The screening of rice germplasm, including those transgenic rice lines ...

International Journal of Food Science and Technology 2005, 40, 563–569

Short communication The screening of rice germplasm, including those transgenic rice lines which accumulate b-carotene in their polished seeds, for their carotenoid profile Jing Tan, Niranjan Baisakh, Norman Oliva, Vilas Parkhi, Mayank Rai, Lina Torrizo, Karabi Datta & Swapan K. Datta* Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines (Received 5 May 2003; Accepted in revised form 20 January 2005)

Keywords

Accessions, endosperm, golden rice, whole grain.

Introduction

Carotenoids are a large group of lipid-soluble pigments synthesized in plants, bacteria, fungi and algae. They are also found in animals. In plants, carotenoids function as accessory pigments in photosynthesis, serve as photo-protective compounds and are also responsible for the red, yellow and orange colours in flowers and fruits. In humans and animals, an important role of certain carotenoids is to act as provitamin A compounds. Of over 600 carotenoids from natural resources, about fifty can serve as precursors of vitamin A. Major provitamin A carotenoids in mammals, including humans, are b-carotene, b-cryptoxanthin and a-carotene (Harborne, 1998). Nonprovitamin A carotenoids, such as lutein and zeaxanthin have other health benefits (Sommerburg et al., 1998). Malnutrition has been recognized as a major health problem of the world, and vitamin A, iron and iodine deficiencies are the major issues (Gopalan, 1977). Vitamin A deficiency (VAD), which is common in developing countries, causes severe visual impairment and correlates with fatal infections and death from diarrhoea, respiratory diseases and measles (West et al., 1989). In Southeast Asia alone, an estimated five million children *Correspondent: Fax: 63 2 580 5699; e-mail: [email protected] doi:10.1111/j.1365-2621.2005.00971.x 2005 Institute of Food Science and Technology Trust Fund

each year suffer from the eye disease xerophthalmia caused by VAD (Sommer, 1988). Rice, an important staple crop, is produced around the world and consumed by half of the world’s population. Asian countries are much more dependent on rice for food energy and protein intake than other countries. But starchy rice, together with other staples, is not a good source of micronutrients especially after milling (Gregorio, 2002). Milling also causes a heavy loss in minerals (Vasconcelos et al., 2003). As milled rice is commonly consumed and there is no carotenoid precursor in the endosperm (Burkhardt et al., 1997), bioengineering seems a practical and effective approach to increase or add micronutrients (Datta & Bouis, 2000). Moreover, studies show synergism between vitamin A and zinc intake (Christian & West, 1998) and that synergy between b-carotene and iron uptake is very likely (Garcia-Casal et al., 1998). Therefore, consumption of transgenic rice with increased b-carotene has the potential to alleviate both VAD and iron deficiency anaemia which is prevalent in riceconsuming countries where resource-poor people are solely dependent on rice as a major source of calories and energy. Bioengineering the entire b-carotene biosynthetic pathway into a japonica rice Taipei 309 endosperm has been achieved and resulted in Ôgolden riceÕ (Ye et al., 2000). At the International Rice Research Institute (IRRI), in the Philippines, research on

563

564

HPLC carotenoid profile of rice germplasm J. Tan et al.

engineering the b-carotene synthesis into tropical indica rice, the common staple in Asian countries, has been successful and a further improvement is under way (Datta et al., 2003; Krishnan et al., 2003). The present paper describes the screening of a large number of exotic and commercial rice cultivars to explore the carotenoid profile of rice germ plasm. These data were compared with data on the carotenoid profile of some transgenic plants where the genes for the entire biochemical pathway used to synthesize b-carotene had been engineered into the plant in such a way that it was hypothesized that b-carotene would be detectable in polished seeds. Normally, polished seeds do not contain b-carotene. Materials and methods

Eighty-eight rice accessions (non-transgenic unless indicated) were used for the screening (Table 1). Seventy-nine were obtained from IRRI germ plasm bank and were grown under the same field conditions at IRRI. Nine (Basmati 370, BR28, BR29, BR-R, Kranti, Limpopo, Mahsuri, NDR359 and Pantdhan4) were collected from the origin countries. Seeds were polished with Kett grain polisher (Kate Co., Tokyo, Japan) for at least 1 min. One gram of rice grains was ground with a cyclone sample mill (UDY Corporation, Fort Collins, CO, USA). For extraction without saponification, 2 mL of acetone was added to rice powder and mixed well in the dark at 4 C. The mixture was centrifuged and the supernatant was collected. The procedure was repeated until complete decolorization. To the pooled extract, a half volume of petrol-ether/ diethyl ether (1:1) was added, followed by water until phase separation. The epiphase was dried down in a Speed-Vac dryer (Maxi Dryer Plus, Heto, Allerod, Denmark), then stored at )20 C or dissolved in 50 lL methanol or hexane for HPLC analysis (40 lL was injected). For extraction with saponification, 500 lL of 40% methanolic KOH was added to 1.0 g of rice powder and vortexed vigorously. The paste was incubated at 56 C for 20 min. One drop (20 lL) of water was added, followed by 10 mL of hexane. The mixture was vortexed and the extract was collected. The pooled extract was dried down for storage or HPLC analysis as mentioned.

At the beginning of the study, dehulled seeds were extracted without selection, in other words, mature and immature (not fully ripened) seeds were mixed for screening. However, as we realized that chlorophyll and carotenoids in the green part of the immature seeds would interfere with the result, only mature dehulled seeds were selected. Extraction with saponification was also chosen in order to eliminate the interfering components. When mature unpolished ÔAmarillo CubaÕ and ÔIR64Õ seeds were tested under both extraction methods (with one or two repeats), lutein and b-carotene peaks were compatible, respectively, in terms of separation, characterization and peak areas. The HPLC analysis was done with a Waters Alliance 2690 Separation Module (Waters Corporation, Milford, MA, USA) that was equipped with a Waters 996 photodiode array detector and Waters Millennium32 Chromatography Manager. Samples were separated on a Waters Symmetry C18 column (4.6 · 250 mm, 5 lm) after passing a guard column of the same material (3.9 · 20 mm, 5 lm) and eluted with one of the two solvent systems. Earlier samples were eluted with solvent systems A1 (methanol:water, 16:1) and B1 (tert-butylmethyl ether:methanol, 1:1) at a flow rate of 1 mL min)1. The column was developed with 100% A1 for 25 min, followed by a linear gradient to 100% B1 in 50 min. The final condition was maintained for 10 min before the 15 min reequilibration. As this HPLC condition was time consuming and chloroform, which gave a high solvent front, was not an ideal solvent, we later switched to use hexane or methanol as solvent and a time-saving HPLC (30 min) system. This system included A2 (acetonitrile:tetrahydrofuran:water, 10:4:6) and B2 (acetonitrile:tetrahydrofuran:water, 10:8.8:1.2). The column was developed with 100% A2 for the first 3 min, then a linear gradient to 100% B2 in 7 min and remained at B2 for 20 min. In order to compare these two systems, unpolished mature seeds of IR68899B, extracted with acetone and ether, were analysed under both HPLC conditions. Lutein, b-carotene and other major peaks could be well separated. Peak identification was based on retention time, main absorption maxima and spectral shape as compared with the corresponding standards under


2005 Institute of Food Science and Technology Trust Fund


Table 1 Rice cultivars screened for b-carotene and other carotenoids

Variety name

Country of origin

IRGC no. if known

FAO designated

FAO date

Agno (up) Amarillo Amarillo Azucena Banjar Kuning (up) Basmati 370 Beaq Kuning Jaru Bencer Binalasang (up) Bongkitan BR 28 (up) BR 11 BR 29 BR-R (elite line) Burik Burungan C4 63G Calibo Cempo Kuning (up)

Philippines Cuba Philippines Philippines Indonesia India Indonesia Philippines Philippines Philippines Bangladesh Bangladesh Bangladesh Bangladesh Philippines Indonesia Philippines Philippines Indonesia

88282 51117 47120 328 43338

No Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14

43341 44309 5284 52998 IRTP1801 53458 IRTP15241

Yes Yes Yes Yes No Yes No

44367 27099 16331 3929 43366

Yes Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14

Dinorado

Philippines

30333

Yes

1994.09.14

Dudemasino Elon Elon (up) Gedabung Kuning Ginobierno (up) IR51500-AC11-3 IR64 IR68144 IR68899B IR72 Jao Leuang 27-3-19 Jarom Emas 156 (up) Ketan Kuning Khao Dawk Mali 105 Khao Tah Tong Klemas Kranti Kuning Leuang Awn (up) Leuang Awn 4-8-134 Leuang Bang Bai Leuang Bun-Mah Leuang Gra-Dahd (up) Leuang Khamin Leuang Lek 30-1-57 Leuang Meuang Gahn Leuang Noi 31-1-39 Leuang Nuan 807 (up) Leuang Pra1 Leuang Pra2 Leuang Pra3 Leuang Rai (up) Leuang Rai La Gwian

India Philippines Indonesia Philippines Philippines Philippines Philippines Philippines Philippines Thailand Malaysia Indonesia Thailand Thailand Indonesia India Indonesia Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand

45569 5193 43698 47214 76994 66970

Yes Yes Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14

76330 44138 40684 25429 27748 47856 38596 IRTP2543 43715 40666 44155 47858 36687 40667 47859 44156 47792 44157 36615 47793 47794 47795 40668 47796

Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14

Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14


1994.09.14 1994.09.14 1994.09.14 1994.09.14

Specificity

Blast resistant Moderately salt tolerant

Moderately salt tolerant Blast resistant; red seed coat

1994.09.14

Moderately salt tolerant

Moderately salt tolerant; red seed coat Aromatic; blast resistant; vigorous under drought; red seed coat Moderately blast resistant Extra vigorous under drought Moderately salt resistant

Moderately blast resistant Blast resistant Moderately salt resistant

Moderately salt resistant

Moderately blast resistant


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Table 1 Continued

Variety name

Country of origin

IRGC no. if known

FAO designated

FAO date

Leuang Tah Aiam Leuang Tah Leuam 8-16-176 (up) Leuang Tawng 105-6-101 Leuang Tawng 105-6-8 (up) Leuang Tawng Kam Leuang Yai 17-26-81 (up) Leuang Yai 74-4-20 Limpopo Mahsuri Malagkit Songsong Mamina Mimis Mot Bui Nagpili

Thailand Thailand Thailand Thailand Thailand Thailand Thailand Mozambique India Philippines India Philippines Vietnam Philippines

47797 36616 44158 36618 47798 36620 44159

Yes Yes Yes Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14

IRTP20196 599

Yes

1994.09.14

52937 79075 24187

Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14

Nahalin (up) NDR 359 Nep Hoa Vang Nang Huong Cho Dao (NHCD) Padi Jarum Emas Padi Kuning Padi Payo Kuning Panci Kuning Pantdhan 4 (p) Perurutong NB

Philippines India Vietnam Vietnam Indonesia Indonesia Indonesia Indonesia India Philippines

81704

Yes

1996.03.07

40748

Yes

1994.09.14

43499 43504 43510 43730

Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14

566

Yes

1994.09.14

Purtok (up) Pusa-Basmati 1 Raden Kuning Sahm Ruang Leuang Sahm Ruang Leuang 14-33-46 (up) Salumpikit (up) Serendah Kuning Sirendah Kuning Sonapatnai Swarna Taipei 309 Tulsi (up)

Philippines India Indonesia Thailand Thailand Philippines Indonesia Indonesia India India China India

44707 78422 43557 47817 36660 5423 47740 43649 46695 IRTP12715 42576 16943

Yes Yes Yes Yes Yes Yes Yes Yes Yes

1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14 1994.09.14

Yes Yes

1994.09.14 1994.09.14

Specificity

Aromatic; cold tolerant

Blast resistant; extra vigorous under drought; red seed coat

Cold tolerant Aromatic Vigorous under drought

Bacterial blight resistant; vigorous under drought; purple seed coat Extra vigorous under drought Extra vigorous under drought

Moderately resistant to blast

Vigorous under drought

Note: Varieties are screened under both polished (p) and unpolished (up) conditions unless otherwise indicated.

the same separation conditions. Typical data were given in Table 2. Lutein and b-carotene standards were from Sigma (St Louis, MO, USA). All chemicals were from J.T. Baker (Phillipsburg, NJ, USA). Results and discussion

Among the eighty-eight non-transgenic rice accessions (including BR-R, an elite restorer line), sixtynine cultivars were screened using both polished

and unpolished samples and nineteen cultivars were screened for unpolished seeds only. Fortynine cultivars were analysed using the 85-min HPLC system; these were unpolished seeds (randomly selected). Forty accessions, both polished and unpolished, were run using the 30-min HPLC method. None of the polished cultivars was identified as containing b-carotene when both HPLC conditions were used. When unpolished, a majority of the rice accessions showed both lutein and b-carotene peaks but some had only one




24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 85 85 85 85 85 85 85 85 85 85 85 85 85 85

Lutein standard b-Carotene standards Amarillo Cuba (unpolished) Amarillo Cuba (polished) Dudemasino (unpolished) Dudemasino (polished) IR68899B (unpolished) IR68899B (polished) Sirendah Kuning (unpolished) Sirendah Kuning (polished) Swarna (unpolished) Swarna (polished) Leuang Meuang Gahn (unpolished) Leuang Meuang Gahn (polished) Golden Indica Rice Nang Huong Cho Dao 3 (polished) Golden indica rice IR64E26 (polished) Lutein standard b-Carotene standards Bongkitan (unpolished) Bongkitan (polished) Calibo (unpolished) Calibo (polished) Khao Dawk Mali 105 (unpolished) Khao Dawk Mali 105 (polished) Klemas (unpolished) Klemas (polished) Limpopo (unpolished) Limpopo (polished) Golden Rice T309 (polished) Golden Indica Rice KDGR29–104 (polished)

1 – 1 – 1 – 1 – 1 – 1 – 1 – 1 1 1 – 1 – – – 1 – 1 – – – – 1

Peak

445.3, 473.1

444.1, 471.9 444.1, 473.1

445.3, 473.1

18.89 18.37

17.66

450.1, 477.9

10.50

17.57

450.1, 479.2

10.47

450.1, 477.9 451.4, 479.2 444.1, 473.1

450.1, 477.9

10.52

10.83 11.15 17.62

450.1, 479.2

10.34

456.2, 482.8

450.1, 477.9

10.41

10.27

450.1, 477.9

Abmax (nm) (peak II, III)

11.10

Rta (min)

b

Retention time. Absorbance maximum. Main absorbance maximum (peak II) is shown in bold. c Ratio of the height of the main absorbance maximum (II) to the longest wave maximum (III).

a

HPLC (min)

Sample

1.10

1.08

1.09

1.08

1.11 1.11 1.09

1.11

1.10

1.11

1.12

1.10

1.10

1.11

Ratioc (II/III)

Lutein

Lutein

Lutein

Lutein

Lutein Lutein Lutein

Lutein

Lutein

Lutein

Lutein

Lutein

Lutein

Peak identification

2 2 – 2 – 2 – 2 – 2 – 2 – 2 2 – 2 2 – 2 – 2 – 2 – – – 2 2

Peak

453.8, 479.2

62.03

452.6, 479.2

452.6, 479.2 450.1, 477.9

62.36

61.22 61.62

452.6, 479.2

452.6, 479.2 451.4, 481.6

61.52 61.77

62.02

457.4, 484.0 457.4, 482.8

457.4, 488.8

457.4, 485.2

456.2, 488.8

458.6, 484.0

457.4, 485.2 457.4, 484.0

Amax (nm) (peak II, III)

19.33 19.84

19.35

19.74

19.80

19.40

20.83 19.53

Rt (min)

Table 2 Peak retention times and spectral characteristics of lutein and b-carotene standards, selected non-transgenic and transgenic rice cultivars

1.13 1.17

1.14

1.12

1.12

1.13 1.14

1.12

1.10

1.13

1.18

1.13

1.12 1.11

Ratio (II/III)

b-Carotene b-Carotene

b-Carotene

b-Carotene

b-Carotene


b-Carotene

b-Carotene

b-Carotene


Peak identification



567

568


(Table 1 and 2). Chlorophyll and other unidentified peaks were eluted between lutein and b-carotene in immature seeds. As a contrast, Chromatogram (450 nm)

Name

transgenic rice contained not only b-carotene and lutein but also other carotenoids after polishing (Fig. 1). In the future, a more detailed and Spectrum of peak 1

0.045

457.4

450.1

0.040

Standards of Lutein (peak 1) and β-carotene (peak 2)

485.2

477.9

0.035

1

0.030 0.025 U A

0.020 0.015 0.010

2

0.005 0.000 2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes

350.00

400.00

0.0050

450.00 nm

500.00

380.00

550.00

400.00

420.00

440.00

460.00

480.00

500.00

520.00

480.00

500.00

520.00

540.00

nm

450.1

0.0045

NHCD unpolished

Spectrum of peak 2

457.4 477.9

0.0040

1

0.0035 0.0030 0.0025 0.0020

U A

0.0015 0.0010 0.0005

2

0.0000 –0.0005 –0.0010 2.00

4.00

6.00

8.00

10.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

380.00

400.00

420.00

440.00

460.00 nm

540.00

0.0050 0.0045 0.0040

NHCD polished

0.0035 0.0030 0.0025 U A

0.0020 0.0015 0.0010 0.0005 0.0000 2.00

4.00

6.00

8.00

10.00

0.026

Golden indica rice NHCD 3 polished

457.4

450.1

0.024

477.9

0.022

484.0

0.020

1

0.018 0.016 0.014

2

0.012

U A

0.010 0.008 0.006 0.004 0.002 0.000 2.00

4.00

6.00

8.00

12.00 14.00 Minutes

10.00

16.00

18.00

20.00

22.00

24.00

350.00

400.00

450.00

500.00

550.00

350.00

400.00

450.00

500.00

550.00

nm

nm

0.0050

461.0

452.6

0.0045

485.2

479.2

0.0040

1

0.0035

IR64 unpolished

0.0030 0.0025 U A

0.0020 0.0015

2

0.0010 0.0005 0.0000 –0.0005 2.00

4.00

6.00

8.00

10.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

380.00

400.00

420.00

440.00

460.00 nm

480.00

500.00

520.00

540.00

0.0050 0.0045 0.0040

IR64 polished

0.0035 0.0030 0.0025 U A

0.0020 0.0015 0.0010 0.0005 0.0000 2.00

4.00

6.00

8.00

12.00 14.00 Minutes

10.00

16.00

18.00

20.00

22.00

24.00

0.010

450.1

Golden indica rice IR64E26 polished

458.6

477.9

0.009 0.008

484.0

1

0.007 0.006 0.005

U A

2

0.004 0.003 0.002 0.001 0.000

2.00

4.00

6.00

8.00

12.00 14.00 Minutes

10.00

16.00

18.00

20.00

22.00

24.00

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

0.0040 450.1

0.0035

477.9

0.0030

BR29 unpolished

1

0.0025 0.0020 U A

0.0015 0.0010 0.0005 0.0000 542.2

–0.0005

2.00

4.00

6.00

8.00

10.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

0.0040 0.0035 0.0030

BR29 polished

0.0025 0.0020 U A

0.0015 0.0010 0.0005 0.0000

2.00

4.00

6.00

8.00

10.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

0.030

Golden indica rice KDGR29-111-5 polished

450.1

1

0.020

U A

457.4 477.9

0.025

0.015 0.010

2

0.005 0.000 2.00

4.00

6.00

8.00

10.00

12.00 14.00 Minutes

16.00

18.00

20.00

22.00

24.00

350.00

400.00

450.00

nm

500.00

550.00

360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 540.00 nm

Figure 1 HPLC chromatograms and spectra of identified peaks for carotenoid standards, selected rice accessions (polished and unpolished) and polished transgenic rice cultivars under the 30-min run.




quantitative study of the presence of carotenoids in brown rice could be done for cultivars with particular traits. Hence, the current screening showed a different carotenoid profile between polished and unpolished non-transgenic seeds. Some of these exotic germplasm lines, containing lutein or other carotenoids, could be used as parental lines for further breeding or bioengineering to improve their carotenoid contents. Transgenic rice contain carotenoids including b-carotene, even after polishing, could also be used directly for consumption as milled rice is still widely accepted by consumers. Acknowledgments

USAID is gratefully acknowledged for its financial support. We thank Drs B. Sivakumar, National Institute of Nutrition, India, and Peter Beyer, University of Freiburg, Germany, for their valuable suggestions on carotenoid studies and Bill Hardy for editorial assistance. References Burkhardt, P.K., Beyer, P., Wu¨nn, J. et al. (1997). Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis. The Plant Journal, 11, 1071–1078. Christian, P. & West, K.P. Jr (1998). Interactions between zinc and vitamin A: an update. American Journal of Clinical Nutrition, 68, 435S–441S.


Datta, S.K. & Bouis, H.E. (2000). Application of biotechnology to improving the nutritional quality of rice. Food and Nutrition Bulletin, 21, 451–456. Datta, K., Baisakh, N., Oliva, N. et al. (2003). Bioengineereed ÔgoldenÕ indica rice with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnology Journal, 1, 81–90. Garcia-Casal, M.N., Layrisse, M., Solano, L. et al. (1998). Vitamin A and b-carotene can improve nonheme iron absorption from rice, wheat and corn by humans. Journal of Nutrition, 128, 646–650. Gopalan, C. (1977). Malnutrition: the major health problem of the world. International Journal of Health Education, 20, 74–78. Gregorio, G.B. (2002). Progress in breeding for trace minerals in staple crops. Journal of Nutrition, 132, 500S–502S. Harborne, J.B. (1998). The terpenoids. In: Phytochemical Methods: a Guide to Modern Techniques of Plant Analysis, 3rd edn (edited by J.B. Harborne). Pp. 138–140. London: Chapman and Hall. Krishnan, S., Datta, K., Baisakh, N., de Vasconcelos, M. & Datta, S.K. (2003). Tissue-specific localization of b-carotene and iron in transgenic indica rice (Oryza sativa L.). Current Science, 84, 1232–1234. Sommer, A. (1988). New imperatives for an old vitamin (A). Journal of Nutrition, 119, 96–100. Sommerburg, O., Keunen, J.E.E., Bird, A.C. & van Kuijk, F.J.G.M. (1998). Fruits and vegetables that are sources for lutein and zeaxanthin: the macular pigment in human eyes. British Journal of Opthamology, 82, 901–907. Vasconcelos, M., Datta, K., Oliva, N. et al. (2003). Enhanced iron and zinc accumulation in transgenic rice with ferritin gene. Plant Science, 164, 371–378. West, K.P. Jr, Howard, G.R. & Sommer, A. (1989). Vitamin A and infection: public health implications. Annual Review Nutrition, 9, 63–86. Ye, X., Al-Babili, S, Klo¨ti, A. et al. (2000). Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid free) rice endosperm. Science, 287, 303–305.


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