2004 – item 3a...enprima 0.33 84.8 8.5 17 delft 0.35 90.8 6.9 10 miracle 0.38 88.8 0.9 12...

S U P P O R T I N G

D A T A A N D E V I D E N C E

F O R T H E P R O P O S E D R U L E S

C H A N G E S 2 0 0 4 –

I T E M 3 A

FOR INFORMATION AND CONSIDERATION AT THE ORDINARY MEETING 2004

SUPPORTING DATA AND EVIDENCE FOR THE PROPOSED RULES CHANGES 2004 - ITEM 3A

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SUPPORTING DATA AND EVIDENCE FOR PROPOSED RULES CHANGES - ITEM 3A: CHANGES TO THE UNIFORM BLOWING METHOD FOR POA PRATENSIS VARIETIES

PURITY ANALYSIS OF SMALL SEEDED VARIETIES OF POA PRATENSIS

TABLE OF CONTENTS 0. Summary 2 1. Introduction 3 2. Preliminary work before 1998 4 3. Working group 1998-2002 6 4. Working group 2003-2004 8 5. Conclusions 13 6. Proposed Rules Change 2004 13 7. Literature 15 8. Acknowledgements 15 9. Annex 1: Statistical analyses blower speeds in

purity assessments of Poa pratensis 16


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0. Summary

In 1994 the ISTA Purity Committee received information that seed companies had problems with the purity test of small seeded varieties of Poa pratensis. Internal work in seed companies in the early 1990s had revealed that in the purity test of small seeded varieties of Poa pratensis many seeds with caryopsis were blown into the light fraction, and classified as inert matter according to the ISTA Rules.

Work carried out by the ISTA Working Group on blowing in the late 1990s indicated that the percentage of pure seed in the light fraction was 65% (average of 10 small seeded varieties). If the blower setting was lowered to 0.90, the light fraction still contained more than 40% of pure seeds.

The subject was discussed at the ISTA Extra Ordinary Meetings in 2002 and in 2003. The discussion in 2003 revealed that several items needed to be evaluated further, and it was decided to start a new series of experiments. Laboratories from Denmark, the Netherlands and the USA were involved. This report summarises the results from the previous years and present the results received from Denmark and the Netherlands in 2003. USA and Canada (Representing ISTA and AOSA) participated in planning of the new experiment and in discussion of the results.

When accepting the factor 0.82 for Poa pratensis in 1992 the results of the blowing by this factor were compared with the hand method. For that reason it was decided that the main principle for accepting a new blowing method in the ISTA Rules should be the comparison of the new blowing factor with the hand method (based on PSD41 in the ISTA Rules).

If the amount of particles classified wrongly is the same for both the heavy and the light fractions, it is assumed that the result of the hand method will be the same as the result obtained by means of the blower. The equivalence in number of misplaced seeds in the heavy and the light fraction is at present used when calibrating the seed blowers with the ISTA calibration samples.

In the examinations in 2003 participated 5 seed testing laboratories. In total 32 samples of 10 small seeded varieties were blown at the blower settings 1.00, 0.90 (only 22 samples), 0.82, 0.70, and 0.60.

The results of the tests revealed that: • It was possible to obtain similar results when identical samples are blown with

different blower settings at four seed laboratories. • Identical results were obtained when seed samples of five varieties with different

1000 seed weight were re-analysed at the same seed laboratory. • Due to variable results between samples of the same variety and between varieties it

is concluded that the pure live seed (PLS) is not suited for selection of the blower setting of Poa pratensis.

• The equivalence of g misplaced seeds in the heavy and in the light fraction provided uniform results for all small seeded varieties tested (1000 seed weight from 0.22-0.34 g).

• The factor 0.82 was the optimal blower setting, and it represents the best possible compromise between the increase in % pure seed and the decrease in % germination, when the blower settings are reduced from 1.00 to 0.60.

• The factor 0.82 has already been in use for Poa trivialis at the ISTA seed laboratories for more than 10 years.

• It is, accordingly, proposed that the factor 0.82 should be included in the ISTA Rules for blowing of all varieties of Poa pratensis with a 1000 seed weight <0.35 g.


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1. Introduction Breeding of Poa pratensis has diverted in two directions. One direction is aiming at high yielding varieties for forage purposes. Another direction is aiming at varieties for amenity purposes, leading to finer leafed types that grow slower.

It appears that leaf size and plant growth are correlated with seed size.

As a consequence forage types are getting heavier seeds, and amenity types are moving towards smaller and lighter seeds.

In the 1980s new small seeded varieties of Poa pratensis have entered the markets in Europe. Presently seed companies are already testing varieties having a 1000 seed weight as low as 0.19g!

The low seed yield of these varieties is compensated by a premium for good genetic quality. In the cleaning plant it appeared that the purities requested in the market could only be reached when heavy seed losses during cleaning were accepted.

Internal work in seed companies in the early 1990s revealed that of these low 1000 seed weight varieties many seeds with caryopsis (‘pure seed’) are blown into the light fraction, and classified as inert matter according to the ISTA Rules. Seed yields of these varieties in terms of kilograms per hectare are low as well.

As a consequence of this, good varieties have not reached the market, because on top of an already low yield of raw material too many valuable seeds had to be cleaned out in order to reach the purity level as assessed by the present purity test.

In 1994 the ISTA Purity Committee received information that seed companies had problems with the purity test of small seeded varieties of Poa pratensis. Evidence was presented indicating that when samples of such varieties were blown on blowers calibrated with the ISTA calibration sample, a considerable amount of pure seeds were blown into the light fraction and accordingly considered as inert matter.

In 1996 a questionnaire regarding problems with purity analysis of small seeded varieties of Poa pratensis was sent out to Danish and Dutch seed companies. The replies were that they had problems with in total 15 small seeded varieties of Poa pratensis.

The received information lead to some activity late 1996, when Hans Arne Jensen (chairman of the ISTA Purity committee), Danish Plant Directorate sent a draft working plan to

R. Minnaar of NAK in the Netherlands on how to identify the correct blower setting for such varieties. A student did some preliminary work, but this was never published.

Due to change of personnel, not much happened during the following year or two. After the ISTA Congress in 1998 in South Africa, when the results of the preliminary work was presented by Harry Nijënstein during the meeting of the ISTA Purity Committee, a working group started some activities in the field of this subject. Leadership of the working group was taken over by Anny van Pijlen of NAK.

Anny van Pijlen sent an enquiry to all members of the purity working group: Ken Allison (Canada), Sharon Davidson (USA), Ola Kristian Dille (Norway), Hans Arne Jensen (Denmark), Maria Rosaria Mannino (France), Harry Nijënstein (the Netherlands), Arnold Larsen (USA), Elzbieta Malluzynska (Poland).

The question asked was: “Do you have any problems with purity analysis of small seeded varieties of Poa pratensis? And what actions did you take?”


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Table 1.1: The results of the enquiry of 1998. Country Answer The Netherlands Some private companies have problems Norway No problems Denmark Some private companies have problems Poland No problems France No problems USA No problems

The reasons for not having problems in some countries are probably twofold: In some countries little or no production of Poa pratensis takes place (Norway, France, Poland). In the USA seed companies did not produce many small seeded varieties at that time.

However, because it was quite clear that seeds were classified wrongly in some important growing areas for Poa pratensis, it was decided to continue the work.

The results of the working group were available in 2001. However, due to communicational problems, these data were never presented to the purity committee and ISTA meetings in time for change of the ISTA Rules.

The problems with purity tests of small seed varieties and proposals for change of the ISTA Rules were discussed at ISTA Extraordinary Meetings in 2002 and in 2003.

During the ISTA EOM in Zürich, Switzerland the latest results of the working group were presented. As it became obvious that the proposed factor 0.90 did not solve the problem and number of new questions were asked and it was realized that harmonisation with the AOSA Rules should be examined, a new series of experiments were set up.

This paper wraps up activities and results from: Before1998, from 1998-2002, and from 2003-2004.

2. Preliminary work before 1998 In the 1940s some research on climax blowing point has been carried out. Leggett (1941) suggested ‘that considerations should be given to a redefinition of pure seed according to a standard synthetic sample prepared to conform to the “climax” blowing point.

However, there is no proof that these discussions have lead to adoption of this principle for acceptance of new methods for the ISTA Rules. On the contrary: the paper of Jensen & Bülow-Olsen (1992) indicates that for adopting the Poa trivialis blower settings in the ISTA Rules, the results of the blower were compared with those of the hand method.


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The entire purity chapter of the ISTA Rules is based on pure seed definitions. Purity determinations of other Poa species like Poa secunda/ampla, Poa annua, Poa compressa, Poa nemoralis, Poa palustris are based on Pure Seed Definition (PSD) 41:

“ Spikelet, with lemma and palea enclosing a caryopsis, with or without awn, plus attached sterile floret. Floret, with lemma and palea enclosing a caryopsis, with or without awn. Caryopsis. Piece of caryopsis larger than one-half the original size. “

In the present experiments PSD41 is used for classifying seeds.

In the early 1990s, when seed companies encountered problems in reaching the requested purities for small seeded Poa pratensis, these companies did several experiments in order to find the cause for these problems. This paragraph summarises the results of these experiments.

Analyses of small seeded varieties of Poa pratensis revealed that a considerable percentage of pure seeds (according to PSD 41) were blown into the light fraction (see Table 2.1).

Table 2.1: Experiment carried out by Cebeco Seeds in 1994. Germination percentage of pure seeds (PSD 41) from light fraction. Results are averages of two seed lots per variety, blown at 2*0.5g.

Variety Weight of 1000 seeds

Purity % Pure seed in light fraction

% Germination of pure seed in light

Limousine 0.23 87.9 12.5 51 Ampellia 0.29 86.9 9.2 21

Julia 0.29 86.6 7.9 15 Enprima 0.33 84.8 8.5 17

Delft 0.35 90.8 6.9 10 Miracle 0.38 88.8 0.9 12

LSD (p<0.05) 5.9 7

The percentage of pure seeds in the light fraction is considerable for all varieties showing a weight of 1000 seeds of 0.35g or lower.

The germination percentages of the pure seed in the light fraction show the same pattern as the pure seed percentages in the light fraction: higher germination percentages for varieties with low weight of 1000 seeds.

The rather low purity percentages reflect the market situation in Europe in the late 1980s and early 1990s. Presently the purity levels are much higher (see Table 4.1).

Work done in another company in 1995 confirmed the earlier results (see Table 2.2). Instead of germinating only the pure seeds from the light fraction, now all ‘seeds’ (containing either caryopsis or being empty) from the entire light fraction were germinated.

As expected, germination levels were lower compared to those in Table 2.1, but a similar trend was obtained, because a considerable part of the light fraction consisted of seeds able to germinate.

The germination percentage reflects the numbers of pure seeds in the light fraction.


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Table 2.2 Average seed weights, purity and germination percentages of seeds from the light fraction of eight varieties. Experiments by Mommersteeg in 1995, 2*0.5g.

Variety Number of samples

Weight of 1000 seeds

Purity Germination of total light

fraction

Variation in germ. light

fraction

Germination heavy

fraction Monopoly 19 0.26 91.3 9.9 05 – 20 92.4 Parade 10 0.33 91.3 3.4 01 – 07 89.8 Cynthia 14 0.33 93.5 18.2 08 – 30 92.6 Asset 11 0.36 90.8 10.1 02 – 23 88.8

Geronimo 25 0.37 92.5 16.2 03 – 36 94.0 Saskia 13 0.37 94.1 5.9 01 – 16 86.2 Minstrel 10 0.39 92.5 2.3 00 – 06 85.2 Cocktail 12 0.43 91.7 1.3 01 - 04 83.1

In a third experiment the relation between purity and germination of Poa pratensis varieties was investigated (Table 2.3).

Although the purity is increased by 3.2% on average at a lower blower setting, germination percentage is hardly influenced.

The percentage PLS increases from 79% to 81% when the blower setting is increased.

Table 2.3 Relation between purity and germination percentages of 6 varieties, blown at 4 different blower settings at 2*0.5g. Results are averages of two samples per variety. Blower setting figures 2.9

3.0

3.1

3.2

Variety Weight of 1000 seeds

Purity Germ. Purity Germ. Purity Germ Purity Germ

Limousine 0.23 94.2 86 91.6 87 88.6 89 88.3 87 Ampellia 0.29 89.6 88 88.2 88 86.9 89 85.8 90

Julia 0.29 91.6 85 90.5 86 89.5 86 88.8 86 Enprima 0.33 91.0 88 89.8 88 88.9 88 88.3 88

Delft 0.35 92.9 87 91.6 86 90.7 89 89.9 91 Miracle 0.38 97.4 90 96.9 87 96.6 87 96.2 87 average 92.8 87 91.4 87 90.2 88 89.6 88 % PLS 81 80 79 79

3. Working group 1998 – 2002 The ISTA working group for blowing consisted of members from the following countries: Canada, USA, Norway, Denmark, France, the Netherlands and Poland.

The results in the following tables are the averages of work carried out by five laboratories: three ISTA accredited laboratories in three countries (UK, Denmark, the Netherlands), and two company laboratories from the Netherlands.


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Table 3.1. Percentage of pure seed blown into the light fraction and percentages of empty seeds blown in the heavy fraction. Per variety on sample was subdivided in five sub-samples, and send to five laboratories: three ISTA accredited laboratories in three countries (UK, Denmark, the Netherlands), and two company laboratories from the Netherlands. Each lab blew the samples at 1*0.5g (submitted sample).

Weight of

Blower setting

0.90

factor

0.90

Blower setting

1.00

factor

1.00

Variety 1000 seeds

Pure in light Empty in heavy

Pure in light Empty in heavy

Unique NL 0.20 22.1 12.2 36.5 14.4 Unique USA 0.26 34.5 0.1 72.8 0.0

Barblue 0.26 57.9 5.3 63.2 6.8 Barmax 0.26 83.3 4.0 92.5 8.1

Limousine 0.26 27.5 4.2 42.5 6.1 Washington 0.28 55.4 0.2 86.6 0.2 Monopoly 0.29 16.7 0.1 84.8 0.0

Julia 0.31 51.4 0.0 68.5 0.0 Midnight 0.33 69.5 0.1 74.3 0.2

Viva 0.47 0.0 0.6 29.0 0.6 average 0.29 41.8 2.7 65.1 3.6

At normal blower settings (1.00) the light fraction contained on average 65.1% of pure seeds. At a blower setting of 0.90 of the normal setting for Poa pratensis, the light fraction still contains 41.8% of pure seeds (Table 3.1.). In the average figures also the heavy variety number 2 (1000 seed weight=0.47g) is included. Averages for pure seeds blown into the light fraction of small seeded varieties only would even be higher.

Although a blower setting of 0.90 improves the situation considerably, still far too many good seeds are blown into the light fraction and valued as inert matter.

Table 3.2. Percentage germination of pure seeds after blowing at two different settings. Per variety on sample was subdivided in five sub-samples, and send to five laboratories: three ISTA accredited laboratories in three countries (UK, Denmark, the Netherlands), and two company laboratories from the Netherlands. Each lab blew the samples at 1*0.5g (submitted sample).

Weight of blower setting 0.90 blower setting 1.00 Variety 1000 seeds. % germination % germination

Unique NL 0.20 66 69 Unique USA 0.26 83 82

Barblue 0.26 69 78 Barmax 0.26 83 84

Limousine 0.26 87 89 Washington 0.28 90 92 Monopoly 0.29 95 93

Julia 0.31 89 89 Midnight 0.33 88 78

Viva 0.47 92 92 average 0.29 84 85


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In another experiment the effect of blower settings on the germination level was examined. On average hardly any difference exist between the two blower settings. Varieties number 5 and 4 deviate. The reason for this is not known (Table 3.2.).

In the enquiry of 1996, 15 varieties of Poa pratensis in Europe with a weight of 1000 seeds lower than 0.35g were reported to provide problems with the purity tests. In 2003 this number in Europe had increased to 57 (Table 3.3.), and at present more than half of the varieties in Europe belongs to the group of small seeded varieties.

Apart from the number of small seeded varieties, also the weight of 1000 seeds of newly breed varieties appears to decrease.

In Europe the weight of 1000 seeds in new varieties tested now are as low as 0.19g.

Table 3.3. Number of normal (=>0.35g)and of small seeded (<0.35g) varieties that were identified in a survey in 2003. The Europe figures are based on information from DLF Trifolium, Barenbrug and Cebeco Seeds. Varieties

1000 seed Varieties

1000 seed

weight >=0.35g weight <0.35g Origin number % number % Europe 29 34 57 66

USA 20 70 9 31 total 49 43 66 57

Unfortunately the results in Table 3.1, 3.2 and 3.3 were not presented to ISTA before the ISTA EOM of 2003.

4. Working group 2003-2004

As a follow up on the discussions during the ISTA EOM of 2003 it was decided to establish a new working group. The following laboratories assisted in the work carried out, and presented in table 4.1: Danish Plant Directorate (Denmark), DLF (Denmark), NAK (Netherlands), Advanta Seeds (Netherlands), Cebeco Seeds (Netherlands).

USA and Canada (representing ISTA and AOSA) have also been involved in planning and discussion of these experiments.

The Standard Operating Procedure (SOP) was distributed as a draft in July, 2003. After some discussions the final SOP was distributed on August 8th. (Major changes in the final version, compared to the first draft are underlined; See annex 1):

• Test one replicate of 1 gram (instead of two reps of 0.5 gram), which allowed for calculation of intra-lab variation.

• Splitting al fractions into pure seed and inert matter (based on PSD41) was made optional instead of obligatory, because of the amount of work involved.

• Include blower setting of 0.90 (was left out in the preliminary draft, because it was proven by the first working group that this blower setting could not solve the problem completely).

As some laboratories started working using the draft-SOP, not all have used one replicate of 1g. Some have used two replicates of 0.5g. This is indicated in the bottom part of the table.


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The aim of the new experiment was to elucidate:

• Can similar results be obtained when testing seeds from same seed sample in different seed laboratories?

• Can similar results be obtained when re-testing seed samples of different varieties in the same seed laboratory?

• Which blower settings provide both optimal and uniform results when blowing small seeded varieties of Poa pratensis?

• Can all small seeded varieties be blown at the same blowing point or is it necessary to have f. inst. one blower setting for varieties with a weight of 1000 seeds between 0.34 and 0.30g and another blower setting for varieties with weight of 1000 seeds <0.30g?

The evaluation of the results of the Poa pratensis experiment was not finally decided during the ISTA Meeting in Zurich. During the discussion it was, however, pointed out that when accepting the blowing factor 0.82 for Poa trivialis, comparison was made with the hand method, which was the ISTA method for purity analysis of Poa trivialis before change of the ISTA Rules in 1992 (see Jensen & Bülow-Olsen 1992).

For that reason it was decided – as an optional method – to analyze the amount of misplaced seeds (i.e. inert matter (seed without caryopsis) in the heavy fraction and ‘pure seeds’ (seeds with caryopsis) in the light fraction).

The blowing factor giving the approximately same amount of misplaced seeds in the heavy and in the light fraction was assumed to be the optimal blowing point. The equivalence in misplaced seeds in the heavy and in the light fraction is also used when calibrating the seed blowers with the ISTA calibration samples.

During the discussion in Zurich it was agreed to include germination of at least seeds from the heavy fraction as well, which would allow calculation of % pure live seed (%PLS) for seed samples blown by different blower settings. Calculation of PLS was also included in the experiment on Poa trivialis, mentioned above (Jensen & Bülow-Olsen 1992).

The method used in the present experiments, comparing the amount of ‘seeds’ wrongly classified, can be taken as an adapted method for comparing the blowing method to the hand method.

It appeared to be impossible to meet the dates for submitting new proposals for change of the AOSA Rules (October 15th=) as set in the program of this working group (see annex 1). The program involved a considerable amount of time consuming purity analysis, followed by germination tests that take more than four weeks.

The first germination test results became available in the last week of September, the last ones on October 8th, 2003.

Results and discussion.

Table 4.1 contains the results of all laboratories analysis, carried out in 2003. The table is divided into three parts:

• The upper part gives the purities and germinations results, leading to the calculated percentage of pure live seeds (PLS). The blower setting for each variety/sample providing the highest content of PLS is printed in bold.

• The middle part gives the grams pure seed in the light fraction, and the gram empty particles in the heavy fraction. The results of the blowing with the lowest difference in the misplaced seeds are printed in bold.

• The bottom part shows some additional information on the seed lot.


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The experiment comprised in total 32 samples distributed on 10 varieties of Poa pratensis. Table 4.2 Key figures extracted from table 4.1 Variety Weight of

1000 seeds Number of samples

Number of seed lots

Laboratory Number

Harmony 0.23 4 2 4 Mardonna 0.24 4 2 4 Unique 0.24-0.25 2 1 1,3 Julia 0.26 1 1 1 Platini 0.26-0.28 4 2 4 Sobra 0.27-0.30 4 2 4 Eprima 0.32-0.34 4 4 1,2,3,5 Cynthia 1 1 5 Saskia 1 1 5 Balin 0.31-0.34 4 2 4 Evora 0.45-0.48 3 1 1,2,3 The overall averages from Table 4.1 show that the percent of pure seed increase from 94.6 to 99.8 when the blower setting is changed from 1.00 to 0.60. The increase in percent pure seed is specific obvious when the blowing factor is changed from 1.00 to 0.82 (Table 4.3).

For germination on average 9% lower germination is obtained when the blower setting is changed from 1.00 to 0.60. As Poa pratensis in general have a low germination, the blowing factors 0.60 and 0.70, which give the lowest germination, should be avoided (Table 4.3).

Table 4.3: Averages for the heavy fraction: Percentage germination and percentage PLS. In the right part of the table, all samples of which the blower setting of 0.90 was not tested, were excluded from calculation of the averages. See Table 4.2. for information on varieties, number of samples etc.

Pure live seeds

Excluding 0,90

Pure live seeds

Including 0,90 blower setting

Heavy %

Germination %

(PLS) %

blower setting

Heavy%

Germination %

(PLS) %

0.60 99.8 79 79 0.60 99.9 80 80 0.70 99.4 80 80 0.70 99.7 81 80 0.82 98.2 83 81 0.82 98.8 82 81 0.90 97.7 84 82 0.90 97.7 84 82 1.00 94.6 88 83 1.00 95.5 86 83

The calculated content of pure live seeds (PLS) shows a considerable variation between samples of same variety as well as between varieties (see Table 4.1). The statistical analysis revealed that the differences were significant (1% level). This factor is, accordingly, not suited for establishment of the optimal blowing factor for Poa pratensis.


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The uniformity of testing samples from the same variety was examined for the variety Enprima. Identical samples were analyzed at four different seed laboratories (gram pure seeds in light fractions and gram empty seeds in heavy fractions was only recorded at three laboratories) For this variety the results at blower speed 0.82 showed no significant differences among laboratories for g light seed in the heavy fraction and g heavy seed in the light fraction, except for % heavy seeds, where the amount of was slightly less for laboratory 3 than for laboratory 2 and 5. The result at laboratory 1 may seem high, but this is most probably because this laboratory received seeds from a different sample. The blower speed 0.82 had on the average the best balance between g pure seeds in light fraction and g empty seeds in heavy fraction. This was also the case for 7 out of the 11 varieties. For the remaining 4 varieties the absolute difference for the 0.82 blower speed was only significant larger than the blower speed with best balance for one variety, Julia (where it was significant at the 4% level). The variance between repeated analyses of the same sample are for most variables smaller for data from blower speed 0.82 than for all data. This indicates that the variability between repeated analyses is lower for this blower speed than for the others. For % heavy seeds the standard deviation is *0.017=0.13 which show that the probability of exceeding the ISTA tolerances will be very low. The ability to re-test seed samples of five different varieties with different seed weight was examined by one seed laboratory (laboratory no. 4). The results of the re-test for blower setting 0,82 are presented in table 4.4. In the table is the differences are the differences between the analysis results compared with the ISTA Tolerances. Table 4.4: Differences between two purity tests on same submitted sample of five varieties compared with the ISTA Tolerances, Table 3.1. Blower setting 0,82. Laboratory 4. Variety Pair % Heavy seed 1. test 2. test average difference ISTA Tolerance Harmony I 99.6 99.6 99.6 0.0 0.5 II 99.2 99.2 99.2 0.0 0.7 Maradonna I 97.7 97.9 97.8 0.2 1.1 II 99.1 99.1 99.1 0.0 0.7 Platini I 99.0 99.2 99.1 0.2 0.7 II 99.4 99.4 99.4 0.0 0.6 Sobra I 99.0 99.0 99.0 0.0 0.8 II 98.1 98.1 98.1 0.0 1.0 Balin I 98.9 99.2 99.1 0.3 0.7 II 98.2 98.6 98.4 0.4 1.0 It appears from table 4.4, that the reproducibility within laboratory 4 is good when blowing small seeded varieties at blower setting 0,82. The differences between pair of purity analyses were all within the ISTA Tolerances.

If looking at the blowing level where the grams of pure seed in the light fraction are equal to or nearly equal to the grams of empty seeds in the heavy fraction, a consistent picture appears (see table 4.1). All varieties up to Enprima show an optimal blowing point of 0.82. For Balin the blowing point can either be 0.82 (2 samples) or 0.90 (2 samples).


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The variety Evora has a weight of 1000 seeds >0.35 and shall, accordingly, be blown at a blower setting 1.00.

Returning to the hand method is not an option. The blowing method is both faster and gives more uniform results than the hand method (Nelson, 1970; Porter & Leggatt, 1942; Leggatt, 1941)

The present ISTA Poa pratensis calibration samples are based on a mixture of commonly grown varieties with a weight of 1000 seeds ranging from 0.31 to 0.40 g (average 0.35 g). Care was taken that the new calibration samples gave the same purity results as those produced in the Netherlands 10-15 years ago.

The differences in seed weight between Poa pratensis and Poa trivialis did for many years prevent the ISTA stations from using the blowing method for Poa trivialis. AOSA was the pioneer in solving the problem and introduced the multiplication factor 0.82, when blowing Poa trivialis on a blower, calibrated with a Poa pratensis calibration sample. Within the ISTA Purity Committee a study was initiated, and this study confirmed the AOSA factor 0.82 (Jensen & Bülow-Olsen, 1992). The factor of 0.82 for Poa trivialis was introduced into the ISTA Rules in 1992.

If we look at the 1000 seed weight’s of the small seeded varieties tested in these experiments, they appear to be on the same level as the weight of 1000 seeds for Poa trivialis. Data received from the Netherlands and Denmark give the following weight of 1000 seeds for varieties of Poa trivialis: Sabre: 0.24 (two lots), Solo: 0.21 (six lots), and Dasas: 0.24 (ten lots).

Therefore, it is not a surprise that the suggested optimal blower setting 0.82 for small seeded Poa pratensis varieties is the same as the one already in use for Poa trivialis.

Choosing the already existing blower setting for Poa trivialis has the additional advantage that no extra blowing points have to be added. This makes it easier for all involved to implement the blowing factor also for small seed varieties of Poa pratensis.


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5. Conclusions Based on previous results and the results from this study, it can be concluded that:

• It was possible to obtain similar results when identical samples are blown with different blower settings at four seed laboratories.

• Identical results were obtained when seed samples of five varieties with different 1000 seed weight were re-analysed at the same seed laboratory.

• Due to variable results between samples of the same variety and between varieties it is obvious that Pure Live Seed (PLS) is not suited for selection of the blower setting of Poa pratensis.

• The equivalence of g misplaced seeds in the heavy and in the light fraction provided uniform results for all small seeded varieties tested (1000 seed weight from

0.22-0.34 g). • The factor 0.82 was the optimal blower setting for all small seeded varieties tested,

and it represents the best possible compromise between the increase in % pure seed and the decrease in % germination when the blower settings are reduced from 1.00 to 0.60.

• The factor 0.82 has already been in use for Poa trivialis at the ISTA seed laboratories for more than 10 years.

• It is, accordingly, proposed that the factor 0.82 should be included in the ISTA Rules for blowing of all varieties of Poa pratensis with a 1000 seed weight <0.35 g.

6. Proposed Rules Change 2004 3.5.2.A.5. Uniform blowing method. This method is obligatory for Poa pratensis, Poa trivialis and Dactylis glomerata. The working sample size is 1g for Poa pratensis and Poa trivialis and 3g for Dactylis glomerata. The blowing pressure is determined for Poa pratensis and Dactylis glomerata by means of a calibration samples issued under the authority of the International Seed Testing Association. The blowing pressure for the varieties of Poa pratensis listed below with an average weight of 1000 seeds <0.35g is obtained by multiplying the blower setting for Poa pratensis by 0.82 (applies only for General Seed Blowers). Seed identified as ‘variety not stated’ is excluded from the list. The blowing pressure for Poa trivialis is obtained by multiplying the blower setting for Poa pratensis by 0.82 (applies only for General Seed Blowers). Prior to calibration both the calibration and working samples must be exposed to room conditions. For those not having a General Seed Blower, please contact the International Seed Testing Association Secretariat.


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List of varieties of Poa pratensis with an average weight of 1000 seeds <0.35g. Variety 1000 seed weight

Balin 0.34g Compact 0.34g Julia 0.33g Limousine 0.33g Enprima 0.32g Oxford 0.32g Ikone 0.31g Sobra 0.31g Pegasus 0.29g Platini 0.29g Slezanka 0.28g Mardona 0.27g Tommy 0.26g Lato 0.24g Harmony 0.23g Add to: 1.2 Guidelines for the introduction of new species and methods into the Rules

Small seeded varieties of Poa pratensis: Before a small seeded variety is included in 3.5.2.A.5, a determination of the 1000 seed weight must be performed on at least 20 samples from different seed lots, representing seeds grown either in two different harvest years or in two different countries. The determination of the 1000 seed weight shall be carried out on pure seeds, obtained by blowing a 1g sample of Poa pratensis using the standard blower setting (factor 1.00). Only seed remaining in the heavy fraction shall be used for the 1000 seed weight. See Chapter 10 for the weight determination procedure. Seed identified as ‘variety not stated’ is excluded from the list. Results shall be submitted to the ISTA Purity Committee with a request to change the ISTA Rules.


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7. Literature

Brown … , Porter … (1935). An improved method of testing seeds of Kentucky Bluegrass (Poa pratensis L.). Proceedings AOSA 27: 44-49.

Jensen, H.A. & Bülow-Olsen, A. (1992) Comparison of purity analysis results from Poa trivialis L. samples tested according to the ISTA and the AOSA method. Seed Science & Technology 20: 655-661.

Leggatt, C.W. (1941) The “Climax” blowing point in the testing of grass seeds for percentages of pure live seed. Contr.: No. 657. Division of Botany and Plant Pathology, Science Service, D. of A. Ottawa, Canada.

Nelson, B. (1970) Uniform Blowing Procedure for Canada and Rough Bluegrass Subcommittee. Proceedings of the Association of Official Seed Analysts. 60: 24-25.

Porter, R.H., Leggatt CW (1942) A new concept of pure seed as applied to seed technology. Scientific Agriculture 23(2): 80-103.

8. Acknowledgements The working group acknowledges the analytical work performed by the participating seed laboratories, the valuable comments received during preparation of the manuscript and the statistical analysis carried out by Dr. Kristian Kristensen, Biometry Research Unit, Danish Institute of Agricultural Sciences.


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r s

r

Annex 1:

Statistical analyses blower speeds in purity assessments of Poa pratensis Kristian Kristensen

Biometry Research Unit, Department of Animal Breeding and Genetics Danish Institute of Agricultural Sciences

November 2003

Method Each variable were first analysed in an analysis that took into account the effects of variety, laboratory and blower speed as well as all two-way interactions and the three-way interaction. The residual effects were separated into 1) within sample variation and 2) between sample variation. Mathematically the model may be written as:

( ) ( ) ( ) ( )where

is the observed value for variety at laboratory with blower speed for replicate of sample is the random effect of sam

vlbsr v l b vl vb lb vlb vs vlbsr

vlbsr

vs

Y D E

Y v l bD

µ α β γ αβ αγ βγ αβγ= + + + + + + + + +

2

2

ple in variety is the random effect within sample

and are asumed to be independently normally distributed with mean zero and vaiances

and , respectivelyThe greeck letters

vlbsr

vs vlbsr D

E

s vED E σ

σare the fixed effects.

Next all data with blower speed 0.82 were analysed in a model that took into account the effects of variety and laboratory as well as the two-way interaction. The residual effects were separated into 1) within sample variation and 2) between sample variation. Mathematically the model may be written as:

( )where

is the observed value for variety at laboratory with blower speed 0.82 for replicate of sample

is the random effect of sample in variety

vlsr v l vl vs vlsr

vlsr

vs

vlsr

Y D E

Y v ls

D s vE

µ α β αβ= + + + + +

2 2

is the random effect within sample and are asumed to be independently normally distributed with mean zero and vaiances

and , respectivelyThe greeck letters are the fixed effects.

vs vlsr

D E

D Eσ σ

The set of varieties analysed differed from laboratory to laboratory in such a way that the table variety by laboratory were disconnected. Therefore, in order to estimate the effects of the factors (variety, blower speed and laboratory) it was necessary to separate the data into two groups: 1) data from laboratory 4 and 2) data from laboratory 1, 2, 3 and 5. In addition it was necessary to delete the blower speed 0.90 in the second group (laboratory 1, 2, 3 and 5). In the estimation also all interactions with laboratory were excluded (they were non-significant or almost non-significant for all variables (see table 1 and 4)).


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Results Model check The model was checked for the underlying assumptions using graphical methods. The graphics were constructed to check whether the variability dependent on the response, whether the residuals looked normally distributed and whether deviating observations were present. This check revealed that rather large discrepancies were present for the two replicates of Mardona for blower speed 1 at laboratory 4. However, the discrepancies were judged to be of a size that could have happened by change, so the observations were kept in the analyses. All data The analysis showed that there were a significant interaction between blower speed and variety for all characters except for % pure life seeds. The blower speed had a significant effect on all variables. For only one variable, g empty seeds in heavy fraction, significant differences between laboratories were found. See table 1. The last column, Balance, is the difference between g pure seeds in light fractions and g empty seeds in heavy fractions. The variance components are shown in the bottom part of table 1. The residual variance is the within samples variation. The sample component is the additional variation when the results comes from different samples. They were for most variables of similarly size - only for the variables balance and g pure seed in light fraction was the component for between sample considerable less that the component for within sample. For those two variables most of the variance were caused by the within sample variability. For the other variables the variance between two observed values was approximately doubled if the observed values come from different samples compared to observed values from the same sample. The effect of Blower speed is shown in table 2. The effect of blower speed was of the same type for both groups of laboratories although the effects were lager for laboratory 1, 2, 3 and 5 than for laboratory 4. For the variable g empty seeds in heavy fraction the magnitude of the figures were considerable larger for laboratory 1, 2, 3 and 5 than for laboratory 4. However, this was mainly caused by the variety Julia (see table 7). The blower speed 0.82 had on the average the best balance between g pure seeds in light fraction and g empty seeds in heavy fraction (table 2). This was also the case for 7 out of the 11 varieties (table 7). For the remaining 4 varieties the absolute difference for the 0.82 blower speed was only significant larger than the blower speed with best balance for one variety, Julia (where it was significant at the 4% level).

Table 1 Significance of fixed effects and estimated variance components for each variable using all data Effect name % heavy

seeds Germination percent

% pure live seeds

g pure in light frac.

g empty in heavy frac.

Balance, g

Significance of fixed effects*

Variety ** ** * *** ** Laboratory *** Blowers. *** *** *** *** *** *** V×L * V×B *** ** (*) *** *** *** L×B V×L×B Estimated random effects Sample 0.647 5.698 8.515 0.000016 0.000003 0.000008 Residual 0.607 9.001 7.995 0.000045 0.000005 0.000058 *) One, two of three asterisks denote that the effect is significant at the 5%, 1% or 0.1% level, respectively


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Table 2 Estimated effects of Blower speed Blower speed % heavy seeds Germinatio

n percent % pure live seeds

mg pure in light frac.

mg empty in heavy frac.

Balance, mg

Laboratory 4

0.60 100.0 79.3 79.3 0.0 7.0 -7.00.70 99.8 80.4 80.2 0.1 6.1 -6.00.82 98.9 81.0 80.2 1.7 2.0 -0.30.90 97.8 82.7 81.0 7.5 0.3 7.21.00 95.5 85.6 81.9 28.7 0.0 28.7Laboratory 1, 2, 3 and 5 0.60 99.4 77.7 77.3 0.0 25.2 -25.50.70 98.7 80.0 79.0 0.4 22.4 -22.10.82 96.9 85.1 82.5 5.8 14.7 -9.01.00 91.7 90.0 82.6 35.2 11.1 23.9 For the variable g empty seeds in heavy fraction there was a significant effect of laboratory. This was mainly caused by laboratory 1 that had a high value and laboratory 4 that had a low value (table 3). However, it should be noted that the difference between laboratory 4 and the other three couldn't be separated from the effect of the groups of varieties analysed at the two laboratories.

Table 3 Effect of laboratory for the variable mg empty seeds in heavy fraction Laboratory Lab 4 Lab 1,2,3,5 1 253 134 3 5 17 Data at blower speed 0.82 When analysing data where seeds were cleaned at the blower speed 0.82 significant effects of variety were found for most variables. For the same data significant effects of laboratory were found for 3 variables (see table 4). I addition a specific test for differences between laboratories for the variety Enprima was added (Enprima was the only variety analysed by all samples). Significant differences between laboratories were only found for % heavy seeds and were mainly caused by the relative low value at laboratory 3 (note: laboratory 1 received a different sample). The variance components (bottom part of figure 4) are for most variables smaller for data from blower speed 0.82 than for all data. This indicates the variability between repeated analyses are lower for this blower speed than for the others. For % heavy seeds the standard deviation is √0.017=0.13 which show that a difference larger than 0.6 between two samples will be very rare.

Table 4 Significance of fixed effects and estimated variance components for each variable using only data from blower speed 0.82. Effect name % heavy


% pure live seeds

g pure in light frac.

g empty in heavy frac.

Balance, g

Significance of fixed effects*

Variety ** ** ** *** *** Laboratory *** *** ** V×L * Laboratories for Enprima

*

Estimated random effects Sample 0.309 1.100 1.827 0.000002 0.000003 0.000005 Residual 0.017 11.450 11.256 0.000003 0.000001 0.000038 *) One, two of three asterisks denote that the effect is significant at the 5%, 1% or 0.1% level, respectively


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Table 5 Estimated effects of varieties for blower speed 0.82 Blower speed % heavy


% pure live seeds



Balance, mg

Laboratory 4

Balin 98.7 81.5 80.5 0.9 6.2 -5.3Harmony 99.4 86.5 86.0 0.2 0.3 -0.1Mardona 98.5 62.5 61.5 2.3 1.5 0.8Platini 99.2 84.5 83.9 4.0 0.6 3.4Sobra 98.5 90.3 88.9 0.9 1.3 -0.4Laboratory 1, 2, 3 and 5 Cynthia 95.5 87.0 83.1 5.5 2.3 3.1Enprima 97.7 88.0 85.9 2.0 3.1 -1.2Evora 98.0 93.3 91.4 -1.7 4.1 -5.8Julia 95.2 84.5 80.4 13.8 66.3 -53.0Saskia 98.7 86.0 85.0 1.7 3.2 -1.6Unique 95.9 69.2 66.3 14.3 8.3 6.0 The estimated effects for each variety are shown in table 5. For germination and % pure live in seeds the varieties Mardona and Unique had significant low values. For the three variables mg pure seeds in light fraction, mg empty in heavy fraction and balance Julia had values that deviated from most other varieties. The variety Unique had a very high value for the variable mg pure in light fraction. The estimated effects for each laboratory are shown in table 6. For the variable % heavy seeds laboratory 4 and 1 had higher values than the other, bur only laboratory 1 and 3 differed significantly (laboratory 4 can not be compared statistically with the other laboratories). For the variables mg empty seeds in heavy fraction and balance the values at laboratory 4 are much closer to zero, than for the other 3 laboratories. However, the estimates for laboratory 4 cannot be compared statistically with the values for the other laboratories.

Table 6 Estimated effects of laboratories for blower speed 0.82 Blower speed % heavy


% pure live seeds



Balance, mg

Laboratory 4

4 98.9 81.1 80.2 1.7 2.0 -0.3Laboratory 1, 2, 3 and 5 1 97.4 81.2 79.2 8.4 21.7 13.22 96.7 82.0 79.3 3 96.2 85.8 82.6 5.0 9.3 -4.35 96.9 89.7 86.9 4.2 12.7 -8.5


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Table 7 Estimated blower by variety effects for two groups of laboratories

Laboratory 4 Laboratory 1, 2, 3 and 5 Blower speed Balin Harmony Mardona Platini Sobra Cynthia Enprima Evora Julia Saskia Unique

% heavy seeds 0.60 100 100 100 100 100 99.4 99.8 99.1 98.7 99.9 99.60.70 99.6 99.8 99.9 99.9 99.7 98.1 98.9 98.8 98.1 99.5 98.70.82 98.7 99.4 98.5 99.3 98.5 95.5 97.6 98.0 95.7 98.7 95.90.90 97.2 99.1 96.1 98.8 97.8 . . . . . .1.00 95.1 98.0 89.8 97.9 96.6 88.5 95.2 96.3 84.7 97.0 88.5

Germination 0.60 78.3 86.0 62.2 83.2 87.0 80.9 83.7 87.4 73.6 85.9 54.70.70 80.8 86.3 62.5 84.3 88.3 78.9 84.5 87.4 80.6 90.9 57.70.82 81.5 86.5 62.5 84.5 90.2 89.9 88.7 92.4 82.6 88.9 68.20.90 84.8 87.0 62.2 86.0 93.5 . . . . . .1.00 87.0 88.8 73.0 86.5 92.8 90.9 94.5 93.7 89.6 90.9 80.2

% pure live seeds 0.60 78.2 86.0 62.3 83.3 87.0 80.4 83.6 86.6 72.8 85.8 54.60.70 80.4 86.1 62.4 84.2 88.0 77.3 83.6 86.3 79.2 90.4 57.00.82 80.5 86.0 61.5 83.9 88.9 85.7 86.7 90.5 79.2 87.7 65.40.90 82.4 86.2 60.0 84.9 91.5 . . . . . .1.00 82.7 87.0 65.6 84.7 89.6 80.2 90.0 90.2 76.2 88.1 71.1

mg pure seeds in light fraction 0.60 0.1 -0.0 0.0 -0.0 0.1 2.1 0.0 -3.0 -2.7 2.0 0.60.70 0.1 0.1 0.2 -0.0 0.1 2.0 0.2 -2.5 0.6 2.0 0.40.82 1.0 0.3 2.3 4.0 0.9 5.8 2.0 -2.2 13.3 2.0 14.10.90 5.5 3.0 18.5 6.0 4.7 . . . . . .1.00 20.7 13.0 79.1 14.8 16.0 39.6 13.5 5.6 98.1 9.7 44.6

mg empty seeds in heavy fraction 0.60 12.5 4.1 5.8 3.2 9.4 6.1 16.2 15.7 91.1 4.9 17.10.70 11.1 1.9 8.1 2.5 6.7 6.6 9.4 10.4 96.8 3.9 7.40.82 6.3 0.3 1.5 0.6 1.3 1.9 3.0 4.9 67.1 2.8 8.50.90 1.1 0.0 0.2 0.1 0.2 . . . . . .1.00 0.0 -0.0 -0.0 0.0 0.0 1.5 1.0 -4.8 64.0 1.4 3.7

Balance, mg 0.60 -12 -4.2 -5.7 -3.2 -9.4 -3.7 -16 -19 -94 -2.6 -170.70 -11 -1.8 -7.9 -2.5 -6.6 -4.3 -9.4 -13 -97 -1.6 -7.20.82 -5.3 -0.1 0.8 3.4 -0.4 4.2 -1.2 -7.5 -54 -0.5 5.40.90 4.4 2.9 18.3 5.9 4.5 . . . . . .1.00 20.7 13.0 79.1 14.8 16.0 38.4 12.3 10.0 33.7 8.6 40.7

2004 – item 3a...enprima 0.33 84.8 8.5 17 delft 0.35 90.8 6.9 10 miracle 0.38 88.8 0.9 12...

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