Iron biofortification of myanmar rice

Abstract

Iron (Fe) deficiency elevates human mortality rates, especially in developing countries. In Myanmar, the prevalence of Fe-deficient anemia in children and pregnant women are 75 and 71%, respectively. Myanmar people have one of the highest per capita rice consumption rates globally. Consequently, production of Fe-biofortified rice would likely contribute to solving the Fe-deficiency problem in this human population. To produce Fe-biofortified Myanmar rice by transgenic methods, we first analyzed callus induction and regeneration efficiencies in 15 varieties that are presently popular because of their high-yields or high-qualities. Callus formation and regeneration efficiency in each variety was strongly influenced by types of culture media containing a range of 2,4-dichlorophenoxyacetic acid concentrations. The Paw San Yin variety, which has a high-Fe content in polished seeds, performed well in callus induction and regeneration trials. Thus, we transformed this variety using a gene expression cassette that enhanced Fe transport within rice plants through overexpression of the nicotianamine synthase gene HvNAS1, Fe flow to the endosperm through the Fe(II)-nicotianamine transporter gene OsYSL2, and Fe accumulation in endosperm by the Fe storage protein gene SoyferH2. A line with a transgene insertion was successfully obtained. Enhanced expressions of the introduced genes OsYSL2, HvNAS1, and SoyferH2 occurred in immature T2 seeds. The transformants accumulated 3.4-fold higher Fe concentrations, and also 1.3-fold higher zinc concentrations in T2 polished seeds compared to levels in non-transgenic rice. This Fe-biofortified rice has the potential to reduce Fe-deficiency anemia in millions of Myanmar people without changing food habits and without introducing additional costs.

Keywords: Myanmar rice; OsYSL2; anemia; biofortification; ferritin; iron; nicotianamine; rice transformation.

Figure 1

Callus induction and regeneration efficiency in rice varieties Paw San Yin (V3) and Tsukinohikari in different medium combinations. (A) Tissue culture of Paw San Yin (V3) in the N6D-N6D-MSre-MS medium combination. (B) Tissue culture of Paw San Yin (V3) in the 2N6-2N6-MSre-MS medium combination. (C) Tissue culture of Tsukinohikari in the N6D-2N6-MSre-MS medium combination. See “Media Used for Callus Induction and Regeneration Test” in the Section “Materials and Methods” for medium combination. DAG, days after germination; DAT, days after transferring. The numerals shown inside parentheses mean DAG or DAT when photograph was taken on each medium.

Figure 2

Identification of the best medium combination for each variety. V1–V15 shown on upper right part of each picture represent the variety numbers described in Table 1 and TK means Tsukinohikari. The media combination described under each picture showed the best media combination for each variety. Pictures of regenerated plants were taken at 3 days after acclimation. Pictures of V5 and V7 were not shown as regenerated plants were not obtained from these two varieties.

Figure 3

Regeneration efficiency of Myanmar rice varieties and the Tsukinohikari variety. V1–V15 under horizontal bar represent the rice variety numbers described in Table 1 and TK means Tsukinohikari. Numbers on vertical bars are quotients of total numbers of regenerated plants divided by numbers of callus-induced seeds, which represent regeneration efficiency.

Figure 4

Durations of time required for green spot appearance in Myanmar rice varieties and in the variety Tsukinohikari. V1–V15 on vertical bar represent the variety numbers described in Table 1 and TK means Tsukinohikari. Durations of time from transfer to MSre media to green spot appearance are indicated. Regenerated plants were obtained from these green spots.

Figure 5

Transformation of Paw San Yin-Fer-NAS-YSL2. (A) Callus induction (at 29 DAG). (B) Pre-incubation (at 8 DAT). (C) Three days following Agrobacterium-infection. (D) Calli on 1st selection medium of N6D-CH10 (at 7 DAT). (E) Calli on 2nd selection medium of N6D-CH30 (at 14 DAT). (F) Calli on 3^rd selection medium of N6D-CH50 (at 7 DAT). (G) Calli on MS regeneration medium (MSre-CH50) (at 20 DAT). (H) Calli on MSre-CH50 (at 32 DAT). (I) Green spots appearance of calli and shoots emergence on MS medium (at 4 DAT). (J) Regenerated plants on MS medium. (K) Greenhouse-grown T₀ plants. Left panels of (C–F) show transformation with Agrobacterium at a concentration of OD = 0.01; right panels of (C–F) shows transformation at a concentration of OD = 0.1. The numerals shown with DAG or DAT mean DAG or DAT when photograph was taken on each medium.

Figure 6

Confirmation of gene insertion in transgenic Paw San Yin-Fer-NAS-YSL2 line 1. NT, non-transgenic Paw San Yin line; L1, Paw San Yin-Fer-NAS-YSL2 line 1; Vec, Fer-NAS-YSL2 vector (positive control). OsGlb promoter-SoyferH2, soybean Ferritin gene SoyferH2 with promoter region of the 26 kDa OsGlb1 gene; OsAct promoter-HvNAS1, barley nicotianamine synthase 1 gene with promoter region of the rice OsActin1 gene; HPT, hygromycin phosphotransferase gene; NPTII, neomycin phosphotransferase II gene; iGUS, β-glucuronidase gene with an intron; OsActin1, endogenous rice actin gene.

Figure 7

Metal concentrations in T₁ polished seeds of Paw San Yin-Fer-NAS-YSL2. (A) Fe concentration. (B) Zn concentration. NT, non-transgenic Paw San Yin. L1, Paw San Yin-Fer-NAS-YSL2 transgenic line 1. Bars represent means ± SE, n = 3. Asterisks (**) above the bars indicate significant differences at P < 0.01 between NT and L1 (determined byt-test).

Figure 8

Greenhouse-grown T₁ plants. Photograph was taken during tillering stage at 30 days after transplanting. NT, non-transgenic Paw San Yin. L1 sublines, Paw San Yin-Fer-NAS-YSL2 transgenic line 1 sublines.

Figure 9

Quantitative real-time RT-PCR analysis of HvNAS1, OsYSL2, and SoyferH2. (A) HvNAS1, (B) OsYSL2, and (C) SoyferH2 expression levels. T₁ plants were cultivated in commercial soil (Bonsolichigou) in a greenhouse. Total RNA was extracted from immature T₂ seeds (seeds at the early milky stage, 10 days after fertilization) of each line (n = 3). NT, non-transgenic Paw San Yin. L1-1, L1-2, and L1-3, Paw San Yin-Fer-NAS-YSL2 transgenic line 1 sublines. Bars represent means ± SE of three independent real-time RT-PCR reactions. Asterisks (*) and (**) above the bars indicate significant differences at P < 0.05 and P < 0.01, respectively, between NT and L1 sublines (determined by t-test). n.d., not detected.

Figure 10

Metal concentrations in T₂ polished and brown seeds of Paw San Yin-Fer-NAS-YSL2. (A) Fe concentration in T₂ polished seeds. (B) Zn concentration in T₂ polished seeds. (C) Fe concentration in T₂ brown seeds. (D) Zn concentration in T₂ brown seeds. NT, non-transgenic Paw San Yin. L1-1, L1-2, and L1-3, Paw San Yin-Fer-NAS-YSL2 transgenic line 1 sublines. Bars represent means ± SE, n = 3. Asterisks (*) and (**) above the bars indicate significant differences at P < 0.05 and P < 0.01, respectively, between NT and L1 sublines demonstrated by t-tests.

Figure 11

Fe and Zn content per T₂ seed of Paw San Yin-Fer-NAS-YSL2. (A) Fe content in husk, bran, and endosperm per T₂ seed. (B) Zn content in husk, bran, and endosperm per T₂ seed. NT, non-transgenic Paw San Yin; L1-1 and L1-2, Paw San Yin-Fer-NAS-YSL2 transgenic line 1 sublines. Metal content of polished seed is shown as endosperm. Metal content of bran is calculated by subtracting metal content of polished seed from that of brown seed.