Radicle emergence test in relation to

emergence, seed storage and other

vigour tests

Stan Matthews

University of Aberdeen

United Kingdom

• The germination progress curve

• Mean germination time (MGT)

• MGT in relation to:

– seed age

– seed performance (vigour)

– radicle emergence (RE)

• RE and seed performance (vigour)

• RE as a vigour test

• RE in relation to other vigour tests

• Explanation of relationships

• Concluding comments

Outline

0

20

40

60

80

100

0 24 48 72 96 120 144

Time from set to germinate (h)

Germ

ination (

%)

F

H

I

Lag period

Longer lag period

Germination progress curves to radicle emergence (RE)

Mean germination time (MGT) = Ʃnt / Ʃn

n = number of newly germinated seeds at time t

t = days from when seeds were set to germinate.

MGT= Mean length of lag period

Mean length of lag period differs between commercial seed lots

Why do the lag periods differ between lots?

Maize

y = -0.40x + 72.01

R² = 0.850***

30

35

40

45

60 70 80 90 100

MG

T a

t 2

0°C

(h

ou

rs)

Total germination after controlled deterioration (%).

MGT and seed age: oilseed rape

High MGT

Slower germination

Long lag period

Low MGT

Faster germination

Short lag period

More aged

Less aged

As seeds age, the lag period to RE increases (higher MGT)

2012: Seed Sci. &Technol., 40, 413-424

MGT and seed age

Seed age measured by AA or CD negatively correlates ( r) and predicts (R2) MGT

significantly (mostly p< 0.01 or p< 0.001)

Species (number of lots) r R2

Oilseed rape (9) -0.92 0.85

Maize (9) -0.79 0.62

Radish (10) -0.89 0.80

Cotton (17) -0.91 0.83

Pepper (11) -0.97 0.95

Watermelon (10) -0.95 0.90

Melon (10) -0.75 0.57

Cucumber (9) -0.94 0.88

Viola (9) -0.91 0.83

The more aged the seed, the longer the lag period

For references see STI (2012), 142, 31-35

Relates to ageing tests

0

20

40

60

80

100

0 24 48 72 96 120 144

Time from set to germinate (h)

Germ

ination (

%)

F

H

I

Lag period

Longer lag period

Germination progress curves

MGT = Ʃnt / Ʃn

n = number of newly germinated seeds at time t

t = days from when seeds were set to germinate.

MGT= Mean length of lag period

• Mean length of lag period differs between commercial seed lots

• Ageing increases MGT and spread of timing of germination

Ageing

Maize

MGT and seed performance (vigour)

• Rate of emergence

• Final emergence

• Spread of emergence

MGT predicts rate of and final emergence: oilseed rape

y = -5.88x + 271.79

R² = 0.930***

y = -2.34x + 165.39

R² = 0.745**

20

40

60

80

100

30 35 40 45

Fie

ld e

mer

gen

ce (

%)

MGT at 20°C (hours)

Final emergence

Low MGT

Short lag period

Early emergence

High final emergence

High MGT

Long lag period

Late emergence

Low final emergence

7 day emergence

2012: Seed Sci. & Technol., 40, 413-424

MGT (R2) predicts rate of, and final, emergence in many species.

All R2 significant: mostly at P< 0.01 or P<0.001

Longer delay to RE; higher MGT

Slower and lower emergence

Low

vigour

Species (lots) Rate of

emergence

Final emergence

(%)

Oil seed rape (9) 0.93 0.75

Radish (9) 0.92 0.86

Maize (9) 0.62 0.72

Cotton (13) - 0.83

Pepper (11) 0.94 0.45 (modules)

Watermelon (10) 0.83 0.81

Melon (10) 0.74 0.70

Cucumber (9) 0.76 0.64

Viola (9) 0.91 0.92

References, see STI (2011), 142, 31-35

Maize: MJGT and MGT relate to seedling size and uniformity in lab

(14 d, 13oC)

MJGT = 3.52 days

MGT = 4.80 days

MJGT = 4.82 days

MGT = 5.84 days

Timing and spread of emergence: maize

High MGT indicates slower and more spread emergence

Relates to seedling growth tests

Timing and spread of emergence

Important for vegetable production from modules

Cabbage High MGT Low MGT

SO FAR:

MGT from germination curves relates to vigour

BUT:

Frequent RE counts not convenient for a routine test

Are there alternatives?

M.-H. Wagner – Seed Symposium – IPK Gatersleben 11/04/18

Automated monitoring since 2005

• Digital imaging upon Jacobsen incubators

• High throughput, thermal precision, repeatability

• Top of paper germination

Demilly et al., 2014

30 hours

Differences seen in

early radicle emergence

counts (30 hours)

Oilseed rape

Automated germination progress curves (GEVES)

2012: Seed Sci. & Technol., 40, 413-424

y = -0.17x + 49.92

R² = 0.910***

30

35

40

45

40 50 60 70 80 90 100

MG

T a

t 2

0°C

(h

ou

rs)

Radicle emergence (%) at 30 hours

Oilseed rape: Single count of RE predicts MGT

2012: Seed Sci. & Technol., 40, 413-424

Low MGT,

Short lag period

High RE

High MGT,

Long lag period

Low RE

Early single counts of RE (top of paper) predict MGT in 17 species

Automated digital imaging (GEVES)

All significant;

13/17, R2 >0.90

Temp Species No. of

lots

analysed

MGT (h) range RE count (h)

best related to

MGT

Highest

R2

20o C

OSR 68 28-60 34 0.87

Maize 35 49-78 60 0.83

Radish 6 29-56 40 0.98

Wheat 15 29-72 50 0.97

Cabbage 40 23-60 36 0.90

Onion 32 33-103 72 0.87

Tomato 56 52-125 82 0.92

Sugarbeet 17 49-75 64 0.67

Lucerne 30 18-39 30 0.91

Lettuce 8 23-28 24 0.94

Carrot 8 41-69 56 0.97

25oC

Sunflower 7 28-38 36 0.97

Eggplant 8 76-96 90 0.99

Turnip 8 16-20 18 0.97

Chicory 8 23-31 40 0.93

Parsley 8 123-161 130 0.95

Cotton 5 46-61 52 0.99

y = 1.07x - 24.75

R² = 0.961***

y = 0.40x + 48.67

R² = 0.713**

20

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80

100

20 40 60 80 100

% R. E. 30h at 20°C

Fie

ld e

mer

gen

ce (

%)

7d

Max

Oilseed rape: Single count of RE predicts field emergence

2012: Seed Sci. & Technol., 40, 413-424

Low RE

Slow emergence

Lower final emergence

High RE

Faster emergence

Higher final emergence

Species Lots (n) Time of RE count r R2 Significance

( p< )

Maize 9 66h , 20oC

6d, 13oC

0.76

0.85

0.58

0.72

0.02

0.01

Oilseed rape 9 30h, 20oC 0.84 0.71 0.01

Radish 11 48h, 20oC 0.95 0.90 0.001

Cotton 10 3d, 18oC 0.87 0.76 0.001

Rice 13 54h, 25oC 0.90 0.81 0.001

Watermelon 11 68h, 25oC 0.81 0.66 0.01

Melon 10 44h, 25oC 0.74 0.55 0.05

Cucumber 10 48h, 25oC 0.84 0.70 0.05

Viola 9 48h, 20oC 0.82 0.67 0.01

Also seen in wheat, alfalfa, onion and forage crops

Early single RE counts

correlate ( r) and predict (R2) final emergence in many species

For rice: Luo et al. (2017), Seed Sci. & Technol., 45, 1-8

All other species see STI (2011), 142, 4- 48

RE vigour test and ISTA: Validation then ISTA Rules

• Test relates to performance (vigour)

• Convenient for routine use – single early RE count

• Repeatable

• Reproducible

y = 0.86x + 16.95

R² = 0.95***

0

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0 20 40 60 80 100

Lab

ora

tory

B

rad

icle

emer

gen

ce (

%)

Laboratory A radicle emergence (%)

y = 1.012x - 2.65

R² = 0.96***

0

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0 20 40 60 80 100

Lab

ora

tory

C r

adic

le

emer

gen

ce (

%)

Laboratory A radicle emergence (%)

y = 1.126x - 19.82

R² = 0.94***

0

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100

0 20 40 60 80 100

Lab

ora

tory

C r

adic

le

emer

gen

ce

( %

)

Laboratory B radicle emergence (%)

Repeatability of RE in different laboratories : oilseed rape

Validated for oilseed rape:2014

ISTA Proficiency test 2016

57 labs; 70% satisfactory

Matthews, Wagner, Kerr, McLaren and Powell (2012) Seed Sci. & Technol, 40, 413-424

Species Germination

medium

Replication Germination

temperature

Criterion of radicle

emergence

Timing of radicle

emergence count

Zea mays Paper towels 8 replicates

of 25 seeds

20 ± 1oC

or 13 ± 1oC

Production of 2mm

radicle

66h ± 15 min at 20 ± 1oC

144h ± 1 h at 13 ± 1oC

Brassica

napus

Pleated

papers

2 replicates

of 100 seeds

20 ± 1oC Appearance of a

radicle after breaking

through the seed

coat. Seeds in which

the seed coat has

split, but no radicle

has emerged, must

not be included.

30h ± 15 min

Raphanus

sativus

Top of paper 4 replicates

of 50 seeds

20 ± 1oC Production of 2mm

radicle

48h ±15 min

Specific directions for RE test in ISTA Rules

Note: alternative germination

media not acceptable for RE test Precision essential

Lag period and the cold test: maize

SLOW FAST

y = 0.86x + 11.62

R² = 0.96

p < 0.001

0

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20 40 60 80 100

Germination after 6 days at 13°C

Ge

rmin

ati

on

in

co

ld t

es

t

(% n

orm

al s

ee

dlin

gs

)

Low RE

Long lag period

Low cold test germination

High RE

Short lag period

High cold test germination

RE

Seed Sci. & Technol.(2010), 38, 379-389

RE relates to cold test

Significance of the lag period

T.A Villiers, 1970’s : Lettuce

Dormant seeds stored in the dark for 12 months

Dry seeds Fully imbibed seeds (in air, moist filter paper)

Germinated in light Germinated in light

Low germination (RE)

Many chromosome aberrations

Many abnormal seedlings

High germination (RE)

Few chromosome aberrations

Few abnormal seedlings

Villiers hypothesis: metabolic repair of membranes and DNA when stored imbibed

D. Osborne: 1980’s and 90’s: rye

• Evidence of DNA repair in lag period before radicle emergence

• Supporting and detailed evidence for DNA repair from Arabidopsis mutants

(Waterworth et al., 2007 to date)

• Some evidence for membrane repair

y = 0.492x + 11.83

R² = 0.850***

30

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40 50 60 70 80 90 100 110 120 130 140 150

MG

T (

hours

)

EC µS cm-1 g-1 after 3h (EC3)

A

Evidence for membrane damage

Radish and bulk EC 100 seeds in 40ml water

Longer lag period (high MGT)

In lots with higher leakage (EC)

Mavi et al., (2016) Seed Sci. & Technol., 44. 393-409

RE relates to EC test

Evidence of membrane damage and repair

Radish: single seed EC and RE performance

(Amalgamated data from 20 seeds from each of 10 seed lots)

EC measured after 5 hours in water at 20oC, followed by germination on moist paper at 10oC

All

seeds

Dead

seeds

Seeds that produce: Seed producing normal

seedlings with:

Abnormal

seedlings

Normal

seedlings

Early RE

<66h

Late RE

>66h

Number of seeds

199 22 14 165 73 92

Mean EC (5h)

6.88 14.24 11.99

(MGT: 122h)

5.79

(MGT: 91h) 4.25 6.26 * **

Also:

• 5h hydration in air drying back

• Significant reduction in leakage and lag period

• Evidence of metabolic repair.

Mavi et al. (2016) Seed Sci. & Technol., 44, 393-409

Concluding comments

• As seeds age the lag period to RE increases (higher MGT)

• As MGT increases – Rate and final emergence decrease

– Longevity in store decreases

• Higher MGT (longer lag period) smaller and less uniform seedlings

• An early count of RE (manual or automated) – Predicts MGT

– Predicts seed vigour – validated for maize, oilseed rape, radish

– Evidence for its potential for many other species

• Hypothesis: as seeds age, the need for repair and lag period increase

• Hypothesis explains connections with other vigour tests – AA, CD, seedling growth, cold and EC tests