Training ManualFor professional staff

Seed Quality Evaluation(Vegetable Crops) By

Muhammad Boota Sarwar

Published by

Facilitation Unit for Participatory Vegetable Seed And Nursery Production Program

Ministry of Food, Agriculture & Livestock, IslamabadCONTENTSSr. Chapters No . 1 Introduction 2 Objective of Seed Quality EvaluationAccuracy of the Results of Seed Analysis Recognized Standard Methods Reporting Results

Pag e No. 6 66 7 7

3

Seed SamplingDefinitions used in Seed Sampling Size of Seed Lot Preconditions for Seed Sampling Table-1: Maximum Seed Lot Weight of Vegetable Crops Table-2: Submitted Sample Weight of Vegetable Crops

88 9 9 10 11

4

Methods for Drawing Primary Seed SamplesA. Seeds in Bulk Table-3: Required Intensity of Primary Seed Samples for Seeds in Bulk B. Seeds in Sacks C. Seeds in Small Containers Table - 4: Required Intens i t y of Pr imary Seed Samples Seeds in Sacks or Mult ip l e Containers of 15 kg to 100 kg capaci ty D. Seeds in Processing or Conditioning Machines Table-5: Required Intensity of Primary Seed Samples for Seeds during Seed Processing E. GENERAL

1212 12 12 13 13 for

13 14 14

5

Preparation of Working Sample in the LaboratoryMinimum Weight of Working Sample Obtaining the Working Sample Storage of Seed Samples Table-6: Working Sample Weight of Vegetable Crops

1515 15 15 16 2

6

Mixing and Dividing ProceduresMechanical Divider Method a. Centrifugal Divider b. Soil Divider Hand Mixing or Spoon Method

1717 17 18 20

7

Purity Analysis1. Objectives 2. Definitions: Seed Pure Seed Other Crop Seed Weed Seed Inert Matter 3. General Principles 4. Apparatus 5. Procedure for Separation of Components Table-7: Minimum number of decimal places required for weighment of components 6. Calculation and Expression of Results 7. Reporting of Results Table-8. Tolerances for comparing percentage of purity analysis components, to determine if two tests from same submitted sample are compatible

2222 22 22 22 25 25 25 29 30 30 30 31 31 32

8

Determination of Other Seeds by NumberObjective Procedure Calculation and Expression of Results Reporting of Results

3434 34 34 34

9

Verification of Species and CultivarsObjective Preconditions for the Test General Principles Apparatus and Facilities Weights of Submitted Sample Table-9A: Minimum weight of Submitted Samples for Determination of Species / Cultivars Examination of seeds Examination of seedlings Examination of plants in glasshouse or growth chamber Examination of plants in field plots Calculating and expression of results Reporting results

3535 35 35 36 36 36 37 37 38 38 39 39

10

Determination of Moisture ContentDefinition

4141 3

Sample Procedure: A: Constant temperature oven method Calculation of Results Tolerance B: Electric Moisture Meter

41 41 41 43 44 44

11 12

Determination of 1,000 Seed WeightDefinition Procedure

4545 45

Purity Procedures for Coated SeedsNote Definitions Obtaining the working sample Foreign seed determination

4747 47 47 48

13

The Germination TestIntroduction Methods to be used Definitions Preparation of Seeds for Germination Tests Germination Conditions Germination Methods Table-10. Germination Methods for Vegetable Crop Species Special Germination Procedures Beta spp. Phaseolus vulgaris Vicia faba Coated seeds Tetrazolium testing Use of fungicides Treatments for Promoting Germination of Dormant Seeds Counts and Duration of Test Calculation and Reporting of Germination Results Retesting Evaluation of Tests Germination Tolerances Table-11. Maximum tolerated ranges in germination percentages of replicates for deciding when to retest Table-12. Maximum tolerated differences in germination percentages of tests for deciding which tests to average Seedling Descriptions: Family Azoaceae - New Zealand spinach Family Asteraceae - Lettuce Family Asteraceae Kinds other than lettuce Family Brassicaceae Cabbages, Radish, Turnip

4949 49 50 51 52 57 58 61 61 61 61 62 62 63 63 66 67 69 70 72 74 77 79 80

4

Family Chenopodiaceae Beet, Spinach Family Cucurbi taceae Melons, Gourds, Squashes Family Fabaceae Beans, Cowpea Family Fabaceae Peas, Lathyrus Family Fabaceae enugreek F Family Liliaceae Asparagus Family Liliaceae Onion, Leek, Chives Family Malvaceae Okra Family Apiaceae & Solanaceae Carrot, Tomato, Chili, Eggplant etc

82 84 86 88 90 92 95 97 100 101 103 104

14

Biochemical tests for viabilityObjectives Principle Reagent Procedure Calculation and Expression of Results Reporting Results

107107 107 108 108 108 109

15

Seed Health TestingObjective Definitions Principle Procedure Calculation of Results Reporting Results

110110 110 111 111 114 114

16

Specific InstructionsVegetable Crops of Family Solanaceae; Tomato, Chil i , Sweet pepper, Eggplant: Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne tomato pathogens with common names of the diseases they cause The main seed-borne eggplant pathogens with common names of the diseases they cause The main seed-borne pepper pathogens with common names of the diseases they cause Method for the Detection of Xanthomonas campestr i s pv. Vesicator i a on Pepper seed Method for the Detection of Tobamoviruses on Pepper seed (ISF)

120 122 122 122 123 124 125

5

Method for the Detect ionXanthomonas of campestr i s pv. vesicatoria on Pepper and Tomato seed (STA selective media assay) Method for the Detection of Xanthomonas campestris pv. vesicatoria on Pepper and Tomato seed (ISHI- vegetable selective media assay) Method for the Detect ionClav of ibacte r michiganensissubsp. michiganensis on Tomato seed Method for the Detection of Tobamoviruses on Tomato seed (Adapted from ISF) Method for the Detection of Pepino Mosaic Virus on Tomato seed (Adapted from ISF)

130 133

137 145 149

17

Specific InstructionsVegetable Crops of Family Cucurbitaceae Melons, Gourds, Squashes Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne watermelon pathogens with common names of the diseases they cause The main seed-borne cucumber pathogens with common names of the diseases they cause The main seed-borne melon pathogens with common names of the diseases they cause The main seed-borne squash pathogens with common names of the diseases they cause

154154 154 154 154 155 158 158 158 159 159

18

Specific InstructionsVegetable Crops of Family Brassicaceae Cabbages, Cauliflower, Turnip, Radish Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Brassica pathogens with

160160 160 160 160 161 164 164

6

common names of the diseases they cause The main seed- borne radish pathogens with common names of the diseases they cause Detect ion of Phoma l i ngam on Brassica spp. Detect ion of Xanthomonas campestr i s pv. campestris on Brassica spp. Method for the Detection of Xanthomonas campestris pv. campestris in disinfested seed of Brassica spp.

164 165 171 188

19

Specific InstructionsVegetable Crops of Family Fabaceae Peas, Beans 193 Seed unit 193 Submitted Sample Weight 193 Working Sample Weight 193 Purity Analysis 193 Germination 194 SEED HEALTH TESTING: 204 The main seed- borne peas pathogens with common 204 names of the diseases they cause 205 The main seed- borne Phaseo lu spathogens with common names of the diseases they cause The main seed- borne Broad beanpathogens with 207 common names of the diseases they cause Detect ion of Ascochyta pisi on Pisum sativum (Pea) [ISTA Validated Method] 208 Detection of Colletotrichum lindemuthianum on Phaseolus vulgaris (Bean) 216 Detection of Pea Early-Browning Virus and Pea Seed-borne Mosaic Virus on Pisum sativum 222 Detection of Pseudomonas savastanoi pv. phaseolicola on Phaseolus vulgaris Detection of Xanthomonas axonopodis pv. phaseoli 230 and Xanthomonas axonopodis pv. phaseoli var. fuscans on Phaseolus vulgaris 247

20

Specific InstructionsVegetable Crops of Family Apiaceae Carrot Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination

265265 265 265 265

7

SEED HEALTH TESTING: The main seed- borne carrot pathogens with common names of the diseases they cause The main seed- borne Parsn ip pathogens with common names of the diseases they cause The main seed- borne Pars l e y pathogens with common names of the diseases they cause The main seed- borne Ce le ry pathogens with common names of the diseases they cause Blot te r method for the detect ion Alteof rnar i a dauci on Daucus carota [ISTA] Detection of Xanthomonas hortorum pv. carotae on Daucus carota [ISTA] Alternaria dauci: ISTA Freeze Blotter test Alternaria dauci: Blotter/Pathogenicity test

268268 268 269 269 270 277 312 313

266

21

Specific InstructionsVegetable Crops of Family Chenopodiaceae Beetroot, Swiss chard, Spinach Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Beetroot pathogens with common names of the diseases they cause The main seed-borne Spinach pathogens with common names of the diseases they cause

315315 315 315 315 316 319 320

319

22

Specific InstructionsVegetable Crops of Family Malvaceae Okra Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Okra pathogens with common names of the diseases they cause

321321 321 321 321 321 323

23

Specific InstructionsVegetable Crops of Family Asteraceae Lettuce Seed unit

324

8

Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed- borne le t tuce pathogens with common names of the diseases they cause

324 324 324 324 327

24

Specific InstructionsVegetable Crops of Family Asteraceae Lettuce Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne lettuce pathogens with common names of the diseases they cause

328328 328 328 328 329 333

9

INTRODUCTIONPrime objective of seed quality evaluation is to make available high quality seeds and planting material having capacity to produce a plentiful crop of the required cultivar. Various evaluation and seed testing processes have been developed to assess the quality of seeds before sowing. The concept of seed quality may vary from the perspective of seed producer, Seed Company involved in seed marketing, the end user (farmer) and the government but anyhow the main objective of seed quality evaluation is to determine the value of seed for planting. Seed is a living biological product hence its behavior is not predictable with as sureness as is possible in case of nonliving inert products hence methods used for seed quality evaluation should be based on scientific knowledge of seed and the evaluation results should be reproducible universally within acceptable variance. Although broader definition of seed includes all parts of the plant which can be readily used as planting material but for the purpose of this manual the definition of seed is delimited to true seeds and seed units (fruits) marketed as seed. The text of the Training Manual that follows has been developed for professionals already acquainted with basics of seed testing procedures; to provide updated knowledge of the techniques used for evaluation of vegetable seeds of commerce.

10

It is acknowledged that Technical resources of ISTA, OECD and AOSA have been utilized to develop this Training Manual to present information on seed quality evaluation of vegetable crops ensuring credibility for seed transactions in international trade; with the hope that professional Seed Analysts working in public and private sector seed testing laboratories will benefit from the handbook.

PART-I

Basic Concepts of Seed Testing11

1.OBJECTIVE OF SEED QUALITY EVALUATIONThe objective of all methods and procedures used to analyze a representive sample of the seed lot is to get analytical information regarding the factors addressed in the specific seed standards and can include, depending on the crop and category of seed: 1. numbers per unit weight (per 25 g, per Kg, etc.) of other crop seeds, weed seeds and inert matter such as ergot/sclerotia; 2. percentages by weight of the components of the seed sample (pure seed, other crop kinds, weed seeds, inert matter, etc.); 3. moisture content in percentage; 4. percentages by number of germinable seeds, diseased seeds and seeds of other species.

Accuracy of the Results of Seed Analysis

12

Subject to the natural variations which occur in random sampling, the accuracy with which the results of seed analyses will represent a lot of seed depends on: a. The thoroughness of the blending of the lot of seed from which the sample is drawn; b. The care used in drawing the sample; c. The care with which a number of small samples drawn from several containers are mixed to form a composite sample representing a lot of seed or, when the lot is not homogeneous, the care taken in keeping unlike samples separate; d. The care with which the working sample is taken; e. The technical knowledge, skill and accuracy of the analyst; f. The condition of the equipment used.

Recognized Standard Methods: procedures set out in the:

The methods and

a. Procedures and Direct i ons for

b. c. d. e.

Seed Cert i f i c a t i on System in Pakis tan not i f i ed by Food and Agr icu l t u re Div is i on , Government of Pakis tan Rules for test i ng seed publ i shed by the US Associa t i on of Off i c i a l Seed Analyst (AOSA) Rules for Seed Test ing In ternat i ona l Rules for seed test publ i ng i shed by In ternat i ona l Seed Test ing Associa t i on ( ISTA) OECD Schemes for the Var ie ta l Cert i f i c a t i on or the Contro l of Seed Moving in In te rnat i ona l Trade Canadian Methods and Procedures for Test ing Seed

Reporting Results The Report of Analysis must contain the following information:o

Ident i f i c a t i on number. This must be a unique laborato ry - ass igned number which i s ident i ca l13

to

o o o o

o o

o o

the sample number and the identification number of any related worksheets; Lat in ame n of crop kind on which the reported analyses were conducted; Name of the laborato ry i ssu ing the Report ; A lo t ident i f i e r , such as seed lo t number, i f known Federa l Seed Cert i f i c a t i on and Regis t ra t i on Department (FSC&RD) Fie ld Inspect i on Report Number, i f known; Analyt i ca l resu l t s as requi red for speci f i c category of seed; Test method(s) used i f di f f e ren t f rom those prescr ibed in Methods and Procedures not i f i ed by Government of Pakis tan ; Signature of Accredi ted Analyst for the crop kind under analys i s ; Date test completed.

2. SEED SAMPLINGSeed quality evaluation requires inspection of seeds in a specific seed lot; whereas it is not practically possible to check each seed in the lot hence a representative sample of the seed lot is drawn and checked to assess quality of the whole seed lot. Statistical models developed on the basis of 99% probability of accuracy provide protocols for drawing a seed sample representing the seed lot in a reduced form. The seed sample is then subjected to various visual examinations and tests for assessing planting quality of the seed lot. Therefore accuracy of seed testing results directly depends on accuracy of protocol followed for drawing and dispatch of seed sample.

Definitions used in Seed Sampling:

14

1. True Seed or Botanica l Seed : Mature ovule is called true seed; without attachment of any other part of the flower or fruit. 2. Seed Units: Fruits, with or without other parts of the flower, commercially marketed as seed are called seed units as is the case of caryopsis, achenes, schizocarps etc. 3. Seed Lot: A seed lot is a specified quantity of seed, physically identifiable, in respect of which reproducible results can be drawn on the basis of sampling. 4. Primary Sample: A primary sample is a small portion taken from one point in the lot. 5. Composite Sample: The composite sample is formed by combining and mixing all the primary samples taken from the lot. 6. Submitted Sample: A submit ted sample is a sample that is to be submitted to the testing laboratory and may comprise either the whole of the composite sample or a sub-sample thereof. The submitted sample may be divided into sub-samples packed in different material meeting conditions for specific tests (e.g. moisture or health). 7. Working Sample: The working sample is the whole of submitted sample or a sub-sample taken from the submitted sample in the laboratory on which one of the quality tests is made. 8. Sub-sample: A sub-sample is a portion of a sample obtained by reducing the sample in a random manner. 9. Duplicate sample: A duplicate sample is another sample obtained for submission from the same composite sample and marked Duplicate sample. 10. Sealed: Sealed means that the container or individual containers in which seed is held are enclosed in such a way that they cannot be opened to gain access to the seed and closed again without either destroying the seal or leaving evidence of tempering. This definition refers to the sealing of seed lots and seed samples.

15

11. Sel f - seal i ng conta iners : The valve-pack bag is a specific type of self sealing container. It is filled through a sleeve-shaped valve which is automatically closed by the completion of filling the bag. 12. Marked/labeled: A container of a seed lot can be considered as marked or labeled when there is a unique identification mark on the container, which defines the seed lot to which the container belongs. All containers of a seed lot must be marked with the same unique seed lot designation (numbers, characters or combination of both). Marking of sub-samples must ensure that there is always an unambiguous link between the seed lot and the sub-samples. 13. Coated seeds: Coated seeds are seeds covered with material that may contain pesticides, fungicides, dyes or other additives. The following types of coated seeds are defined: 14. Seed pellets: More or less spherical units, usually incorporating a single seed with the size and shape of the seed no longer readily evident. 15. Encrusted seed: Units more or less retaining the shape of the seed with the size and weight changed to a measurable extent. 16. Seed granules: Units more or less cylindrical, including types with more than one seed per granule. 17. Seed tapes: Narrow bands of material, such as paper or other degradable material, with seeds spaced randomly, in groups or in a single row. 18. Seed mats: Broad sheets of material, such as paper or other degradable material, with seeds placed in rows, groups or at random throughout the sheets. 19. Treated seed: Seeds with treatments, which have not resulted in a significant change in size, shape or addition to the weight of the original seed. Size of Seed Lot: The quantity of a seed lot has been restricted for each crop based upon statistical calculations to ensure that the sample

16

stays to be representative of the particular seed lot. Ignoring this limit of lot size may lead to disingenuous results. The maximum seed lot weight permitted in respect of various vegetable crops is provided in Table-1. Preconditions for Seed Sampling: 1. Seed lot must be within range of maximum seed lot weight; and larger seed lots must be divided to more number of seed lots to confirm to maximum seed lot weight; and properly demarked and labeled accordingly prior to sampling. 2. Seed lot must be reasonably homogenous; and if there is a doubt in this respect, the seed lot may be well mixed to make it homogenous. The sample drawn from a heterogeneous seed lot is generally not representative of the seed lot. 3. Appropriate record of origin of the seed lot will be required in case of formal certification and labeling; however for seed packing under Truthful Labeling no such record is required. 4. When seeds are in sacks or in small containers, each sack or container should be theoretically and practically accessible for drawing primary samples. Sampling will not be representative if some portions of the seed lot are inaccessible. 5. Seed sample is drawn from a particular seed lot in two stages; first primary samples are drawn from the seed lot in a prescribed manner and then primary samples are mixed and reduced to form Submitted Sample for dispatch to seed testing laboratory. The prescribed quantity of Submitted Sample in respect of vegetable crops is detailed in Table-2. Marking/labeling and sealing of containers: The seed lot shall be in marked/labeled containers which are self-sealing, sealed (or capable of being sealed) or under the control of the seed sampler.

17

Where the seed lot is already marked/labeled and sealed before sampling, the seed sampler must verify marking/labeling and sealing on every container. Otherwise the sampler has to mark/label the containers and must seal every container before the seed lot leaves his/her control. The samplers are personally responsible for the seals, labels and bags supplied to them and it is their duty to ensure that primary, composite or submitted samples shall never be left in the hands of persons not authorized by the seed testing laboratory unless they are sealed in such a way that they cannot be tampered with.

Table- 1: MaximumSeed Lot Weight of Vegetable Crops, subject to a tolerance of 5%Sr. No . 1 2 3 4 5 6 7 8 9 10 Species Abelmoschus esculentus Allium cepa Beta vulgaris L. (all varieties) Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Coriandrum sativum Cucumis melo (all varieties) Cucumis sativus Crop Okra Onion Beet Cauliflower, Cabbage, etc Turnip, Chinese cabbage Pepper Watermelon Coriander Muskmelon, Long melon Cucumber Maximum Seed Lot Weight (MT) 20 10 20 10 10 10 20 20 10 10 18

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lactuca sativa Lagenaria siceraria Luffa acutangula Luffa aegyptica Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Praecitrullus fistulosus Raphanus sativus Solanum melongena Spinacia oleracea

Pumpkin Red gourd Marrow Cluster bean Carrot Lettuce Bottle gourd Ribbed gourd Sponge gourd tomato Bitter gourd Pea Tinda Radish Eggplant Spinach

20 10 20 20 10 10 10 20 20 10 10 20 10 10 10 10

Note: Seed pel le t s , seed granules , seed tapes or seed mats. The maximum number of seeds that a seed lo t of seed pel l e t s , seed granules , seed tapes or seed mats may conta in i s 1,000,000,000 (10,000 uni ts of 100,000) except that the weight of the seed lo t , inc lud ing the coat ing mater ia l ma not exceed 40,000 kg subject to a to le rance of 5% (42,000 kg) . When seed lo t s ize i s expressed in uni ts the tota l weig of the seed lo t must be given on the ISTA In te rnat i ona l See Lot Cert i f i c a t e . Maximum lot size for treated and encrusted seeds is defined by applying the quantities indicated in Table-1 to the seeds without coating material.

Table-2: Submitted Sample Weight of Vegetable CropsSPECIES CROP Submitte d Sample (g) 1000

Abelmoschus esculentus

okra

19

Allium cepa Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Praecitrullus fistulosus Cucumis melo Coriandrum sativum Cucumis sativus Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lagenaria siceraria Lactuca sativa Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Raphanus sativus Solanum melongena Spinacia oleracea

onion Cauliflower, Cabbage, etc Turnip, Chinese cabbage pepper watermelon tinda muskmelon coriander cucumber Pumpkin Red gourd marrow Cluster bean carrot Bottle gourd lettuce tomato Bitter gourd pea radish eggplant spinach

80 70 70 150 1000 150 150 500 150 1000 350 1000 800 30 700 700 15 700 1000 300 150 250

Methods for Drawing Primary Seed SamplesA. Seeds in Bulk: Port i ons of seeds shal l

be taken wit a st i ck sampler f rom at leas t the number of posi t i ons ind i ca ted below in Table- 3, selec ted randomly and inc lud ing both vert i ca l and hor i zonta l posi t i ons .

Table-3: Required Intensity of Primary Seed Samples for Seeds in Bulk

20

Sr. No . 1 2 3 4 5

Size of Lot (KG) Up to 50 51-1,500 1,501-3,000 3,001-5,000 5,001-20,000

Number of Positions To be Sampled Not less than 3 Not less than 5 At least 1 for each 300 kg Not less than 10 At least 1 for each 500 kg

B. Seeds in Sacks:

1. When seeds are in sacks they shall be sampled at random and samples are taken from the top, middle and the bottom of each selected sack. The position from which the seeds are taken shall be different from sack to sack and samples shall be drawn from different horizontal positions of the lot. 2. Wherever pract i cab le , seed shal l be sampled with a metal spear , tr i e r or probe wit a sol i d point which shal be of suf f i c i en t length to reach beyond the middle of the sack when inser ted f rom the s ide and shal l have an oval aperture so placed that the ins t rument removes port i ons of seeds of equal volume f rom each part of the sack through which i t trave l s . The ins t rument should be inser ted in to the sack in an upward di rec t i on at an angle of approx imately o 30 to the horizontal, with its aperture downwards until the aperture reaches center of the sack, then rotated to make aperture upwards and withdrawn at a uniform speed. 3. A stick sampler with more than one aperture and transverse partitions can be used through inserting it diagonally in closed position, then opened, gently agitated to fill completely, closed again and withdrawn. 4. Required intensity of primary seed samples for seeds in sacks or small containers is provided below in Table-4. C. Seeds in Small Containers:

21

1. When seeds are in smal l

conta iners ( less than 15 kg) a maximum 100 kg weight of seed i s taken as the basic uni t . Conta iners are combined to form sampl ing uni ts of 100 kg ( fo r example 20 conta iners each of 5 kg form one uni t ) . 2. Containers are properly shaken before drawing primary samples. 3. Samples are drawn at random in accordance with required intensity provided in Table-4. Table- 4: Required Intensity of Primary Seed Samples for Seeds in Sacks or Multiple Containers of 15 kg to 100 kg capacity (inclusively) . Sr. No. of Sacks or No Units of . Containers 1 1 to 5 2 6 to 14 3 4 5 15 to 30 31 to 49 50 or above Numbers to be Sampled Each sack or unit of containers Not less than 5 sack or unit of containers At least 1 for each sack or unit of containers Not less than 10 sack or unit of containers At least 1 in 5 sack or unit of containers

D. Seeds in Processing or Conditioning Machines:

Port i ons of seeds shal l be drawn dur ing seed process ing or condi t i on ing to get ent i re cross sect ion of the seed st ream at regular in te rva l s with sampl ing f requency as provided in Table- 5 below. Table-5: Required Intensity of Primary Seed Samples for Seeds during Seed Processing22

Sr. Size of Lot No (KG) . 1 Up to 50 2 51-1,500 3 1,501-3,000 4 5 3,001-20,000 20,001 and above

Number of Positions To be Sampled Not less than 3 Not less than 5 At least 1 for each 300 kg, But not less than 5 At least 1 for each 500 kg, but not less than 10 At least 1 for each 700 kg, but not less than 40

E. GENERAL:1. The quant i t y

of pr imary samples may be reduced to the quant i t y requi red as submit ted sample through mixing and halv ing method with hands. I f the pr imary samples appear uni fo rm they are combined to form the composi te sample, i f not , the sampl ing procedure must be stopped. When pr imary samples are col l ec ted di rec t l y in to one conta iner , the content of th i s conta in shal l be regarded as the composi te sample only i f i t appears uni fo rm, i f not , i t must not be used for obta in ing a submitted sample. 2. Portions of seed for moisture test shall be drawn separately in such a way as to prevent exposure to the environment and packed in air-tight containers for dispatch to the laboratory. 3. Seed samples should be proper ly labe with led the same ident i f i c a t i on as the seed in lo prescr t ibed manner before dispatch to the laboratory .

Preparation of Working Sample in the Laboratory

23

1.

Minimum Weight of Working Sample : Minimum Weight of Working Sample for var ious vegetable crops i s provided below in Table- 6. Obtaining the Working Sample : The submit ted sample f i r s t be thoroughly mixed and then Working Sample i s obta ined ei ther by repeated having or by abstrac t i ng and subsequent ly combining smal l random port i ons .

2.

Storage of Seed Samples1.

Before Testing: Every ef fo r t must be made to star t test i ng a sample on the day of receip t . I f delay i s unavoidable , the sample shal l be stored in cool wel l vent i l a t ed room in such a way that changes in the qual i t y of seed are minimized.

2.

After Testing:The pr imary aim of storage of samples after test i ng i s to be able to repeat the or ig ina l test carr i ed out on the submitted sample. Therefore , storage condi t i ons should be such that changes in the seed qual i t y tra i t s tested are minimal . E.g . in the cas of pur i t y test or other seed count , the sample should be stored in such a way that the physica l ident i t y i s kept . In the case of germinat ion , viab i l i t y or heal th t in orthodox seeds the sample should be stored under cool and dry condi t i ons . For such tests in reca lc i t r an t and in termediate seeds of trop i ca l and sub- t rop i ca l species , long term storage i s not poss ib le . For such seed of temperate species storab i l i t y i s depending on the fungal status and to some extent whether the seed i s dormant or not . Al l fac to rs perta in i ng to storage need to be determined on a species basis . Protect i on against insects and rodents may be necessary .

24

When a re-test in a different testing laboratory is required, a portion shall be drawn from the stored sample in accordance with prescribed method and submitted to the designated testing laboratory. The remainder shall be retained in store. Table-6: Working Sample Weight of Vegetable CropsSPECIES CROP Workin g sample (g) 140 8 7 7 15 250 70 70 50 70 700 180 700 80 3 70 70 7 70 900 30 15 25

Abelmoschus esculentus Allium cepa Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Praecitrullus fistulosus Cucumis melo Coriandrum sativum Cucumis sativus Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lagenaria siceraria Lactuca sativa Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Raphanus sativus Solanum melongena Spinacia oleracea

okra onion Cauliflower, Cabbage, etc Turnip, Chinese cabbage pepper watermelon tinda muskmelon coriander cucumber Pumpkin Red gourd marrow Cluster bean carrot Bottle gourd lettuce tomato Bitter gourd pea radish eggplant spinach

25

Mixing and Dividing Procedures1. Mechanical Divider Method This method is suitable for most kinds of seeds. The apparatus divides a sample into two approximately equal parts. The submitted sample is mixed by passing it through the divider, recombining the two parts and passing the whole sample through a second time and similarly a third time. The sample then is reduced by passing the seed through repeatedly and removing one-half on each occasion. This process of successive halving is continued until a working sample of the required size is obtained Use of compressed air is highly recommended for cleaning mechanical dividers. The dividers described below are examples of suitable equipment. Other devices may be used if it can be demonstrated that they provide an unbiased sub-sample.a.

Centri fugal Divider (Gamet type) This divider is suitable for all kinds of seed except oilseeds (rapeseed, canola, mustards, flax, etc.) and kinds susceptible to damage (peas, soybeans, etc.) and the extremely chaffy types. This divider makes use of centrifugal force to mix and scatter seeds over the dividing surface. The centrifugal divider tends to give variable results when not carefully operated. i. Preparation of the apparatus: Level the div ider using the adjustab le feet .

26

Check the div ider and four conta iners for clean l i ness . Note that seeds can be t rapped under the spinner and become a source of contaminat ion .

ii.

Sample mixing: Place a conta iner under each spout . Feed the whole sample in to the hopper; when f i l l i n g the hopper, the seed must always be poured centra l l y . After the sample has been poured in to the hopper, the spinner i s operated and the seed passes in to the two conta iners . Turn of f spinner . Ful l conta iners are replaced by empty conta iners . The contents of the two fu l l conta iners are fed centra l l y in to the hopper together , the seed being al l owed to blend as i t f l ows in . The spinner i s operated. The sample mixing procedure i s repeated at leas t once more. Sample reduction: The two fu l l conta iners are replaced by two empty conta iners . The contents of one fu l l conta iner are set aside and the contents of the other conta iner are fed in to the hopper. The spinner i s operated. The success ive halv ing process i s cont inued unt i l the working sample(s ) of not less than the minimum weight (s ) requi red as set out in Table 1 are obta ined. Ensure that the div ider and conta iners are clean after each mixing operat i on .

iii.

b.

Soil divider (r i f f l e divider)

27

This divider is suitable for most kinds of seed, including peas, beans, soybeans, etc., provided the fall of the seed is not such that the seed will be damaged. For round-seeded kinds such as Brass i ca , the rece iv ing pans should be covered to prevent the seeds f rom bouncing out . This divider consists of a hopper with attached channels or ducts, a frame to hold the hopper, four collecting pans and a pouring pan. Ducts or channels lead from the hopper to the collecting pans, alternate ones leading to opposite sides. Riffle dividers are available in different sizes for different sizes of seed. The width and number of channels and spaces are important. The width of the channels must be at least two times the largest diameter of the seed or any possible contaminants being mixed. This apparatus, similar to the centrifugal divider, divides the sample into approximately equal parts. i. Preparation of the apparatus: Place the r i f f l e div ider on a f i rm, leve l clean sur face . Ensure the div ider i s leve l . Ensure that the div ider and the 4 sample col l ec t i on conta iners are clean. Check al l channels , jo in t s and seams of the div ider and col l ec t i on conta iners to ensure there are no seeds or other plant matter present before each use. Two clean empty col l ec t i on conta iners shal l be placed under the channels to receive the mixed seed. Sample mixing: Pour the whole sample in to the div ider by running the seed backwards and forwards along the edge of the div ider so that al l the channels and spaces of the div ider receive an equal amount of seed.28

ii.

The two fu l l conta iners shal l be rep laced with two clean empty col l ec t i on conta iners . The contents of one fu l l col l ec t i on conta iner shal l be poured in to the div ider by hold ing the long edge of the pan against the long edge of the r i f f l e hopper and then rota t i ng the bottom up so that the seeds pour across al l channels at the same t ime. Repeat the procedure with the other fu l l conta iner . This process of mixing the ent i re submit ted sample shal l be repeated at leas t 2 more t imes before success ive halv ing begins .

iii.

Sample reduction: The contents of one fu l l col l ec t i on conta iner are set aside. Empty col l ec t i on conta iners are placed under each channel , and the contents of the other conta iner i s poured in to the hopper by hold ing the long edge of the pan against the long edge of the r i f f l e hopper and then rotat i ng the bottom up so that the seeds pour across al l channels at the same t ime. The success ive halv ing process i s cont inued unt i l the working sample(s ) of not less than the minimum weight (s ) requi red as set out in Table 1 are obta ined. Ensure that the div ider and col l ec t i on conta iners are clean after each mixing operat i on . Check al l channels of the div ider , the jo i n t s and seams.

2. Hand Mixing or Spoon Method This method should only be used for samples of a single small-seeded species that are smaller than Tr i t i c umspp. , very chaf fy species or uncleaned seed where i t i s demonstrated that one of the mechanica l div iders wi l l not take a representat i ve working sample(s ) . The sample i s poured uni fo rmly over a t ray with a s ide to s ide swinging motion. The receiv i ng pan should be kept leve l . This mixing29

procedure is repeated a minimum of three times. A tray, a spatula and a spoon with a straight edge are required. After the preliminary mixing, pour the seed evenly over the tray with a side-to-side swing, alternately in one direction and at right angles to it. The depth of the seed in the pan shall not exceed the height of the vertical sides of the spoon. Do not shake the tray thereafter. With the spoon in one hand, the spatula in the other, and using both, remove small portions of seed from not less than five random places on the tray. Sufficient portions of seed are taken to constitute a working sample(s) of the required size as set out in Table-6.

30

PURITY ANALYSIS1. Objective The objective of purity analysis is to determine (a) the percentage composition by weight of the sample being tested and by inference the composition of the seed lot, and (b) the identity of the var ious species of seeds and iner t part i c l e s const i t u t i ng the sample. 2. Definitions 2.1 Seed: A seed, in laboratory practice, is defined as "a structure which contains at least one ripened ovule with or without accessory parts". In many crop plants, the structure commonly regarded as the seed is botanically a fruit. Thus, in addition to true seeds, the foregoing definition includes florets and caryopses in the Poaceae, achenes, cypselas, schizocarps, mericarps, nutlets, one- and two-seeded pods of small-seeded legumes, seed balls, or portions thereof, in Beta spp. , f ru i t s with enclos ing calyx as in Tetragonia tetragonioides and bulblets as in Poa bulbosa. It also includes coated seed. Very often structures which do not strictly comply with the above definition are included in the pure seed because the analyst cannot tell whether or not the ripened ovule is present. 2.2 Pure Seed:

31

The pure seed shall include seed of the crop kind (or kinds) under analysis which must be named in labelling, and includes small, immature, shrivelled, cracked, insect damaged, diseased, sprouted, or otherwise injured seeds, provided that: In the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s ize shal l be cons idered pure seed except that seeds of the Fabaceae and Brass i caceae with thei r seedcoats ent i re l y removed shal l be regarded as iner t matter . For separated coty ledons of seeds of the Fabaceae refer to def in i t i o n of Iner t Matter . b. Intact seed units (commonly found dispersal units such as achenes and similar fruits, schizocarps and mericarps with or without perianth and regardless of whether they contain a true seed) shall be considered pure seed unless it is readily apparent that no true seed is present.a.

(The term "readily apparent" shall be interpreted to mean that the purity analyst should not use a diaphanoscope, stereoscopic microscope, hand lens, pressing or other special equipment or means to detect whether true seeds are present.) c. In the case of the florets and caryopses of Poaceae, pure seed shall consist of: i. Broken florets or free caryopses, provided they are larger than one-half the original size; ii. Entire florets and one-seeded spikelets with an obvious caryopsis containing endosperm,32

as determined by the use of slight pressure or by examination over light; iii. Floret s of the crop genera Lol i um and Festuca in which the caryopsis is at least one-third the length of the palea as measured from the base of the rachilla; iv. In the case of the Festuca spp., Agropyron cristatum or Agropyron desertorum, attached sterile florets which do not extend to or beyond the tip of the fertile floret shall be left attached and considered part of the pure seed. The length of an awn shall be disregarded when determining the length of a sterile floret; v. Where the uniform blowing method is used, all material of the kind of seed under analysis which remains in the heavy portion after blowing according to the instructions for that kind of seed, not including: Broken florets or free caryopses which are one-half or less than one-half of the original size, Other crop seeds, Weed seeds, Heavy inert matter, In the case of Dactylis glomerata, one-fifth the weight of multiple florets (see section 3.7). vi. Florets with fungus bodies, such as ergot (Claviceps

33

purpurea) , ent i r e l y enclosed with in lemma and palea, and vii. Four- f i f t h s the weight of mult ip l e f l o re t s remain ing in the heavy port i on in the case of Dacty l i s glomerata .

2.3 Other Crop Seeds: Seeds of crop kinds listed in the Procedures and Direct i ons for Seed Cert i f i c a t i on System in Pakis andtan found as contaminants in a sample shal l be cons idered other crop seeds, except that certa in seeds and st ruc tures as descr ibed in the Defin i t i o ns here be cons idered iner t matter . 2.4 Weed Seeds:

Seeds of plants not listed as crop kinds in the Procedures and Direct i ons for Seed Cert i f i c a t i on System in shal Pakis l tan be cons idered weed seeds, except certa in seeds and st ruc tures as descr ibed in the Defin i t i o ns shal l be considere iner t matter . The class i f i c a t i on of seeds as noxious or othe weeds shal l be accord ing to Procedures the and Directions for Seed Certification System in Pakistan. Structures such as the bulblets of Poa bulbosa and Allium vineale and tubers of Cyperus esculentus shall be considered weed seeds. Stolons (including pieces) of various grass species capable of germination shall be treated as weeds (and not as inert matter). 2.5 Inert Matter from the Crop Kind(s) Under Analysis: a. Pieces of broken or damaged seeds: i. ii. One-half the original size or less; Seeds of Fabaceae and Brassicaceae with the seedcoats entirely removed;34

Separated cotyledons of Fabaceae, irrespective of whether or not the radicle-plumule axis and/or more than half of the seed coat may be attached; iv. Structures in which it is readily apparent that no true seed is present. 2.6 Inert Matter from Weed Plants and Other Crop Plants Found as Contaminants: All broken seeds, florets or free caryopses which are half or less than one-half the original size, including structures which, by visual examination (including dissection and the use of the diaphanoscope) can be definitely demonstrated as falling within the categories listed below. Doubtful structures shall be classed as weed seeds or other crop seeds as the case may be. a. Seeds and structures of Poaceae 1. Florets or free caryopses, with more than one-half the radicle-plumule axis missing, 2. Glumes and empty florets when unattached to fertile florets, 3. Attached ster i l e f l o re t s and basal appendages which must be removed f rom the fer t i l e f l o re t s and considered part of the iner t matter except , in the fo l l ow ing cases: o Attached ster i l e f l o re t s and basal appendages are not removed f rom: Agrost i s spp. ; Alopecurus spp.; Arrhenatherum elatius; Dactylis glomerata; Phalaris arundinacea; Poa spp.; and o Attached sterile florets which do not extend to or beyond the tip of the fertile floret are not35

iii.

removed from Festuca spp. ; Agropyron cristatum, or Agropyron desertorum. The length of an awn shall be disregarded when determining the length of a sterile floret. 4. Immature florets of Elytrigia repens with a caryopsis less than one-third the length of the palea, measured from the base of the rachilla, 5. Free undamaged caryopses of Agropyron spp. 2 mm or less in length, 6. Florets containing fungus bodies, such as ergot (Claviceps purpurea). b. Seeds of families other than Poaceae Seeds that are devoid of embryo. Seeds of Fabaceae, and Brassicaceae with the seed coat entirely removed. iii. Separated cotyledons of Fabaceae, irrespective of whether or not the radicle-plumule axis and/or more than half of the seed coat may be attached. iv. Empty fruits (seeds) such as occur in the following plant families: Asteraceae, Convolvulaceae, Cyperaceae, Polygonaceae, Solanaceae, etc. v. Seeds of Cuscuta spp. which are either fragile or ashy gray to creamy white in colour. vi. Seeds of Plantago lanceolata which are black, with no brown colour present, whether shrivelled or plump. The colour of questionable seeds should be determined under a magnification of approximately 10x with strong light. i. ii.

36

Dehul led Ambrosia spp. ( invo luc re and per icarp absent) . viii. Ind iv i dua l seeds Juncus of tenuis or other species Juncus of having seeds of a similar size shall be considered inert matter. Clusters or capsules of Juncus spp. shall be left intact, counted and included with the weed seeds. ix. Multiple structures, capsules, pods, heads, etc., are opened, the seeds are removed, and the non-seed material is placed with the inert matter, except as noted in part viii above for Juncus spp..vii.

2.7 Inert Material other than Seeds: The following materials shall be classed as inert matterin all cases: a. Nematode galls, including galls enveloped by the lemma and palea of grass florets, b. Fungus bodies such as ergot (Claviceps purpurea) and other sclerotia, and smut balls except as defined in section 3.2.2 c.vi, c. Chaff, stems, leaves, stone cells, stones, sand, soil particles, dust, and any other material not seeds, and d. Seeds (crop and weed seeds) in which larvae occupy one-half or more of the seed unit. 3. General Principles The working sample is separated into three components (pure seed, other seeds and inert matter) in ISTA Scheme, into four four components (pure seed, other crop seeds, weed seeds and inert matter) in Canadian Scheme and into five components (pure seed, other varieties seeds, other37

crop seeds, weed seeds and inert matter ) in Pakis tan i Scheme; and percentage of each part i s determined by weight . Al l species of seed and al l kinds of iner t matter present shal l be ident i f i ed as far as poss ib le and, i f requi r for report i ng , i t s percentage by weight shal l be determined. 4. Apparatus Aids such as transmitted light, sieves and blowers may be used in separating the component parts of the working sample.5.

Procedure for separation of components

The working sample shall be separated into its component parts based on an examination of each particle in the sample, but in certain cases special procedures are obligatory, such as uni fo rm blowing or s iev ing . The separation of the pure seed must be on such a basis that i t can be made by vis ib l e seed character i s t i c s , mechanica l aids or using pressure without impai r i ng the capaci ty for germinat ion . After separation, each component part and any species of seed or seeds of other varieties or kind of inert matter for which a percentage is to be reported, shall be weighed in grams to the minimum number of decimal places necessary to calculate the percentage to one decimal place; as detailed below in Table-7.

Table-7: Minimum number of decimal places required for weighment of components Weight of Weigh the working sample and its Working Sample components to the following number (in grams) of decimal places

38

Less than 1 1 to 9.999 10 to 99.99 100 to 999.9 1000 or more

4 3 2 1 0

6. Calculation and Expression of Results The percentage by weight of each of the component parts shall be calculated to one decimal place. Percentages must be based on the sum of the weight of the components, not on the original weight of the working sample, but the sum of the weight of the components must be compared with the original weight as a check against loss of material or other error. If a duplicate analys i s i s made on two hal f working samples , the di f f e rence between the two must not exceed the to le rance between dupl i ca te analyses given in TableWhen two or more purity analyses of whole working samples are made, the results must be expressed as a weighted average percentage.

7. Reporting Results The results of a purity analysis shall be given to one decimal place and the percentage of al l components must tota l 100.

39

components of less than 0.05% shall be reported as Traces. If the result for a component is nil, this must be shown as 0.0 in the appropriate space. The Latin names of the species of pure seed, other crop seeds, weed seeds; and kinds of inert matter must be reported on the Analysis Certificate / Report. Percentage of a particular species of other crop seed or weed seed or kind of inert matter, at the request of the sender, must be shown on the Analysis Certificate / Report.

Table- 8. Tolerances for comparing percentage of purity analysis components, to determine i f two tests from same submitted sample are compatible.Average of two working samples (%) 99.95-100.00 99.90-99.94 99.85-99.89 99.80-99.84 99.75-99.79 99.70-99.74 99.65-99.69 99.60-99.64 99.55-99.59 99.50-99.54 99.40-99.49 0.00-0.04 0.05-0.09 0.10-0.14 0.15-0.19 0.20-0.24 0.25-0.29 0.30-0.34 0.35-0.39 0.40-0.44 0.45-0.49 0.50-0.59 Tolerance of Nonchaffy seeds 0.14 0.23 0.28 0.33 0.36 0.39 0.43 0.46 0.48 0.51 0.54 Tolerance of Chaffy seeds 0.16 0.24 0.30 0.35 0.39 0.42 0.46 0.49 0.52 0.54 0.58

40

99.30-99.39 99.20-99.29 99.10-99.19 99.00-99.09 98.75-98.99 98.50-98.74 98.25-98.49 98.00-98.24 97.75-97.99 97.50-97.74 97.25-97.49 97.00-97.24 96.50-96.99 96.00-96.49 95.50-95.99 95.00-95.49 94.00-94.99 93.00-93.99 92.00-92.99 91.00-91.99 90.00-90.99 88.00-89.99 86.00-87.99 84.00-85.99 82.00-83.99 80.00-81.99 78.00-79.99 76.00-77.99 74.00-75.99

0.60-0.69 0.70-0.79 0.80-0.89 0.90-0.99 1.00-1.24 1.25-1.49 1.50-1.74 1.75-1.99 2.00-2.24 2.25-2.49 2.50-2.74 2.75-2.99 3.00-3.49 3.50-3.99 4.00-4.49 4.50-4.99 5.00-5.99 6.00-6.99 7.00-7.99 8.00-8.99 9.00-9.99 10.00-11.99 12.00-13.99 14.00-15.99 16.00-17.99 18.00-19.99 20.00-21.99 22.00-23.99 24.00-25.99

0.59 0.63 0.67 0.71 0.76 0.84 0.91 0.97 1.02 1.08 1.13 1.18 1.25 1.33 1.41 1.48 1.59 1.72 1.83 1.94 2.04 2.18 2.34 2.49 2.61 2.73 2.83 2.93 3.01

0.63 0.67 0.71 0.75 0.81 0.89 0.97 1.04 1.09 1.15 1.20 1.26 1.33 1.41 1.50 1.57 1.68 1.81 1.93 2.05 2.15 2.30 2.47 2.62 2.76 2.88 2.99 3.09 3.18

41

72.00-73.99 70.00-71.99 65.00-69.99 60.00-64.99 50.00-59.99

26.00-27.99 28.00-29.99 30.00-34.99 35.00-39.99 40.00-49.99

3.09 3.16 3.26 3.37 3.46

3.26 3.33 3.44 3.55 3.65

Taken from Table P11, 5% probability, Handbook of Tolerances (S.R. Miles, Proceedings of the International Seed Testing Association, Volume 28, Number 3).

Determination of Other Seeds by NumberObjective: The objective of the determination is to estimate the number of seeds of other species (for example objectionable weeds or noxious species) required under the notified standards or requested by the sender. Procedure: The determination is made by count and expressed as number of seeds found in the quantity examined. When seeds found cannot be identified with certainity to the species level, it is permitted to report the genus name only.

42

The working sample for determination by count shall be the whole submitted sample or equal to submitted sample if duplicate sample is drawn for the purpose. If a species designated by the sender is difficult to ident i f y , only a minimum one f i f t h of the prescr ibed working sample for counts need be examined for that part i cu l a r species . Calculation and Expression of Results: The result is expressed as the number of seeds belonging to each designated species or category found in actual quantity found. In addition number per unit weight (eg per kilogram) may be calculated. Reporting Results: The actual weight of seed examined, and the Latin name and number of seeds of each species sought and found in this weight, alongwith calculated count per unit weight, shall be reported on the Analysis Certificate / Report.

Verification of Species and Cultivars And Separation of Other VarietiesObjective: The objective is to to determine the the extent to which the submitted seed sample conform to the species (us ing methods not permiss ib l e in pur i t y test ) or cul t i va r cla imed and extent of other species or other cul t i va r contaminants . Precondition for the Test: The determination is valid only if the species or cultivar is stated by the sender of the sample and if an authentic standard sample the species or cultivar is available for43

comparison. The characters compared may be morpholog ica l , physio log i ca l , cyto log i ca l or chemical . General Principles: The determination is carried out, depending on the species or cultivar in question, on (a) Seeds, (b) Seedlings or more mature plants grown in a laboratory, a glasshouse, a growth chamber or field plots. Normally seeds are compared with seeds from the authentic sample and seedlings and plants are compared with seedlings and plants at the same stage of development grown from the authentic sample contemporaneously, nearby and in identical environmental conditions. Exceptionally, depending on the certainity of the determination (eg ploidy), comparison with the authentic control sample is not necessary. In the case of species or cul t i va r s that are suf f i c i en t l y uni fo rm as to one or more diagnost i c characters (eg in sel f pol l i na ted species ) , a count i s made of the number of seeds, seedl ings or plants that are not genuine. I f the species or cul t i va r i s not suf f i c i en t l y uni fo rm (eg in cross - pol l i na ted species ) , a count i s made of any obvious of f - types and a genera l judgement i s expressed as to the genuineness of the sample under test . Apparatus and Facilities: A determination shall be made only if apparatus, equipment and other facilities are available for the character to be stud ied , and in genera l as fo l l ows ; a) In the laboratory apparatus and reagents for morphological, physiological and cytological

44

examinations, chemical tests and germination of seeds as appropriate. b) In glasshouse and growth chambers provision of controlled environmental conditions adequate to induce the development of diagnostic characters. c) In f i e l d plots cl imat i c , edaphic and cul tu ra l condi t i o to permit normal development of the diagnost i c characters and suf f i c i en t protect i on against pests and diseases . In all cases, the determination shall be made by a specialist familiar with the morphological, physiological or other characters of the species or cultivar in question. Weight of Submitted Samples: Table-9A: Minimum weight of Submitted Samples for Determinat ion of Species / Cult i va r sSr. No . 1 2 3 4 Species Laborator y Only (g) Field Plot & Lab (g) 2,000 1,000 500 250

Pisum, Phaseolus , Vic ia , Lup inus Zea, , Glyc ine and other species 1,000 with seeds of similar size Hordium, Avena, Triticum, Secale and other species with seeds of 500 similar size Beta and other spec ies with seeds of 250 s imi la r s ize All other genera with smaller 100 seeds

Examination of Seeds: 1. Working sample: Not less than 400 seeds, taken at random from a sub-sample drawn in accordance with the procedure followed for drawing the working sample. 2. Determinations:

45

a. For morpholog ica l

characters , the seeds shal l be examined with the aid of sui tab le magnify ing apparatus when necessaryBecause . of the s imi la r i t y in appearance among the species of Brass i caand Sinapis, all portions of the Brassica and Sinapis samples must be examined using a stereoscopic microscope, except that for samples of Sinapis alba, a microscope need not be used. b. For color characters , the seeds may be examined under fu l l dayl igh ort l i gh t of l imi ted spect rum, eg ul t rav i o l e t . c. For chemical characteristics, the seeds shall be treated with the appropriate reagent and the reaction of each seed noted. Examination of Seedlings:1. Working sample: Not less than 400 seeds (or for

plo idy in i t i a l l y 100 with a fur ther 100 i f determinat ion inconc lus i ve ) , taken at random f rom a sub- sample drawn in accordance with the procedure fo l l owed for drawing the working sample. The seeds shal l be germinated in repl i ca tes of not more than 100, on an appropr ia te medium. When the seedl i ngs have reached a sui tab le stage of development , they are examined in whole or in part , with or without fur ther treatment . For a determinat ion of plo idy , root t ip or other t i s sue i s excised and processed for microscopic examinat ion .

2. Determinat ion :

Examination of Plants in Glasshouse or Growth Chamber: 1. Working sample: Sufficient seeds to produce not less than 100 plants, but this number may be reduced in the case of climbing or creeping species. The seeds shall be taken at random from a sub-sample drawn in

46

accordance with the procedure followed for drawing the working sample.2. Determinat ion :

The seeds shal l be sown in sui tab le conta iners and mainta ined in the envi ronmental condi t i ons necessary for the development of diagnost i c characters . When the plants have reached a sui tab le stage of development, the cr i t i c a l characters shal l be observed on each plant and noted.

Examination of Plants in Field Plots: The submitted sample shall be sown (in whole or in part) as soon as pract i cab le after rec iept . Each sample shall be sown in at least two replicate plots. As insurance against failure the replicates should be situated in different fields or different parts of the same field. The plots may be of any convenient size that will provide enough plants for the determination to be the accuracy required. If the seed is sown in situ, it shall be sown in rows, mechanically if possible. Spacing between rows and between plants shall be sufficient to allow development of the characters to be examined. Both transplanting and thinning are possible source of error and the sowing rate shall be adjusted to produce approximately the same number of plants in the test and control plots. When absolutely necessary, thinning or transplanting of seedlings from elsewhere in the plot is permissible.

Observations shall be made during the whole growing per iod , but part i cu l a r l y at the stages for appearance of diagnost i c characters and deviat i ons f rom the contro l sample recorded. Plants recognizeab le as belonging to another cul t i va r or species or as aberrant (eg spel to i d wheat) shal l counted and recorded. Ei ther an actua l count or an est imate of the number of plants in the plot shal l be made at the t im of plant examinat ion .

47

Calculations and Expression of Results: When not more than 2,000 seeds, seedl ings or plants are examined, the number found to be not genuine i s computed as a percentages without decimals . I f more than 2,000 are examined, the number i s computed as a percentages with one decimal place. In determination of seedlings, the results are expressed as percentage of normal seedlings examined; and abnormal seedlings are excluded from the computation. In case of field plot examination, the number of plants of genuine species / cultivar, other species / cultivars and aberrants shall be calculated as percentages of total number of plants examined.

When characters are measured, the mean and other stat i s t i c s may be calcu la ted . Cult i va r s of cross - fer t i l i z i n g species often show var iab i l i t y of plant character i s t i c s to s a degree that i t i s di f f i c u l t to def ine accurate ly al l of f - ty in such cases, any calcu la t i ons of percentage impur i t i e s sha be supplemented by appropr ia te comments about the genuineness of the test sample. Reporting Results:

For laboratory, glasshouse or growth chamber tests, the number of seeds, seedlings or plants examined shall be stated and the results be reported as the percentage of extraneous seeds or seedlings. If none are found, the results shall be reported as follows: The laboratory (or glsshouse) examination for genuineness of th i s sample revealed noth ing to ind i ca te that the species and cul t i va r stated by the sender i s incor rec t .

48

In case of a fluorescence test of Lol i um, the resu l t s shal l reported as fo l l ows : Of seeds producing normal seedlings, % react positively and % react negatively to ultraviolet light.

be

The result of a field plot examination, whenever possible, shall be reported as percentage of each other species, other cul t i va r s or aberrants found. I f a sample i s found to be a cul t i va r other than that stated by the sender , th i s resu l t s be repor ted . I f the proport i on of plants of other cul t i va r s present in sample exceeds 15%, the repor t shal l state addi t i ona l l y that , The sample cons is t s of a mixture of di f f e ren t cul t i va r s . If none other cultivar found, the results shall be reported as follows: The field plot examination for genuineness of this sample revealed nothing to indicate that the species and cultivar stated by the sender is incorrect.

49

Determination of Seed Moisture ContentDefinition: Moisture content of a sample is the loss in weight when it is dried without oxidation, decomposition or the loss of other volatile substances. Sample weight: 100 g for species that have to be ground and 50 g for all other species. When moisture meters are to be used for testing, a larger sample size may be necessary. Sample condit ion: Samples for moisture tests must be received in airtight, moisture proof, and undamaged containers, which must be filled with seed to ensure that as much air as possible has been excluded. The sample should not be used for purity or germination purposes due to the nature of the container and the sub-sampling method. Tests should commence as soon as possible after receipt and exposure of the seed to the ambient air should be kept to a minimum. When the above requirements have not been met, new samples must be requested. Procedure: Either of the following methods may be used for moisture test determinat ion . A. Constant Temperature Oven Method: The determination shall be started as soon as possible after reciept of the sample. For species that do not require50

grinding, no more than two minutes may elapse from the time the sample is removed from the container in which it was received untill the working sample is enclosed in the drying container and weighed. Weighing: Weighing shall be in grams to three decimal places. Working sample: the determination shall be carried out in duplicate on two independently drawn working samples each of the following weight, depending on the diameter of the containers used: Less than 8 cm diameter: 4 to 5 g 8 cm diameter or larger: 10 g before the working sample is drawn the submitted sample shall be thoroughly mixed without exposure to the atmosphere and working sample should be prepared swiftly within 30 seconds to avoid exposure. Grinding: Larger seeds must be ground before drying unless their high oil content makes them difficult to grind or (particularly in seed of Linum with oi l of a high iod ine number) l i ab l e to gain in weight through oxidat i on . Grinding of fol lowing vegetable species is obligatory: Cit ru l l u s lanatus , Lathyrus spp. , Phaseolus spp. , Pisum sat i vum, Vic ia spp. Pre-drying: I f the species i s one for which gr ind ing i s necessary and the moisture content i s more than 17% (or 10% in the case of Glyc ine max and 13% in the case of Oryza sative), pre-drying is obligatory. Two sub-samples, each weighing at least 25g are placed in weighed containers and then dried to reduce moisture content to less than the limit. After pre-drying the sub-samples are reweighed in their containers to determine the loss in weight. Immediately51

thereafter the two partly dried sub-samples are separately ground and subjected to following prescribed methods:

Vegetable species for which low constant temperature oven method shall be used: Allium spp., Brassica spp., Capsicum spp., Raphanus sat i vus and Solanum melongena Vegetable species for which high constant temperature oven method shall be used: Asparagus officinalis, Beta vulgaris, Citrullus lanatus, Cucumis spp. , Cucurbi ta spp. , Daucus carota , Lactuca sat i va , Lathyrus spp. , Lycopers i con lycopers i cum, Phaseolus spp. , Pisum sat i vum, Spinac ia oleracea, Vic ia spp. Low constant temperature oven method: The working sample must be evenly distributed over the surface of the container. Weigh the container and its cover before and after filling. Place the container rapidly, on top of its cover, in an oven maintained at a temperature of 1032 oC and dry for 171 hour. The drying period begins at the time the oven returns to the required temperature. At the end of the prescribed period cover the container and place in a desiccator to cool for 30-40 minutes. After cooling Weigh the container with its cover and contents. The relative humidity of the ambient air in the laboratory must be less than 70% when the determination is carried out. High constant temperature oven method: The procedure is same as described above except that the oven is maintained at a temperature of 130-133oC and dried52

for four hours for Zea mays, two hours for other cereals and one hour for other species (including all vegetable crops). No condition of relative humidity in the laboratory.

Calculation of Results: The moisture content as a percentage by weight shall be calculated to one decimal place by means of the following formula: (M2 M3) X 100 (M2 M1) Where M1 is the weight in grams of the container and its cover, M2 is the weight in grams of the container and its cover and its contents before drying, and M3 is the weight in grams of the container and its cover and its contents after drying. If the material is pre-dried (first stage), the original moisture content of the sample is calculated to one decimal place by means of the following formula: S1 + S2 - S1 X S2 100 Where S1 is the moisture lost in in thhe first stage, and S2 is the moisture lost in in thhe first stage. Tolerance: Take as the result the arithmetic mean of the duplicate determinations carried out on a sample if the difference

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between the two determinations does not exceed 0.2%. otherwise repeat the determination in duplicate. B. Electric Moisture Meter The electric moisture meter of var ious models can be used i f cal i b ra t i on i s cert i f i e d by a competant agency. Seed test i ng laborato r i es using the Model elect r i c moisture meter must have appropr ia te convers ion charts for the crop kind under test . In addi t i on , they must per iod i ca l l y compare tests with elect r i c moisture meter with constant temperature oven method for standard i za t i on purposes.

Determination of 1,000 Seed WeightDefinition: Weight of 1,000 seeds in grams calculated from the average of 8 replications of 100 seeds drawn manually at random from the working sample; or f rom count ing whole working samle by the machine. The results shall be expressed to the number of decimal places used in purity determinations. Procedure: Working sample: The working sample shall be the entire pure seed fraction of a purity analysis. Counting the entire working sample: Put the whole working sample through the machine, and read vthe number of seeds on the ind i ca to r . After count ing weigh the sample in grams to the same number of decimal places as in the pur i t y analys i s ; then calcu la te the weight of 1,000 seeds accord ing ly .

Counting replicates: From the working sample count out at random, by hand or with a germination counter, eight repl i ca tes , each of 100 seeds. Weigh each rep l i ca te in grams54

to the same number of decimal places as in the purity analysis. Calculate the variance, standard deviation and coefficient of variation as follows: Variance = n(x2) - (x)2 n(n-1) where x = weight of each replicate in grams n = number of replicates = sum of ________ standard deviation (s) = variance coeffient of variation = (s/x) X 100 where x = mean weight of 100 seeds if the coefficient of variation does not exceed 6.0 for chaffy grass seeds, or 4.0 for other seeds, the results of the determination can be calculated; otherwise count and weigh a further 8 replicatesand calculate the standard deviation for the 16 replicates. Discard any replicate that diverge from the mean by more than twice the standard deviation so calculated.

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Purity Procedures for Coated Seed

NOTE: Standards and labe l l i n g requi rements for coated seed are not yet not i f i ed under Seed the Act , 1976 . The catagory of the seed or seed mixture must be estab prior l i shed to coating. However, the percentage by weight of seed must appear on the label. Because of the nature of the coated seed product and the procedures for testing, it is not valid to compare percentage by weight values determined by testing de-coated seed, with the standards notified in the rules. Procedures for determining percentages by weight of coated seed are therefore not given here. If necessary for information purposes, the procedures for testing coated seed given in the current Rules for Testing Seeds, published by the Association of Official Seed Analysts (USA), should be used. The following procedure may be used to determine if56

imported seed meets the minimum standards for foreign seeds. Definition Coated seed is a seed unit covered with any substance which changes the size, shape, or weight of the original seed. Seeds coated with ingredients such as, but not limited to, rhizobia, dyes, and pesticides are excluded. Obtaining the working sample The minimum submitted sample size for samples of coated units to be submitted for a foreign seed determination should be approximately 50,000 units. Due to variation in weight of coating materials, the size or weight of the working sample shall be determined separately for each lot. This weight shall be determined by weighing 100 completely coated units and calculating the weight of 25,000 units for the foreign seed determination. Use the methods described under purity analysis to obtain the working sample. Table-9B: Sample sizes of pelleted seeds in number of pellets

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Sr. Determinations No. 1 2 3 4 5 6 Purity analysis (including verification of species) Weight determination Germination

Submitted samples not less than 7,500 7,500 7,500

Working Samples not less than 2500 Pure pellet fraction 400 7500 25000 2000

Determination of other seeds 10,000 Determination of other seeds (encrusted seeds and seed granules) Size grading 25,000 10,000

Table-9C: Sample sizes of seeds tapes Sr. Determinations No. 1 Verification of species 2 4 5 Germination 2,500 Purity Analysis (if required) Determination of other seeds 2,500 10,000 400 2,500 7,500 2,500 seeds 100 seeds Submitted Working samples not Samples less than not less than

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Foreign seed determination To determine the number of foreign seeds (noxious weeds, other weeds and other crops) per unit weight, examine approximately 25,000 units which have been de-coated. Remove the coating material from the seed by washing with water or other solvents such as, but not limited to, vinegar (5% acetic acid) or dilute sodium hydroxide. Use of fine mesh sieves is recommended for this procedure and stirring or shaking the coated units may be necessary to obtain decoated seed. Spread on blotters or filter paper in a shallow container. Air dry overnight at room temperature.

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The Germination Test1. Introduction The object of testing for germination is to determine the maximum germination potential of the seed. Testing under field conditions is normally unsatisfactory, as the results cannot be repeated with reliability. Laboratory methods have, therefore, been evolved in which the external conditions are controlled to give the most regular, rapid and complete germination for the majority of samples of a particular species. The conditions have been standardized to enable the test results to be reproduced within limits as near as possible to those determined by random sample variation. This chapter provides the methods and procedures to be used for germination testing for the purpose of grading seed. 2. Methods to be used 2.1 Prescribed methods The laboratory methods prescribed in section 6.2 (Table- 10) shal l be used when germinat ion tests are to be used as the basis for grading seed underSeeds the Regulat i ons , except that modified methods may be used according to section 2.2. 2.2 Modified methods On those exceptional occasions when samples do not respond to the prescribed methods to give the germination potential, an appropriate modified method may be used as follows:

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a. The analys t

b. c.

d. e.

must be certa in that the prescr i bed methods of Table- 10 do not produce resu l t s which tru l y ref l ec t the maximum germinat ion potent ia l of the sample or samples under test . The modified method must enable the analyst to report germination as defined in section 3.1, i.e. the condition of essential seedling structures must be assessed. The analyst must have reasonable grounds to expect that the method to be used is appropriate for the specific problem at hand and gives reproducible results. Published methods should be used wherever possible. The analyst must be competent in the use of the method. The method used must be clearly indicated on the Report of Analysis.

3. Definit ions 3.1 Seed germination In seed laboratory practice, germination is defined as the emergence and development from the seed embryo of those essential structures which, for the kind of seed under test, are indicative of the ability to produce a useful, mature plant under favorable field conditions. In a laboratory germination test, the plant-producing potential of a sample of seed is evaluated. 3.2 Normal seedlings Seedlings possessing the essential structures that are indicative of their ability to produce useful mature plants under favorable field conditions. Detailed seedling descr ip t i ons are given in sect i on 14. 3.3 Abnormal seedlings All seedlings which cannot be classified as normal seedlings. Detailed descriptions of abnormal seedlings are given in section 14.61

3.4 Fresh seeds Seeds which have failed to germinate but have imbibed moisture and appear firm, fresh, and capable of germination at the end of the prescribed test period and under the prescribed test conditions. Such seeds may be viable but dormant. 3.5 Dormant seeds Viable seeds, other than hard seeds, which fail to germinate when provided the prescribed germination conditions. 3.6 Hard seeds Seeds which remain hard at the end of the prescribed test period because their impermeable seed coats prevent the absorption of water. See section 12.7. 3.7 Dead seeds Seeds which at the end of the test period are neither hard nor dormant nor have produced any part of a seedling. 4. Preparation of Seeds for Germination Tests 4.1 Source of seeds for germination

a. From the pure seed separation: For germination of grasses, seeds shall be taken, without discrimination, from the pure seed separated in the purity test. b. From the submit ted sample: For germinat ion of al l kinds other than those mentioned in sect i on 4.1.a . , seeds shal l be taken di rec t l y f rom the submit ted sample after carefu l mixing. The seeds for germinat ion shal l be taken without discr iminat i on except that they must be "pure seed" as def ined under pur i t y analys i s . The fo l l ow ing seed st ruc tu res are cons idered as seed uni t s and need not be separated for germinat ion : mult ip l e f l o re t s in oats , ent i r e spike le t s in emmer and spel t , an ent i re schizocarps of the Apiaceae.62

4.2 Number of seeds for germination Individual kinds of seeds: Four hundred seeds of any kind may be planted in replicates of 100, 50 or 25 seeds as appropr ia te accord ing to s ize of seeds. 5. Germination Conditions 5.1 Planting of seeds Seeds should be adequately and uniformly spaced so that contact of adjacent seeds is avoided. Counting and planting may be done by hand, ensuring seed selection is random and in accordance with the pure seed definitions, or with planting aids provided these do not introduce a bias when the seed is selected. If a vacuum counter is used, the head must be held flat and completely covered with seed before the vacuum is turned on to avoid biased selection of seed. 5.2 Substrata and moisture All Substrata, containers and moistening agents must be non-phytotoxic. New shipments of Substrata must be tested for phytotoxicity according to section 5.6. The Substrata must have a pH value within the range 6.0 to 7.5 when moistened. The substratum must be moist enough to supply the needed moisture to the seeds at all times. Avoid supplying excessive moisture which will restrict aeration of the seeds. Except as provided for those kinds requiring high moisture levels, the substratum should never be so wet that a film of water is formed around the seeds. For most kinds of seeds, blotters or other paper Substrata should not be so wet that by pressing, a film of water forms around the finger. The initial quantity of water to be added will depend on the nature and dimensions of the substratum and also on the size and species of the seed to be tested. The optimum amount should be determined by experiment.

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Subsequent watering should be avoided wherever possible as it is likely to increase variability between replicates and between tests. Since the rate of evaporation will depend on the relative humidity of the air, it is desirable to keep water in the germination chambers or to provide other means of supplying a relative humidity of approximately 95%. Germination tests should be inspected at frequent intervals to insure that an adequate moisture supply is available at all times.a. Sand. Sand for

germinat ion tests should be pract i ca l l y f ree of organic matter , solub le sal t s , clay or f i ne s i l t , and should be composed of part i c l e s al l of which wi l l pass through a 2 mm round- holed s ieve, and not more than 25% wi l l pass through a 0.5 mm s ieve. Sand graded at 24- mesh i s sui tab le . To improve the waterhold ing capaci ty of the sand, vermicul i t e may be added. Sand i s used for the fo l l ow ing methods: 1. S ( in sand) . Seeds are placed on a leve l layer of moist sand and covered with a layer of uncompressed sand to a depth of 10 to 20 mm, depending on the s ize of the seed. The quant i t y of water added wi l l depend on the part i c l e s i ze of the sand, the character i s t i c s of the vermicu l i t e when added, and the character i s t i c s of the seeds to be planted. The opt imum amount of water to add should be determined by exper iment . As a guidel i ne , for 24- mesh sand with no vermicul i t e added, add 125 mL water per l i t r e of sand, or add water unt i l the sand can be formed in to a bal l when squeezed in the palm of the hand, the bal l breaking f ree ly when pressed between two f i ngers . 2. TS (top of sand).Seeds are pressed in to the sur face of the sand. No cover ing layer i s added. Moisture leve l s are def ined as fo l l ows : Standard: Two or three drops of water show when conta iner i s t ipped to 45 angle . Light : No f ree water shows when conta iner i s

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tipped. Heavy: Small pool of water shows when container is tipped.b. Paper. Blot te r s ,

towels , f i l t e r paper and cel l uco t ton must conform to the genera l speci f i c a t i ons for paper Substra ta as out l i ned in the current ISTA In te rnat i ona l Rules for Seed Test. ing For blot te r tests moisture leve l s are def ined as fo l l ows : Standard: Soak until saturated. Drain until begins to drip. Light: Soak until saturated. Drain thoroughly and press against a dry absorbent surface to remove excess moisture. Heavy: Soak until saturated. Do not drain. Paper is used in the following methods:1. BB (between blotters). Blot te r s

of an appropr ia te s i ze for the crop kind under analys i s are used, the seeds being spaced on one hal f of the moist blot te r before the other hal f i s fo lded over . In the case of wheat, for example, blot te r s measuring 16x28 cm fo lded to 16x14 cm would be appropr ia te ) . 2. RB (raised blotters) . The blot te r s are prepared as for BB, but to improve aerat i on of the seeds, the top blot te r i s ra i sed f rom the seed by means of corks , via l tops , narrow st r i p s of blot te r , etc . 3. TB (top of blotters)Seeds . are placed on top of standard germinat ion blot te r s or f i l t e r paper . 4. RT (rol led towels).Seeds are evenly spaced on two sheets of standard weight (38 lb ) or a s ing le sheet of heavy weight (76 lb ) germinat ion paper towel , then covered with a s ing le sheet . The towels are ro l l ed and placed in an upr ight posi t i on . The paper towel l i ng should be moistened unt i l i t s

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wet weight is about three times that of its dry weight. 5. PP (pleated paper). The seeds are placed in a pleated , accord ion- l i ke f i l t e r paper st r i p with 50 pleats , usual l y two to a pleat . This method may be used as an al te rnat i ve where any paper substra tum i s prescr i bed .

c. Soil . Samples may be retes ted in a soi l

mix to conf i rm tests made by other methods, for example test i ng samples which produced seedl ings showing symptoms of phytotox ic i t y when germinated on paper or in sand. Soi l should be a good qual i t y , organic soi l - less pott i ng mix. The soi l should have a pH value with in the range between 6.0 and 7.5. Water should be added unt i l the soi l can be formed in to a bal l when squeezed in the palm of the hand, the bal l breaking f ree ly when pressed between two f i ngers .

5.3 Water The water used to moisten the Substrata shall be reasonably free from organic or inorganic impurities. If the usual water supply is not satisfactory, distilled or de-ionized water may be used. The pH value should be within the range between 6.0 and 7.5. 5.4 Temperature Temperatures are prescribed in Table- 10 and should be determined at the leve l of the seeds on the substra tum. The temperature should be as uni fo rm as poss ib l e throughout the germinat ion or prechi l l chamber. Care should be taken that heat generated by the l i gh t source does not cause the temperature to exceed the temperature leve l prescr ibed . The temperature ind i ca ted should be regarded as a maximum and var ia t i on due to the apparatus should not be more than 2C.66

Single numerals in the temperature column of Table-9 indicate that the seed should be germinated at a constant temperature. Two numerals separated by a dash indicate that the seed should be germinated with an alternation of temperatures, the test being held at the lower temperature for approximately 16 hours per day, and at the higher temperature for 8 hours per day. When testing seeds that are dormant, it is essential that the change-over of temperature be accomplished in one hour or less. If the tests are not given alternating temperatures over weekends and on public holidays, they may be held at the lower temperatures during such times. When prechilling (rechilling or midchilling) is required for breaking dormancy, a constant temperature of not less than 5C or more than 10C shall be selected, maintained and used as described in section 8.1. 5.5 Light

Seeds of most of the species in Table- 10 wi l l germinate ei ther in l i gh t or in darkness . However, i l l uminat i on i s genera l l y recommended, as better developed seedl ings are produced which are more easi l y evaluated. I f l i gh t i s known to be necessary or benef i c i a l to induce complete germinat ion of any given kind of seed, i t s use i s ind i ca ted in Table- 9 in the columns "Addi t i ona l Direct i ons" . Light should be provided by a cool white fluorescent source for 8 hours in every 24. Where the seeds are germinated at alternating temperatures, they should be illuminated during the high temperature period. Seeds for which light is prescribed in Table-9 should be germinated on top of the substratum, at an intensity of 750 to 1200 lux. Iluminance of seed for which light is not prescribed may be as low as 250 lux. 5.6 Phytotoxicity Testing

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A phytotoxicity test must be conducted to compare a substratum of unknown quality with one in stock of acceptable quality. For this test, seeds of certain species which are known to be sensitive to toxic substances in the substratum are used: e.g. Phleum pratense , Agrostis gigantea, Festuca rubra var. commutata, Allium cepa, Apium graveolens, Cichorium intybus and Lepidium sativum. At least two species must be included in the test. The evaluation is made by comparing the root development of the seedlings of the two species grown on the two sources of substratum. The evaluation should be made on or before the days specified in Table-9 for first count of the species used for the test, because symptoms due to toxic substances are more pronounced at an early stage of root growth. Such symptoms are shortened roots and sometimes discolored root tips, roots raised from the substratum, and root hairs "bunched". In grasses, coleoptiles may be flattened and shortened. 6. Germination Methods 6.1 Outline of germination methods The prescribed methods as given in Table-10 must be used. When alternate methods are indicated, one of them (any combination of substratum and temperature) may be used. If a sample does not respond satisfactorily to the method selected, it may be retested by one or more of the alternate methods or by a modified method as described in section 2.2. The sequence of alternate methods in Table-9 does not indicate any preference. Methods in the "General Requirements" column of Table-10 must be used. Methods in the "Fresh or Dormant Seeds" column may be used when dormant seed is known or suspected to be present. These are described in section 8 and may be applied to the original test, or to retests.

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When a kind of seed for which a method is not given in Table-10 is received for germination, the current ISTA In ternat i ona l Rules for Seed Test or ing AOSA Rules for Testing Seeds are to be consulted for an appropriate method. For kinds for which there is no published method, the method for a closely related species should be followed. The method used must be clearly indicated on the Report of Analysis.

The abbreviations used in Table-10 are defined as follows:

BB - Between blotters (see section 5.2.b.1) RB - Raised blotters (see section 5.2.b.2) TB - Top of blotters (see section 5.2.b.3) RT - Rolled towels (see section 5.2.b.4) PP - Pleated paper (see section 5.2.b.5) S - In sand (see section 5.2.a.1) TS - Top of sand (see section 5.2.a.2) KNO3 - Use solution of potassium nitrate instead of water. (see section 8.2) GA3 - Use solution of gibberellic acid instead of water. (see section 8.4) TZ - Tetrazolium (see section 7.7)

6.2 Table- 10. Germination Methods for Vegetable Crop SpeciesKind of Seed Number Substrata of seeds BB; S; RT BB; S; RT BB; S; RT BB; TS; RT TS; BB; RT First Final Temperature Count Count (C) (days) (days) 20 20 20 20-30 20-30 7 7 7 7 7 14 14 14 21 21 Light Prechill Additional Directions General Requirements Lightly covered (sand test) Lightly covered (sand test) Light moisture; Lightly covered (sand test) Additional Dir