Seed deterioration caused by ageing in
production and storage and its repair
during early germination determine the
performance of crop seeds.
Stan Matthews and Alison Powell
University of Aberdeen
Scotland
Outline
• Rate of radicle emergence (RE)as a vigour
test
• Ageing / repair explanation
• Prospects for molecular methods
• Avoiding production of low vigour seeds
Definitions of germination
• Just germination (JG): radicle appears through seed coat/pericarp (RE)
– mostly cell extension only
• Physiological germination (G): radicle at least 2mm (RE)
– Cell extension and cell division
• Normal germination:
– Includes mobilisation of food reserves
Imbibed JG Just germinated
Radicle emergence
(RE)
Maize: Stages of germination
G Germinated
Radicle emergence (2mm)
(RE)
Measurement of rate
0
20
40
60
80
100
0 24 48 72 96 120 144
Time from set to germinate (h)
Germ
ination (
%R
E)
F
H
I
MGT =∑ (nt) / ∑ n
Maize: 3 seed lots A
E
20o C
Germination progress curves
0
20
40
60
80
100
0 12 24 36 48 60 72
Time after sowing (hours)
Rad
icle
em
erg
en
ce (
%) A
B
C
D
E
F
G
H
I
OSR: Germination progress curves (9 lots cv Vision). Automated computer vision (SNES, France)
20o C
Mean germination time (MGT)
• G = germinated (2mm radicle; RE)
• MGT =∑ nt / ∑ n
n = number of seeds newly germinated at time t
t = hours or days from when set to germinate.
• MET same calculation for emergence
Ellis and Roberts, 1980
OSR: MGT predicts field emergence
Matthews, et al. 2012
Slow
MGT relates to emergence
(Slow and low)
MGT relates to emergence
Species Country (n) Significance
Maize Italy (24) ***
Argentina (6) ***
Cotton Australia (13) ***
Pepper Turkey (11) *
Watermelon Turkey (10) ***
Melon Turkey (10) ***
Cucumber Turkey (9) ***
Oilseed rape Scotland (9) ***
MGT also relates to MET
0
20
40
60
80
100
0 24 48 72 96 120 144
Time from set to germinate (h)
Germ
ination (
%)
F
H
I
66
A
E
Measurement of rate
Maize: 3 seed lots
Germination progress curves
20o C
Maize: Single counts predict MGT
SLOW FAST FAST SLOW
% RE ) % RE )
R2 values confirm that single counts predict MGT
All R2 P< 0.001
Crop Source Lots
(n)
Conditions % standard
germination
Maize Denmark 7 0.98 (66h, 20oC) 0.95 (6d, 13oC)
> 90% Austria 9 0.95 (66h, 20oC) 0.95 (6d, 13oC)
Watermelon Turkey 10 0.83 (68h, 25oC) >98%
Cucumber Turkey 9 0.97 (48h, 25oC) >98%
Radish Turkey 9 0.94 (48h, 20oC) >80%
Cotton Australia 13 0.96 (3d 18°C) >82%
Test based on single counts completed more quickly
Species Lots
(n)
Time of count
and
temperature
Significance
Maize 9 66h, 20oC *
9 6d, 13oC **
Cotton 13 3d, 18oC ***
Watermelon 10 68h, 25oC *
Melon 10 44h, 25oC *
Cucumber 9 48h, 25oC **
Oil seed rape 9 30h, 20oC **
Canola 19 2d, 20-30oC ***
Viola 9 2d, 20oC **
Single counts correlate with final emergence
Earlier RE, higher emergence
R2 = 0.94
R2 = 0.91
R2 = 0.71
0
20
40
60
80
100
0 20 40 60 80 100
% Germination
% F
ield
em
erg
en
ce (
Co
ol)
% Cold test (12d)
% 5d 13 o C
% 54h 20 o C
Maize: Rate relates to emergence (Argentina)
Courtesy of Augusto Martinelli and colleagues (2007)
Relative rate the same at 13oC and 20oC
FAST SLOW
Cold
wet
sowing
Normal seedlings
Radicle
Emergence (RE)
(RE)
y = 0.608x + 39.202
R20.847 =
y = 1.4364x - 43.311
R20.9859 =
0
20
40
60
80
100
120
40 50 60 70 80 90 100
Mean % site emergence (6 lots)
% F
ield
em
erg
ence
Lot 1
Lot 5
Maize in Argentina: Effect of cold, wet soil
Low temperature and oxygen: drastic effect on low vigour lot 5
10d after sowing
Av 12oC soil
Total 32mm rain
10d after sowing
Av 18oC soil
Total 8mm rain
y = 0.608x + 39.202
R2 = 0.847***
y = 1.4364x + 43.311
R2 = 0.986***
Lot %
SG
% RE %
CT 13oC 20oC
1 99 86 80 98
5 91 3 21 67
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
Maize: MGT related to shoot length and variation
in the field (after 3 weeks in Iran)
Mean Shoot Length (3 weeks)
R2 = 0.68*
20
25
30
35
40
45
50
55
60
5 5.5 6 6.5 7 7.5
MGT in Lab (days)
Sh
oo
t L
en
gth
(m
m)
Variation in Shoot Length (3 weeks)
R2 = 0.77**
20
25
30
35
40
45
50
5 5.5 6 6.5 7 7.5
MGT in Lab (days)
C.V
. S
ho
ot
Le
ng
th (
%)
Khajeh Hosseini, Lomholt and Matthews, 2009
Oil seed rape emergence (sown 30 September 2011)
High vigour
F
Low vigour
C
26 March 2012 9 April 2012 23 April 2012
F C
C
C F
F
Comment
• Early RE counts indicate
– relative field emergence (rate and final)
– seedling size in maize and oilseed rape
• Also applies to other species
• Worth trying on grasses, clover and spinach
• Faster than other measures of vigour
– Early count of normals
– Seedling growth test
– Ageing tests (AA and CD)
Outline
• Rate of radicle emergence (RE)as a vigour
test
• Ageing / repair explanation
• Prospects for molecular methods
• Avoiding production of low vigour seeds
Period of
ageing
Accelerated ageing:
Seeds aged (72h) in 100% RH, 41oC; germination: validated for soyabean
Controlled deterioration:
Seeds aged (24h) at 20% MC, 45oC; germination: validated for Brassica
spp.
Ageing based tests
y = -0.40x + 72.01
R² = 0.850***
30
35
40
45
60 70 80 90 100
MG
T a
t 20°C
(hours
)
Total germination after controlled deterioration (%).
Oilseed rape
Low vigour
i.e. aged seeds
Slow
Faster High vigour
i.e. less aged seeds
Matthews, Wagner et al. 2012
The greater seed age, the later RE (slower germination)
Species A: Rate of
emergence
B: Final
emergence
(%)
C: germination
(%) after AA or
CD test
Maize * ** (9) * (9)
Cotton ND *** (13) *** (13)
Pepper *** * (11) ** (5)
Watermelon ** *** (10) *** (10)
Melon ** *** (10) *** (10)
Cucumber ** *** (9) *** (9)
Oil seed rape *** ** (9) *** (9)
Viola *** *** (9) *** (9)
Significance of correlations between MGT and
A) rate of, and B) final emergence, and C) measure of seed age
Physiologically older seed
(more deteriorated)
Slower to germinate and emerge
Lower final emergence
Evidence for ageing / repair
• Ageing tests (AA and CD) –validated by ISTA
Measure levels of deterioration (age)
Age related to MGT
• AA increases MGT (i.e. slows rate of RE) - examples
Wheat (Guy and Black, 1998)
Sunflower (Bailly et al, 2002)
Further evidence for ageing / repair
• Ranking of MGT of lots
Same at all temperatures (Matthews et al. 2011)
Low temperature and O2 increase MGT (slower) (Bradford et al., 2007)
• Hydration / drying improves seed
Increases rate of RE (reduces MGT) (Matthews and Khajeh Hosseini, 2007)
Reduces level of deterioration (Powell et al, 2000)
Seen in priming
0
20
40
60
80
100
0 24 48 72 96 120 144
Time from set to germinate (h)
Germ
ination (
%)
F
H
I
Germination at 20oC Lag period
Longer lag period
66
A
E
At 13oC:
•slower
•more separated
Germination progress curves
Ageing / repair explanation
• Aged seeds are low in vigour
• Metabolic repair during the lag period
before RE needs a longer time for aged low
vigour seed
• MGT is greater, i.e. germination is slower
Ageing / Repair explains all vigour tests!
Vigour test Species (V or NV) Result reported Interpretation on the basis of aging
/ repair hypothesis
Conductivity test Garden pea (V),
Soybean (V),
Phaseolus spp (V)
Conductivity of seed leachate
(µS cm-1g-1)
Leakage from dead seeds and dead
/ damaged tissue following aging
and imbibition damage
Accelerated aging
(AA)
Soybean (V)
Other species (NV)
% Normal seedlings
Ageing and position of seed lot on
the survival curve
Saturated salt AA Small seeded species
(NV)
% Normal seedlings
Controlled
deterioration
Brassica spp (V) % total germinated seeds
(normal plus abnormal
seedlings)
Tetrazolium staining Soybean (V in
progress)
Summary of staining patterns
(see references)
Pattern of living / dead tissue
Radicle emergence Maize (V
Brassica spp (V in
progress)
% seeds with emerged radicle Time for repair of aging
Cold test (10oC) Maize (NV)
Other species (NV)
% normal and abnormal
seedlings; % dead seeds
Incomplete repair at low
temperature (and especially in
anaerobic conditions) increases
abnormal seedlings
Seedling size and
uniformity
Various species (NV) Varies e.g. seedling dry
weight, length or uniformity
Differences in the timing of RE
Cool test (18oC) Cotton (NV) % based on number of
normal seedlings with
hypocotyl + root length >4cm
Incomplete repair at lower
temperature slows RE giving
smaller seedlings
Vigour tests summarised and explained
Prospects for molecular methods
• Cytological evidence of DNA damage (Roberts et al)
Aged seeds
• More cells with broken chromosomes
• More abnormal seedlings
• Evidence of repair early in germination
Organelles and membranes in maize (Berjak and Villiers, 1972)
DNA in lag period of aged rye embryos (Osborne, 1983)
Radiation damaged chromosomes repaired in imbibed dormant lettuce seed (Villiers and Edgecumbe, 1972)
DNA repair in Arabidopsis
• Early detection of DNA damage (G1 of cell cycle)
– Increases in specific DNA damage responsive transcripts in first 6h
– Possible indicators of chromosomal breakage
• Phosphorylations to provide energy (G1)
– For synthesis e.g. of nucleotides
– O2 required for ATP production
• Repair of double strand breaks (DSBs) by ligases (G1)
– Mutants lacking ligases have longer lag periods
– This results in:
• reduced seedling size
• reduced ability to repair after storage
• Detection, phosphorylation and repair need time
– Lag period to RE is longer in aged seeds
– Especially so in mutants lacking ligases Waterworth, West et al. 2010
Observations on crop seeds corresponding with
Arabidopsis
• As seed ageing increases (naturally or imposed) the longer the lag
period
• Low O2 and temperature increase lag period and reduce germination
and emergence
• Increase in abnormals (DSBs) in cold test (low temp and O2)
• Seedling size and variation determined by timing of RE
• Limited hydration (and drying), e.g. in priming, reduces deterioration
level
Subcellular evidence in crop seeds needed – in progress
• A search for cellular markers before RE to rapidly quantify quality
Link with cell wall loosening to achieve RE
Comments on production
• Avoid deterioration from pre-harvest to sowing
– Low MC crucial pre-harvest – suitable production
areas help
– Minimise deterioration before storage; dry (12-13%
MC) and cool down quickly
– Keep MC and temperature down during storage and
distribution
– Monitor quality and limit time in storage
• Germination stays high, but vigour falls – seen in maize, cotton,
melon and OSR
Conclusions
• Rate of radicle emergence (RE)as a vigour test
– Potential test for all species
• Ageing / repair explanation
– Explains all vigour tests
• Prospects for molecular methods
– Rapid, early assessment of vigour?
• Avoiding production of low vigour seeds
– Avoiding seed ageing crucial
Acknowledgements
We would like to acknowledge the contribution of many
colleagues in many countries from –
• Seed companies
• Seed testing laboratories, and
• Universities
For names, see list of references
Avoiding production of low
vigour seed
The seed journey: parent plant to crop
establishment
Pre-harvest
Harvest
Post-harvest
•Processing
Drying / cooling
Cleaning
Grading
Chemical treatment
Packaging (bagging)
•Storage
•Distribution
•Sowing
Pre-harvest and Harvest
Mass / physiological maturity
(seeds at maximum dry weight)
Harvest maturity
(seeds closer to storage moisture content)
Critical period
for deterioration
Deterioration problems
1. Water stress – premature drying Deterioration
(green soybeans / OSR / vegetable seeds)
2. Rain, humidity, high temperature (weathering) during slow drying
• on plants Deterioration
• in windrows plus fungi
Seed production areas
Favoured areas within
•Countries
•Continents
•Worldwide
•Warm dry summers – little or no rain during
pre-harvest period
•Irrigated crops in dry climate – water under control
Areas of seed production
