accelerated ageing vigour test...the test was first developed by delouche (1965) to estimate the...

ACCELERATED AGEING

VIGOUR TEST

JULIO MARCOS-FILHO

SEED TECHNOLOGY

DEPT. OF CROP SCIENCE

UNIVERSITY OF SÃO PAULO/ESALQ

BRAZIL

INTRODUCTION

The accelerated aging (AA) vigour test provides

valuable information on both seed storability and field

seedling emergence potentials

It is validated by ISTA and included in the International

Rules for Seed Testing since 2001

BASIS (PRINCIPLE) OF THE TEST

The development of the AA test came from observations

of Crocker and Groves (1915). They suggested that seed

deterioration during storage was caused by protein

coagulation and that this process was accelerated by

increases in seed mass temperature

They proposed a germination test after exposing seeds

from 50oC to 100oC for short periods to estimate the

storability of seed lots

The test was first developed by Delouche (1965) to estimate

the longevity of seeds stored in a warehouse.

Progress of these studies: protocol for the AA test by

Delouche and Baskin (1973). The current recommended

procedure is based on McDonald and Phannendranath

(1978).


The proposal considered that the decline in germination

following AA is proportional to the initial physiological

potential of the seed lot: higher vigour seed lots age

slower than lower vigour seed lots

The AA stress exposes seeds for relatively short

periods to high temperature (41oC) and relative

humidity (≈ 95%) followed by a germination test

Higher vigour seed lots are more tolerant to these

stressful conditions and produce a higher percentage of

normal seedlings (> germination percentage after AA)


This combination of raised seed water content and high

temperature causes rapid seed ageing

Seedlings from high vigour seeds

Photo: França-Neto


Seedlings from seeds of different vigour levels

Photo: França-Neto


APPARATUS

- Balance: capable of weighing to the nearest 0.001g

- Plastic AA box (11,0cm x 11,0cm x 3,5cm) with a lid, into

which is placed a plastic or a wire tray mesh screen

- Bottle-top dispensette: range from 0-100 ml

- Ageing chamber

- Water: deionized or distilled

- Water content test facilities

- Germination test facilities

- Thermometers

VERY IMPORTANT: cleanliness of materials and equipment

AGEING CHAMBER

Capable of maintaining a constant temperature of 41+0.3oC

A water jacketed ageing chamber is recommended

ISTA VALIDATED AA

PROCEDURE FOR SOYBEANS

PROCEDURE

1. Preparation of the sample

Determine the water content (fw) of the submitted

sample: must be between 10.0% and 14.0%

If not, it must be adjusted (directions in ISTA Rules

for Seed Testing)

Check the temperature in the ageing chamber:

must be calibrated at 41+0.3oC

PROCEDURE

2. Preparation of the plastic AA boxes and seed sample

Add 40 ml of water and place a dry wire mesh tray

to each plastic AA box

Photos: Riad Baalbaki

X

PROCEDURE


Weigh a seed sample of 42 + 0.5g and draw

randomly to the surface of the screen tray: seeds

should be placed in a single layer deep onto

screen to assure even water uptake by seeds

X Photo: Riad Baalbaki

PROCEDURE


The AA plastic boxes and wire mesh trays need to

be washed prior to each test run in a 1.0%

solution of sodium hypochlorite (clorox) to

prevent fungal contamination

After distributing seeds in the respective tray

(more than one box for large-seeded cultivars or

species), place a lid on each plastic AA box, but

do not seal edges

PROCEDURE

3. Ageing the seeds

The AA boxes with the seed samples should be placed

on a shelf, allowing an air space of + 2,5 cm between

the boxes to assure proper air circulation and

temperature uniformity inside the chamber, thus

avoiding water condensation inside the AA boxes

Place the shelves holding the AA boxes + samples into

the aging chamber, being careful not to splash water

onto seeds during handling

PROCEDURE

3. Ageing the seeds

Keep the outer chamber door opened as briefly as

possible to minimize heat loss and temperature

recovery time

The aging chamber should not be opened during

the 72 h ageing period ( 1 min ➔ decrease of

2 – 3oC inside the boxes and around 30 min to

recover to 41oC)

PROCEDURE

3. Ageing the seeds

At the conclusion of the AA period, immediately

weight the aged control sample. If this weight is

lower than 52.0g or higher than 55.0g, the test

results may not be accurate and it is necessary

to retest

The final seed water content for soybeans after a

72 h ageing period is usually 27 – 30% (AOSA,

2009)

PROCEDURE

4. Testing for germination

Set up a germination test using four 50-seed

replicates for each sample within 1.0 h after

removal from the chamber, according to the

procedures outlined in the ISTA Rules for Seed

Testing

Make soybean counts after four days

PROCEDURE


Photos: França-Neto

PROCEDURE


Results of germination from aged seeds are

expressed in percentage of normal seedlings

It must be verified if the difference between

replicates does not exceed the maximum tolerance

value (Tables 15F, 15G or 15H of ISTA Rules)

The seed water content before the test must be

reported

PROCEDURE

4. Testing for germination: tolerances

Average percentage

germination

Maximum

range

Average percentage

germination

Maximum

range

99 2 -- 86-88 13-15 12

98 3 -- 83-85 16-18 13

97 4 6 79-82 19-22 14

96 5 7 74-78 23-27 15

95 6 8 68-73 28-33 16

93-94 7-8 9 55-67 34-46 17

91-92 9-10 10 51-54 4-50 18

89-90 11-12 11 ---- ---- ----

Table 15F. Tolerated ranges between two replicates of 100 seeds in one AA test (ISTA Rules)

Table 15 G refers to two AA tests on the same submitted sample; Table 15H, for

different AA tests on different submitted samples

MAJOR FACTORS AFFECTING THE RESULTS

The AA test has advantages of being inexpensive and

requiring no additional technical training to the

analysts familiar with the germination test.

However, certain precautions need to be taken to

reduce variability in test results

1. Temperature


The AA test is performed at a precise 41oC

A variation of no more than 0.3oC should be

permitted to avoid effects on germination

results after AA

1. Temperature


Seed

Lot

Initial standard

germination (%)

Accelerated aging germination (%)

February March April June July

1 92 86 82 86 91 91

2 92 68 70 60 84 66

3 96 90 93 89 92 83

4 96 84 83 89 96 89

Mean 82 82 81 91 82

Effect of temperature variation during the AA test in a six month storage of four

soybean seed lots (TeKrony, 2003)

2. Seed water content


If seed water content is below 10.0% or above 14.0%

it must be adjusted between these values by

moistening or drying. See ISTA Rules for details

Large soybean seeds usually have a lower final seed

water content and higher germination after AA than

smaller seeds when size differences are greater than

1.0mm

3. Seed size


Seeds to be aged should not be treated with

fungicide(s), if possible.

4. Fungicide treatment

However, if seeds are already treated for marketing,

treated seeds may be tested

It is preferable not to compare in the same test seed

samples treated with different products

5. Genotype


The tolerance of the seeds to the conditions of the

AA test depends on the genotype, because there

are more sensitive cultivars to high temperatures.

It is more convenient to compare seed lots of the

same cultivar

6. Conditions for germination

The germination test after ageing must be performed

according to the procedures outlined in the ISTA

Rules for Seed Testing

RELATIONSHIP OF AA RESULTS AND SEEDLING

EMERGENCE AND SEED STORABILITY

1. Field Seedling Emergence

Year

Number

of

samples

Mean (%)

Germination

Time 1 of sowing Time 2 of sowing

Standard

germination

Accel.

ageing

Std

germination

Accel.

ageing

1980 40 82 0.45 ** 0.90** 0.60 0.92**

1984 29 89 0.41** 0.64** 0.59 0.55

1987 16 91 0.90** 0.88** 0.87** 0.96**

1988 33 91 0.84** 0.80** 0.85** 0.80**

1991 15 95 0.49 0.74** 0.48 0.70**

1992 38 94 0.58** 0.90** 0.52 0.91**

1993 20 90 0.89** 0.91** --- ---

Correlation of standard germination (SG) and accelerated ageing germination (AA)

and seedling field emergence in soybeans (Egli and TeKrony, 1995)

Seed lots Standard

germination (%)

Accelerated

ageing (%)

Field

emergence (%)

1 89 a 87 a 94 a

2 92 a 82 b 94 a

3 81 c 84 b 90 b

4 70 d 78 c 89 b

5 77 c 77 c 88 b

6 86 b 68 d 84 c

1. Field Seedling Emergence

Means of standard germination, accelerated ageing and seedling

field emergence from six soybean seed lots (Schuab et al., 2007)

2. Seed storage potential

Cultivar Seed lot Germination (%) Initial Accelerated

Ageing (%) Initial 9-month storage

Williams

1 94 68 34

2 91 73 74

3 92 59 44

Cutler 71

1 88 43 63

2 79 83 76

3 98 68 75

Dare

1 91 45 50

2 95 84 78

3 97 87 84

York

1 96 90 89

2 98 91 89

3 85 68 74

Utilisation of the AA test to identify differences in storability of soybean seed lots

(Egli et al, 1979).

Seed lots

Initial 4-month storage

Germin.

(%)

TAA

(%)

Emerg.

(%)

Germin.

(%)

Emerg.

(%)

1 91 84 87 86 82

2 94 85 91 91 93

3 81 74 89 81 72

4 90 80 86 86 81

5 90 78 92 85 94

6 84 84 90 87 88

2. Seed storage potential

Germination, accelerated ageing, and field seedling emergence from six soybean seed lots, cv.

BRS 184, before storage and germination and seedling emergence from seeds stored for four

months (Yaguchi et al., 2014)

POTENTIAL USES FOR OTHER SPECIES

In addition to the validation by ISTA to assess

soybean seed vigour, there are AA test procedures

available for more than 40 species, including

grain, vegetable, forage, and forestry crops.

Protocols for the AA test were reported by Marcos-Filho (2016)

in Seed Physiology of Cultivated Plants. Londrina/BR, Abrates,

616 p.

It has been successfully used by seed companies to

compose “in house” seed quality control programs

SATURATED SALT

ACCELERATED AGEING (SSAA)

The results of the AA test for small-seeded crops

sometimes are not successful because those

seeds absorb water more rapidly resulting in large

variation in seed water content and lack uniformity

among seed samples and in deterioration after the

artificial ageing (Powell, 1995)

SATURATED SALT


Jianhua and McDonald (1996) proposed the SSAA, using the

same apparatus and procedure for the traditional AA,

except for a substitution of water for a saturate salt

solution (usually NaCl: R.H.% inside the ageing box =

76%).

The results have shown reduction in the rates of seed water

uptake and deterioration, and additional advantages of

simplicity, lower growth of fungi, that represent a concern

in a traditional AA, without reducing the test accuracy.

SATURATED SALT



SATURATED SALT ACCELERATED AGEING (SSAA)



Photo: Riad Baalbaki

Species Temp /

Duration

Initial Water

Content (%)

Final Water Content (%)

Trad. AA SSAA

Brocolli 41oC / 72 h 8,0 - 10,0 27,0 – 37,0 9,5 -11,0

Onion 41oC / 72 h 6,5 - 7,5 35,5 – 39,5 10,2 -12,6

Carrot 41oC / 72 h 7,7 – 8.0 39,5 – 48,5 10,0 -11,0

Melon 41oC / 72 h 7,0 - 7,5 27,5 - 30,5 9,0 – 10,0

Sweet corn 41oC / 72 h 8,0 - 9,0 27,0 – 28,5 20,0 - 21,5

Bell pepper 41oC / 72 h 6,8 - 8,6 29,5 – 37,8 10,0 - 12,0

Soybean 41oC / 72 h 12,0 - 13,0 27,0 – 30,0 12,0 - 12,5

Tomato 41oC / 72 h 8,0 -8,5 37,0 – 41,0 9,0 - 10,0

Temperature, duration of ageing and initial and final seed water

content of various crops in the SSAA (in Marcos-Filho, 2016)


Seed lots

Initial 4-month storage

Germin.

(%)

TAA

(%)

SSAA

(%)

Emerg.

(%)

Germin.

(%)

Emerg.

(%)

1 91 84 94 87 86 82

2 94 85 94 91 91 93

3 81 74 79 89 81 72

4 90 80 82 86 86 81

5 90 78 92 92 85 94

6 84 84 93 90 87 88

Seed storage potential

Germination, accelerated ageing, saturated salt accelerated ageing and field seedling

emergence from six soybean seed lots, cv. BRS 184, before storage and germination and

seedling emergence from seeds stored for four months (Yaguchi et al., 2014)

Seedling emergence

Cultivar Traditional AA Saturated Salt AA

BRS 184 0.839** 0.875**

M-Soy 7908 RR 0.588** 0.670**

Both cultivars 0.702** 0.779**

Coefficient of correlation (r) of data from seedling emergence, TAA, and

SSAA from two soybean cultivars, each represented for six seed lots

stored for four months (Yagushi et al., 2014)

FINAL COMMENTS

In comparison to standard germination (SG)

results of the same seed lot prior to aging:

a) The AA germination may be similar to SG:

the seed lot will be identified as HIGH VIGOUR

b) The AA germination may be lower to SG:

the seed lot will be identified as MEDIUM to

LOW VIGOUR

FINAL COMMENTS

The larger the difference between the SG test

and the AA results, the lower the seed vigour

level of that sample

Seeds that perform as well under the AA stress

conditions as under the optimum conditions of

the SG test are expected to perform well at a

wide range of field and storage conditions

THANK YOU FOR

YOUR ATTENTION

E-mail: [email protected]

Phone: 55 (19) 3429-4220

accelerated ageing vigour test...the test was first developed by delouche (1965) to estimate the...

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