Report from the Germination Committee Sylvie Ducournau

Sylvie Ducournau 1

Committee report 2013 - 2016

Agenda

Germination committee members Rules development

New methods New species Special projects Proposals for rules changes

Publications Workshops Future work of the committee

Committee membership Chair Sylvie Ducournau France

Vice-Chair Gillian McLaren United Kingdom

Members Ignacio Aranciaga Argentina

Sarah Dammen United States Ronald Don United Kingdom Ending 2016

Fabio Gorian Italy

Christine Herzog Switzerland David Johnston United States

Andrea Jonitz Germany

Jin Wook Kim South Korea Ending 2016

Augusto Martinelli Argentina

Lea Mazor Israel Ending 2016

Harry Nijenstein The Netherlands

Takayuki Okuda Japan Ending 2016

Rita Zecchinelli Italy

Many thanks to…

Ronnie Don Lea Mazor

Takayuki Okuda Jin Wook Kim

Pernilla Andersson

Aidin Hamidi

New applicants…

Meriam Dekalo-Keren

Rules development – New methods Validation study for a revised method of the germination of Dactylis glomerata and Festuca arundinacea – Harry Nijenstein

Objective: to shorten the duration of the germination test Experiment: 8 participants, 4 seed lots per species Conclusion: due to significant differences between the last two counts (14 and 21 days for Dactylis ; 10 and 14 days for Festuca), the current durations will not be changed

Addition of soaking treatment for Momordica charantia seed germination – Yu Sung

Objective: to improve germination by soaking seeds for 10 minutes at 60°C Experiment: 3 participants, 3 samples Conclusion: the method could not be validated due to high variability of the results

Rules development – New methods Alternative germination substrates (i.e. agar) for conservation programs, seed banks, small seed lots – David Johnston

Work in conjunction with FTS Committee for Pinus germination testing Presentations showing the benefits of using agar

Comparative tests AOSA / SCST on tomato and corn Repeatability and reproductibility of the results comparable between agar and

current AOSA methods

Study in one laboratory, comparison between agar and all the ISTA substrates for corn germination (24 samples tested) The results indicate that the media are equivalent

Conclusion

Agar as a media demonstrates very good repeatability and reproducibility Agar is a viable media for corn germination testing There are opportunities for testing agar media on other species

Rules development – New species Germination method for Chenopodium quinoa – Lesly Gonzales

Experiment: 6 laboratories, 3 samples, methods tested: TP and BP; 20 and 20 < = > 30°C; no pre-treatment – KNO3 – prechilling Results are still to be analyzed

Rules development – New species Germination method for Carica papaya – Maggie Lin

Experiment: 7 laboratories, 3 samples, methods tested: S and BP; 20 < = > 30°C; no soaking – soaking in water for 16h – soaking in water + GA3 All the methods give comparable results But repeatability and reproducibilty are better with sand media

%

Ger

min

atio

n

BP G

A3_W

ater

soa

king

BP_N

o so

akin

g

BP_W

ater

soa

king

Sand

GA3

_Wat

er s

oakin

g

Sand

_No

soak

ing

Sand

_Wat

er s

oakin

g

50

60

70

80

90

100

50

60

70

80

90

100

Conclusion The methods proposed are:

S, 20 < = > 30°C ; 12 - 28 days ; with a pre-treatment consisting of soaking the seeds in water, with or without GA3 added in the substrate.

Rules development – New species Germination method for Brassica carinata – Rita Zecchinelli

Test plan validated in 2015 and experiments started in 2016 6 laboratories 6 samples (3 from Italy, 3 from Canada) Methods

TP, 20°C, 5 – 7 days TP, 20 < = > 30°C, 5 – 7 days BP, 20°C, 5 – 7 days BP 20 < = > 30°C, 5 – 7 days

IDSeeds - GEVES

Rules development – New species

Germination method for Salvia hispanica – Sarah Dammen Test plan in preparation with

3 samples 2 temperatures (20°C and 20 < = > 30°C) with and without prechilling

IDSeeds - GEVES

Questions & Specific projects Temperature regime for Raphanus germination

Question raised by Pernilla Andersson 4 seed lots had poor germination results using 20°C and better results with

20 < = >30°C

Christine Herzog repeated the tests for the same seed lots

TP, 20°C PP, 20°C PP, 20 < = > 30°C BP, 20 < = > 30°C Tests with 7 days of prechilling at 10°C Non germinated seeds checked for their viability with TZ

Questions & Specific projects

0102030405060708090

10020 20

20-3

0

20-3

0 20 20

20-3

0

20-3

0 20 20

20-3

0

20-3

0 20 20

20-3

0

20-3

0 20 20

20-3

0

20-3

0

TP PP PP BP TP PP PP BP TP PP PP BP TP PP PP BP TP PP PP BP

1 1 1 1 2 2 2 2 4 4 4 4 5 5 5 5 Std Std Std Std

% Normal seedlings % Fresh seeds

Conclusion Temperature 20°C gave poor results compared to 20 < = > 30°C, on all the substrates, but were worst with TP. The reason being fresh seeds. Remove 20°C ? With or without validation study ? Add recommendation in table 5A ?

Questions & Specific projects Duration of the germination test for Helianthus annuus

Question was raised regarding the fact that germination test for sunflower is often ended after 7 days instead of 10 days A short survey was completed by the Germination committee to identify the most commonly used duration 7 members answered

Duration varies from 7 to 8, 9 or 10 days It is usually possible to end the test after 7 days, except when seeds are dormant, or when

primary leaves are difficult to evaluate, or for some ornamental species Reducing the standard duration to 7 days, would necessitate the inclusion on the certificate

of results obtained at 7 days in case of prolongation

Conclusion Keep the current duration for sunflower (10 days) Make the Rules clearer about the conditions for reducing the duration of the tests. Should the decision be left to the laboratory alone, or should evidence be required to justify the shortening of the test period.

Questions & Specific projects Root system evaluation of Spinach seedlings

The lack of criteria about root length is causing significant variability in germination results between laboratories Tests were organized by Lucile Daron from Enza Zaden 3 samples, 9 to 10 laboratories, 3 ring tests

Ring test no.

Germination method

Repetitions (no. of seeds)

Pre- treatment

Germination temperature

(°C)

Water supply during

germination test (mL)

Light regime

(light/dark)

Germination assessment (days after

sowing)

Root criteria

1 variable variable variable variable variable variable variable Variable

2 Pleated Paper

(PP) 2*100 none 10 40 12h/12h 14 variable

3 Pleated Paper

(PP) 2*100 none 10 40 12h/12h 14

Root/Shoot < 1;

priority to root length

Conclusion Method used and criteria for seedling evaluation are both equally important Need to harmonize the criteria for short root evaluation

Questions & Specific projects Root system evaluation of Spinach seedlings

A plan has been proposed for a new validation study based on the ISTA Method validation programme With the aim of proving that the lack of criteria about root length is causing significant variability in germination results A test plan has been produced and is being reviewed

Germination in the dark

Germination 12h light/12h dark

Picture source: Naktuinbouw, light testing

Picture source: Enza Zaden, 1st ring test, lot 1

Proposals for rules changes Clarification about the procedures for promoting germination of dormant seed

…. More complete germination may be obtained by retesting after one or a combination of the procedures listed in 5.6.3.1, 5.6.3.2 and 5.6.3.3. These procedures may be applied to the original test, if dormancy is suspected, even if there is no specific procedure indicated in column 6 of table 5A. Recommended procedures are indicated in column 6 of Table 5A, but this does not prevent the use of other procedures listed in 5.6.3.1, 5.6.3.2 and 5.6.3.3. ….

The reporting of multigerm seeds At the moment, there are different recommendations in paragraphs “ 5.6.5.2. "Multigerm seed units” “5.8 "Calculation and expression of results” “5.9 "Reporting results” The number or the proportion of normal seedlings produced by 100 units, or The number or the proportion of units producing one, two or more than two normal seedlings

Proposals for rules changes Re-introduction of tolerance tables for comparing results between laboratories

5.11.1 Tolerances in one laboratory Table 5B … 5.11.2 Tolerances in the same laboratory Table 5C … 5.11.3 Tolerances in different laboratories Table 5F. Tolerances between results of two tests made in different laboratories on the same or different samples from the same seed lot (2 way test at 5% significance level) on 400 seed tests. In accordance with Miles (1963), column C, 400 seed tests.

Average germination percentage of 2 tests

Tolerance

51-100%

0-50%

99 2 2 97-98 3-4 3 94-96 5-7 4 91-93 8-10 5 87-90 11-14 6 82-86 15-19 7 76-81 20-25 8 70-75 26-31 9 60-69 32-41 10 51-59 42-50 11

Proposals for rules changes Chapter 13 - Testing seeds by weighed replicates - Expression of the results

Change “seeds germinated” by “normal seedlings” in order to comply with the practice in seed testing laboratories and to harmonize with the reporting of the results of a germination test.

Germination method for Carica papaya

Publications Amendments to the Handbook for Seedling Evaluation A description of counting “by hand” A description of methods for seed disinfection Precision regarding the storage duration of KNO3 and GA3 solutions Water Retention Protocol

Correction of formulas Change of the duration of the draining (from 12 to “a minimum of 16

hours to a maximum of 24 hours”) Objective of the water retention measurement for germination testing

Cleanliness and Innocuity Protocol The germination of sensitive species has to be evaluated after the

final count The list of sensitive species is extended

Publications Amendments to the Handbook for Seedling Evaluation

Work with FTS Committee on photos of tree and shrub groups To update photos of seedlings for species already in the Handbook and to add photos of new species

Fagus Ulmus

Publications Amendments to the Handbook for Seedling Evaluation

In Section 18: evaluation of loops and spirals on hypocotyl for Phaseolus vulgaris

If there is no damage causing the loop on the hypocotyl (on the upper or lower part), then the seedling has to be considered as normal.

To be sure, the seedlings may be left in light for one more day. The loops usually disappear.

In Section 18: evaluation of root system of ornamental and flower species of Lupinus

Ornamental and flower species of Lupinus do not form a significant amount of secondary roots during the germination period, compared to Lupinus albus. However, some secondary roots do form when the primary root is damaged and the seedling develops normally.

Lupinus albus Lupinus hartwegii

Publications Amendments to the Handbook for Seedling Evaluation

In Section 18: evaluation of the root system of Glycine max

A seedling with a defective primary root is classed as normal, if sufficient normal secondary roots have developed.

The lack of precise criteria for sufficient causes discrepancies in normal seedling classification

A working group has been set up by Ignacio Aranciaga Proposals concerning different aspects of seedling evaluation (root

system for instance) Proposals concerning defects affecting species other than soybean

Sylvie Ducournau

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Soybean seedling evaluation

Workshop Germination Workshop – Estonia – 11-13 June 2016

Seed Testing Laboratory of the ARC (Agricultural Research Center) in Saku 35 participants from 22 countries Programme

Principles of the germination test Seedling evaluation (grasses, clovers, protein species, sunflower, brassica, carrot) Results calculation and reporting Quality management in germination testing

Sylvie Ducournau 25

Workshop Seed Analyst Training Workshop (BSC, PUR, GER) – INDIA Programme in preparation

Sampling, Purity analysis, Determination of Other Seeds by Number, Germination test, Accreditation

Species

Wheat, rice, corn, sorghum, pigeon pea, mung bean, cotton, sunflower, tomato, chillie, gram, soybean

Dates: 5-9 December 2016 Location: Hyderabad

Sylvie Ducournau 27

Future work of the committee Method validation Ending ongoing validation studies

Chenopodium quinoa, Brassica carinata, Salvia hispanica Starting new studies

Organic growing media for Phaseolus vulgaris and Glycine max, Agar media for Pinus, Agar and BP for Lolium, KNO3 for Lactuca sativa, TP for Zea mays, Spinach root evaluation, temperature for Raphanus sativus…

Publications Give details on light conditions and requirements for germination tests in the Handbook Complete the Tree and Shrub sections with updated photos in the Handbook

Thanks to all the members of the committee, the Statistics committee and the ISTA Secretariat

Thank you for your attention