AN EVALUATION OF GERMINATION TOLERANCES FOR CORN AND

SOYBEANS ANDand

DEVELOPMENT OF POSSIBLE TOLERANCES FOR VIGOR

HONGYU LIU AND L. O. COPELAND

1999

BACKGROUND

Factors affecting seed test results

ò Homogeneity of the seed lotò Sampling technique

ò Testing techniqueò Interpretation of results

Variation in seed testing

ò Test variation is unavoidable.

ò It is accounted for by the use of ‘Tolerances’ in the seed industry and for enforcement of labeling laws.

Definition

ToleranceTolerance----The maximum The maximum limit by which a second test limit by which a second test

result may differ from the result may differ from the first result, or first result, or labeledlabeled quality quality and still be considered not and still be considered not

significantly different.significantly different.

Objectives of study

�Evaluate the tolerances for germination in the Rules for Seed Testing by measuring the variation in germination test results.

�Measure the extent of the variation in tests for seed vigor.

�Suggest NEW tolerances needed to cover the variation in both germination and seed vigor test results.

EXPERIMENT ONE

GERMINATION TEST

VARIATION

BACKGROUND

Past Research on Tolerances for Germination

òò RodewaldRodewald in 1889. in 1889.

òò First tolerances in official Rules In 1917. First tolerances in official Rules In 1917.

ò Miles in 1960s. ò In 1963, Miles’ tolerance tables were

accepted into ISTA Rules, but not AOSA Rules.

The questions:

Are these tolerances for germination test results still valid?

Are they valid for all species, especially for corn and soybeans?

Sample sizes in germination testing

òòRodewaldRodewald in 1889. in 1889.

òòNo record of experimental evidence.No record of experimental evidence.

òòMany labs in the U.S. conduct ‘official’ Many labs in the U.S. conduct ‘official’ germination tests on fewer than 400 germination tests on fewer than 400 seeds (e.g., 200 or even 100 seeds).seeds (e.g., 200 or even 100 seeds).

Thus, the second question is

Could smaller sample sizes (fewer seeds) be used in routine germination testing without sacrificing service?

We recognize that some precision would be lost by testing fewer seeds.

RESEARCH METHODS

PART ONE

ò Conventional Germination Referee (CGR) was conducted.

òò 24 seed lots of corn and soybeans 24 seed lots of corn and soybeans were tested by 16were tested by 16--46 AOSA and 46 AOSA and SCST labs in the Midwest and Upper SCST labs in the Midwest and Upper Great Lakes Region from 1994 to Great Lakes Region from 1994 to 19961996..

PART TWOPART TWOBlind Germination Referee (BGR)Blind Germination Referee (BGR)

Five seed lots of corn and soybeans Five seed lots of corn and soybeans representing different levels of germination representing different levels of germination from the from the lowlow--70s70s to the to the midmid--90s90s were tested were tested by 25 labs.by 25 labs.

Eight 100Eight 100--seed replicates, comprising a total seed replicates, comprising a total of forty (40) 100of forty (40) 100--seed samples for each crop seed samples for each crop were tested. The 40 100were tested. The 40 100--seed samples were seed samples were anonymously labeled.anonymously labeled.

STATISTICAL CALCULATIONS

ò Correlation coefficients among reps within individual laboratories.

ò Subsets of data for different numbers of reps.

ò Standard deviation as a function of numbers of reps for each seed lot.

ò Tolerance = f × σ1 × tα (Miles’ method).

Construct subsets of data for various numbers of replications

x11Lab 1

Lab 2

Lab n

x11

x21

xn1

xmn

xn4

xij

xn4xn1

Subset with 1 rep

Subset with 2 reps

RESULTS

-1.00

-0.50

0.00

0.50

1.00

Individual laboratory

Cor

rela

tion

coef

ficie

nt

-1.00

-0.50

0.00

0.50

1.00

Individual laboratory

Co

rrel

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n c

oef

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Individual Labs

Cor

rela

tion

coef

fici

ent

0

-0.5

-1

0.5

1

-1

-0.5

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0.5

1

Individual Labs

Soybean BGR testsSoybean CGR tests

Distribution of correlation coefficient among replicates within a laboratory for germination results

Mean correlation coefficient for germination tests

Test Corn Soybean

CGR 0.37 0.36

BGR -0.01 0.03

Relationship among # of replicates and standard deviation for soybean BGR

tests conducted in 1997

0

1

2

3

4

5

6

7

8

1(94) 2(89) 3(85) 4(78) 5(73)

Seed lot (Germination %)

Sta

ndar

d de

viat

ion

One rep Two repsThree reps Four repsFive reps Six repsSeven reps Eight reps

Tolerance estimates for germination tests with four 100-seed replicates

Germina t ion S o y b e a n C o r n A O S A I S T AL e v e l B G R t e s t C G R t e s t B G R t e s t C G R t e s t R u l e s R u l e s

%99 1 1 2 1 5 298 2 2 3 2 5 397 3 3 3 3 5 396 3 3 4 3 5 495 4 4 4 4 6 494 4 4 5 4 6 493 5 5 5 5 6 592 5 5 5 5 6 591 5 6 6 6 6 590 6 6 6 6 6 6

Tolerance = f × σ1 × tα (Miles’ method)

Tolerance estimates for germination tests with four 100-seed replicates

Germination Soybean Corn AOSA ISTALevel BGR test CGR test BGR test CGR test Rules Rules

%89 6 7 6 7 7 688 6 7 6 7 7 687 7 8 7 7 7 686 7 8 7 8 7 785 7 8 7 8 7 784 8 9 7 9 7 783 8 9 8 9 7 782 8 10 8 10 7 781 9 10 8 10 7 880 9 10 8 11 7 8

Tolerance = f × σ1 × tα (Miles’ method)

CONCLUSIONS

èA positive inter-replicate bias existed in CGR tests, but not in BGR tests, for both corn and soybean.

The Implication: Replicates were not independent of each other in the CGR tests.

èStandard deviation decreased with the increase in germination level and the number of replicates.

èThe big decrease of the standard deviation was observed when number of replicates went up from 1 to 2.

èFor germination above 90%, the tolerances in the AOSA Rules may be too wide.

èFor germination below 85%, the tolerances in both Rules may be too restrictive.

EXPERIMENT TWO

TOLERANCES for VIGOR

BACKGROUND

WHY VIGOR TEST?ò WG tests are successful in predicting

seed quality in at least two aspects: high level of repeatability & information about the potential germination of seed lot under optimum conditions.

ò The major problem: inability to detect potential differences in performance among higher germinating seed lots under adverse conditions.

VIGOR TEST METHODS

ò Many methods for testing vigor were developed in the past 40 years, although only very few are in international usage.

ò None of them is considered or applied as a standard method.

WHAT ARE THE PROBLEMS FOR VIGOR TESTS

“Test repeatability, sensitivity, relevance, and a lack of

understanding of the variables and assumptions involved”

(Handbook of vigor test methods by Hampton, 1995).

Conventional Wisdom about vigor tests

uVigor test methodology is not as standardized as germination.

uThus, vigor test results are not as repeatable as those for germination.

uConsequently, tolerances are not appropriate for comparing vigor test results.

COLD TEST

ò Routinely conducted on almost all corn and much of the soybean seed sold in the United States.

ò The test result is not officially labeled on the seed offered for sale.

OBJECTIVES

¶ to determine the extent of the variability among cold test results of corn and soybean seeds in routine tests;

· to compare the variation in the cold test vs. the WG test; and

¸ to develop suggested tolerances if possible.

MATERIALS AND METHODS

ò Three year data (1993-95) of corn and soybean cold referee test results were collected from the AOSA/SCST.

ò From tests conducted in 11-51 labs and on 13-14 different seed lots.

STATISTICAL ANALYSES

ò Standard deviation for each lot and the data distribution.

ò Subsets of data for different number of replications (constructed the same way as for germination test study).

ò Standard deviation with different numbers of replications for each seed lot.

ò Tolerance = f × σ1 × tα (Miles’ method).

Germination of Seed Lots Used

u Conventional germination referee (CGR)– Corn - 23 seed lots germinating 93-98% and one (1)

seed lot germinating 81%.– Soybean - 23 seed lots germinating 88-97% and one

(1) seed lot germinating 69%.

u Blind germination referee (BGR)– Corn - 5 seed lots germinating 71, 82, 86, 90 & 94%.– Soybean - 5 seed lots - 74, 80, 85, 91 & 95%.

RESULTS

Percentage of data distribution of test results, compared with warm germination (WG) test results

Corn SoybeanCold Test WG Cold Test WG

Data range

50-seed 100-seed 50-seed 100-seed

± 1σ 49.1 20.8 35.4 15.7 9.0 30.5± 2σ 76.4 45.4 61.1 29.9 25.3 56.8± 3σ 87.3 60.8 82.4 48.8 34.3 75.7± 4σ 96.4 76.2 92.2 65.4 48.8 87.4± 5σ 98.2 84.6 95.8 74.8 60.2 94.8± 6σ 98.2 90.0 97.8 84.3 70.5 96.3± 7σ 98.2 93.8 98.5 90.6 79.5 97.4± 10σ 100.0 96.9 100.0 100.0 90.4 100.0

Relationship among number of 50-seed replicates and standard

deviation for corn cold vigor tests

0

2

4

6

8

10

12

14

1(95) 2(93) 3(91) 4(90) 5(59)Seed lot (Vigor level %)

Sta

ndard

devi

atio

n

One repTwo repsThree repsFour reps

Tolerance comparisonsTolerance forGermination

or vigor level Corn cold test(four 50-seedreplicate test)

Warm germination test (four 100-seedreplicate test) from or in

(%) CGR AOSA Rules ISTA Rules

99 1 1 5 298 2 2 5 397 2 3 5 396 3 3 5 495 4 4 6 494 4 4 6 493 5 5 6 592 6 5 6 591 6 6 6 590 7 6 6 6

Tolerance = f × σ1 × tα (Miles’ method)

Tolerance comparisons

Tolerance = f × σ1 × tα (Miles’ method)

Tolerance forGerminationor vigor level Corn cold test

(four 50-seedreplicate test)

Warm germination test (four 100-seed replicatetest) from or in

(%) BGR CGR AOSA ISTA

99 1 2 1 5 298 2 3 2 5 397 2 4 3 5 396 3 4 3 5 495 4 4 4 6 494 4 5 4 6 493 5 5 5 6 592 6 6 5 6 591 6 6 6 6 590 7 6 6 6 6

CONCLUSIONS

èThe cold test accuracy for corn was much better than that for soybean. Variation in soybean cold test results was at a point where we believe it’s not reasonable to suggest tolerances.

èVariation for 50-seed corn cold tests was equal to or less than that for warm germination test.

èBased on this study, a four 50-seed replicate cold test is suggested for corn, along with its tolerances for vigor levels of 90% or above.

èThe cold test for soybean was considered to be far from standardized based on the variation we observed. However, the status of the standardization was much better for corn.