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From Soil to Seed

The Quest for Zinc-Enriched Rice

Sarah E.J. Beebout September 22, 2011

Why is zinc important for poverty mitigation?

Rice productivity Zn deficiency: Stunting, poor tillering Poor grain filling Low yields

Prevalence? Up to 50% of rice soils are Zndeficient (White and Zasoski, 1999)

2 Human nutrition
Zn deficiency: children Impairs cognitive development Causes growth stunting Impairs immune system function Greater susceptibility to respiratory infections, diarrhea

all ages

(Sandstead, 1989, 1994; Black, 2001)

Burden of Disease: Least Developed Sub-region
Risk factors Underweight Zinc deficiency Iron deficiency Vitamin A deficiency Unsafe sex Sanitation Cooking stove pollution Blood pressure Cholesterol Tobacco 0 2 4 6 8 10 12 14 16


DALY=disability adjusted life years; econometric index of time lost to disease

% Attributable DALYs

(Ezzati et al., 2004)

Brief history of Zn research on rice

Zn-deficiency identified as a yield-limiting factor in some soils (Katyal & Ponnamperuma, 1974) Zn fertilizer recommendations developed (Castro, 1977) Began screening germplasm for Zn-deficiency tolerance (Ponnamperuma, 1977)

1980s and 1990s:

More detailed chemical and physiological mechanisms identified (Sajwan & Lindsay, 1986; Beggi et al., 1994) Zn fertilizer recommendations improved (Savithri et al., 1999) Germplasm tolerance to moderate Zn-deficiency improved (e.g. IR34, IR36, and many others) Yield-limiting Zn deficiency: SOLVED by breeding
Inconsistent response to Zn fertilizer application Very poor Zn fertilizer use efficiency (<1%)

Zinc fertilizers not very popular with farmers

Starting in the late 1990s: biofortification=enriching food with human nutrients during plant growth
(in contrast to fortification=adding nutrients during food processing)

New goal: breeding plants to improve human nutrition Zn, Fe, vitamin A CGIAR Micronutrients Project multiple crops HarvestPlus Challenge Program

Biofortification: Complements other approaches to human zinc nutrition

Dietary diversification Biofortification: Increasing nutritional value of staple foods



Biofortification: Why rice? Why zinc?

Rice now: Average 16 ppm Zn (white) 40-60% of calories for Asian poor 10-20 % of daily Zn requirement High-Zn rice: Natural variability up to 30 ppm Zn (unlike Fe) 60-70% of daily Zn requirement Improve Zn status of 500 million people

(Hunt et al., 2002; Meenakshi et al., 2007)

Zinc target levels

seed brown (de-hulled) white (polished)

30 ppm
from 20 most of Zn is retained (unlike Fe)

24 ppm
from 16

Convergence of Interests (2007)

Plant Zn nutrition with increasing rice yields:
Zn deficiency was noticeable again removal of biomass was mining soil Zn

Biofortification Zn fertilizer application enriched grain Zn in wheat (Cakmak, 2007)

desire to improve identification and correction of Zn deficiency

desire in HarvestPlus to explore Zn fertilizer application to other crops

First soil science objective for biofortification collaboration:

Manage rice for better zinc uptake and higher grain zinc

Test Zn deficiency management recommendations on grain Zn

From Zn deficiency literature

Plant symptoms were worse with: long-term flooding (Forno et al., 1975) high soil organic matter use up O2 in soil=redox potential decreases but Zn2+ does not change form with redox changes (unlike Fe3+ to Fe2+)

Zn fertilizer has limited usefulness in flooded soils:

Available soil Zn (mg/kg)

60 50 40 30 20 10 0 -5 15 35

Low (native) Medium (10 kg/ha) High (50 kg/ha)




(Johnson-Beebout et al., 2009)

Hypotheses applying Zn-deficiency research to biofortification

1. Higher grain Zn if soil is non-flooded (than if continuously flooded) 2. Higher grain Zn with later (than basal) Zn fertilizer application 3. Higher grain Zn of genetically biofortified lines with optimized agronomic management (than without)

Greenhouse experiment (2008)

Objective: To test the effect of the timing of Zn fertilizer application on grain Zn content, relative to: Plant growth stage Flooding/draining periods

Design and management: Jack Jacob, Efren Laureles, Oliver Castillo Implementation and data collection: Jerone Onoya, Max Alumaga, Angel Bautista, Briccio Salisi

Results: Soil
Available soil Zn (mg/kg)

40 30 20 10 0 -20 0 20 40 60 80 100

Flooded Mid-season drainage Late-season drainage


Days after transplanting

Zn fertilization

1-week drainage periods

Soil Zn is highest during drainage periods, regardless of when applied (as expected based on Zn-def research)

Results: Grain Zn (IR69428)

Zn concentration (mg/kg)


brown rice

continuous flooding mid-season drainage late-season drainage



25 Z0 ZB ZM ZL

IR69428-6-1-1-3-3 (biofortified) was above target Zn level under all conditions of this experiment Zn was increased from 31 to 37 ppm by adding Zn during a late season drainage period

Field experiment in Zn-deficient field in Pila,Laguna (DS 2009)

Objective: To test the effect of the different amounts of Zn fertilization, with: Continuous flooding Alternate wetting and drying (AWD)
Farmer: Domingo Nido Design and management: Wency Larazo, Oliver Castillo, Dennis Tuyogon Implementation and data collection: Jerone Onoya, Angel Bautista, Ike Reyes, Juan Puno, Briccio Salisi

Results: Soil
Redox potential
600 500 400 300 200 100 0 -100 -200 -300 Alternate Wetting and Drying Continuous Flooding

Eh (mV)

AWD vs. CF: higher redox (i.e. more oxidized) higher Zn availability

Soil Zn availability

soil Zn (mg/kg)

4 3 2 1 0

Results: Grain Zn, NSIC 158

Zn concentration (mg/kg) brown rice
a b

bc c ab a




15 Z0 Z1 Z2 Z3

water management effect : AWD>CF by 2 ppm small Zn fertilizer effect (<1 ppm), only at highest Zn application rate

Aha! moments
water management (only as dry as safe AWD) affected grain Zn of a non-biofortified variety investigate AWD further even with optimized management, NSIC 158 only had 23 ppm Zn, compared with 37 for IR69428 management cant replace breeding

Discussion with breeders

We have further increased grain Zn content by improving water and zinc fertilizer management But only in 1 (biofortified) genotype and the soil science hypotheses have been confirmed, as well as their relevance to grain Zn. But only in 1 genotype

New objective for biofortification collaboration:

Understand how genotypes differ in grain Zn in differing soil environments

Test Zn management strategies on multiple genotypes to determine basic physiological Zn uptake characteristics

Greenhouse experiment with 2 biofortified genotypes (2009)

Objective: To test the effects of Zn fertilizer and water management (without late season drainage) on: IR69428 (as in earlier greenhouse experiment) IR68144
Design and management: Oliver Castillo, Efren Laureles Data analysis: Francis Rubianes Implementation and data collection: Jerone Onoya, Max Alumaga, Angel Bautista, Ike Reyes, Briccio Salisi

Results: Grain Zn
40 IR69428 IR68144

a b c cd

Zn conc (mg/kg ) brown rice


c d


25 no added Zn Zn at mid-tillering Zn at heading

no effect of 1-week mid-season drainage on grain Zn this time IR69428 responded to late-season Zn application IR68144 responded to vegetative stage Zn application Implication: IR69428 continues taking up Zn during grain-filling IR68144 does not

Remobilization vs. Root Uptake


Zn Get lots of Zn into plant early Ensure sufficient Zn supply during grain-filling

Previous lit: no remobilization in rice (unlike wheat)

Two aerobic rice genotypes: Zn-65 tracer studies showed no movement of labeled Zn from leaf to grain (Jiang et al., 2007) Nipponbare: Zn-62 tracer imaging study showed Zn moving from root to grain in 3.5 h, bypassing the flag leaf (Tsukamoto et al., 2006)

Physiologists join the collaborative biofortification effort

Starting point: Several genotypes tolerant of extreme Zn deficiency, with physiological data for some (RK Singh, Glenn Gregorio, Matthias
Wissuwa, Abdel Ismail)

but unknown grain Zn Several genotypes with high grain Zn

(Parminder Virk, Tajinder Singh, Deepinder Grewal)

but unknown Zn-deficiency tolerance

Post-doctoral work of Somayanda Impa

Root Zn uptake mechanisms active at Genotype category Zn-deficiency tolerant Zn-biofortified: remobilization Zn-biofortified: direct uptake Seedling *** *** *** Grain-filling

Management and Data Collection: MJ Morete, Randell Eusebio

Results preview
Early season Zn efficiency (%) 88 49 58 100 98 93 75 90 Grain Zn attributable to different uptake mechanisms (%) Remobilization 11 32 100 100 100 0 0 31 Direct Uptake 89 68 0 0 0 100 100 69

Genotype RIL-46 IR74 IR68144 IR82247 SWHOO IR69428 Joryoongbyeo IR64

Category Zn-deficiency tolerant Zn-deficiency susceptible Zn-biofortified: remobilization Zn-biofortified: direct uptake Popular check

Foliar Zn application (DS2011)

Physiology and Zinc Management
Rationale: Foliar Zn application would bypass soil chemistry challenges Hypotheses:
1) Remobilization genotypes are more likely than direct uptake to respond to foliar Zn by increasing grain Zn 2) Any genotype is more likely to respond in a Zn-deficient soil

Design and management: Ranee Mabesa Implementation and data collection: Jerone Onoya, Rowell Mayores, Nards Baclao

Results: Grain Zn
Zn-sufficient site Genotype NSIC222 IR64 IR68144 IR83668 IR85800 IR91152AC-79 IR83317AC-124 IR91143AC-4 IR83317AC-25 IR64196 IR75862 Zn-deficiency tolerant Biofortification: unknown Popular check Biofortification: remobilization Category >30 ppm Zn? No Almost (+spray) Yes (+spray) Yes No Yes Yes (+spray) Yes (+spray) No No (not planted) Responsive to foliar Zn? No Yes Yes No No No Yes Yes No No Zn-deficient site >30 ppm Zn? No No Almost (+spray) No No Responsive to foliar Zn? No Yes Yes No No

(no filled grains) No No No Yes Yes No

(not planted) No No

Results: Grain Zn
Zn-sufficient site Genotype NSIC222 IR64 IR68144 IR83668 IR85800 IR91152AC-79 IR83317AC-124 IR91143AC-4 IR83317AC-25 IR64196 IR75862 Zn-deficiency tolerant Biofortification: unknown Popular check Biofortification: remobilization Category >30 ppm Zn? No Almost (+spray) Yes (+spray) Yes No Yes Yes (+spray) Yes (+spray) No No (not planted) Responsive to foliar Zn? No Yes Yes No No No Yes Yes No No Zn-deficient site >30 ppm Zn? No No Almost (+spray) No No Responsive to foliar Zn? No Yes Yes No No

(no filled grains) No No No Yes Yes No

(not planted) No No

Discussion with breeders: Divided by a common language

G (genotype) x E (environment) interactions
Soil scientists: opportunity for optimizing management (goal: absolute yield target)

Plant breeders: inconsistent genotype performance (goal: find best-performing genotypes)

(Brooks, 2010)

HarvestPlus Rice Meeting (2009)

One breeder presented:
grain Zn data from 12 sites soil data from each site for every routinely measured parameter (pH, texture, exchangeable bases, etc. ) no correlations of grain Zn with any of the soil data

Aha! moments

routine soil lab testing is not helpful because it doesnt measure redox-relevant parameters there must be a better way to assess E

Another new objective for biofortification collaboration

Determine which E variables have greatest impact on GxE patterns

New approach
Design GxE trials collaboratively, with locations chosen for Zn characteristics

Zinc GxE experiments (DS2011)

Five locations in Philippines, representing:
Zn-depleted (IRRI) Excessively submerged (Bukidnon, Mindanao) Peaty (Bay, Laguna) Calcareous (Bohol) Zn-sufficient (PhilRice, Nueva Ecija)

Experiment design: Deepinder Grewal, Glenn Gregorio, Sarah Beebout Experiment management: Eric Clutario, Andy Sajise, Francis Rubianes

At each site:
Two sets of plant breeding trials:
Zn biofortification advanced lines, with and without Zn fertilizer (from Deepinder) Zn-deficiency tolerant advanced lines (from Glenn)

Environmental data collection:

Water status throughout Background soil characterization Detailed soil monitoring in lab

Plant data collection:

Grain Zn Grain yield

No results yet (data being compiled)

Stages of interdisciplinary collaboration

1. Discussion of experiment results:
seminars, project meetings aha! moments that helped set research objectives

2. Input into experiment design:

breeders nominating genotypes for Zn management trials soil scientists recommending Zn fertilizer and water management for plant breeding trials
(Brooks and Johnson-Beebout, in press)

3. Joint experiments:
soil scientists influence design of breeding trials (location, + Zn fertilizer, data collection) breeders choose genotypes by standard breeding protocols (compromising on the number) division of labor for implementation

Challenges of stage 3
time: coordinating three research groups money: sorting out budgets fairly respect: sufficient mutual understanding of the other disciplines to listen to design ideas

Conclusions about plant breeding strategies

1. Pre-release varietal GxE trials: use sites differing in Zn availability
minimum set: depleted, submerged, calcareous

2. Screen popular (old) varieties at same sites to rank Zn-deficiency tolerance 3. Biofortification: select for both remobilization and direct uptake
improve GxE performance make management more flexible

Conclusions about Zn management

1. Trend towards drier water management will improve Zn uptake 2. Standard Zn fertilizer recommendation (10 kg Zn/ha applied basally) is not helpful
Better for researchers: apply Zn to non-flooded soil at maximum tillering Better for farmers: ??? first need is to predict likelihood of Zn deficiency

Funding: HarvestPlus; Swiss Agency for Development and Cooperation (SDC), through their Research Fellow Partnership Program (Impas post-doctoral fellowship) Interactive discussions about Zn research: Gerard Barry, Achim Dobermann, Matthias Wissuwa, Ismail Cakmak, Wolf Pfeiffer, Untung Susanto, Andrew Green Jason and Miriam Soils Group

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