PHENOTYPING PIPELINE FORTRANSGENIC WHEAT AND BARLEY

6

PHENOTYPING PIPELINE FOR TRANSGENIC WHEAT AND BARLEY

LemnaTec Workshop PAG 2013

Factors limiting productivity in low yielding environments Heat

UV

Frost

Drought Wind

0.25 – 0.50 cm

from P. Rengasamy

Al tolerance screens

Al tolerance of barley transgenics 30

Root re-growth (mm) after relief from Al stress in ALMT1 transgenic barley

25

20

15

T1 barley transgenics 10

5

0

Nick Collins and Perry Gustafson

Frost tolerance Transgenics control

AGRF Frost Simulation Chamber (Australian Genome Research Facility)

Chen et al. (2009a) Theor. Appl. Genet. 119: 685-694

Frost screen • Vegetative frost tolerance is not correlated with reproductive tolerance • Screening requires synchronised flowering • Field evaluation only possible at special sites and once per year

Moisture stress - drought Pot

Big bins

Field

Evaluation of transgenics T0 – Screen for transgenic copy number

T1 – Transgene expression – Zygocity – Abnormal phenotypes

T2 – Stress phenotyping

T3 – Small scale field trials

0.6 0.4 0.2 0 -0.2 -15

-10

-5

0

Soil water potential (bar)

Tamaroi

Sokoll

RAC875

Kukri

Krichauff

Jandaroi

Gladius

Excalibur

0.8

Drysdale

1

Berkut

1.2

sensitivity to SWP (0.1MPa-1)

Relative Growth Rate

1.4

Bobwhite S26

1.6

Growth response to drought

Bobwhite Halle

Plant growth under moisture stress

0 -0.02 -0.04 -0.06 -0.08 -0.1 -0.12 -0.14 -0.16 -0.18 -0.2

Variation between cultivars

Stable drought -0.5 bar -3 bar -6 bar

Source: Boris Parent

0.14

Correlations Platform/Field

RGR (Platform)

60

0.12

Digital ground cover

Atil (Durum) Triticum dicoccum

40

0.1 0.08 0.06

R2 = 0.18 p = 1.05 10-6

20

0.04 0 0

10 20 Day after germination

30

0.02 0.02

0.07

RGR (Field)

0.12

150 Lines Population Atil / Dicoccum (Durum wheat / Emmer wheat) Grown under well-watered conditions with 2 replicates in the field (Mexico) or partially replicated in the platform.

RGR was calculated by fitting logistic curves on leaf area in the platform or on NDVI in the field during the vegetative phase.

Correlations Platform/ Field

All traits

First set Gladius / Drysdale (flowering time ~ 20d) Field

Platform

WW WW Platform

D1 D2 WW

Field

D

lem1WW.dry.w.tx lem1WW.dry.w.K lem1WW.area.Yf lem1WW.area.tx lem1WW.area.K lem1WW.SWP lem1WW.dry.w lem1WW.area lem1WW.transpi.tot lem1WW.transpi.area.rate lem1WW.WUE lem1WW.dry.w.size lem1WW.dry.w.growth lem1WW.dry.w.rgr lem1WW.area.size lem1WW.area.growth lem1WW.area.rgr lem1D1.dry.w.tx lem1D1.dry.w.K lem1D1.area.Yf lem1D1.area.tx lem1D1.area.K lem1D1.SWP lem1D1.dry.w lem1D1.area lem1D1.transpi.tot lem1D1.transpi.area.rate lem1D1.WUE lem1D1.dry.w.size lem1D1.dry.w.growth lem1D1.dry.w.rgr lem1D1.area.size lem1D1.area.growth lem1D1.area.rgr lem1D2.dry.w.tx lem1D2.dry.w.K lem1D2.area.Yf lem1D2.area.tx lem1D2.area.K lem1D2.SWP lem1D2.dry.w lem1D2.area lem1D2.transpi.tot lem1D2.transpi.area.rate lem1D2.WUE lem1D2.dry.w.size lem1D2.dry.w.growth lem1D2.dry.w.rgr lem1D2.area.size lem1D2.area.growth lem1D2.area.rgr pol1WW.flow.day pol1WW.head.day pol1WW.one.seed.w pol1WW.seed.nb.spk pol1WW.seed.w.spk pol1WW.til.nb.plant pol1WW.spk.nb.plant pol1WW.seed.w.plant pol1WW.yield pol1WW.one.stem.w pol1WW.stem.w.plant pol1WW.Biomass.plant pol1WW.HI pol1D.flow.day pol1D.head.day pol1D.one.seed.w pol1D.one.seed.vol pol1D.seed.nb.spk pol1D.seed.w.spk pol1D.til.nb.plant pol1D.spk.nb.plant pol1D.seed.w.plant pol1D.yield pol1D.one.stem.w pol1D.stem.w.plant pol1D.Biomass.plant pol1D.HI

D1

D2

WW

D

lem1WW.dry.w.tx lem1WW.dry.w.K lem1WW.area.Yf lem1WW.area.tx lem1WW.area.K lem1WW.SWP lem1WW.dry.w lem1WW.area lem1WW.transpi.tot lem1WW.transpi.area.rate lem1WW.WUE lem1WW.dry.w.size lem1WW.dry.w.growth lem1WW.dry.w.rgr lem1WW.area.size lem1WW.area.growth lem1WW.area.rgr lem1D1.dry.w.tx lem1D1.dry.w.K lem1D1.area.Yf lem1D1.area.tx lem1D1.area.K lem1D1.SWP lem1D1.dry.w lem1D1.area lem1D1.transpi.tot lem1D1.transpi.area.rate lem1D1.WUE lem1D1.dry.w.size lem1D1.dry.w.growth lem1D1.dry.w.rgr lem1D1.area.size lem1D1.area.growth lem1D1.area.rgr lem1D2.dry.w.tx lem1D2.dry.w.K lem1D2.area.Yf lem1D2.area.tx lem1D2.area.K lem1D2.SWP lem1D2.dry.w lem1D2.area lem1D2.transpi.tot lem1D2.transpi.area.rate lem1D2.WUE lem1D2.dry.w.size lem1D2.dry.w.growth lem1D2.dry.w.rgr lem1D2.area.size lem1D2.area.growth lem1D2.area.rgr pol1WW.flow.day pol1WW.head.day pol1WW.one.seed.w pol1WW.seed.nb.spk pol1WW.seed.w.spk pol1WW.til.nb.plant pol1WW.spk.nb.plant pol1WW.seed.w.plant pol1WW.yield pol1WW.one.stem.w pol1WW.stem.w.plant pol1WW.Biomass.plant pol1WW.HI pol1D.flow.day pol1D.head.day pol1D.one.seed.w pol1D.one.seed.vol pol1D.seed.nb.spk pol1D.seed.w.spk pol1D.til.nb.plant pol1D.spk.nb.plant pol1D.seed.w.plant pol1D.yield pol1D.one.stem.w pol1D.stem.w.plant pol1D.Biomass.plant pol1D.HI 0 # 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0 # # 0 0 # 0 0 0 0 0 0 1 0 0 # # 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 # # # 0 # # # 0 0 0 0 0 # # # 0 # 0 0 # # 0 0 # # 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 # # # 0 # # # 0 0 0 0 0 # # # 0 # 0 0 # # 0 0 # # 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 # # # 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 # # 0 0 # # 0 0 # # 0 0 0 0 # # 0 0 0 # 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 # 0 # # # 0 0 0 1 0 0 1 0 1 0 0 0 # 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 1 1 # # 0 0 0 # 0 0 # # 0 0 0 0 # # 0 0 0 # 0 1 0 0 1 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 0 1 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 1 0 # 0 0 0 0 0 0 0 # # # 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 # # 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 # # # # # 1 1 0 0 1 1 1 0 0 0 # 0 0 0 0 0 # 0 0 1 0 0 1 0 0 0 1 0 0 # # # # 0 # # 0 0 # # # 0 # # 0 0 0 0 # 0 0 0 # # 0 0

All traits

Correlations Platform/ Field Second set Gladius / Drysdale, 150 Lines (flowering time ~ 9d) Platform

WW

WW Platform

D WW Field

D

lem2WW.dry.w.tx lem2WW.dry.w.K lem2WW.area.Yf lem2WW.area.tx lem2WW.area.K lem2WW.SWP lem2WW.dry.w lem2WW.area lem2WW.transpi.tot lem2WW.transpi.area.rate lem2WW.WUE lem2WW.dry.w.size lem2WW.dry.w.growth lem2WW.dry.w.rgr lem2WW.area.size lem2WW.area.growth lem2WW.area.rgr lem2D.dry.w.tx lem2D.dry.w.K lem2D.area.Yf lem2D.area.tx lem2D.area.K lem2D.SWP lem2D.dry.w lem2D.area lem2D.transpi.tot lem2D.transpi.area.rate lem2D.WUE lem2D.dry.w.size lem2D.dry.w.growth lem2D.dry.w.rgr lem2D.area.size lem2D.area.growth lem2D.area.rgr pol2WW.head.day pol2WW.flow.day pol2WW.spk.plant pol2WW.biomass.plant pol2WW.grain.w.plant pol2WW.HI pol2WW.seed.plant pol2WW.seed.spk pol2WW.one.seed.w pol2WW.yield pol2D.head.day pol2D.flow.day pol2D.spk.plant pol2D.biomass.plant pol2D.grain.w.plant pol2D.HI pol2D.seed.plant pol2D.seed.spk pol2D.one.seed.w pol2D.yield

Field

D

WW

D

lem2WW.dry.w.tx lem2WW.dry.w.K lem2WW.area.Yf lem2WW.area.tx lem2WW.area.K lem2WW.SWP lem2WW.dry.w lem2WW.area lem2WW.transpi.tot lem2WW.transpi.area.rate lem2WW.WUE lem2WW.dry.w.size lem2WW.dry.w.growth lem2WW.dry.w.rgr lem2WW.area.size lem2WW.area.growth lem2WW.area.rgr lem2D.dry.w.tx lem2D.dry.w.K lem2D.area.Yf lem2D.area.tx lem2D.area.K lem2D.SWP lem2D.dry.w lem2D.area lem2D.transpi.tot lem2D.transpi.area.rate lem2D.WUE lem2D.dry.w.size lem2D.dry.w.growth lem2D.dry.w.rgr lem2D.area.size lem2D.area.growth lem2D.area.rgr pol2WW.head.day pol2WW.flow.day pol2WW.spk.plant pol2WW.biomass.plant pol2WW.grain.w.plant pol2WW.HI pol2WW.seed.plant pol2WW.seed.spk pol2WW.one.seed.w pol2WW.yield pol2D.head.day pol2D.flow.day pol2D.spk.plant pol2D.biomass.plant pol2D.grain.w.plant pol2D.HI pol2D.seed.plant pol2D.seed.spk pol2D.one.seed.w pol2D.yield 0 ## ## 0 ## 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ## 0 1 1 1 1 0 1 0 1 1 0 0 1 1 0 0 1 0 0 0 ## 0 1 0 ## 0 0 0 0 0 0 0 0 0 0 0 ## 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## 0 0 ## 0 0 0 0 ## ## 0 0 ## 0 0 ## 0 0 ## ## 0 0 ## 1 ## 0 0 ## 0 0 0 1 1 0 1 1 0 1 0 1 1 0 1 0 1 1 1 1 0 1 1 0 0 ## ## 0 0 0 0 0 0 0 0 ## ## 0 0 0 0 ## 0 0 ## ## ## ## ## 0 ## ## 0 0 ## 0 0 0 0 1 1 0 0 1 0 1 0 0 0 0 1 1 1 1 1 1 1 0 0 ## 0 0 0 0 0 0 0 1 0 ## 0 0 0 1 ## 0 ## ## 0 0 ## 0 0 0 ## ## ## 0 0 ## 0 0 1 1 0 1 0 0 0 1 1 0 0 0 1 1 1 1 1 0 1 0 0 ## 0 0 ## ## ## ## ## 0 ## ## 0 ## ## 0 ## 0 ## ## 0 0 ## ## ## 0 1 ## ## ## ## ## ## 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 ## 0 0 ## ## ## ## 1 1 ## ## 0 ## ## 1 ## ## 0 ## ## ## 0 0 ## 0 ## 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 0 1 1 1 1 0 0 1 0 1 ## 0 0 ## ## ## ## 1 1 ## ## 0 ## ## 1 ## ## 0 ## 0 ## 0 0 ## 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 0 1 1 1 1 0 0 1 0 0 ## 0 1 ## 0 0 ## ## 0 0 ## 0 0 0 0 ## 0 ## ## 0 ## ## ## 0 ## 0 ## 0 ## ## 0 ## 0 1 1 1 1 1 1 0 1 1 1 1 1 1 0 0 1 1 1 0 0 1 1 0 1 1 0 0 1 ## 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 ## 0 ## 0 0 0 0 1 0 0 1 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 ## 0 0 0 0 0 0 1 ## 0 ## ## ## 1 ## 0 0 ## 0 ## ## 0 0 ## 0 0 0 1 1 1 1 1 1 1 1 0 1 0 0 1 0 1 0 1 1 0 1 ## 0 0 ## ## ## ## 1 1 ## ## 0 ## ## 1 ## ## 0 ## ## ## 0 0 ## 0 ## 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 0 1 1 1 1 0 0 1 0 0 ## ## 0 ## ## ## ## 0 0 ## ## ## ## ## 0 0 ## 0 0 ## ## 0 0 0 0 ## 0 0 0 0 0 0 0 1 0 1 1 1 1 0 1 1 0 1 0 1 1 1 1 0 1 1 0 0 ## ## 1 0 0 0 0 0 0 0 ## ## 0 0 ## 0 1 ## 0 0 ## 0 0 ## 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 0 1 0 0 0 1 1 1 0 1 1 0 1 0 1 ## 0 0 ## ## ## ## 1 1 ## ## 0 ## ## 1 ## ## 0 ## 0 ## 0 0 ## 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 0 1 1 1 1 0 0 1 0 0 ## ## 0 ## ## ## 0 1 0 ## ## ## ## ## 0 0 ## 0 0 ## ## 0 0 0 1 ## 0 0 0 0 0 0 0 1 0 1 1 1 1 0 1 1 0 1 0 1 1 1 1 0 1 1 ## 0 ## ## 1 0 0 0 0 0 0 0 ## ## 0 0 ## 0 0 0 0 0 ## 0 0 ## 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 0 1 0 0 0 1 1 1 0 1 0 0 1 0 1 1 0 0 0 0 0 0 1 1 0 0 1 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 ## 0 0 1 0 1 1 0 1 ## ## 0 0 0 0 1 1 0 0 1 1 1 0 1 1 0 1 1 1 0 0 0 1 1 1 1 1 1 1 0 1 0 0 0 1 1 0 0 1 1 0 1 1 ## 0 1 0 1 0 1 1 1 1 0 0 0 1 0 0 0 1 1 0 1 1 1 1 1 0 1 0 0 0 1 1 0 1 0 0 1 1 1 0 1 1 1 0 0 1 0 1 0 0 1 0 0 1 0 1 0 0 ## 0 0 0 ## ## 1 1 0 1 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 0 1 1 1 0 1 1 0 1 1 ## 0 0 0 0 0 0 0 1 1 0 1 1 1 0 0 0 1 0 0 0 1 1 0 1 0 0 1 0 1 1 1 0 0 1 0 1 0 0 0 1 1 1 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 ## 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 ## ## 0 1 1 0 1 1 1 0 0 ## 1 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 0 0 ## 0 ## 0 0 0 1 1 1 1 0 0 0 1 1 0 1 1 1 1 1 1 1 0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 ## 0 0 0 ## ## 0 0 1 1 0 1 0 1 0 1 ## 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 0 1 1 1 0 1 1 0 1 1 ## 0 0 0 0 0 0 0 1 1 0 1 1 1 0 0 0 1 0 1 0 0 0 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 0 0 1 1 1 1 0 0 1 0 0 1 0 0 ## 0 1 0 1 0 1 0 1 1 0 0 1 0 0 ## 1 0 0 0 0 0 1 0 0 1 1 1 1 1 1 1 1 0 1 1 0 0 0 1 0 0 1 1 1 1 0 1 1 1 0 1 1 0 ## 0 1 0 1 0 1 0 1 1 0 0 1 0 0 ## 1 1 0 0 1 1 0 1 1 0 0 0 0 1 1 0 0 1 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 1 0 0 1 1 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 1 1 0 0 1 0 0 1 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 0 0 0 0 0 ## 0 ## 0 0 0 0 1 0 1 0 0 0 0 1 1 0 1 0 0 1 0 1 0 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 0 1 1 1 0 1 1 0 1 0 0 0 0 0 1 0 ## 0 0 0 0 0 0 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 0 0 0 1 1 0 1 1 0 0 0 1 0 1 ## 1 1 1 1 ## ## 0 ## 1 0 0 0 0 1 1 1 1 1 0 0 0 1 0 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 ## 0 0 0 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 0 1 0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 1 1 1 1 0 0 0 0 1 0 0 1 0 ## ## 0 0 0 0 ## 0 0 1 0 1 1 0 0 1 1 0 0 1 0 0 1 0 1 0 1 1 0 1 0 0 1 0 1 0 1 0 0 1 0 0 1 0 0 0 1 ## 0 ## 0 0 0 0 0 0 0 ## 0 0 0 0 0 1 0 1 0 0 0 0 1 1 0 1 0 0 1 0 1 0 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 0 1 1 1 0 1 1 0 1 0 0 0 0 0 1 0 ## 0 0

Drought test in large boxes -0.5 bar

Water potential

Well-watered

Well-watered

Moderate water deficit

-6 bar

Severe water deficit

-15 bar

Cyclic drought

Severe drought

Boris Parent, Nataliya Kovalchuk

Watering: Toro system Environment monitoring: MEA system Both connected to a computer

Design

120cm

Stage 1. Multi – Early Phenotyping: 1 Bin: 80 plants 8 lines, 1 WT control, 1 null control 8 plants per line Measure: Vigour, flowering time, height Leaf sampling: RNA Stage 2. Phenotyping for WW/Drought: 1 Bin: 10 plots.

3 lines, 1 WT control, 1 null control x 2 rep Randomised block design -Flowering time

-Height -Biomass -Spike number(per plant) -Seed number(per plant) -Seed weight (per plant) -Seed number(per spike)

80cm

120cm

80cm

Small scale field trials – Glenthorne, SA

 Wheat  Barley  Rainout shelters for drought

POT1::YFP

Bright field

Salinity trials – Kunjin, WA 2011 High salt Water logging Poor soil structure High salt

Mod salt Mild salt

ď ś Gradient of salt down field High salt

Low- mod. salt

Conclusions • Diverse phenotyping platforms needed to cover a range of stresses • Assay for traits in individual plants versus plant community • Specialist field sites – Regulatory requirements for field trials

Acknowledgments Aluminium Nick Collins Perry Gustafson Frost Nick Collins Sergiy Lopato Drought Boris Parent Sergiy Lopato Natalya Kovalchuk Salt Mark Tester Stuart Roy Transformation Serik Eliby Ainur Ismagul