Tetrazolium Committee

Transcription

1 Tetrazolium Committee Chair: Stefanie Krämer Committee report

2 Committee Membership 1 Mrs. Stefanie Krämer Germany Chair 2 Mrs. Karen A. Hill Australia Vice chair 3 Mr. Ignacio Aranciaga Argentina 4 Mrs. María Belén Aranguren Argentina 5 Mrs. Valerie Blouin France 6 Mrs. Sharon Davidson USA 7 Mr. Gary Duffy Ireland 8 Mr. Jose B. Franca- Neto Brazil 9 Mrs. Linda Maile United Kingdom 10 Mr. Augusto Martinelli Argentina 11 Mr. Sergio Pasquini Italy 12 Mrs. Anny van Pijlen Netherland 13 Mr. José Manuel Chávez Bravo* Mexico Mrs. Irena Jumburga** Mrs. Lindsay Robb** * new member ** resigned members Latvia Canada

3 RULES Development v Composition of a guidance for the Tetrazolium testing of treated seeds chapter 11

4 11.6 The tetrazolium test Object The objects are the same as defined in Definitions The definitions are the same as prescribed in General principles The general principles are the same as described in Reagent The reagents are the same as described in 6.4

5 Procedure Coated seed units (seed pellets, encrusted seeds or seed granules): Four replicates of 100 coated seed units are washed to remove the coating mass. Depending on the consistency of the coating mass, it may be necessary to agitate whilst soaking, to release seeds from the coating. The duration of the washing should not take longer than the premoistening period prescribed in Table 6A.

6 The number of seeds detected in each replicate of 100 coated seed units (of the species stated by the applicant) must be reported as an average of all four replicates. If there are more than 100 seeds in each replicate of coated units only 100 seeds per replicate will be used for the Tetrazolium testing. Coated units without seeds (e.g. empty pellets) are deemed to be non viable seeds. The test procedure of the washed, uncoated seeds then continues with the premoistening or if the total premoistening time is achieved, with the preparation for the staining step as prescribed in Table 6A.

7 Seed tapes: The number of seeds (of the species stated by the applicant) per metre has to be detected and reported. To complete the test, 400 seeds must be extracted from the seed tape. The test procedure then continues with the premoistening step as prescribed in table 6A.

8 Seed mats: The number of seeds (of the species stated by the applicant) per seed mat has to be determined and reported (in large seed mats the number of seeds per square metre). To complete the test it is necessary to extract 400 seeds from the seed mat. The test procedure then continues with the premoistening step as prescribed in Table 6A Calculation, expression of results and tolerances The same criteria are valid as prescribed in 6.6

9 Reporting results The result of a tetrazolium test on coated seeds must be reported as follows: - Following the species name, the words seed pellets, encrusted seeds, seed granules, seed tapes or seed mats, as applicable, must be clearly entered.

10 The following additional information must be reported under Other determinations : - The statement Number of seeds (of the species stated by the applicant) included in 100 seed pellets ( or encrusted seeds or seed granules ) - or the statement Number of seeds (of the species stated by the applicant) included in one metre seed tape - or the statement Number of seeds (of the species stated by the applicant) included in one seed mat or in one square metre of seed mat - The statement Tetrazolium test: % were viable must be entered.

11 In cases where the testing procedure deviates from that prescribed in Table 6A, any deviating procedure must also be reported. The only areas where deviations from procedures given in Table 6A are permitted are for premoistening time, tetrazolium concentration, staining temperature and staining time. For precise guidance about the limitation of the deviations permitted see 6.5

12 - If individual seeds are tested at the end of the germination test, the result must be reported in accordance with 5.9 In addition, in the case of species of the Fabaceae, one of the following and only one, must be reported: either (in cases where the percentage of the viability of hard seed is not determined) Tetrazolium test:...% of seeds were viable,...% of hard seeds found in the test or (in cases where the percentage of the viability of hard seed is determined) Tetrazolium test:...% of seeds were viable,...% of hard seeds included in the percentage of viable seed

13 Additions for Chapter 1: Tetrazolium test on coated seeds the same text as in will be inserted

14 v Composition of a guidance for the Tetrazolium testing of seed mixtures, chapter 18

15 18.7 Tetrazolium test For species representing more than 5% of the seed mixture, four replicates of 100 seeds are tested from the pure seed of each component species. If insufficient seed is available from the pure seed fraction the test will be carried out on (in order of the priority) two replicates of 100 seeds, one replicate of 100 seeds or on all pure seed of the species in the pure seed fraction, depending on the seed availability.

16 For species representing 5% or less of the mixture a tetrazolium test will not be carried out except at the specific request of the customer. In this case the tetrazolium test is carried out on two replicates of 100 seeds, one replicate of 100 seeds or on all pure seed of the species in the pure seed fraction, depending on the availability of seed.

17 18.9 Reporting results Tetrazolium Test The Tetrazolium results for each component species are reported under Other determinations. The results are reported as a percentage and the number of seeds tested is also reported. When fewer than 100 seeds are tested, the number of viable seeds is reported together with the total number of seeds tested.

18 Additions for Chapter 1: Tetrazolium Test the same text as in will be inserted

19 vbrachiaria spp. Brachiaria spp. BP/16; W/6 BP/16; W/6 Remove glumes, cut transversely near embryo Cut longitudinall y through embryo and 3/4 of endosperm Observe external embryo surface. 1 2 Observe cut surface. ⅓ radicle ⅓ radicle Date - Presenter Name

20 vhelianthus spp. Helianthus spp. W/18 Remove pericarp and seed coats from the seed. 1 3 Cut longitudinally through the cotyledons and the radiclehypocotyl axis. Observe both sides of the seed. ⅓ radicle measured from radicle tip, ⅓ of distal end of cotyledons if pervading, ½ of distal end of the cotyledons if superficial

21 Introduction of new species: no progress

22 Updating and reorganisation of Tetrazolium Handbook no progress, completed?

23 Workshops and Seminars September 2012 Future worshops? ISTA Workshop on Moisture, Vigor and Tetrazolium of agricultural seeds in Mellbourne, Australien Tetrazolium part was dealing with Desmodium, Cucumis, Festuca, Helianthus, Lolium, Medicago, Panicum, Phleum and Trifolium

24 Other projects: Storage experiment of durability of Tetrazolium solution Start in May 2012 End in April 2013

25 Test principles: Once a month 100 seeds were tested a ph check before and after use Storing in a dark bottle and at 5 C to 10 C

26 Participants Brand name, date of expiry, consistence Testspecies Garry Duffy Fisher brand exp date 2014/08/23 Hordeum vulgare Jose Franca Neto Karen Hill Brand: Vetec-Química Fina validity: 10/2015 SIGMA 2,3,5-Triphenylterazolium chloride >95%, MW 334.8g/mol powdery Glycine max Chloris gayana Linda Maile Molekula, no expiry date, powdery Brassica napus Sergio Pasquini Fluka without any expiry date, powdery Fagus sylvatica Tetrazolium Red; TTC, Reagent CAS Lolium sp., Sharon Festuca Assay Min 98%,F.W Davidson arundinacea, F.W Poa pratensis Valerie Blouin sygma aldrich 2013 Carpinus betulus Stefanie Krämer Merck,2013/10/31.powdery Trifolium pratense

27 Month Result Comments May % Normal staining patterns observed June % Normal staining patterns observed July % Normal staining patterns observed Aug 12 94% Normal staining patterns observed Sep 12 92% Normal staining patterns observed October % Normal staining patterns observed Nov 12 94% Normal staining patterns observed December % Normal staining patterns observed Janurary % Normal staining patterns observed February % Normal staining patterns observed March % Normal staining patterns observed Apr 13 95% Normal staining patterns observed All tests conducted by the same two Seed Analysts, Maury Conway and Garry Duffy. The solution was stored at 5 C +/- 2 C in a dark glass bottle. Species Hordeum vulgare. The same sample was used throughout lab id PA Seed Testing Laboratory, Backweston Laboratory Campus, Celbridge, Co. Kildare. Ireland. Ph

28 April 2013 Normal Staining

29 Concentrati on/ without buffer Concentrati on/ without buffer Date Remarks ph with ph meter 1,00% 0,10% May 16, 2012 Perfect staining constant June 15, 2012 Perfect staining constant July 16, 2012 Perfect staining constant Aug. 18, 2012 Perfect staining constant Sept. 14, 2012 Perfect staining constant Oct. 18, 2012 Perfect staining constant Nov. 21, 2012 Perfect staining constant Dec. 17, 2012 Perfect staining constant Jan. 25, 2013 Perfect staining constant Feb. 19, 2013 Perfect staining constant Mar. 13, 2013 Perfect staining constant Apr. 14, 2013 Perfect staining constant May 17, 2013 Perfect staining constant Jose Franca - Neto, Glycine max Vetec - Expiry date Oct/ Powdery 10 C, dark, in a brown glasbottle

30 QSTL TZ Storage experiment - Chloris gayana Month Ph before Ph after Method Result % May 7 7 Dipstick 59 June 7 7 Dipstick 57 July 7 7 Dipstick 54 August 7 7 Dipstick 56 September 7 7 Dipstick 60 October 7 7 Dipstick 57 November 7 7 Dipstick 52 December 7 7 Dipstick 58 January 7 7 Dipstick 54 February 7 7 Dipstick 55 March 7 7 Dipstick 58 April 7 7 Dipstick 54 Karen Hill

31 Date ph, before ph, after Remark (Meter or staining staining paper) ######## 6,98 7,55 ph meter 98% ######## 7,04 7,72 ph meter 96% ######## 7,05 7,5 ph meter 96% ######## 7,04 7,38 ph meter 99% ######## 7,07 7,5 ph meter 96% ######## 7,12 7,26 ph meter 99% ######## 7,2 7,41 ph meter 98% ######## 7,32 7,51 ph meter 96% ######## 7,06 7,28 ph meter 98% ######## 6,88 6,55 ph meter 99% ######## 6,8 6,68 ph meter 97% ######## 6,72 6,92 ph meter 97% New bottle TZ /same batch Linda Brassica Maile napus The TZ solution was made 22/05/12 and stored at 7.5C The brassica sample was stored at 10C The ph meter was calibrated Sunday each time 16 with June ph 2013, 4 and Stefanie ph 7 solutions Krämer 1% Tetrazolium solution used

32 Date PH, before staining PH, after staining tested phmeter) ,09 6,92 tested with digital phmeter Crison Basic ,02 6,50 " " ,00 6,82 " " ,05 6,73 " " ,02 6,90 " " ,01 6,82 " " ,06 6,91 " " ,03 6,88 " " ,00 6,92 " " ,01 6,90 " " ,01 6,91 " " ,06 6,92 " " Fluka without any expired date, powdery Test species: Fagus sylvatica Sergio Pasquini Storage conditions = 10C Dark, brown bottle

33 100 seed TZ, ISTA study Autor Sharon Davidson date PH before staining Poa F. PH after staining remark pratensis arundinacea L. perenne ,76 6,83 PH meter ,85 6,87 PH meter ,98 7,03 PH meter ,88 6,91 PH meter ,86 6,9 PH meter ,89 6,93 PH meter ,87 6,93 PH meter ,89 6,9 PH meter ,85 6,94 PH meter ,85 6,9 PH meter ,81 6,86 PH meter ,84 6,86 PH meter Spectrum Chemical, no experiation, powder, Prep date = May 2012 Start of the test = May 17, 2012 End of the test - April 19, 2013 Storage conditions = 10C Dark, brown bottle

34 Valerie Blouin PH, before staining PH, after staining results TZ/100 seeds on the same sample of Carpinus betulus viable seeds June ,8 6, July ,9 6, Aug 12 6,8 6, Sep 12 6,9 6, non viables seeds October , Nov 12 6, December , January , Fevruary ,8 6, March , Apr , May , May ,8 6, new solution made in may year conserved solution

35 Stefanie Krämer, Trifolium pratense, TEZ solution with buffer Date PH, before staining PH, after staining Method May Dipstick June Dipstick July Dipstick August Dipstick September Dipstick October Dipstick November Dipstick December Dipstick January Dipstick Febrary Dipstick March Dipstick April Dipstick

36 Stefanie Krämer, Trifolium pratense TEZ solution with tap water Date PH, before staining PH, after staining Method May 7,5 7,5 Dipstick June 7,5 7,5 Dipstick July 7,5 7,5 Dipstick August 7,5 7,5 Dipstick September 7,5 7,5 Dipstick October 7,5 7,5 Dipstick November 7,5 7,5 Dipstick December 7,5 7,5 Dipstick January 7,5 7,5 Dipstick Febrary 7,5 7,5 Dipstick March 7,5 7,5 Dipstick April 7,5 7,5 Dipstick

37 One year old TEZ - solution Fresh TEZ - solution

38 Fresh solution Old solution made with buffer Old solution made with tap water

39 Conclusion: The storage of Tetrazolium solution of one year under the conditions 5 C to 10 C in a brown or dark bottle does not effect the chemical reactivity. Rules Proposal for 2014 and included in The results of the experiment will be published on the ISTA web side

40 Future project: Updating of the ISTA web site section TEZ Committee: v Providing of several guidelines, which normally are included in the handbook, e.g. checking of the TEZ solution. v Frequent questions v...

41 Questions to the Committee Date Subject Country Sep 12 Arbutus unedo, Arbutus x andrachnoides Greece Arbutus andrachne Oct 2012 Solution check Australia Dez 2012 Calluna vulgaris Germany Jan 2013 Sorghum spp. Germany May 2013 Cannabis sativa Canada

42 Proficiency Tests PT12-1 Sorghum PT12-2 Phleum pratense PT13-3 Brassica napus 2012 PUR, TZ, GER 2012 PUR, TZ, GER 2013 PUR, OSD,GER, MOI, TZ

43 Thank you to all who supported in any way our work!