ICAR-Indian Grassland and Fodder Research Institute · 2019-01-09 · Sanjay Kumar A. Maity D....
88
Seed Testing Laboratory Division of Seed Technology ICAR-Indian Grassland and Fodder Research Institute Gwalior Road, Jhansi-284 003 (U.P.)
Transcript of ICAR-Indian Grassland and Fodder Research Institute · 2019-01-09 · Sanjay Kumar A. Maity D....
Seed Testing LaboratoryDivision of Seed Technology
ICAR-Indian Grassland and Fodder Research InstituteGwalior Road, Jhansi-284 003 (U.P.)
Seed Testing LaboratoryDivision of Seed Technology
ICAR-Indian Grassland and Fodder Research InstituteGwalior Road, Jhansi-284 003 (U.P.)
Compiled and edited bySanjay Kumar
A. MaityD. Vijay
Manjunatha N.C.K. Gupta
V.K. WasnikM. Tomar
D. DebS.K. SinghR.P. Nagar,
Maneet Rana and
V.K. Yadav
Seed Testing LaboratoryDivision of Seed Technology
ICAR-Indian Grassland and Fodder Research InstituteGwalior Road, Jhansi-284 003 (U.P.)
Compiled and edited bySanjay Kumar
A. MaityD. Vijay
Manjunatha N.C.K. Gupta
V.K. WasnikM. Tomar
D. DebS.K. SinghR.P. Nagar,
Maneet Rana and
V.K. Yadav
FOREWORD
India faces acute shortage of green fodder due to many reasons, unavailability of quality seeds is
one of them. Currently, there is deficit of approx. 30% in availability of cultivated fodder seed.
Quality seed play important role in agriculture and by using quality seed, productivity can be
increased by 10-20%. Use of quality seeds being the potent source to bring the change in farmers
economy is now of prime importance in modern agriculture as well as precision agriculture. Now
a days the huge quantity of spurious seed are available in the market resulting into crop failure
and under performance of seed. Under the Seed Act all samples of notified and TL seeds being
sold will be required to be correctly labelled about seed quality attributes so that buyers can
evaluate the physical aspects of the planting seed he purchases. To maintain the quality of seed
sold out in market suitable seed testing protocol need to be developed for fodder and forage crops
so that seed quality assurance can be delivered to farmer through seed inspector and seed
certification.
Indian Grassland and Fodder Research Institute (IGFRI), a premier institute under umbrella of
Indian Council of Agriculture Research constantly strive to bring cutting-edge research and
technological interventions in forage crops. It has developed many technologies for seed testing
of forage and fodder crops.
I am happy that this publication will fulfill long pending demand of policy makers as well as
researchers.
The compilation on “Procedure for Forage Seed Testing” is an endeavor to bring different seed
testing protocols and methods to increase the availability of quality seed in the country. I feel that
the present compilation will be beneficial to all the stakeholders engaged in seed industry. I find
the laboratory manual very exhaustive with treasure of information covering all aspects of forage
seed testing for which I express my appreciation to lead author, Dr. Sanjay Kumar and his team
for bringing out this useful publication.
DirectorICAR-IGFRI, Jhansi
Laboratory Manual 2018
Citation:
Published on:
Published by:
Cover page design:
November, 2018
Sanjay Kumar, Maity A., Vijay D., Manjunatha N., Gupta C.K., Wasnik V.K., Tomar M., Deb D., Singh S.K., Nagar R.P., Rana Maneet and Yadav V.K. (2018) Procedure for Forage Seed Testing. ICAR-Indian Grassland and Fodder Research Institute, Jhansi. Pp 83.
November, 2018
DirectorICAR-Indian Grassland and Fodder Research InstituteJhansi- 284003, Uttar Pradesh, India.
© 2018 All right reserved. No part of this publication may be reproduced or transmitted in any form by any means, electronic or mechanical photocopy, recording or any information storage and retrieval system without the permission in writing from the copyright owners.
Sanjay Kumar
Printed at:Darpan Printers & Lamination, Agra
FOREWORD
India faces acute shortage of green fodder due to many reasons, unavailability of quality seeds is
one of them. Currently, there is deficit of approx. 30% in availability of cultivated fodder seed.
Quality seed play important role in agriculture and by using quality seed, productivity can be
increased by 10-20%. Use of quality seeds being the potent source to bring the change in farmers
economy is now of prime importance in modern agriculture as well as precision agriculture. Now
a days the huge quantity of spurious seed are available in the market resulting into crop failure
and under performance of seed. Under the Seed Act all samples of notified and TL seeds being
sold will be required to be correctly labelled about seed quality attributes so that buyers can
evaluate the physical aspects of the planting seed he purchases. To maintain the quality of seed
sold out in market suitable seed testing protocol need to be developed for fodder and forage crops
so that seed quality assurance can be delivered to farmer through seed inspector and seed
certification.
Indian Grassland and Fodder Research Institute (IGFRI), a premier institute under umbrella of
Indian Council of Agriculture Research constantly strive to bring cutting-edge research and
technological interventions in forage crops. It has developed many technologies for seed testing
of forage and fodder crops.
I am happy that this publication will fulfill long pending demand of policy makers as well as
researchers.
The compilation on “Procedure for Forage Seed Testing” is an endeavor to bring different seed
testing protocols and methods to increase the availability of quality seed in the country. I feel that
the present compilation will be beneficial to all the stakeholders engaged in seed industry. I find
the laboratory manual very exhaustive with treasure of information covering all aspects of forage
seed testing for which I express my appreciation to lead author, Dr. Sanjay Kumar and his team
for bringing out this useful publication.
DirectorICAR-IGFRI, Jhansi
Laboratory Manual 2018
Citation:
Published on:
Published by:
Cover page design:
November, 2018
Sanjay Kumar, Maity A., Vijay D., Manjunatha N., Gupta C.K., Wasnik V.K., Tomar M., Deb D., Singh S.K., Nagar R.P., Rana Maneet and Yadav V.K. (2018) Procedure for Forage Seed Testing. ICAR-Indian Grassland and Fodder Research Institute, Jhansi. Pp 83.
November, 2018
DirectorICAR-Indian Grassland and Fodder Research InstituteJhansi- 284003, Uttar Pradesh, India.
© 2018 All right reserved. No part of this publication may be reproduced or transmitted in any form by any means, electronic or mechanical photocopy, recording or any information storage and retrieval system without the permission in writing from the copyright owners.
Sanjay Kumar
Printed at:Darpan Printers & Lamination, Agra
PREFACE
Seed, being basic unit of agriculture act as carrier of technology for a given crop and to upkeep its
quality in the form of genetic vigor is paramount. Simultaneously, productivity and availability
of quality seed in the market is essential to channelize the effort of researchers who develops the
variety and its production packages. Currently, India is facing huge problem of marketing of poor
seed quality which leads to lower productivity and production. The purity of seed in sense of
physical and genetic is very important in agriculture therefore, in any situation make sure that
seed should be pure and getting of pure quality seed is right of farmer. To provide quality seed to
farmers different organization viz., certification agencies, department of agriculture and seed
testing laboratories are working in this direction according to Seed Act 1966. Recently, efforts
made by the Division of Seed Technology at Indian Grassland and Fodder Research Institute
(IGFRI) has led to development of seed testing laboratory where farmers can be easily get the
quality of seed purchased from market. The present laboratory manual is being brought to fulfill
long pending demand of policy makers as well as researchers. The authors are highly thankful to
all the stakeholders who raised various queries at various form regarding seed testing protocol
for fodder seed marketed in India. We are also grateful to Director, IGFRI for his valuable input,
continuous guidance and all support to bring this publication in present form.
Authors
PREFACE
Seed, being basic unit of agriculture act as carrier of technology for a given crop and to upkeep its
quality in the form of genetic vigor is paramount. Simultaneously, productivity and availability
of quality seed in the market is essential to channelize the effort of researchers who develops the
variety and its production packages. Currently, India is facing huge problem of marketing of poor
seed quality which leads to lower productivity and production. The purity of seed in sense of
physical and genetic is very important in agriculture therefore, in any situation make sure that
seed should be pure and getting of pure quality seed is right of farmer. To provide quality seed to
farmers different organization viz., certification agencies, department of agriculture and seed
testing laboratories are working in this direction according to Seed Act 1966. Recently, efforts
made by the Division of Seed Technology at Indian Grassland and Fodder Research Institute
(IGFRI) has led to development of seed testing laboratory where farmers can be easily get the
quality of seed purchased from market. The present laboratory manual is being brought to fulfill
long pending demand of policy makers as well as researchers. The authors are highly thankful to
all the stakeholders who raised various queries at various form regarding seed testing protocol
for fodder seed marketed in India. We are also grateful to Director, IGFRI for his valuable input,
continuous guidance and all support to bring this publication in present form.
Authors
Contents
S.No. Content Page No.
1. Importance of quality seed and seed quality assurance in India 1-7
2. International and National seed organizations 8-13
3. Seed sampling and procedures 14-19
4. Physical purity testing 20-24
5. Seed germination testing 25-34
6. Seed moisture testing 35-39
7. Seed viability determination 40-44
8. Seed vigour determination 45-49
9. Testing of pelleted and coated seeds 50-53
10. Variety identification test 54-73
Contents
S.No. Content Page No.
1. Importance of quality seed and seed quality assurance in India 1-7
2. International and National seed organizations 8-13
3. Seed sampling and procedures 14-19
4. Physical purity testing 20-24
5. Seed germination testing 25-34
6. Seed moisture testing 35-39
7. Seed viability determination 40-44
8. Seed vigour determination 45-49
9. Testing of pelleted and coated seeds 50-53
10. Variety identification test 54-73
1. IMPORTANCE OF QUALITY SEED AND QUALITY ASSURANCE IN INDIA
Among the all inputs used in agriculture, quality seed of improved varieties is the most effective
input for improving crop productivity. The high quality seed are expected to respond better for
other inputs like fertilizers, irrigation and plant protection chemicals. The concept of seed quality
and importance of its each components are discussed below.
Characteristics/attributes of quality seed
1. Genetic purity: Genetic purity refers to the percentage of true to types of seed mentioned
on seed lot. In general, the genetic purity of the seed is decreasing with each stage of seed
multiplication/generation, hence genetic purity should be 100% for Breeder seed, 99%
for Foundation seed and 98% for certified seed.
2. Physical purity : Physical purity of seed is the proportion of pure seed component present
in a seed lot apart from other crop seed, weed seed and inert matter. Physical purity for
most of the forage and fodder crops should be 98% for selling of seed in the market.
3. Germination percentage : The quality seed should have germination percentage
according to the Indian Minimum Seed Certification Standards (IMSCS) so that farmers
can get optimum plant stand in the field.
4. Vigour : Seed vigour is an important quality parameter which needs to be assessed to
supplement germination and viability tests to gain insight into the performance of a seed
lot in the field or in storage. It is the sum total of those properties of the seed which
determine the level of activity and performance of the seed or seed lot during germination
and seedling emergence.
5. Viability : The viability of the seed is a measure of seed aliveness. The seed should be
viable so that it can develop into plants and reproduce themselves under appropriate
conditions. The seed viability can be measured through tetrazolium chloride test (Tz).
6. Moisture content: The moisture content of seed is the amount of water present inside the
seed which is usually expressed as a percentage on dry weight basis. A small change in
seed moisture content has a large effect on the storage life of the seeds. Therefore, it is
important to have following optimum moisture content in good quality seed. eg. Cereals:
10-12%, Pulses: 7-9% and Oilseeds:6-7%, Vegetables: 5-6%.
Importance of quality seed : Seed is a vital input in crop production and following are the
different major role of improved seeds in agriculture
1. It is the cheapest input in crop production and key to agriculture progress.
2. Response of other inputs in crop production depends on quality seed material used.
3. The cost of seed required for raising the crop is very small compared to other inputs.
1
1. IMPORTANCE OF QUALITY SEED AND QUALITY ASSURANCE IN INDIA
Among the all inputs used in agriculture, quality seed of improved varieties is the most effective
input for improving crop productivity. The high quality seed are expected to respond better for
other inputs like fertilizers, irrigation and plant protection chemicals. The concept of seed quality
and importance of its each components are discussed below.
Characteristics/attributes of quality seed
1. Genetic purity: Genetic purity refers to the percentage of true to types of seed mentioned
on seed lot. In general, the genetic purity of the seed is decreasing with each stage of seed
multiplication/generation, hence genetic purity should be 100% for Breeder seed, 99%
for Foundation seed and 98% for certified seed.
2. Physical purity : Physical purity of seed is the proportion of pure seed component present
in a seed lot apart from other crop seed, weed seed and inert matter. Physical purity for
most of the forage and fodder crops should be 98% for selling of seed in the market.
3. Germination percentage : The quality seed should have germination percentage
according to the Indian Minimum Seed Certification Standards (IMSCS) so that farmers
can get optimum plant stand in the field.
4. Vigour : Seed vigour is an important quality parameter which needs to be assessed to
supplement germination and viability tests to gain insight into the performance of a seed
lot in the field or in storage. It is the sum total of those properties of the seed which
determine the level of activity and performance of the seed or seed lot during germination
and seedling emergence.
5. Viability : The viability of the seed is a measure of seed aliveness. The seed should be
viable so that it can develop into plants and reproduce themselves under appropriate
conditions. The seed viability can be measured through tetrazolium chloride test (Tz).
6. Moisture content: The moisture content of seed is the amount of water present inside the
seed which is usually expressed as a percentage on dry weight basis. A small change in
seed moisture content has a large effect on the storage life of the seeds. Therefore, it is
important to have following optimum moisture content in good quality seed. eg. Cereals:
10-12%, Pulses: 7-9% and Oilseeds:6-7%, Vegetables: 5-6%.
Importance of quality seed : Seed is a vital input in crop production and following are the
different major role of improved seeds in agriculture
1. It is the cheapest input in crop production and key to agriculture progress.
2. Response of other inputs in crop production depends on quality seed material used.
3. The cost of seed required for raising the crop is very small compared to other inputs.
1
4. It is estimated that good quality seeds of improved varieties can enhance yield about 20-
25% and good crop yield can be obtain through use of quality seed under less favorable
conditions.
5. The improved seed is the carrier of new technology and also tool for secure food supply.
6. The quality seed is the medium for rapid rehabilitation of agriculture after natural
disasters.
Ecological factors:
The growth of the plant and the seed quality are strongly influenced not only by the genetic factors alone but also by the environment prevailing during the seed formation, maturation and growth. The important ecological factors which governs the seed quality include: soil, adaptability, wind velocity and rains; light intensities and temperature conditions; insect activities during flowering; pests and pathogens. These factors may favorably or unfavorably affect the seed quality depending upon the situation.
Agronomic packages and production technology:
Application of the prescribed agronomical packages is utmost important for raising a good seed crop. The cultural practices required for raising the seed crop are different to those of raising the general crop. Timely operations during the crop management is essential aspect for quality seed production of desired genetic purity and physical purity. Seed source, time of sowing, land preparation, isolation distance, plant protection, weed control, rouging and nutritional management affecting the quality of seed.
Harvest and post-harvest handling
Harvesting and post-harvest handling of the seed crop are essential component of a seed certification programme for obtaining the quality seed. This is an integral part of a seed certification programme. The time, energy and money spend in growing genetically pure and disease-free seed crop may go waste, if harvested seed crop is left to the vagaries of the weather. Seed quality is very much affected during threshing, drying, cleaning, grading, packing and transportation of seed lots if adequate technology and care is not provided to the harvested produce.
Quality assurance
This is the responsibility of the seed corporation/organization and the seed companies to provide assurance about the quality of the seed offered by them for marketing or distribution. There would not be any problem to the seed producing organizations in providing the assurance of the seed quality, if they are conscious about the maintenance of the quality of their produce. In the absence of the system of quality maintenance, it would be rather impossible to provide the assurance of the seed quality. Without observing the quality maintenance, if quality assurance is provided by an organization in the form of a declaration or label containing the details of the physical purity and germination of seed, the organization may face heavy penalties under the provisions of the seed legislation of 'Seed Act' or else will lose the faith of the consumer.
Accordingly, it would be desirable on the part of an organization or seed company involved in the seed trade that before providing the assurance relating to the identity of the variety and the quality of the seed offered by them, they must ensure to get the seed tested in the seed testing laboratory.
Seed quality control
Quality control is an important component of the seed programme and the essence of any seed programme lies in the quality control. Seed quality control is a system which ensures to govern
Differences between seed and grain
Seed Grain
It should be a viable and vigorous one Need not to be a viable one
It should be physically and genetically pure Not so
Should satisfy Indian Minimum Seed No such requirementsCertification Standards (IMSCS)
It can be treated with pesticide /fungicide It should never be treated with any to protect against storage pests and fungi chemicals, since it is used for consumption
Respiration rate and other physiological No such specificationsand biological processes should be kept at low level during storage
Should be compulsorily certified No such condition in grain production
It should satisfy all the quality norms Not consideredSeed can be utilized as grain purpose if it Grain never can be converted into seedis not treated with poisonous chemicals
Factors affecting the seed quality:
Seed quality is influenced by a variety of factors imposed to the seed during its formation,
development, maturation, growth, harvesting, threshing/extraction, drying, cleaning, grading,
packing, storage and marketing. The factors which governs the seed quality can be broadly
grouped into 4 categories:
1. Genetic factors
2. Ecological factors
3. Agronomic packages and production technology
4. Harvesting and post-harvest handling
Genetic factors:
The quality of seed declines during the seed production stages due to combined effects of
ecological and genetic factors. This is, especially true in situations where same sources of the
seeds is continuously used over the years.
32
4. It is estimated that good quality seeds of improved varieties can enhance yield about 20-
25% and good crop yield can be obtain through use of quality seed under less favorable
conditions.
5. The improved seed is the carrier of new technology and also tool for secure food supply.
6. The quality seed is the medium for rapid rehabilitation of agriculture after natural
disasters.
Ecological factors:
The growth of the plant and the seed quality are strongly influenced not only by the genetic factors alone but also by the environment prevailing during the seed formation, maturation and growth. The important ecological factors which governs the seed quality include: soil, adaptability, wind velocity and rains; light intensities and temperature conditions; insect activities during flowering; pests and pathogens. These factors may favorably or unfavorably affect the seed quality depending upon the situation.
Agronomic packages and production technology:
Application of the prescribed agronomical packages is utmost important for raising a good seed crop. The cultural practices required for raising the seed crop are different to those of raising the general crop. Timely operations during the crop management is essential aspect for quality seed production of desired genetic purity and physical purity. Seed source, time of sowing, land preparation, isolation distance, plant protection, weed control, rouging and nutritional management affecting the quality of seed.
Harvest and post-harvest handling
Harvesting and post-harvest handling of the seed crop are essential component of a seed certification programme for obtaining the quality seed. This is an integral part of a seed certification programme. The time, energy and money spend in growing genetically pure and disease-free seed crop may go waste, if harvested seed crop is left to the vagaries of the weather. Seed quality is very much affected during threshing, drying, cleaning, grading, packing and transportation of seed lots if adequate technology and care is not provided to the harvested produce.
Quality assurance
This is the responsibility of the seed corporation/organization and the seed companies to provide assurance about the quality of the seed offered by them for marketing or distribution. There would not be any problem to the seed producing organizations in providing the assurance of the seed quality, if they are conscious about the maintenance of the quality of their produce. In the absence of the system of quality maintenance, it would be rather impossible to provide the assurance of the seed quality. Without observing the quality maintenance, if quality assurance is provided by an organization in the form of a declaration or label containing the details of the physical purity and germination of seed, the organization may face heavy penalties under the provisions of the seed legislation of 'Seed Act' or else will lose the faith of the consumer.
Accordingly, it would be desirable on the part of an organization or seed company involved in the seed trade that before providing the assurance relating to the identity of the variety and the quality of the seed offered by them, they must ensure to get the seed tested in the seed testing laboratory.
Seed quality control
Quality control is an important component of the seed programme and the essence of any seed programme lies in the quality control. Seed quality control is a system which ensures to govern
Differences between seed and grain
Seed Grain
It should be a viable and vigorous one Need not to be a viable one
It should be physically and genetically pure Not so
Should satisfy Indian Minimum Seed No such requirementsCertification Standards (IMSCS)
It can be treated with pesticide /fungicide It should never be treated with any to protect against storage pests and fungi chemicals, since it is used for consumption
Respiration rate and other physiological No such specificationsand biological processes should be kept at low level during storage
Should be compulsorily certified No such condition in grain production
It should satisfy all the quality norms Not consideredSeed can be utilized as grain purpose if it Grain never can be converted into seedis not treated with poisonous chemicals
Factors affecting the seed quality:
Seed quality is influenced by a variety of factors imposed to the seed during its formation,
development, maturation, growth, harvesting, threshing/extraction, drying, cleaning, grading,
packing, storage and marketing. The factors which governs the seed quality can be broadly
grouped into 4 categories:
1. Genetic factors
2. Ecological factors
3. Agronomic packages and production technology
4. Harvesting and post-harvest handling
Genetic factors:
The quality of seed declines during the seed production stages due to combined effects of
ecological and genetic factors. This is, especially true in situations where same sources of the
seeds is continuously used over the years.
32
the quality of the seed through checks, certification and official regulations (legislation). Seed quality control itself is a system which ensures the interest of the farmers and to avoid hazards in the crop production. It is the responsibility of the government to enforce the measures for controlling the quality of the seed being marketed in the country. This is usually accomplished through the legislation in the form of a law or Act of Parliament. Essentially, there are two components in a seed quality control system in India; the seed certification and labelling. Seed certification may not be compulsory but labeling is usually a compulsory provision of any seed legislation. To accomplish the task of seed legislation or Seed Act, framing the rules and regulations pertaining the legislation and their scope is the pre-requisite. In addition, certain basic infra-structural facilities are also required. This includes, the notification of the seed standards, kind or varieties expected to be governed by the legislation, establishment of seed certification agency, seed testing laboratories, variety notification and authorization of seed inspectors.
The following are the essential components of a seed quality control programme:
1. Quality control of breeder seed 2. Seed certification
3. Seed legislation 4. Seed standards
5. Seed testing
In India, seed quality control programme was initiated during 1953 with the establishment of the National Seeds Corporation, which was the single agency for production, certification and marketing of the seed throughout the country. However, at a later stage, during 1970's and 1980's state seed certification agencies were established in most of the states, which are responsible to carry forward the seed certification programme in the respective state.
which have now been revised. The statutory provisions of the Indian seeds act, have got strong
linkages with the various components of the seed programme, such as variety notification, seed
production, seed certification and seed marketing.
Seed Standards
Seed quality control measures also depends on stipulated seed standards for labeling and
certification. The standards should be based on the analytical data generated by the seed
testing laboratories and should be realistic and achievable. In laying down the seed standards,
the standards of the other countries having similar socio-economic conditions may be
consulted. It would be desirable that before obtaining the final approval of the standard by the
competent authority, the standards should be discussed thoroughly by a committee, which is
represented by the seed producers, certification and seed law enforcement officials. To
maintain the optimum quality of seed, state certification agency should evaluate the track of
seed from production to marketing. Therefore, seed certification agency laid down the
standards for both field and seed which should be reviewed at timely intervals in order to make
them realistic.
Stages of Seed Multiplication
The benefits of an improved variety are not realized unless enough, pure seed has been produced
for its commercial spread. The initial amount of pure seed which is limited in quantity is
multiplied under various stages or classes.
a. Nucleus seed b. Breeders seed c. Foundation seed d. certified seed
Nucleus seed :
It is the initial amount of pure seed of an improved variety available with plant breeder who has
developed it. The nucleus seed is in very limited quantity hence, its genetic and physical purity
should be maintained 100%.
Breeder's seed :
It is the seed obtained from the progeny of nucleus seed which is directly supervised by a breeder
of concern crop. The genetic and physical purity of breeder seed should be 100% to avoid
degeneration of variety during seed multiplication. Breeder seed tag colour is golden yellow.
Foundation seed :
Foundation seed is produced from breeder seed under direct supervision of NSC, SSC and SCA.
The bags of foundation seed sealed with white colored label.
Certified seed :
It is progeny of foundation class of seed and produced under direct supervision of state seed
certification agency. The certified seed bags are sealed with blue colored tag. The certified seed is
directly used for commercial crop production.
Figure 1a. Flow chart for local and formal seed distribution system
Figure 1b. Breeder, foundation and certified seed production system in India.
Seed Act was passed in India during 1966 which is essentially a 'truthful' labeling act and
applicable to the notified kind/varieties. For the regulation of the seeds act and the seed
certification programme in the country, two separate sets of the standard namely, minimum
labelling standards and minimum seed certification standards were prescribed during 1970
54
the quality of the seed through checks, certification and official regulations (legislation). Seed quality control itself is a system which ensures the interest of the farmers and to avoid hazards in the crop production. It is the responsibility of the government to enforce the measures for controlling the quality of the seed being marketed in the country. This is usually accomplished through the legislation in the form of a law or Act of Parliament. Essentially, there are two components in a seed quality control system in India; the seed certification and labelling. Seed certification may not be compulsory but labeling is usually a compulsory provision of any seed legislation. To accomplish the task of seed legislation or Seed Act, framing the rules and regulations pertaining the legislation and their scope is the pre-requisite. In addition, certain basic infra-structural facilities are also required. This includes, the notification of the seed standards, kind or varieties expected to be governed by the legislation, establishment of seed certification agency, seed testing laboratories, variety notification and authorization of seed inspectors.
The following are the essential components of a seed quality control programme:
1. Quality control of breeder seed 2. Seed certification
3. Seed legislation 4. Seed standards
5. Seed testing
In India, seed quality control programme was initiated during 1953 with the establishment of the National Seeds Corporation, which was the single agency for production, certification and marketing of the seed throughout the country. However, at a later stage, during 1970's and 1980's state seed certification agencies were established in most of the states, which are responsible to carry forward the seed certification programme in the respective state.
which have now been revised. The statutory provisions of the Indian seeds act, have got strong
linkages with the various components of the seed programme, such as variety notification, seed
production, seed certification and seed marketing.
Seed Standards
Seed quality control measures also depends on stipulated seed standards for labeling and
certification. The standards should be based on the analytical data generated by the seed
testing laboratories and should be realistic and achievable. In laying down the seed standards,
the standards of the other countries having similar socio-economic conditions may be
consulted. It would be desirable that before obtaining the final approval of the standard by the
competent authority, the standards should be discussed thoroughly by a committee, which is
represented by the seed producers, certification and seed law enforcement officials. To
maintain the optimum quality of seed, state certification agency should evaluate the track of
seed from production to marketing. Therefore, seed certification agency laid down the
standards for both field and seed which should be reviewed at timely intervals in order to make
them realistic.
Stages of Seed Multiplication
The benefits of an improved variety are not realized unless enough, pure seed has been produced
for its commercial spread. The initial amount of pure seed which is limited in quantity is
multiplied under various stages or classes.
a. Nucleus seed b. Breeders seed c. Foundation seed d. certified seed
Nucleus seed :
It is the initial amount of pure seed of an improved variety available with plant breeder who has
developed it. The nucleus seed is in very limited quantity hence, its genetic and physical purity
should be maintained 100%.
Breeder's seed :
It is the seed obtained from the progeny of nucleus seed which is directly supervised by a breeder
of concern crop. The genetic and physical purity of breeder seed should be 100% to avoid
degeneration of variety during seed multiplication. Breeder seed tag colour is golden yellow.
Foundation seed :
Foundation seed is produced from breeder seed under direct supervision of NSC, SSC and SCA.
The bags of foundation seed sealed with white colored label.
Certified seed :
It is progeny of foundation class of seed and produced under direct supervision of state seed
certification agency. The certified seed bags are sealed with blue colored tag. The certified seed is
directly used for commercial crop production.
Figure 1a. Flow chart for local and formal seed distribution system
Figure 1b. Breeder, foundation and certified seed production system in India.
Seed Act was passed in India during 1966 which is essentially a 'truthful' labeling act and
applicable to the notified kind/varieties. For the regulation of the seeds act and the seed
certification programme in the country, two separate sets of the standard namely, minimum
labelling standards and minimum seed certification standards were prescribed during 1970
54
Table 1. Indian minimum seed certification standards (IMSCS) for forage and grasses.
(F-Foundation class, C-Certified class; *400m Isolation distance from Sorghum helpense)
Crops Isolation Pure seed Other crop Total weed OWS Germi-distance (no/kg) seed(no/kg) (no/kg) nation %
F C F C F C F C F C F C
Berseem 400 100 98 98 10 20 10 20 5 10 80 80
Oats 3 3 98 98 10 20 10 20 2 5 85 85
Sorghum 200* 100* 97 97 5 10 5 10 0.02% 0.04% 75 75
Maize 400 400 98 98 5 5 - - - - 80 80
Guar 10 5 98 98 10 20 - - - - 70 70
C. ciliaris 20 10 80 80 20 40 20 40 - - 30 30
Dharaf 20 10 80 80 20 40 20 40 - - 15 15
Dinanath 20 10 95 95 20 40 20 40 - - 50 50
Guinea 20 10 80 80 20 40 20 40 - - 20 20
Senji 50 25 98 98 10 20 10 20 - - 65 65
Lucerne 400 100 98 98 10 20 10 20 5 10 80 80
Marvel 20 10 90 90 10 20 10 20 - - 40 40
Napier 10 10 99.5 98.8 - - - - - - - -
Rice bean 50 20 98 98 0 0 5 10 - - 70 70
Setaria 400 200 95 95 20 40 20 40 - - 50 50
Stylo 50 25 90 90 10 20 10 20 - - 40 40
Teosinte 200 100 98 98 5 10 0 0 - - 80 80
Difference between certified seed and truthfully labelled seed
Certified Seed: It is the progeny of foundation seed produced under direct supervision of SSCA.
The seed certification agency inspects the seed plots during crop growth and seed processing.
After processing seed samples are drawn by seed certification officer and sent to seed testing
laboratory for testing. The seed lots which meets the IMSCS further processed for bagging and
tagging with blue colour tag. The validity period of seed certification is only for 9 months after
that seeds are tested again and if found fit according to IMSCS then certification will be extended
to 6 more months. Therefore, the certification is the third party (SSCA) guarantee for quality of
seed selling by any organization and if any problem occurred regarding quality of seed then
SSCA is whole responsible agency.
Truthful labelled seed (TLS): TL seed is a class of seed which is produced and marketed by any
organization under truthfully labelled category. The information written (seed standards) on seed
bag should be true and as per the producer/marketing organization. If any default will be there
then producer is responsible for seed quality attributes.
Figure 2. Breeder, foundation, certified andtruthfully labelled seed tags
76
Table 1. Indian minimum seed certification standards (IMSCS) for forage and grasses.
(F-Foundation class, C-Certified class; *400m Isolation distance from Sorghum helpense)
Crops Isolation Pure seed Other crop Total weed OWS Germi-distance (no/kg) seed(no/kg) (no/kg) nation %
F C F C F C F C F C F C
Berseem 400 100 98 98 10 20 10 20 5 10 80 80
Oats 3 3 98 98 10 20 10 20 2 5 85 85
Sorghum 200* 100* 97 97 5 10 5 10 0.02% 0.04% 75 75
Maize 400 400 98 98 5 5 - - - - 80 80
Guar 10 5 98 98 10 20 - - - - 70 70
C. ciliaris 20 10 80 80 20 40 20 40 - - 30 30
Dharaf 20 10 80 80 20 40 20 40 - - 15 15
Dinanath 20 10 95 95 20 40 20 40 - - 50 50
Guinea 20 10 80 80 20 40 20 40 - - 20 20
Senji 50 25 98 98 10 20 10 20 - - 65 65
Lucerne 400 100 98 98 10 20 10 20 5 10 80 80
Marvel 20 10 90 90 10 20 10 20 - - 40 40
Napier 10 10 99.5 98.8 - - - - - - - -
Rice bean 50 20 98 98 0 0 5 10 - - 70 70
Setaria 400 200 95 95 20 40 20 40 - - 50 50
Stylo 50 25 90 90 10 20 10 20 - - 40 40
Teosinte 200 100 98 98 5 10 0 0 - - 80 80
Difference between certified seed and truthfully labelled seed
Certified Seed: It is the progeny of foundation seed produced under direct supervision of SSCA.
The seed certification agency inspects the seed plots during crop growth and seed processing.
After processing seed samples are drawn by seed certification officer and sent to seed testing
laboratory for testing. The seed lots which meets the IMSCS further processed for bagging and
tagging with blue colour tag. The validity period of seed certification is only for 9 months after
that seeds are tested again and if found fit according to IMSCS then certification will be extended
to 6 more months. Therefore, the certification is the third party (SSCA) guarantee for quality of
seed selling by any organization and if any problem occurred regarding quality of seed then
SSCA is whole responsible agency.
Truthful labelled seed (TLS): TL seed is a class of seed which is produced and marketed by any
organization under truthfully labelled category. The information written (seed standards) on seed
bag should be true and as per the producer/marketing organization. If any default will be there
then producer is responsible for seed quality attributes.
Figure 2. Breeder, foundation, certified andtruthfully labelled seed tags
76
2.INTERNATIONAL AND NATIONAL SEED TESTING ORGANIZATION
Due to living nature of seed, the testing methods must be developed based on scientific
knowledge of seed and should be accurate and reproducible. At nationally and internationally
following different organizations are working towards standardization and development of new
protocols for evaluation of seed quality attributes.
1. National Seed Research & Training Center (NSRTC): The Govt. of India, Ministry of
Agriculture & Farmers Welfare, Department of Agriculture and Cooperation, New Delhi has
started the National Seed Research and Training Centre (NSRTC)at Varanasi during October
2005. The prime objective of establishment of NSRTC is to have a Central Seed Testing
Laboratory (CSTL) and to organize the national trainings, workshops & congress for the
benefit of personnel who are involved in seed development and quality control programme.
Central Seed Testing Laboratory (CSTL): CSTL is maintaining the uniformity in seed
testing result among all state seed testing laboratories at the national level. It acts as
referral laboratory for court referred seed samples and also a member laboratory of
International Seed Testing Association (ISTA), Switzerland. In addition, CSTL monitor
the quality of marketed seeds through purchasing the samples from various seed selling
points and its testing. Apart from this under the 5% re-testing programme, CSTL is testing
5% samples from notified state seed testing laboratories across the country free of cost to
know the efficiency of state seed testing laboratory.
2. Indian Institute of Seed Science (IISS): After realizing the importance of seed, the ICAR
launched the AICRP on seed in 1979 at Seed Technology Division, IARI, New Delhi as a
National Seed Project. Based on the overall progress and development of the NSP and
growing importance of seed in modern agriculture, the IACR has upgraded the Project
Coordinator Unit of NSP to the status of the Project Directorate in X Plan and named
asDirectorate of Seed Research (DSR). DSR started operating since 31 December 2004
from Kushmaur village in the district Maunath Bhanjan, UP. In 2017 institute was
upgraded and its name was changed to Indian Institute of Seed Science (IISS).
3. International Seed Testing Association (ISTA):The International Seed Testing
Association was founded in 1924 during the 4th International Seed Testing Congress held
in Cambridge, United Kingdom.The headquarters of the association is located in
Switzerland and managed by the Secretary General. ISTA membership consists of
member laboratories and sampling entities, personal members, associate members and
industry members from more than 80 countries/distinct economies around the world.
More than 130 of the ISTA Member Laboratories are accredited by ISTA and entitled to
issue ISTA International Seed Analysis Certificates. The followings are two important
objectives of organization;
·The primary purpose of the Association is to develop, adopt and publish standard
procedures for sampling and testing seeds, and to promote uniform application of these
procedures for evaluation of seeds moving in the international trade.
·The secondary purposes of the association are to promote research in all areas of seed
science and technology, including sampling, testing, storing, processing, certification and
marketing. Apart from this ISTA also promote to participate in conferences/training
courses and to establish and maintain liaison with other organizations having related
interests in seed.
ISTA is best known for developing and publishing the international rules for seed testing
which is a comprehensive set of instructions and techniques and used by seed analysts
throughout the world. The rules include prescriptions for seed sampling, physical purity
test, germination, viability, variety identification, seed health and seed moisture test. The
ISTA rules published in the three official ISTA languages (English, French and German)
and have been widely translated to other languages. Although the ISTA rules are used
throughout the world for testing seeds which are sold in domestic markets but from the
ISTA point of view these rules are mainly for ISTA seed analysis certificates, which are
widely used for international seed trade. There are two types of ISTA seed certificates:
1. Orange International Seed Lot Certificate
2. Blue International Seed Sample Certificate
Orange International Certificate: To issue this certificate authority of ISTA accredited
member laboratory should officially draw the sample from seed lot and carried out the test.
The colour of this certificate is orange.
Blue International Certificates: In this certificate ISTA accredited laboratory is
responsible only for testing the sample as submitted. It is not responsible for the
relationship between the sample and any seed lot from which it may have been derived.
The colour of this certificate is blue. ISTA requires that its member laboratories wishing to
issue ISTA Certificates should fulfill the ISTA Accreditation Standard.
Procedure for seed testing laboratories to get ISTA Accreditation:
1. ISTA Membership: Laboratories intending to become accredited have to become ISTA
member first. They should contact the ISTA Secretariat for the necessary application
forms and complete the form (Form D) available from the Secretariat. The ISTA Executive
Committee will then decide about the application and grant membership.
2. ISTA Proficiency Testing Program: All accredited laboratories and those interested to be
accredited have to participate successfully in the ISTA interlaboratory proficiency testing
program, which consist at least three referee test rounds per year. Seed samples of known
quality are sent to the laboratories to carry out the examination according to the ISTA
Rules and results have to be reported within a period of three months. The participating
98
2.INTERNATIONAL AND NATIONAL SEED TESTING ORGANIZATION
Due to living nature of seed, the testing methods must be developed based on scientific
knowledge of seed and should be accurate and reproducible. At nationally and internationally
following different organizations are working towards standardization and development of new
protocols for evaluation of seed quality attributes.
1. National Seed Research & Training Center (NSRTC): The Govt. of India, Ministry of
Agriculture & Farmers Welfare, Department of Agriculture and Cooperation, New Delhi has
started the National Seed Research and Training Centre (NSRTC)at Varanasi during October
2005. The prime objective of establishment of NSRTC is to have a Central Seed Testing
Laboratory (CSTL) and to organize the national trainings, workshops & congress for the
benefit of personnel who are involved in seed development and quality control programme.
Central Seed Testing Laboratory (CSTL): CSTL is maintaining the uniformity in seed
testing result among all state seed testing laboratories at the national level. It acts as
referral laboratory for court referred seed samples and also a member laboratory of
International Seed Testing Association (ISTA), Switzerland. In addition, CSTL monitor
the quality of marketed seeds through purchasing the samples from various seed selling
points and its testing. Apart from this under the 5% re-testing programme, CSTL is testing
5% samples from notified state seed testing laboratories across the country free of cost to
know the efficiency of state seed testing laboratory.
2. Indian Institute of Seed Science (IISS): After realizing the importance of seed, the ICAR
launched the AICRP on seed in 1979 at Seed Technology Division, IARI, New Delhi as a
National Seed Project. Based on the overall progress and development of the NSP and
growing importance of seed in modern agriculture, the IACR has upgraded the Project
Coordinator Unit of NSP to the status of the Project Directorate in X Plan and named
asDirectorate of Seed Research (DSR). DSR started operating since 31 December 2004
from Kushmaur village in the district Maunath Bhanjan, UP. In 2017 institute was
upgraded and its name was changed to Indian Institute of Seed Science (IISS).
3. International Seed Testing Association (ISTA):The International Seed Testing
Association was founded in 1924 during the 4th International Seed Testing Congress held
in Cambridge, United Kingdom.The headquarters of the association is located in
Switzerland and managed by the Secretary General. ISTA membership consists of
member laboratories and sampling entities, personal members, associate members and
industry members from more than 80 countries/distinct economies around the world.
More than 130 of the ISTA Member Laboratories are accredited by ISTA and entitled to
issue ISTA International Seed Analysis Certificates. The followings are two important
objectives of organization;
·The primary purpose of the Association is to develop, adopt and publish standard
procedures for sampling and testing seeds, and to promote uniform application of these
procedures for evaluation of seeds moving in the international trade.
·The secondary purposes of the association are to promote research in all areas of seed
science and technology, including sampling, testing, storing, processing, certification and
marketing. Apart from this ISTA also promote to participate in conferences/training
courses and to establish and maintain liaison with other organizations having related
interests in seed.
ISTA is best known for developing and publishing the international rules for seed testing
which is a comprehensive set of instructions and techniques and used by seed analysts
throughout the world. The rules include prescriptions for seed sampling, physical purity
test, germination, viability, variety identification, seed health and seed moisture test. The
ISTA rules published in the three official ISTA languages (English, French and German)
and have been widely translated to other languages. Although the ISTA rules are used
throughout the world for testing seeds which are sold in domestic markets but from the
ISTA point of view these rules are mainly for ISTA seed analysis certificates, which are
widely used for international seed trade. There are two types of ISTA seed certificates:
1. Orange International Seed Lot Certificate
2. Blue International Seed Sample Certificate
Orange International Certificate: To issue this certificate authority of ISTA accredited
member laboratory should officially draw the sample from seed lot and carried out the test.
The colour of this certificate is orange.
Blue International Certificates: In this certificate ISTA accredited laboratory is
responsible only for testing the sample as submitted. It is not responsible for the
relationship between the sample and any seed lot from which it may have been derived.
The colour of this certificate is blue. ISTA requires that its member laboratories wishing to
issue ISTA Certificates should fulfill the ISTA Accreditation Standard.
Procedure for seed testing laboratories to get ISTA Accreditation:
1. ISTA Membership: Laboratories intending to become accredited have to become ISTA
member first. They should contact the ISTA Secretariat for the necessary application
forms and complete the form (Form D) available from the Secretariat. The ISTA Executive
Committee will then decide about the application and grant membership.
2. ISTA Proficiency Testing Program: All accredited laboratories and those interested to be
accredited have to participate successfully in the ISTA interlaboratory proficiency testing
program, which consist at least three referee test rounds per year. Seed samples of known
quality are sent to the laboratories to carry out the examination according to the ISTA
Rules and results have to be reported within a period of three months. The participating
98
laboratories get a detailed evaluation sheet that shows their performance and if results are
out of tolerance, then participating laboratory gets a test leader report which advising the
possible corrective actions to enhance the laboratory's performance continuously.
3. Establishment of Quality Assurance Program: A laboratory that wishes to be accredited
has to set up its own quality assurance program including quality documentation which
should follow the ISTA accreditation standard and its bylaws. These standards are based
on ISO-25 guide, but has been especially amended to meet the needs of seed testing
laboratories.
4. ISTA Audit: Prior to accreditation, and every three years thereafter, the laboratories are
audited by two ISTA auditors (system and technical auditor). Based on the auditor's
recommendation and the performance in the referee tests, accreditation is granted to
member laboratory. Prior to the audit, the laboratories are requested to submit the quality
documents translated into one of the official languages of ISTA to the Secretariat. The
auditors will check the appropriateness of these documents prior to the audit and if
documents are found appropriate for auditing a date will be arranged accordingly with the
laboratory. The quality documents should at least contain a description of the quality
assurance system, standard operational procedures (SOPs) and working instructions for
all test methods carried out in the laboratory.
5. Authorization to issue ISTA Certificates: After having successfully fulfilled the
requirements of accreditation, authorization to issue ISTA Certificates is obtained through
the agreement of the government of the respective country. Therefore, the applicant is
requested to complete Annex D of the application form by the respective government.
6. Establishment of Monitoring System: Upon decision of the government of each country
a monitoring system could be established for company laboratories.
The ISTA Accreditation is valid for three years and after this period a re-accreditation audit
has to take place to maintain the accreditation status and for authorization to issue ISTA
Certificates.
4. Association of Official Seed Analysts (AOSA) : The AOSA is an organization of
member laboratories across the United States and Canada to assure a high standard of seed
quality. Many individuals within the AOSA member laboratories have acquired AOSA
certified seed analyst status through extensive training followed by a mandatory
certification testing process. AOSA was formed in 1908 with headquarter at Washington
(USA) in response to initial attempts by individual states to develop seed laws. This was
the beginning of regulated seed commerce in the United States. Initial priorities included,
as was defined in the constitution, an attempt to seek uniformity and accuracy in methods,
results, and reports. It set as its objective an effort to perfect and make publicly known,
through publication, uniform rules for seed testing.
Relationships between ISTA, AOSA and OECD: ISTA is one of a number of organizations in
the seed world. To begin with, there is another organization, the Association of Official Seed
Analysts (AOSA), which is involved in the standardization of seed testing procedures in the USA
and Canada, and publishes its own seed testing rules especially adapted for the market in North
America. Nowadays, ISTA and the AOSA have a joint committee on the harmonization of rules
and work together in a number of other ways. ISTA provides testing services for companies
trading seed internationally and so it is important that we have a good working relationship with
the International Seed Trade Federation (FIS). The OECD Seed Schemes provide a system for
the assurance of varietal purity and identity of seed moving in international trade, and are
normally used in tandem with ISTA seed lot certificates, which carry the results of other quality
tests. For this reason, ISTA and OECD have always worked closely together. Some of the recent
discussions in OECD have centered on the possibility that seed tests could be made by seed
testing laboratories other than ISTA members.
Table 2. List of seed testing laboratories, seed certification agency and state seed corporation in Uttar Pradesh.
S.N. Seed Testing Laboratories
1. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, Barabanki (UP)
2. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, 9515, Civil Line, Jhansi (UP)
3. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, Infront of Block Officer, Meerut. (UP)
4. Seed Testing Laboratory, UP State Seed Certification Agency, Govt. Garden Campus, Kariappa Road, Alambagh, Lucknow (UP)
5. Regional Agriculture Seed Testing & Demonstration Station, Department of Agriculture, Azamgarh (UP)
6. Seed Testing Laboratory, UP State Seed Certification Agency, 35-C/6, Rampur Bagh, Bareilly (UP)
7. Regional Agriculture Seed Testing & Demonstration Center,Department of Agriculture, 32-8, Civil Line, Mathura (UP)
8. Regional Agriculture Seed Testing & Demonstration Center,Department of Agriculture, Station Road, Hardoi (UP)
9. Seed Testing Laboratory, Department of Seed Technology, C.S. Azad University of Agriculture & Technology, Kanpur (UP)
1110
laboratories get a detailed evaluation sheet that shows their performance and if results are
out of tolerance, then participating laboratory gets a test leader report which advising the
possible corrective actions to enhance the laboratory's performance continuously.
3. Establishment of Quality Assurance Program: A laboratory that wishes to be accredited
has to set up its own quality assurance program including quality documentation which
should follow the ISTA accreditation standard and its bylaws. These standards are based
on ISO-25 guide, but has been especially amended to meet the needs of seed testing
laboratories.
4. ISTA Audit: Prior to accreditation, and every three years thereafter, the laboratories are
audited by two ISTA auditors (system and technical auditor). Based on the auditor's
recommendation and the performance in the referee tests, accreditation is granted to
member laboratory. Prior to the audit, the laboratories are requested to submit the quality
documents translated into one of the official languages of ISTA to the Secretariat. The
auditors will check the appropriateness of these documents prior to the audit and if
documents are found appropriate for auditing a date will be arranged accordingly with the
laboratory. The quality documents should at least contain a description of the quality
assurance system, standard operational procedures (SOPs) and working instructions for
all test methods carried out in the laboratory.
5. Authorization to issue ISTA Certificates: After having successfully fulfilled the
requirements of accreditation, authorization to issue ISTA Certificates is obtained through
the agreement of the government of the respective country. Therefore, the applicant is
requested to complete Annex D of the application form by the respective government.
6. Establishment of Monitoring System: Upon decision of the government of each country
a monitoring system could be established for company laboratories.
The ISTA Accreditation is valid for three years and after this period a re-accreditation audit
has to take place to maintain the accreditation status and for authorization to issue ISTA
Certificates.
4. Association of Official Seed Analysts (AOSA) : The AOSA is an organization of
member laboratories across the United States and Canada to assure a high standard of seed
quality. Many individuals within the AOSA member laboratories have acquired AOSA
certified seed analyst status through extensive training followed by a mandatory
certification testing process. AOSA was formed in 1908 with headquarter at Washington
(USA) in response to initial attempts by individual states to develop seed laws. This was
the beginning of regulated seed commerce in the United States. Initial priorities included,
as was defined in the constitution, an attempt to seek uniformity and accuracy in methods,
results, and reports. It set as its objective an effort to perfect and make publicly known,
through publication, uniform rules for seed testing.
Relationships between ISTA, AOSA and OECD: ISTA is one of a number of organizations in
the seed world. To begin with, there is another organization, the Association of Official Seed
Analysts (AOSA), which is involved in the standardization of seed testing procedures in the USA
and Canada, and publishes its own seed testing rules especially adapted for the market in North
America. Nowadays, ISTA and the AOSA have a joint committee on the harmonization of rules
and work together in a number of other ways. ISTA provides testing services for companies
trading seed internationally and so it is important that we have a good working relationship with
the International Seed Trade Federation (FIS). The OECD Seed Schemes provide a system for
the assurance of varietal purity and identity of seed moving in international trade, and are
normally used in tandem with ISTA seed lot certificates, which carry the results of other quality
tests. For this reason, ISTA and OECD have always worked closely together. Some of the recent
discussions in OECD have centered on the possibility that seed tests could be made by seed
testing laboratories other than ISTA members.
Table 2. List of seed testing laboratories, seed certification agency and state seed corporation in Uttar Pradesh.
S.N. Seed Testing Laboratories
1. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, Barabanki (UP)
2. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, 9515, Civil Line, Jhansi (UP)
3. Regional Agriculture Seed Testing & Demonstration Station,Department of Agriculture, Infront of Block Officer, Meerut. (UP)
4. Seed Testing Laboratory, UP State Seed Certification Agency, Govt. Garden Campus, Kariappa Road, Alambagh, Lucknow (UP)
5. Regional Agriculture Seed Testing & Demonstration Station, Department of Agriculture, Azamgarh (UP)
6. Seed Testing Laboratory, UP State Seed Certification Agency, 35-C/6, Rampur Bagh, Bareilly (UP)
7. Regional Agriculture Seed Testing & Demonstration Center,Department of Agriculture, 32-8, Civil Line, Mathura (UP)
8. Regional Agriculture Seed Testing & Demonstration Center,Department of Agriculture, Station Road, Hardoi (UP)
9. Seed Testing Laboratory, Department of Seed Technology, C.S. Azad University of Agriculture & Technology, Kanpur (UP)
1110
Table 3. List of ISTA accredited laboratories in India
S.N. Accredited laboratories Accredited tests
1. Bejo Sheetal Seeds Pvt. Ltd., Sampling, physical Purity,Mantha Road, Jalna, Maharashtra, India germination and moisture content
2. Indo-American Hybrid Seeds (India) Pvt Ltd Sampling, physical purity, Seed Laboratory, Bangalore-560 098 germination viability, weight and
moisture content
3. Mahyco Pvt. Ltd., Dawalwadi, Sampling, physical purity,Jalna Maharashtra, India germination viability, weight, seed
health, seed vigour, verification of species and moisture content
4. Namdhari Seeds Pvt. Ltd., 1st phase, Sampling, physical purity, Ideal Homes Township, Rajarajeshwari Nagar germination and moisture contentBangalore
5. Nuziveedu Seeds Limited, Kompally Sampling, physical purity, Quthbullapur Mandal, Secunderabad India germination and moisture content
6. Seed Testing Laboratory, Department of Physical purity and germinationSeed Certification Lawley road, G.C.T., Tamilnadu 641013, Coimbatore, India
6. National Seeds Corp. Ltd., Beej Bhavan, Pusa Complex, New Delhi
7. Rasi Hyveg (P) Ltd., 126/sector-8, IMT-Manesar, Gurgaon, India
8. Rasi Seeds (P) Ltd., Cadalur Main Road, Thulukanur, Attur, Salem (TN)
9. Seed Testing Laboratory, Department of Agriculture, Old Pedgaon Road,Laxminagar, Parbhani (MH)
10. Telangana Seed Certification Agency, Rajendranagar, Hyderabad (AP)
11. United Phosphorous Limited, ANR Center, Banjara Hills, Hyderabad (AP)
12. AICRP on Seed Tech., UAS, Gandhi Krishi Vignana Kendra, Bangalore
Research journal published in Seed Science and Technology:
1. Seed Science Research: The journal is published by International Society for Seed Science
(ISSS), Scotland, United Kingdom. This is a leading international journal featuring high-
quality original papers and review articles on the fundamental aspects of seed science,
reviewed by internationally
distinguished editors. The
emphasis is on the physiology,
biochemistry, molecular
biology and ecology of seeds.
2. Seed Science and Technology:
The journal is published by
International Seed Testing
Association (ISTA), Bassersdorf,
Switzerland.
3. Seed Research: The journal is
published by Indian Society of
Seed Technology (ISST),
New Delhi.
4. Research Journal of Seed
Science: Research Journal of
Seed Science is an international
journal publ ishes pee r
reviewed research work in the
form of research articles,
reviews and/or mini-reviews
and short communications
dealing with recent and
exciting developments in the
field of seed science.
Seed certification agency
1. Uttar Pradesh State Seed Certification Agency, A-284, Sector 5, Indira Nagar Lucknow (UP)
State seed corporation
1. Uttar Pradesh Beej Vikas Nigam, C-973/74 B, Mahanagar, Faizabad Road, Lucknow-226006 (UP)
Table 4. List of ISTA Non-accredited laboratory (ISTA member) in India
S.N. ISTA Non-accreditedLaboratory
1. Bayer Bioscience Pvt. Ltd, Toopran, Medak District, Telangana, India
2. Seed Testing Laboratory, Division of Seed Science &Technology, IARI-110012New Delhi
3. J.K. Agri Genetics Ltd., Varun Towers, Begumpet, Hyderabad, Andhra Pradesh
4. Kaveri Seed Pvt Ltd., Pamulaparthy, Medak, Andhra Pradesh, India
5. CSTL, National Seed Research and Training Centre, G.T.Road, Collectry Farm,Varanasi (U.P) Figure 3. Map showing number of seed testing laboratories in
different states of India and UP
1312
Table 3. List of ISTA accredited laboratories in India
S.N. Accredited laboratories Accredited tests
1. Bejo Sheetal Seeds Pvt. Ltd., Sampling, physical Purity,Mantha Road, Jalna, Maharashtra, India germination and moisture content
2. Indo-American Hybrid Seeds (India) Pvt Ltd Sampling, physical purity, Seed Laboratory, Bangalore-560 098 germination viability, weight and
moisture content
3. Mahyco Pvt. Ltd., Dawalwadi, Sampling, physical purity,Jalna Maharashtra, India germination viability, weight, seed
health, seed vigour, verification of species and moisture content
4. Namdhari Seeds Pvt. Ltd., 1st phase, Sampling, physical purity, Ideal Homes Township, Rajarajeshwari Nagar germination and moisture contentBangalore
5. Nuziveedu Seeds Limited, Kompally Sampling, physical purity, Quthbullapur Mandal, Secunderabad India germination and moisture content
6. Seed Testing Laboratory, Department of Physical purity and germinationSeed Certification Lawley road, G.C.T., Tamilnadu 641013, Coimbatore, India
6. National Seeds Corp. Ltd., Beej Bhavan, Pusa Complex, New Delhi
7. Rasi Hyveg (P) Ltd., 126/sector-8, IMT-Manesar, Gurgaon, India
8. Rasi Seeds (P) Ltd., Cadalur Main Road, Thulukanur, Attur, Salem (TN)
9. Seed Testing Laboratory, Department of Agriculture, Old Pedgaon Road,Laxminagar, Parbhani (MH)
10. Telangana Seed Certification Agency, Rajendranagar, Hyderabad (AP)
11. United Phosphorous Limited, ANR Center, Banjara Hills, Hyderabad (AP)
12. AICRP on Seed Tech., UAS, Gandhi Krishi Vignana Kendra, Bangalore
Research journal published in Seed Science and Technology:
1. Seed Science Research: The journal is published by International Society for Seed Science
(ISSS), Scotland, United Kingdom. This is a leading international journal featuring high-
quality original papers and review articles on the fundamental aspects of seed science,
reviewed by internationally
distinguished editors. The
emphasis is on the physiology,
biochemistry, molecular
biology and ecology of seeds.
2. Seed Science and Technology:
The journal is published by
International Seed Testing
Association (ISTA), Bassersdorf,
Switzerland.
3. Seed Research: The journal is
published by Indian Society of
Seed Technology (ISST),
New Delhi.
4. Research Journal of Seed
Science: Research Journal of
Seed Science is an international
journal publ ishes pee r
reviewed research work in the
form of research articles,
reviews and/or mini-reviews
and short communications
dealing with recent and
exciting developments in the
field of seed science.
Seed certification agency
1. Uttar Pradesh State Seed Certification Agency, A-284, Sector 5, Indira Nagar Lucknow (UP)
State seed corporation
1. Uttar Pradesh Beej Vikas Nigam, C-973/74 B, Mahanagar, Faizabad Road, Lucknow-226006 (UP)
Table 4. List of ISTA Non-accredited laboratory (ISTA member) in India
S.N. ISTA Non-accreditedLaboratory
1. Bayer Bioscience Pvt. Ltd, Toopran, Medak District, Telangana, India
2. Seed Testing Laboratory, Division of Seed Science &Technology, IARI-110012New Delhi
3. J.K. Agri Genetics Ltd., Varun Towers, Begumpet, Hyderabad, Andhra Pradesh
4. Kaveri Seed Pvt Ltd., Pamulaparthy, Medak, Andhra Pradesh, India
5. CSTL, National Seed Research and Training Centre, G.T.Road, Collectry Farm,Varanasi (U.P) Figure 3. Map showing number of seed testing laboratories in
different states of India and UP
1312
The reliability of seed quality test of a seed sample depends mainly on two components; the
accuracy with which the sample represents the lot and the accuracy & precision of the laboratory
test. It is observed in many cases that the variations in test results are due to the variation in the
sampling. Hence, seed sampling is one of the basic components responsible for the accurate seed
testing results. Therefore, utmost care is required during drawing the sample and it should be
accurately represent the composition of the seed lot.
Seed Lot : It is a specified quantity of seed, physically identifiable, in respect of which a seed test
certificate can be issued.
Types of seed sample: There are four major type of seed samples used in seed testing laboratory.
1. Primary sample : Small portion taken from one point in the lot.
2. Composite sample : It is formed by combining &mixing all primary samples taken from
the seed lot.
3. Submitted sample : This is the sample which are submitted to seed testing laboratory for
seed quality analysis. The size of the submitted sample is specified in ISTA rules for
different species.
4. Working sample : It is a sub sample taken from submitted sample with the help of seed
divider, on which seed quality test is made.
General Principles of Sampling :
During the seed sampling the followings care should be taken to avoid any missampling
1. Under seed law enforcement programme only trained and experienced officials are
authorize to undertake sampling and he has to give notice to such intention to the
person from whom he intends to take sample.
2. At least two persons should be present during sampling and obtain the signature of
both the witnesses on form VIII of the seed rules.
3. As per the Seed Act 1966, sampler must verify the information provided on seed bag
viz., kind, variety, lot number, date of test and seller's name and address.
4. In case of certified seed lots sampler should check some additional information's on
seed certification tag viz.,name & address of certification agency, name &address of
certified seed producer, class & designation of seed and date of issue of the certificate
& its validity.
5. While checking the quality of marketed seed, seed inspector should take three
representative samples in the prescribed manner followed by marking and sealing.
·One sample to be delivered to the person from whom it has been taken.
·Second to be sent seed testing laboratory for analysis.
·Third to be retained for any legal proceedings in future.
Upon the request of the sampler, the owner shall provide full information regarding bulking and
mixing of the seed lot. When there is definite evidence of heterogeneity sampling shall be
refused. If the nature of the presentation of the seed lot or container makes it impossible to
adequately apply these procedures, then the sampling shall not be undertaken, and alternative
presentation of the seed lot should be sought. The size of the seed lot should not exceed to the
maximum size as prescribed in the rules subject to 5% of tolerance.
3. SEED SAMPLING AND PROCEDURE
Table 5. Minimum sampling intensity of forage seed lots (ISTA, 2015)
Minimum sampling intensity for seed lots when seed in bulk
Upto 500 At least 5 primary samples
501 - 3,000 One primary sample for each 300 kg but not less than 5
3,001 - 20,000 One primary sample for each 500 kg but not less than 10
>20,001 One primary sample for each 700 kg but not less than 40
Minimum sampling intensity for seed lots in containers
Containers No. of Primary Samples
1- 4 3 samples from each container.
5 - 8 2 samples from each container
9 - 15 1 samples from each container
16 - 30 15 samples in total
31 - 59 20 samples in total
>60 30 samples in total
For seed lots in containers smaller than 15 kg capacity, containers shall be combined to sampling
units not exceeding 100kg and sampling units shall be regarding as containers in the sampling
scheme above.
Equipments used for seed sampling:
1. Sleeve or Stick type trier (Bag trier): This trier is generally used to obtain samples from
bags. In this triertube and sleeves have open slots in their walls. When tube is turned the slot
in tube and sleeves becomes in line so that seed can flow into the cavity of the tube.
2. Bin trier: It is larger than sleeve trier which is constructed on the same principle. It is
used for sampling from heaps and godowns.
Nobbe trier: Nobbe trier have a long pointed tube which can reach upto the center of bag.
The trier have an oval hole near the pointed end for filling the seed inside the tube.
1514
The reliability of seed quality test of a seed sample depends mainly on two components; the
accuracy with which the sample represents the lot and the accuracy & precision of the laboratory
test. It is observed in many cases that the variations in test results are due to the variation in the
sampling. Hence, seed sampling is one of the basic components responsible for the accurate seed
testing results. Therefore, utmost care is required during drawing the sample and it should be
accurately represent the composition of the seed lot.
Seed Lot : It is a specified quantity of seed, physically identifiable, in respect of which a seed test
certificate can be issued.
Types of seed sample: There are four major type of seed samples used in seed testing laboratory.
1. Primary sample : Small portion taken from one point in the lot.
2. Composite sample : It is formed by combining &mixing all primary samples taken from
the seed lot.
3. Submitted sample : This is the sample which are submitted to seed testing laboratory for
seed quality analysis. The size of the submitted sample is specified in ISTA rules for
different species.
4. Working sample : It is a sub sample taken from submitted sample with the help of seed
divider, on which seed quality test is made.
General Principles of Sampling :
During the seed sampling the followings care should be taken to avoid any missampling
1. Under seed law enforcement programme only trained and experienced officials are
authorize to undertake sampling and he has to give notice to such intention to the
person from whom he intends to take sample.
2. At least two persons should be present during sampling and obtain the signature of
both the witnesses on form VIII of the seed rules.
3. As per the Seed Act 1966, sampler must verify the information provided on seed bag
viz., kind, variety, lot number, date of test and seller's name and address.
4. In case of certified seed lots sampler should check some additional information's on
seed certification tag viz.,name & address of certification agency, name &address of
certified seed producer, class & designation of seed and date of issue of the certificate
& its validity.
5. While checking the quality of marketed seed, seed inspector should take three
representative samples in the prescribed manner followed by marking and sealing.
·One sample to be delivered to the person from whom it has been taken.
·Second to be sent seed testing laboratory for analysis.
·Third to be retained for any legal proceedings in future.
Upon the request of the sampler, the owner shall provide full information regarding bulking and
mixing of the seed lot. When there is definite evidence of heterogeneity sampling shall be
refused. If the nature of the presentation of the seed lot or container makes it impossible to
adequately apply these procedures, then the sampling shall not be undertaken, and alternative
presentation of the seed lot should be sought. The size of the seed lot should not exceed to the
maximum size as prescribed in the rules subject to 5% of tolerance.
3. SEED SAMPLING AND PROCEDURE
Table 5. Minimum sampling intensity of forage seed lots (ISTA, 2015)
Minimum sampling intensity for seed lots when seed in bulk
Upto 500 At least 5 primary samples
501 - 3,000 One primary sample for each 300 kg but not less than 5
3,001 - 20,000 One primary sample for each 500 kg but not less than 10
>20,001 One primary sample for each 700 kg but not less than 40
Minimum sampling intensity for seed lots in containers
Containers No. of Primary Samples
1- 4 3 samples from each container.
5 - 8 2 samples from each container
9 - 15 1 samples from each container
16 - 30 15 samples in total
31 - 59 20 samples in total
>60 30 samples in total
For seed lots in containers smaller than 15 kg capacity, containers shall be combined to sampling
units not exceeding 100kg and sampling units shall be regarding as containers in the sampling
scheme above.
Equipments used for seed sampling:
1. Sleeve or Stick type trier (Bag trier): This trier is generally used to obtain samples from
bags. In this triertube and sleeves have open slots in their walls. When tube is turned the slot
in tube and sleeves becomes in line so that seed can flow into the cavity of the tube.
2. Bin trier: It is larger than sleeve trier which is constructed on the same principle. It is
used for sampling from heaps and godowns.
Nobbe trier: Nobbe trier have a long pointed tube which can reach upto the center of bag.
The trier have an oval hole near the pointed end for filling the seed inside the tube.
1514
Methods for obtaining working samples:
Submitted seed samples received in the seed testing laboratory are required to be reduced to
obtain working samples for carrying out various test. To obtain working sample following
different methods are generally used.
1. Seed divider method: To avoid any variability in the results, different types of dividers are
used to obtain working sample from submitted sample.
A. Conical divider: This type of seed divider is suitable to obtain working sample for large
seeded crops. In this divider there are 19 channels and 19 spaces (each 25.4 mm wide)
are designed for passing the equal amount of seed.
B. Soil divider: It is simple divider, which is built on the same principle as the conical
divider. The channels are here arranged in a straight row instead of a circle as in the
conical divider. The soil divider consists of a hopper with attached channels or ducts, a
frame to hold the hopper, two receiving pans and a pouring pan. This divider is suitable
for large and chaffy seeds.
C. Centrifugal divider/ Gamete divider : This divider makes use of centrifugal force to
mix and scatter the seeds over the dividing surface. In this divider the seed flows
downward through a hopper into a shallow rubber cup or spinner. Upon rotation of the
spinner by an electric motor the seeds are thrown out by centrifugal force and fall
downward. The circle where the seeds fall is equally divided into two parts by a
stationary baffle so that approximately half the seeds fall in one spout and half in the
other spout.
Figure 4. Seed sampling devices (A) Sleeve trier, (B) Bin trier (C) Nobbe trier (Source: ISTA)
Figure 5.(A) Seed label on which detailed information about seed lot (B) representation primary and composite samples
Figure 6. (A) Method of obtaining primary samples (B) Empting the trier into bucket (C) Checking of seed lot and drawing primary sample (Source: ISTA)
A B C
Figure 7. Different types of seed divider (A) Conical seed divider (B) Soil seed divider (C) Centrifugal seed divider
A B C
A
B
C
1716
Methods for obtaining working samples:
Submitted seed samples received in the seed testing laboratory are required to be reduced to
obtain working samples for carrying out various test. To obtain working sample following
different methods are generally used.
1. Seed divider method: To avoid any variability in the results, different types of dividers are
used to obtain working sample from submitted sample.
A. Conical divider: This type of seed divider is suitable to obtain working sample for large
seeded crops. In this divider there are 19 channels and 19 spaces (each 25.4 mm wide)
are designed for passing the equal amount of seed.
B. Soil divider: It is simple divider, which is built on the same principle as the conical
divider. The channels are here arranged in a straight row instead of a circle as in the
conical divider. The soil divider consists of a hopper with attached channels or ducts, a
frame to hold the hopper, two receiving pans and a pouring pan. This divider is suitable
for large and chaffy seeds.
C. Centrifugal divider/ Gamete divider : This divider makes use of centrifugal force to
mix and scatter the seeds over the dividing surface. In this divider the seed flows
downward through a hopper into a shallow rubber cup or spinner. Upon rotation of the
spinner by an electric motor the seeds are thrown out by centrifugal force and fall
downward. The circle where the seeds fall is equally divided into two parts by a
stationary baffle so that approximately half the seeds fall in one spout and half in the
other spout.
Figure 4. Seed sampling devices (A) Sleeve trier, (B) Bin trier (C) Nobbe trier (Source: ISTA)
Figure 5.(A) Seed label on which detailed information about seed lot (B) representation primary and composite samples
Figure 6. (A) Method of obtaining primary samples (B) Empting the trier into bucket (C) Checking of seed lot and drawing primary sample (Source: ISTA)
A B C
Figure 7. Different types of seed divider (A) Conical seed divider (B) Soil seed divider (C) Centrifugal seed divider
A B C
A
B
C
1716
2. Spoon method: This method is useful to obtain working sample for small-seeded grasses,
moisture determination and for seed health testing. In this method small portions of seed
from five random places are drawing by using both spoon and spatula followed by mixing of
samples and constituting a sub-sample of the required size.
3. Hand halving method: This method is the most satisfactory method for chaffy, tree and
shrub seed. In this method seeds are poured evenly into a smooth clean surface and mound
the seeds with a flat-edged spatula. This seed mound is divided into half and each half is
halved again, which will give four portions. Each of the four portions is halved again giving
eight portions which should be arranged in two rows of four each. Combine and retain the
alternate portions of seed and remove the remaining four portions. Combine the first and
third portions in the first row with the second and fourth in the second row. The same steps
are repeated by using the retained portions until the desired weight of sample is obtained.
4. Random cups method: In this method a special tray is used to obtain working sample size.
In this tray there are different types of cups are fitted to distribute the seed over the tray.
54321
4321
Figure 8. Spoon method steps (1) distributing the seed over the pan (2) one spoon is pushed vertically into the seed layer (3) with the second spoon the seed in front of the vertical spoon is collected
(4) both spoons are removed from the seed (5) the seed sample is transferred to a collection pan (Source: ISTA).
Figure 9. Figure showing different steps of hand halving method to obtain working sample(Source: ISTA).
1 32
4 65
Figure10. Random cup method steps (1)Cups of different size in one set (2) A tray with cups and distributing a sample over the tray (3) The tray with the total sample distributed over the tray
(4) The cups removed from the tray and emptied into a glass vessel.(Source: ISTA)
Table 6. Maximum seed lot size & working sample weight of forage crops(ISTA, 2015)
S.N. Crops Max. seed lot (kg) Min. weight of sample (g)
Submitted Working
1. Berseem 10000 60 6
2. Cenchrus ciliaris 10000 60 6
3. Cenchrus setigers 20000 150 15
4. Sorghum 30000 900 90
5. Cluster bean 20000 1000 100
6. Guinea (Panicum maximum) 10000 20 2
7. Senji (Melilotus spp) 10000 50 5
8. Lucerne (Rijka) (Medicago sativa) 10000 50 5
9. Oats (Avena sativa L.) 30000 1000 120
10. Nandi grass (Setaria italica L.) 10000 90 9
11. Stylo santhes spp. 10000 70 7
12. Cowpea (Vigna unguiculata (L.) 30000 1000 400
13. Para grass (Brachiaria mutica) 10000 30 3
14. Brachiaria ruziziensis 20000 150 15
15. Bothriochloa insculpta 10000 20 2
16. Festuca arundinacea 10000 50 5
17. Lolium spp. 10000 60 6
18. Zea mays L. 40000 1000 900
19. Teosinite 20000 1000 900
20. Chickling vetch (Lathyrus sativus) 20000 1000 450
1918
2. Spoon method: This method is useful to obtain working sample for small-seeded grasses,
moisture determination and for seed health testing. In this method small portions of seed
from five random places are drawing by using both spoon and spatula followed by mixing of
samples and constituting a sub-sample of the required size.
3. Hand halving method: This method is the most satisfactory method for chaffy, tree and
shrub seed. In this method seeds are poured evenly into a smooth clean surface and mound
the seeds with a flat-edged spatula. This seed mound is divided into half and each half is
halved again, which will give four portions. Each of the four portions is halved again giving
eight portions which should be arranged in two rows of four each. Combine and retain the
alternate portions of seed and remove the remaining four portions. Combine the first and
third portions in the first row with the second and fourth in the second row. The same steps
are repeated by using the retained portions until the desired weight of sample is obtained.
4. Random cups method: In this method a special tray is used to obtain working sample size.
In this tray there are different types of cups are fitted to distribute the seed over the tray.
54321
4321
Figure 8. Spoon method steps (1) distributing the seed over the pan (2) one spoon is pushed vertically into the seed layer (3) with the second spoon the seed in front of the vertical spoon is collected
(4) both spoons are removed from the seed (5) the seed sample is transferred to a collection pan (Source: ISTA).
Figure 9. Figure showing different steps of hand halving method to obtain working sample(Source: ISTA).
1 32
4 65
Figure10. Random cup method steps (1)Cups of different size in one set (2) A tray with cups and distributing a sample over the tray (3) The tray with the total sample distributed over the tray
(4) The cups removed from the tray and emptied into a glass vessel.(Source: ISTA)
Table 6. Maximum seed lot size & working sample weight of forage crops(ISTA, 2015)
S.N. Crops Max. seed lot (kg) Min. weight of sample (g)
Submitted Working
1. Berseem 10000 60 6
2. Cenchrus ciliaris 10000 60 6
3. Cenchrus setigers 20000 150 15
4. Sorghum 30000 900 90
5. Cluster bean 20000 1000 100
6. Guinea (Panicum maximum) 10000 20 2
7. Senji (Melilotus spp) 10000 50 5
8. Lucerne (Rijka) (Medicago sativa) 10000 50 5
9. Oats (Avena sativa L.) 30000 1000 120
10. Nandi grass (Setaria italica L.) 10000 90 9
11. Stylo santhes spp. 10000 70 7
12. Cowpea (Vigna unguiculata (L.) 30000 1000 400
13. Para grass (Brachiaria mutica) 10000 30 3
14. Brachiaria ruziziensis 20000 150 15
15. Bothriochloa insculpta 10000 20 2
16. Festuca arundinacea 10000 50 5
17. Lolium spp. 10000 60 6
18. Zea mays L. 40000 1000 900
19. Teosinite 20000 1000 900
20. Chickling vetch (Lathyrus sativus) 20000 1000 450
1918
Physical purity analysis tells us the proportion of pure seed component in a seed lot as well as the
proportion of other crop seed, weed seed and inert matter by weight in percentage for which seed
standards have been prescribed. The testing of physical purity helps in the following ways:
1. It improve the pure plant establishment by eliminating other crop seed and weed seeds.
2. Physical purity helps to raise a disease free-crop by removing inert matter from the seed.
3. Physical purity analysis is a pre-requisite for germination test because 'pure seed'
component is used for germination testing.
4. Physical purity improve the uniformity of seed which make the sowing appropriate
through seed drill machine.
A. Pure Seed: The pure seed shall refer to the species stated by the sender, or found to
predominate in the test, and shall include all botanical varieties and cultivars of that species.
(If immature, undersized, shriveled, germinated and diseased seeds identified as stated
species then these should be counted as pure seed component). Apart from this during
physical purity determination following different facts should be considered.
1. Seed units of families Leguminaceae, Cruciferae, Cupressaceae, Pinaceae and
Taxodiaceae with the seed coat entirely removed shall be regarded as inert matter.
2. Separated cotyledons of leguminaceae are regarded as inert matter, irrespective of
whether or not the radicle-plumule axis and/or more than half of the testa may be
attached.
3. In Lolium, Festuca and Elytrigiarepens a floret with a caryopsis less than 1/3 the length
of the palea is regarded as inert matter.
4. In certain species the separation of pure seed and inert matter is done by uniform blowing
procedure and this method is obligatory for Poa pratensis and Dactylis glomerata.
5. Multiple seed unit (MSU) are left intact in the pure seed fraction in case of Dactylis and
Festuca.
6. In case of Arrhenatherium, Avena, Chloris, Dactylis, Festuca, Holens, Poa, Sorghum
and Triticum spelta attached sterile florets are not removed and it should be included in
the pure seed fraction.
B. Other Crop Seed: Other crop seed shall include seed units of any plant species other than of
pure seed grown as crops. Multiple structures, capsules, pods are opened and the seeds are
taken out and the non-seed material is placed in the inert matter.
C. Weed Seeds: Seeds, bulblets or tuber of plants recognized by laws, official regulations or by
general usage shall be considered as weed seeds.
D. Inert matter: Inert matter shall include seed units and all other matter and structures not
defined as pure seed excluding other crop seed and weed seeds.
General Principles: As per ISTA Rules, the working sample is separated into four components
i.e. pure seeds, other seed, weed seeds and inert matter. The percentage of each part is determined
by weight. All species of seed and each kind of inert matter present shall be identified as far as
possible and if required for reporting, its percentage by weight shall be determined.
Equipments : The following different types of equipment's are required for physical purity test
·Seed dividers
·Balance: Electric balance are better due to their accuracy and quickness
·Seed blowers
·Diaphnoscope: Reflected light are used to separate empty florets of grasses (IM).
·Sieves
·Sample pans, dishes, forceps, spatula and hand lens
·Seed herbarium of crop and weed seed
4. PHYSICAL PURITY TEST
C
A B
BA
Figure 11. Equipment's used for physical purity testing (A) Seed blower (B) Seed sieving (C) Lens physical purity work board
Figure 12. (A) Aluminium spatula & forceps (B) Light physical purity work board
2120
Physical purity analysis tells us the proportion of pure seed component in a seed lot as well as the
proportion of other crop seed, weed seed and inert matter by weight in percentage for which seed
standards have been prescribed. The testing of physical purity helps in the following ways:
1. It improve the pure plant establishment by eliminating other crop seed and weed seeds.
2. Physical purity helps to raise a disease free-crop by removing inert matter from the seed.
3. Physical purity analysis is a pre-requisite for germination test because 'pure seed'
component is used for germination testing.
4. Physical purity improve the uniformity of seed which make the sowing appropriate
through seed drill machine.
A. Pure Seed: The pure seed shall refer to the species stated by the sender, or found to
predominate in the test, and shall include all botanical varieties and cultivars of that species.
(If immature, undersized, shriveled, germinated and diseased seeds identified as stated
species then these should be counted as pure seed component). Apart from this during
physical purity determination following different facts should be considered.
1. Seed units of families Leguminaceae, Cruciferae, Cupressaceae, Pinaceae and
Taxodiaceae with the seed coat entirely removed shall be regarded as inert matter.
2. Separated cotyledons of leguminaceae are regarded as inert matter, irrespective of
whether or not the radicle-plumule axis and/or more than half of the testa may be
attached.
3. In Lolium, Festuca and Elytrigiarepens a floret with a caryopsis less than 1/3 the length
of the palea is regarded as inert matter.
4. In certain species the separation of pure seed and inert matter is done by uniform blowing
procedure and this method is obligatory for Poa pratensis and Dactylis glomerata.
5. Multiple seed unit (MSU) are left intact in the pure seed fraction in case of Dactylis and
Festuca.
6. In case of Arrhenatherium, Avena, Chloris, Dactylis, Festuca, Holens, Poa, Sorghum
and Triticum spelta attached sterile florets are not removed and it should be included in
the pure seed fraction.
B. Other Crop Seed: Other crop seed shall include seed units of any plant species other than of
pure seed grown as crops. Multiple structures, capsules, pods are opened and the seeds are
taken out and the non-seed material is placed in the inert matter.
C. Weed Seeds: Seeds, bulblets or tuber of plants recognized by laws, official regulations or by
general usage shall be considered as weed seeds.
D. Inert matter: Inert matter shall include seed units and all other matter and structures not
defined as pure seed excluding other crop seed and weed seeds.
General Principles: As per ISTA Rules, the working sample is separated into four components
i.e. pure seeds, other seed, weed seeds and inert matter. The percentage of each part is determined
by weight. All species of seed and each kind of inert matter present shall be identified as far as
possible and if required for reporting, its percentage by weight shall be determined.
Equipments : The following different types of equipment's are required for physical purity test
·Seed dividers
·Balance: Electric balance are better due to their accuracy and quickness
·Seed blowers
·Diaphnoscope: Reflected light are used to separate empty florets of grasses (IM).
·Sieves
·Sample pans, dishes, forceps, spatula and hand lens
·Seed herbarium of crop and weed seed
4. PHYSICAL PURITY TEST
C
A B
BA
Figure 11. Equipment's used for physical purity testing (A) Seed blower (B) Seed sieving (C) Lens physical purity work board
Figure 12. (A) Aluminium spatula & forceps (B) Light physical purity work board
2120
Procedures:
1. Homogenize the submitted sample: Boerner or soil type seed divider should be used to
homogenize the submitted sample before reducing it to the size of working sample.
2. Working sample preparation: The working sample shall be either a weight estimated to
contain at least 2,500 seed units or not less than weight indicated i.e. 6 g for berseem.
3. Separation of pure seed components: The separation of pure seed from inert matter and
weed seed should be completed under following different stages:
·Before starting separation carefully examine the working sample to determine the use of
particular aid such as blower or sieves for making initial separation.
·After preliminary separation with the help of sieves or blower, place and spread the
retained or heavier portion on the purity work board for final separation. Based on
external appearance (shape, size, colour, gloss, surface texture) and seed appearance in
transmitter light pure seeds are separated with the help of spatula and forceps.
·Separate out impurities such as other crop seeds, weed seeds and inert matter and place
the impurities separately in purity dishes, leaving only the pure seed on the purity board.
·After separation, identify the other crop seed, weed seed and record their names on the
analysis card. The kind of inert matter present in the sample should also be identified and
recorded.
·Weight each component, pure seed, other crop seed, weed seed and inert matter in grams
to the number of decimal places shown below:
physical purity including the presence of a weed seeds therefore, authentic identification of weed
seeds in crop varieties is vital. An in depth knowledge of botany of a plant as well as its seed is
necessary, for correct identification of a particular species and weed seed should be identify up to
the species.
·Calculate the percentage by weight of each component to one decimal place only, basing
the percentage on the sum of the weight of all the four components. If any component is
less than 0.05% record it as 'Trace'. Component of 0.05% to 0.1% are reported as 0.1%.
Identification of crop and weed seeds: Increase in seed trade has promoted increased transfer
of seeds of crop varieties from one region and country to other. Association of seeds of other
varieties and weeds in a seed lot is a well-known. Before seeds are marketed within or to outside
the country, it's testing is mandatory. In seed testing it is essential to test the seed samples for
Table 7. Decimal place required for weighing of physical purity components
S.N. Wt. of working sample(g) No. of decimal place required Example
1. Less than 1 4 0.9025
2. 1 to 9.990 3 9.025
3. 10 to 99.99 2 90.25
4. 100 to 999.9 1 902.5
5. 1000 or more 0 1025
Table 8. Objectionable weed seed species in grasses and fodder crops
S.N. Crops Objectionable weed name
Common English Scientific
1. Berseem Kasni Chicory Chicorium intybus L.
2. Lucerne Amarbel Dodder Cuscuta spp.
3. Oat Hirankhuri Wild morning Convulvulus arvensis L.
Jangali jae Wild oat Avena fatua L.
4. Napier grass - Canada thistle Cirsium arvense L.
Amarbel Dodder Cuscutta spp.
Baru Johnson grass Sorghum halpense L.
- Quack grass Agropyron repens L.
2322
Procedures:
1. Homogenize the submitted sample: Boerner or soil type seed divider should be used to
homogenize the submitted sample before reducing it to the size of working sample.
2. Working sample preparation: The working sample shall be either a weight estimated to
contain at least 2,500 seed units or not less than weight indicated i.e. 6 g for berseem.
3. Separation of pure seed components: The separation of pure seed from inert matter and
weed seed should be completed under following different stages:
·Before starting separation carefully examine the working sample to determine the use of
particular aid such as blower or sieves for making initial separation.
·After preliminary separation with the help of sieves or blower, place and spread the
retained or heavier portion on the purity work board for final separation. Based on
external appearance (shape, size, colour, gloss, surface texture) and seed appearance in
transmitter light pure seeds are separated with the help of spatula and forceps.
·Separate out impurities such as other crop seeds, weed seeds and inert matter and place
the impurities separately in purity dishes, leaving only the pure seed on the purity board.
·After separation, identify the other crop seed, weed seed and record their names on the
analysis card. The kind of inert matter present in the sample should also be identified and
recorded.
·Weight each component, pure seed, other crop seed, weed seed and inert matter in grams
to the number of decimal places shown below:
physical purity including the presence of a weed seeds therefore, authentic identification of weed
seeds in crop varieties is vital. An in depth knowledge of botany of a plant as well as its seed is
necessary, for correct identification of a particular species and weed seed should be identify up to
the species.
·Calculate the percentage by weight of each component to one decimal place only, basing
the percentage on the sum of the weight of all the four components. If any component is
less than 0.05% record it as 'Trace'. Component of 0.05% to 0.1% are reported as 0.1%.
Identification of crop and weed seeds: Increase in seed trade has promoted increased transfer
of seeds of crop varieties from one region and country to other. Association of seeds of other
varieties and weeds in a seed lot is a well-known. Before seeds are marketed within or to outside
the country, it's testing is mandatory. In seed testing it is essential to test the seed samples for
Table 7. Decimal place required for weighing of physical purity components
S.N. Wt. of working sample(g) No. of decimal place required Example
1. Less than 1 4 0.9025
2. 1 to 9.990 3 9.025
3. 10 to 99.99 2 90.25
4. 100 to 999.9 1 902.5
5. 1000 or more 0 1025
Table 8. Objectionable weed seed species in grasses and fodder crops
S.N. Crops Objectionable weed name
Common English Scientific
1. Berseem Kasni Chicory Chicorium intybus L.
2. Lucerne Amarbel Dodder Cuscuta spp.
3. Oat Hirankhuri Wild morning Convulvulus arvensis L.
Jangali jae Wild oat Avena fatua L.
4. Napier grass - Canada thistle Cirsium arvense L.
Amarbel Dodder Cuscutta spp.
Baru Johnson grass Sorghum halpense L.
- Quack grass Agropyron repens L.
2322
Figure 13. (A)Dinanath panicle (B) Dinanath fluffed seed (C) Dinanath naked seed (D) Guinea grass panicle (E) Guinea grass seed (F) Bracharia grass panicle (G) Cenchrus setigerus panicle (H) Cenchrussetigerus seed
(I) Cenchrus ciliaris seed
A B C
D E F
G H I
In seed testing germination has been defined as “the emergence and development of the seedling
to a stage where the aspects of its essential structures indicates whether or not it is able to develop
further into a satisfactory plant under favorable conditions in soil” (ISTA, 2015). Hence, the
ultimate aim of seed testing in laboratory is to obtain information about the planting value of the
seed sample and by inference the quality of the seed lot.
Essential equipments for germination test : The following pieces of equipment and materials
are essential to carry out the germination tests in the seed testing laboratories.
1. Seed Germinator : The seed germinators are the essential requirement for germination
testing for maintaining the specific conditions of temperature, relative humidity and light.
The seed germinators are generally of two types, namely: cabinet germinator and walk in
germinator. The germinators should maintain correct temperature and RH (90-98%)
according to species under investigation. The germinators should be disinfected periodically
by flushing with hot water or with potassium permanganate solution.
2. Counting devices : The counting devices includes counting boards, automatic seed counter
and vacuum seed counter. These devices are required to aid germination testing by
minimizing the time spent on planning the seeds as well as to provide proper spacing of the
seed on germination substrata. Counting boards are suitable for medium and bold sized
seeds, while vacuum counter can be used for small sized seeds. In case of grasses seed where
seeds are fluffy and small in size, counting of seed should be accomplished by manually.
3. Germination substrata: The accuracy and reproducibility of the germination result are very
much dependent on the quality of the substrata (paper and sand) used for germination testing.
Generally followings different types of germination substrata are used for testing the forage
seeds.
A. Paper substrata: The paper substrata are used in the form of top of paper (TP), pleated
paper (PP) and between paper (BP). In most of the laboratories, paper-toweling method
(roll towel test) is most commonly used for medium and bold seeds. The paper substrata
are not reusable.
B. Sand substrata: The sand substrata have advantage of being relatively less expensive
and reusable. The results in sand media are more accurate and reproducible in
comparison with 'roll towel' tests especially in case of seed lots that are aged or heavily
treated with chemicals. In sand germination the sand should be reasonably uniform and
free from very small and large particles. It should not contain toxic substances and its pH
should be within the range of 6.0- 7.5. The sand should be washed, sterilized and graded
with a sieve set having holes of 0.8 mm diameter (upper sieve) and 0.05 mm diameter
(bottom sieve). The sand retained on the bottom sieve should only be used.
5. SEED GERMINATION TEST
2524
Figure 13. (A)Dinanath panicle (B) Dinanath fluffed seed (C) Dinanath naked seed (D) Guinea grass panicle (E) Guinea grass seed (F) Bracharia grass panicle (G) Cenchrus setigerus panicle (H) Cenchrussetigerus seed
(I) Cenchrus ciliaris seed
A B C
D E F
G H I
In seed testing germination has been defined as “the emergence and development of the seedling
to a stage where the aspects of its essential structures indicates whether or not it is able to develop
further into a satisfactory plant under favorable conditions in soil” (ISTA, 2015). Hence, the
ultimate aim of seed testing in laboratory is to obtain information about the planting value of the
seed sample and by inference the quality of the seed lot.
Essential equipments for germination test : The following pieces of equipment and materials
are essential to carry out the germination tests in the seed testing laboratories.
1. Seed Germinator : The seed germinators are the essential requirement for germination
testing for maintaining the specific conditions of temperature, relative humidity and light.
The seed germinators are generally of two types, namely: cabinet germinator and walk in
germinator. The germinators should maintain correct temperature and RH (90-98%)
according to species under investigation. The germinators should be disinfected periodically
by flushing with hot water or with potassium permanganate solution.
2. Counting devices : The counting devices includes counting boards, automatic seed counter
and vacuum seed counter. These devices are required to aid germination testing by
minimizing the time spent on planning the seeds as well as to provide proper spacing of the
seed on germination substrata. Counting boards are suitable for medium and bold sized
seeds, while vacuum counter can be used for small sized seeds. In case of grasses seed where
seeds are fluffy and small in size, counting of seed should be accomplished by manually.
3. Germination substrata: The accuracy and reproducibility of the germination result are very
much dependent on the quality of the substrata (paper and sand) used for germination testing.
Generally followings different types of germination substrata are used for testing the forage
seeds.
A. Paper substrata: The paper substrata are used in the form of top of paper (TP), pleated
paper (PP) and between paper (BP). In most of the laboratories, paper-toweling method
(roll towel test) is most commonly used for medium and bold seeds. The paper substrata
are not reusable.
B. Sand substrata: The sand substrata have advantage of being relatively less expensive
and reusable. The results in sand media are more accurate and reproducible in
comparison with 'roll towel' tests especially in case of seed lots that are aged or heavily
treated with chemicals. In sand germination the sand should be reasonably uniform and
free from very small and large particles. It should not contain toxic substances and its pH
should be within the range of 6.0- 7.5. The sand should be washed, sterilized and graded
with a sieve set having holes of 0.8 mm diameter (upper sieve) and 0.05 mm diameter
(bottom sieve). The sand retained on the bottom sieve should only be used.
5. SEED GERMINATION TEST
2524
C. Soil/Organic media : Soil is generally not recommended as primary growing medium
since it is generally difficult to obtain consistency in soil. As the soil from different part
varies widely, to have uniform results across laboratories also it is not recommended. It
may be used as an alternative of seeds is in doubt in sand/paper. If it is used it should meet
the general specification of growing media.
Toxicity determination of germination substrata: The quality of the paper highly influences
the test results hence care should be taken while selecting the paper and it is necessary to conduct
biological test to check for the existence of toxic materials in unknown quality paper. The testing
paper should be cut into pieces and place in the petri plate. The two thickness of such paper
should be used for cutting and saturated with tap water or distilled water. Seeds of brassica
species or onion are place on the top of paper and evaluation may be done by comparing the
seedling development with seedlings grown on the known quality paper. The evaluation is done
after 3 days in Brassica and 6 days in onion. If unknown quality paper contains any toxicity the
root tips gets shortened, discolored, root hairs brunched and sometimes plumule gets shortened.
The similar toxicity test should be conducted for sand medium also where, sand should be placed
in boxes as 2 cm deep uniform layer and moistened to its 50% water holding capacity.
Test for capillary rise in germination paper : The test paper is cut into ten strips of 10mm wide
and out of ten strips five are cut in the machine direction of the paper and remaining five in the 0
cross machine direction. Immerse these strips in distilled water at 27±2 C, to a depth of 20 mm
from the end of the strips. After 2 min. measure the height to which the water has risen in the strips
to nearest 1 mm. The average of five strips in machine direction and cross direction is calculated.
The lower average is considered as the capillary rise of the paper and it should meet the
prescribed standard.
1. Other equipments : The other equipments required for germination testing include the
refrigerators, scarifier, hot water bath, incubator, forceps, spatula, germination boxes, plastic
plates, roll towel stands and plastic or surgical trays, etc. A large oven with temperature oranging 100 -200 C is also required for sterilizing the sand. Germination paper (Creppe Kraft
paper or towel paper, sunlit filter paper and blotters) and sand are the basic supplies required
for germination tests.
Table 9. Specific characteristics of germination paper
Characters Filter paper Towel paper2
Mass (g/m ) 130-135 95-1002
Bursting strength (Kg/cm ) minimum 1.0 2.0
Capillary rise (mm) minimum 30 30
pH 6.0-7.5 6.0-7.5
Ash, % by mass (maximum) 1.20 1.50
Germination test conditions:
1. Temperature : The temperature is one of the most important and critical factors for the
laboratory germination tests. The temperature requirement for germination is specific
according to the kind of crop or species. During the germination test temperature should be
uniform throughout the period and temperature variation inside the germinator should not be
more than 1°C. The prescribed temperature for germination can be broadly classified into
two groups, viz. constant temperatures and alternate temperatures.
A. Constant temperature : In this type temperature should be uniform during the entire
germination period.
B. Alternate temperature : In the alternate temperature conditions, the lower temperature
should be maintained for 16 hours and the higher for 8 hours. The gradual changeover
lasting 3 hours is usually satisfactory for non-dormant seeds. However, a sharp change
over lasting 1 hour or less, or transfer of test to another germinator at lower temperature,
may be necessary for seeds, which are likely to be dormant.
2. Light : Seeds of most of the species can germinate either in light or darkness. It is always
Figure 14. Seed germination testing equipments (A) Automatic seed counter (B) Seed counting board (C) Seed germinator
A
B
C
2726
C. Soil/Organic media : Soil is generally not recommended as primary growing medium
since it is generally difficult to obtain consistency in soil. As the soil from different part
varies widely, to have uniform results across laboratories also it is not recommended. It
may be used as an alternative of seeds is in doubt in sand/paper. If it is used it should meet
the general specification of growing media.
Toxicity determination of germination substrata: The quality of the paper highly influences
the test results hence care should be taken while selecting the paper and it is necessary to conduct
biological test to check for the existence of toxic materials in unknown quality paper. The testing
paper should be cut into pieces and place in the petri plate. The two thickness of such paper
should be used for cutting and saturated with tap water or distilled water. Seeds of brassica
species or onion are place on the top of paper and evaluation may be done by comparing the
seedling development with seedlings grown on the known quality paper. The evaluation is done
after 3 days in Brassica and 6 days in onion. If unknown quality paper contains any toxicity the
root tips gets shortened, discolored, root hairs brunched and sometimes plumule gets shortened.
The similar toxicity test should be conducted for sand medium also where, sand should be placed
in boxes as 2 cm deep uniform layer and moistened to its 50% water holding capacity.
Test for capillary rise in germination paper : The test paper is cut into ten strips of 10mm wide
and out of ten strips five are cut in the machine direction of the paper and remaining five in the 0
cross machine direction. Immerse these strips in distilled water at 27±2 C, to a depth of 20 mm
from the end of the strips. After 2 min. measure the height to which the water has risen in the strips
to nearest 1 mm. The average of five strips in machine direction and cross direction is calculated.
The lower average is considered as the capillary rise of the paper and it should meet the
prescribed standard.
1. Other equipments : The other equipments required for germination testing include the
refrigerators, scarifier, hot water bath, incubator, forceps, spatula, germination boxes, plastic
plates, roll towel stands and plastic or surgical trays, etc. A large oven with temperature oranging 100 -200 C is also required for sterilizing the sand. Germination paper (Creppe Kraft
paper or towel paper, sunlit filter paper and blotters) and sand are the basic supplies required
for germination tests.
Table 9. Specific characteristics of germination paper
Characters Filter paper Towel paper2
Mass (g/m ) 130-135 95-1002
Bursting strength (Kg/cm ) minimum 1.0 2.0
Capillary rise (mm) minimum 30 30
pH 6.0-7.5 6.0-7.5
Ash, % by mass (maximum) 1.20 1.50
Germination test conditions:
1. Temperature : The temperature is one of the most important and critical factors for the
laboratory germination tests. The temperature requirement for germination is specific
according to the kind of crop or species. During the germination test temperature should be
uniform throughout the period and temperature variation inside the germinator should not be
more than 1°C. The prescribed temperature for germination can be broadly classified into
two groups, viz. constant temperatures and alternate temperatures.
A. Constant temperature : In this type temperature should be uniform during the entire
germination period.
B. Alternate temperature : In the alternate temperature conditions, the lower temperature
should be maintained for 16 hours and the higher for 8 hours. The gradual changeover
lasting 3 hours is usually satisfactory for non-dormant seeds. However, a sharp change
over lasting 1 hour or less, or transfer of test to another germinator at lower temperature,
may be necessary for seeds, which are likely to be dormant.
2. Light : Seeds of most of the species can germinate either in light or darkness. It is always
Figure 14. Seed germination testing equipments (A) Automatic seed counter (B) Seed counting board (C) Seed germinator
A
B
C
2726
better to illuminate the seeds for the proper growth of the seedlings. Under the situations
where light is essential for germination, tests should be exposed to the natural or artificial
source of light. However, care must be made to ensure that an even intensity is obtained over
the entire substrate and any heating from the source does not affect the prescribed
temperature. Seeds that require light for germination must be illuminated with cool
fluorescent light for at least 8 hours in every 24 hours cycle. Under the situation where testing
of the seed is required to be undertaken at alternate temperatures along with light, the tests
should be illuminated during high temperature period.
3. Number of Replications : Four replication of 100 seeds or under unavoidable situations
eight replications of 50 seeds or sixteen replication of 25 seeds according to the kind and size
of containers may be used for germination test.
4. Duration of testing : The duration of the test is determined by the time prescribed for final
count (ISTA Seed Testing Rules) but chilling periods before or during the test which is
required to break dormancy is not included in the test period. If at the end of the prescribed
test period some seeds have just started to germinate the test may be extended for an
additional period up to 7 days.
Principles for germination test:
1. For germination test a true representative sample should be draw from the seed lot. To obtain
a random sample for testing it is always best to take samples from different parts of the bag or
container. If the seed to be tested from a seed lot that contains more than one bag then samples
must be taken from several bags. A Thumb rule for determining number of bags for drawing
primary sample is that the number of bags should represents the square root of the lot size.
For example if the lot contains nine bags, then sample at least three bags. If the lot contains
100 bags, then sample at least 10 bags. The sample thus drawn is further divided and the
required numbers of seeds are the taken to perform the actual test.
2. The working sample for germination test consists of 400 pure seeds randomly drawn either
manually or with the help of counting devices.
3. After placing the seeds on the prescribed substrata, the test should be transferred to the
controlled temperature condition maintained in the cabinet or walk-in-germinator for
prescribed period (ISTA, 2015).
4. A test may be terminated prior to the prescribed time when the analyst is satisfied that the
maximum germination of the sample has been obtained. The time for the first count is
approximate and a deviation of 1-3 days is permitted.
5. The germination tests need to be evaluated on the expiry of the germination period, which
varies according to the kind of seed. However, the seed analyst may terminate the
germination test on or before the final count day or extend the test beyond the period
depending on the situation.
6. First and second counts are usually taken in case of Top of Paper (TP) and Between Paper (BP) media however a single final count is made in case of sand tests.
Procedure of germination test:
A. Between Paper (BP) Media (Roll Towel Test):
1. Soak the towel paper in water and after its wetting (10-15min) take out the paper from water and remove extra moisture by pressing the soaked paper by hand and holding it in plastic trays.
2. Placed this wet paper over the wax paper/butter paper and write the test number, crop, variety and date of test.
3. Arrange the sufficient number of seeds on the wet paper with proper spacing followed by placing of another layer of wet towel paper over the seed.
4. Roll firmly both wet and wax paper from left to right and secure with rubber band in the center.
5. Place this roll towel in slanting position in the seed germinator maintained at the desired temperature and relative humidity.
B. Top of Paper (TP) Media:
1. For the top of paper test quality paper such as 'sunlit' or 'whatman' filter paper should be used. Crepe kraft (towel) paper or blotter paper of unknown quality should not be used for top of paper tests.
2. This filter paper should be cut in the form of circles or rectangles according to the size and shape of petridish/container.
3. Put two layer of filter paper in the petridish/germination box and note down the date of test and crop name on filter paper with help of pencil. Put enough water to moisten the filter paper followed by holding the petridish /germination box in slanting position in order to drain out the extra moisture.
4. Space the counted seeds on the moist blotter/filter paper and cover the air tight lid.
5. Transfer the petridishes in the germinator maintained at the desired temperature.
Table 10. Germination test procedure of different forage crops (ISTA, 2015)
Crop Substrate Temp. First count Final count Treatmentso( C) (days) (days)
Berseem TP, BP 20 3 7 -
Maize TP, BP, S 25, 20-30 4 7 -
Cowpea BP, S 25, 20-30 5 8 -
Oat BP, S 20 5 10 Preheat o(30-35 C)
& Prechill
2928
better to illuminate the seeds for the proper growth of the seedlings. Under the situations
where light is essential for germination, tests should be exposed to the natural or artificial
source of light. However, care must be made to ensure that an even intensity is obtained over
the entire substrate and any heating from the source does not affect the prescribed
temperature. Seeds that require light for germination must be illuminated with cool
fluorescent light for at least 8 hours in every 24 hours cycle. Under the situation where testing
of the seed is required to be undertaken at alternate temperatures along with light, the tests
should be illuminated during high temperature period.
3. Number of Replications : Four replication of 100 seeds or under unavoidable situations
eight replications of 50 seeds or sixteen replication of 25 seeds according to the kind and size
of containers may be used for germination test.
4. Duration of testing : The duration of the test is determined by the time prescribed for final
count (ISTA Seed Testing Rules) but chilling periods before or during the test which is
required to break dormancy is not included in the test period. If at the end of the prescribed
test period some seeds have just started to germinate the test may be extended for an
additional period up to 7 days.
Principles for germination test:
1. For germination test a true representative sample should be draw from the seed lot. To obtain
a random sample for testing it is always best to take samples from different parts of the bag or
container. If the seed to be tested from a seed lot that contains more than one bag then samples
must be taken from several bags. A Thumb rule for determining number of bags for drawing
primary sample is that the number of bags should represents the square root of the lot size.
For example if the lot contains nine bags, then sample at least three bags. If the lot contains
100 bags, then sample at least 10 bags. The sample thus drawn is further divided and the
required numbers of seeds are the taken to perform the actual test.
2. The working sample for germination test consists of 400 pure seeds randomly drawn either
manually or with the help of counting devices.
3. After placing the seeds on the prescribed substrata, the test should be transferred to the
controlled temperature condition maintained in the cabinet or walk-in-germinator for
prescribed period (ISTA, 2015).
4. A test may be terminated prior to the prescribed time when the analyst is satisfied that the
maximum germination of the sample has been obtained. The time for the first count is
approximate and a deviation of 1-3 days is permitted.
5. The germination tests need to be evaluated on the expiry of the germination period, which
varies according to the kind of seed. However, the seed analyst may terminate the
germination test on or before the final count day or extend the test beyond the period
depending on the situation.
6. First and second counts are usually taken in case of Top of Paper (TP) and Between Paper (BP) media however a single final count is made in case of sand tests.
Procedure of germination test:
A. Between Paper (BP) Media (Roll Towel Test):
1. Soak the towel paper in water and after its wetting (10-15min) take out the paper from water and remove extra moisture by pressing the soaked paper by hand and holding it in plastic trays.
2. Placed this wet paper over the wax paper/butter paper and write the test number, crop, variety and date of test.
3. Arrange the sufficient number of seeds on the wet paper with proper spacing followed by placing of another layer of wet towel paper over the seed.
4. Roll firmly both wet and wax paper from left to right and secure with rubber band in the center.
5. Place this roll towel in slanting position in the seed germinator maintained at the desired temperature and relative humidity.
B. Top of Paper (TP) Media:
1. For the top of paper test quality paper such as 'sunlit' or 'whatman' filter paper should be used. Crepe kraft (towel) paper or blotter paper of unknown quality should not be used for top of paper tests.
2. This filter paper should be cut in the form of circles or rectangles according to the size and shape of petridish/container.
3. Put two layer of filter paper in the petridish/germination box and note down the date of test and crop name on filter paper with help of pencil. Put enough water to moisten the filter paper followed by holding the petridish /germination box in slanting position in order to drain out the extra moisture.
4. Space the counted seeds on the moist blotter/filter paper and cover the air tight lid.
5. Transfer the petridishes in the germinator maintained at the desired temperature.
Table 10. Germination test procedure of different forage crops (ISTA, 2015)
Crop Substrate Temp. First count Final count Treatmentso( C) (days) (days)
Berseem TP, BP 20 3 7 -
Maize TP, BP, S 25, 20-30 4 7 -
Cowpea BP, S 25, 20-30 5 8 -
Oat BP, S 20 5 10 Preheat o(30-35 C)
& Prechill
2928
Guar BP 20-30 5 14 -
Sorghum BP, TP 25, 20-30 4 10 Prechill
Guinea TP 20-30 10 28 KNO &3
Prechill
Senji TP, BP 20 3 14 -
Lucerne TP, BP 20 4 10 Prechill
Setaria TP, BP 20-30 4 10 -
Stylo TP, BP 20-35 4 10 Cut seed
d) Seedlings which are seriously decayed by fungi or bacteria, but only when it is clearly evident that the parent seed is not source of infection and it can be determined that all the essential structures were present.
B. Abnormal seedlings : Abnormal seedlings are those which do not show the capacity for continued development into normal plants when grown in good quality soil and under favorable conditions of water, temperature and light. Seedlings with the following defects shall be included as abnormal seedlings:
·Damaged seedlings : Seedlings with no cotyledons, seedlings with constrictions, splits, cracks or lesions which affect the conducting tissues of the epicotyl and hypocotyl. Seedlings without a primary root for those species where a primary root is an essential structure except for Pisum, Vicia, Lupinus, Vigna, Glycine, Arachis, Gossypium, Zea and all species of Cucurbitaceae where several vigorous secondary roots have developed to support the seedlings in the soil.
·Deformed seedlings : Seedlings with weak or unbalanced development of the essential structures such as spirally twisted or stunted plumules, hypocotyls or epicotyls swollen shoots and stunted roots, split plumules or coleoptiles without a green leaf, watery and glassy seedlings or without further development after emergence of the cotyledons.
·Decayed seedlings : Seedlings with any of the essential structures so diseased or decayed that normal development is prevented except when there is clear evidence to show that the cause of injection is not the seed itself.
C. Fresh ungerminated seeds : Seeds which are neither hard nor have germinated but remain firm and apparently viable at the end of the test period.
D. Hard seeds: Seeds which do not absorb moisture till the end of the test period and remain hard eg. seed of leguminaceae and malvaceae
E. Dead seeds : Seeds at the end of the test period are neither hard or nor fresh or have produced any part of a seedling. Generally dead seeds collapse and milky paste comes out when pressed at the end of the test.
Evaluation of germination test : In evaluating the germination test the seedlings and seeds are categorized into following different categories.
A. Normal seedlings: Seedlings which show the capacity for continued development into normal plants when grown in good quality soil and under favorable conditions of water supply temperature and light. The seedlings must possess followings essential structures to be considered as normal seedling.
·A well-developed root system including a primary root.
·A well-developed &intact hypocotyl without damage to the conducting tissues.
·An intact plumule with a well-developed green leaf or an intact epicotyl with a normal plumular bud.
·One cotyledon for seedlings of monocotyledons and two fordicotyledons.
·Seedlings with slight defects but showed good vigour and balanced development of the other essential structures also considered as normal seedling.
a) Seedlings of Pisum, Vicia, Phaseolus, Lupinus, Vigna, Glycine, Arachis, Gossypium, Zea and all species of Cucurbitaceae, with a damaged primary root but with several secondary roots of sufficient length and vigour to support the seedlings in soil.
b) Seedlings with superficial damage or decay to the hypocotyl, epicotyl or cotyledons, which is limited in area and does not affect the conducting tissues.
c) Seedlings of dicotyledons with only one cotyledon.
Figure 15. Germination test substrata (A) Filter paper (B) Roll towel paper (C) Sand tray
Figure 16. (A) TP test (B) BP test (C) Oat normal seedlings (D) oat abnormal seedlings
A
A
B
B
C
C
D
3130
Guar BP 20-30 5 14 -
Sorghum BP, TP 25, 20-30 4 10 Prechill
Guinea TP 20-30 10 28 KNO &3
Prechill
Senji TP, BP 20 3 14 -
Lucerne TP, BP 20 4 10 Prechill
Setaria TP, BP 20-30 4 10 -
Stylo TP, BP 20-35 4 10 Cut seed
d) Seedlings which are seriously decayed by fungi or bacteria, but only when it is clearly evident that the parent seed is not source of infection and it can be determined that all the essential structures were present.
B. Abnormal seedlings : Abnormal seedlings are those which do not show the capacity for continued development into normal plants when grown in good quality soil and under favorable conditions of water, temperature and light. Seedlings with the following defects shall be included as abnormal seedlings:
·Damaged seedlings : Seedlings with no cotyledons, seedlings with constrictions, splits, cracks or lesions which affect the conducting tissues of the epicotyl and hypocotyl. Seedlings without a primary root for those species where a primary root is an essential structure except for Pisum, Vicia, Lupinus, Vigna, Glycine, Arachis, Gossypium, Zea and all species of Cucurbitaceae where several vigorous secondary roots have developed to support the seedlings in the soil.
·Deformed seedlings : Seedlings with weak or unbalanced development of the essential structures such as spirally twisted or stunted plumules, hypocotyls or epicotyls swollen shoots and stunted roots, split plumules or coleoptiles without a green leaf, watery and glassy seedlings or without further development after emergence of the cotyledons.
·Decayed seedlings : Seedlings with any of the essential structures so diseased or decayed that normal development is prevented except when there is clear evidence to show that the cause of injection is not the seed itself.
C. Fresh ungerminated seeds : Seeds which are neither hard nor have germinated but remain firm and apparently viable at the end of the test period.
D. Hard seeds: Seeds which do not absorb moisture till the end of the test period and remain hard eg. seed of leguminaceae and malvaceae
E. Dead seeds : Seeds at the end of the test period are neither hard or nor fresh or have produced any part of a seedling. Generally dead seeds collapse and milky paste comes out when pressed at the end of the test.
Evaluation of germination test : In evaluating the germination test the seedlings and seeds are categorized into following different categories.
A. Normal seedlings: Seedlings which show the capacity for continued development into normal plants when grown in good quality soil and under favorable conditions of water supply temperature and light. The seedlings must possess followings essential structures to be considered as normal seedling.
·A well-developed root system including a primary root.
·A well-developed &intact hypocotyl without damage to the conducting tissues.
·An intact plumule with a well-developed green leaf or an intact epicotyl with a normal plumular bud.
·One cotyledon for seedlings of monocotyledons and two fordicotyledons.
·Seedlings with slight defects but showed good vigour and balanced development of the other essential structures also considered as normal seedling.
a) Seedlings of Pisum, Vicia, Phaseolus, Lupinus, Vigna, Glycine, Arachis, Gossypium, Zea and all species of Cucurbitaceae, with a damaged primary root but with several secondary roots of sufficient length and vigour to support the seedlings in soil.
b) Seedlings with superficial damage or decay to the hypocotyl, epicotyl or cotyledons, which is limited in area and does not affect the conducting tissues.
c) Seedlings of dicotyledons with only one cotyledon.
Figure 15. Germination test substrata (A) Filter paper (B) Roll towel paper (C) Sand tray
Figure 16. (A) TP test (B) BP test (C) Oat normal seedlings (D) oat abnormal seedlings
A
A
B
B
C
C
D
3130
Figure 17. (A) Oat between paper test (B) Berseem normal seedlings (C) Berseem abnormal seedlings
Calculation of result:After the completion of seed germination duration the results are expressed as percentage by
number.Number of seeds germinated
Germination % X 100Number of seeds planted
The average percentage is calculated to the nearest whole number. The total % of all the category
of seeds (normal, abnormal. dead hard, fresh ungerminated) should be 100.
Reporting of result:
The following items shall be entered in the appropriate space of the analysis certificate when reporting the result of a germination test:
·Kind &variety
·Date of testing
·Duration of test
·Percentage of normal seedlings, abnormal seedlings, hard seeds, fresh seeds and dead seeds. If the result for any of these categories is found to be nil, it shall be entered as 0 (zero).
Methods to improve germination/ Dormancy breaking treatments:
1. Hard seeds: For many species where hard seeds occurs some special treatments are essential
for the germination of seed. These treatments may be applied prior to the commencement of
the germination test or if it is suspected that the treatment may adversely affects the non-hard
seeds then these should be carried out on the hard seeds remaining after the prescribed test
period.
·Soaking: Seeds with hard seed coats may germinate more readily after soaking in water
for 24-48hours. The germination test is commenced immediately after soaking eg.
Acacia spp.
·Mechanical scarification : Careful piercing, chipping, filing or sand papering of the
seed coat maybe sufficient to break the coat imposed seed dormancy condition. Care
must be taken to scarify the seed coat at a suitable part in order to avoid damaging the
embryo. The best site for mechanical scarification is seed coat immediately above the
tips of the cotyledons.
·Acid scarification: Treating with concentrated sulphuric acid (H SO ) is effective 2 4
treatment for breaking the physical dormancy in some species viz.,Macroptilium and
Sesbania. The seeds are soaked into mild concentrated acid until its seed coat becomes
pitted. Digestion may be rapid or take more than one hour, but the seeds should be
examined every few minutes. After digestion, seeds must be thoroughly washed in
running water before the germination test is commenced.
2. Inhibitory Substances: Naturally occurring substances in the pericarp or seed coat, which
act as inhibitors for seed germination may be removed by washing the seeds in running water oat a temperature of 25 C before the germination test is made. After washing, the seeds should
be dried back to original moisture content at a maximum temperature of 25°C before
germination test. Germination of certain species like grasses are promoted by removing
outer structures such as involucre of bristles or lemma and palea.
3. Prechilling: Seeds which are having physiological dormancy are required pre-chilling
treatment to induce the germination. Replicates for germination are placed in contact with
the moist substratum and kept at a low temperature for an initial period before they are
removed to the recommended temperature prescribed for germination test. Generally seeds
are kept at a temperature between 5°C and 10°C for an initial period of 7 days whereas, tree
seeds are kept at a temperature between 3°C and 5°C for a period varying with the species
from 7 days to 12 months. In some cases, it may be necessary to extend the prechilling period
or to rechill. The prechilling period is not included in the germination test period but both the
duration and the temperature should be reported on the analysis card eg. Oat, sorghum,
guinea and lucerne.
4. Pre-drying: The replicates for germination should be heated at a temperature not exceeding o40 C with free air circulation for a period of 7 days before they are placed under the
prescribed germination conditions. In some cases it may be necessary to extend the pre-
drying period. Both the duration and the temperature should be reported on the Analysis
Certificate.
3332
A B
C
Figure 17. (A) Oat between paper test (B) Berseem normal seedlings (C) Berseem abnormal seedlings
Calculation of result:After the completion of seed germination duration the results are expressed as percentage by
number.Number of seeds germinated
Germination % X 100Number of seeds planted
The average percentage is calculated to the nearest whole number. The total % of all the category
of seeds (normal, abnormal. dead hard, fresh ungerminated) should be 100.
Reporting of result:
The following items shall be entered in the appropriate space of the analysis certificate when reporting the result of a germination test:
·Kind &variety
·Date of testing
·Duration of test
·Percentage of normal seedlings, abnormal seedlings, hard seeds, fresh seeds and dead seeds. If the result for any of these categories is found to be nil, it shall be entered as 0 (zero).
Methods to improve germination/ Dormancy breaking treatments:
1. Hard seeds: For many species where hard seeds occurs some special treatments are essential
for the germination of seed. These treatments may be applied prior to the commencement of
the germination test or if it is suspected that the treatment may adversely affects the non-hard
seeds then these should be carried out on the hard seeds remaining after the prescribed test
period.
·Soaking: Seeds with hard seed coats may germinate more readily after soaking in water
for 24-48hours. The germination test is commenced immediately after soaking eg.
Acacia spp.
·Mechanical scarification : Careful piercing, chipping, filing or sand papering of the
seed coat maybe sufficient to break the coat imposed seed dormancy condition. Care
must be taken to scarify the seed coat at a suitable part in order to avoid damaging the
embryo. The best site for mechanical scarification is seed coat immediately above the
tips of the cotyledons.
·Acid scarification: Treating with concentrated sulphuric acid (H SO ) is effective 2 4
treatment for breaking the physical dormancy in some species viz.,Macroptilium and
Sesbania. The seeds are soaked into mild concentrated acid until its seed coat becomes
pitted. Digestion may be rapid or take more than one hour, but the seeds should be
examined every few minutes. After digestion, seeds must be thoroughly washed in
running water before the germination test is commenced.
2. Inhibitory Substances: Naturally occurring substances in the pericarp or seed coat, which
act as inhibitors for seed germination may be removed by washing the seeds in running water oat a temperature of 25 C before the germination test is made. After washing, the seeds should
be dried back to original moisture content at a maximum temperature of 25°C before
germination test. Germination of certain species like grasses are promoted by removing
outer structures such as involucre of bristles or lemma and palea.
3. Prechilling: Seeds which are having physiological dormancy are required pre-chilling
treatment to induce the germination. Replicates for germination are placed in contact with
the moist substratum and kept at a low temperature for an initial period before they are
removed to the recommended temperature prescribed for germination test. Generally seeds
are kept at a temperature between 5°C and 10°C for an initial period of 7 days whereas, tree
seeds are kept at a temperature between 3°C and 5°C for a period varying with the species
from 7 days to 12 months. In some cases, it may be necessary to extend the prechilling period
or to rechill. The prechilling period is not included in the germination test period but both the
duration and the temperature should be reported on the analysis card eg. Oat, sorghum,
guinea and lucerne.
4. Pre-drying: The replicates for germination should be heated at a temperature not exceeding o40 C with free air circulation for a period of 7 days before they are placed under the
prescribed germination conditions. In some cases it may be necessary to extend the pre-
drying period. Both the duration and the temperature should be reported on the Analysis
Certificate.
3332
A B
C
5. Chemical Treatments:
Potassium nitrate (KNO ): The germination substratum may be moistened with a 0.2% 3
solution of KNO . The substratum is saturated at the beginning of the test but water is used 3
for moistening it thereafter. The use of this treatment should be noted on the analysis
certificate.
Gibberellic acid (GA ): Moisten the germination substratum with 50 ppm solution of GA 3 3
which can be prepared by dissolving 50 mg of GA in 1000 ml of water. Place the seed for 3
germination under prescribed temperature conditions.
The seed moisture content (mc) is the amount of water present inside the seed which is expressed as a percentage on wet weight basis. The seed moisture content is the most vital quality parameter and it is closely associated with different aspects of seed physiology viz., seed maturity, optimum harvesting time, mechanical damage, seed longevity and pest infestation.
Determination of seed moisture content:
The determination of seed moisture content is imperative since a small change in seed moisture content has a large effect on the storage life of the seeds. Therefore, it is important to know the moisture content in order to make a reasonably accurate prediction of the possible storage life of seed. The ideal method of seed moisture estimation should be reproducible, require less time, economically feasible and it should be adopted to all kind seed. However, it is impossible to combine all the separameters in any one single method but, in order to measure the moisture content of seeds, methods can be broadly grouped into two categories:
1. Direct method: Direct methods measure the seed moisture content directly by loss or gain in seed weight. The followings are the different direct methods used for moisture determination.
a. Desiccation method
b. Phosphorus pentaoxide method
c. Oven-drying method
d. Vacuum drying method
e. Distillation method
f. Karl Fisher's method
g. Direct weighing balance
h. Microwave oven method
2. Indirect method: These methods are convenient and quick in use but they measure only approximate value of seed moisture content. Indirect methods measure the seed moisture content based on some physical parameters like electrical conductivity or electrical resistance of the moisture present in the seed followed by these values are transformed into seed moisture content with the help of calibration charts for each species, against the standard air-oven method or basic reference method. This types of methods are frequently used in processing plants. The Karl-Fisher's method has been considered as the most accurate and the basic reference method for standardizing of other methods of seed moisture determination e.g. Digital moisture meter.
Oven drying method: The constant temperature oven drying method is the only practical method, approved by International Seed Testing Association (ISTA) and other organization to be used for routine seed moisture determination in a seed-testing laboratory. The constant temperature oven drying method is broadly grouped into two categories:
6. SEED MOISTURE TESTING
3534
5. Chemical Treatments:
Potassium nitrate (KNO ): The germination substratum may be moistened with a 0.2% 3
solution of KNO . The substratum is saturated at the beginning of the test but water is used 3
for moistening it thereafter. The use of this treatment should be noted on the analysis
certificate.
Gibberellic acid (GA ): Moisten the germination substratum with 50 ppm solution of GA 3 3
which can be prepared by dissolving 50 mg of GA in 1000 ml of water. Place the seed for 3
germination under prescribed temperature conditions.
The seed moisture content (mc) is the amount of water present inside the seed which is expressed as a percentage on wet weight basis. The seed moisture content is the most vital quality parameter and it is closely associated with different aspects of seed physiology viz., seed maturity, optimum harvesting time, mechanical damage, seed longevity and pest infestation.
Determination of seed moisture content:
The determination of seed moisture content is imperative since a small change in seed moisture content has a large effect on the storage life of the seeds. Therefore, it is important to know the moisture content in order to make a reasonably accurate prediction of the possible storage life of seed. The ideal method of seed moisture estimation should be reproducible, require less time, economically feasible and it should be adopted to all kind seed. However, it is impossible to combine all the separameters in any one single method but, in order to measure the moisture content of seeds, methods can be broadly grouped into two categories:
1. Direct method: Direct methods measure the seed moisture content directly by loss or gain in seed weight. The followings are the different direct methods used for moisture determination.
a. Desiccation method
b. Phosphorus pentaoxide method
c. Oven-drying method
d. Vacuum drying method
e. Distillation method
f. Karl Fisher's method
g. Direct weighing balance
h. Microwave oven method
2. Indirect method: These methods are convenient and quick in use but they measure only approximate value of seed moisture content. Indirect methods measure the seed moisture content based on some physical parameters like electrical conductivity or electrical resistance of the moisture present in the seed followed by these values are transformed into seed moisture content with the help of calibration charts for each species, against the standard air-oven method or basic reference method. This types of methods are frequently used in processing plants. The Karl-Fisher's method has been considered as the most accurate and the basic reference method for standardizing of other methods of seed moisture determination e.g. Digital moisture meter.
Oven drying method: The constant temperature oven drying method is the only practical method, approved by International Seed Testing Association (ISTA) and other organization to be used for routine seed moisture determination in a seed-testing laboratory. The constant temperature oven drying method is broadly grouped into two categories:
6. SEED MOISTURE TESTING
3534
5. After weighing, remove the cover or lid of the weighing bottles and place the weighing bottles inside an oven, which is already maintaining the desired temperature, for the recommended period.
1. Low constant temperature oven method: This method has been recommended for seed of the species which are rich in oil content or volatile substances. In this method, the pre-weighed moisture bottles along with seed material are placed in an oven maintaining a
otemperature of 103 C. Seeds are dried at this temperature for 17±1hr. The relative humidity of the ambient air in the laboratory must be less than 70 per cent when the moisture determination is carried out.
2. High constant temperature oven method: The procedure is the same as above except that othe oven is maintained at a temperature of 130-133 C. The sample is dried to a period of four
hours for Zea mays, two hours for other cereals and one hour for other species (Table). In this method, there is no special requirement pertaining to the relative humidity of the ambient air in the laboratory during moisture determination.
Essential equipments:
1. Constant temperature precision hot-air electric oven
2. Weighing bottles/ Moisture containers
3. Desiccator with silica gel
4. Analytical balance capable of weighing up to 1 mg
5. Seed grinder
6. Tong/Heat resistant gloves/A steel brusho
Seed drying period: The prescribed period of seed drying shall be 17±1 hrs at 103 C under low o
constant and 1to 4 hrs at 130-133 C under high constant temperatures. Maize seed should be dried for 4 hrs, cereals and other millets for 2 hrs and the remaining species for 1 hr. Seeds rich in oil content or with volatile substances should be dried for 17±1 hrs under low constant temperature. Seed drying period should be begins from the time when oven maintained the desired temperatures.
Sample size: The ISTA rules recommend that two replicates, each with 4 gm of seed should be used for determination of seed moisture content. In seed gene banks to avoid any depletion of seed resources sample weight may be modified to 0.2 to 0.5 gm per replicate, with precise weighing.
Seed moisture testing procedure:
1. Seed moisture determination should be carried out in duplicate on two independently drawn working samples.
2. Weight each bottle with its cover with an accuracy of 1 mg or 0.1 mg.
3. Grind the seed material, evenly by using any grinder that does not cause heating or loss of moisture content (if needed).
. Mix thoroughly the submitted sample and by using spoon transfer the small portions (4 to5 gm) of seed samples into weighing bottles which should be evenly distributed inside the bottom of containers.
Figure 18. Seed moisture testing equipments (A) Desiccator (B) Different moisture testing boxes (C) Seed grinder (D) Digital seed moisture meter (E) Hot air oven
A
B
C
D
E
3736
5. After weighing, remove the cover or lid of the weighing bottles and place the weighing bottles inside an oven, which is already maintaining the desired temperature, for the recommended period.
1. Low constant temperature oven method: This method has been recommended for seed of the species which are rich in oil content or volatile substances. In this method, the pre-weighed moisture bottles along with seed material are placed in an oven maintaining a
otemperature of 103 C. Seeds are dried at this temperature for 17±1hr. The relative humidity of the ambient air in the laboratory must be less than 70 per cent when the moisture determination is carried out.
2. High constant temperature oven method: The procedure is the same as above except that othe oven is maintained at a temperature of 130-133 C. The sample is dried to a period of four
hours for Zea mays, two hours for other cereals and one hour for other species (Table). In this method, there is no special requirement pertaining to the relative humidity of the ambient air in the laboratory during moisture determination.
Essential equipments:
1. Constant temperature precision hot-air electric oven
2. Weighing bottles/ Moisture containers
3. Desiccator with silica gel
4. Analytical balance capable of weighing up to 1 mg
5. Seed grinder
6. Tong/Heat resistant gloves/A steel brusho
Seed drying period: The prescribed period of seed drying shall be 17±1 hrs at 103 C under low o
constant and 1to 4 hrs at 130-133 C under high constant temperatures. Maize seed should be dried for 4 hrs, cereals and other millets for 2 hrs and the remaining species for 1 hr. Seeds rich in oil content or with volatile substances should be dried for 17±1 hrs under low constant temperature. Seed drying period should be begins from the time when oven maintained the desired temperatures.
Sample size: The ISTA rules recommend that two replicates, each with 4 gm of seed should be used for determination of seed moisture content. In seed gene banks to avoid any depletion of seed resources sample weight may be modified to 0.2 to 0.5 gm per replicate, with precise weighing.
Seed moisture testing procedure:
1. Seed moisture determination should be carried out in duplicate on two independently drawn working samples.
2. Weight each bottle with its cover with an accuracy of 1 mg or 0.1 mg.
3. Grind the seed material, evenly by using any grinder that does not cause heating or loss of moisture content (if needed).
. Mix thoroughly the submitted sample and by using spoon transfer the small portions (4 to5 gm) of seed samples into weighing bottles which should be evenly distributed inside the bottom of containers.
Figure 18. Seed moisture testing equipments (A) Desiccator (B) Different moisture testing boxes (C) Seed grinder (D) Digital seed moisture meter (E) Hot air oven
A
B
C
D
E
3736
6. Dactylis glomerata No High 1 -
7. Festuca spp. No High 1 -
8. Lathyrus spp. Coarse High 1 To 17% MC or less
9. Lolium spp. No High 2 -
10. Macroptilium atropurpureum Coarse High 1 To 17% MC or less
11. Medicago spp. No High 1 -
12. Melilotus spp. No High 1 -
13. Paspalum spp. No High 1 -
14. Setaria spp. No High 1 -
15. Trifolium spp. No High 1 -
16. Vigna spp. Coarse High 1 To 17% MC or less
17. Zea mays Fine High 4 To 17% MC or less
Table 11. Details of seed moisture determination for forage species (ISTA, 2015)
S.N.Crop/species Grinding / Method Drying at Pre drying cutting high temp. requirement
1. Avena spp. Coarse High 2 To 17% MC or less
2. Brachiaria spp. No High 1 -
3. Bromus spp. No High 1 -
4. Cenchrus spp. No High 1 -
5. Chloris gayana No High 1 -
6. At the end of seed drying period, weighing bottles should be closed with its lid and transfer the weighing bottles to the desiccators having silica gel(blue colour), to cool down for 40 to
3938
6. Dactylis glomerata No High 1 -
7. Festuca spp. No High 1 -
8. Lathyrus spp. Coarse High 1 To 17% MC or less
9. Lolium spp. No High 2 -
10. Macroptilium atropurpureum Coarse High 1 To 17% MC or less
11. Medicago spp. No High 1 -
12. Melilotus spp. No High 1 -
13. Paspalum spp. No High 1 -
14. Setaria spp. No High 1 -
15. Trifolium spp. No High 1 -
16. Vigna spp. Coarse High 1 To 17% MC or less
17. Zea mays Fine High 4 To 17% MC or less
Table 11. Details of seed moisture determination for forage species (ISTA, 2015)
S.N.Crop/species Grinding / Method Drying at Pre drying cutting high temp. requirement
1. Avena spp. Coarse High 2 To 17% MC or less
2. Brachiaria spp. No High 1 -
3. Bromus spp. No High 1 -
4. Cenchrus spp. No High 1 -
5. Chloris gayana No High 1 -
6. At the end of seed drying period, weighing bottles should be closed with its lid and transfer the weighing bottles to the desiccators having silica gel(blue colour), to cool down for 40 to
3938
Germination test is the standard test to know the potential of a given seed lot for emergence under
field conditions. It takes from days to weeks, and in some cases even months to complete. There
have been approaches since long for the search of biochemical methods for assessing viability of
seeds (planting value) without standard germination test. Over the years different tests have been
developed to assess the seed viability. First time Lakon in 1942has reported that tetrazolium salt
can be used to assess the viability of seeds which is commonly known as the T test. Z
Synonymously it is also termed as Topographic Tetrazolium Chloride Test (TTC) or Excised
Embryo Test (EET). The T test is a biochemical test which has been developed to furnish quick Z
estimates of seed germinability based on living tissue respiration.
Principles : The main principles is that living tissues are having a group of enzymes known as
dehydrogenases which can reduce the colorless solution of 2, 3, 5-triphenyl tetrazolium chloride
or bromide into a non-diffusable red colored compound known as formazon. These enzymes are
involved in H-transfer during respiratory activity of biological system and the reaction takes
place within the living cells. The formazon is non-diffusible chemical which clearly define the
topography of living (red) and non-living areas (white) within the seed. In addition to this stained
seeds may also developed varying proportions of necrotic tissues within the stained area. The
positions andsize of the necrotic areas also determine viability of seed.
Reagent : 1% solution of 2, 3, 5-triphenyl tetrazolium chloride within a pH range of 6.5-7.5 is
generally used for quick viability assessment.
Chemical Reaction : The colourless solution of 2, 3, 5-triphenyl tetrazolium chloride changed
into an insoluble red colored substance known as formazon in the presence of certain
dehydrogenase enzymes(released during respiration by living tissue). Since the tissues within a
seed could be at different states of viability so they would be stained differently. Moore (1973)
described the use of T more efficiently on the basis of the topographic pattern of the seed.Z
Methodology: The regular germination tests are easy to conduct and interpret but process and
interpretation of seed viability through Tz test are more difficult therefore, the following points to
be considered.
1. Working Sample: A set of 100 seeds should be tested in replicates of 50 each or less. For an
accurate assessment the test is conducted in 4 x 10 seeds. The seeds should be randomly
drawn from the pure seed component and counted in replicates before conditioning.
2. Pre-moistening: In order to ensure the contact of the tetrazolium solution with the embryo,
some conditioning and preparatory steps may be essential. These depend on the type of the
seed, its permeability and thickness of the seed coat and location of the embryo.
A. For faster conditioning, seeds should be place in water to allow complete hydration of all
the tissues without damages to cotyledons and embryo axes.
B. Some seeds can be directly placed in water, whereas, others must be moistened slowly.
For slow moistening seeds are conditioned either by placing the seed on top of paper or
in between the moist blotter paper. Slow moistening is generally practiced for large
seeded legumes or for the seed samples, which are dry, brittle or aged to avoid tissues
damage (due to rapid intake of water).
3. Preparation for Staining: In addition to moistening, most kinds of seed require some
preparatory steps before the staining to assure the adequate penetration of the staining
solution into the seed, accelerate the rate of staining and to facilitate the evaluation. Thus,
depending upon the kind of the seed, time available, degree of accuracy desired and the
experience of the analyst, one the following methods may be adopted:
·No moistening or preparation (small seeded legumes with soft coats)
·Slow moistening without any preparation (large seeded with soft coats)
·Piercing, puncturing or cutting of the seed coat (small seeded grasses)
·Cutting the seeds longitudinally through the midsection of the embryo and through part
of the endosperm, leaving the two halves attached at the base or slitting the seed
completely, keeping only half for the staining, discard the other
·Cutting the seeds longitudinally slightly off-center to avoid cutting into the embryo
·Removing the seed coat (with forceps/needle/razor blade etc.) with minimum injury to
the tissues. Sometimes, a thin membrane adheres to the cotyledons even after removal of
theseed coat, this be removed by a sliding motion, after an additional 30 min of soaking
(dicots with hard seed coats).
4. Staining: Seeds can be stained in watch glass, petri dishes or beakers and sufficient solution
is used to cover the seeds and to allow its absorption. As a thumb rulea concentrated (1.0%)
solution can be used for legumes and grasses that are not bisected through the embryo
whereas, a dilute (0.25% or 0.50%) solution should be used for grasses and cereals that are o
bisected through the embryo. In general, seeds are placed in the solution and held at 30 C for
7. SEED VIABILITY DETERMINATION
4140
Germination test is the standard test to know the potential of a given seed lot for emergence under
field conditions. It takes from days to weeks, and in some cases even months to complete. There
have been approaches since long for the search of biochemical methods for assessing viability of
seeds (planting value) without standard germination test. Over the years different tests have been
developed to assess the seed viability. First time Lakon in 1942has reported that tetrazolium salt
can be used to assess the viability of seeds which is commonly known as the T test. Z
Synonymously it is also termed as Topographic Tetrazolium Chloride Test (TTC) or Excised
Embryo Test (EET). The T test is a biochemical test which has been developed to furnish quick Z
estimates of seed germinability based on living tissue respiration.
Principles : The main principles is that living tissues are having a group of enzymes known as
dehydrogenases which can reduce the colorless solution of 2, 3, 5-triphenyl tetrazolium chloride
or bromide into a non-diffusable red colored compound known as formazon. These enzymes are
involved in H-transfer during respiratory activity of biological system and the reaction takes
place within the living cells. The formazon is non-diffusible chemical which clearly define the
topography of living (red) and non-living areas (white) within the seed. In addition to this stained
seeds may also developed varying proportions of necrotic tissues within the stained area. The
positions andsize of the necrotic areas also determine viability of seed.
Reagent : 1% solution of 2, 3, 5-triphenyl tetrazolium chloride within a pH range of 6.5-7.5 is
generally used for quick viability assessment.
Chemical Reaction : The colourless solution of 2, 3, 5-triphenyl tetrazolium chloride changed
into an insoluble red colored substance known as formazon in the presence of certain
dehydrogenase enzymes(released during respiration by living tissue). Since the tissues within a
seed could be at different states of viability so they would be stained differently. Moore (1973)
described the use of T more efficiently on the basis of the topographic pattern of the seed.Z
Methodology: The regular germination tests are easy to conduct and interpret but process and
interpretation of seed viability through Tz test are more difficult therefore, the following points to
be considered.
1. Working Sample: A set of 100 seeds should be tested in replicates of 50 each or less. For an
accurate assessment the test is conducted in 4 x 10 seeds. The seeds should be randomly
drawn from the pure seed component and counted in replicates before conditioning.
2. Pre-moistening: In order to ensure the contact of the tetrazolium solution with the embryo,
some conditioning and preparatory steps may be essential. These depend on the type of the
seed, its permeability and thickness of the seed coat and location of the embryo.
A. For faster conditioning, seeds should be place in water to allow complete hydration of all
the tissues without damages to cotyledons and embryo axes.
B. Some seeds can be directly placed in water, whereas, others must be moistened slowly.
For slow moistening seeds are conditioned either by placing the seed on top of paper or
in between the moist blotter paper. Slow moistening is generally practiced for large
seeded legumes or for the seed samples, which are dry, brittle or aged to avoid tissues
damage (due to rapid intake of water).
3. Preparation for Staining: In addition to moistening, most kinds of seed require some
preparatory steps before the staining to assure the adequate penetration of the staining
solution into the seed, accelerate the rate of staining and to facilitate the evaluation. Thus,
depending upon the kind of the seed, time available, degree of accuracy desired and the
experience of the analyst, one the following methods may be adopted:
·No moistening or preparation (small seeded legumes with soft coats)
·Slow moistening without any preparation (large seeded with soft coats)
·Piercing, puncturing or cutting of the seed coat (small seeded grasses)
·Cutting the seeds longitudinally through the midsection of the embryo and through part
of the endosperm, leaving the two halves attached at the base or slitting the seed
completely, keeping only half for the staining, discard the other
·Cutting the seeds longitudinally slightly off-center to avoid cutting into the embryo
·Removing the seed coat (with forceps/needle/razor blade etc.) with minimum injury to
the tissues. Sometimes, a thin membrane adheres to the cotyledons even after removal of
theseed coat, this be removed by a sliding motion, after an additional 30 min of soaking
(dicots with hard seed coats).
4. Staining: Seeds can be stained in watch glass, petri dishes or beakers and sufficient solution
is used to cover the seeds and to allow its absorption. As a thumb rulea concentrated (1.0%)
solution can be used for legumes and grasses that are not bisected through the embryo
whereas, a dilute (0.25% or 0.50%) solution should be used for grasses and cereals that are o
bisected through the embryo. In general, seeds are placed in the solution and held at 30 C for
7. SEED VIABILITY DETERMINATION
4140
complete coloration. After a period of staining (as per ISTA rules) seeds are rinsed 2-3 times
in water and then evaluated. During evaluation, the seeds should be left with little amount of
water to prevent these from drying. If seeds are not to be evaluated immediately, these seeds o
should be kept in the refrigerator (5 to 10 C) with little amount of water.
5. Interpretation of T Test: To interpret the staining pattern of seed correctly, as viable or Z
non-viable (dead), the seed analyst must understand correctly the following essential structures of the embryo and their role in the growth of seedling.
1. Dicotyledonous seed : Main root, cotyledons and plumule
2. Monocotyledonous seed: Seminal roots (secondary roots), joining region of scutellum, behind the embryonic axis against the endosperm (acts as single cotyledon to bring the starchy food reserves to the young seedling) and plumule.
Advantages and disadvantages of T Test: Z
1. Advantage: Quick and fairly accurate, through which we can also determine the viability of a dormant seed lot in a short time. Seeds are not damages (in dicots) and can be germinated.
2. Disadvantages
·Distinction between normal and abnormal seedlings difficult.
·Cannot differentiate between dormant and non-dormant seeds.
·Correct evaluation is possible only after prolonged experience.
·Microorganisms harmful for seedling emergence remain undetected.
Tetrazolium Test for Vigour Assessment : Kittock and Law (1968) described a method for estimating the seed vigour on the basis of colorimetric determination of formazon and colour intensity of stained embryo or seed. The method is described below:
·Prepare tetrazolium solution as described earlier and put 100 seeds in a Petri dish lined o
with moist filter papers and keep at 20±1 C for overnight.
·Excise the embryonic axes and add 1 ml of tetrazolium solution to 25 axes in three replication.
o·Incubate in dark at 30 C for 4 hrs. The incubation time varies with the crop species. However, for maintaining uniformity, duration should be kept constant for a crop under study.
·Drain out the excess solution and washing in distilled water followed by soaking of embryonic axes in 10 ml of methyl cellulose for 4 to 6 hrs with occasional stirring until the extraction of red colored formazon i.e., the axes become colourless.
·Decant the extract and read the intensity of the colour at 480 nm in a spectrophotometer by using methyl cellulose as the blank.
·The higher the intensity of formazon or colour development are classified as more vigorous seed.
Figure 19. (A) Tetrazolium salt (B) Brachiaria viable seed (C) Maize Viable seed
Figure 20. (A) Viable seed of Red clover (B) non-viable seed of Red clover
A B C
A B
Table 12. Standard procedure for tetrazolium testing of forage and fodder crops (ISTA, 2015)
nS.N. Species Premoistening Preparation Sol Staining Preparation before % period beforestaining (h) evaluation
1. Avena spp. Remove glumes Cut seed 1 18 Extract embryo and and BP/18; W/18 transversely observe embryo
near embryo surface includingback of scutellum
2. Brachiaria spp. BP/18; W/6 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
3. Bromus spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
4. Chloris gayana Remove glumes Cut transversely 1 6 Observe surface of and BP/16 at near embryo embryo and10°C; W/3 scutellum
5. Dactylis spp. BP/18; W/2 Remove glumes, 1 18 Observe externalcut transversely embryo surfacenear embryo
4342
complete coloration. After a period of staining (as per ISTA rules) seeds are rinsed 2-3 times
in water and then evaluated. During evaluation, the seeds should be left with little amount of
water to prevent these from drying. If seeds are not to be evaluated immediately, these seeds o
should be kept in the refrigerator (5 to 10 C) with little amount of water.
5. Interpretation of T Test: To interpret the staining pattern of seed correctly, as viable or Z
non-viable (dead), the seed analyst must understand correctly the following essential structures of the embryo and their role in the growth of seedling.
1. Dicotyledonous seed : Main root, cotyledons and plumule
2. Monocotyledonous seed: Seminal roots (secondary roots), joining region of scutellum, behind the embryonic axis against the endosperm (acts as single cotyledon to bring the starchy food reserves to the young seedling) and plumule.
Advantages and disadvantages of T Test: Z
1. Advantage: Quick and fairly accurate, through which we can also determine the viability of a dormant seed lot in a short time. Seeds are not damages (in dicots) and can be germinated.
2. Disadvantages
·Distinction between normal and abnormal seedlings difficult.
·Cannot differentiate between dormant and non-dormant seeds.
·Correct evaluation is possible only after prolonged experience.
·Microorganisms harmful for seedling emergence remain undetected.
Tetrazolium Test for Vigour Assessment : Kittock and Law (1968) described a method for estimating the seed vigour on the basis of colorimetric determination of formazon and colour intensity of stained embryo or seed. The method is described below:
·Prepare tetrazolium solution as described earlier and put 100 seeds in a Petri dish lined o
with moist filter papers and keep at 20±1 C for overnight.
·Excise the embryonic axes and add 1 ml of tetrazolium solution to 25 axes in three replication.
o·Incubate in dark at 30 C for 4 hrs. The incubation time varies with the crop species. However, for maintaining uniformity, duration should be kept constant for a crop under study.
·Drain out the excess solution and washing in distilled water followed by soaking of embryonic axes in 10 ml of methyl cellulose for 4 to 6 hrs with occasional stirring until the extraction of red colored formazon i.e., the axes become colourless.
·Decant the extract and read the intensity of the colour at 480 nm in a spectrophotometer by using methyl cellulose as the blank.
·The higher the intensity of formazon or colour development are classified as more vigorous seed.
Figure 19. (A) Tetrazolium salt (B) Brachiaria viable seed (C) Maize Viable seed
Figure 20. (A) Viable seed of Red clover (B) non-viable seed of Red clover
A B C
A B
Table 12. Standard procedure for tetrazolium testing of forage and fodder crops (ISTA, 2015)
nS.N. Species Premoistening Preparation Sol Staining Preparation before % period beforestaining (h) evaluation
1. Avena spp. Remove glumes Cut seed 1 18 Extract embryo and and BP/18; W/18 transversely observe embryo
near embryo surface includingback of scutellum
2. Brachiaria spp. BP/18; W/6 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
3. Bromus spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
4. Chloris gayana Remove glumes Cut transversely 1 6 Observe surface of and BP/16 at near embryo embryo and10°C; W/3 scutellum
5. Dactylis spp. BP/18; W/2 Remove glumes, 1 18 Observe externalcut transversely embryo surfacenear embryo
4342
6. Festuca spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
7. Lolium spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
8. Medicago spp. W/18 Leave seed 1 18 Remove seed coatintact to expose embryo
9. Melilotus spp. W/18 Leave seed 1 18 Remove seed coatintact to expose embryo
10. Panicum spp. BP/18; W/6 Remove glumes, 1 18 Expose embryo and cut transversely observe external near embryo surface
11. Poa spp. BP/16; W/2 Pierce near 1 18 Remove lemma to embryo expose embryo
12. Setaria spp. Remove glumes Cut transversely 1 16 Observe externaland W at 7 °C/5 near embryo embryo and
longitudinally cutembryo.
13. Sorghum spp. W at 7 °C/18 Cut longitudinally 1 3 Observe cut surfacethrough embryo and ¼ of endosperm
14. Trifolium W/18 Leave seed intact 1 18 Remove seed coatto expose embryo
15. Zea mays W/18 Cut longitudinally 1 2 Observe cut through embryo surfaces*and ¾ of endosperm
BP- Between paper; W- Water
Seed vigour is an important quality parameter which needs to be assessed to supplement germination and viability tests to gain insight into the performance of a seed lot in the field or in storage. Seed vigour is still a concept rather thana specific property of a seed or seed lot.
Definition: Seed vigour is the sum of those properties which determine the potential level of activity and performance of the seed or seed lot during germination and seedling emergence (ISTA, 2015). Virtually all vigor tests can be classified into one of the following categories:
A. Seedling growth and evaluation tests
B. Stress tests
C. Biochemical tests
A. Seedling growth and evaluation tests : Growth tests are based on the principle that vigorous seeds grow at a faster rate than poor vigour seeds even under favorable environments. Vigorous seeds rapidly germinate, metabolize and establish in the field therefore, any method which used to determine the rapidity of growth of the seedling will give an indication of seed vigour level. Under growth test following different procedures are generally used for estimation of seed vigour:
1. First count : The test is done along with the regular germination test. The number of normal seedlings, germinated on the first count day, as specified in the germination test for each species, are counted. The number of normal seedlings gives an idea of the level of seed vigour in the sample. Higher the number of normal seedlings greater is the seed vigour.
2. Seedling length : For growth test seedlings are grown in between rolled towel paper in such a way that the micropyle are oriented towards bottom to avoid root twisting. The rolled towel papers are kept in the germinator maintained at a temperature recommended for crop in reference. After a specified period of time (5-10 days) towel papers are removed and ten seedlings are selected, their length is measured and mean seedling length is calculated. Seed lots producing the taller seedlings are considered more vigorous than the seed lots producing shorter seedlings. For dry weight determination, the seedlings are removed and dried in an air oven at 100°C temperature for 24 hours. The seedling dry weight provides additional information for assessing seed vigour.
3. Mean emergence time (MET) : It is reciprocal of the rate of germination and shown to be highly indicative of emergence performance in seed lots. Lower vigour seed lot will take more MET compared to higher vigour lot. The MET is calculated according to following equation of Ellis and Roberts, 1981.
Where n is the number of seeds newly germinated on day D, (2 mm radicle emergence) and D is the number of days calculated from the beginning of emergence.
8. SEED VIGOUR TESTING
4544
6. Festuca spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
7. Lolium spp. BP/16; W/3 Remove glumes, 1 18 Observe external cut transversely embryo surfacenear embryo
8. Medicago spp. W/18 Leave seed 1 18 Remove seed coatintact to expose embryo
9. Melilotus spp. W/18 Leave seed 1 18 Remove seed coatintact to expose embryo
10. Panicum spp. BP/18; W/6 Remove glumes, 1 18 Expose embryo and cut transversely observe external near embryo surface
11. Poa spp. BP/16; W/2 Pierce near 1 18 Remove lemma to embryo expose embryo
12. Setaria spp. Remove glumes Cut transversely 1 16 Observe externaland W at 7 °C/5 near embryo embryo and
longitudinally cutembryo.
13. Sorghum spp. W at 7 °C/18 Cut longitudinally 1 3 Observe cut surfacethrough embryo and ¼ of endosperm
14. Trifolium W/18 Leave seed intact 1 18 Remove seed coatto expose embryo
15. Zea mays W/18 Cut longitudinally 1 2 Observe cut through embryo surfaces*and ¾ of endosperm
BP- Between paper; W- Water
Seed vigour is an important quality parameter which needs to be assessed to supplement germination and viability tests to gain insight into the performance of a seed lot in the field or in storage. Seed vigour is still a concept rather thana specific property of a seed or seed lot.
Definition: Seed vigour is the sum of those properties which determine the potential level of activity and performance of the seed or seed lot during germination and seedling emergence (ISTA, 2015). Virtually all vigor tests can be classified into one of the following categories:
A. Seedling growth and evaluation tests
B. Stress tests
C. Biochemical tests
A. Seedling growth and evaluation tests : Growth tests are based on the principle that vigorous seeds grow at a faster rate than poor vigour seeds even under favorable environments. Vigorous seeds rapidly germinate, metabolize and establish in the field therefore, any method which used to determine the rapidity of growth of the seedling will give an indication of seed vigour level. Under growth test following different procedures are generally used for estimation of seed vigour:
1. First count : The test is done along with the regular germination test. The number of normal seedlings, germinated on the first count day, as specified in the germination test for each species, are counted. The number of normal seedlings gives an idea of the level of seed vigour in the sample. Higher the number of normal seedlings greater is the seed vigour.
2. Seedling length : For growth test seedlings are grown in between rolled towel paper in such a way that the micropyle are oriented towards bottom to avoid root twisting. The rolled towel papers are kept in the germinator maintained at a temperature recommended for crop in reference. After a specified period of time (5-10 days) towel papers are removed and ten seedlings are selected, their length is measured and mean seedling length is calculated. Seed lots producing the taller seedlings are considered more vigorous than the seed lots producing shorter seedlings. For dry weight determination, the seedlings are removed and dried in an air oven at 100°C temperature for 24 hours. The seedling dry weight provides additional information for assessing seed vigour.
3. Mean emergence time (MET) : It is reciprocal of the rate of germination and shown to be highly indicative of emergence performance in seed lots. Lower vigour seed lot will take more MET compared to higher vigour lot. The MET is calculated according to following equation of Ellis and Roberts, 1981.
Where n is the number of seeds newly germinated on day D, (2 mm radicle emergence) and D is the number of days calculated from the beginning of emergence.
8. SEED VIGOUR TESTING
4544
4. Speed of germination (SG): Speed of germination is used to calculate by using following formula, of Association of Official Seed Analysts (AOSA, 1983).
No. of emerged seeds No. of seeds emergedSG = + - - - - + Day of first count Days of final count
5. Seed Vigour Index: According to Abdul-Baki and Anderson (1973) seed vigour index are generally calculated by following two formulas;
·Seed vigour index-I=Germination % ×Seedling length (cm)
·Seed vigour index-II=Germination % ×Seedling dry weight (mg)
6. Radicle emergence test: The radicle emergence test for Zea mays is an ISTA validated
vigour test. In this test single count of radicle emergence is calculated from routine
germination test and mean just germination time (MJGT) is calculated. Higher the MJGT
lower the seed vigour and vice-versa.
B. Stress Test: A good correlation between the results of a standard germination test and field
emergence usually exists when field conditions are favorable. Under unfavorable conditions
this relationship is not found. Under unfavorable conditions high vigor seeds have a greater
potential for emergence. Consequently a number of vigor tests, which assess performance
under stress conditions, have been developed.
1. Accelerated ageing test (AAT): This test was originally developed to determine the
storage potential of seeds but studies have shown that results are correlated with
emergence of cotton, peas, beans and soybeans. It is recommended by the ISTA Seed
Vigour Testing Committee as a vigor test for soybeans. In this test seeds are exposed to oan elevated temperature (40-45 C) and high relative humidity (>90%) for 72 hours,
depending on the species. Under these conditions seed deterioration is accelerated, with
the least damage in high vigor seeds. After this treatment a standard germination test is
conducted and the results compared with those from an untreated control.
Apparatus and equipments: Accelerated aging chamber, equipment for germination
test, seed samples, tight jar, muslin cloth and wire mesh. The following step should be
followed for accelerated ageing test.
·Prepare a desiccator and filled the lower part of desiccator with water.
·One hundred seeds each in four replications are tied in a fine muslin cloth and placed
in desiccator on a wire mesh. Care should be taken to avoid a direct contact between
water and the seed.
·The desiccator is covered with the lid and sealed with parafin wax to make it air tight.
·The desiccator is then placed in the accelerated aging chamber maintained at 45.2°C
temperature and >90% RH for 3-5 days.
·The desiccator is removed after this period and the seeds are cooled in another
desiccator. The seeds are then tested for normal germination test specific to different
crops. After the AAT germination % of artificial ageing gives the level of seed vigour
and higher the germination % greater is the vigour of the seed lot.
2. Cold Test: The cold test has been developed in USA to evaluate the seed vigour of maize (corn). In USA when the corn is planted in late spring, the soil is humid and cold. The weak seeds do not germinate and establish. Therefore. To simulate the actual field conditions witnessed at the time of corn planting, cold test has been developed. The main aim of test is to differentiate between weak and vigorous seed lots by subjecting them to low temperature prior to germination at optimum temperature. The following steps are taken to conduct this test.
·After grinding and properly sieving the soil is filled into tray upto 2 cm depth.
·Fifty seeds are placed over the sand and covered with another 2 cm thick layer of soil.
·The soil is compacted and enough water is added to make the soil about 70% of its owater holding capacity. The temperature of the water should be 10 C.
·After watering the tray are covered with polythene bags and placed in the o
refrigerator maintained at 10 Ctemperature for one week.
·After one week the trays are removed and placed in the germinator maintained at 25°C temperature.
·Germination percentage is computed by counting the number of normal seedlings as in germination test. Higher the germination parentage greater is the seed vigour.
3. Paper Piercing Test: The principle of paper piercing test is similar to that of brick gravel test. High vigour seed lots are expected to produce strong seedlings which can pierce a particular type of paper while seedlings of poor vigour lots may not be able to pierce the paper. Therefore, the seedlings which emerge by piercing the paper are more vigorous than those which are not able to emerge through the paper.
Apparatus and equipment: All the material required for conducting germination test in sand boxes or trays plus the special paper which should have the following characteristics:
2@Basic weight =90 g/m
@Thickness =0.4 mm
@Bulk =42
@Dry bursting strength =0.3 kg/cm
@Breaking length =1000-5000mm
@Filtering speed =500 ml/minute
@Wet bursting strength =150 mm
@Ash content =0.1%
4746
4. Speed of germination (SG): Speed of germination is used to calculate by using following formula, of Association of Official Seed Analysts (AOSA, 1983).
No. of emerged seeds No. of seeds emergedSG = + - - - - + Day of first count Days of final count
5. Seed Vigour Index: According to Abdul-Baki and Anderson (1973) seed vigour index are generally calculated by following two formulas;
·Seed vigour index-I=Germination % ×Seedling length (cm)
·Seed vigour index-II=Germination % ×Seedling dry weight (mg)
6. Radicle emergence test: The radicle emergence test for Zea mays is an ISTA validated
vigour test. In this test single count of radicle emergence is calculated from routine
germination test and mean just germination time (MJGT) is calculated. Higher the MJGT
lower the seed vigour and vice-versa.
B. Stress Test: A good correlation between the results of a standard germination test and field
emergence usually exists when field conditions are favorable. Under unfavorable conditions
this relationship is not found. Under unfavorable conditions high vigor seeds have a greater
potential for emergence. Consequently a number of vigor tests, which assess performance
under stress conditions, have been developed.
1. Accelerated ageing test (AAT): This test was originally developed to determine the
storage potential of seeds but studies have shown that results are correlated with
emergence of cotton, peas, beans and soybeans. It is recommended by the ISTA Seed
Vigour Testing Committee as a vigor test for soybeans. In this test seeds are exposed to oan elevated temperature (40-45 C) and high relative humidity (>90%) for 72 hours,
depending on the species. Under these conditions seed deterioration is accelerated, with
the least damage in high vigor seeds. After this treatment a standard germination test is
conducted and the results compared with those from an untreated control.
Apparatus and equipments: Accelerated aging chamber, equipment for germination
test, seed samples, tight jar, muslin cloth and wire mesh. The following step should be
followed for accelerated ageing test.
·Prepare a desiccator and filled the lower part of desiccator with water.
·One hundred seeds each in four replications are tied in a fine muslin cloth and placed
in desiccator on a wire mesh. Care should be taken to avoid a direct contact between
water and the seed.
·The desiccator is covered with the lid and sealed with parafin wax to make it air tight.
·The desiccator is then placed in the accelerated aging chamber maintained at 45.2°C
temperature and >90% RH for 3-5 days.
·The desiccator is removed after this period and the seeds are cooled in another
desiccator. The seeds are then tested for normal germination test specific to different
crops. After the AAT germination % of artificial ageing gives the level of seed vigour
and higher the germination % greater is the vigour of the seed lot.
2. Cold Test: The cold test has been developed in USA to evaluate the seed vigour of maize (corn). In USA when the corn is planted in late spring, the soil is humid and cold. The weak seeds do not germinate and establish. Therefore. To simulate the actual field conditions witnessed at the time of corn planting, cold test has been developed. The main aim of test is to differentiate between weak and vigorous seed lots by subjecting them to low temperature prior to germination at optimum temperature. The following steps are taken to conduct this test.
·After grinding and properly sieving the soil is filled into tray upto 2 cm depth.
·Fifty seeds are placed over the sand and covered with another 2 cm thick layer of soil.
·The soil is compacted and enough water is added to make the soil about 70% of its owater holding capacity. The temperature of the water should be 10 C.
·After watering the tray are covered with polythene bags and placed in the o
refrigerator maintained at 10 Ctemperature for one week.
·After one week the trays are removed and placed in the germinator maintained at 25°C temperature.
·Germination percentage is computed by counting the number of normal seedlings as in germination test. Higher the germination parentage greater is the seed vigour.
3. Paper Piercing Test: The principle of paper piercing test is similar to that of brick gravel test. High vigour seed lots are expected to produce strong seedlings which can pierce a particular type of paper while seedlings of poor vigour lots may not be able to pierce the paper. Therefore, the seedlings which emerge by piercing the paper are more vigorous than those which are not able to emerge through the paper.
Apparatus and equipment: All the material required for conducting germination test in sand boxes or trays plus the special paper which should have the following characteristics:
2@Basic weight =90 g/m
@Thickness =0.4 mm
@Bulk =42
@Dry bursting strength =0.3 kg/cm
@Breaking length =1000-5000mm
@Filtering speed =500 ml/minute
@Wet bursting strength =150 mm
@Ash content =0.1%
4746
Procedure :
·The cereal seeds are placed on 1.5 cm moist sand in a tray or sand box.
·The seeds are covered with specially selected dry filter paper which is thencovered with
2 cm of moist sand.
·After this the sand boxes/trays are kept in a germinator maintained at 20°C temperature
for 8 days.
·After 8 days sand boxes/trays are taken out and seedlings emerging above the paper are
counted. A seed lot having maximum number of seedlings coming out of paper is
considered to be most vigorous. The test is highly dependent on the quality of paper and
should be used when such papers are available.
1. Hiltner Test (Brick gravel test) : The test was developed by Hiltner in Germany in 1917. He
observed that the seeds of cereal crops affected by fusarium disease were able to germinate in
regular test but were not able to emerge from brick gravels of 2-3 mm size. Later on Roberts
(1972) observed that healthy seeds were able to emerge from the brick gravel. The principle
is that the weak seedlings are not able to generate enough force to overcome the pressure of
brick gravels, so this method can be used to differentiate vigour levels in cereal seeds. Perry
(1984b) found this method reproducible and associated with field emergence in case of
wheat.
Apparatus and equipment : Germination box, aluminium tray, sand, sandmarker brick
gravel of 2-3 mm size, germinator, seed sample.
Procedure:
·The sand is sieved, moistured and filled in the germination box leaving about 3 cm
empty at the top followed by sowing of 100 seeds are placed in each box.
·After this 2-2.5 cm of porous brick gravel is spread over the seeds and box is kept in the
germinator at appropriate temperature.
·After the period required for germination, the box is removed and the seedlings which
have emerged through the brick gravel layer are counted.
·The percentage of emerged seedlings are used to compare seed vigour of different lots.
The test should be repeated 3-4 times to get authentic value.
2. Controlled deterioration test : As the name suggested the test involves the deterioration of
samples of seeds from seed lots in a precise and controlled manner at an elevated moisture o
content (depending on the species, often 20%) and temperature (45 C) for a defined duration;
for convincing 24h is preferred.
·Weigh the four replicates of 100 seeds and determine the initial moisture content of seed.
·Place the seed sample on moist germination paper and allow to imbibe the water.
Imbibition of water should be taken on filter paper due to consistent and reproducible
result. Because precision is needed in the adjustment of the MC, since a 1% difference in
MC has a clear and striking effect on the final germination. o o·When desired MC has been reached, seal the seeds in foil packet and hold at 7 ±2 C
overnight. o
·Place the seed packets into water bath at 45 C for 24 hrs. After incubation period
removes the seeds from water bath and cool under running water.
·Setup a germination test for each lot (100 seeds from each replicate) and assess the total
germination after appropriate time.
C. Biochemical tests:
1. Electrical conductivity test: The electrical conductivity (EC) was recommended by
ISTA for testing vigour for garden peas (Pisum sativum). In this test sample of 50 seeds
are soaked in deionized water and the EC of the soak water is measured after 24 hours.
The technology of seed vigour testing has not been perfected so far, so much so that there
is not a single universally accepted seed vigour test method. Research is needed to
further refine the current seed vigour test methods and to develop new methods which
are more related to field/storage conditions.
Figure 21. (A) Electrical conductivity meter (B) Graph depict that during the seed deterioration vigour declined preceding the seed germination(Julio, 2015)
4948
Procedure :
·The cereal seeds are placed on 1.5 cm moist sand in a tray or sand box.
·The seeds are covered with specially selected dry filter paper which is thencovered with
2 cm of moist sand.
·After this the sand boxes/trays are kept in a germinator maintained at 20°C temperature
for 8 days.
·After 8 days sand boxes/trays are taken out and seedlings emerging above the paper are
counted. A seed lot having maximum number of seedlings coming out of paper is
considered to be most vigorous. The test is highly dependent on the quality of paper and
should be used when such papers are available.
1. Hiltner Test (Brick gravel test) : The test was developed by Hiltner in Germany in 1917. He
observed that the seeds of cereal crops affected by fusarium disease were able to germinate in
regular test but were not able to emerge from brick gravels of 2-3 mm size. Later on Roberts
(1972) observed that healthy seeds were able to emerge from the brick gravel. The principle
is that the weak seedlings are not able to generate enough force to overcome the pressure of
brick gravels, so this method can be used to differentiate vigour levels in cereal seeds. Perry
(1984b) found this method reproducible and associated with field emergence in case of
wheat.
Apparatus and equipment : Germination box, aluminium tray, sand, sandmarker brick
gravel of 2-3 mm size, germinator, seed sample.
Procedure:
·The sand is sieved, moistured and filled in the germination box leaving about 3 cm
empty at the top followed by sowing of 100 seeds are placed in each box.
·After this 2-2.5 cm of porous brick gravel is spread over the seeds and box is kept in the
germinator at appropriate temperature.
·After the period required for germination, the box is removed and the seedlings which
have emerged through the brick gravel layer are counted.
·The percentage of emerged seedlings are used to compare seed vigour of different lots.
The test should be repeated 3-4 times to get authentic value.
2. Controlled deterioration test : As the name suggested the test involves the deterioration of
samples of seeds from seed lots in a precise and controlled manner at an elevated moisture o
content (depending on the species, often 20%) and temperature (45 C) for a defined duration;
for convincing 24h is preferred.
·Weigh the four replicates of 100 seeds and determine the initial moisture content of seed.
·Place the seed sample on moist germination paper and allow to imbibe the water.
Imbibition of water should be taken on filter paper due to consistent and reproducible
result. Because precision is needed in the adjustment of the MC, since a 1% difference in
MC has a clear and striking effect on the final germination. o o·When desired MC has been reached, seal the seeds in foil packet and hold at 7 ±2 C
overnight. o
·Place the seed packets into water bath at 45 C for 24 hrs. After incubation period
removes the seeds from water bath and cool under running water.
·Setup a germination test for each lot (100 seeds from each replicate) and assess the total
germination after appropriate time.
C. Biochemical tests:
1. Electrical conductivity test: The electrical conductivity (EC) was recommended by
ISTA for testing vigour for garden peas (Pisum sativum). In this test sample of 50 seeds
are soaked in deionized water and the EC of the soak water is measured after 24 hours.
The technology of seed vigour testing has not been perfected so far, so much so that there
is not a single universally accepted seed vigour test method. Research is needed to
further refine the current seed vigour test methods and to develop new methods which
are more related to field/storage conditions.
Figure 21. (A) Electrical conductivity meter (B) Graph depict that during the seed deterioration vigour declined preceding the seed germination(Julio, 2015)
4948
Seed enhancements are the post–harvest treatments that improve germination and seedling growth to facilitate the delivery of seeds and other materials required at the time of sowing. Seed enhancements are the post–harvest treatments that improve germination and seedling growth to facilitate the delivery of seeds and other materials required at the time of sowing. Seed coating is one of the most useful areas of seed enhancements and an economical approach for improving the seed performance. Seed placement and performance can be greatly enhanced by altering the shape of seeds or placing the chemicals on seed coat which regulate and improve germination. A wide range of materials are used to coat seeds as individuals in discrete units as in case of pellets or spaced in strips or sheets. However, seed treated in traditional ways with pesticides alone should be tested according to the methods prescribed for normal uncoated seeds. The followings are different types of seed enhancement types:
1. Seed coating: Seed coating is the formation of thin layer of polymer containing different additives. It maintain the shape and size of seed and doesn't increase the weight of seed.
2. Seed pellets: These are more or less spherical units developed for precision sowing, usually incorporating a single seed with the size and shape of the seed no longer readily evident. The pellet, in addition to the pelleting material, may contain pesticides, dyes or other additives.
3. Encrusted seed: Units more or less retaining the shape of the seed with the size and weight changed to a greater or less extent. The encrusting material may contain pesticides, fungicides, dyes or other additives.
4. Seed granules: Units more or less cylindrical, including types with more than one seed joined together. The granule, in addition to the granulating material, may contain pesticides, dyes or other additives.
5. Seed tapes: Narrow bands of material, such as paper or other degradable material, with seeds spaced randomly, in groups or in a single row.
6. Seed mats: Broad sheets of material, such as paper or other degradable material, with seeds placed in rows, groups or at random throughout the sheets.
The followings four different tests are usually performed on coated and pelleted seeds
A. Physical purity analysis
B. Number of other seeds
C. Germination test
A. Physical purity analysis :
1. Size of lot : If the lot is reasonably homogenous, the maximum weight of lot may be as great as the maximum weight of lot for which sampling procedures are prescribed as in case of uncoated seeds subject to the tolerance of 5% and subject to the seed number limitation
prescribed below: The maximum number of seeds that a lot of seed pellets, encrusted seeds, seed granules, seed tapes or seed mats may contain is 1,000,000,000 (10,000 units of 100,000) except that the weight of lot, including the coating material may not exceed 42,000 kg (40,000 kg plus 5%). When lot size is expressed in units the total weight of the lot must be given on the certificate.
2. Sample size: The submitted sample should be according to Table. 1. As submitted samples of coated seeds normally contain fewer seeds than corresponding samples of uncoated seeds therefore, special care is necessary in drawing the sample to ensure that it is representative of the lot. Precautions are necessary to avoid damage or change in the pellets or seed tape during drawing, handling and transport, and samples must be submitted in suitable containers. To obtain working sample from submitted sample seed divider should be used but the distance of fall must never exceed that 250 mm. For seed tapes take pieces of tape at random, to provide sufficient seeds for the test.
3. Purity analysis: Purity analysis is not obligatory test but if it is requested by the applicant, then a purity analysis on depelleted seeds or seeds removed from the tape may be carried out in accordance with method prescribed for the uncoated seeds. The working sample of pellets is separated into the following three components: pure pellets, unpelleted seed and inert matter, and the percentage of each part is determined by weight. The working sample of not less than
9. TESTING OF COATED AND PELLETED SEEDS
Figure 22. Coated and pelleted seed of cowpea and Dinanath grass
5150
Seed enhancements are the post–harvest treatments that improve germination and seedling growth to facilitate the delivery of seeds and other materials required at the time of sowing. Seed enhancements are the post–harvest treatments that improve germination and seedling growth to facilitate the delivery of seeds and other materials required at the time of sowing. Seed coating is one of the most useful areas of seed enhancements and an economical approach for improving the seed performance. Seed placement and performance can be greatly enhanced by altering the shape of seeds or placing the chemicals on seed coat which regulate and improve germination. A wide range of materials are used to coat seeds as individuals in discrete units as in case of pellets or spaced in strips or sheets. However, seed treated in traditional ways with pesticides alone should be tested according to the methods prescribed for normal uncoated seeds. The followings are different types of seed enhancement types:
1. Seed coating: Seed coating is the formation of thin layer of polymer containing different additives. It maintain the shape and size of seed and doesn't increase the weight of seed.
2. Seed pellets: These are more or less spherical units developed for precision sowing, usually incorporating a single seed with the size and shape of the seed no longer readily evident. The pellet, in addition to the pelleting material, may contain pesticides, dyes or other additives.
3. Encrusted seed: Units more or less retaining the shape of the seed with the size and weight changed to a greater or less extent. The encrusting material may contain pesticides, fungicides, dyes or other additives.
4. Seed granules: Units more or less cylindrical, including types with more than one seed joined together. The granule, in addition to the granulating material, may contain pesticides, dyes or other additives.
5. Seed tapes: Narrow bands of material, such as paper or other degradable material, with seeds spaced randomly, in groups or in a single row.
6. Seed mats: Broad sheets of material, such as paper or other degradable material, with seeds placed in rows, groups or at random throughout the sheets.
The followings four different tests are usually performed on coated and pelleted seeds
A. Physical purity analysis
B. Number of other seeds
C. Germination test
A. Physical purity analysis :
1. Size of lot : If the lot is reasonably homogenous, the maximum weight of lot may be as great as the maximum weight of lot for which sampling procedures are prescribed as in case of uncoated seeds subject to the tolerance of 5% and subject to the seed number limitation
prescribed below: The maximum number of seeds that a lot of seed pellets, encrusted seeds, seed granules, seed tapes or seed mats may contain is 1,000,000,000 (10,000 units of 100,000) except that the weight of lot, including the coating material may not exceed 42,000 kg (40,000 kg plus 5%). When lot size is expressed in units the total weight of the lot must be given on the certificate.
2. Sample size: The submitted sample should be according to Table. 1. As submitted samples of coated seeds normally contain fewer seeds than corresponding samples of uncoated seeds therefore, special care is necessary in drawing the sample to ensure that it is representative of the lot. Precautions are necessary to avoid damage or change in the pellets or seed tape during drawing, handling and transport, and samples must be submitted in suitable containers. To obtain working sample from submitted sample seed divider should be used but the distance of fall must never exceed that 250 mm. For seed tapes take pieces of tape at random, to provide sufficient seeds for the test.
3. Purity analysis: Purity analysis is not obligatory test but if it is requested by the applicant, then a purity analysis on depelleted seeds or seeds removed from the tape may be carried out in accordance with method prescribed for the uncoated seeds. The working sample of pellets is separated into the following three components: pure pellets, unpelleted seed and inert matter, and the percentage of each part is determined by weight. The working sample of not less than
9. TESTING OF COATED AND PELLETED SEEDS
Figure 22. Coated and pelleted seed of cowpea and Dinanath grass
5150
2500 pure pellets is depelleted by shaking in fine mesh sieves immersed in water (0.5 mm sieve size).The pelleting material is dispersed in the water and the remaining seed material is dried overnight on filter paper and then in an air oven. After drying, the material must be subjected to a purity analysis in accordance with the procedure prescribed for uncoated seeds. The component parts (pure seed, other seeds and inert matter) shall be reported as percentages of their total weight, ignoring the pelleting material. The percentage of pelleting material shall be reported separately only on request. When a purity test on seeds removed from tapes is requested, the tape material of the working sample with paper tapes is cautiously separated and stripped off. Water soluble tape material is moistened until the seeds come free. When pelleted seeds are found in the tapes, follow the procedure prescribed above. The moistened seeds must be dried and the freed seed material must be subjected to a purity test as above.
Verification of species : In order to check that the seed in the pellets is largely of the species stated by the applicant, it is obligatory to remove the pelleting material from 100 pellets taken from the pure pellet fraction of the purity test and determine the species of each seed. The percentage by weight of each of the component parts shall be calculated to one decimal place based on the sum of the weights of the components. The result of a purity analysis shall be given to one decimal place and the percentage of all components must total 100. Components of less than 0.05% shall be reported as trace. The name and number of seeds of each species found in the examination of the 100 seeds removed from pellets or tapes shall be reported on certificate under 'other determinations'.
A. Number of other seeds : The determination to estimate the number of seeds of other species is carried out only at the request of the applicant. The determination is made by a count of seeds of the species designated by the applicant, and the result is expressed as a number of seeds found in the weight and approximate numbers of pellets examined or for tapes in the length of tape (or area of mat) examined. The pelleting material or tape material shall be removed as prescribed for purity analysis, but drying is not obligatory. The working sample is searched either for seeds of all other species or of certain designated species, as required by the applicant. The result is expressed as the scientific name and number of seeds belonging to each designated species or category found in the actual weight and approximate number of pelleted seeds examined or length of tape examined for seed tapes(eg. per kilogram, per meter or per square meter).
B. Germination Test : Germination tests on pelleted seeds shall be made with pellets from the pure pellet fraction of a purity test. The pellets/seed tape shall be placed on the substrate in condition in which they are received (e.g. without rinsing or soaking). Paper, sand and in certain situations soil are permissible as substrates but for pelleted seed the use of pleated paper and for seed tapes a between paper method of which the upright rolled towel has proved satisfactory in many cases, is recommended.
·Working sample : The pure pellets shall be well mixed and 400 pellets counted at random in replicates of 100. The working sample from seed tapes shall consist of randomly taken pieces of tape to make up four replicates of at least 100 seeds each.
·Test conditions : Methods, substrates, temperatures, light conditions and special treatments will remain the same as in case of normal seeds.
·Duration of test : Extension beyond the period prescribed for uncoated seeds may be necessary. However, slow germination may be an indication that test conditions are not optimum and a germination test of seeds removed from the covering material may be made as a check.
·Evaluation : Evaluation of seedlings as normal or abnormal shall be in accordance with criteria laid down for uncoated seeds.
·Multiple seed structures : Multiple seed structures may occur in pellets or in tapes or more than one seed may be found in a pellet. In either case these shall be tested as single seeds. The result of the test indicates the percentage of structures or pellets which have produced at least one normal seedling. Pellets or seeds in tapes producing two or more such seedlings are counted and their number recorded.
·Calculation and expression of results : Results are expressed as percentage by number. In addition, for taped seeds the total length of tape used in the germination test is measured and the total number of normal seedlings is noted followed by number of normal seedlings per meter is calculated.
Table 13. Sample size of pelleted seed for physical purity analysis (ISTA, 2015).
S.N. Determinations Submitted Working samples samples
Sample sizes of pelleted seeds in number of pellets
1. Purity analysis (Verification of species) 7500 2500
2. Weight determination 7500 Pure pellet fraction
3. Germination 7500 400
4. Determination of other seeds 10000 7500
5. Determination of other seeds 25000 25000 (In encrusted seeds and seed granules)
6. Size grading 10000 2000
Sample sizes of seed tapes
1. Verification of species 2500 seeds 100 seeds
2. Germination 2500 seeds 400 seeds
3. Purity analysis (if required) 2500 seeds 2500 seeds
4. Determination of other seeds 10000 seeds 7500 seeds
5352
2500 pure pellets is depelleted by shaking in fine mesh sieves immersed in water (0.5 mm sieve size).The pelleting material is dispersed in the water and the remaining seed material is dried overnight on filter paper and then in an air oven. After drying, the material must be subjected to a purity analysis in accordance with the procedure prescribed for uncoated seeds. The component parts (pure seed, other seeds and inert matter) shall be reported as percentages of their total weight, ignoring the pelleting material. The percentage of pelleting material shall be reported separately only on request. When a purity test on seeds removed from tapes is requested, the tape material of the working sample with paper tapes is cautiously separated and stripped off. Water soluble tape material is moistened until the seeds come free. When pelleted seeds are found in the tapes, follow the procedure prescribed above. The moistened seeds must be dried and the freed seed material must be subjected to a purity test as above.
Verification of species : In order to check that the seed in the pellets is largely of the species stated by the applicant, it is obligatory to remove the pelleting material from 100 pellets taken from the pure pellet fraction of the purity test and determine the species of each seed. The percentage by weight of each of the component parts shall be calculated to one decimal place based on the sum of the weights of the components. The result of a purity analysis shall be given to one decimal place and the percentage of all components must total 100. Components of less than 0.05% shall be reported as trace. The name and number of seeds of each species found in the examination of the 100 seeds removed from pellets or tapes shall be reported on certificate under 'other determinations'.
A. Number of other seeds : The determination to estimate the number of seeds of other species is carried out only at the request of the applicant. The determination is made by a count of seeds of the species designated by the applicant, and the result is expressed as a number of seeds found in the weight and approximate numbers of pellets examined or for tapes in the length of tape (or area of mat) examined. The pelleting material or tape material shall be removed as prescribed for purity analysis, but drying is not obligatory. The working sample is searched either for seeds of all other species or of certain designated species, as required by the applicant. The result is expressed as the scientific name and number of seeds belonging to each designated species or category found in the actual weight and approximate number of pelleted seeds examined or length of tape examined for seed tapes(eg. per kilogram, per meter or per square meter).
B. Germination Test : Germination tests on pelleted seeds shall be made with pellets from the pure pellet fraction of a purity test. The pellets/seed tape shall be placed on the substrate in condition in which they are received (e.g. without rinsing or soaking). Paper, sand and in certain situations soil are permissible as substrates but for pelleted seed the use of pleated paper and for seed tapes a between paper method of which the upright rolled towel has proved satisfactory in many cases, is recommended.
·Working sample : The pure pellets shall be well mixed and 400 pellets counted at random in replicates of 100. The working sample from seed tapes shall consist of randomly taken pieces of tape to make up four replicates of at least 100 seeds each.
·Test conditions : Methods, substrates, temperatures, light conditions and special treatments will remain the same as in case of normal seeds.
·Duration of test : Extension beyond the period prescribed for uncoated seeds may be necessary. However, slow germination may be an indication that test conditions are not optimum and a germination test of seeds removed from the covering material may be made as a check.
·Evaluation : Evaluation of seedlings as normal or abnormal shall be in accordance with criteria laid down for uncoated seeds.
·Multiple seed structures : Multiple seed structures may occur in pellets or in tapes or more than one seed may be found in a pellet. In either case these shall be tested as single seeds. The result of the test indicates the percentage of structures or pellets which have produced at least one normal seedling. Pellets or seeds in tapes producing two or more such seedlings are counted and their number recorded.
·Calculation and expression of results : Results are expressed as percentage by number. In addition, for taped seeds the total length of tape used in the germination test is measured and the total number of normal seedlings is noted followed by number of normal seedlings per meter is calculated.
Table 13. Sample size of pelleted seed for physical purity analysis (ISTA, 2015).
S.N. Determinations Submitted Working samples samples
Sample sizes of pelleted seeds in number of pellets
1. Purity analysis (Verification of species) 7500 2500
2. Weight determination 7500 Pure pellet fraction
3. Germination 7500 400
4. Determination of other seeds 10000 7500
5. Determination of other seeds 25000 25000 (In encrusted seeds and seed granules)
6. Size grading 10000 2000
Sample sizes of seed tapes
1. Verification of species 2500 seeds 100 seeds
2. Germination 2500 seeds 400 seeds
3. Purity analysis (if required) 2500 seeds 2500 seeds
4. Determination of other seeds 10000 seeds 7500 seeds
5352
Trade Related Aspects of Intellectual Property Rights Agreement(TRIP ) was negotiated at the S
end of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) in 1994 and it is administered by the WTO. The formation of TRIPs agreement resulted in shifting of controlled access to plant genetic resources from free exchange and unhindered exploitation. Similarly UN Convention on Biological Diversity (CBD) 1993 recognizes the sovereignty of nations over their plant genetic resources and rights of farmers to receive compensation for direct and indirect commercial exploitation of traditional varieties. To comply with these international developments, India has enacted Protection of Plant Varieties and Farmers' Rights Act (PPV&FR) (Sui generis system means 'of its own kind') to provide legal framework for plant breeder's and farmer's rights. Novelty, distinctness, uniformity and stability are the essential requirements for grant of protection to all the varieties. Therefore, a perfect system for identification of varieties and cultivars is the fundamental requirement to enforce this protection. Conventionally, morphological descriptors are routinely used for establishing the identity of varieties but these descriptors are highly influenced by many factors viz., impact of environment on trait expression, epistatic interactions, pleiotrophic effects, labour intensive, time consuming and required large area to perform the test. Electrophoresis of seed proteins and isozymes analysis has overcome these limitations to some extent but now many powerful DNA based techniques are available to overcome these drawbacks. The followings are different systems through which we can identify the variety or species in particular.
1. Morphological markers
2. Chemical markers
3. Biochemical markers
4. Molecular markers
Submitted sample : The testing laboratory must ensure that the size of the submitted sample is sufficient to perform the tests as requested by the applicant (Table).
A. Morphological Markers : The species or cultivar identification can be done through
different morphological characters. Generally, samples should be compared with respective
authentic sample for correct identification. If more than one submitted sample is to be
verified for the same cultivar/species, it is sufficient to include at least one authentic sample
as a control for every 15 working samples from the submitted samples.
1. Examination of seeds: The variety can be identify based on seed colour, shape, size and
weight. For testing morphological traits, the seeds must be examined with the aid of a
suitable magnifying apparatus. In Avenasativaa useful character is grain colour, which
maybe white, yellowish grey or black.
2. Examination of seedlings: The seeds must be germinated on an appropriate medium and
when the seedlings have reached a suitable stage of development, they are examined in
whole or in part, with or without further treatment. The ISTA has recommended
following different tests for identification of variety based on seedling characters.
@Coleoptiles pigmentation
@Seedling colour test in Beta spp: Some varieties can be distinguished by seedling
colour, which may be white, yellow, pale red, or red. Sow the seeds in damp sand in
dishes placed in subdued daylight at room temperature. After seven days the
seedlings are examined for hypocotyl colour. For sugar beet and white fodder beet,
the proportion of white to pale-red seedlings gives some indication of the
genuineness of the cultivar.
@Colour of cotyledons in Brassica spp.
@Fluorescence test in Lolium spp and Festuca spp:
Lolium : In most varieties of Lolium multiflorum the root traces of the majority of
the seedlings show fluorescence under ultra-violet light, whereas in most varieties of
Lolium perenne the root traces of the majority of the seedlings do not show
fluorescence. The fluorescence test alone, however, is not always a sufficient basis
for the identification of a species or a cultivar, because many of the varieties grown
contain a certain number of plants which do not give the typical reaction for the
species. Further, several of the hybrid forms between the two species may give an
intermediate reaction. For determination of the reaction of seedling root traces to
ultra-violet light, the seeds are placed on suitable non-fluorescent white filter paper
moistened with water for germination within the 20–30°C range using either an
alternating or constant 20°C temperature, in darkness or weak light (not more than
250 lux), under conditions where no drying occurs; the seeds are spaced and
arranged so as to prevent entwining of the roots and confusion of the fluorescent
traces. The examination should be made only when the roots are sufficiently well
developed, which may not be until the fourteenth, or in the case of dormant seed the
eighteenth day. The seedlings are examined under ultra-violet light from a lamp
Table 14. Submitted sample size for variety and species identification (ISTA, 2015)
S.N. Species Laboratory Field plot andonly (g) laboratory (g)
1. Glycine, Lupinus, Phaseolus, Pisum, Vicia, Zea and 1000 2000species of other genera with seeds of similar size
2. Avena, Hordeum, Secale, Triticum and species of 500 1000other genera with seeds of similar size
3. Beta and species of other genera with seeds of 250 500similar size
4. All smaller seeded species 100 250
10. VARIETY IDENTIFICATION TEST
5554
Trade Related Aspects of Intellectual Property Rights Agreement(TRIP ) was negotiated at the S
end of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) in 1994 and it is administered by the WTO. The formation of TRIPs agreement resulted in shifting of controlled access to plant genetic resources from free exchange and unhindered exploitation. Similarly UN Convention on Biological Diversity (CBD) 1993 recognizes the sovereignty of nations over their plant genetic resources and rights of farmers to receive compensation for direct and indirect commercial exploitation of traditional varieties. To comply with these international developments, India has enacted Protection of Plant Varieties and Farmers' Rights Act (PPV&FR) (Sui generis system means 'of its own kind') to provide legal framework for plant breeder's and farmer's rights. Novelty, distinctness, uniformity and stability are the essential requirements for grant of protection to all the varieties. Therefore, a perfect system for identification of varieties and cultivars is the fundamental requirement to enforce this protection. Conventionally, morphological descriptors are routinely used for establishing the identity of varieties but these descriptors are highly influenced by many factors viz., impact of environment on trait expression, epistatic interactions, pleiotrophic effects, labour intensive, time consuming and required large area to perform the test. Electrophoresis of seed proteins and isozymes analysis has overcome these limitations to some extent but now many powerful DNA based techniques are available to overcome these drawbacks. The followings are different systems through which we can identify the variety or species in particular.
1. Morphological markers
2. Chemical markers
3. Biochemical markers
4. Molecular markers
Submitted sample : The testing laboratory must ensure that the size of the submitted sample is sufficient to perform the tests as requested by the applicant (Table).
A. Morphological Markers : The species or cultivar identification can be done through
different morphological characters. Generally, samples should be compared with respective
authentic sample for correct identification. If more than one submitted sample is to be
verified for the same cultivar/species, it is sufficient to include at least one authentic sample
as a control for every 15 working samples from the submitted samples.
1. Examination of seeds: The variety can be identify based on seed colour, shape, size and
weight. For testing morphological traits, the seeds must be examined with the aid of a
suitable magnifying apparatus. In Avenasativaa useful character is grain colour, which
maybe white, yellowish grey or black.
2. Examination of seedlings: The seeds must be germinated on an appropriate medium and
when the seedlings have reached a suitable stage of development, they are examined in
whole or in part, with or without further treatment. The ISTA has recommended
following different tests for identification of variety based on seedling characters.
@Coleoptiles pigmentation
@Seedling colour test in Beta spp: Some varieties can be distinguished by seedling
colour, which may be white, yellow, pale red, or red. Sow the seeds in damp sand in
dishes placed in subdued daylight at room temperature. After seven days the
seedlings are examined for hypocotyl colour. For sugar beet and white fodder beet,
the proportion of white to pale-red seedlings gives some indication of the
genuineness of the cultivar.
@Colour of cotyledons in Brassica spp.
@Fluorescence test in Lolium spp and Festuca spp:
Lolium : In most varieties of Lolium multiflorum the root traces of the majority of
the seedlings show fluorescence under ultra-violet light, whereas in most varieties of
Lolium perenne the root traces of the majority of the seedlings do not show
fluorescence. The fluorescence test alone, however, is not always a sufficient basis
for the identification of a species or a cultivar, because many of the varieties grown
contain a certain number of plants which do not give the typical reaction for the
species. Further, several of the hybrid forms between the two species may give an
intermediate reaction. For determination of the reaction of seedling root traces to
ultra-violet light, the seeds are placed on suitable non-fluorescent white filter paper
moistened with water for germination within the 20–30°C range using either an
alternating or constant 20°C temperature, in darkness or weak light (not more than
250 lux), under conditions where no drying occurs; the seeds are spaced and
arranged so as to prevent entwining of the roots and confusion of the fluorescent
traces. The examination should be made only when the roots are sufficiently well
developed, which may not be until the fourteenth, or in the case of dormant seed the
eighteenth day. The seedlings are examined under ultra-violet light from a lamp
Table 14. Submitted sample size for variety and species identification (ISTA, 2015)
S.N. Species Laboratory Field plot andonly (g) laboratory (g)
1. Glycine, Lupinus, Phaseolus, Pisum, Vicia, Zea and 1000 2000species of other genera with seeds of similar size
2. Avena, Hordeum, Secale, Triticum and species of 500 1000other genera with seeds of similar size
3. Beta and species of other genera with seeds of 250 500similar size
4. All smaller seeded species 100 250
10. VARIETY IDENTIFICATION TEST
5554
transmitting radiations between 300 nm and 400 nm, with maximum radiation
between 360 nm and 370 nm, and a trace of visible light. The examination must be
made in such a manner that all seedlings, whose roots produce traces with
fluorescence to any degree can be detected. Seedlings not showing fluorescent root
traces should be pulled off the filter paper while under the ultra-violet light in order
to detect fluorescence from roots which may have grown into the paper. The
numbers of fluorescent and non-fluorescent seedlings and the number of normal
seedlings are recorded for each replicate. The results should be reported in whole
numbers.
Festuca : Festuca rubra L. and Festuca ovina L. can be distinguished in the same
way as Lolium spp. The roots themselves show fluorescence in an atmosphere
containing ammonia; those of F. rubra are yellow-green in ultra-violet light while
the roots of F. ovina are bluish green. The seeds are germinated using the method
described for Lolium spp. with the count being made after 14 days or 21 days if
development is not sufficient at 14 days. While under UV light the roots on the
substratum are treated with ammonia gas produced from a plastic bottle ('spraying
bottle') partially filled with a solution of ammonia in water. A weak solution
(producing enough ammonia to smell) is adequate for this purpose.
3. Examination of plants in glasshouse or growth chamber: The seeds must be sown in
suitable containers and maintained in environmental conditions necessary for the
development of the traits. When the plants have reached a suitable stage of development,
the traits must be observed on each plant.
4. Examination of plants in field plots: When plants are tested in field plots, each working
samplemust be sown in at least two replicate plots. Observations must be made during
the whole growing period for morphological descriptors of the varieties.
B. Chemical Test : Various chemical tests are being used to reveal differences among the seeds
or seedlings of different genotypes. These chemical tests are quick, easy to carry out,
reproducible and can be undertaken throughout the year under controlled laboratory
conditions. They require virtually no technical expertise or training and can be completed in
a relatively short time. The results of these tests are usually distinct, easily interpreted and
helps in grouping of any crop genotypes based on chemical nature of protein which are
exposed products of genes. Some of the sensitive analytical techniques employed in the
laboratory are phenol test, modified phenol test, ferrous sulphate test, potassium hydroxide
test, sodium hydroxide test and GA test.3
C. Biochemical Markers : ISTA published the first protocol for variety testing using
polyacrylamide gel electrophoresis (PAGE) for wheat and barley in 1987. Since then the
technique has been refined and modified suitably for a number of species viz., Triticum,
Hordeum, Lolium, Pisum, Zea, Helianthus, Avena etc. Work is underway to examine
application of these for several other crops by the ISTA Working Group on Variety Testing.
UPOV has recommended electrophoresis techniques for establishing distinctness of
varieties of wheat, barley, maize, soybean and sunflower. In Germany electrophoresis
characteristics (particularly IEF) is used for the verification of species and varieties of wheat,
barley, oat, maize, pea, mustard and lolium. It can also be used effectively for verification of
hybridity in maize, sunflower (ISTA, LUFA and Germany), tomato (LUFA), cotton, castor
(IARI, New Delhi) and a number of crops. In common use, electrophoresis refers to
movement of ions through a matrix which acts as a molecular sieve in which charged
molecules are separated on the basis of charge/mass. Based on the solubility properties,
proteins are classified into 4 classes.
Table 15. Different biochemical tests recommended by ISTA and AOSA for variety identification
S.N. Species Test Recommendation
1. Wheat Phenol ISTA
2. Lupin Lugol
3. Barley, pea, maize, sunflower and lolium Electrophoretic
4. Sweet clover CuSO -NH AOSA4 3
5. Oat HCl
6. Soybean Peoxidase
7. Rice and sorghum KOH
8. Wheat NaOH
Table: 16. Different types of proteins and their characteristics
S.N. Protein Nature of protein
1. Albumin Soluble in water. Mostly enzymes
2. Globulin Soluble in dilute salt solutions. Mostly storage bodies
3. Prolamins Soluble in aqueous alcoholic solutions. Storage proteins
4. Glutenins Soluble in acidic or alkaline solutions. Mainly, structural proteins
As proteins carry a net charge at any pH, other than their isoelectric point, they migrate in an
electric field, the rate of migration being dependent on the charge density. By altering the pore
size of the gel (using different concentrations of polymers and cross linkers) and the charge on the
molecule (by altering the pH of the system) a high degree of resolution can be achieved. This
results in a characteristic protein banding pattern for a cultivar/species. This property is
effectively used for variety testing with necessary modification, viz., using a specific protein
fraction or isoenzyme etc.
5756
transmitting radiations between 300 nm and 400 nm, with maximum radiation
between 360 nm and 370 nm, and a trace of visible light. The examination must be
made in such a manner that all seedlings, whose roots produce traces with
fluorescence to any degree can be detected. Seedlings not showing fluorescent root
traces should be pulled off the filter paper while under the ultra-violet light in order
to detect fluorescence from roots which may have grown into the paper. The
numbers of fluorescent and non-fluorescent seedlings and the number of normal
seedlings are recorded for each replicate. The results should be reported in whole
numbers.
Festuca : Festuca rubra L. and Festuca ovina L. can be distinguished in the same
way as Lolium spp. The roots themselves show fluorescence in an atmosphere
containing ammonia; those of F. rubra are yellow-green in ultra-violet light while
the roots of F. ovina are bluish green. The seeds are germinated using the method
described for Lolium spp. with the count being made after 14 days or 21 days if
development is not sufficient at 14 days. While under UV light the roots on the
substratum are treated with ammonia gas produced from a plastic bottle ('spraying
bottle') partially filled with a solution of ammonia in water. A weak solution
(producing enough ammonia to smell) is adequate for this purpose.
3. Examination of plants in glasshouse or growth chamber: The seeds must be sown in
suitable containers and maintained in environmental conditions necessary for the
development of the traits. When the plants have reached a suitable stage of development,
the traits must be observed on each plant.
4. Examination of plants in field plots: When plants are tested in field plots, each working
samplemust be sown in at least two replicate plots. Observations must be made during
the whole growing period for morphological descriptors of the varieties.
B. Chemical Test : Various chemical tests are being used to reveal differences among the seeds
or seedlings of different genotypes. These chemical tests are quick, easy to carry out,
reproducible and can be undertaken throughout the year under controlled laboratory
conditions. They require virtually no technical expertise or training and can be completed in
a relatively short time. The results of these tests are usually distinct, easily interpreted and
helps in grouping of any crop genotypes based on chemical nature of protein which are
exposed products of genes. Some of the sensitive analytical techniques employed in the
laboratory are phenol test, modified phenol test, ferrous sulphate test, potassium hydroxide
test, sodium hydroxide test and GA test.3
C. Biochemical Markers : ISTA published the first protocol for variety testing using
polyacrylamide gel electrophoresis (PAGE) for wheat and barley in 1987. Since then the
technique has been refined and modified suitably for a number of species viz., Triticum,
Hordeum, Lolium, Pisum, Zea, Helianthus, Avena etc. Work is underway to examine
application of these for several other crops by the ISTA Working Group on Variety Testing.
UPOV has recommended electrophoresis techniques for establishing distinctness of
varieties of wheat, barley, maize, soybean and sunflower. In Germany electrophoresis
characteristics (particularly IEF) is used for the verification of species and varieties of wheat,
barley, oat, maize, pea, mustard and lolium. It can also be used effectively for verification of
hybridity in maize, sunflower (ISTA, LUFA and Germany), tomato (LUFA), cotton, castor
(IARI, New Delhi) and a number of crops. In common use, electrophoresis refers to
movement of ions through a matrix which acts as a molecular sieve in which charged
molecules are separated on the basis of charge/mass. Based on the solubility properties,
proteins are classified into 4 classes.
Table 15. Different biochemical tests recommended by ISTA and AOSA for variety identification
S.N. Species Test Recommendation
1. Wheat Phenol ISTA
2. Lupin Lugol
3. Barley, pea, maize, sunflower and lolium Electrophoretic
4. Sweet clover CuSO -NH AOSA4 3
5. Oat HCl
6. Soybean Peoxidase
7. Rice and sorghum KOH
8. Wheat NaOH
Table: 16. Different types of proteins and their characteristics
S.N. Protein Nature of protein
1. Albumin Soluble in water. Mostly enzymes
2. Globulin Soluble in dilute salt solutions. Mostly storage bodies
3. Prolamins Soluble in aqueous alcoholic solutions. Storage proteins
4. Glutenins Soluble in acidic or alkaline solutions. Mainly, structural proteins
As proteins carry a net charge at any pH, other than their isoelectric point, they migrate in an
electric field, the rate of migration being dependent on the charge density. By altering the pore
size of the gel (using different concentrations of polymers and cross linkers) and the charge on the
molecule (by altering the pH of the system) a high degree of resolution can be achieved. This
results in a characteristic protein banding pattern for a cultivar/species. This property is
effectively used for variety testing with necessary modification, viz., using a specific protein
fraction or isoenzyme etc.
5756
S.N. Starch Gel Polyacrylamide Gel
1. It is used for isoenzyme It is used for both proteins & isoenzyme
2. Cheap & easy to analyze a number Costly but long shelf life of stock solutionof enzymes
3. Difficult to store & low in resolution higher resolution & greater polymorphism
4. It provide poor homogeneity & varying pore size but toxic limited pore size
The mobility of a protein molecule increases as the pH of the buffer is farther from its isoelectric point, therefore, a pH should be chosen that maximizes mobility differences among the components of protein mixture. As a general rule, basic proteins are better separated at an acidic pH, while most of the proteins having isoelectric points within pH 4.0 are best separated under alkaline condition of pH 8-9. In common practice, PAGE is carried out at a pH range of 3 to 10.
The ionic strength of the buffer system should be such that the sample is kept in solution with adequate buffering capacity. The electrical resistance is lower at higher ionic concentrations of the buffer, which results in higher current and greater heat generation, though the bands are generally sharper (if the heat can be dissipated) at higher concentrations of buffer. Generally, buffers within the range of 0.05 to 0.1 M are found most suitable, though lower or higher ionic strengths can also be used.
Polyacrylamide gel electrophoresis of denatured proteins (SDS-PAGE):
Separation of proteins on the basis of their molecular mass is performed by denaturing proteins in the presence of sodium dodecyl sulphate (SDS) a strong anionic detergent and a thiol agent (e.g. 2-mercaptoethanol) and then subjecting it to polyacrylamide gel electrophoresis (i.e. SDS-PAGE). Most proteins bind SDS in a constant weight ratio (1.4 g of SDS per g of protein). Due to strong negative charge on SDS, the SDS-bound denatured protein molecules carry identical charge density and therefore, migrate in polyacrylamide gels on the basis of the size of the molecule. The discontinuous SDS-PAGE of Laemmli (1971), with required modification is by far the most commonly used method for denatured proteins. Besides these two common polyacrylamide gel electrophoretic procedures, there are other techniques, such as isoelectric focusing (IEF) and two dimensional gel electrophoresis which offer even greater resolution for separating proteins in a mixture.
Starch gel electrophoresis: Non-toxic nature of starch, speed and low cost of analysis are the major factors favouring its use for routine isoenzyme analysis. The gel is prepared in a horizontal tray using electrophoresis grade starch suspension solution. Crude sample extract are used for analysis and after the run is over, several (upto 6) horizontal slices are cut from the gel for the purpose of staining for different enzyme systems. Isoenzyme pattern is revealed as stained zone(s). The resolution of bands is slightly low in starch gel as compared to polyacrylamide.
Detailed procedures for PAGE of wheat and barley and SDS-PAGE of Pea and Lolium are given. These can also be employed with minor modifications for variety identification in other crops. The extraction buffer and procedure also needs to be modified according to the component to be analyzed. Generally, the pH of the extraction buffer is either near-neutral or closer to the gel and electrophoresis buffer, though a lower ionic strength. The sample of total proteins or any fraction of seed proteins required for electrophoresis is normally smaller than for isoenzymes. Concentration of proteins should ideally be about 1000 µg per ml, of which 50-200 µg protein per well may be loaded for different isoenzymes depending on the expected activity level in the given tissue sample. For total proteins a sample of upto 10 µg is normally sufficient. In a discontinuous system even relatively dilute samples can give good resolution, as the sample gets
PAGE : The polyacrylamide gel is formed by the vinyl polymerisation of acrylamide monomers into long polyacrylamide chains and cross linking these by the inclusion of an bifunctional monomer N, N' methylene-bis-acrylamide (bis). The polymerisation reaction produces random chains of polyacrylamide incorporating a small proportion of bis molecules which, in turn, react with groups in other chains forming cross-links that results in a three dimensional network. The concentration of acrylamide determines the polymer chain length, while the concentration of bis determines the extent of cross-linking. Thus, the gel density, elasticity, mechanical strength and pore size are determined by the concentration of acrylamide and bis used.
The pore size of the gels is greatly influenced by the acrylamide concentration. The effective pore size decrease with the increasing acrylamide concentration. The total acrylamide+bis concentration in a gel mixture is represented by % T while the concentration of the cross-linking monomer (ie. Bis) is represented by % C. The pore size increases with the increase in % C. Gels ranging from 3 to 30% acrylamide concentration can be made and used for separating molecules
6of size upto 1 x 10 daltons. Gels with linear gradients of increasing acrylamide concentration are also used for a greater resolution, particularly when analyzing a mixture of proteins of very low to very high molecular weights.
To initiate the process of polymerization of acrylamide, a catalyst, such as ammonium persulphate or riboflavin is added along with an accelerator e.g. N, N, N'N'-tetra methylenediamine (TEMED). TEMED catalyzes the formation of free radicals from persulphate, while these radicals initiate the polymerization reactions. In a riboflavin-TEMED system, on the other hand, though the photo-oxidation of riboflavin can produce free radicals necessary for polymerization, TEMED is also added to ensure complete and uniform polymerization.
Selection of a suitable gel and buffer system:
The mixture of proteins to be analyzed by PAGE consists of molecules differing in size and net charge. The degree of separation of these proteins is greatly influenced by different conditions of pH, concentrations of acrylamide and bis, ionic strength, potential gradient, strength of the electric field (i.e. current/volts applied), running duration, temperature etc. Selection of optimum conditions depends on the type of the sample to the examined. Thus, for a sample of proteins with high molecular weights, a large pore gel (low acrylamide concentration) will be suitable. However, when the separation is based mainly on charge difference, gels with large pores are used even for smaller molecules of proteins.
5958
S.N. Starch Gel Polyacrylamide Gel
1. It is used for isoenzyme It is used for both proteins & isoenzyme
2. Cheap & easy to analyze a number Costly but long shelf life of stock solutionof enzymes
3. Difficult to store & low in resolution higher resolution & greater polymorphism
4. It provide poor homogeneity & varying pore size but toxic limited pore size
The mobility of a protein molecule increases as the pH of the buffer is farther from its isoelectric point, therefore, a pH should be chosen that maximizes mobility differences among the components of protein mixture. As a general rule, basic proteins are better separated at an acidic pH, while most of the proteins having isoelectric points within pH 4.0 are best separated under alkaline condition of pH 8-9. In common practice, PAGE is carried out at a pH range of 3 to 10.
The ionic strength of the buffer system should be such that the sample is kept in solution with adequate buffering capacity. The electrical resistance is lower at higher ionic concentrations of the buffer, which results in higher current and greater heat generation, though the bands are generally sharper (if the heat can be dissipated) at higher concentrations of buffer. Generally, buffers within the range of 0.05 to 0.1 M are found most suitable, though lower or higher ionic strengths can also be used.
Polyacrylamide gel electrophoresis of denatured proteins (SDS-PAGE):
Separation of proteins on the basis of their molecular mass is performed by denaturing proteins in the presence of sodium dodecyl sulphate (SDS) a strong anionic detergent and a thiol agent (e.g. 2-mercaptoethanol) and then subjecting it to polyacrylamide gel electrophoresis (i.e. SDS-PAGE). Most proteins bind SDS in a constant weight ratio (1.4 g of SDS per g of protein). Due to strong negative charge on SDS, the SDS-bound denatured protein molecules carry identical charge density and therefore, migrate in polyacrylamide gels on the basis of the size of the molecule. The discontinuous SDS-PAGE of Laemmli (1971), with required modification is by far the most commonly used method for denatured proteins. Besides these two common polyacrylamide gel electrophoretic procedures, there are other techniques, such as isoelectric focusing (IEF) and two dimensional gel electrophoresis which offer even greater resolution for separating proteins in a mixture.
Starch gel electrophoresis: Non-toxic nature of starch, speed and low cost of analysis are the major factors favouring its use for routine isoenzyme analysis. The gel is prepared in a horizontal tray using electrophoresis grade starch suspension solution. Crude sample extract are used for analysis and after the run is over, several (upto 6) horizontal slices are cut from the gel for the purpose of staining for different enzyme systems. Isoenzyme pattern is revealed as stained zone(s). The resolution of bands is slightly low in starch gel as compared to polyacrylamide.
Detailed procedures for PAGE of wheat and barley and SDS-PAGE of Pea and Lolium are given. These can also be employed with minor modifications for variety identification in other crops. The extraction buffer and procedure also needs to be modified according to the component to be analyzed. Generally, the pH of the extraction buffer is either near-neutral or closer to the gel and electrophoresis buffer, though a lower ionic strength. The sample of total proteins or any fraction of seed proteins required for electrophoresis is normally smaller than for isoenzymes. Concentration of proteins should ideally be about 1000 µg per ml, of which 50-200 µg protein per well may be loaded for different isoenzymes depending on the expected activity level in the given tissue sample. For total proteins a sample of upto 10 µg is normally sufficient. In a discontinuous system even relatively dilute samples can give good resolution, as the sample gets
PAGE : The polyacrylamide gel is formed by the vinyl polymerisation of acrylamide monomers into long polyacrylamide chains and cross linking these by the inclusion of an bifunctional monomer N, N' methylene-bis-acrylamide (bis). The polymerisation reaction produces random chains of polyacrylamide incorporating a small proportion of bis molecules which, in turn, react with groups in other chains forming cross-links that results in a three dimensional network. The concentration of acrylamide determines the polymer chain length, while the concentration of bis determines the extent of cross-linking. Thus, the gel density, elasticity, mechanical strength and pore size are determined by the concentration of acrylamide and bis used.
The pore size of the gels is greatly influenced by the acrylamide concentration. The effective pore size decrease with the increasing acrylamide concentration. The total acrylamide+bis concentration in a gel mixture is represented by % T while the concentration of the cross-linking monomer (ie. Bis) is represented by % C. The pore size increases with the increase in % C. Gels ranging from 3 to 30% acrylamide concentration can be made and used for separating molecules
6of size upto 1 x 10 daltons. Gels with linear gradients of increasing acrylamide concentration are also used for a greater resolution, particularly when analyzing a mixture of proteins of very low to very high molecular weights.
To initiate the process of polymerization of acrylamide, a catalyst, such as ammonium persulphate or riboflavin is added along with an accelerator e.g. N, N, N'N'-tetra methylenediamine (TEMED). TEMED catalyzes the formation of free radicals from persulphate, while these radicals initiate the polymerization reactions. In a riboflavin-TEMED system, on the other hand, though the photo-oxidation of riboflavin can produce free radicals necessary for polymerization, TEMED is also added to ensure complete and uniform polymerization.
Selection of a suitable gel and buffer system:
The mixture of proteins to be analyzed by PAGE consists of molecules differing in size and net charge. The degree of separation of these proteins is greatly influenced by different conditions of pH, concentrations of acrylamide and bis, ionic strength, potential gradient, strength of the electric field (i.e. current/volts applied), running duration, temperature etc. Selection of optimum conditions depends on the type of the sample to the examined. Thus, for a sample of proteins with high molecular weights, a large pore gel (low acrylamide concentration) will be suitable. However, when the separation is based mainly on charge difference, gels with large pores are used even for smaller molecules of proteins.
5958
Chemicals: All chemicals should be of 'analytical reagent' grade or equivalent·Bisacrylamide ('specially purified for electrophoresis') (BIS)
·Tris (Tris [hydroxymethyl] methylamine)
·Glycine
·Hydrochloric acid
·Sodium dodecyl sulphate (SDS)\
·Glycerol
·2-mercaptoethanol
·Dimethylformamide
·Ammonium persulphate (APS) (or riboflavin)
·NNN'N'-tetramethylethylenediamine (TEMED)
·Methanol
·Glacial acetic acid
·Trichloroacetic acid (TCA)
·Bromophenol Blue
·PAGE Blue G-90 (or PAGE Blue 83), (or any reagent equivalent to the 'Coomassie Brilliant Blue' G or Rseries of dyes)
Solution preparation: The following different solution are required to conduct PAGE in Lolium.
1. Stock gel buffer (main or resolving gel) : To prepare 1M Tris HCl (pH 8.8) 121.1 g of Tris is dissolved in about 750 ml of distilled water, adjusted to pH 8.8 with hydrochloric acid (use 1 M [approximately 90 ml concentrated HCl per L distilled water] HCl solution) and made up to 1 L and can be stored at 4°C.
2. Stock gel buffer (stacking gel) : To prepare 1M Tris HCl (pH 6.8) 30.3g of Tris is dissolved in 200 mL of distilled water and adjusted to pH 6.8 with hydrochloric acid and made up to 250 ml. This can be stored at 4°C.
3. Stock SDS solution: 10 g of SDS is dissolved in distilled water (this requires gentle stirring and heating) and made volume up to 100 ml. This should be stored at room temperature. If the SDS comes out of solution, it can be re-dissolved by gentle heating.
4. 1% ammonium persulphate solution : 0.1 g of ammoniumpersulphate is dissolved in 10 ml of distilled water. This must be prepared freshly on each occasion, immediately prior to use.
5. Stock sample extraction buffer solution : To 12.5 mLof stacking gel buffer is added 20 mL glycerol, 24.1 mL of distilled water, 4 g of SDS and 12 mg bromophenol blue (optional). This is mixed and stored at room temperature. If the SDS comes out of solution, it can be re-dissolved by gentle heating.
concentrated when passed through the stacking gel. If the sample concentration is too high, a continuous system gives better results. The choice of buffer system depends on the nature of protein under analysis.
Figure: 23. Polyacrylamide Gel Electrophoresis gel schematic representation.
Standard reference method for the verification of varieties of Lolium by Polyacrylamide
Gel Electrophoresis (PAGE):
Principle: The standard reference method for the verifying varieties of Pisum and Lolium is by
polyacrylamide gel electrophoresis (PAGE). Seed proteins are extracted from individual Pisum
seeds or from a meal of Lolium seeds, treated with SDS and separated using a discontinuous
SDS-PAGE procedure. The pattern of protein bands found on the gel is characteristic of a variety.
As a guideline for Pisum, it is recommended that 100 individual seeds are used. Very precise
estimates of varietal purity may require a larger sample. If a comparison batches of 50 seeds can
be undertaken in order to minimize the workload. A simple check on the identity of a single major
constituent of a seed lot can be done using less than 50 seeds. For Lolium, a bulk sample of seeds
is analysed. In most cases, whilst this will serve to verify seed lots, it will not permit the detection
of admixtures of two or more varieties.
6160
Chemicals: All chemicals should be of 'analytical reagent' grade or equivalent·Bisacrylamide ('specially purified for electrophoresis') (BIS)
·Tris (Tris [hydroxymethyl] methylamine)
·Glycine
·Hydrochloric acid
·Sodium dodecyl sulphate (SDS)\
·Glycerol
·2-mercaptoethanol
·Dimethylformamide
·Ammonium persulphate (APS) (or riboflavin)
·NNN'N'-tetramethylethylenediamine (TEMED)
·Methanol
·Glacial acetic acid
·Trichloroacetic acid (TCA)
·Bromophenol Blue
·PAGE Blue G-90 (or PAGE Blue 83), (or any reagent equivalent to the 'Coomassie Brilliant Blue' G or Rseries of dyes)
Solution preparation: The following different solution are required to conduct PAGE in Lolium.
1. Stock gel buffer (main or resolving gel) : To prepare 1M Tris HCl (pH 8.8) 121.1 g of Tris is dissolved in about 750 ml of distilled water, adjusted to pH 8.8 with hydrochloric acid (use 1 M [approximately 90 ml concentrated HCl per L distilled water] HCl solution) and made up to 1 L and can be stored at 4°C.
2. Stock gel buffer (stacking gel) : To prepare 1M Tris HCl (pH 6.8) 30.3g of Tris is dissolved in 200 mL of distilled water and adjusted to pH 6.8 with hydrochloric acid and made up to 250 ml. This can be stored at 4°C.
3. Stock SDS solution: 10 g of SDS is dissolved in distilled water (this requires gentle stirring and heating) and made volume up to 100 ml. This should be stored at room temperature. If the SDS comes out of solution, it can be re-dissolved by gentle heating.
4. 1% ammonium persulphate solution : 0.1 g of ammoniumpersulphate is dissolved in 10 ml of distilled water. This must be prepared freshly on each occasion, immediately prior to use.
5. Stock sample extraction buffer solution : To 12.5 mLof stacking gel buffer is added 20 mL glycerol, 24.1 mL of distilled water, 4 g of SDS and 12 mg bromophenol blue (optional). This is mixed and stored at room temperature. If the SDS comes out of solution, it can be re-dissolved by gentle heating.
concentrated when passed through the stacking gel. If the sample concentration is too high, a continuous system gives better results. The choice of buffer system depends on the nature of protein under analysis.
Figure: 23. Polyacrylamide Gel Electrophoresis gel schematic representation.
Standard reference method for the verification of varieties of Lolium by Polyacrylamide
Gel Electrophoresis (PAGE):
Principle: The standard reference method for the verifying varieties of Pisum and Lolium is by
polyacrylamide gel electrophoresis (PAGE). Seed proteins are extracted from individual Pisum
seeds or from a meal of Lolium seeds, treated with SDS and separated using a discontinuous
SDS-PAGE procedure. The pattern of protein bands found on the gel is characteristic of a variety.
As a guideline for Pisum, it is recommended that 100 individual seeds are used. Very precise
estimates of varietal purity may require a larger sample. If a comparison batches of 50 seeds can
be undertaken in order to minimize the workload. A simple check on the identity of a single major
constituent of a seed lot can be done using less than 50 seeds. For Lolium, a bulk sample of seeds
is analysed. In most cases, whilst this will serve to verify seed lots, it will not permit the detection
of admixtures of two or more varieties.
6160
Note: If de-gassing of the gel mixture is a problem, it is possible to eliminate this step and use a 3 times higher concentration of APS (i.e. 3.75 ml of a 3% solution (0.3 g dissolved in 10 ml of distilled water).
2. Stacking gel: The overlaid water (or isopropanol) is removed from the surface of the main gel with a Pasteur pipette and the gel surface is rinsed briefly with diluted stacking gel buffer, (stock buffer diluted 1:8) and then carefully drained and dried using filter paper.
To make sufficient stacking gel for 4 slab gels the following quantity of solutions are required:
Ø10 ml 1M Tris buffer pH 6.8
Ø67.2 ml gel solution (4.0 g of acrylamide + 0.07 g BIS, made upto 67.2 ml with distilled water); De-gas (in a Buchner flask) and then add.
Ø3.0 ml 1% APS
Ø0.8 ml 10% SDS
Ø80 ml TEMED (full strength, straight out of the bottle)
The stacking gel is poured (using a syringe, as before, if appropriate) to the top of the gel cassette and an acrylic well-forming 'comb' is inserted, ensuring that no air-bubbles are trapped beneath the 'teeth'. The gel is allowed to polymerise (about 1 hour). Again, degassing can be omitted if a higher concentration of APS is used. It is recommended that 3.0 ml of a 2% solution (0.2 g in 10 ml of distilled water) should be sufficient. As an alternative polymerization system for the stacking gel, it is possible to use 0.008% riboflavin solution (freshly prepared), in place of APS. Polymerization should occur if the gels are left in the light, but it may be necessary to use a UV lamp. The precise volume to use should be determined by experimentation, to give a polymerization time of 30-60 minutes. However, as a guide, about 7.5 ml of riboflavin should be used per 50 ml of stacking gel mixture.
Electrophoresis: The electrophoresis tank buffer should be prepared by using 3.0 g Tris, 14.1 g glycine, 1.0 g SDS and made volume up to 1 litre with distilled water (it may be necessary to warm the solution gently to dissolve the SDS). A sufficient volume to fill the electrophoresis apparatus in use (top and bottom chambers) should be freshly prepared. The acrylic comb is removed from the stacking gel (with care; this gel is rather soft) and the resultant wells are washed and partially filled with tank buffer (as above). The samples are loaded into the wells, using a syringe. The gel thickness and the size of the wells largely determine the volume of extract which is loaded. As a guide, between 5 and 15 ml is appropriate in most cases. If required, bromophenol blue (5 µl of a 1% aqueous solution containing 10% glycerol) can be added to a few wells to act as a marker (this can also be incorporated into the sample extraction buffer). If the gel cassette is sealed with adhesive type, this is removed from the lower (bottom) side only. The wells are filled with tank buffer while taking care of do not disturb the samples. The gel is placed in the tank and electrophoresis carried out at 25 mA per gel until the tracking dye has migrated through the stacking gel and then at 45mA per gel until the bromophenol blue is at the bottom of the gel.
6. Gel fixing solution : To 400 mL methanol, 100 mL glacialacetic is added and made up to 1 L with water. About 200 mL is needed per gel.
7. Gel staining solution: 200 mL of following (1) and 10 mL of (2) solution is sufficient for staining one gel.
(1) 15 % Trichloroacetic acid (TCA) (375 g made up to 2.5 L with water)
(2) 1 % PAGE Blue or equivalent in methanol (1 g in 100 ml of methanol)
Procedure : Seed meals for analysis are prepared by passing 0.5–2.0 g of seed through a hammer mill. If preferred, a rotor-type electric coffee grinder or other blender can be used. Diluted extraction buffer is prepared by diluting the stock sample extraction bufferin the ratio 17 buffer : 6 mercaptoethanol : 10 dimethylformamide :17 distilled water (make up only a volume of this extractant sufficient to be used within a day). The seed meal is extracted with diluted sample extraction buffer in the ratio of 80 mg/1.0 ml using, 1.5 mL polypropylene micro-centrifuge tubes. The samples are left for about 1 h at room temperature, resuspended using a vortex mixer and heated for 10 min in a boiling water bath. (A small slit can be made in the caps of the tubes to prevent build-up of pressure.) After cooling, the tubes are centrifuged at 1800× g for 5 min and the clarified supernatants used for electrophoresis.
Gel preparation: The clean and dry gel cassettes are assembled according to the type of equipment in use. Note that if adhesive sealing tape is used in the system, it is advisable to prepare the cassettes at least one day in advance to allow the tape to 'age' and adhere more tightly. Many types of vertical electrophoresis equipment have been found to be suitable. It is strongly recommended that a gel of thickness of 1.5 mm or less is used, as this seems to give better results. The following instructions are for the preparation of a 12.5% acrylamide main gel and a 5% stacking gel
1. Main (resolving) gel : To make 4 slab gels (180 x 140 x 1.5 mm), the following is required:
Ø56.4 ml 1 M Tris pH 8.8
Ø86.25 ml gel solution (19.6 g acrylamide + 0.26 g BIS, made up to 90 ml with distilled water); De-gas (in a Buchner flask) these solutions &then add
Ø3.75ml 1% APS
Ø1.5 ml 10% SDS
Ø75 ml TEMED (full strength, straight out of the bottle)
After careful mixing (do not cause 'foaming') the gel is slowly poured. If appropriate to the type of equipment, a 25 ml disposable syringe and needle can be used to pour the gel mixture into the cassette. The gel should be poured to a height which leaves room for a 3-4 cm layer of stacking gel. The gel surface is carefully overlaid with a 1 cm layer of distilled water (or isopropanol) using a Pasteur pipette or syringe, and the gel is then left to polymerize (about1 hour).
6362
Note: If de-gassing of the gel mixture is a problem, it is possible to eliminate this step and use a 3 times higher concentration of APS (i.e. 3.75 ml of a 3% solution (0.3 g dissolved in 10 ml of distilled water).
2. Stacking gel: The overlaid water (or isopropanol) is removed from the surface of the main gel with a Pasteur pipette and the gel surface is rinsed briefly with diluted stacking gel buffer, (stock buffer diluted 1:8) and then carefully drained and dried using filter paper.
To make sufficient stacking gel for 4 slab gels the following quantity of solutions are required:
Ø10 ml 1M Tris buffer pH 6.8
Ø67.2 ml gel solution (4.0 g of acrylamide + 0.07 g BIS, made upto 67.2 ml with distilled water); De-gas (in a Buchner flask) and then add.
Ø3.0 ml 1% APS
Ø0.8 ml 10% SDS
Ø80 ml TEMED (full strength, straight out of the bottle)
The stacking gel is poured (using a syringe, as before, if appropriate) to the top of the gel cassette and an acrylic well-forming 'comb' is inserted, ensuring that no air-bubbles are trapped beneath the 'teeth'. The gel is allowed to polymerise (about 1 hour). Again, degassing can be omitted if a higher concentration of APS is used. It is recommended that 3.0 ml of a 2% solution (0.2 g in 10 ml of distilled water) should be sufficient. As an alternative polymerization system for the stacking gel, it is possible to use 0.008% riboflavin solution (freshly prepared), in place of APS. Polymerization should occur if the gels are left in the light, but it may be necessary to use a UV lamp. The precise volume to use should be determined by experimentation, to give a polymerization time of 30-60 minutes. However, as a guide, about 7.5 ml of riboflavin should be used per 50 ml of stacking gel mixture.
Electrophoresis: The electrophoresis tank buffer should be prepared by using 3.0 g Tris, 14.1 g glycine, 1.0 g SDS and made volume up to 1 litre with distilled water (it may be necessary to warm the solution gently to dissolve the SDS). A sufficient volume to fill the electrophoresis apparatus in use (top and bottom chambers) should be freshly prepared. The acrylic comb is removed from the stacking gel (with care; this gel is rather soft) and the resultant wells are washed and partially filled with tank buffer (as above). The samples are loaded into the wells, using a syringe. The gel thickness and the size of the wells largely determine the volume of extract which is loaded. As a guide, between 5 and 15 ml is appropriate in most cases. If required, bromophenol blue (5 µl of a 1% aqueous solution containing 10% glycerol) can be added to a few wells to act as a marker (this can also be incorporated into the sample extraction buffer). If the gel cassette is sealed with adhesive type, this is removed from the lower (bottom) side only. The wells are filled with tank buffer while taking care of do not disturb the samples. The gel is placed in the tank and electrophoresis carried out at 25 mA per gel until the tracking dye has migrated through the stacking gel and then at 45mA per gel until the bromophenol blue is at the bottom of the gel.
6. Gel fixing solution : To 400 mL methanol, 100 mL glacialacetic is added and made up to 1 L with water. About 200 mL is needed per gel.
7. Gel staining solution: 200 mL of following (1) and 10 mL of (2) solution is sufficient for staining one gel.
(1) 15 % Trichloroacetic acid (TCA) (375 g made up to 2.5 L with water)
(2) 1 % PAGE Blue or equivalent in methanol (1 g in 100 ml of methanol)
Procedure : Seed meals for analysis are prepared by passing 0.5–2.0 g of seed through a hammer mill. If preferred, a rotor-type electric coffee grinder or other blender can be used. Diluted extraction buffer is prepared by diluting the stock sample extraction bufferin the ratio 17 buffer : 6 mercaptoethanol : 10 dimethylformamide :17 distilled water (make up only a volume of this extractant sufficient to be used within a day). The seed meal is extracted with diluted sample extraction buffer in the ratio of 80 mg/1.0 ml using, 1.5 mL polypropylene micro-centrifuge tubes. The samples are left for about 1 h at room temperature, resuspended using a vortex mixer and heated for 10 min in a boiling water bath. (A small slit can be made in the caps of the tubes to prevent build-up of pressure.) After cooling, the tubes are centrifuged at 1800× g for 5 min and the clarified supernatants used for electrophoresis.
Gel preparation: The clean and dry gel cassettes are assembled according to the type of equipment in use. Note that if adhesive sealing tape is used in the system, it is advisable to prepare the cassettes at least one day in advance to allow the tape to 'age' and adhere more tightly. Many types of vertical electrophoresis equipment have been found to be suitable. It is strongly recommended that a gel of thickness of 1.5 mm or less is used, as this seems to give better results. The following instructions are for the preparation of a 12.5% acrylamide main gel and a 5% stacking gel
1. Main (resolving) gel : To make 4 slab gels (180 x 140 x 1.5 mm), the following is required:
Ø56.4 ml 1 M Tris pH 8.8
Ø86.25 ml gel solution (19.6 g acrylamide + 0.26 g BIS, made up to 90 ml with distilled water); De-gas (in a Buchner flask) these solutions &then add
Ø3.75ml 1% APS
Ø1.5 ml 10% SDS
Ø75 ml TEMED (full strength, straight out of the bottle)
After careful mixing (do not cause 'foaming') the gel is slowly poured. If appropriate to the type of equipment, a 25 ml disposable syringe and needle can be used to pour the gel mixture into the cassette. The gel should be poured to a height which leaves room for a 3-4 cm layer of stacking gel. The gel surface is carefully overlaid with a 1 cm layer of distilled water (or isopropanol) using a Pasteur pipette or syringe, and the gel is then left to polymerize (about1 hour).
6362
·Ampholytes: pH 2–4, pH 4–6, pH 5–8 and pH 4–9
·Ammonium peroxydisulphate (APS)
·N,N,N',N'-Tetramethylethylenediamine (TEMED)
·Urea
·L-Aspartic acid
·L-Glutamic acid
·L-Arginine (base)
·L-Lysine
·Ethylenediamine
·Trichloroacetic acid (TCA)
·Coomassie Brilliant Blue G 250 (or equivalent)
·Coomassie Brilliant Blue R 250 (or equivalent)
·Ethanol (96%)
·Acetic acid (99%)
·Gel-Slick' (or 'Repelsilane', or equivalent)
Solutions:
1. Extraction solution: 30 % (v/v) 2-chloroethanol (30 mL 2-chloroethanol made up to 100 mL with distilled water). This solution can be stored for at least two weeks at room temperature. Optional extraction solution: distilled water.
2. Anode solution: L-aspartic acid (0.83 g) and L-glutamic acid (0.92 g) are dissolved in hot distilled water and diluted to 250 ml. This solution can be stored for two weeks at 4°C.
3. Cathode solution: L-arginine (base) (1.18 g), L-lysine (0.91 g) and ethylene diamine (30.00 mL) are dissolved in distilled water and diluted to 250 mL. This solution can be stored for two weeks at 4°C.
4. Stock gel solution: Acrylamide (16.57 g) and bisacrylamide (0.43 g) are dissolved in distilled water and diluted to 250 mL. This solution can be stored for up to two weeks at 4°C.
5. Gel fixing solution:Trichloroacetic acid (TCA)(approx. 12 % (w/v) solution). Stock solution: 1 kg TCA dissolved in 450 mL distilled water. Before use, 120 mL stock solution is diluted with 880 mL distilled water, to give an approximately 12 % TCA solution. About 400 mL is needed for one gel, and the solution can be used up to three times.
6. Gel staining solution: Coomassie Blue R 250 (0.45 g), Coomassie Blue G 250 (1.35 g), acetic acid (330 mL) and ethanol (540 mL) are mixed and made up to 3000 mL with distilled water; 400 mL is sufficient for staining one gel.
oThe temperature should be maintained at 15-20 C, if possible, by circulating tap-water (or coolant) through the tank buffer.
Fixing and staining: Several different approaches can be used for fixing and staining the proteins. If results are not required very rapidly, then at the end of electrophoresis, the gel is removed from the tank, taken from the cassette and incubated in fixing solution, with slow shaking, for at least 1 hour. The gel is rinsed in distilled water (5 min), and then stained by incubation (at least 2 hrs, usually overnight) in gel staining solution. When properly stained, the gel is rinsed in distilled water for 2-3 hours (TCA can be added if the background is very blue) and then sealed in a polythene bag for examination or photography. Gels can be stored
ofor many months at 4 C, if sealed properly. For more rapid staining, the gel can be fixed and
ostained at a higher temperature (80 C) in an oven for 30 min and then, following cooling, destained in a solution containing 10% glacial acetic acid and 35% ethanol for a further 30-60 min, with shaking.
Evaluation of results: The methods are mostly used in a comparatively way, i.e. is the protein pattern of the sample identical to that of the authentic reference variety? It is also useful to include on each gel a sample of a known variety with a well-described and established protein banding pattern. This can serve as a quality standard for gels – if the banding pattern of the reference variety is clearly observed, then the gel can be analyzed to provide useful information. In addition, gels can be 'calibrated' by the inclusion of standard proteins of known molecular weights on each gel, which allows the calculation of the molecular weights of protein bands of interest.
Standard procedure for verifying varieties of Zea mays (Maize):
The standard reference method for measuring the hybrid purity and verifying varieties of Zea mays (maize) is by ultrathin-layer isoelectric focusing (UTLIEF). The alcohol- soluble proteins (zeins) or water soluble proteins are extracted from individual maize seeds and separated by isoelectric focusing (IEF) in ultrathin-layer gels. The pattern of protein bands found on the gel is characteristic for a variety or an inbred line. Also, it is generally possible to estimate the purity of hybrid samples by finding one or more zein bands in the male parent that are lacking in the female parent (and present in the hybrid). These bands can be used as marker bands for the verification of hybrids and as a means of estimating hybrid purity. Ultrathin gels can be run at higher voltages with shorter running times, and stain more quickly than conventional gels.
Apparatus: Any suitable horizontal electrophoresis apparatus with a cooling system and high voltage power supply may be used.
Chemicals: All chemicals should be of 'analytical reagent' grade or equivalent.
·2-Chloroethanol
·Acrylamide ('specially purified for electrophoresis')
·Bisacrylamide (BIS) ('specially purified forelectrophoresis')
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·Ampholytes: pH 2–4, pH 4–6, pH 5–8 and pH 4–9
·Ammonium peroxydisulphate (APS)
·N,N,N',N'-Tetramethylethylenediamine (TEMED)
·Urea
·L-Aspartic acid
·L-Glutamic acid
·L-Arginine (base)
·L-Lysine
·Ethylenediamine
·Trichloroacetic acid (TCA)
·Coomassie Brilliant Blue G 250 (or equivalent)
·Coomassie Brilliant Blue R 250 (or equivalent)
·Ethanol (96%)
·Acetic acid (99%)
·Gel-Slick' (or 'Repelsilane', or equivalent)
Solutions:
1. Extraction solution: 30 % (v/v) 2-chloroethanol (30 mL 2-chloroethanol made up to 100 mL with distilled water). This solution can be stored for at least two weeks at room temperature. Optional extraction solution: distilled water.
2. Anode solution: L-aspartic acid (0.83 g) and L-glutamic acid (0.92 g) are dissolved in hot distilled water and diluted to 250 ml. This solution can be stored for two weeks at 4°C.
3. Cathode solution: L-arginine (base) (1.18 g), L-lysine (0.91 g) and ethylene diamine (30.00 mL) are dissolved in distilled water and diluted to 250 mL. This solution can be stored for two weeks at 4°C.
4. Stock gel solution: Acrylamide (16.57 g) and bisacrylamide (0.43 g) are dissolved in distilled water and diluted to 250 mL. This solution can be stored for up to two weeks at 4°C.
5. Gel fixing solution:Trichloroacetic acid (TCA)(approx. 12 % (w/v) solution). Stock solution: 1 kg TCA dissolved in 450 mL distilled water. Before use, 120 mL stock solution is diluted with 880 mL distilled water, to give an approximately 12 % TCA solution. About 400 mL is needed for one gel, and the solution can be used up to three times.
6. Gel staining solution: Coomassie Blue R 250 (0.45 g), Coomassie Blue G 250 (1.35 g), acetic acid (330 mL) and ethanol (540 mL) are mixed and made up to 3000 mL with distilled water; 400 mL is sufficient for staining one gel.
oThe temperature should be maintained at 15-20 C, if possible, by circulating tap-water (or coolant) through the tank buffer.
Fixing and staining: Several different approaches can be used for fixing and staining the proteins. If results are not required very rapidly, then at the end of electrophoresis, the gel is removed from the tank, taken from the cassette and incubated in fixing solution, with slow shaking, for at least 1 hour. The gel is rinsed in distilled water (5 min), and then stained by incubation (at least 2 hrs, usually overnight) in gel staining solution. When properly stained, the gel is rinsed in distilled water for 2-3 hours (TCA can be added if the background is very blue) and then sealed in a polythene bag for examination or photography. Gels can be stored
ofor many months at 4 C, if sealed properly. For more rapid staining, the gel can be fixed and
ostained at a higher temperature (80 C) in an oven for 30 min and then, following cooling, destained in a solution containing 10% glacial acetic acid and 35% ethanol for a further 30-60 min, with shaking.
Evaluation of results: The methods are mostly used in a comparatively way, i.e. is the protein pattern of the sample identical to that of the authentic reference variety? It is also useful to include on each gel a sample of a known variety with a well-described and established protein banding pattern. This can serve as a quality standard for gels – if the banding pattern of the reference variety is clearly observed, then the gel can be analyzed to provide useful information. In addition, gels can be 'calibrated' by the inclusion of standard proteins of known molecular weights on each gel, which allows the calculation of the molecular weights of protein bands of interest.
Standard procedure for verifying varieties of Zea mays (Maize):
The standard reference method for measuring the hybrid purity and verifying varieties of Zea mays (maize) is by ultrathin-layer isoelectric focusing (UTLIEF). The alcohol- soluble proteins (zeins) or water soluble proteins are extracted from individual maize seeds and separated by isoelectric focusing (IEF) in ultrathin-layer gels. The pattern of protein bands found on the gel is characteristic for a variety or an inbred line. Also, it is generally possible to estimate the purity of hybrid samples by finding one or more zein bands in the male parent that are lacking in the female parent (and present in the hybrid). These bands can be used as marker bands for the verification of hybrids and as a means of estimating hybrid purity. Ultrathin gels can be run at higher voltages with shorter running times, and stain more quickly than conventional gels.
Apparatus: Any suitable horizontal electrophoresis apparatus with a cooling system and high voltage power supply may be used.
Chemicals: All chemicals should be of 'analytical reagent' grade or equivalent.
·2-Chloroethanol
·Acrylamide ('specially purified for electrophoresis')
·Bisacrylamide (BIS) ('specially purified forelectrophoresis')
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3. Electrophoresis: The gel is placed on the pre-cooled (10°C) cooling plate of the
horizontal electrophoresis apparatus. To ensure better gel adhesion and cooling, a thin
layer of water is placed between the gel and the plate. The electrode wicks are soaked in
the appropriate solution and placed at either end of the gel. Samples (approx. 22 ìL) are
loaded in the applicator strip 0.5 cm below the bufferwick of the anode and focusing
carried out at 2500 V, 15 mA, 40 W for about 70 min until completion (for one gel).
a. Double-focusing is possible with this method and it is then necessary to place the
cathode wick in the centre of the gel, with anode wicks at both ends.
b. The precise conditions and times required for focusing will vary depending on the
dimensions of the gel, the type of maize hybrid, inbred line etc., and may need to be
determined empirically.
4. Fixing and staining: At the end of the electrophoresis, the gel is removed andincubated
in fixing solution with slow shaking, for at least 20 min. The gel is then stained by
shaking in gel staining solution for 50 min. Destaining takes about 15 min, followed by
brief rinsing in distilled water. The gel is dried overnight at room temperature or in an
oven at 70°C for 20 min and then can be sealed with adhesive film. Gels can be stored for
many years at room temperature.
Standard procedure for verifying varieties of Avena sativa (oats):
Principle: The standard reference method for verifying varieties of Avena sativa (oats) is by
polyacrylamide gel electrophoresis (PAGE). The urea/ethylene glycol-soluble proteins
(primarily avenins) are extracted from seeds and separated by PAGE at pH 3.2. The pattern of
protein bands can be considered as a 'fingerprint' of a variety. The 'fingerprints' can be used to
identify (to verify the identity of) unknown samples and mixtures, by single seed analysis. As
a guideline, it is recommended that 100 seeds are used or sequential testing using subsamples
of 50 seeds can be undertaken in order to minimize the workload. A simple check of the
identity of a single major constituent of a seed lot can be done using less than 50 seeds.
Chemicals required: The following different chemicals (analytical reagent grade) are
required
·Acrylamide (specially purified for electrophoresis)
·Bisacrylamide ('specially purified for electrophoresis')
·Urea
·Glacial acetic acid
·Glycine
·Ferrous sulphate
·Hydrogen peroxide
·Pyronin G or Pyronin Y
7. Gel destaining solution: Ethanol (750 mL) and acetic acid (125 mL), made up to 2500 ml with distilled water.
Procedure: The following different steps are to performed to conduct the IEF in maize.
1. Protein extraction: A single dry seed is bisected longitudinally and one half of this seed crushed to a fine powder with pestle and mortar. Approximately 50 mg of the seed meal is extracted with 0.2 mL of extraction solution in a microtitre plate or a microcentrifuge tube. The samples are left for about 1 h at 20°C. After this time, the microtitre plateor microtube is treated with ultrasound for 30s and then centrifuged at 2000 × g for 5 min. The clarified supernatant is used for electrophoresis. Frozen at –20 °C, the remaining extract will keep for up to 3 months.
2. Gel preparation: Gels are made up either directly between two thin glass plates or between a polyester carrier sheet held on glass and a glass plate. The plates or sheets must be treated before use, one (carrier) with a silylating reagent to make the gel adhere and one (cover) with 'Gel-Slick' (or equivalent) to prevent gel adhesion. Commercially available prepared gel carrier sheets (e.g. 'Gel-Bond') can also be used. The clean and dry gel cassettes are assembled, according to the type of equipment in use. A gel thickness of 0.12 mm is recommended, which can be achieved by the use of a defined thickness of adhesive tape as a spacer. The following are taken and mixed:
·0 mL stock gel solution
·16 g urea
·0.55 mL ampholytes (pH 2–4)
·0.55 mL ampholytes (pH 4–6)
·1.40 mL ampholytes (pH 5–8)
·1.90 mL ampholytes (pH 4–9)
Optional gel solution:
·50 mL stock gel solution
·16 g urea
·1.5 g taurin
·mL ampholytes (pH 5–8)
·1.50 mL ampholytes (pH 2–11)
Note: dissolve taurin first into the stock gel solution due to its slow solubility
For polymerization, 0.35 mL APS (20 % w/v solution freshly prepared) and 0.05 mL TEMED (full strength) are added carefully, to avoid introducing excessive amounts of air. This will be sufficient for 10 gels of the dimensions 240 × 180 × 0.12 mm (one gel requires 6.5 mL). After careful mixing, the gel is poured onto the carrier plate/ sheet, the cover plate is carefully lowered and the gel'cassette' left to polymerise for at least 45 min. Gels not immediately used can be stored wrapped at 4°C for at least one week.
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3. Electrophoresis: The gel is placed on the pre-cooled (10°C) cooling plate of the
horizontal electrophoresis apparatus. To ensure better gel adhesion and cooling, a thin
layer of water is placed between the gel and the plate. The electrode wicks are soaked in
the appropriate solution and placed at either end of the gel. Samples (approx. 22 ìL) are
loaded in the applicator strip 0.5 cm below the bufferwick of the anode and focusing
carried out at 2500 V, 15 mA, 40 W for about 70 min until completion (for one gel).
a. Double-focusing is possible with this method and it is then necessary to place the
cathode wick in the centre of the gel, with anode wicks at both ends.
b. The precise conditions and times required for focusing will vary depending on the
dimensions of the gel, the type of maize hybrid, inbred line etc., and may need to be
determined empirically.
4. Fixing and staining: At the end of the electrophoresis, the gel is removed andincubated
in fixing solution with slow shaking, for at least 20 min. The gel is then stained by
shaking in gel staining solution for 50 min. Destaining takes about 15 min, followed by
brief rinsing in distilled water. The gel is dried overnight at room temperature or in an
oven at 70°C for 20 min and then can be sealed with adhesive film. Gels can be stored for
many years at room temperature.
Standard procedure for verifying varieties of Avena sativa (oats):
Principle: The standard reference method for verifying varieties of Avena sativa (oats) is by
polyacrylamide gel electrophoresis (PAGE). The urea/ethylene glycol-soluble proteins
(primarily avenins) are extracted from seeds and separated by PAGE at pH 3.2. The pattern of
protein bands can be considered as a 'fingerprint' of a variety. The 'fingerprints' can be used to
identify (to verify the identity of) unknown samples and mixtures, by single seed analysis. As
a guideline, it is recommended that 100 seeds are used or sequential testing using subsamples
of 50 seeds can be undertaken in order to minimize the workload. A simple check of the
identity of a single major constituent of a seed lot can be done using less than 50 seeds.
Chemicals required: The following different chemicals (analytical reagent grade) are
required
·Acrylamide (specially purified for electrophoresis)
·Bisacrylamide ('specially purified for electrophoresis')
·Urea
·Glacial acetic acid
·Glycine
·Ferrous sulphate
·Hydrogen peroxide
·Pyronin G or Pyronin Y
7. Gel destaining solution: Ethanol (750 mL) and acetic acid (125 mL), made up to 2500 ml with distilled water.
Procedure: The following different steps are to performed to conduct the IEF in maize.
1. Protein extraction: A single dry seed is bisected longitudinally and one half of this seed crushed to a fine powder with pestle and mortar. Approximately 50 mg of the seed meal is extracted with 0.2 mL of extraction solution in a microtitre plate or a microcentrifuge tube. The samples are left for about 1 h at 20°C. After this time, the microtitre plateor microtube is treated with ultrasound for 30s and then centrifuged at 2000 × g for 5 min. The clarified supernatant is used for electrophoresis. Frozen at –20 °C, the remaining extract will keep for up to 3 months.
2. Gel preparation: Gels are made up either directly between two thin glass plates or between a polyester carrier sheet held on glass and a glass plate. The plates or sheets must be treated before use, one (carrier) with a silylating reagent to make the gel adhere and one (cover) with 'Gel-Slick' (or equivalent) to prevent gel adhesion. Commercially available prepared gel carrier sheets (e.g. 'Gel-Bond') can also be used. The clean and dry gel cassettes are assembled, according to the type of equipment in use. A gel thickness of 0.12 mm is recommended, which can be achieved by the use of a defined thickness of adhesive tape as a spacer. The following are taken and mixed:
·0 mL stock gel solution
·16 g urea
·0.55 mL ampholytes (pH 2–4)
·0.55 mL ampholytes (pH 4–6)
·1.40 mL ampholytes (pH 5–8)
·1.90 mL ampholytes (pH 4–9)
Optional gel solution:
·50 mL stock gel solution
·16 g urea
·1.5 g taurin
·mL ampholytes (pH 5–8)
·1.50 mL ampholytes (pH 2–11)
Note: dissolve taurin first into the stock gel solution due to its slow solubility
For polymerization, 0.35 mL APS (20 % w/v solution freshly prepared) and 0.05 mL TEMED (full strength) are added carefully, to avoid introducing excessive amounts of air. This will be sufficient for 10 gels of the dimensions 240 × 180 × 0.12 mm (one gel requires 6.5 mL). After careful mixing, the gel is poured onto the carrier plate/ sheet, the cover plate is carefully lowered and the gel'cassette' left to polymerise for at least 45 min. Gels not immediately used can be stored wrapped at 4°C for at least one week.
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the equipment used). Samples (18 ìL) are loaded into the wells and the gel cassettes are placed in the tank, ensuring that the sample wells are completely filled. Electrophoresis is carried out 10 min at 200 V and then at 500 V (constant voltage) for twice the time taken for the pyronine G marker dye to leave the gel. Water should be circulated through the buffer tank to maintain the temperature at 15–20°C.
4. Fixing and staining: The gel cassette is removed from the tank, opened and the gel placed in a plastic box containing about 200 mL fixing and staining solution. Staining is complete in one day. When properly stained, the gel is rinsed in distilled water for 2–3 h, and can then be photographed. Any blue background in the gel is removed by rinsing in 10 % TCA solution.
Nomenclature of avenin bands: The method is best used comparatively i.e., by comparing the avenin profile of an unknown sample with that of authentic reference samples extracted and analysed at the same time. There is no internationally agreed system of nomenclature for avenins, and bands are usually numbered sequentially or their relative mobilities measured.
A. Molecular Markers:
The Genetic purity refers to the percentage of contamination by seeds or genetic materials of other varieties or species. The genetic purity test is necessary for seed certification of different categories of seed. The genetic purity is deteriorating in each stage of seed multiplication and subsequent handling. Even then, the genetic purity of produced seeds gets deteriorated because of mechanical admixtures, out crossing, residual segregation, ecological adaptation of cultivar and mutation, which at times are unavoidable. Therefore, utmost care is taken to maintain the genetic purity and identity of seeds during multiplication. Hence, it is necessary to ensure the genetic purity and identity of seeds before it reaches the farmers. In those cases the molecular markers will be of immense utility for varietal identity and purity analysis. The emergence of DNA-based markers has changed the practice of variety identification techniques. The dramatic advances in molecular genetics over the last few years have provided workers involved in the conservation of plant genetic resources with a range of new techniques for easy and reliable identification of plant species. Properties desirable for ideal DNA markers include highly polymorphic nature, codominant inheritance (determination of homozygous and heterozygous states of diploid organisms), frequent occurrence in the genome, selective neutral behavior (the DNA sequences of any organism are neutral to environmental conditions or management practices), easy access (availability), easy and fast assay, high reproducibility, and easy exchange of data between laboratories. Molecular markers may be broadly classified into three categories in the chronological order of their development.
1. First generation of markers was the hybridization based markers. These are so called because the DNA profile is visualized through hybridization of DNA with radioactively labeled probes of known sequence e.g. RFLP.
·Trichloroacetic acid
·Ethylene glycol
·Methanol
·Coomassie Brilliant Blue G 250 or Serva Blue G (or equivalent)
Solutions: The following different solutions are need to prepared to run the PAGE
1. Extraction solution: Pyronin G (or Pyronin Y) (0.05 %(w/v) in 3M urea (18 % w/v) in a 75:25 (v/v) mixture of ethylene glycol and water (keep cold or prepare fresh).
2. Stock tank buffer solution: Glacial acetic acid (4 mL) and glycine (0.4 g), made up to 1 L with water; keep cold.
3. Stock gel buffer solution: Glacial acetic acid (20 mL)and glycine (1.0 g), made up to 1 L with water; keep cold.
4. Fixing and staining solution: Coomassie Blue G 250 or Serva Blue G (1 g) in methanol (250 mL) + 100 g trichloroacetic acid dissolved in 800 mL water.
Procedure
1. Protein extraction: The lemma and palea are removed, then samples are crushed with pliers and milled to a fine powder using a pestle and mortar (an electric blender may be used).The meal is extracted in 1.5 mL polypropylene centrifuge tubes. Extraction solution (0.1 mL per ground seed) is added, the contents of the tubes are thoroughly mixed with a vortex mixer and the tubes are allowed to stand for 2h or overnight at room temperature. The tubes are centrifuged for 15 min at 14 000 × g and the supernatants used for electrophoresis.
2. Gel preparation: Clean and dry cassettes are assembled, according to the design of the equipment. Treating the glass plates with silicon prior to assembly can facilitate subsequent removal of the gel. The gel cassettes can incorporate a plastic backing sheet (e.g. 'Gel Bond PAG', FMC Corporation). This supports the gel during subsequent operations. To make 2 slab gels (160 × 180 × 1.5 mm), the following is required: stock gel buffer (approx. 60 mL) and the following added- acrylamide (12.5 g), bisacrylamide (0.4 g), urea (6.0 g), ascorbic acid (0.1 g), ferrous sulphate (0.005 g). The solution is stirred and made up to 100 mL with stock gel buffer solution. Freshly prepared 0.6 % (v/v) hydrogen peroxide solution (0.2 mL per 100 mL of gel solution) is added, mixed quickly and the gel poured. Note that the gel mixture can be cooled to near freezing prior to the addition of the peroxide. An acrylic 'comb' is placed in the top of the cassette, to make wells in the gel. The gel mixture should over-fill the cassette, or be overlaid with water, to ensure satisfactory polymerization of the upper surface. Polymerization should be complete in 5–10 min.
3. Electrophoresis: The acrylic comb is removed from the gel and the sample wells filled with tank buffer. The tank is filled with an appropriate volume of buffer (depending on
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the equipment used). Samples (18 ìL) are loaded into the wells and the gel cassettes are placed in the tank, ensuring that the sample wells are completely filled. Electrophoresis is carried out 10 min at 200 V and then at 500 V (constant voltage) for twice the time taken for the pyronine G marker dye to leave the gel. Water should be circulated through the buffer tank to maintain the temperature at 15–20°C.
4. Fixing and staining: The gel cassette is removed from the tank, opened and the gel placed in a plastic box containing about 200 mL fixing and staining solution. Staining is complete in one day. When properly stained, the gel is rinsed in distilled water for 2–3 h, and can then be photographed. Any blue background in the gel is removed by rinsing in 10 % TCA solution.
Nomenclature of avenin bands: The method is best used comparatively i.e., by comparing the avenin profile of an unknown sample with that of authentic reference samples extracted and analysed at the same time. There is no internationally agreed system of nomenclature for avenins, and bands are usually numbered sequentially or their relative mobilities measured.
A. Molecular Markers:
The Genetic purity refers to the percentage of contamination by seeds or genetic materials of other varieties or species. The genetic purity test is necessary for seed certification of different categories of seed. The genetic purity is deteriorating in each stage of seed multiplication and subsequent handling. Even then, the genetic purity of produced seeds gets deteriorated because of mechanical admixtures, out crossing, residual segregation, ecological adaptation of cultivar and mutation, which at times are unavoidable. Therefore, utmost care is taken to maintain the genetic purity and identity of seeds during multiplication. Hence, it is necessary to ensure the genetic purity and identity of seeds before it reaches the farmers. In those cases the molecular markers will be of immense utility for varietal identity and purity analysis. The emergence of DNA-based markers has changed the practice of variety identification techniques. The dramatic advances in molecular genetics over the last few years have provided workers involved in the conservation of plant genetic resources with a range of new techniques for easy and reliable identification of plant species. Properties desirable for ideal DNA markers include highly polymorphic nature, codominant inheritance (determination of homozygous and heterozygous states of diploid organisms), frequent occurrence in the genome, selective neutral behavior (the DNA sequences of any organism are neutral to environmental conditions or management practices), easy access (availability), easy and fast assay, high reproducibility, and easy exchange of data between laboratories. Molecular markers may be broadly classified into three categories in the chronological order of their development.
1. First generation of markers was the hybridization based markers. These are so called because the DNA profile is visualized through hybridization of DNA with radioactively labeled probes of known sequence e.g. RFLP.
·Trichloroacetic acid
·Ethylene glycol
·Methanol
·Coomassie Brilliant Blue G 250 or Serva Blue G (or equivalent)
Solutions: The following different solutions are need to prepared to run the PAGE
1. Extraction solution: Pyronin G (or Pyronin Y) (0.05 %(w/v) in 3M urea (18 % w/v) in a 75:25 (v/v) mixture of ethylene glycol and water (keep cold or prepare fresh).
2. Stock tank buffer solution: Glacial acetic acid (4 mL) and glycine (0.4 g), made up to 1 L with water; keep cold.
3. Stock gel buffer solution: Glacial acetic acid (20 mL)and glycine (1.0 g), made up to 1 L with water; keep cold.
4. Fixing and staining solution: Coomassie Blue G 250 or Serva Blue G (1 g) in methanol (250 mL) + 100 g trichloroacetic acid dissolved in 800 mL water.
Procedure
1. Protein extraction: The lemma and palea are removed, then samples are crushed with pliers and milled to a fine powder using a pestle and mortar (an electric blender may be used).The meal is extracted in 1.5 mL polypropylene centrifuge tubes. Extraction solution (0.1 mL per ground seed) is added, the contents of the tubes are thoroughly mixed with a vortex mixer and the tubes are allowed to stand for 2h or overnight at room temperature. The tubes are centrifuged for 15 min at 14 000 × g and the supernatants used for electrophoresis.
2. Gel preparation: Clean and dry cassettes are assembled, according to the design of the equipment. Treating the glass plates with silicon prior to assembly can facilitate subsequent removal of the gel. The gel cassettes can incorporate a plastic backing sheet (e.g. 'Gel Bond PAG', FMC Corporation). This supports the gel during subsequent operations. To make 2 slab gels (160 × 180 × 1.5 mm), the following is required: stock gel buffer (approx. 60 mL) and the following added- acrylamide (12.5 g), bisacrylamide (0.4 g), urea (6.0 g), ascorbic acid (0.1 g), ferrous sulphate (0.005 g). The solution is stirred and made up to 100 mL with stock gel buffer solution. Freshly prepared 0.6 % (v/v) hydrogen peroxide solution (0.2 mL per 100 mL of gel solution) is added, mixed quickly and the gel poured. Note that the gel mixture can be cooled to near freezing prior to the addition of the peroxide. An acrylic 'comb' is placed in the top of the cassette, to make wells in the gel. The gel mixture should over-fill the cassette, or be overlaid with water, to ensure satisfactory polymerization of the upper surface. Polymerization should be complete in 5–10 min.
3. Electrophoresis: The acrylic comb is removed from the gel and the sample wells filled with tank buffer. The tank is filled with an appropriate volume of buffer (depending on
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@No prior sequence information is needed.
@Many polymorphic markers can be generated with a single primer.
Disadvantages:
@Reproducibility is low as the results vary greatly with different reaction conditions because the conditions used for PCR, like annealing temperatures, random primers and concentration of reagents etc. is not stringent.
@They are dominant markers.
C. Amplified Fragment Length Polymorphism (AFLP):
This method was first shown by Vos et al., in 1995.This marker system combines the use of restriction enzymes and PCR. The AFLP fragments are obtained by digestion of DNA with restriction enzymes followed by the ligation of digested products with oligonucleotide adapters of known sequences and their subsequent amplification by PCR. The amplified products may be 80 to 500 bp in size.
Advantages:
@A large number of polymorphic fragments can be obtained in a single experiment.
Disadvantages:
@The process is complex as compared to the more advanced techniques of marker development (discussed ahead).
@The fragments are mostly scored as dominant markers.
D. Simple Sequence Repeats (SSR):
Microsatellites or SSR are tandem repeats of 1 – 6 nucleotides. For example, (A) n, (AT) n, (ATG) n, (GATT) n, (CTACG) n, (TACGAC) n, and so on. They are abundant in genomes of all organisms. The sequence of unique flanking regions of SSR can be used to design primers and carry out PCR to amplify SSR containing sequences. The polymorphism can be detected by agarose gel electrophoresis if differences are large enough (agarose gels can detect differences greater than 10 base pair), or polyacrylamide gel electrophoresis or capillary electrophoresis (sensitive enough to detect differences as low as 1 to 2 bases).
Advantages:
@They are usually codominant.
@The method is easy and quick.
@Automation can be done using fluorescently labeled primers which allow detection by a technique called capillary electrophoresis, thus omitting the use of agarose of polyacrylamide gels.
@Low amount of DNA is required.
@Reproducibility is high.
@These markers can be easily exchanged between different laboratories.
2. The second generation of markers were the PCR based markers, as their assay was carried out through amplification using either arbitrary or sequence specific primers. eg. RAPD, AFLP, SSR, SCAR and VNTR.
3. The third generation markers are the most recent ones, called as SNPs. Their detection requires sequence information. With the advancement in the field of DNA sequencing, SNPs have become very popular in the last few years.
The followings are the description for different types of molecular markers:
A. Restriction Fragment Length Polymorphism (RFLP): RFLP was the first molecular marker system to be used. Its application was first shown by Botstein et al. (1980) for linkage mapping in humans. Later, it was a widely accepted system for use in plants. The restriction enzymes recognize and cut DNA at specific sites called restriction sites. Polymorphism can be detected if there is any variation in any two individuals at the restriction sites of any particular restriction enzyme. Such polymorphism can be visualized as variation in length after cutting the DNA with the enzyme and carrying out Southern blotting. RFLP analysis involves digestion of DNA by restriction enzyme followed by separation of the fragments using agarose gel electrophoresis and detection by Southern hybridization using a labeled probe.
Advantages:
@It is a codominant marker.
@The results are highly reproducible.
Disadvantages:
@The process is long and tedious.
@Large amount of DNA is required.
@Use of radioactively labeled probes.
B. Random Amplified Polymorphic DNA (RAPD):
It was first shown by Welsh and McClelland (1990). If the length of primers used for PCR, is shorter (9-10nt) than that normally used (18-25nt) and their sequence is arbitrary and the amplification is not done under stringent conditions, several loci are amplified which are unique for a genotype and can be used for detection of polymorphism. For RAPD analysis, PCR is carried out using short (generally around 10 nucleotides long) arbitrary (any random sequence) primers. They serve as both forward and reverse primer and amplify many genomic fragments simultaneously. Amplified fragments usually range from 500 base pair to 5 kb. They can be separated and visualized using agarose gel electrophoresis.
Advantages:
@The method is quick and easy.
@Amount of DNA required is very less.
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@No prior sequence information is needed.
@Many polymorphic markers can be generated with a single primer.
Disadvantages:
@Reproducibility is low as the results vary greatly with different reaction conditions because the conditions used for PCR, like annealing temperatures, random primers and concentration of reagents etc. is not stringent.
@They are dominant markers.
C. Amplified Fragment Length Polymorphism (AFLP):
This method was first shown by Vos et al., in 1995.This marker system combines the use of restriction enzymes and PCR. The AFLP fragments are obtained by digestion of DNA with restriction enzymes followed by the ligation of digested products with oligonucleotide adapters of known sequences and their subsequent amplification by PCR. The amplified products may be 80 to 500 bp in size.
Advantages:
@A large number of polymorphic fragments can be obtained in a single experiment.
Disadvantages:
@The process is complex as compared to the more advanced techniques of marker development (discussed ahead).
@The fragments are mostly scored as dominant markers.
D. Simple Sequence Repeats (SSR):
Microsatellites or SSR are tandem repeats of 1 – 6 nucleotides. For example, (A) n, (AT) n, (ATG) n, (GATT) n, (CTACG) n, (TACGAC) n, and so on. They are abundant in genomes of all organisms. The sequence of unique flanking regions of SSR can be used to design primers and carry out PCR to amplify SSR containing sequences. The polymorphism can be detected by agarose gel electrophoresis if differences are large enough (agarose gels can detect differences greater than 10 base pair), or polyacrylamide gel electrophoresis or capillary electrophoresis (sensitive enough to detect differences as low as 1 to 2 bases).
Advantages:
@They are usually codominant.
@The method is easy and quick.
@Automation can be done using fluorescently labeled primers which allow detection by a technique called capillary electrophoresis, thus omitting the use of agarose of polyacrylamide gels.
@Low amount of DNA is required.
@Reproducibility is high.
@These markers can be easily exchanged between different laboratories.
2. The second generation of markers were the PCR based markers, as their assay was carried out through amplification using either arbitrary or sequence specific primers. eg. RAPD, AFLP, SSR, SCAR and VNTR.
3. The third generation markers are the most recent ones, called as SNPs. Their detection requires sequence information. With the advancement in the field of DNA sequencing, SNPs have become very popular in the last few years.
The followings are the description for different types of molecular markers:
A. Restriction Fragment Length Polymorphism (RFLP): RFLP was the first molecular marker system to be used. Its application was first shown by Botstein et al. (1980) for linkage mapping in humans. Later, it was a widely accepted system for use in plants. The restriction enzymes recognize and cut DNA at specific sites called restriction sites. Polymorphism can be detected if there is any variation in any two individuals at the restriction sites of any particular restriction enzyme. Such polymorphism can be visualized as variation in length after cutting the DNA with the enzyme and carrying out Southern blotting. RFLP analysis involves digestion of DNA by restriction enzyme followed by separation of the fragments using agarose gel electrophoresis and detection by Southern hybridization using a labeled probe.
Advantages:
@It is a codominant marker.
@The results are highly reproducible.
Disadvantages:
@The process is long and tedious.
@Large amount of DNA is required.
@Use of radioactively labeled probes.
B. Random Amplified Polymorphic DNA (RAPD):
It was first shown by Welsh and McClelland (1990). If the length of primers used for PCR, is shorter (9-10nt) than that normally used (18-25nt) and their sequence is arbitrary and the amplification is not done under stringent conditions, several loci are amplified which are unique for a genotype and can be used for detection of polymorphism. For RAPD analysis, PCR is carried out using short (generally around 10 nucleotides long) arbitrary (any random sequence) primers. They serve as both forward and reverse primer and amplify many genomic fragments simultaneously. Amplified fragments usually range from 500 base pair to 5 kb. They can be separated and visualized using agarose gel electrophoresis.
Advantages:
@The method is quick and easy.
@Amount of DNA required is very less.
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Disadvantages:
@The cost of development of SSR markers is high if sequence information of any
organism is not already available.
E. Single Nucleotide Polymorphism (SNP):
Single nucleotide polymorphism or SNPs are single base substitutions. SNP is the most
abundant type of molecular marker in all organisms (for example, one SNP about every 20 bp
in wheat in some regions of the genome). If SNPs can be detected between any two
individuals, they can be used as molecular markers. SNPs may be detected using many
different techniques. An easy way of detection is available when SNP causes variation in the
restriction site of an enzyme. In such a case, difference in the recognition of template DNA of
Figure 24. The PCR machine, gel electrophoresis and schematic representation of DNA amplification.
different individuals, by the restriction enzyme, may lead to different patterns of bands when
DNA is digested, resulting in polymorphism.
Advantages:
@SNPs are abundant in genomes so one can generate a large number of molecular markers
@There are many methods available for detection of SNPs and they can be automated.
Disadvantages:
@The costs for marker development and genotyping are very high.
@Technical expertise is required.
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Disadvantages:
@The cost of development of SSR markers is high if sequence information of any
organism is not already available.
E. Single Nucleotide Polymorphism (SNP):
Single nucleotide polymorphism or SNPs are single base substitutions. SNP is the most
abundant type of molecular marker in all organisms (for example, one SNP about every 20 bp
in wheat in some regions of the genome). If SNPs can be detected between any two
individuals, they can be used as molecular markers. SNPs may be detected using many
different techniques. An easy way of detection is available when SNP causes variation in the
restriction site of an enzyme. In such a case, difference in the recognition of template DNA of
Figure 24. The PCR machine, gel electrophoresis and schematic representation of DNA amplification.
different individuals, by the restriction enzyme, may lead to different patterns of bands when
DNA is digested, resulting in polymorphism.
Advantages:
@SNPs are abundant in genomes so one can generate a large number of molecular markers
@There are many methods available for detection of SNPs and they can be automated.
Disadvantages:
@The costs for marker development and genotyping are very high.
@Technical expertise is required.
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NOTES NOTES
NOTES NOTES
NOTES
NOTES
Published by:
Director
ICAR-Indian Grassland and Fodder Research Institute
Gwalior Road, Jhansi-284003 (U.P.)
Ph: 0510-2730666
Website: www.igfri.res.in;
e-mail: [email protected]
