From Soil to Seed

The Quest for Zinc-Enriched Rice

Sarah E.J. BeeboutSeptember 22, 2011

Why is zinc important for poverty mitigation?

1 Rice productivity•Zn deficiency:

•Stunting, poor tillering•Poor grain filling•Low yields

Prevalence?“Up to 50%” of rice soils are Zn-deficient (White and Zasoski, 1999)

2 Human nutrition

•Zn deficiency:

•Impairs cognitive development•Causes growth stunting

•Impairs immune system function•Greater susceptibility to respiratory infections, diarrhea

children

all ages

(Sandstead, 1989, 1994; Black, 2001)

(Ezzati et al., 2004)

0 2 4 6 8 10 12 14 16

TobaccoCholesterol

Blood pressureCooking stove pollution

SanitationUnsafe sex

Vitamin A deficiencyIron deficiencyZinc deficiency

Underweight

% Attributable DALYs

Burden of Disease: Least Developed Sub-region

Risk factors

Malnutrition

Prevalence?

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

Brief history of Zn research on rice1970’s: • 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)

1980’s and 1990’s: • 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 1990’s: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• CGIAR Micronutrients Project• HarvestPlus Challenge Program

Zn, Fe, vitamin Amultiple crops

Biofortification: Complements other approaches to human zinc nutrition

Dietary diversification

Fortification Supplementation

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 poor10-20 % of daily Zn requirement

High-Zn rice:Natural variability up to 30 ppm Zn (unlike Fe)60-70% of daily Zn requirementImprove Zn status of 500 million people

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

Zinc target levels

seed

brown(de-hulled)

white(polished)

30 ppmfrom 20

24 ppmfrom 16

•most of Zn is retained (unlike Fe)

Convergence of Interests (2007)

Plant Zn nutrition

• with increasing rice yields:• Zn deficiency was

noticeable again• removal of biomass

was “mining” soil Zn

• desire to improve identification and correction of Zn deficiency

Biofortification• Zn fertilizer application

enriched grain Zn in wheat (Cakmak, 2007)

• 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

Approach:

Test Zn deficiency management recommendations on grain Zn

From Zn deficiency literature

Plant symptoms were worse with:•long-term flooding•high soil organic matter

(Forno et al., 1975)

use up O2 in soil=redox potential decreases

•but Zn2+ does not change form with redoxchanges (unlike Fe3+ to Fe2+)

Zn fertilizer has limited usefulness in flooded soils:

0102030405060

-5 15 35 55 75 95

Low (native)

Medium (10 kg/ha)

High (50 kg/ha)

Avai

labl

e so

il Zn

(mg/

kg)

Days

(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

0

10

20

30

40

-20 0 20 40 60 80 100 120Avai

labl

e so

il Zn

(mg/

kg)

Days after transplanting

Flooded

Mid-season drainage

Late-season drainage

Zn fertilization1-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)

25

30

35

40

Z0 ZB ZM ZL

Zn c

once

ntra

tion

(mg/

kg) continuous flooding

mid-season drainagelate-season drainage

•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

brow

n ric

e

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

Farmer: Domingo NidoDesign and management: WencyLarazo, Oliver Castillo, Dennis Tuyogon

Implementation and data collection:Jerone Onoya, Angel Bautista, Ike Reyes, Juan Puno, Briccio Salisi

Objective:To test the effect of the different amounts of Zn

fertilization, with:– Continuous flooding– Alternate wetting and drying (AWD)

Results: Soil

-300-200-100

0100200300400500600

Eh (m

V)

Redox potentialAlternate Wetting and DryingContinuous Flooding

0

1

2

3

4

5Soil Zn availability

soil

Zn (m

g/kg

)

AWD vs. CF:

•higher redox (i.e. more oxidized)•higher Zn availability

Results: Grain Zn, NSIC 158

15

20

25

AWD CF

a

b

15

20

25

Z0 Z1 Z2 Z3

aabc

bc

Zn co

ncen

trat

ion

(mg/

kg)

brow

n ric

e

•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 can’t 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 biofortificationcollaboration:

Understand how genotypes differ in grain Zn in differing soil environments

Approach:

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

Greenhouse experiment with 2 biofortified genotypes (2009)

Design and management:Oliver Castillo, Efren LaurelesData analysis: Francis Rubianes

Implementation and data collection: Jerone Onoya, Max Alumaga, Angel Bautista, Ike Reyes, Briccio Salisi

Objective:To test the effects of Zn fertilizer and water

management (without late season drainage) on:– IR69428 (as in earlier greenhouse experiment)– IR68144

Results: Grain Zn

25

30

35

40

no added Zn Zn at mid-tillering Zn at heading

Zn c

onc

(mg/

kg )

brow

n ric

eIR69428

IR68144

ab

c c cd

d

•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

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

Hypotheses:Root Zn uptake mechanisms

active at

Genotypecategory

Seedling Grain-filling

Zn-deficiency tolerant ***

Zn-biofortified:remobilization ***

Zn-biofortified: direct uptake ***

Management and Data Collection: MJ Morete, Randell Eusebio

Results preview

Early season Znefficiency

(%)

Grain Zn attributable to different uptake mechanisms

(%)

Genotype Category RemobilizationDirect Uptake

RIL-46 Zn-deficiency tolerant 88 11 89

IR74 Zn-deficiency susceptible 49 32 68

IR68144 Zn-biofortified: remobilization

58 100 0

IR82247 100 100 0

SWHOO 98 100 0

IR69428 Zn-biofortified: direct uptake

93 0 100

Joryoongbyeo 75 0 100

IR64 Popular check 90 31 69

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 Zn2) 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 ZnZn-sufficient site Zn-deficient site

Genotype Category >30 ppm Zn?Responsive to foliar Zn?

>30 ppmZn?

Responsive to foliar Zn?

NSIC222Popular check

No No No No

IR64 Almost (+spray) Yes No Yes

IR68144 Biofortification: remobilization Yes (+spray) Yes Almost

(+spray) Yes

IR83668

Biofortification: unknown

Yes No No No

IR85800 No No No No

IR91152AC-79 Yes No (no filled grains)

IR83317AC-124 Yes (+spray) Yes No Yes

IR91143AC-4 Yes (+spray) Yes No Yes

IR83317AC-25Zn-deficiency

tolerant

No No No No

IR64196 No No (not planted)

IR75862 (not planted) No No

Results: Grain ZnZn-sufficient site Zn-deficient site

Genotype Category >30 ppm Zn?Responsive to foliar Zn?

>30 ppmZn?

Responsive to foliar Zn?

NSIC222Popular check

No No No No

IR64 Almost (+spray) Yes No Yes

IR68144 Biofortification: remobilization Yes (+spray) Yes Almost

(+spray) Yes

IR83668

Biofortification: unknown

Yes No No No

IR85800 No No No No

IR91152AC-79 Yes No (no filled grains)

IR83317AC-124 Yes (+spray) Yes No Yes

IR91143AC-4 Yes (+spray) Yes No Yes

IR83317AC-25Zn-deficiency

tolerant

No No No No

IR64196 No No (not planted)

IR75862 (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)

“Aha!” momentsHarvestPlus 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

•routine soil lab testing is not helpful because it doesn’t 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 BeeboutExperiment 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

AcknowledgementsFunding: HarvestPlus; Swiss Agency for Development and

Cooperation (SDC), through their Research Fellow Partnership Program (Impa’s 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