Electrical conductivity test: physiological basis and

applicationHULYA ILBI

EGE UNIVERSITY, IZMIR, TURKEY

OUTLINES

Background to why the test was initially developed

Solute leakage and vigour

Causes of differences in solute leakageElectrical conductivity testMethodCritical pointsRelationship to vigour

Potential for other species

Background to test development

1960’s, UK◦ Market for frozen garden peas◦ Demand for regular sowing to achieve regular timed harvests◦ Required early sowings in wet cold conditions◦ Poory emergence of high germination lots at early sowings◦ No problems at later sowings

Highlighted the vigour problem in peas

Observation: Low vigour seeds- pre-emergence mortality due to infection by Pythium spp

1968; Plant Pathology, 17, 11-17 and Proc. Int. Seed Test. Assoc., 32, 553-563.1970: Horticultural Research, 10, 50-58; 1971: Ann. Appl. Biol., 68, 177-83.

Why was there greater incidence of fungal infection ?• Observation of differences in solute leakage from high and low

vigour lots • High vigour: low leakage; • Low vigour: high leakage

• What leaks out?• Amino acids, sugars, ions (e.g. K+)

• Leakage occurs from dead tissue• Sugars act as food source for fungi• Leakage plus dead tissue influences susceptibility to fungal

infection

Method for detecting solute leakage: ElectricalConductivity

K+ major constituent of leakage

K+ determines electrical conductivity of seed soak water

Conductivity reveals total solute leakage

Solute Leakage associated with vigour

High vigour seeds◦ Low leachate conductivity◦ Low predisposition to infection by fungi◦ Good emergence even in poor conditions

Low vigour seeds◦ High leachate conductivity◦ Greater predisposition to infection by fungi◦ Good emergence in non-stressful conditions ◦ Fungal infection and poor emergence in stressful conditions

Electrical Conductivity Test as vigour test

Pisum sativum: garden pea

Glycine max: soybean

Phaseolus vulgaris: green / common / snap / garden bean Cicer arietinum:Chickpea

Raphanus sativus:Radish

1984: Seed Sci. & Technol., 12, 659-668; 1986: J. agric. Sci. Camb., 106, 419-425 ; 1991: J. agric. Sci., Camb. 116, 259-264; 2014:Seed Sci. & Technol., 42, 76-86

What are the causes of differences in leakage?

Seed ageing

Imbibition damage

Ageing

80% germination

Increased leakage

XX

Imbibition damage

•Rapid water uptake in absence of testa•Increased dead tissue•Increased leakage•Physical damage to membrane

Plus testa

Minus testa

Imbibition plus or minus testa then TZ staining

1978: J. exp. Bot., 29, 1215-1229

Why does rapid water uptake occur in intact seeds?

• Cracks in the seed coat - peas, soybean• More rapid water uptake

• Imbibition damage reduced vigour

• Loosely adhering seed coat• white seeded cultivars of green bean, chickpea, cowpea, longbean• white seeded cultivars have higher EC than coloured.• chickpea – white seeded Kabuli type EC > coloured Desi type EC

1979: J. exp. Bot., 30, 193-197; 1980: J. agr. Sci. Camb., 95, 35-38; also see 2006: review Seed vigour and its assessment in Handbook of Seed Science and Technology, Haworth Press. .

Electrical conductivity (EC) test • Measures differences in solute leakage•EC validated and in ISTA Rules for:

• Grain legumes• garden pea, soybean, Phaseolus vulgaris, chickpea

• Radish

•EC test shown to be:• repeatable and reproducible• related to an expression of vigour

•Basic method the same, but specific differences

Materials required for EC test•Conductivity meter: cell constant = 1.0•Water: deionised or distilled, < 5 µS cm-1

•Flasks / beakers / tubes of specified size•Precision balance for weighing seeds•Facilities to maintain 20oC•Facilities for MC determination

Species Containers to be used

Sample size

Seed moisture content

Water volume

Temperature Soak time

15A.1Cicer arietinumGlycine max, Phaseolus vulgaris, Pisum sativum(garden peas only, excluding petit-poisvarieties)

Erlenmeyer flasks or beakers, capacity 400-500ml with a base diameter of 80 mm (±5mm)

4 weighed replicates of 50 seeds

Adjust to 10 – 14%

250 ml 20oC 24h

15A.2Raphanus sativus Tubes 7-8 cm high

with a diameter of 4cm

4 weighed replicates of 100 seeds

No change 40ml 20oC 17h

Materials and conditions for conductivity test

Preparation for the test •Calibrate electrode / conductivity cell (K = 1.0)

• Use standard solutions or 0.1M KCl

•Prepare seed material• MC must be 10-14%; adjust if necessary

• Raise MC in moist cloths• Reduce MC at 30o C• Calculate desired weight at adjusted MC : • (100 - initial seed MC / 100 – desired MC) x weight of sub-

sample

•Seal seed in moisture-proof containers

•Hold at 5-10oC for 12-18h for MC to equilibrate

Add 250 ml water to each pair of flasks selected

Check conductivity – it must be

Setting up the test• Add 250ml water to all flasks; include 2 controls per test run• Cover flasks• Leave 18-24h at 20oC

Weigh 4 replicates of 50 seeds / lot (0.01g)

Count 4 replicates of 50 seeds from pure seed fraction or from sub-sample with adjusted MC

Add one replicate of seeds to each flask, swirl

Cover the flasks and hold at 20oC for 24h

• Check conductivity of the control flasks it must be < 5µS cm-1

Taking the conductivity reading

• Mix the leachate:

Swirl seeds and leachate around Decant through plastic sieveOr stir with glass rod

Or

Take reading; do not place electrode on peas

Wash electrode between readings

Calculation of results

Subtract reading of control

Divide each reading by seed weight

Mean of replicates = conductivity cm-1g-1

Are the 4 replicates in tolerance?◦ If not, repeat the test!

Critical aspects of the test

•Calibration of meter (K = 1)•Water quality (≤ 5 μS cm-1)•Cleanliness•Seed moisture content (10.0-14.0%) for grainlegumes

•Temperature (20 ± 2oC)•Timing: setting up and reading (± 15 mins)

Soyabean

Relationship of test results to vigourPhaseolus

White seed coat Brown or black seed coat

1984: Seed Sci. & Technol, 12, 659-668 1986: J. agric.Sci., Camb., 106, 419-425

r= -0.87***

all lots R ² = 0.877***excluding R7 R 2 = 0.856**

y = -0.2235x + 123.32

40

50

60

70

80

90

100

90 140 190 240 290

Seed

ling

emer

genc

e (%

)

EC after 17 hours (µS cm-1 g-1)

A

Radish

Emergence

2014: Seed Sci. & Technol., 42, 76-86

Storage potential: 12 months storage at 25oC

all lots R ² = 0.865***excluding R7 R 2 = 0.789**

y = -0.3167x + 131.05

0

20

40

60

80

100

90 140 190 240 290

Stan

dard

ger

min

atio

n af

ter s

tora

ge (%

)

EC after 17 hours (µS cm-1 g-1)

B

Grafik1

193

98

117

144

172

181

290

241

116

Module

EC after 17 hours (µS cm-1 g-1)

Seedling emergence (%)

A

all lots R ² = 0.877***excluding R7 R 2 = 0.856**

y = -0.2235x + 123.32

80

96

95

94

90

90

50

74

94

İstatistik

Başlangıç çimlenmeKontrollü bozulma

İnitial germSowing 1Sowing 2CD germination

Toplam% NormalMGT saatNormal çıkMETNormal çıkMETToplam% NormalFirst countEC 17EC 24

949447886268102.3806812197.1241

84824588638897603212189.5235

88864568638094.9844824193.3195

94924581637998803216224

100100389661848998929699.8108.2

100100391005910084.592789296.9112.7

1009839100619686.494809498.4106.2

100983999609387988698109

1001004196589286643652120137.8

1001004296559680.6604056117.4123.1

1001004296589282765636114.7139.7

1001004296579383402848134

98944296598878.3766668144.3171.3

92923696599285847682143.9172.4

96923696619678.7826676143.4171.6

100963596609281867280172

94905688719281.4403628173.6185.6

94906088789296383018171.2185.1

96947196918497.1443420172.4196.9

98965491808992443618189

98926196798899.1502432193.7194.1

96905592788493.8542442168205.6

98946092798896521434180.8214.5

98966593798796501434205

88741104018848155.3626281.2325

96801063616448152.614210298.9316

96861235616568159.6422290319

907612744172551561046320

989666688984116261624240.8251

969473728376115.4261214241261

989267649076121.1201420240.9259

969273688779118341826265

100984592589288.7927684121117.7

100984396649688.3766064111.3120.3

1001004096648891.8725668116.2112.2

1001004095629290604456117

Makale için hazırlananlar

Table 2

Normal seedling emergence (%)

Sowing 1Sowing 2EC

MeanSeed lotsEmergenceMETEmergenceMET17 h24 h

80R181637998193224

96R29960938798109

95R396579383117134

94R496609281144172

90R591808992172189

90R693798796181205

50R74417255156290320

74R8688779118241257

94R995629290116117

table 3

 Normal seedling emergence (%)

ModuleFieldStorageEC

Seed lotsMETEmergenceMETEmergence(12 months)17 h

R357 a96 b83 ab93 a93117

R260 a99 a87 bc93 a9198

R962 a95 b90 c92 a93116

R460 a96 b81 a92 a87144

R887 c68 e118 f79 b58241

R580 b91 c92 cd89 a72172

R679 b93 b96 de87 ab84181

R163 a81 d98 e79 b82193

R7172 d44 f156 g55 c28290

Seed lotsProduction yearS.m.c. (%)NormalAbnormalMGTECEC

17 h24 hDead+ab

R320085.11000 b421171340

R220085.7990 b39981091

R920085.4991 b421161171

R420085.1943 b371441726

R820085.7943 b702412576

R520085933 b601721897

R620085.1935 b601812057

R120085.5891 b4619322411

R720075.37914 a11729032021

MGTCDEC

(d)% TG17 h

399698

4275116

4260117

3782144

6042172

6052181

4676193

7027241

1179290

Makale için hazırlananlar

Module

EC after 17 h (µScm-1g-1)

Seedling emergence (%)

y = -0,2235x + 123,32

Field

EC at 17 h (µScm-1g-1)

EC 17 h

EC after 17 h (µScm-1g-1)

Standart germination after storage (%)

y = -0,3167x + 131,05

Dead+Abnormal (%)

EC 17 h (µScm-1g-1)

Normal (%)

EC 17 h (µScm-1g-1)

EC at 17 h (µScm-1g-1)

MGT (h)

y = 0,3582x - 4,78

% Total germination after CD

MGT (h)

B

y = -0,8038x + 103,35 all lots R² = 0,800minus R7 R2 = 0,838

% Total germination after CD

EC at 17 h (µScm-1g-1)

A

y = -1,9303x + 283,76all lots R² = 0,741**minus R7 R2 = 0,558

Guidelines for assessment of risk: UK Range of conductivity

valuesVigour

43 μS-1 g-1 Seed not suitable for sowing -

•Why does EC work for grain legumes and radish? •Would it work for other species?

Maize

Rice Carrot

Triticale Lolium

Pea Phaseolus

Embryo – living

Endosperm – deadSmall living embryo

Seed structure determines whether conductivity might be applicable

Diagrams from Tetrazolium Handbook

Large living cotyledons• Reduced living tissue reflected in leakage from whole seed e.g. grain legumes, radish

• EC a useful test

Small living embryo (large dead endosperm)• When changes in living tissue occurSmall changes in leakageMost likely reduced viabilitye.g rice, maize, grass species, carrot, lettuce ……

• EC not appropriate

But: Potential for application to small seeded vegetable species

• Radish (Raphanus sativus) In ISTA Rules • Relatively large, living cotyledons – like grain legumes • Potential for other Brassicaceae

To Conclude:

Conductivity as a vigour test is:◦ Quick◦ Easy◦ Repeatable◦ Applies to grain legumes and radish◦ Has potential for other Brassiceae

Thanks for your attention

Electrical conductivity test: physiological basis and applicationSlayt Numarası 2Background to test developmentWhy was there greater incidence of fungal infection ?�Method for detecting solute leakage: Electrical Conductivity Solute Leakage associated with vigourElectrical Conductivity Test as vigour testWhat are the causes of differences in leakage? Ageing Imbibition damageWhy does rapid water uptake occur in intact seeds?Electrical conductivity (EC) test Materials required for EC testSlayt Numarası 14Preparation for the test Add 250 ml water to each pair of flasks selectedSetting up the testWeigh 4 replicates of �50 seeds / lot (0.01g)�Cover the flasks and hold at 20oC for 24h�Check conductivity of the control flasks �it must be < 5S cm-1Take reading; do not place electrode on peasCalculation of resultsCritical aspects of the testSlayt Numarası 24Slayt Numarası 25Guidelines for assessment of risk: UK Slayt Numarası 27Slayt Numarası 28Slayt Numarası 29�But: Potential for application to small seeded vegetable species�Slayt Numarası 31Slayt Numarası 32