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ISTA SEED MOISTURE, GERMINATION AND VIGOUR WORKSHOP HELD ON 3RD TO 7TH
NOVERMER 2008 AT KEPHIS NAKURU - KENYA.
1. WORKSHOP PROGRAMME DATE TIME ACTIVITY FACILITATOR
3rd November 2008
8.30 – 8.45a.m. Workshop registration at KEPHIS Nakuru Conference Hall
Aguyo/Zeddy
8.45 – 9.00a.m. Welcome note by Regional Manager/Local Organizer
Dr. Ahenda
9.00 – 9.15a.m. Official Opening speech by Board Director, KEPHIS, Dr. Vasey Nyamu Mwaja
Dr. Ahenda
9.15 – 9.30a.m. Remarks by Provincial Director of Agriculture, Rift Valley, Mr. P.L.O Nyambuya
Dr. Ahenda
9.30 – 10.00 a.m.
Registration of expectations of participants
ISTA
10.00 – 10.30a.m.
Coffee break and group photo
10.30am – 12.30p.m.
ISTA Rules Chapter 9 Moisture (lecture/discussion) Principles and methods of seed moisture determination, including the oven moisture test and grinding and tolerances (lecture/discussion and practical work) Quality Assurance in moisture determination (lecture/discussion and practical work) New species for the Rules (lecture/discussion and practical work) Work of the Moisture Committee (discussion)
Craig McGill
12.30 – 1.30p.m. Lunch
1.30 – 3.30p.m. Moisture: Continue
Craig McGill
3.30 – 4.30p.m. General introduction to Seed Sampling and Purity. (lecture/discussion)
Anny van Pijlen
4.30 – 5.00 p.m. General evaluation of the theory and practical training (questions/answers)
4th November 2008
8.30-10.00a.m. ISTA Rules Chapter 5 Germination (lecture/discussion). Theory Germination testing. Practicals and theory on selected species for Germination Testing. Work of the Germination Committee (discussion)
Anny van Pijlen
10.00 – 10.30 a.m.
Coffee Break
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DATE TIME ACTIVITY FACILITATOR
10.30am – 12.30p.m.
Practical work: evaluation of Allium cepa, Daucus carota, Helianthus annuus
Gillian McLaren/Anny van Pijlen
12.30 – 1.30 p.m.
Lunch
1.30 – 3.00p.m. Continue: Practical work evaluation of Allium cepa, Daucus carota, Helianthus annuus
Gillian McLaren/Anny van Pijlen
3.00 – 3.30p.m. Coffee break
3.30 – 4.30 p.m. How to use the Seedling evaluation Handbook. Seedling evaluation handbook (lecture/discussion)
Gillian McLaren
4.30 -5.00p.m. General evaluation of practical training (questions/answers)
5th November 2008
9.00 – 10.00a.m Visit to Kenya Seed Company Nakuru Dr. Ahenda/Kibet 10.00 – 10.30p.m.
Coffee break
10.30 – 12.00 pm
A visit to Menengai crater Dr. Ahenda/Kibet
12.30 – 1.00 pm Lunch 1.00 – 6.00 pm Excursion to Lake Nakuru National Park (Those interested)
6th November 2008
8.30 – 10.00 a.m. Calibration of equipment (lecture/discussion)
Gillian McLaren
10.00 – 10.30a.m.
Coffee Break
10.30 a.m -12.30p.m.
Practical work: evaluation of Desmodium spp., Arachis hypogaea, phaseolus vulgaris
Gillian McLaren/Anny van Pijlen
12.30 – 1.30p.m. Lunch
1.30 – 3.00p.m. The concept of vigour (Lecture) Vigour tests (Lecture)
Gillian McLaren
3.00 – 3.30 p.m. Coffee break
3.30 – 4.30p.m. ISTA Accreditation (lecture/discussion)
Anny van Pijlen
4.30 – 5.00p.m. General evaluation of practical training (questions/answers)
7th November 2008
8.30 – 10.00 a.m. Quality assurance in seed testing, Quality Assurance in Germination testing (lecture/discussion)
Anny van Pijlen
10.00 – 10.30a.m.
Coffee Break
10.30 – 11.30a.m.
Training of staff (lecture/discussion). Craig McGill
11.30 a.m. – 12.30 p.m.
General evaluation of the workshop and evaluation of expectations of the participants
ISTA
12.30 – 1.30p.m. Lunch
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DATE TIME ACTIVITY FACILITATOR
1.30 – 2.30 p.m. Official closing with the presentation of the certificates GM – I.O. /GM PI 2.30 – 4.00 pm. Tree planting session and tour of the station Regional Manager 6.00 – 8.00 pm. Closing Dinner at Cool Rivers Hotel
2. LIST OF PARTICIPANTS
No. Title
Name
Position Company Address Country Phone Email
1
Mr Cham Puro
FAO United Nations Sudan
c/o P.O. Box 30470-00100
Kenya 00256 477120470
2
Mr Njaga John Njagi
FRESHCO (K)LTD
27659 Kenya 0733-370526
3
Mr Goma Baymolo
Seeds Officer
Seed Control and Certification Institute Official Seed Testing Station
Mount Makulu, P.O. Box 350199
Zambia 00260 211 278236
4
Mr Andriamainty Marius
Seed Multiplication Specialist
AVRDC/vBSS-Madagascar
Ampandrianomby 1690
Madagascar
00261330555514
5
Mrs Nyabvure Christine
Seed Analyst
Zimbabwe Seed Testing Section, Seed Services
550 Causeway, fifth st. ext.
Zimbabwe 002634720370
7
Mr Wathiru James
Seed Quality control and research supervisor
Pollen Ltd
Ruiru, Kenya
Kenya
00254722724255863
8
Mr Gupta Arnab
VIBHA SEEDS GROUP "INSPIRE"
Plot No 21, Sector 1, Huda Techno Enclave, Hitechcity Road
India
00914065264638
9
Dr. Singh Pankaj
VIBHA SEEDS GROUP "INSPIRE"
Plot No 21, Sector 1, Huda Techno Enclave, Hitechcity Road
India
00914065264638
10 Mr Okayo
Robert Inspector KEPHIS
80126 Kenya 00254723
748287 [email protected]
11
Mr Muchiri Josphat
KEPHIS
18288
Kenya 00254722665224
Page 4 of 64
No. Title
Name
Position Company Address Country Phone Email
12
Mr Wachira Ephraim
KEPHIS
49592
Kenya 00254722481334
13
Mrs Shitabule Eveline Wanguba
Inspector KEPHIS 7094-40100 Kenya 0733859765
14 Mr Osoro
Timothy Analytical Chemist
KEPHIS 249 Kitale Kenya 0203597211
15
Mr Oganda James Kefa
Seed Analyst
KEPHIS
1679
Kenya 254723881081
16
Mrs Mutete Kavu Carol
Seed inspector
KEPHIS
49592
Kenya 2540203597202
17
M/s Zivanai Blessing
Seed Analyst
Zimbabwe Seed Testing Section, Seed Services
550 Causeway, fifth st. ext.
Zimbabwe 002634720370
18
M/s Abdalla Amina Nasser
Analyst SGS Limited Kenya
19
Mr Kamau Mary
Seed Quality control and research supervisor
Pollen Ltd
Ruiru, Kenya
Kenya
00254722724255863
20
Mr Miruka George
Hygrotech
BOX 1484
Kenya
21
M/s Kingori Jane Wanjiru
Simlaws
Box 40042
Kenya
FACILITATORS
1 Anny Van Pijlen Nertherlands NAK/AGRO
2 Craig McGill New Zealand Massey university;
3 Gillean Mclaren Scotland SASA
4 Simeon Kibet Kogo Kenya Kephis; Kenya [email protected]
RAPPOTEURS
1 jacob cheptaiwa Kenya [email protected]
2 Wilson Sitienei Kenya [email protected]
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GROUP PHOTO OF ISTA SEED MOISTURE, GERMINATION AND VIGOUR WORKSHOP
PARTICIPANTS
First row from left: Gillean Mclaren, Craig Mcgill,Dr Vassey N.Mwaja,P.L.O Nyambuya,Anny Van Piljen,Dr Joseph Ahenda,Stephen Ithili Second row from left:, Andriamainty Marius, James Wathiru, Amina Abdalla, Evaline Shitabule, Carolin Kavu, Jane Kingori, Timothy Osoro, Z. Kinyanjui Third row from left: J.Cheptaiwa, Simeon Kibet, Miruka George, F.Nganga, R.Okayo, Mary Kamau, Kefa Oganda, Fourth row from left: J.Muchiri, W.Sitienei, Cham Puro,Ephraim Wachira , M.Kiptoo
Page 6 of 64
3. WORKSHOP OPENING REMARKS
ISTA SEED MOISTURE, GERMINATION AND VIGOUR WORKSHOP LOCAL ORGANIZER, Dr
J.O.Ahenda (GENERAL MANAGER PLANNING & IMPLEMENTATION-KEPHIS)
The workshop begun at 9.15 A.M. with a word of prayer from one of the participants. Dr. Ahenda the local
organizer gave a background of the workshop and welcomed the participants and facilitators to the workshop; H e
observed that the workshop was going to take longer than the previous purity workshop held in the same venue. He
also recognized the presence of KEPHIS BOARD DIRECTOR, Dr Mwaja, PDA Rift valley and Kephis General
Manager Support Operations. The participants then introduced themselves. Dr Ahenda gave a short introduction of
KEPHIS Nakuru, the workshop venue and the seed testing laboratory and said that the station is bordered by
agricultural development corporation (ADC) to the south, staff quarters to the west, Kenya Agricultural Research
institute (KARI) to north, and Dundori hills to the east. He added that the station has post control plots, seed testing
and variety laboratories.
On the seed testing laboratory, he said that it is an active ISTA member, attend ISTA congresses and as participated
in tropical crops method validation. He said that the laboratory has 55 seed samplers, 14 seed analysts, and 12
support staff. He gave the laboratory’s scope of accreditation as sampling, purity, and other seed determination,
germination, moisture and tetrazolium tests, which covers over 100 species. He also added that the scope is to
increase in the next ISTA audit to include seed health and seed vigour and all these is aimed at enhancing seed
trade.
GENERAL MANAGER SUPPORT OPERATION, KEPHIS, Mr. S.Ithili
The GM-SO, Mr S. Ithili recognized the presence of the Kephis Director, The PDA Rift Valley, Anny and her team
and the participants, and took the opportunity to welcomed them all to the workshop .He said was happy with every
visitor and thanked KEPHIS- Nakuru staff for the good work they did in preparation for the workshop. He said the
meeting will improve seed quality and boost international trade
PROVINCIAL DIRECTOR OF AGRICULTURE (PDA) FOR RIFT VALLEY PROVINCE Mr. P.L.O
Nyambuya.
The PDA thanked KEPHIS management for hosting the workshop and participants for their attendance. He said
that he was attending the workshop as the farmers’ representative and as chair of seed allocation panel, which made
him work closely with KEPHIS to give farmers high quality seeds. As a result crop production in the province as
improved (cited an increase of 11 to 24 million bags between the period 2006 and 2008 in the region). He also said
that Rift valley province was leading in horticulture and wheat production, produced three quarters of national food
requirements in maize and said was making plans to meet national food requirements. He also mentioned that the
region is the base of seed production in the country.
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He added that he was happy to note that ISTA has a membership of over 176 countries, with over 100 seed testing
laboratories and has done a lot in hosting member states and seed labs to improve uniformity in seed testing. He
informed the participants that CAP 326 of the laws of Kenya deals with seeds and KEPHIS was formed to ensure
compliance with the laws. In conclusion, he thanked Kephis for hosting the workshop then welcomed Dr Mwaja to
make his remarks and officially open the workshop.
BOARD DIRECTOR, KEPHIS,Dr. Vasey Nyamu Mwaja
Dr Mwaja who is a member of the board of directors of KEPHIS gave the following opening speech.
“Good morning ladies and gentlemen,
I take this opportunity to welcome you to Kenya and specifically to KEPHIS Nakuru. Ladies and gentlemen, first
and foremost, I take this opportunity to convey the pleasure and appreciation of the board of directors of KEPHIS,
senior management and staff to ISTA for giving KEPHIS the opportunity to host this workshop on ‘seed moisture,
germination and vigour testing’ here in KEPHIS Nakuru regional office. Our hope is that the objectives of the
workshop will be met and your participation will enhance your capacity for the good and benefit of your respective
countries and humanity in general.
Before we start the workshop, I find it necessary for me to give you a short history about Kenya plant health
inspectorate service (KEPHIS) seed testing laboratory (formerly called national seed quality control service). This
is important to enable all of us appreciate the steps and the entire journey that Kenya as a country has taken in
terms of having in place an effective seed certification and testing system.
The first seed-testing laboratory in Kenya was established in 1944 at the national agricultural laboratory (NAL) Kabete,
Nairobi. Subsequently, the seed certification in Kenya started with the formation of Kenya inspection service for
seeds (KIS) in 1964 and in the same year, Kenya became a member of ista and hosted an ista meeting on seed testing
and a training course.
Thereafter in 1965, an improved seed-testing laboratory was put up at the agricultural laboratories, Nairobi with the
support of the government of Netherlands to enforce the seed ordinance of 1962. The laboratory was then serving
mostly the European large-scale farmers who wanted to determine the quality of cereals and grass seeds before
exporting.
The seed testing laboratory was moved from NARL to Lanet, Nakuru (where we are today) in 1979. It was then
renamed national seed quality control service and designated as the official seed testing station. The official seed testing
laboratory was then modernized and joined with the expanded field seed certification program. In addition the station
was also mandated to carry out crop variety testing which included distinctness, uniformity and stability (DUS) and
national performance trials (NPT).
In 1980, the official seed testing laboratory was further expanded to include seed pathology tests (viz. virus,
bacteria and fungi tests). These tests were intended to support seed inspectorate in identifying seed borne diseases
and to safeguard against importation of seed borne diseases and pests. The establishment of the sub-section within
the official seed testing laboratory has gone a long way in enhancing the quality of seeds being accessed by the
Page 8 of 64
farming community. The activity also falls within the mandate of KEPHIS, which includes undertaking
inspections, testing, certification, quarantine control, variety testing and descriptors of seeds and plant materials.
The laboratory has so far undergone two successful accreditation audits (viz; 2002 and 2005) hence has been a very
active ista member laboratory. The next audit of the laboratory is expected in January 2009 where the laboratory
will expand its scope to include seed health and vigour testing.
The referee test results from ista have so far been very encouraging since the grades obtained have ranged from ‘A’
to ‘B ’. It is also noteworthy that the laboratory is the only ista accredited laboratory in east Africa region though
Kenya is encouraging the neighboring countries of Uganda and Tanzania to join ista membership.
in terms of capacity, ladies and gentlemen, the laboratory is currently testing on average 3000 seed samples and
issuing about 150 ista certificates for seed moving in the international market per year. The laboratory has on
several occasions carried out ‘ring tests’ with other ista member laboratories and the results have been very
comparable.
However, the board and management of KEPHIS is aware that the potential of the laboratory is 10,000 seed
samples per annum. We are therefore committed to ensuring that the potential is realized in the near future though
we recognize that this is definitely a challenge that calls for concerted and focused efforts on our part.
Ladies and gentlemen, I find it necessary to specifically mention in summary the number of samples tested during
the last three years. In 2006, 3452 samples were tested for 30 companies; in 2007 there was an increase in both the
numbers of companies and samples to 3881 for 32 companies. In 2008, 33 companies have been inspected and the
number of samples so far is 2663 (exclusive of October and November). This is an indication that we have a
daunting task an institution in terms of the need to facilitate the growth of the seed sector in the country.
Ladies and gentlemen, as we host this workshop we are conscious of the many benefits that we derive from ista
membership. These include among others:
• The seed industry having access of their seed to international markets.
• Accessing the worlds’ seed standard requirements and information.
• Attending ista meetings and trainings hence adding value to the seed analysts’ in terms of acquired
knowledge (i.e. the current training in Kenya).
• Accessing ista information on seed technology through ista publications and journals.
• Ability to participate in ista committees hence able to suggest the inputting of new seed crops.
Thus, ladies and gentlemen, as we welcome you to this ista workshop, we are exceptionally pleased since in this
millennium, this workshop will be the second to be hosted by Kenya through KEPHIS, the first of which was held
in July 2006 (ISTA purity workshop). We therefore hope that the workshop will generate contributions and gains
that will impact on Africa’s agriculture and thus food security for all its nations.
we are also conscious that after 44 years since the country joined ista, our systems have been sustained and
improved upon which has made it possible for this workshop to be held in Kenya. We are in that respect
appreciative of all the support that ista has accorded the country and KEPHIS in particular over the years. We look
forward to continued support and collaboration.
Page 9 of 64
Ladies and gentlemen, it is now my pleasure to declare this workshop officially opened and further to invite you all
to take time and visit parts of the country to enjoy the beautiful natural resources as well as the company of the
people of Kenya.
Thank you very much″
Anny van Pijlen -ON BEHALF OF THE FACILITATORS
She said that they were happy to be in Kenya and thanked KEPHIS for the support in organizing the workshop.
Workshop Introduction, By Anny van Pijlen
She introduced the workshop program, which is attached and added that there will be a visit to Menengai crater,
lake Nakuru National park and evening dinner. She also gave the main topics of the workshop as moisture,
germination and vigour and encouraged the participants to ask questions to make the workshop interactive.
REGISTERING EXPECTATION OF PARTICIPANTS, By Anny van Pijlen
Participants were requested to mention what they expected from the workshop and the expectations were registered
as below.
Expectations of participants
• Procedures for moisture testing
• Introduction of new species into ISTA Rules
• Sampling techniques/skills
• Procedures for germination and vigour testing
• Accreditation procedures
• Quality systems in a seed testing laboratory
• Sampling sizes
• Relationship between germination, moisture and vigour
• Moisture in treated seeds
• Germination and vigour-how the two results relate to each other and in relation to field emergence
Page 10 of 64
4. WORKSHOP PROCEEDINGS
4.1 ISTA RULES CHAPTER 9 MOISTURE, (By Craig McGill)
Principles of the oven moisture test
ISTA rules chapter 9.1.2 Definition
The moisture content of a sample is the loss in weight when it is dried in accordance with this chapter. It is
expressed as a percentage of the weight of the original sample.
Question
Why is it important to determine seed moisture? (From one participant)
Answer from Craig
Seed moisture determination is important for the following reasons;
1. Storage- To reduce attack by pests
2. Affect seed viability (Germination)
3. To know optimal harvest time
4. For seed drying and processing
5. Seed treatment
6. For vigour testing
7. For seed trade
Process
Receive sample Working samples Grind? Pre-dry? Cut? Weigh 2 x 4.500 �0.500g seeds Place
in oven y hrs at z temperature Cool in desiccators Weigh again Calculate Result on certificate
Pre-drying
1. Weigh two containers.
2. Mix the submitted sample thoroughly and draw two sub samples of 25 � 5g. Place the sub samples in the
weighed containers.
3. Dry the sub samples at 130°C for 5–10 minutes to reduce the moisture to below that allowed.
4. Leave the open containers exposed in the laboratory for two hours.
5. If the moisture content is above 30% the sub samples should be dried overnight in a warm place
6. Reweigh the containers and draw the working sample for grinding
Question on pre-drying
What is the temperature for pre-drying of samples for moisture determination?
Answer from Craig: Pre-drying is done at low temperature like 25oC and the final MC is obtained by adding
together the pre-dry moisture content and the oven moisture content.
Question on moisture meters
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1. Do moisture meters give the correct value?
Answer from Craig; Moisture meters moisture content of the environment surrounding the seeds and they requires
calibration
2. What is the relationship between moisture meters and the oven method?
Answer from Craig; Moisture content of species varies depending on the oil content.
WORKING SAMPLE
1. Mix the submitted sample thoroughly either in its container with a spoon or by pouring the sample back and
forth between the original container and a similar container.
2. Take a minimum of three sub samples with a spoon from different positions in the bag.
3. Combine them to form a working sample of the required size.
4. Work quickly and systematically, the seed must not be exposed to the air for more than 30 seconds during the
sample reduction.
5. Repeat the steps above to obtain the second working sample.
Grinding
_ make sure that the mill is clean
_ grind the first working sample
_ grind the second working sample
_ work quickly and systematically
_ clean the mill with a brush and/or Vacuum cleaner.
Species for which grinding is required:
_Cereals,Peas,Soybean,Beans,Corn,Cotton
Cutting
1. Draw one subsample approximately equal to the weight of ten intact seeds.
2. Quickly cut the subsample into small pieces of less than 7 mm.
3. Mix the cut subsample with a spoon and divide into two working samples.
4. Place the working samples into weighed containers and weigh.
5. Work quickly and systematically; exposure to the atmosphere should not exceed four minutes.
Weighing
First working sample A:
Repeat procedures 1-3 on the second working sample
1. Quickly mix the seed ground material.
2. Weigh the container and lid.
3. Weigh 4.5 � 0.5g.
Second working sample B:
Weigh 4.5 �0.5 g to three decimal places
For species that require grinding weigh to three decimal places after grinding.
Drying
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Low constant temperature:
101 - 105°C for 17 ± 1 hours
High constant temperature:
130 – 133°C for 1 hour (�3 minutes), 2 hours (�6 minutes) or 4 hours (�12 minutes), depending upon species
High constant temperature
Species Time(Hours)
Corn 4
Cereals 2
Peas and beans 1
Grass species 1
Many vegetable species 1
Small seeded legumes 1
Low constant temperature Species:
Brassica sp.,Soy bean,Cotton,Onion,Mustard,Sesame
Time
Time is measured from the time when the oven has returned to the set temperature (103°C or 130°C)
Desiccators
Remove the samples from the oven, cover quickly with the lid, and put in desiccators for cooling to ambient
temperature.
Reweigh again to 3 decimal places
M1: is the weight of the container and its lid
M2: is the weight of the container, its lid and the seed before drying
M3: is the weight of the container, its lid and the seed after drying.
Calculate for both working samples A and B
Moisture content: (M2 - M3) x 100 .
(M2-M1)
The difference between A and B must not exceed 0.2 %.Calculate the moisture content as the average of A and B
For two stage moisture there is an extra step before calculating the average of both working samples A and B. You
have the pre-drying moisture (S1) and the oven drying moisture (S2). S1 and S2 are both calculated using the
formula
Moisture content: (M2 - M3) x 100 .
(M2-M1)
Then the moisture content of each working sample is calculated using the formula
Page 13 of 64
Moisture content: S1+ S2-( S1x S2)
100
The difference between A and B must not exceed 0.2%
Calculate the moisture content as the average of A and B
Report the result to one decimal place
Calculating the Moisture Content
Example
Example Moisture content percent
A 10.093
B 10.086
Difference between A and B 0.006
Average 10.090
Rounding to 1 decimal 10.01
Result for Example 1
Example 1 Moisture
Working sample Content in percent
A 10.454
B 10.245
Difference 0.209
Difference rounded off 0.2
10.3495
Result 10.3
Result for Example 1
Example 1 Moisture content in percent
Working sample Before rounding After rounding
A 10.454 10.5
B 10.245 10.2
Difference 0.209 0.3
Tolerance and reporting
Page 14 of 64
• The difference between the two determinations must not exceed 0.2 %
• The moisture content must be reported to the nearest 0.1%.
Calculation of moisture if a sample has been pre-dried
(S1 + S 2) – (S1 x S2)
100
S1 is the pre-drying moisture
S2 is the oven moisture
Results for a sample that required pre-drying
Moisture before pre-drying(2 x 25 g), first stage Moisture content
A, S1 10.672
B, S1 10.588
Moisture after pre-drying
(2 x 4.5 g) second stage
A, S2 8.115
B, S2 8.077
To calculate the moisture for working sample A
(10.672 + 8.115) – (10.672 x 8.115) =
100
17.921 % moisture for working sample A
Result after pre-drying
Moisture A 17.921
Moisture B 17.810
Difference 0.111
Average 17.866
Result for reporting 17.9
Result for a sample requiring re-testing
A 13.949
B
13.649
Page 15 of 64
Difference
0.300
Result
13.799
RESULT 1
13.8
Tolerance exceeded.
Addition of new species into Chapter 9; Determination of Moisture Content Background. Need to have a method to determine the”correct” moisture content. This method shall be compared with the oven method for deciding which temperature and time is needed as well as to decide the necessity for grinding. Basic reference method 103°C for 17 hours- Compulsory to test for necessity for grinding Not compulsory to test for shorter drying time at higher temperature. Validation Protocol At least 3 labs, up to a maximum of 6. Each sample is tested in one of two ways, whole or ground. At least two moisture levels that are representative of the species and routinely used. Moisture levels shall be at least 5% apart. At least two, and up to four at a maximum, samples per moisture level Two working samples on each sample received. Grinding tested shall be fine and coarse unless 55% of the seed passes through a 2.0 mm round holed sieve when only fine grinding is required. All test to be performed at 103°C. In each laboratory all the samples shall be tested at the same time For Solanum nigrum, for three moisture levels and four samples (seed lots), the number of samples per laboratory is: 4 (samples) x 2 (moisture contents) x 2 (fine ground and whole) x 2 working samples = 32 containers 4 (samples) x 3 (moisture contents) x 2 (fine ground and whole) x 2 working samples = 48 containers To test for 130°C and a shorter drying time Each sample is tested in one of two ways, whole or ground. At least 3 labs up to a maximum of 6. At least two moisture levels. Moisture levels shall be at least 5% apart At least two, and up to four at a maximum, samples per moisture level Grinding tested shall be fine and coarse unless 55% of the seed passes through a 2.0 mm round holed sieve when only fine grinding is required. All test to be performed for 17 hours at 103°C and 1, 2, 3 and 4 hours at 130°C. In each laboratory all the samples shall be tested at the same time. Evaluation of the results 1. Calculate the means for each laboratory per sample for each method (e.g. grinding or 1, 2, 3 or 4 hours at 130°C). 2. Calculate the means for each laboratory per sample for the reference method. 4. Calculate the difference between 1 and 3. 3. Calculate the sample means for the reference method. Tolerance – acceptance criteria 1. The acceptable difference in moisture determined between 1 and 2 is � 0.3%. 2. The maximum percentage of deviating results (i.e. greater than 3% difference) is 25%. 3. If 75 % of the differences for the samples are less than 0.3% then the whole seed method is accepted. 4. If 75 % of the differences for the samples are less than 0.3% then the corresponding 130°C method may be used as an alternative to 17 hours at 103°C whole seed method is accepted Can we use whole seeds for a species giving these results?
Page 16 of 64
Difference between whole seed and seeds ground at coarse grinding Results: difference between the two methods, whole and coarse grinding - tolerance is 0.3%. 0.10 0.00 -0.30 0.10
0.60 0.40 0.37 0.37 0.07 -0.23 0.03 0.47 -0.07 0.03 0.33 0.03 0.13 0.23 13 out of 18 results are within tolerance = 72.2%. This means that the seeds have to be ground. Q uestion: The recommendation that 50% of ground seed pass through the specific size of sieves is not always achieved. Answer: Other labs have experienced the same situation. The moisture committee has also discussed the issue but the rules do not need to be changed since clause comes from ISTA. The experience of the lab in Massey University, New Zealand was to make adjustments in the grinder to attempt to obtain 50% seed passing through the grinder. Question: Is the repeatability of the sample possible when it’s out of tolerance especially for results, which require heating for 17hrs. Answer: It is possible because reporting is not limiting but generally it should be as soon as possible. The seed is supposed to be kept in the lab for as long as 1 year in the lab store. The seed should be kept in a suitable place within the 24hrs within the retest period. 4.2 GENERAL INTRODUCTION TO SEED SAMPLING AND MONITORING OF SAMPLERS (By Anny van Pijlen-NAK AGRO) Sample = A certain quantity from a lot taken in such a way that it forms a reliable average of the whole lot. Representative Sample The number of random samples influences how representative the submitted sample will be because most biological properties follow a normal distribution. The more samples that are taken, the greater the probability of obtaining a sample mean close to the population mean. Definitions
• Seed lot • Primary sample • Composite sample • Submitted sample • Duplicate sample
In the ISTA Rules • Sampling rules • Rules and methods for seed testing • Maximum seed lot sizes • Sample sizes Administrative matters Sampling form – Name of person/company – Type of sample – Species – Variety – Lot number – Number of units to be sampled – Tests to be carried out
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Checking seed lot data Uniformity of the lot- At time of sampling the lot shall be subjected to, mixing, blending and processing techniques so that it is as uniform as practicable. Visual check before sampling: - Seed size - Seed colour - Technical purity - Admixtures Marking, sealing and packing the sample • Samplers are personally responsible for seals, labels and bags supplied to them. • It is their duty to ensure that no unauthorized persons have access to them. • On no account shall primary, composite or unsealed Submitted samples come into hands of unauthorized Persons. Procedure for sampling the seed lot • Only done by trained persons • Recognized by seed lab • Or employed by other (semi)official organization recognized by head of seed lab. Procedure for sampling the seed lot • The seed lot shall be arranged so that each container or part of the lot is accessible. • When there is evidence of heterogeneity, either physical or documentary, sampling shall be refused. ISTA Rules 2.5.1.1
• Enough space • Enough space
Maximum Seed Lot Size Grasses and clovers 10.000 kg Fine seeded species 10.000 kg Beet seed 20.000 kg Cereals, peas and beans 25.000 kg Zea mays 40.000 kg Panicum spp 10.000 kg Seed Lot Size See ISTA Rules Table 2A part 1, 2 and 3 For maximum weight of the seed lot and minimum weight of: • Submitted sample • Working sample for purity • Working sample for count of other species (OSD) Sampling equipment Samplers – dynamic spear – stick sampler Sampling by hand Automatic sampling Sampling equipment Stick or slieve, triers Spear or Nobbe triers • Trier in use at an angle of about 30º to the horizontal. • The hole facing downwards untill it reaches the centre of the bag. • Trier is turned and the hole is facing upwards than. Sampling a Container Soil divider processing plant Seed processing Plant Summary Sampling Procedure • Administration (application for sampling) • Accessibility of seed lot
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• Sampling equipment • Sampling • Marking, sealing and packing of the sample Dispatch of submitted sample Shall be dispatched by seed sampler to the seed lab without delay. Shall never be left in the hands of persons not authorized by sampling agency or by the seed testing lab unless
they are sealed in such a way that they cannot be tampered with. Where the seed has been treated with chemicals, the name of the treatment shall be given.
Conditions for licensing seed samplers The samplers shall: • have the technical qualifications – Theoretical + practical training – Official examination – Certificate • derive no private gain from sampling • carry out sampling according to ISTA Rules + National Regulations QA in Seed Sampling • QMS (Quality Management System) • Job description + responsibilities • ISTA Rules + sampling instructions • Proper technical equipment • Automatic sampler must be recognized Instructions • Application form for sampling • Sampling + reducing the sample • Packaging + administration • Sending samples to the laboratory Instructions for sampling • Checking seed lot identity • How to check homogeneity • Type of sampling equipment to be used • Number of primary samples as described by ISTA Rules • In which cases sampling should be refused • How to reduce the composite sample Sampling bags Automatic sampling Instruction for sending sample • Identification of the sample – Unique sample identification number – Species + variety name – Category – Indication that seed is treated • Type of bag to be used • Sealing of sample bag Sealing the submitted sample Dividing down the sample in the lab Training (initial period) • Attend number of samplings by qualified samplers • Number of samples to be taken under supervision of qualified sampler • Attending instruction sessions • Formal qualification Monitoring of samplers • Supervision by senior seed sampler • Audits • Check sampling (not needed in case of automatic sampling)
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Audits • Regular • Planning + appointment • Checklist • Non- conformities previous audits • Audit report Check sampling • Percentage • Preferably after sampling by licensed sampler • Same method + equipment • Both samples tested in same laboratory • Results compared (ISTA Tolerance tables) Maintaining proficiency • Take samples regularly • Regular training • Supervision by senior sampler • Audits Question. Can a duplicate sample be used in the moisture test? Answer. No Moisture samples are sampled separately. Question. Is colour or dye in seed a factor to be considered while sampling. Answer. Yes. 4.3 GENERAL INTRODUCTION TO PURITY ACCORDING TO ISTA RULES (By Anny van Pijlen-NAK
AGRO)
Why??
• Cleaned seed should not contain a high percentage of other particles
• Cleaning machines leave impurities
Objectives of the purity test:
• Determine composition of sample by weight
• Determine identity of other particles
• Determine the quality of the seed lot
Size of submitted sample
See: table 2A of ISTA rules for details:
• Usually between 60g and 1000 gram
• Sample must be sufficient for all tests
• Second test or re-test
• Samples stored for 1 year
Lolium perenne as an example.
Maximum weight of the seed lot 10.000kg
Minimum weights:
60g - Submitted sample
6g - Working sample for purity
60g - Working sample other species
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Expensive seed lots:
Weigh the submitted sample and if it is according to the ISTA Rules:
- Continue
Not according to the ISTA Rules:
- Ask for a new sample or
- Report the weight of the sample on the certificate under “Other determinations”
Preparation working sample
• ISTA rules give detailed prescriptions two half working samples
• Mechanical dividing, spoon dividing, hand dividing
• Equipment:
- Divider (soil, Boerner, conical)
- Spoons / spatula
- Shallow metal tray
- Balance with weighing beaker
Purity definitions
• Pure seed
• Inert matter
• Other seeds: Other crop seeds, weed seeds
Pure seed
• Species stated by the sender or species that predominates in the test
• Includes all botanical varieties/ cultivars, intact seeds, Pieces more than one half of original size
The following structures even if they are: immature; undersized; shriveled; diseased or germinated providing they
can be definitely identified as that species unless transformed into visible fungal sclerotia.
Inert matter
- Soil/earth particles, sand, Straw or chaff, stems, leaves, ect, Nematode galls, Sclerotinia spp/claviceps purpurea,
All non seed matter.
- Seed and seedlike structures like: Pieces of broken or damaged seeds half and less than half of the original size,
Caryopsis half and less than half
- If it’s readily apperent that there is no true seed present
Other seeds:
Shall include seeds and seed-like structures of any plant species other than that of pure seed: weed seeds, crop
seeds
Equipment
• Purity table: special created form with diaphanoscope
• Balance (analytical or precision)
• Weighing table (thick marble or stone plate)
• Binocular microscope/magnifying glass
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• Metal containers/sieves/funnel
• Spoons /spatulas /needles /shallow trays
• Blowers: South Dakota/General/Franken/Hearson
• Dividers: soil divider, Boerner, Gamet
Purity test procedure
Working sample: working sample is taken from submitted sample
- not less than indicated in table 2A column 4 of ISTA Rules
- Weight based on 2500 seeds number count test = 25.000 seeds
- 2 half working samples (drawn independently)
ISTA Rules: Weighing 3.5.1.A
Weight of sample Number of decimal (at least)
in grams places for each component
Less than 1, 0000 4
1,000 to 9,999 3
10, 00 to 99, 99 2
100, 0 to 999, 9 1
1000 or more 0
Tolerance:
- Not exceeding ISTA tolerance: If yes: repeat the test
- Appeal/re-test: asked for by the company or farmer
Reporting results
Reporting results
• Results of purity analysis shall be given to 1 decimal place.
• Percentage of all components must be total 100.
• Components less than 0.05 shall be reported as “Trace”.
• % of pure seed, other seed and inert matter must be reported in the spaces provided on the ISTA Certificate.
• If the result for a component is nil, this must be shown as “0.0” in the appropriate space on the certificate.
• Scientific name of the species of pure seed must be reported on certificate.
• Also the kind of inert matter.
• Other seed reported with scientific name according to table 2A or current ISTA List of Stabilized Plant Names.
MSU (PSD 33) or seed with appendages (PSD 15, 38, 46 and 62) attached must be shown on ISTA certificate only
if requested by applicant.
Other seeds by number
A complete test is one in which the whole work sample is searched for all other seeds present.
A limited test is one in which the search is restricted to stated species in the whole work sample.
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• A reduced test is one in which only part of the work sample is examined.
• A reduced-limited test is one in which less than the prescribed weight of seed for a working sample is examined
for the stated species only.
Special tests
Pelleted (encrusted) seed
Normal purity procedure, fractions are: pure pellets, unpelleted seed and inert matter
• Soak 100 pellets in water.
• Check if there is pure seed, weed seed or other particles inside the pellets.
Other seed detemination (OSD): Test for other species and/or insects, sand for seed law of specific countries
1000 kernel weight: Count a number (8x100 seeds) of pure seeds weight of seed divided by the number of seeds
Certificates
ISTA Orange-Official seed sampler and laboratory from the same country or an other country.
ISTA Blue- Anybody can take/draw the sample, no relation with the seed lot. Only the sample is tested according
to the ISTA Rules.
National certific
Question. How do you do an appeal in purity yet the pure seed component is sent for germination
Answer. The option is to use the remnant seed and indicate not done according to ISTA or resample the lot for
retesting or refer to the reference sample.
Question. Which species of seed are tested using diaphanoscope.
Question. Can an assistant do sampling?
Answer. Possible if they are trained
4.4. ISTA RULES CHAPTER 5 GERMINATION
Germination testing.
Work of the germination committee.
(Lecture notes missing)
Desmodium Germination Evaluation By Simon K. Kogo-KEPHIS-NAKURU
Scientific Classification
• Kingdom: Plantae
• Division: Magnoliophyta
• Class: Magnoliopsida
• Sub class: Rosidae
• Order: Fabales
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• Family: Fabaceae
• Sub family: Faboideae
• Tribe: Desmodieae
• Genus: Desmodium
• Species: Intorture
Botany
Desmodium is an inconspicuous legume of which few have bright or large flowers. Some species can become
sizeable plants, but most of them are herbs or small shrubs. The fruit are tomants (viz meaning that each seed
is dispersed individually enclosed in its segment. This makes it a tenacious plants hence can be considered as a
weed.
Desmodium species
USES:
• As a nitrogen fixer
• As livestock feed
• As biological weed control method
• As an insect repeller
Laboratory Testing Table Part 1 Of Ista Rules
• Substrate TP
• Temperature (°C) 20 – 30
• First count 4 days
• Field count 10 days
Acid Scarification
• Digestion is concentrated acid (H2SO4) is effective. The seeds are soaked in acid until the seed coats are
pilled (soften viz 10 minutes in our lab).
• Digestion may be rapid, or take more than one hour, but the seeds should be examined every few minutes.
After the digestion, seeds must be thoroughly washed in running water before the germination test
commences.
Desmodium gangeticum Desmodium intortum Desmodium triforum
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• Additional directions include recommendations for breaking dormancy. Use of H2S04
Evaluation
The shoot system consists of the elongated hypocotyl and two cotyledons with the terminal bud lying between
them. There is no epicotyl elongation within the test period; epicotyl and terminal bud are not usually discernible.
The root system consists of the primary root, usually with root hairs, which much be well developed. Secondary
roots may occasionally develop during the test period, but they are not taken into account in seedling evaluation.
Normal seedlings
Seedling as a whole
All essential structures are normal, as detailed in the following: Root system The primary root Is intact or
Shows acceptable defects: • Discolored or necrotic spots • Healed cracks and splits • Superficial cracks and splits1
Shoot system
The hypocotyl Is intact or Shows acceptable defects:
• Discolored or necrotic spots • Healed cracks and splits • Superficial cracks and splits1 • Loose twists
The terminal bud and surroundingtissue
(Usually not visible) Is intact
The cotyledons Are intact Or Show acceptable defects:
• Up to 50% of tissue not functioning normally • Only one (intact) cotyledon • Three cotyledons
Abnormal seedlings
Seedling as a whole
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The seedling Is abnormal if it • Is deformed • Is fractured • The cotyledons emerge before the primary root from the seed
coat • Consists of fused twin seedlings • is yellow or white • is spindly • is glassy • is decayed as a result of primary infection
one or more of the essentialstructures
Are abnormal as detailed in the following:
root system
The primary root is defective if it • is stunted or stubby • is retarded • is missing • is broken • is split from the tip • is trapped in the seed coat* • shows negative geotropism • is constricted • is spindly • is glassy • is decayed as a result of primary infection
A seedling is classed as abnormal, if the primary root is defective, even if secondary roots have developed. A seedling with its primary root trapped in the seed coat is considered normal, if by the end of the test the root tip has found its way out of the seed coat.
Shoot system
The hypocotyls Is defective if it • is too short and thick • is deeply cracked or broken1 • is split right through • is missing • is bent over or forming a loop • is tightly twisted or forming a spiral • is constricted • is spindly • is glassy • is decayed as a result of primary infection
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Damage or decay of the cotyledons at thepoint of attached to the seedling axis or nearthe terminal bud render as seedling abnormal –irrespective of the 50% rule
The terminal bud or surround tissues (Usually not visible) Is defective
The cotyledons Are defective if they • are defective to such an extent, that less than 50%
of the original tissue or estimated tissue) isfunctioning or curled
• are swollen or curled • are deformed • are broken or otherwise damaged • are separate or missing • are discolored or necrotic • are glassy • are decayed as a result of primary infection
4.5 HOW TO USE THE ISTA HANDBOOK FOR SEEDLING EVALUATION (By Gillian McLaren-SASA)
Purpose of the Seedling Evaluation Handbook
Providing trainee seed analysts with a general botanical overview that is necessary for an understanding of the
work they will be doing
Provide all analysts with Guidance on the interpretation of the ISTA Rules and
Additional information on the ways rule requirement may be satisfied
General Botanical Overview
Section 1
Part 1 Life
Part 2 Systematic and Nomenclature
Part 3 Life Processes
_ Photosynthesis
_ Respiration
_ Growth
_ Reproduction
General Botanical Overview
Section 1 Part 3 Reproduction
Life cycle of a flowering plant
Seed Germination Fertilization
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Environmental requirements
_Water
_Temperature
_Gaseous environment
Attributes of the seed
_Chemical deficiency
_Weather experienced during development
_Maturity
_Mechanical damage
_Heat damage
_Chemicals treatments
_Insects and mites
_Seed-borne pathogens
_Seed Longevity
Physiology of Dormancy
_ Innate
_ Induced or secondary dormancy
_ Enforced
Innate Dormancy
_Immaturity of the embryo
_After-ripening requirements
– Low temperature imbibition
– Dry storage
_Specific Environmental stimulus required
– Mechanical
– Light
– Alternating temperatures
Mature pliant
Seedling Flowers
Vegetative growth
Reproductive phase
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– Potassium nitrate
– Ethylene and carbon dioxide
– Pre-washing
Agriculture and seed testing
The importance of seed in agriculture and the importance of seed testing.
Laboratory Conditions for Seedling Evaluation
Laboratory Equipment
_ Temperature Control
_ Light
_ Humidity
Growing Media Specifications
_ Water retention
_ PH
_ Conductivity
_ Cleanliness and freedom from toxicity
Dormancy breaking treatments
Pre-treatments
_ Pre-heating
_ pre-chilling
_ Pre-washing
_ Removal of seed structures
_ soaking
_ Mechanical or chemical scarification
Treatments during the germination test
_ KNO3
_ GA3
_ sealed polythene envelopes
_ alternating temperatures
_ Light
Nomenclature of the various seedling parts:
_Root system
_Seedling axis and terminal bud
_Cotyledon(s)
Seed Testing deals with 8 morphologically distinct types of seedlings:
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Seedling
Type
Mono/Di
Cotyledon
Germ Type Green Area
Representative
Genus
A
Mono Epigeal Cotyledon Allium
B
Mono Hypogeal Primary Leaf Freesia
C Mono Hypogeal Epicotyl/
Terminal Bud
Asparagus
D
Mono Hypogeal Primary leaf
Poaceae
E
Dicot Epigeal Hypocotyl/
2 Cotyledons
Brassica
F
Dicot Epigeal Hypocotyl/
2 Cotyledons
Epicotyl/ Primary Leaf
Phaseolus
G Dicot Hypogeal Epicotyl/ Primary
leaves
Pisum
H
Conifers Epigeal Hypocotyl
Pinus
For the evaluation of the seedlings, there are 3 basic elements in the ISTA Handbook:
1. The definition of Normality / Abnormality and the codification
1.1 Definition of Normality and Abnormality (Section 8: Seedling
Evaluation)
1.2 Codification of Abnormalities (Appendix 3: Index of Seedling
Abnormalities)
2. The guidelines for the evaluation of certain types of seedlings and anomalies (e.g. 50 % rule, loops and spirals) –
Section 8.4
3. The assignment of the genera to categories and groups
3.1 The criteria of assignment (Section 7 : Evaluation of Seedlings of Particular Genera)
3.2 The seedling groups and their representative genera (Appendix 2: Index of the Seedling Groups)
The definition of Normal/Abnormal & the Codification
Normal and Abnormals are defined by the ISTA Rules. The Handbook, Section 8 gives a detailed description
of:
• Normal seedlings
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• Intact seedlings
• Seedlings regarded as slightly defective
• Secondary infected seedlings
• Abnormal seedlings
Normal / Abnormal seedlings
• “Normal Seedlings” as defined by the ISTA Rules:
“show the potential for continued development into satisfactory plants when grown in good quality soil and under
favorable conditions of moisture, temperature and light”
• “Abnormal Seedlings” as defined by the ISTA Rules:
“do not show the potential for continued development into satisfactory plants when grown in good quality soil and
under favorable conditions of moisture, temperature and light”
Codification of Abnormalities
Appendix 3: Index of the Seedling Abnormalities
Codification
The code has 3 positions e.g.
1 1 / 0 1
Part of the seedling Organ affected Reference number of the
Abnormality
Seedling 0 Seedling 0
Root system 1 Hypocotyl 1 Short 01
Shoot system 2 Terminal Bud 2 Split 04
Experience and the results of investigations have led to the development of some specific guidelines for the
evaluation of certain types of seedlings, irrespective of their Seedling Type or Group.
There are guidelines for the evaluation of:
• Damaged cotyledons and primary leaves – the 50% rule
• Seedlings with necrosis of the cotyledons
• Seedlings with splits and cracks
• Seedlings with loops and spirals
• Seedlings where secondary roots are taken into consideration when the primary root is damaged or missing
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• Seedlings exhibiting negative geotropism
• Multigerm seeds
• Samples where a large number of doubtful seedlings are produced
The guidelines for the evaluation of certain types of seedlings and anomalies
Example 1:
Damaged cotyledons and primary leaves – the 50% rule (Section 8.4.1)
Example 2:
Seedlings with splits and cracks (Section 8.4.3)
A split is considered a hindrance to the development of the seedling when it reaches the central cylinder or it affects
the transport of sap –so a transverse cut is made:
• If the split does not reach the central cylinder, the seedling is considered normal
• If the split reaches the outer layers of the central cylinder:
• It is normal if healing has occurred provided there is no deformation of the
epicotyl or hypocotyl
• It is abnormal if no healing has occurred
• If the split reaches or crosses the central cylinder it is abnormal, irrespective of whether healing has taken place
or not
Example 3
Seedlings with loops and spirals (twists) – (Section 8.4.4)
A loop or spiral in the hypocotyl or epicotyl is considered a hindrance to the development of a seedling. These
could have been caused by laboratory testing and its important to distinguish such apparent abnormalities from
naturally occurring defects
The assignment of the genera to categories &groups (Section 7)
• A description of the seedling and the criteria for their evaluation according to systematic families is not possible
• The seedlings of different genera have different morphologies and for this reason they are assigned to different
categories and groups for assessment purpose
• In practice, seedlings or their genera are arranged into groups with:
• Similar morphologies
• Evaluated using similar criteria
• Peas and Beans belong to the same family: the Fabaceae but they have different morphologies
• Cucumis and Gossypium belong to 2 different families but the
Seedlings show similar patterns. They are grouped in the same group for assessment
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Criteria of assignment Section 7.1.1
In this Handbook genera have been classified into 2 categories:
• Agricultural and horticultural plants, represented by the letter A
• Trees and shrubs, represented by the letter B
Categories A and B is then sub- divided into groups according to the following criteria:
Systematic class 1 monocotyledons
2 dicotyledons
3 conifers
Germination mode 1 epigeal germination
2 hypogeal germination
Shoot development 1 without epicotyl elongation
2 with epicotyl elongation
3 no shoot elongation;shoot apex
enclosed with a sheath (coleoptile)
4 Tuberous hypocotyl
Root system and its
Significance for evaluation 1 primary root essential
2 secondary roots may compensate
For the primary root
3 several equal seminal roots
-The letters and numbers are combined to form Group number for Example; Group A-2-1-2-2 comprises
seedlings:
• A – 2 – 1 – 2 – 2 of agricultural or horticultural plants
• A – 2 – 1 – 2 – 2 belonging to the dicotyledons
• A – 2 – 1 – 2 – 2 with epigeal germination
• A – 2 – 1 – 2 – 2 with epicotyl elongation
• A – 2 – 1 – 2 – 2 with secondary roots that are taken into account if the primary root is defective
The seedling groups and their representative genera
How do you find the seedling type & group?
•Go to Appendix 2: Index of the seedling groups e.g. Lactuca
The seedling type and seedling group of Lactuca is: Type E – Group A-2-1-1-1
How do you find the descriptions and illustrations for Lactuca?
• Go to the relevant section
• The Type E – Seedling Group A-2-1-1-1 is at the bottom of the pages within the Handbook
Seedling Type E – Seedling Group A-2-1-1-1
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Appendix 1: Glossary
Appendix 4: Systematics of the ISTA families
Lets do some practising!
_ Question 1: groups and representative genera In the category of trees and shrubs………….
How many groups are there?
Which are the representative genera of trees and shrubs?
Answers!
- Go to the table of contents _ Section 7, 7.2.2 Seedling Groups
- ANSWER = 3
Which are the representative genera of trees and shrubs?
- ANSWER = Robinia / Quercus / Abies, Pinus
_ Question 2: Codification of Abnormalities
What is the code of this abnormal seedling?
The kind of abnormality is a ‘loop of the hypocotyl’
Go to Appendix 3: Index of the Seedling Abnormalities, table 2 Abnormalities of the shoot system
• Shoot system – code 2
• Hypocotyl defective - code 1
• Is bent over or forming a loop - code 06
2 1 06
Shoot Hypocotyl Loop
- ANSWER = 21/06
Last Question!
_ Question 3: Seedling evaluation according to groups
How do I assess seedlings of Beta infected by fungi – How and where do I find the relevant information in the
Handbook?
Answers!
- Beta vulgaris
- Go to Handbook – Appendix 2: Index of the Seedling Groups
- Seedling Type E – A – 2 – 1 – 1 – 1
Refer to the bottom of the pages and go to Seedling Type E Seedling Group A-2-1-1-1 (Brassica) 15 – 5; Go
to the Supplementary Remarks
- ANSWER = Information on Beta vulgaris:
4.6. GERMINATION PRACTICALS ACCORDING TO ISTA RULES 2008
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(By Gillian Mclaren and Anny van Pijlen-SASA / NAK AGRO)
Practicals
Germination According to ISTA Rules 2008 • Phaseolus vulgaris • Arachis hypogaea • Allium cepa • Daucus carota • Helianthus annuus • Desmodium spp Growth and development of seedlings Dicotyledons Primary leafs, Hypocotyl, Secondary roots, Primary root, Cotyledons, Epicotyl, Cotyledons, Primary leafs Plumula Monocotelydons Second leaf, First leaf, Coleoptile, Mesocotyl, Secondary roots, Primary root Methods ISTA Rules Daucus carota ISTA Method: • Substrate: TP; BP • Temperature: 20-30/20 • Counting days: 7 =14 • Pretreatment: none Seedling Type E Seedling group A-2-1-1-1 Daucus carota Lactuca; Zinnia / Asteraceae Beta; Spinacea/ Chenopodiaceae Brassica / Brassicaceae Lycopersicon esculentum/ Solanaceae Helianthus annuus Daucus carota Seedlings as a whole are normal if: All essential structures are normal, as detailed in the following Root system Primary root -is intact or shows acceptable flaws: _ discolored or necrotic spots _ healed cracks and splits _ Superficial cracks and splits Seedling as a whole is abnormal, because it _ is deformed _ is fractured _ the cotyledons emerge before the primary root from the seed coat _ consists of fused twin seedlings _ is yellow or white _ is spindly or glassy is decayed as a result of primary infection A seedling is classed as abnormal, if the primary root is defective, even if secondary roots have developed. A seedling with its primary root trapped in the seed coat is considered normal, if by the end of the test the root tip has found its way out of the seed coat. Allium cepa ISTA Method: • Substrate: TP; BP; S • Temperature: 20/15 • Countingdays: 6 =12 • Pretreatment: prechill
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Seedling Type A Seedling group A-1-1-1-1 Allium cepa/Liliaceae Monocotyledons Normal seedlings: Intact root, shoot and root system Normal seedlings always have to have a “knee” and a primary root. Acceptable defects like: loose twists, discolored or necrotic spots Helianthus annuus ISTA Method: • Substrate: BP; S • Temperature: 20-30/25/20 • Counting days: 4 =10 • Pretreatment: Preheat; prechill Seedling Type E Seedling group A-2-1-1-1 Helianthus annuus Daucus carota Lactuca; Zinnia / Asteraceae Beta; Spinacea/ Chenopodiaceae Brassica / Brassicaceae Lycopersicon esculentum/ Solanaceae Normal seedlings: • Intact: hypocotyl, cotyledons, terminal bud. • Primary root • 50% rule • Intact root, shoot and root system • Acceptable defects like: loose twists, discolored or necrotic spots A seedling is classed as abnormal, if the primary root is defective, even if secondary roots have developed. A seedling with its primary root trapped in the seed coat is considered normal, if by the end of the test the root tip has found its way out of the seed coat. Phaseolus vulgaris ISTA Method: • Substrate: BP; S • Temperature: 20-30/25/20 • Counting days: 5 =9 • Pretreatment: none Section 18 Type: F Group: A-2-1-2-2 Family: Fabaceae Dicotyledons Phaseolus vulgaris also Arachis hypogaea, Glycine max • Primary root: sufficient secondary roots is also considered as a normal seedling. • Intact: hypocotyl, first leaves, cotyledons, terminal bud. • Acceptabel defects e.g. losely twisted hypocotyl, healed cracks; 3 cotyledons; 50% functioning of the cotyledons and first leaves. • Apply the 50% rule if the leaves are deformed e.g. white leaves, necroses, primary infection. Phaseolus vulgaris 25% rule • If the leaves have been developed “normal” consider that the leaves must be larger than 25% of the normal growth (= normal). • To evaluate you have to calculate if the leaves are more 25% of the normal growth. Calculate the length and the width of the leaves.
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• In practice: take a normal developed leaf and compare the leaves of which you are uncertain and judge if they are larger or smaller than 25 % of the normal leaf. Arachis hypogagea ISTA Method: • Substrate: BP; S • Temperature: 20-30/25 • Countingdays: 5 =10 • Pretreatment: Remove sells; Preheat 40ºC and see disinfection Section 18 Type : F Group : A-2-1-2-2 Family : Fabaceae Dicotyledons Phaseolus vulgaris Arachis hypogaea Glycine max Desmodium spp ISTA Method: • Substrate: TP • Temperature: 20-30 • Countingdays: 4 =10 • Pretreatment: H2SO4 Desmodium triflorum Seedling type: E Seedling group: A-2-1-1-1 The same group: Dianthus Clarkia Geranium Family: Fabaceae Desmodium triflorum
Question 1. W e (Seed testing lab Nakuru) have been experiencing a problem with secondary infections in Arachis in our laboratory; how can we control these infections? Answer from Gillian: You can do a re-test in soil or use different fungicides treatment because of development of resistance by the pathogens.
4.7.CALIBRATION, TEMPERATURE CONTROL, ENTRANCE CONTROL AND SHELF LIFE (By
Anny van Pijlen-NAK AGRO)
Calibration and checking temperature
• Dividers
• Balances
• Seed counter
• Germination cabinets
• Oven
• Thermometers
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• Data loggers
Entrance control shelf live
• Filter paper
• Sand/soil/organic growing media
• TZ salt/GA3/ KNO3
• New equipment
Dividers
• In the seed laboratory
• At the seed processing plant
NB: For the Gamet divider it is not necessary to calibrate anymore
Calibration dividers
1x per year all qualified staff must show that they can make a correct dividing of a sample with the soil divider.
Example
Calibration dividers
A check sample can be composed of 500g coarse seed (e.g. Triticum or Hordeum) and 100g fine seed (e.g
Trifolium or Phleum pratense).
Example
Procedure:
1. Prepare a sample as described in previous sheet
2. Mix the sample thoroughly
3. Ensure the divider is leveled
4. Divide the sample with the divider
5. Weigh the pans and record the weight (here you also can separate the different species by sieving)
6. Repeat this from step 4 and do this 10 times
7. Calculate the 10 steps (lost of weight of both fractions)
8. Use tolerances as defined
9. Sign the file with initials and date
10. Conclude: Approved or not approved
• During the dividing a maximum of 5g seed lost may be accepted.
• Maximum of 5% difference between the right and left pan may be accepted.
• After the calibration: determine the acceptable range in your laboratory.
Check each analyst/sampler who is qualified for dividing and keep records.
Calibration balances
• Inventory of balances.
• Ensure stable place for balances
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• Certified calibration weights
• External calibration
• Procedure
• Logbook/register
Disturbances/Sensibilities
1. Out of horizontal balances, not leveled
2. Electrostatic charges
3. Environmental temperature, temperature differences of samples
4. Humidity
5. Air pressure
6. Vibrations, air flow, mechanical damage, overloading
• Calibration weights e.g. 1g, 5g, 20g, 1kg depending on the range of the balance
• Settlement of warning, action and limiting values
• Given by external calibration institute
• Wear gloves or use tweezers during calibration
Example external maintenance, control or calibration registration form
Identification
number
Date
Signature
Serviceman
Report Remark
Example internal maintenance, control or calibration registration form
Identification number Date
Signature analyst Remark/Comclusion
Calibration balances Minimum Maximum
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Warning value 4,933 5,067
Action value 4,867 5,133
Limit value 4,800 5,200
Calibration seed counter
• Prepare samples of different species
• Normally used for counting
• Count 1000 seeds by hand (check the result)
Procedure (example):
Put the seeds at least 10 times through the seed counter.
If the 10 repetitions are within the pre-set tolerance the counter is accepted for the procedure.
Accepted/not accepted. Add date and initials of the analyst on the record sheet.
Calibration seed counter
• 1 x per week
• Triticum aest.
• Linum us.
• 100 seeds
Action values
< 99
> 101
Temperature control cabinets
How many readings per day:
• Constant temperature equipment at least 3, at regular pre-set times
However ………..
If records show temperature is stable in terms of temperature with variations of less than 1ºC between readings the
recording frequency can be reduced to once per day.
But………………
If there is any indication of a change in performance recording frequency must be increased.
For alternating temperature equipment at least 3 readings must be recorded per day at pre-set times. The timing of
these readings must be such that both temperature phases are monitored.
Different types of temperature measuring equipment
Temperature control cabinets
Number of measuring points:
For Germination /Temperature Rooms or Cabinets with an area of:
• Less than 10m2 there must be at least one measuring point;
• between 10m2 and 20m2 there must be at least two measuring points;
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• 20m2 or more there must at least three measuring points.
Where there is more than one measuring point the difference in temperature between measuring points should be
no more than ±2ºC in the temperature.
When comparing the temperature of the room against the required temperature, the mean of the temperatures
recorded at the measuring points is used.
Temperature oven
If in the oven 3 layers /shelves are used for the moisture determination than at least 3 readings must be recorded
during the calibration. If there is a deviation between one of the shelves, the analyst can decide not to use the shelf
that is exceeding the tolerance.
NB: Notification must be visible on the oven and known by the analysts that are using the oven.
If the oven is in use, daily temperature readings must be recorded.
Calibration of temperature probes
Calibration can be achieved in a number of ways:
– Probes can be calibrated externally
– Externally calibrated reference probes can be used to calibrate working probes
– Probes can be calibrated using ice Steam/Boiling water must NOT be used to calibrate probes.
• External calibration every 5 years
• Is supplemented by annual in-house ice point calibration.
Any probe whose temperature reading differs from the standard by more or less than ±0.5 ºC should be removed
from service.
Use an externally calibrated reference thermometer to calibrate working the thermometers in the laboratory.
• Calibration of Thermometers
• International Ice Point Method
Ice Point Method
• The ice particles should be no more than a few millimeters in diameter.
• The water and ice should be pure or prepared from de-ionized water, which is air saturated. For high precision the
thermometer should be maintained in the mixture for 10 minutes prior to reading.
In theory accuracies of ±0.001ºC may be achieved but in practice ±0.005ºC is more likely.
Calibration of probes should take place at least once a year.
Any probe whose temperature reading differs from the ice point by more or less than ±0.5ºC should be removed
from service. Only probes that can read 0ºC can be calibrated using ice point methodology.
Temperature probes, electronic meters and data loggers
The ISTA Seed Testing Laboratory
Accreditation Standard 9.2 states that:
The laboratory must maintain a record system to suit its particular circumstances.
Records must be kept for a defined period but not less than of six years”
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• Calibration Checks should be recorded and be available for inspection by auditors.
• Keep a Thermometer/Probe Register.
The records who may want to acertain the temperature of a particular piece of apparatus on a specific date should
be archived for a period of at least six years.
Entrance control
Check the old batch against the new batch.
Use one of the species mentioned in the Rules e.g. Lepidium, Festuca.
NB: In tropical areas Brassica nigra, and Brassica campestris is used.
Indicate accepted/not accepted and Date and initials of the analyst.
Shelf life
• TZ salt
• KNO3
• GA3
Shelf life- Stated by the supplier. If not:
• Determine the shelf life by experiment. e.g. use new purchased material against the one in stock.
• Note the date of purchase on the bottle.
• Control sheets must be prepared recording the preparation of the bottles of tetrazolium salt
• Initials of analyst
• Date of preparation, expiry date
• Describe in the SOP how long the solution can de stored and fill out the date on the solution
Questions: Deep freezers –20’c sometimes goes to –23’c what should be done?
Question: How do you calibrate the micro pippete?
Question: How many times do you calibrate the divider?
Answer: It can be once a year for each user
4.8.INTRODUCTION TO VIGOUR TESTS (By Gillian McLaren-SASA)
Outline
• How does vigour relate to germination?
• Definition of vigour
• Why do vigour differences occur?
• What vigour differences mean
• Types of vigour test
• Use of vigour tests
Germination
- in the laboratory test is the emergence and development of the seedling to a stage where the aspect of its essential
structures indicates whether or not it is able to develop further into a satisfactory plant under favorable conditions
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Germination and field emergence of commercial seed lots
Brassica oleracea e.g. cabbage, Brussels sprouts
Ambient = ambient in UK
DH = dehumidified and low temperature store
Crop No of lots Range among lots
Lab germination Field emergence
Garden pea 80 >80 8-85
Soybean 18 83-96 22-90
Green bean 30 75-100 34-93
Onion 12 77-92 46-72
Turnip 30 88-99 36-76
Differences in storage potential
Germination (%) after storage for 2 years
Initial Store 1 Store 2 Store 3
Germination(%) (ambient) (ambient) (DH)
84 28 33 56
97 84 88 91
93 54 63 73
91 17 21 39
• Differences in field emergence and storage potential were not detected by germination tests.
• Led to concept of vigour: an additional aspect to the quality of
germinable seeds
• ISTA definition (2001)
Seed vigour is the sum of those properties that determine the activity and performance of seed lots of acceptable
germination in a wide range of environments
Why do vigour differences occur?
• Differences in the physiological age – the main cause of vigour differences
• Seeds can age during:
– Seed production
• weather conditions, delayed harvest, processing and transport
– Storage
• temperature, seed moisture content, storage relative humidity
Aspects of performance associated with seed vigour
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• Emergence ability of seeds under unfavourable environmental conditions
• Rate and uniformity of seed germination and seedling growth
• Performance after storage, particularly the ability to germinate
What vigour differences mean
Vigour level
High Low
Mean rate of Fast Slow
germination
Spread of Narrow Wide
germination time
Mean seedling size Large, uniform Small, variable
Emergence Good Poorer
Storage potential Good Poorer
Effect of low temperatures at emergence: Maize Argentina October 2007
Seed lots
5 6 4 2 3 1
% ISTA Germination 91 95 94 97 93 99
% Field emergence(Mean of 10
sites)
80 84 84 87 86 92
Warm location(17oC 62MM)
76 81 82 91 85 82
Cool location(12oC 56MM)
32 46 45 58 60 73
Vigour and response to water stress
Brassica juncea Indian mustard(Mehra, Tripathi and Powell,2003)
Water Germination
Potential (normal/abnormals %)
(-MPa)
High vigour lot Low vigour lot
0 93/3 59/18
-0.27 98/1 79/8
-0.45 91/7 66/18
-0.73 90/5 47/23
-1.00 0/19 0/8
High germination retained Germination reduced at
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at reduced water potential reduced water potential
Why do we have vigour tests?
• To provide information about the planting value in a wide range of environments and/or storage potential of seed
lots
• To provide additional information to the standard germination test to assist in differentiation of seed lots of
acceptable germination
Types of vigour tests
• Direct tests
– Environmental stress or other conditions reproduced in lab.
– % emergence or rate of emergence recorded
• Indirect tests
– measure another aspect of seed shown to associated with seedling performance
Types of vigour tests
• Physiological assessments (direct)
– aspects of germination e.g. rate of germination test
– stress tests e.g.cold test, cool germination test
• Biochemical assessments (indirect) e.g.conductivity; tetrazolium
• Application of the ageing process (indirect)
– accelerated ageing
– controlled deterioration
What do vigour tests provide?
• A more sensitive index of seed quality than the standard germination test
• A consistent ranking of seed lots of acceptable germination
• Information on emergence and storage potential of seed lots to plan marketing strategy
Conclusions
• Vigour tests provide information on seed quality that supplements the standard germination test
• Results of tests are valuable to the seed companies and the growers
• Vigour tests are in use by many laboratories
• ISTA Validated tests
– Conductivity test for pea
– Accelerated ageing test for soyabean
• Tests undergoing validation
– Controlled deterioration test for Brassica sp
– Rate of germination test for maize
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• Suggested tests
– Cold test for maize
Conductivity Test
• ISTA validated test for Pisum sativum, applied to other grain legumes, green bean (Phaseolus vulgaris)
Validation in progress. Rules 2009?
– soybean (Glycine max) » Comparative tests completed - validation
• Evidence for relationship between conductivity and vigour
– field bean (Vicia faba)
– chickpea (Cicer arietinum)
– cowpea (Vigna unguiculata)
– long bean (Vigna sesquipedalis)
What does the test measure?
• Electrical current through water
• Influenced by ions e.g. potassium K+
• Dry seed in water leaks sugars, amino acids, K+
• Conductivity of seed soak water assesses relative amount of solute leakage
What influences leakage from seeds? -
The theoretical basis of the test-Membrane deterioration/damage and
Increased dead tissue- Increased leakage
Materials
• Conductivity meter: cell constant = 1.0
• Water: deionised or distilled, < 5 �S cm-1
• Flasks or beakers: 400-500ml, 80 �5mm diameter
• Facilities to maintain 20oC
• Facilities for MC determination
Preparation for the test
• Check seed moisture– 10-14%? If not, adjust it
• Check calibration of conductivity meter
• Check cleanliness of flasks
Setting up the test
Add 250ml water to flasks; include 2 controls per run, leave at 20oC for 18 - 24h
Weigh 4 replicates of 50 seeds / lot (0.01g)
Add one replicate to each flask, cover and leave for 24h at 20oC
Take reading; do not place electrode on peas
4.8.1 ACCELERATED AGEING TEST
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• Validated for soyabean
– Comparative tests on maize completed; validation next
• Predicts relative storability and emergence
• Increases the rate of ageing using:
– High temperature
– High relative humidity
• (increased seed moisture)
Equipment
Water jacketed incubator, Very good temperature control (± 0.3oC), and AA boxes: small boxes with mesh grid to
hold seeds, Accurate, standardized thermometer
Correct weight of seed placed above 40ml water in AA box
Boxes placed at 42o C for 72h
Germination test set up; normals and abnormals counted
AA results
• Germination remains high after AA- High Vigour
• Large decrease in germination after AA- Low Vigour
Conditions for the AA test
Validated
Crop Seed wt(g) No. AA boxes Ageing Temp
(°C)
Ageing Time(h)
Soybean 42
1
41
72
Suggested
Maize 40
2 43 72
4.8.2 CONTROLLED DETERIORATION TEST
• Small- seeded vegetable species e.g. Brassica, Carrot, Onion, Aubergine, Pepper, Watermelon
• Currently in Validation procedure and in Rules Proposal for 2009
Theoretical basis of the test
Manipulation of the rate of ageing; raised moisture content by imbibition and weighing; Place seeds in waterproof
foil packet at High temperature (45oC)
Seal foil packet and hold at 7± 2oC overnight
Set up germination test; Place packets of seeds into water bath at 45oC for 24h
Count total germination (normal + abnormal)
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Assessment of results
• CD germination high, similar to lab germination-HIGH VIGOUR
• CD germination a little lower than lab germination- MEDIUM VIGOUR
• CD germination much below lab germination-LOW VIGOUR
(1) Correlation coefficients (r) between CD germination and field emergence of seed lots from 7 species
Crop No. of lots Correlation
Turnip 30 0.95***
Swede 11 0.89***
Cauliflower 12 0.72**
Brussels sprouts 15 0.79***
Lettuce 11 0.71**
Onion 17 0.77***
4.8.3. GERMINATION RATE AS A VIGOUR TEST
Evidence that germination rate relates to vigour
– Early counts in seed testing
– References for maize, oil seed rape and vegetables
– Use of thermogradient tables for lettuce and other vegetables
Most work on: Maize, Pepper and Cotton
1 23 4
Method
Count germination at specified time
• JG = just germinated (radicle visible)
• G = germinated (2mm radicle)
• MJGT =_ nt / _ n
n = number of seeds newly germinated (just germinated criterion) at time t
t = hours or days from when set to germinate.
• MGT same calculation
• MET same calculation for emergence
Maize: MJGT and MGT relate to seedling size and uniformity in lab
MJGT = 3.52 days
MGT = 4.80 days
MJGT = 4.82 days
MGT = 5.84 days
Maize: MJGT & MGT correlate with performance in soil
Austria Denmark France
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(11 lots)
(7 lots)
(5 lots)
MJGT vs MGT 0.97*** 0.94*** 0.94***
MGT vs Final
emergence
-0.63
-0.63* -0.91*
MGT vs MET 0.75**
0.82*
• Would counts on one day also reflect rate of germination accurately?
• Counts made of physiological germination (2mm radicle)
• Counts after:
– 5 days at 13oC
– 54 hours at 20oC
RGT: a rapid vigour test
Maize
• Rate of germination
– 54 hours at 20oC or 5 days at 13oC
• Cold test
– 7 or 10 days at 10oC, 5 days at 20oC
4.8.4. COLD TEST FOR MAIZE
• Most widely used test for corn
– 86% of labs in N America
– More than 200,000 tests per year in USA
– 50% of labs test >250 samples per year
• Used internationally; Europe, South America
Cold test not standardized between labs
• Containers; trays, boxes (plus and minus airtight covers), rolled towels
• Substrate
– sand alone; warmer zones, silt clay loam, sand + soil mixture, filter paper: plus and minus soil
• Days at 10 oC
– Mostly 7 days: warmer zones
– Some 10 days
– Even 14 days: colder zones
• Moisture
– always wet,sometimes saturated
Rolled towel method
a) Planting of 50 seeds and distribution of soil/sand mixture on the two bottom towels
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b) rolled towels after placement in upright container
c) germinating seedlings
Tray Method
Seeds on crepe- cellulose paper, covered with sand/soil mixture 7 days 10o C, 4 days 25oC
Plastic box method Sand and soil mix
10°C for 7 d, 25°C with light for 6 d
Correlation coefficients (r) between laboratory tests and maize seed lot field performance
SG CT ST AA
CT 0.18
ST 0.42* 0.73***
AA 0.03 0.45** 0.41*
FE 0.16 0.73*** 0.52** 0.53**
SG: standard germination; CT: cold test; ST: soak test;
AA: accelerated ageing; FE: field emergence
* : P ≤ 0.05; ** : P≤ 0.01; *** : P ≤ 0.001
Data from LaRAS, Bologna, Italy, courtesy of Noli et al (2008)
Concluding comments
(1) A range of vigour tests are available
– Conductivity: grain legumes
– Accelerated ageing: soyabean, maize
– Controlled deterioration: small seeded vegetables
– Rate of germination: maize, cotton, peppers (and other vegetables?)
– Cold test: maize
(2) Test conditions must be precise:
– Temperature
– Seed moisture content / relative humidity
– Water quality
– Length of test
(3) Vigour tests:
– can identify differences in the potential performance of seed lots
– are repeatable and reproducible
–
4.8.5. ELECTROCONDUCTIVITY TEST
Materials
_ Conductivity meter: cell constant = 1.0
_Water: deionised or distilled, < 5 �S cm-1
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_ Flasks or beakers: 400-500ml,
80 � 5mm diameter
_ Facilities to maintain 20oC
_ Facilities for MC determination
Preparation for the test
_ Check the seed moisture
– Is it between 10 – 14%?
– If Yes – use it
– If not – adjust it
_ Check calibration of the conductivity meter
_ Check the cleanliness of flasks
SETTING UP THE TEST
Add 250ml water to flasks; include 2 controls per run’ leave at 20oC for 18 - 24h
Weigh 4 replicates of 50 seeds per lot (0.01g)
Add one replicate to each flask, cover and leave for 24 hours at 20oC
Record information
Distilled water check
Enter the:
Conductivity e.g. 1.1 µScm-1
Temperature e.g. 20.2oC
Add in the time the seed was soaked e.g. 11:00
Conductivity meter calibration check
Calibration Solution A & B For each solution:
• Temperature
• Theoretical reading for given temperature
• ±5% of theoretical reading
• Actual measured reading
• Theoretical minus actual readings
Measure the controls Using the conductivity meter:
Measure the control flasks (Control 1 and Control 2)
Temperature (20 ±2°C)
Conductivity ¡Ü 5 µScm-1
Gently swirl the seeds for 15 seconds
Check for hard seed (remove any & weigh)
Take readings
Do not place electrode on seeds
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Recording the results
Add in the results to the appropriate column:
A = Weight of seed (2 decimal places)
B = Count number of hard seeds
C = Weight of hard seed
D = Weight of seeds that imbibe
E = Conductivity of solution
F = Conductivity of replicate minus mean conductivity of control
Conductivity = Column F / Column D
Scottish interpretation of pea conductivity results (for information only – not ISTA)
Electroconductivity
Result (µScm-1)
Interpretation
<25
There is nothing from the electroconductivity test to indicate that the seed
is not suitable for early sowing.
25 - 29
Seed may be suitable for early sowing, but there is a risk of impaired
performance under adverse seed bed conditions
30 - 43
Seed probably not suitable for early sowing especially under adverse
conditions
>43
Seed probably not suitable for sowing..
Question; Any correlation between seed viguor of soil seed crops and their germination.
Answer. Seed viguor in oil crops correlate very well with their germination.
Question: Any reason why de-husked barley are not recommended in seed lot?
Answer: The de-husked barley grains are more exposed to damages and are at risk of not germinating after being
processed.
Question: could seed energy be the same as viguor.
Answer: The seed germination percentage obtained on the first day could refer to viguor and hence energy of seed.
4.9. ACCREDITATION OF LABORATORIES (By Anny VanPijlen)
Two routes:
1. Issuing domestic (national) certificates
2. Issuing international certificates
Same basic requirements in both cases based on QA principles
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The end product of a Seed Testing Laboratory is a test certificate giving the
average quality of the seed lot.
What is Quality Assurance?
QA started in the 20th century in Complex industries with 100% reliability targets
e.g. arms, munitions, computers.
Then spread to mass production industries e.g. cars, and to testing services. Instead of waiting until the product is
made and then checking if it is right QA is used to check all the steps in the process.
If the processes are ok then the product will be ok
Avoiding errors
• Mistakes cost money
• Correcting mistakes wastes too much time
Think first!
• The cheapest mistakes are those that are eliminated before they happen
The Quality revolution
Meeting the quality standard
• The quality standard is achieved when all the customer’s requirements are met.
• Over fulfilling customer requirements costs you money for no extra gain.
• Under fulfilling leaves a dissatisfied customer.
ISO 9001 Certification
• ISO 9001 is the standard used to CERTIFY companies in, for example, manufacturing or service industries.
• Testing laboratories are certified using another standard ISO/IEC 17025.
• ISO 9001 is the basic blueprint for Quality Assurance.
From ISO 9001 to ISO 17025
The ISO 17025 standard is used for the accreditation of testing laboratories e.g. chemistry or molecular biology.
It is based on ISO 9001 but places extra emphasis on:
• Staff competence
• Equipment control and calibration
React
Correct
Backward checks
Act
Prevent
Forward checks
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• Appropriate methods and method development
• Mandatory proficiency tests
From ISO 17025 to the ISTA Standard
The ISTA Standard is adapted from ISO 17025 to meet the specific needs of seed labs.
Specific features of the ISTA Standard include:
• Sampling • Independence of labs
• Use of ISTA Rules • Staff competence
• Mandatory participation in the ISTA proficiency test program
The 5 Ms and 1 E of Quality
Material, Man, Machines, Management, Methods and Environment
All these elements must be under control to get achieve quality
Developing a QA culture
• To introduce QA successfully the organization must develop a “quality culture”
• Staff needs to be convinced of the value of QA
• Once QA systems have been introduced, staff frequently experience greater job satisfaction
QM Principles
Involvement of staff:
• People at all levels are the essence of an organization and their full involvement enables their abilities to be used
for the organization's benefit.
• Motivated, committed and involved people within the organization
• Innovation and creativity in furthering the organization's objectives
• People being accountable for their own performance
• People eager to participate in and contribute to continual improvement
What is a quality system?
_ Say what you do
_ Do what you say and
_ show that you do what you say
Quality manual–General design
Quality objectives
Objectives in relation to the quality of the testing work must be „ SMART „
S = specific
M = measurable
A = achievable
R = relevant
T = time-bound
They are regularly evaluated in a defined process and, if necessary,
• verified
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• Adjusted
• New objectives are set
Elements of a QM system
1. System documentation
Often structured along recognized models such as ISO 9001, ISO/IEC 17025
2. Traceability
Defined interfaces between processes; comprehensive recording
3. Provisions for adaptation
Monitoring, measurements, analyses, improvements, reviews, audits
4. Factual approach
Verification and validation of equipment, methods, processes; systematic procurement of information
Building blocks of a QA system
Q-Manual(Level A)
- Describes the quality system in accordance with the stated quality policy and objectives and the accreditation
standard
(Level B)- Documented quality system procedures (Standard operation procedures) - Describes the activities of
individual functional units
(Level C) -Other quality documents- (Work instructions, forms, reports, etc.)-Consists of detailed work documents
What is Quality Management?
The so called Deming Circle or PDCA principle…
Define targets
and procedures
PLAN DO Implement the
Procedures
Prioritise
opportunities
CHECK
Evaluate
performance
Leads to permanent improvement
Page 55 of 64
STA Accreditation Standard
1. Management Requirements
2. Staff
3. Environment, , equipment and calibration
4. Lot identification, sampling and handling of samples
5. Test Reports and Certificates
6. Records
7. Quality Assurance System
8. Methods and Procedures
Management Requirements
_ Policy and structure
_ Description of the quality system
_ Describe goals for training, education for lab staff
_ Provide a list of species or group of species and analyses for which the lab claims competence for
_ Policy to ensure protection of clients confidential information
_ Appoint a technical manager
_ Demonstrate that the staff have fixed salary
_ Define the organization, management structure of the lab (flowchart can be used)
_ Access to the lab
_ Specify responsibilities and authorities
Staff
• Demonstrate skills of staff (records of training and education)
• Use staffs that are employed by, or under contract of the lab
• Job description of lab staff and samplers (with limitations)
• Proper supervision for testing staff and samplers
Environment, equipment and calibration
• The laboratory must be fit for the purpose of seed testing
• A full range of equipment for the test being done should be provided
• The equipment must be maintained in working order and where necessary, regularly calibrated
Lot identification, sampling and handling of samples
• Number (and type) of containers
• Tests required
• All information on clients demand
• Details of circumstances during seed sampling which can influence the test results
• System for approval of lot identification
• Monitoring the uniformity of the seed lot and refuse of sampling and testing if doubt exist concerning uniformity
• Authorization of samplers
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• Training program
• Up to date lists of samplers
• Monitoring the samplers
• Describe procedure cancellation authorization of samplers
• Name of seed sampler
• Name and address client
• Date of sampling
• Unique number for identification of the seed lot
• Species and where relevant cultivar
• Weight of seed lot
• Procedure present at all stages of obtaining, dispatching, transporting, handling, sub sampling and testing of
samples.
• This to prevent contamination, damage or deterioration which would influence test results.
• Keep records of unusual conditions of the samples by receipt in the lab
• Clear rules for receipt, retention and disposal of samples
• Retention of samples, not less than 1 year after issue of the ISTA Certificate
Methods and Procedures
• Up to date rules, handbooks, manuals, instructions and reference data
• Available for the staff
• Calculations must be checked systematic
Documents, ISTA Rules, handbooks and/or intranet must be up to date
Test Reports and Certificates
• Integrity of data
• Storage and protection of data
• Information of data (to others/clients)
• Access for data (Password computer)
• Only issue certificates on species which are listed in the
Rules
• And for which the lab is accredited
• Signature of responsible person
Records
• Up to date records of staff and training records
• Keep records for at least 6 years
• Use inerasable pen
• Correction of mistakes in records must not be erased but crossed out
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Quality Assurance System
• Document control
• Review by the management
• Review of tenders, requests and contracts
In this lecture:
• Audits
• Proficiency testing
Proficiency testing
is a key element in maintaining the competence of ISTA laboratories
The Rating System
A, B, C and BMP (Below Minimum Performance) for in-round and overall rating
Overall rating will be determined after 6 rounds of participation. Only results of mandatory test rounds are included
in the overall rating
Overall Rating
The same overall rating system is used for Germination, Purity, Moisture content, Tetrazolium and Other Seed
Determination
Score Number give Overall score Threshold
A 5 A 28-30
B 4 B 21-28
C 3 C 16-20
BMP 0 BMP Below16
Consequences
Overal C Warning letter
Overal BMP Suspension
Impact
Confirmation of good performance
Indication of potential systematic errors in testing procedures:
– Defective equipment
– Level of knowledge of staff
– Insufficient documented procedures
– Testing material
Corrective action
• Are deficiencies systematic?
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• Are trends evident?
• Calculation or transcription error?
• Were all items of equipment appropriately maintained and monitored at the time of testing (Maintenance and
monitoring records, etc.)?
• Is staff trained for the particular type of test, have all procedures been adhered to?
• How are the results of repeat tests?
• Was all testing material appropriate?
• Adjusting maintenance of equipment
• Training of staff
• Update and/or amend test procedures
• Carry out ring tests
Proficiency Testing
-120 participating laboratories world wide. The laboratory‘s performance is directly linked to its accreditation
status.
The ISTA Accreditation System:
Accreditation = formal recognition of a laboratory to competently carry out specific tests
Authorisation = agreement of the Designated Authority of the country concerned for the laboratory to issue ISTA
Certificates
Audit procedure
Application for Membership
Membership approved?
Participation in Proficiency Test Programme
Results o.k.?
Stop No
Yes
Corrective actionsNo
Application for accreditation
Yes
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Accreditation procedure
2
Submission of QDocuments to the Secretariat
Documents ok?
Appointment of the audit team
Corrective actions No
Yes
Audit
3
2
Substantial deficiencies?
Approve by EC
Authorization DA?
Corrective actions Yes
Yes
Accreditate but not authorized No
Corrective actions ok?
No
Yes
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ISTA Membership
Accreditation Program
Q-Audit Proficiency Testing
Quality management system according to the ISTA
Accreditation Standard
120 participating laboratories world
wide
ISTA audit every three years
The laboratory‘s performance is
linked to its accreditation status
Audit by two ISTA Three test rounds per year
Advantages of being ISTA accredited
• Prove the lab works successful according to ISTA Rules/Handbooks/Accreditation Standard
• Give status to the lab
• Implementation in the rules of methods developed in a particular laboratory
• In case of problems in testing you can contact the ISTA Secretariat
• Important marketing tool
• Added value of an audit visit
• Possibility to issue ISTA Certificates
Quality assurance is an endless journey of improvement, not a destination.
Question: Do you need a competent person to be checking and signing the certificates?
Answer: Yes the person must have some background of seed testing.
Yes
Accreditate andauthorized
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Question: Can one seek accreditation for specific tests?
Answer: No one can seek accreditation in a package of sampling, purity and germination.
Question: Is it a must that an analyst be a sampler.
Answer: It is not a must. Samplers can be inspectors.
Question: What is the cost implication of being an ISTA member.
4.10. Training of Staff By Craig McGill, Institute of Natural Resources, College of Sciences, Massey University,
New Zealand • QA is a system of management activities that ensure that a process, item, or service is of the type and quality needed by the user www.riskshield.com.au/Glossary.aspx • “failure to invest in staff is a failure to invest.” • training is a critical part of the QA system of the laboratory What do the staff need to be trained in? • training e.g. procedures to both train analysts how to grind a sample and verify analysts are competent to grind a sample • all should understand the context they are working in e.g the role of the laboratory in maintaining seed quality standards within the company, industry and/or company • written procedures e.g. to train analysts and verify their competence in drawing a working sample (Fig 9.6.1. Handbook on Moisture Determination). • will in part depend on their role in the organisation e.g. seed analyst will require different training to the laboratories quality manager Is this enough? Staff also needs training in quality assurance • again will in part depend on their role in the organisation • an seed analyst will require training in quality assurance practices in the laboratory e.g. internal quality control procedures. This can include checking that there are no systematic errors in moisture determination or assessment of a species for germination • a quality assurance manager may require training in quality systems at both the theoretical (workshops, training courses, tertiary training) and practical level e.g. development of quality assurance documentation.
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How to train staff and demonstrate competence. Example: drawing a working sample • can use the triangle system to identify the level of competency the analyst has for drawing the working sample • the first side of the triangle indicates the analyst has been taught to draw the working sample • the second side of the triangle the analyst is practicing drawing working samples with supervision • the third side of the triangle indicates the analyst is competent in the task.
No
The supervisor introduces the analyst to the procedure for drawing a working sample for moisture testing by referring to the relevant sections of the ISTA Rules, ISTA Handbooks, the laboratories quality documents and any other reference material required.
Using a practice sample the supervisor demonstrates the procedure for drawing a working sample (see Chapter 7, Flow Charts 07.3 and 07.3a)
The analyst draws a working sample while being observed by the supervisor. The supervisor provides feed back to the analyst including correction of any mistakes made. The supervisor records the competency stage as. , the analyst has been taught to draw the working sample.
A check test is performed where the supervisor draws a working sample from the same practice sample. The moisture of the samples drawn by the analyst and the supervisor is determined together so any differences out of tolerance found between the samples are likely to be a result of sampling differences not testing.
Has the analyst drawn working samples from the practice samples correctly (i.e. within tolerance) a minimum of seven (7) imes?
Further practice is required
The supervisor records the competency stage as ∧, the analyst is practicing drawing working samples with supervision. The analyst is able to draw a working sample from real sample but is still monitored by the supervisor in the same way as the practice samples.
YES
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Question: Can the staff go through the system at once or at individual case
Answer: Not possible to go group but can handle them in stages by one supervisor
5.0. OFFICIAL CLOSING AT COOL RIVERS HOTEL
Dr. J.O.Ahenda (Local organizer)
He thanked the presenters for the good work they did and the participants for their attendance and active
participation. He then presented certificates.
S. K. Kogo-Regional Manager, KEPHIS Nakuru
The regional manager thanked all the participants, facilitators and ISTA for giving KEPHIS the opportunity to host
the workshop.
Anny van Pijlen on behalf of the lecturers
Anny on behalf of the lecturers said were grateful for invitation to the workshop, was a pleasure for them to be in
Kenya (Nakuru), grateful for the support and organization of the workshop by Kephis and not forgetting the social
programs (excursions to Kenya seed company, Menengai crater, Lake Nakuru national park, Dinner and dancing
at Cool Rivers and speeches by Simeon, Joseph and Arnab Gupta).
She thanked Cool Rivers for their catering service, KEPHIS seed testing laboratory staff, the driver and all staff for
contributing to the success of the workshop and the ISTA secretariat for their support. She pointed out that she will
never forget the workshop because during the workshop Barrack Obama who is the son of Kenya was elected as
the United State of America president and hoped that they will come back for another ISTA event in Nairobi. She
also commented Simeon Kogo for his humor and the participants for their contribution and lively discussions
during the workshop.
6.0. COURSE EVALUATION
Most participants said that the course was well planned, interactive and very helpful. However, most said the
course duration was short (proposed two weeks and doubling time for practical) but they looked forward for more
The analyst is practically examined by drawing working samples from a minimum of seven (7) real samples of a specified group of species for check testing against working samples drawn by the supervisor. The species are chosen so that the supervisor is confident the analyst is fully competent to draw working samples for all species tested in the laboratory.
Has the analyst passed the practical examination i.e. where the analyst and supervisor samples are within tolerance?
YES
The supervisor records the competency as ∧, the analyst is competent to draw a working samples.
NO
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courses in future. In overall, the course was rated excellent by majority of the participants (9 said was Excellent, 5
said was good and 1 did not comment).
