IR68144 seeds showed intense (+++) and medium (++) intensity staining. In contrast, Most of KDML105 seeds were rated low (+) and only some were medium (++). Among the farmers’ rice, there were variations in the intensity of Perl’s Prussian blue reaction within and between seed lots. The difference in grain Fe between seed lots of the same variety name was confirmed by chemical analysis. Progeny testing is now underway to verify genetic basis for major differences in grain Fe concentration within seed lots as indicated by the Perl’s Prussian blue staining.

Media summary

The varieties with high grain Fe content, which is confirmed by Perls’s Prussian blue staining and chemical analysis will be the source of genetic lines with high grain Fe character. Perls’s Prussian blue technique is a simple staining procedure that may be used to quickly screen for high Fe content in large germplasm lots containing hundreds of rice entries, using reaction of genotypes with known grain contents as standards.

Key Words

Highland rice, Oryza sativa, Perls’ Prussian blue, Genetic diversity, Grain iron.

Introduction

The application of the Perls’ Prussian blue (Perls, 1867) has made possible rapid estimation of the amount of non-hemoglobin Fe in the marrow and in the blood of humans (Dorothy and Herrette, 1955) and other vertebrates (Doucet and Viel, 2002). Biologically active Fe is normally very tightly complexed to protein, as in hemoglobin or myoglobin. Fortunately, there are various mechanisms within the tissue, which allow the Fe to be detected, in the form of Fe (III) surrounded by hemosiderin. This Fe is reported to be easily stained with Perls’Prussian blue reaction (Stevens, 1977). The Perls’ Prussian blue technique has recently been used to report the presence of Fe in rice grain within and across genotype (Krishnan et al., 2001; Prom-u-thai et al., 2003). Northern Thailand has been designated as one of the centers of diversity of Oryza sativa (Chang, 1976), a great diversity of traits have been identified among local Thai rice varieties including in the levels of grain Fe concentration (Prom-u-thai and Rerkasem, 2001). Many of these varieties were upland rice from hill-tribe villages. Furthermore, seed lots of these rice local varieties from individual farmers have also been found to be genetically diverse (Chang, 1976 and Dennis, 1987). We have developed an easy and rapid method for assessing Fe content of individual rice grains by staining with Perl’s Prussian blue (Figure 1) (Prom-u-thai et al., 2003). We expected that the variation of Fe content within seed lots from upland varieties may be detected by Perls’ Prussian blue technique. This experiment examined grain Fe content in individual grains and seed lots of some varieties of upland rice from Tee Cha village by staining individual grains with Perl’s Prussian blue. The result was confirmed by chemical analysis.

Methods

Seed of 66 accessions of 17 named varieties of upland rice (Oryza sativa) were collected from Tee Cha village, Sob Moei District, Mae Hong Son, Thailand. Check genotypes included IR68144 (a high Fe content variety from IRRI, 20 mg Fe kg-1) and KDML105 (a low Fe content variety from Thailand, 10 mg Fe kg-1). The Perls’ Prussian blue reaction was determined on individual grains of brown rice, after the husk was removed by hand, with 20 seeds for each accession. Seeds were imbibed in distilled water for 4-5 hours, and were cut in half lengthwise through the embryo with a teflon knife (Advanced Personna Brand) in a Petri dish. The specimens were submerged in freshly prepared Perls’ Prussian blue (Perls, 1867) solution (2% hydrocloric acid mixed with 2% potassium ferrocyanide) for 10 minutes. The seeds were then gently washed continuously in distilled water for 2 minutes. The ferric Fe is released from any attachments to protein by treatment with dilute hydrochloric acid and then reacts with a dilute solution of potassium ferrocyanide to produce an insoluble compound, ferric ferro cyanide (Prussian blue) (Doucet and Viel, 2003). The intensity of staining was rated from 0 (no staining), + (low staining) to +++ (most intense) under a stereo microscope. Seed Fe concentration were determined by dry-ashing and atomic absorption spectrophotometry; AA (Delhaize et al., 1984) in mature grain as whole grain brown rice (palea and lemma removed, complete with embryo

Results and Discussion

Perls’ Prussian blue staining has been recommended as a method for locating Fe (III) in animal tissue because it is fast, reproducible and the reagent penetrates bulky tissue to give a distinctive blue reaction (Baker, 1958). The technique has recently been used to report the presence of iron in the aleurone layer of rice grain (Krishnan et al., 2001) and within part of seed and across varieties (Prom-u-thai et al., 2003). In this study, we have found variation in the intensity of Perls’ Prussian blue staining within and between farmer’s seed lots in 17 upland rice varieties with 66 accessions from Tee Cha village which is supported by chemical analysis data (Table 1).

IR68144, a high Fe variety (20 mg Fe/kg, Prom-u-thai and Rerkasem, 2001), reacted with Perls’Prussian blue staining with +++ and ++ in all of the comparisons. Whereas KDML105, a low Fe variety (10 mg Fe/kg, Prom-u-thai and Rerkasem, 2001), reacted with Perl’s Prussian staining with ++ and +. Between the varieties, Bue Mue Tabong # 5, Bue Bang # 10, # 11, #14 and Bue Goa # 28 had higher grain Fe concentration (range 12.2-14.5 mg Fe kg-1) than Bue Bang # 13 and Bue Goa # 29 (5.6-7.7 mg Fe kg-1) (Table 1). Among the farmers’ rice, variation in the intensity of Perls’ Prussian blue reaction of individual grains was found within each seed lot and between seed lots with the same variety name. The higher frequency of grains with more intense staining in Bue Goa #28 and Bue Bang #10, #11 and #14 were closely associated with higher concentration of Fe determined by chemical analysis. Those seed lots with more grains that stained less intensely, i.e. Bue Goa #29 and Bue Bang #13, on the other hand, turned out be much lower in Fe concentration. Furthermore, 25% of the grains in one seed lot of Bue Mue Tabong, had more intense staining than IR68144.

This study has shown that there is much variation in grain Fe within a local rice germplasm. The Perl’s Prussian blue method has been shown be very effective in detecting variation in grain Fe. Progeny testing is underway to determine the genetic basis especially of grains that stained more intensely than the high Fe check (IR68144) such as those 25% of Bue Mue Tabong #5.

Figure 1. Stereo micrograph showing the intensity of Perls’ Prussian blue staining in embryo of IR68144 (high grain Fe content check) em, embryo; en, endosperm (Prom-u-thai et al., 2003)

Table 1. The percent of grains with different intensity of Perl’ Prussian blue staining in highland varieties relative to low and high grain Fe check varieties of 7 selected accessions with 3 varieties name of rice from Tee Cha village

Variety

Seed lot

Check

Frequency (%)

[Fe]
(mg Fe kg-1)

+++

++

+

0

Bue Mue Tabong

# 5

 

25

65

10

0

14.2

 

IR68144

 

100

     
 

KDML

   

100

   

Bue Bang

#10

 

40

60

0

0

14.5

 

IR68144

100

       
 

KDML

   

100

   

#11

 

65

35

0

0

14.1

 

IR68144

100

       
 

KDML

 

33.3

66.6

   

#13

 

0

10

90

0

5.6

 

IR68144

100

       
 

KDML

   

100

   

#14

 

25

75

0

0

14.0

 

IR68144

66.6

33.3

     
 

KDML

   

100

   

Bue Goa

#28

 

30

70

0

0

12.2

 

IR68144

33.3

66.6

0

   
 

KDML

   

100

   

#29

 

0

0

100

0

7.7

 

IR68144

33.3

66.6

     
 

KDML

   

100

   

+++ = intense intensity, ++ = medium intensity, + = low intensity, 0 = none
Fe concentration in IR68144 20 mg Fe kg-1, in KDML105 10 mg Fe kg-1
Percent from the 20 seeds of Perls’ Prussian blue staining.

Acknowledgement

We acknowledge financial support from the Collaborative Crop Research Program of the McKnight Foundation and Thailand Research Fund. The first author is a recipient of a Royal Golden Jubilee PhD Scholarship. Seeds from hill-tribe village (Tee Cha) were provided by N. Yimyam, KDML105 by the Thai Rice Research Institute and IR68144 by IRRI.

References

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