Even though rice is one of the most important food crops in the world, its micronutrient contents including iron (Fe) is not enough to solve mineral malnutrition which is a significant public health issue in most developing countries (Fitzgerald et al., 2009). Fe deficiency is probably the most widespread micronutrient deficiency in humans. Approximately two billion people suffer from Fe deficiency according to the World Health Organization (WTO, 2015). Fe deficiency causes tiredness, metabolizing problem and anemia. Based on the recorded incidence of anemia, most preschool children and pregnant women in developing countries are suffer from Fe deficiency (Lucca et al., 2006 ; Poletti et al., 2004). Fe supplements is considered as the main way to increase Fe uptake for health care, however it is difficult to intake for people living in developing world. Therefore development of the cereal variety contained high content of Fe would improve Fe nutrient without economic burden (Meng et al., 2005). Experts estimate that a rice based diet should contain 14.5 ppm Fe in an endosperm. However, Cesar P et al reported that average Fe content in a milled rice was 2 ~ 3 ppm and it was 10 ~ 11 ppm in a brown rice (Martínez et al., 2010).

To develop more Fe contained rice, Fe content in endosperm should be analyzed. Fe is micronutrient in rice. Fe content of rice is usually measured by inductively plasma spectrometry (ICP) thus rice sample should be catalyzed with nitric acid solution and be filtered by filter paper. It takes times and could make error while sample processing. Especially Fe content analysis method using ICP is difficult to apply breeding program for screening in high Fe contained rice. To breed high Fe content rice variety, the effective screening method for selecting high Fe contained elite line is essential.

Many researches have been reported that Fe deficiency chlorosis is expressed in the new leaf tissue because Fe is essential micronutrient for plant growth and development (John, 2012; Berry et al., 2006). In this study, we tried to find the effective method in screening high Fe content brown rice by analyzing leaf chlorophyll content after cultivation on Fe limited MS media.

MATERIAL AND METHOD

We firstly investigated the change of leaf chlorophyll content according to Fe content in a brown rice. Transgenic rice, OsNAS3-OX which has high Fe content by over-expressing OsNAS3 (Nicotianamine synthase 3 in Oryza sativa L.) and wild type, Dongjinbyeo were used as materials. Brown rice of each material was sterilized in the 30% sodium hypochlorite for 30 min and planted on a solid MS medium. Fe condition in MS medium were created as five levels - 0, 20, 50, 70 and 100%. The nutrient composition and concentration for solid MS medium for 100% of Fe content was in Table 1. Each nutrient except phytogel was completely dissolved then autoclaved. 50ml of each MS media was poured in a 100 ml of petri dish then dried in a clean bench.

After cultivated for three weeks on solid MS medium, three plants on each solid MS medium were cut to measure leaf chlorophyll content. Leaf chlorophyll content was measured based on Mackinney’s work (Mackinney, 1941). The leaf was cut then put in a 15 ml of tube. The 10 ml of 80% acetone was added in each tube then put at 4? for five days in dark condition. The chlorophyll extracted solution was filtered by the filter paper and chlorophyll content was measured by spectrophotometer at 663 nm and 645 nm.

After confirm the difference of leaf chlorophyll content between transgenic and wild-type plant when cultivated in Fe limited MS media, we analyzed the leaf chlorophyll content of twenty kinds of Korean leading varieties after cultivated on solid MS medium which was contained 0, 5, 10, 15% of Fe (Table 2).

Brown rice of twenty Korean leading varieties were also sterilized in 30% sodium hypochlorite for 30 min and planted on solid MS medium contained different Fe content.

To investigate the Fe content in brown rice, each seed was dried for several days under the natural condition to adjust water content in seed as 16%. Dried seed was grounded. The 0.5 g of each sample was used to measure Fe content with three replications. Fe content analysis was performed by inductively plasma spectrometry (ICP) according to the method reported by Zarcinas et al. (Zarcinas et al., 1987).

SAS version 9.2 (SPSS Inc) was used for data analysis. Duncan’s multiple range test (DMRT) was carried out to identify significant differences (P < 0.05) between individual treatments.

RESULT AND DISCUSSION

Even though development of high Fe contained rice variety is one of the best ways to solve Fe deficiency, it is difficult to measure Fe content in each brown rice for selecting high Fe content brown rice. Therefore more effective method to screen high Fe content brown rice without analysis of Fe content is needed. When Fe content is low in leaves, chlorosis occurs because Fe is required by several enzymes involved in the formation of chlorophyll. We investigated the leaf chlorophyll content after cultivation in the Fe limited MS media to develop an effective screening method of high Fe content brown.

Firstly, we checked the change of leaf chlorophyll content by cultivating on different Fe containing solid MS media. We measured the leaf chlorophyll content of OsNAS3-OX and wild-type plant after cultivated each brown seed on the solid MS medium which was contained various Fe contents for three weeks. The leaf chlorophyll content in both materials showed a significant difference in the solid MS medium contained 0% and 20% of Fe content (Fig. 1(A)). Especially, the difference of each leaf color could be distinguished visually in the solid MS medium contained 0% of Fe. In the 0% of Fe contained solid MS medium, the leaf of OsNAS3-OX showed green color while those of Dongjinbyeo showed yellow and white color since OsNAS3-OX was reported to have about 2.9 time higher Fe content than a wild-type plant (Lee et al., 2009). (Fig. 1(B)). John (2012) and Ramirez et al. (2008) reported that Fe deficiency chlorosis in soybeans was caused by the inability of the plant to utilize Fe in the soil. Similarly Ana et al. (2003, 2006) and Rombola et al. (2006) reported that Fe chlorosis was occurred in orchards and it decreased yield and quality of fruit when Fe content was low in soils. We also considered that OsNAS3-OX might grow better than wild-type plant without serious chlorosis due to high Fe content in a brown rice. However, leaf chlorophyll content of OsNAS3-OX and wild-type plant did not showed significant difference in the solid MS medium contained 40, 60, 80 and 100% of Fe content. Even though the leaf chlorophyll content of OsNAS3-OX were higher than wild-type plant in the 20% Fe containing solid MS medium due to the high Fe content in a brown rice, OsNAS3-OX and wild-type did not show chlorosis in the 20% Fe containing solid MS medium. Therefore we expected the Fe deficiency chlorosis did not occur in over 20% of Fe content in solid MS medium regardless of Fe content in a brown rice. Therefore the Fe content in solid MS media to screening a high Fe content brown rice was considered to be below 20% compare to normal Fe condition in solid MS media.

To investigate the change of leaf chlorophyll content according to Fe content of brown rice in other varieties, we analyzed Fe content of twenty Korean rice varieties (Fig. 2).

Fe contents of materials were between 4.56 mg and 3.05 mg per 100 of a brown rice. Among them, Jogwang showed the highest Fe content with 4.56 mg per 100 g of a brown rice and Ilmibyeo showed the lowest Fe content with 3.05 mg per 100 g of a brown rice. Most varieties contained 3.5 to 3.8 mg Fe per 100 g of brown rice. We classified rice materials as five groups according to Fe content in a brown rice by analyzing Duncan’s multiple range test (DMRT). The Fe content in a brown rice was divided as 7 groups from a to f by DMRT. The only one materials were contained in group a, b, e and f. Therefore we considered a and b as the same group, e and f as the same group to further research. Fe content of group 1 was 4.16 mg ~ 4.56 mg (a and b) which contained high Fe content over 4mg per 100g of a brown rice, group 2 was 3.66 mg ~ 3.85 mg, group 3 was 3.5 mg ~ 3.6 mg, group 4 was 3.32 mg ~ 3.4 mg and group 5 was 3.05 mg ~ 3.23 mg per 100 g of brown rice.

Those twenty kinds of brown rice were cultivated in the solid MS medium contained 0, 5, 10 and 15% of Fe content compare to normal condition. After three weeks, the leaf chlorophyll content was measured. Leaf chlorophyll content among groups showed difference in the solid MS medium contained 0% and 5% of Fe content. However, leaf chlorophyll content in groups did not show significant difference in the solid MS medium contained 10% and 15% Fe content. In the 0% of Fe contained solid MS medium, leaf chlorophyll content of group 1, 2 and 3 showed differences, while leaf chlorophyll content did not show significant difference among group 3, 4 and 5. In the solid MS medium contained 5% of Fe content, leaf chlorophyll content of all groups showed difference. In the solid MS medium contained 10% of Fe content, leaf chlorophyll content of group 1 and 4 showed significant difference while leaf chlorophyll content did not show difference among group 2, 3, 4 and 5. The leaf chlorophyll content of all groups did not show difference in the solid MS medium contained 15% Fe (Table 3).

Though these results, we could consider that 0% of Fe content in a solid MS medium was not enough to screening a brown rice with low Fe containing. And over 10% of Fe content in a solid MS medium was not effective on screening the high Fe contained brown rice.

The correlation between the Fe content of brown rice and leaf chlorophyll content was analyzed. The correlation between Fe content of brown rice and leaf chlorophyll content were 0.66 and 0.79 in the 0% and 5% Fe contained solid MS medium, respectively. In the solid MS medium contained 10% and 15% Fe content, correlation between Fe content of brown rice and leaf chlorophyll content was significantly decreased as 0.39 and 0.037. Though this result, we considered that rice could grow well in the solid MS medium contained 10% and 15% of Fe content without chlorosis regardless of Fe content in a brown rice. While the 0% of Fe content in a solid MS medium might be not enough to grow without leaf chlorosis even though the brown rice had high Fe content. The MS medium containing 5% of Fe content seemed to be the most effective to select a high Fe contained brown rice (Fig. 3).

Through this result, analysis of leaf chlorophyll content after cultivation on solid MS medium containing 5% of Fe content might be effective method to screening a high Fe contained brown rice.

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