Electrical conductivity test: physiological basis and application HULYA ILBI EGE UNIVERSITY, IZMIR, TURKEY

Electrical conductivity test: physiological basis and application HULYA ILBI EGE UNIVERSITY, IZMIR, TURKEY

OUTLINES Background to why the test was initially developed Solute leakage and vigour Causes of differences in solute leakage Electrical conductivity test Method Critical points Relationship to vigour Potential for other species

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. 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

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: Electrical Conductivity 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

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 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

What are the causes of differences in leakage? Seed ageing Imbibition damage

Ageing X X 80% germination Increased leakage

Imbibition damage Imbibition plus or minus testa then TZ staining Plus testa Minus testa Rapid water uptake in absence of testa Increased dead tissue Increased leakage Physical damage to membrane 1978: J. exp. Bot., 29, 1215-1229

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.. 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

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 20 o C Facilities for MC determination

Materials and conditions for conductivity test Species Containers to be used Sample size Seed moisture content Water volume Temperature Soak time 15A.1 Cicer arietinum Glycine max, Phaseolus vulgaris, Pisum sativum (garden peas only, excluding petit-pois varieties) 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 20 o C 24h 15A.2 Raphanus sativus Tubes 7-8 cm high with a diameter of 4cm 4 weighed replicates of 100 seeds No change 40ml 20 o C 17h

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 30 o C Calculate desired weight at adjusted MC : (100 - initial seed MC / 100 desired MC) x weight of subsample Seal seed in moisture-proof containers Hold at 5-10 o C for 12-18h for MC to equilibrate

Check cleanliness of flasks Prepare 4 flasks (replicates) per seed lot Select 2 out of every 10 flasks to be used e.g. 10 lots, i.e. 40 replicates; select 8 flasks Add 250 ml water to each pair of flasks selected Check conductivity it must be <5µS cm -1

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

Count 4 replicates of 50 seeds from pure seed fraction or from sub-sample with adjusted MC Weigh 4 replicates of 50 seeds / lot (0.01g) Add one replicate of seeds to each flask, swirl

Cover the flasks and hold at 20 o C for 24h

Taking the conductivity reading Check conductivity of the control flasks it must be < 5µS cm -1 Mix the leachate: Or Swirl seeds and leachate around Decant through plastic sieve Or stir with glass rod

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 -1 g -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 grain legumes Temperature (20 ± 2 o C) Timing: setting up and reading (± 15 mins)

Relationship of test results to vigour Soyabean Phaseolus r= -0.87 *** White seed coat Brown or black seed coat 1984: Seed Sci. & Technol, 12, 659-668 1986: J. agric.sci., Camb., 106, 419-425

Radish Emergence Storage potential: 12 months storage at 25 o C Seedling emergence (%) 100 90 80 70 60 y = -0.2235x + 123.32 50 all lots R ² = 0.877*** A excluding R7 R 2 = 0.856** 40 90 140 190 240 290 EC after 17 hours (µs cm -1 g -1 ) Standard germination after storage (%) 100 80 60 40 y = -0.3167x + 131.05 20 all lots R ² = 0.865*** B excluding R7 R 2 = 0.789** 0 90 140 190 240 290 EC after 17 hours (µs cm -1 g -1 ) 2014: Seed Sci. & Technol., 42, 76-86

Guidelines for assessment of risk: UK Range of conductivity values <25 μs -1 g -1 Nothing to indicate seed is unsuitable for early sowing or for sowing under adverse conditions Vigour High 25-29 μs -1 g -1 Seed may be suitable for early sowing but there is some risk of poor performance under adverse conditions 30-43 μs -1 g -1 Seed not suitable for early sowing especially under adverse conditions Low >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?

Seed structure determines whether conductivity might be applicable Embryo living Pea Endosperm dead Small living embryo Phaseolus Triticale Maize Lolium Rice Carrot 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 occur Small changes in leakage Most likely reduced viability e.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