speed of seed imbibition related to seedling evaluation...seedling evaluation 30 th ista symposium...

G r o u p e d ’ É t u d e e t d e c o n t r ô l e

d e s V a r i é t é s E t d e s S e m e n c e s

Speed of seedimbibition related to seedling evaluation

3030thth ISTA Symposium ISTA Symposium -- Antalya Antalya -- 14 14 JuneJune 201320131

seedling evaluationWagner MH, Demilly D, Blouin V,

Ducournau S, Léchappé J.



Seed imbibition: first step before

seedling development


Sowing Germination Growth Seedling

Development of oilseed rape seedling



Seed imbibition: first step before

seedling developmentImbibition Germination Growth

Wat

er u

ptak

e


Time

Wat

er

from Bewley & Black, 1994

Aim: test the image analysis parametersobtained before germination

Radicle protusion



• Medicago truncatula

468

10121416

seed

wei

ght(

mg)

seeds at sowing timeseeds during

Seed imbibition measured by image analysis:

seed area or volume is correlated to weight

Demilly et al., 2007


24

4 6 8 10 12 14

seed

seed area (mm²)

seeds during imbibition

4

y = 0.6038x + 0.8853

R² = 0.865

0

2

4

6

8

10

12

14

16

0 5 10 15 20 25

Dry

se

ed

mas

s (m

g)

Dry seed volume on image (mm3)

Demilly et al., 2007

0

50

100

150

0 2 4 6 8

% fr

om in

itial

val

ue

hours after sowing

moisture content (% dry weight)

Area

Volume

• Brassica napus



Two examples of application on

crop species• Hardseededness in lucerne

CuticlePalisadic layerMalpighi cells

Nutritive cells

hard seed section fresh seed section


• Ageing in oilseed rape

Longitudinal sections of Medicago sativa seed coatMO x 63, PTM IMAC, SFR QUASAV

OSRCultivar

Standard germination (%)

Germination rate (%)

1 97 99

2 99 100

3 90 99

4 92 98

5 98 99

hard seed section fresh seed section

Abnormal seedlings usuallyobtained after ageing tests



Power of digital imaging for

studying germination• 15 samples of each species

Range of quality (%) Lucerne Oilseed rape

Standard germination [40-65] [84.5-99.5]

Abnormal seedlings [3-10] [0.5-13]


• Automated imbibition and germination time

courses at 20°C on 1 x 100 seeds (TP) previously

weighed

• Single seedling evaluation after 5 days

Abnormal seedlings [3-10] [0.5-13]

Ungerminated seeds [31-50] [0-2.5]



Single seed analysis linked to

seedling evaluation

Area T0 Mass Initial

Area

increase Germination Seedling

Single seedgermination databaseobtained after image analysis (extract)

Sowing time Image acquisition Image analysis


0

10

20

30

40

50

60

70

80

90

100

0 24 48 72 96 120

Ge

rmin

atio

n (

%)

Time after sowing at 20°C(hours)

11

12

13

21

22

23

31

35

36

41

42

43

51

52

53

Sample date Camera zone seed

Area T0

mm²

Mass

(mg)

Initial

length (mm)

increase

(%)

Germination time (hours)

Seedling type

1 101102 1 hg 11 5.1 2.3 3.7 0.0 56 PA

1 101102 1 hg 12 5.4 2.1 3.9 5.4 SD

1 101102 1 hg 13 5.6 3 3.7 28.3 20 PN

1 101102 1 hg 14 5.4 2.4 3.8 1.4 PN

1 101102 1 hg 15 3.5 1.3 3.1 3.2 36 PA

1 101102 1 hg 16 4.6 1.4 3.7 5.6 SD

1 101102 1 hg 17 4.6 2 3.4 6.5 35 PN

1 101102 1 hg 18 5.2 2.3 3.4 -4.3 SD

1 101102 1 hg 19 4.1 2.2 3.2 6.3 SD

1 101102 1 hg 21 6.0 2.6 4.1 3.1 39 PN

1 101102 1 hg 22 4.6 2.6 3.3 5.6 SD

1 101102 1 hg 23 5.3 2.7 3.8 3.5 SD

1 101102 1 hg 24 4.6 2.2 3.6 6.5 61 PN

1 101102 1 hg 25 4.5 1.5 3.5 4.9 29 PN

1 101102 1 hg 26 6.1 2.1 3.8 46.1 20 PN

1 101102 1 hg 27 3.6 1.3 3.1 6.2 18 PN

1 101102 1 hg 28 4.3 1.4 3.5 6.9 SD

1 101102 1 hg 29 5.5 2.8 3.5 2.0 SD

1 101102 1 hg 31 5.0 2.5 3.7 3.7 SD

1 101102 1 hg 32 5.0 2.1 3.5 63.4 21 PN

1 101102 1 hg 33 5.6 2.7 4.0 3.3 SD

1 101102 1 hg 34 4.1 1.8 3.4 6.3 SD

1 101102 1 hg 35 4.2 1.1 3.3 0.0 55 PN

1 101102 1 hg 36 4.1 1.6 3.4 5.5 PN

1 101102 1 hg 37 3.7 1.0 2.8 32.7 SNG

Automated germination time courses



Seed coat thickness and

hardseededness in Lucerne• X-rays before sowing seeds to detect hard seeds on

4 lots containing more than 40% of hard seeds

Hard seeds sortedafter 8 hours of

HS HS HS

HS HS


0

20

40

60

80

100

1 2 3 4

No

rmal

ge

rmin

atio

n (

%)

Seed lot

No scarification Global scarification Hard seeds scarification

after 8 hours of imbibition

Hard seeds have all adherent testa

HS

HS HS HS HS

HS

HSHSHSHSHS

HS



Imbibition with or without seed

scarification in lucerne

Mean aftermechanicalscarification (25 seeds per lot)

8.0

9.0

10.0

See

d a

rea

me

asu

red

by

imag

e a

nal

ysis

(mm

²)

Imbibition of 15 seedlots at 20°C


Mean beforemechanicalscarification (100 seeds per lot)

3.0

4.0

5.0

6.0

7.0

0 1 2 3 4 5 6

See

d a

rea

me

asu

red

by

imag

e a

nal

ysis

(mm

²)

Time after sowing (hours)



Oilseed rape imbibition at 20°C is

less important but also significant• 15 seed lots from 5 cultivars

9.00

10.00

Sse

ed

vo

lum

e o

bta

ine

d b

y im

age

an

alys

is (

mm

3 ) 11

12

13


4.00

5.00

6.00

7.00

8.00

0 2 4 6 8 10 12

Sse

ed

vo

lum

e o

bta

ine

d b

y im

age

an

alys

is (

mm


13

21

22

23

31

35

36

41

42

43

51

52

53

Cultivar Thousendseedweight (g)

1 5.0

2 4.6

3 4.8

4 4.8

5 6.2



Standard germination test and seedling

evaluation after automated curves

• Oilseed rape germination at 20°C• SG: 200 seeds between paper for 7 days

• Jacobsen system: 100 seeds on top of paper for 5 days

100


50

60

70

80

90

11 12 13 21 22 23 31 35 36 41 42 43 51 52 53

Standard germination

Seedling evaluation in conditions used for image analysis



Imbibition curves at 20°C according

to the seedling type

• Lucerne • Oilseed rape

7

8

See

d a

rea

(mm

²) 8

9

See

d v

olu

me

(m

m3)


3

4

5

6

0 1 2 3 4 5 6 7

See

d a

rea

(mm

²)


Normal seedling Abnormal seedling

Ungerminated seed Hard seed

3

4

5

6

7

0 2 4 6 8 10 12 14

See

d v

olu

me

(m

mTime after sowing (hours)

Normal seedlings Abnormal seedling Ungerminated seed



Imbibition speed and seedling

growth for lucerne

• Imbibition after 4 hours at 20°CSeed and seedling evaluation after 5 days

at 20°C

Seed area at

sowing time

(mm²)

Seed area after

4 h of imbibition (mm²)

Area increase (%)

478 Hard seeds 4.22 c 4.21 d 0 ±0.5


36 Ungerminated seeds 4.97 a 6.00 b 21 ± 6

37 Abnormal seedlings 4.73 b 6.42 a 36 ± 9

949 Normal seedlings 4.37 c 4.96 c 14 ± 2

Means with different letters are significantly differentusing SNK test at the 5% level



Imbibition speed and seedling

growth for oilseed rape

• Imbibition after 6 hours at 20°CSeed and seedling evaluation after 5

days at 20°C

Seed volume at

sowing time (mm3)

Seed volume after

6 h of imbibition (mm3)

Volume increase (%)

58 Ungerminated seeds 5.76 a 7.09 a 23 ± 4


166 Abnormal seedlings 5.85 a 7.15 a 22 ± 2

1275 Normal seedlings 5.57 a 6.61 b 19 ± 1

Means with different letters are significantly different using SNK test (α = 0.05)



Distributions of seed size changes due

to imbibition related to seedling growth

• Imbibition speed relative to dry seed stage

Lucerne Oilseed rape

45

30

Low imbibition speed 85% of normal growth

Less than 20% of imbibition 75% of normal growth


0

5

10

15

20

25

30

35

40

<2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 30% 40% 50% >50%

Pro

po

rtio

n o

f se

ed

s (%

)

Seed area increase after 4h of imbibition

Normal seedlings

Abnormal seedlings

Ungerminated seeds

0

5

10

15

20

25

30

<10% 10% 20% 30% 40% 50% 60% 70% 80% 90% >90%

Pro

po

rtio

n o

f se

ed

s (%

)

Volume increase after 6h of imbibition

Normal seedlings

Abnormal seedlings

Ungerminated seeds



Image analysis and seed imbibition

for practical

• Interesting to check hardseededness in

Legume seed lots

– Early identification, X-rays non invasive


– Early identification, X-rays non invasive

– Better efficiency of scarification when done on

hard seeds only

– Informative for customers and seed processing

plant

– Easy work for seed testing labs



Image analysis and seed imbibition

for further studies• Imbibition speed and seedling growth

– Rapid imbibition leads mainly to abnormal

seedlings or seed death to verify on other species

– BUT no clear threshold limits for a practical use


– BUT no clear threshold limits for a practical use

• Single seed analysis

– seed size measured on image analysis correlated

with seed mass

– useful for genebanks: less seeds, shorter than

standard germination test, automated tools exist

to check seed germination during storage



Many thanks to

• Sandrine Lourdel, Marine Pons, Nathalie Vialles,

students

• Laurence Lecorre, X-rays imaging

• Marjorie Juchaux, Angers University, microscopy


• Marjorie Juchaux, Angers University, microscopy

• Frédéric Corre, Chantal de Walsche, Isabelle

Pauchet, François Poutoire, UFS

• And You!

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speed of seed imbibition related to seedling evaluation...seedling evaluation 30 th ista symposium...

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