Biofortification of Rice

1
Biofortification of Rice

• Gerard Barry
• Golden Rice Network Coordinator
• HarvestPlus Rice Crop Team Leader
• Head of the Intellectual Property Management Unit
• International Rice Research Institute

ASA, etc., Seattle
2
Chronic Undernutrition among Children
3
WORLD CEREAL SITUATION Source food outlook No.
5, Nov. 2003, FAO
Rice consumption has increased from 400 to 416
million tonnes in this period.
Million Tonnes
4
Rice in the Asian Diet(FAOSTAT 2001and 1999)
Total Calories/cap/day 2000 3000 Rice
contributes 30-75 of the Calories
5
The goal of biofortification is to improve the
nutritional values of the crops themselves.

• Target Nutrients (initial)
• Iron
• Zinc
• Betacarotene (Vitamin A)

6
Biofortification will complement ongoing efforts
to improve nutritional well being

• Other approaches
• Improved diet/dietary information always
  needed.
• Supplementation
• vitamin A 1-2 times/year
• iron and zinc needed daily
• Fortification
• Vitamin A oil, sugar, flours
• Iron flours
• especially by reaching rural food production,
• self- and locally sourced and unprocessed foods.
• and will reduce the costs of reaching large
  populations.

7
Some existing Biofortification activities

• Orange-Flesh Sweet Potato
• High-Iron Rice
• High-Betacarotene Maize
• High-Iron Beans

8
Outline High Fe/Zn and Provitamin A Rice

• Breeding/germplasm
• Transgenic approaches
• Genomic opportunities
• Regulatory environment and considerations

9
Estimated Annual Maternal Deaths from Severe
Anemia (Asia)
10
ADB-IRRI-IFPRIBreeding for Iron-Rich Rice to
Reduce Anemia in Asia (2001-2003)

• Implementing Partners
• ADB/IAEA
• DFID
• DANIDA
• USAID
• MI
• Waite Agricultural Research Institute (Aus)
• U Phil/Penn State/Cornell
• UCL/ICDDR, B/Dhaka U.

• Countries
• Bangladesh
• Indonesia
• Philippines
• Viet Nam
• P. R of China
• (Thailand/India)

Joseph Hunt, ADB, October 2003
11
The composition of rice variesiron and zinc
Variety Iron mg/100 g Zinc mg/100
g Ganjay Roozy 2.64 5.89 Zuchem 2.34
5.10 YR 4194 2.32 5.40 Banjaiman 2.27
5.30 Xue Bue Nuo 2.25 4.66 IR 64446 2.22
5.35 Kinmaze 2.17 5.17 Tsuyake 2.12
4.25 CNA 6187 2.07 5.45 Miyazaki 7 2.03
4.25 Ketan Irang 2.02 5.53 CT7127
2.01 4.70 Huri370 1.98 4.57 Lagrosa
1.80 4.82 Ketan Menah 1.61 4.54 IR 10198
1.58 3.79 Skybonnet 1.53 4.13 IR 60864
1.50 4.11 Heibao 1.49 3.16 Alan
1.40 3.92 IR 63877 1.31 3.64 IR 74
1.30 3.64 IR 72 1.17 3.25 IR 36 1.01
3.14
Iron and zinc content of selected varieties
(greenhouse three reps)
Kennedy and Burlingame (2003) Food Chemistry 80
589596
Source adapted from Senadhira et al. (1998)
Paper presented at the Intl. Workshop on
Micronutrient Enhancement of Rice for Developing
Countries, 3 September, Rice Research and
Extension Center, Stuttgart, AK.
12
Breeding for Iron-Rich Rice to Reduce Iron
Deficiency Anemia in Asia

• Some of the objectives
• agronomic research to increase iron/zinc content
  of seeds and
• human nutrition research to test iron/zinc
  bioavailability in rice diets
• develop capacity in Asian agricultural systems
  for adapting and disseminating improved seeds to
  farmers

• Achievements
• Breeding integrates high iron and high yielding
  varieties in NARES w/IRRI support
• Feeding Trial w/ adult women in Phil
• Milling techniques calibrated to measure iron
  content of rice in feeding trial

Joseph Hunt, ADB, October 2003
13
Little iron may be retained following polishing
From Gregorio et al (2000) 21382
14
IR68144-2B-2-2-3-2
Very early maturing, suitable for crop
diversification Moderate resistance to diseases,
except sheath blight

• Maligaya Special Rice 13 released by PhilRice
• Yield potential of at least 5t/ha
• Micronutrient enhancement does not interfere with
  other agronomic traits such as yield
• As acceptable to Philippine consumers as
  comparable rice

Leo Sebastian, PhilRice, October 2003
15
What is the Impact on Nutritional Status?

• 9-month feeding trial in the Philippines was
  recently completed
• Non-anemic, iron-deficient subjects
• Treatment (high-iron rice) and control (low-iron
  rice) groups
• 1.5 mg Fe added to diets due to the high-iron
  rice -- from a base intake of 8.5 mg Fe per day

H. Bouis, Director, HarvestPlus World Food
Prize Symposium 2004
16
Additional iron in human diet from biofortified
rice the results for a study in the Philippines

• There is a significant 3.0 mg net increase in
  daily iron intake from biofortified rice
  (IR68-144)
• This represents a 36 increase of dietary iron in
  diets that are typically low in iron (8-9 mg/day)
• The additional iron narrows the gap towards
  meeting iron requirements by 18, but intakes are
  still 24 below requirements

Jere Haas, Cornell University, October 2003
17
Iron content in some tissues of a transgenic rice
line expressing soybean ferritin cDNA
a Significant or b Non-significant differences
between the mean values were calculated by t-test
(p
SOURCE Goto et.al. (1999) Nature Biotechnology
17 282-286
18
Distribution of iron in the rice seeds
transformed with soybean ferritin cDNA
N4
Fk1

• Iron content of embryo (open rectangular) and
  endosperm (closed rectangular) are shown for 10
  T2 seeds of a transformed (Fk1) and
  nontransformed rice line (N4) are shown.
• Mean values are indicated standard error (n3).
  
• Differences between transformants and
  nontransformants were calculated to be
  significant, with t-test value of p 0.02.

SOURCE Goto et.al. (1999) Nature Biotechnology
17 282-286
19
In situ Western localization of soybean ferritin
in transformed rice seeds.Qu et al. (2003) Plant
Cell Rep 22282285

• The ferritin is localized exclusively in the
  aleurone, sub-aleurone and outer portion of
  starchy endosperm
• It is not found in the embryo.

a Non-transformed seed of cv. Kitaake. b Western
hybridization showing the distribution of
ferritin directed by the GluB-1 promoter in the
transgenic seed of FK22.
20
Iron concentration in IR68144 expressing
transgenic soybean ferritin
SOURCE Vasconcelos et al. (2003) Plant Science
164 371-378
21
Iron detection in transverse sections of
non-transgenic Control (A) and Transgenic (B)
rice grains
The accumulation of iron in the control material
is restricted to the aleurone layer while in
transgenic seeds iron is present in the entire
grain, including the endosperm.
SOURCE Vasconcelos et al. (2003) Plant Science
164 371-378
22
Rice diets were as effective as the FeSO4 diet in
replenishing Hct, Hgb concentration, and liver
iron concentrations.
Source Wendy White, ISU Adapted from
Murray-Kolb et al.(2002) J Nutr. 132957-960.
23
Over-expression of an Arabidopsis zinc
transporter in barley increases seed zinc content
Mean cation content (mg/kg) for groups of 5 seeds
(4-10 groups), total mean zinc and iron content
per seed (g) and mean individual weights (µg) for
T2 seeds used in whole plant growth experiments
(SE)
SOURCE Ramesh et.al. (2004) Plant Molecular
Biology 54 373-385
24
Over-expression of an Arabidopsis zinc
transporter in barley decreases seed weight!!
Mean cation content (mg/kg) for groups of 5 seeds
(4-10 groups), total mean zinc and iron content
per seed (g) and mean individual weights (µg) for
T2 seeds used in whole plant growth experiments
(SE)
SOURCE Ramesh et.al. (2004) Plant Molecular
Biology 54 373-385
25
43 iron homeostasis-related genes in rice
genomic sequence analysisGross et al. (2003)
Genetics and Molecular Biology, 26 477-497

• 18 (10) sequences homologous to the maize
  Fe3-phytosiderophore transporter.
• 2 (1) orthologs to the plant ferric-chelate
  reductases
• 13 (6) paralogs to the ZIP (Zinc regulated
  transporter/Iron regulated transporter Protein)
  family.
• 8 (5) sequences homologous to the NRAMP cation
  transporter family
• 2 (2) sequences homologous to the maize Ferritin
  protein.
• Expressed where and when?
• ( ) number with corresponding cDNAs

26
18 putative genes in the rice genome with
sequence similarity to maize iron(III)-phytosidero
phore transporterKoike et al. (2004) The Plant
Journal (2004), 39, 415424

• OsYSL2 expression induced in leaves by iron
  deficiency.
• Strong OsYSL2 promoter activity in developing
  seeds
• Did not transport iron(III)-phytosiderophore
  (Xenopus assay)
• OsYSL2 - a rice metal-nicotianamine transporter?
  
• phloem transport of iron
• iron translocation into the grain

27
Estimated Annual Number of Child Deaths
Precipitated by Vitamin A Deficiency (Asia)
28
Effects of Malnutrition on Child Survival in
China As Estimated by Profiles

• VAD accounts for 7.5 of estimated deaths of
  children 6-59 months old 206, 000 deaths over
  the past ten years.
• Halving the prevalence would have saved 49,000
  child lives.
• the western provinces, with only 28 of the
  Chinese population, account for over half of
  Vitamin A related child deaths.
• ...most of deaths which had been avoided with
  Vitamin A supplementation were not among children
  with clinical signs of deficiency but among those
  with sub-clinical forms detectable only with
  blood analysis.
• Only population-wide interventions to reduce the
  prevalence of sub-clinical deficiency are likely
  to have any significant impact on the problem...

Ross et al. (2003) Biomedical and Environmental
Sciences 16, 187-193. Source data Lin et al.,
2002 Survey on Vitamin A deficiency in children
under 6-years in China. Chinese Journal of
Preventative Medicine 36 (5) 315-319
29
The damage being inflicted by VM deficiency on
the people and the nation of China has been
estimated as follows

• Approximately 40 to 60 of the nation's 6-to-24
  month-old children at risk of disrupted brain
  development.
• Cause iron deficiency
• The deaths of up to 1,000 young Chinese women
  every year in pregnancy and childbirth.
• Cause severe iron deficiency anaemia
• Approximately 100,000 Chinese infants a year at
  increased risk of death in the period immediately
  before or after birth.
• Cause severe anaemia in mothers

30
Continued

• The deaths of over 20,000 children each year from
  increased susceptibility to infection.
• Cause vitamin A deficiency
• Approximately 12 of China's children growing up
  with lowered immunity, leading to frequent ill
  health and poor growth.
• Cause vitamin A deficiency (the prevalence of
  vitamin A deficiency in children under 6 is
  estimated at 12).

31
Continued

• Lowered productivity of adult work-force. Current
  loss to China is estimated at 0.7 of GDP, and
  future loss is as high as 3.8 of GDP (present
  value loss over the coming 10 years).
• Cause iron and iodine deficiency
• Significant but unmeasured burden on health
  services, educational systems, and on families
  caring for children left disabled or mentally
  impaired.

32
Prevalence of vitamin A deficiency among children
under six years of age by State
Source Ministry of Human Resource Development,
1996 (National Survey of Blindness, 1986-89)
33
Trends in the Prevalence of Vitamin A Deficiency
(by Age, and by Physiological State 1993
1998)
Philippine Nutrition Facts and Figures (National
Nutrition Survey, 1998)
34
Regional Incidence of Iron Deficiency by Group in
the Philippines NNS, 1998
35

• PICTURE of GOLDEN RICE

36
Basic Carotenoid Biosynthetic Pathway
Carotenes
IPP
DMAPP
GGPP (C20)
Phytoene synthase Plant source
Phytoene (C40)
Phytoene desaturase Bacterial source
desaturation
Lycopene (C40)
(Lycopene cyclase) Bacterial source
cyclization
a - carotene
b - carotene
Xanthophylls
b-cryptoxanthin
canthaxanthin
a-cryptoxanthin
zeaxanthin
lutein
astaxanthin
37
Carotenoid profile of Golden Rice

• Transgenic
• Control

?-carotene
lutein
b-cryptoxanthin
a-carotene
Rai et al (unpublished data)
38
What is the contribution to Nutrient Intake?
140
Golden Rice (SGR?)
120
100
Golden Rice (SGR1)
80
60
Plant Sources
40
20
Animal and
0
Fish
Adult
Children
Women
H. Bouis, Director, HarvestPlus World Food
Prize Symposium 2004
39
Carotenoid profile of Golden indica (BR29) rice
(pre- and post-cooking)
?-carotene

• Transgenic
• Control

• Transgenic
• Control

?-carotene
lutein
b
lutein
a
a-carotene
a-carotene
B-cryptoxanthin
B-cryptoxanthin
(a) grains without cooking (b) grains cooked in
excess water
Rai et al (unpublished data)
40
Organization of the Golden Rice Network
Philippines International Rice Research
Institute, National Rice Research Institute
(PhilRice) Vietnam Cuu Long Delta Rice Research
Institute Germany University of Freiburg India
Department of Biotechnology India, Directorate of
Rice Research, Indian Agricultural Research
Institute, University of Delhi - South Campus,
Tamil Nadu Agricultural University, Agricultural
University Pantnagar, University of Agricultural
Sciences Bangalore, Chinsurah Rice Research
Station Bangladesh Bangladesh Rice Research
Institute China Huazhong Agricultural
University, Chinese Academy of Sciences, Yunnan
Academy of Agricultural Sciences Indonesia
Agency for Agricultural Research Development
41
The contexts in which Golden Rice will undergo
regulatory review

• VAD remains a very large problem in the lead
  countries India, China, and the Philippines.
• These countries have approved GM crops for
  commercial production India and Philippines
  1each China 6.
• GM rice has been approved for commercial sale in
  USA, expected for import in EU
• Expect that prior to Golden Rice
• 2(?) GM rice products will be commercialized
• 2(?) GM products substantially equivalent,
  except for the intended changes/ nutritionally
  enhanced will be commercialized
• There is a lot GM rice activity, worldwide.
• Some changes in the regulatory systems may have
  occurred by then

42
LLRICE06, LLRICE62A private sector
exampleAdapted from original documents at
http//agbiosafety.unl.edu/
Rice lines LLRICE06 and LLRICE62 tolerant to
glufosinate ammonium, the active ingredient in
phosphinothricin herbicides (Basta, Rely,
Finale, and Liberty) The gene encoding the
enzyme phosphinothricin-N-acetyltransferase (PAT)
was isolated from the common aerobic soil
actinomycete, Streptomyces hygroscopicus. LLRICE06
and LLRICE62 were tested in field trials to show
that they did not exhibit weedy characteristics,
or negatively affect beneficial or non-target
organisms, and were not expected to impact on
threatened or endangered species. The
nutritional composition of rice was found to be
equivalent to conventional varieties. Assays were
completed for anti-nutritional factors normally
concentrated in the bran fraction of rice, such
as phytic acid, trypsin inhibitor, and lectins.
Summary of Regulatory Approvals Country Environm
ent Food and/or Feed United States 1999
2000 EU Pending (Food/Feed)
43
Number of Transgenic Rice Field Test Applications
Worldwide (USDA and OECD databases, January
2004)
Tests have also been conducted in China 1000
(1995-2003) India 5 (2003-2004) Philippines 1
(2003) Korea 5? (1999- ) Colombia 1
(2001?) Costa Rica 1 (2002?) Based on pers.
commun. secondary and tertiary sources
44
European Network on Safety Assessment of
Genetically Modified Food (ENTRANSFOOD)
Comparative Safety Assessment Approach for GMOs

• Underlying assumption
• Traditionally cultivated crops have gained a
  history of generally accepted use
  (environment/consumer/animals)
• These crops can therefore serve as a baseline for
  the environmental and food/feed safety assessment
  of GM crops
• European Food Safety Authority Stakeholder
  Consultation
• Draft guidance document for the risk assessment
  of genetically modified plants and derived food
  and feed. 25 May 2004, Brussels
• http//www.efsa.eu.int/consultation/483/presentati
  on_gmo_03_intro_hak_may20041.pdf
• Reports published in Food and Chemical
  Toxicology, 42 (7) 2004

45
The contexts in which Golden Rice will undergo
regulatory review

• The same one event will be advanced in all
  countries
• The introduced genes are from a plant and a soil
  bacterium
• The event will be marker-free or use the PMI
  system, will have single-locus, single intact
  insert, genome location mapped
• Some countries may advance the same varieties at
  the same time.

46
A fully integrated approach to the hazard
assessment and characterization of all elements
involved in producing a new GM variety.
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
47
A fully integrated approach to the hazard
assessment and characterization of all elements
involved in producing a new GM variety.
OECD Consensus documents
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
48
A fully integrated approach to the hazard
assessment and characterization of all elements
involved in producing a new GM variety.
Substantially equivalent, except for the intended
changes
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
49
A fully integrated approach to the hazard
assessment and characterization of all elements
involved in producing a new GM variety.
Not event-specific documents can be produced
early
OECD Consensus documents
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
50
A fully integrated approach to the hazard
assessment and characterisation of all elements
involved in producing a new GM variety.
Harmonized already
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
51
A fully integrated approach to the hazard
assessment and characterisation of all elements
involved in producing a new GM variety.
Guidelines evolving
Koenig et al., 2004. Food and Chemical Toxicology
42 10471088
52
Toxicological testing of whole foods which
tests contribute to biosafety evaluation?
SAFOTEST - New methods for the safety testing of
transgenic food Base-case set of studies vs.
Tiered approach 90-day studies for establishing
of safety/wholesomeness of GM foods is not viewed
as being mandatory but to be performed on a
case-by case basis based on the initial data
obtained in a tiered approach. ENTRANSFOOD Sub-c
hronic dietary studies with rats serve as an
indicator that there are no unintended changes in
foods derived from GM crops that might render it
less safe than the comparator. The design should
in principle follow the OECD guidelines for
rodent feeding studies regarding the parameters
to measure (OECD, 1993b).
53

• Thank you!