training manual - nbpgr
TRANSCRIPT
TRAINING MANUAL
International Training Programme
on
MANAGEMENT OF PLANT GENETIC RESOURCES
for
Officers from Directorate of Seed Testing and Certification Ministry of Agriculture,
Baghdad, Republic of Iraq
(15 -20 July 2019)
Programme Director
Dr Kuldeep Singh Director
ICAR-NBPGR
Division of Germplasm Conservation
ICAR-National Bureau of Plant Genetic Resources
Pusa Campus, New Delhi-110 012
Compiled and Edited by:
Chithra Devi Pandey
Anjali Kak Koul
Vimala Devi S
Neeta Singh
J Radhamani
Sushil Pandey
Sherry Rachel Jacob
J Aravind
Padmavati Ganpat Gore Veena Gupta
Published in 2019 by:
Dr Kuldeep Singh, Director
ICAR-National Bureau of Plant Genetic Resources
Pusa Campus, New Delhi-110 012
Acknowledgements are due to Mr. Arup Das and Ms. Sunita for providing technical, secretarial
assistance and designing the cover page.
© All Rights Reserved ICAR-NBPGR
The content of these practical notes are for academic purpose of the training course on
“Management of Plant Genetic Resources “held at ICAR-NBPGR from July 15-20, 2019. The
information contained in the chapters was provided on an “as is” basis with no responsibility and
liability for any errors or omissions on the editors. Some content of chapters are sourced from
prior publications of ICAR-NBPGR.
PREFACE
There is an increasing realization to conserve germplasm in view of many natural and
anthropogenic conditions posing threat to biodiversity. In addition, continuous replacement of
cultivars and landraces with new improved varieties and resultant erosion of plant genetic
resources (PGR). Trusted alternative to secure the PGR through seed banks is vital to provide for
their availability for sustainable use. This ex situ mode of conservation has become not only an
integral part of several national programmes but also an international commitment under the
Convention on Biological Diversity (CBD) and the Global plan of Action (GPA). Hence, there is
need to maintain seed banks as per international standards.
Significant achievements have been made in India with respect to plant genetic resource
conservation, which may not be in place at the same time in many developing countries. ICAR-
National Bureau of Plant Genetic Resources has the mandate of conserving the plant genetic
resources with its national base collection at New Delhi, linked to several medium-term collections
in a system-wide approach in the country. The bureau has the mandate to plan, conduct, promote
and coordinate all activities concerning plant exploration and collection, characterization, safe
conservation and distribution of both indigenous and introduced genetic resources in crop plants
and their wild relatives. It has also been vested with the authority to issue Import Permit and
Phytosanitary Certificate and conduct quarantine checks on all seed materials and plant propagules
(including transgenic material) introduced from abroad or exported for research purpose. It also
provides Human Resource Development in all spheres of PGR management and periodical
reorientation thereof, to the emerging scientific and technological developments from time to time.
With the view to share the knowledge of plant genetic resource conservation, the
Department of Agriculture Research and Education (DARE) and ICAR has entrusted the ICAR-
NBPGR New Delhi with the responsibility to organize the training for the officers from
Directorate of Seed Testing and Certification, Ministry of Agriculture, Baghdad, Republic of Iraq
in the field of Management of Plant Genetic Resources and Conservation. We express our heartfelt
gratitude to Department of Agriculture Research and Education (DARE) and ICAR for selecting
ICAR-NBPGR to conduct this training. We are also grateful to Dr. Kuldeep Singh, Director,
NBPGR for delegating this training to Division of Germplasm Conservation and for permitting
us to plan and execute the training successfully.
The training manual is an excellent source of information, which should be useful to the
genebank managers and curators. The contributors of different chapters are eminent scientists and
experts in their respective field. We are extremely thankful to them for sparing their valuable time
to write respective chapters in spite of their busy schedule.
INTERNATIONAL TRAINING PROGRAMME ON
MANAGEMENT OF PLANT GENETIC RESOURCES
(for officers from Directorate of Seed Testing and Certification Ministry of Agriculture,
Baghdad, Republic of Iraq)
Venue: ICAR-NBPGR, New Delhi
(July 15 -20, 2019)
Programme Director: Dr Kuldeep Singh, Director, ICAR-NBPGR, New Delhi
Programme
Coordinators:
Dr Veena Gupta, Principal Scientist and Head, Division of Germplasm Conservation,
ICAR-NBPGR, New Delhi
Dr Kavita Gupta, Principal Scientist and OIC PME, ICAR-NBPGR, New Delhi
Dr SK Kaushik, Principal Scientist, Coordinator AICRP (Potential Crops) and Nodal Officer
(HRD) ICAR-NBPGR, New Delhi
PROGRAM SCHEDULE
Date Time Topic of the lecture/Practical Resource Person
15.07.2019 09:30 AM - 10:30 AM Introduction & Registration (trainees
expectation)
Dr Neeta Singh, DGC
10:30 AM - 11:00 AM Coffee/Tea Break
11:00 AM - 01:30 PM ICAR-NBPGR Documentary and National
Genebank Visit
Dr Neeta Singh, DGC
01:30 PM - 02:30 PM Lunch Break
02:30 PM - 04:30 PM Visit to ICAR-NBPGR Facilities Dr Smita Lenka Jain, DGC
16.07.2019 09:30 AM - 10:45 AM Exploration & Collection of Plant Genetic
Resources
Dr SP Ahlawat, DPEGC
10:45 AM - 11:00 AM Coffee/Tea Break
11:00 AM - 12:15 PM Conservation of Plant Genetic Resources Dr Neeta Singh, DGC and Dr
Sushil Pandey, DGC
12:15 PM - 01:30 PM Alternative Conservation Strategy for Clonally
Propagated Crops
Dr Ruchira Pandey, Dr R
Gowthami, Dr Era V. Malhotra and
Dr Vartika Srivastava,TCCU
01:30 PM - 02:30 PM Lunch Break
02:30 PM - 04:30 PM Defining conservation strategies for Agri-
horticultural crops
Dr J Radhamani, DGC and
Dr Vimala Devi S, DGC
17.07.2019 09:30 AM - 10:45 AM Quarantine procedures for safe conservation of
Plant Genetic Resources
Dr Kavita Gupta, DPQ
10:45 AM - 11:00 AM Coffee/Tea Break
11:00 AM - 12:15 PM Characterization of Plant Genetic Resources Dr KK Gangopadhyay, DGE
12:15 PM - 01:30 PM Enhancing utilization of conserved Plant
Genetic Resources
Dr Jyoti Kumari, DGE
01:30 PM - 02:30 PM Lunch Break
02:30 PM - 04:30 PM Seed health tesing for pest-free conservation of
Plant Genetic Resources
Dr Jameel Akhtar, DPQ and Dr
Smita Lenka Jain, DGC
18.07.2019
09:30 AM - 11:00 AM Operation and maintenance of Genebank
facility
Dr Rajvir Singh, DGC and Ms
Anjali, DGC
11:00 AM - 11:30 AM Coffee/Tea Break
11:30 AM - 01:30 PM Operation and maintenance of seed dryers/
dehumidifiers/germinators
Mr Satyaprakash, DGC and Mr Lal
Singh, DGC
01:30 PM - 02:30 PM Coffee/Tea Break
02:30 PM - 04:30 PM Modelling and monitoring of seed longevity in
conserved germplasm
Dr Chithra Devi Pandey, DGC and
Dr J Aravind, DGC
19.07.2019 09:30 AM - 11:00 AM Information Management System for Plant
Genetic Resources
Dr Sunil Archak, AKMU, Mr Rajiv
Gambhir, AKMU and Ms Nirmala
Dabral, DGC
11:00 AM - 11:30 AM Coffee/Tea Break
11:30 AM - 12:30 PM IPR issues related to Plant Genetic Resources Dr Vandana Tyagi, GEPU
12:30 PM - 01:30 PM Quick viability test using Tetrazolium salt Dr AD Sharma and Dr Veena
Gupta, DGC
01:30 PM - 02:30 PM Lunch Break
02:30 PM - 04:30 PM Seed viability testing: Principles and practices Dr Anjali Kak Koul, DGC and Dr
Sherry R Jacob , DGC
20.07.2019 09:30 AM - 11:00 AM Feedback Dr Sherry R Jacob, DGC and Dr
Veena Gupta, DGC
11:00 AM - 11:30 AM Coffee/Tea Break
11:30 AM - 01:30 PM Plant Genetic Resources -Indian Perspective Dr Kuldeep Singh, Director
01:30 PM - 02:30 PM Lunch Break
02:30 PM - 04:00 PM Valedictory Function
CONTENTS
Chapter
No.
Title Page No.
Preface
Training Schedule
Lectures
1. Exploration and Collecting Plant Genetic Resources for Food and
Agriculture
1 - 13
2. Conservation Strategies for Agri-Horticultural Crops 14 - 24
3. Conservation of Plant Genetic Resources 25 - 32
4. Alternative Conservation Strategy for Clonally Propagated Crops 33 - 44
5. Guidelines for Sending Germplasm for Pest Free Conservation 45 - 48
6. Seed Health Testing for Pest-Free Conservation of Plant Genetic
Resources
49 - 56
7. Seed Viability Testing: Principles and Practices 57 - 64
8. Quick Viability Test Using Tetrazolium Salt 65 - 70
9. Modelling and Monitoring of Seed Longevity in Conserved Germplasm 71 - 81
10. Operation and Maintenance of Seed Dryers/ De-Humidifiers and Seed
Germinators
82 - 85
11. Information Management System for Plant Genetic Resources 86 - 89
12. Characterization of Plant Genetic Resources 90 - 94
13. Enhancing Utilization of Conserved Plant Genetic Resources 95 - 101
14. Operation and Maintenance of Genebank Facility 102 - 107
15. Intellectual Property Rights (IPR) Issues Related to Plant Genetic
Resources
108 - 114
List of trainees 115
List of faculty and their contact details 116 - 117
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
EXPLORATION AND COLLECTING PLANT GENETIC RESOURCES FOR FOOD
AND AGRICULTURE
SP Ahlawat and KC Bhatt
Division of Plant Exploration and Germplasm Collection, ICAR-National Bureau of
Plant Genetic Resources, New Delhi 110 012
Introduction
Plant genetic resources for food and agriculture are vital to human beings and other animals.
They are source of food, fodder, fuel, fibre and several items of life support. Plant genetic
resources (PGR) consists the diversity of crops and their wild relatives, contributing to
agricultural production. This diversity exists at the ecosystem, species, and genetic level and is
the result of interactions among people and the environment over thousands of years. It is the
source of genetic material that is vital to future generations. We know of 7000 plant species in
the world that are edible, but only 4 species: rice, wheat, maize and potato provide over 50%
of our plant-derived calories. A diverse diet is the basis of food pyramids and nutrition
guidelines around the world. The heavy reliance on a narrow diversity of food crops puts future
food and nutrition security at risk. Diversity in plants can provide a cost-effective way for
farmers to manage pests, diseases and extreme weather events. Plant diversity can provide
options to farmers to manage climate risks particularly, smallholder farmers with more crop
options and help buffer the effects of extreme events such as droughts or floods. Approximately
940 species of cultivated plants are threatened globally (Khoshbakht and Hammer, 2007). In
recent largest survey of 330,000 seed-bearing plant species extinctions published on 10th June,
2019, the world’s plant species have been disappearing at a rate of nearly 3 species a year since
1900, which is up to 500 times higher than would be expected as a result of natural forces alone
(Humphreys, et al. 2019). When a species or the diversity within a species is lost, we also lose
genes that could be important for improving crops, developing resistance to pests and diseases,
or adapt to the changing climate. By 2050, the world’s population will reach 9.1 billion, 34%
higher than now. To feed the growing population, agriculture must provide more food.
Simultaneously, it will be essential to increase its resilience by protecting the plants possessing
with unique traits that survive drought, flood, cyclones, hail-storms. Genetic resources are most
important components of present and future crop breeding programmes. Augmentation of
germplasm is the first and foremost activity in the PGR management system, therefore
meticulous planning and exploration following scientific principles governing the diversity
distribution is crucial. There were reports of sporadic collections of indigenous crop germplasm
during the earlier part of 20th century, for instance, wheat from north-western plains (Howard
and Howard, 1910), jute (Burkill and Finlow, 1907) and few pulses. Dr. BP Pal in his classic
paper ‘Search for new genes” (Pal, 1937) emphasized the importance of germplasm
augmentation in crop improvement programmes. Significant progress has been made
subsequently, in collecting and conservation of indigenous and exotic plant genetic resources
(PGR) of food and agriculture by several countries like, USA, China, India, United Kingdom,
Germany, Japan and international centers of CGIAR like, CYMMET, IRRI, ICARDA,
ICRISAT, CIAT and IITA. By the end of 2016, 4.7 million samples of seeds and other plant
genetic material had been conserved in 602 gene banks across 82 countries and 14 regional and
international centres.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Interdependence on PGR
The diversity in plant genetic resources (PGR) created during thousands of years of evolution
is not equally distributed throughout the globe. In large part, the movement of food crops out
of their native habitats was linked to human movements, with the occasional assistance from
birds, winds, and ocean currents. Gradually, the ad hoc spread of seeds and planting materials
has resulted in deliberate, organized, and formalized systems of acquisition. Plants have been
under free exchange across countries since ages in the form of seeds, vegetative propagules
and whole plants by traders, armies and explorers. Subsequent spread of agriculture and its
networking for food security necessitated the nations to be dependent on each other to meet
their food and other basic requirements. After Convention on Biological Diversity (CBD) in
1993, germplasm was considered as sovereign property of the nation. This demanded the urgent
need of survey, inventorization, collection, conservation and documentation of native PGR.
Crops as botanical immigrants are performing well away from their place of origin, for
example: maize grown in major part of the world, the wheat in Canada, and the potatoes
cultivated on more than 10 million acres in China. None are native to those lands. Directly or
indirectly, therefore, the world’s 7.7 billion people depend on crops and, thus, on genetic
resources that would not normally be found in and are not part of the indigenous flora of their
country.
No country is self-sufficient in its PGR wealth to meet the ever-changing needs of agriculture.
Some regions are endowed with abundant diversity, while other areas are relatively
impoverished. Majority of the world’s productive agricultural systems are dependent on access
to and availability of both native and exotic PGR. Deliberate introductions have not only played
a major role in uplifting the economy of a region but in enhancing the richness of
agrobiodiversity. Some prominent examples of introduction of new crops to a region/ countries
from place of origin are of sunflower to USSR from Central Mexico/USA, Chinese soybean to
North America, Ethiopian coffee to Central and South America, Bahian cacao to West Africa,
Amazonian rubber to Malaysia, African oil palm to Indonesia and Malaysia, Asiatic yams to
tropical America and Africa, wheat from Near East USA; maize, tomato, potato, sweet potato
and groundnut from South America to Europe and the Asian regions. Over the years, there is
much greater interdependence among countries for Plant Genetic Resources for Food and
Agriculture (PGRFA) (excluding industrial products and pharmaceuticals) than for any other
kind of biodiversity (FAO, 1997). Plant breeders, researchers, and other institutional users are
also increasingly interdependent in PGRFA conservation efforts by international distributions
from genebanks. Ever since the establishment of modern genebanks, conservation of PGR and
their flows within and across borders have been tremendous. These collections are the source
for utilization both in traditional breeding programmes and through modern biotechnological
tools.
All crops cultivated now were originally wild plants. Wild plants of food and economic use,
through the process of domestication and cultivation have been transformed into the crops that
we grow and use now. Maxted et al. (2006) defined crop wild relative (CWR) as “a wild plant
taxon that has an indirect use derived from its close genetic relationship to a crop”. They may
include wild forms/populations of crops, wild progenitors and other closely related taxa. Wild
forms of crops are those wild plants belonging to same species under which crop plants fall
(latter evolved from the former). Role of crop wild relative (CWR) in crop improvement (pre-
breeding, possessing biotic resistance, adapted traits in changing climate) are well known.
Need for novel genes, genes for climate resilience, the breakdown of barriers to introgression
through biotechnological tools, increasing pressure on wild species population, meager
collection NGB signifies the importance of collecting germplasm of wild relatives.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Collecting germplasm
Germplasm collection is the first and foremost activity of organizations dealing with
management of plant genetic resources (PGR). This is prerequisite to conserve, utilize these
valuable resources in crop improvement and analyze temporal changes. The requirement for
PGR in general/ or in specific is unpredictable and dynamic. Besides germplasm, a voucher
herbarium specimen is collected, pressed, plant sample deposited for future reference
particularly for the variant type and wild relatives. It supports research work and may be
examined to verify the identity of the specific plant used in a study. In addition to their
taxonomic importance, herbaria are commonly used in the fields of ecology, plant anatomy and
morphology, conservation biology, biogeography, ethnobotany, and paleobotany.
Collection mission for germplasm sample and herbarium specimen requires almost similar and
meticulous preparations from finding the target species and populations to capturing maximum
number of species, diversity for the amount of material collected and resources invested
(Guarino et al., 1995). In a vast and diverse country having temperate to tropics, great specific
and intra-specific variation is expected in various agro-ecological systems and forests. It is
essential to get a fair idea of the extant floristic and genetic diversity, their distribution across
geographical and ecological niches before embarking on actual collection expeditions.
Conducting an ecogeographic survey has been a canonical method for such a preparation
(Maxted et al., 1995). Such surveys help in increasing the emphasis on localized floristic and
germplasm collecting with a focus on specific traits. Step-wise activities of exploration mission
is depicted by flow chart (Fig.1)
1.1. Planning Exploration Mission: Principles and Practices
Since, this activity is being undertaken by organizations with different mandates, the guidelines
have been framed to help the explorers in maintaining standard methods and procedures in
collecting PGR (Arora, 1981, Gautam et al. 1998, Guriano et al. 1995, Hawkes, 1976 and 1980,
Jain and Rao, 1977). An excerpt of guidelines made by Division of Plant Exploration and
Germplasm Collection at ICAR-NBPGR are given here for training purpose.
1.1.1. Conducting Ecogeographic Survey
Prior to explorations, information about distribution of species is collected from secondary
sources and ecogeographic survey is conducted as an essential prerequisite before collecting
genetic resources of cultivated and wild plants and to plan in situ conservation (IBPGR, 1985).
An ecogeographic survey is defined as information gathering and synthesis process on
geographical, ecological, taxonomic and genetic diversity data (Maxted et al., 1995; Castañeda
Álvarez et al., 2011). The survey is generally consists collating information from herbarium
specimens, genebank accessions, PGR databases, in addition to published as well as informal
literature, etc. Outcome of such survey is predictive and can be used in formulation of
collection priorities. Generally, an ecogeographic survey and analysis results in - i. delineation
of priority crop species, ii. identification of areas for germplasm, herbarium collecting and in
situ conservation, iii. identify populations of cultivated and CWR species which are not
conserved in genebank, iv. to plan and execute collection missions. Ecogeographic surveys
provide information on PGR to infer about history of their evolution and adaptation, to assess
status of conservation and to prioritize areas for conservation. Ecogeographic data have also
been employed to define core collections in crop species (Frankel and Brown, 1984) and
identify gaps in collections (Shehadeh et al., 2013). Ecogeographic data are employed only as
a proxy to the genetic diversity data.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
1.1.2. Prioritization of Species and Areas after Gap Analysis
Depending upon the priorities, gathering knowledge of the crops/species before launching
a mission is important.
The areas to be explored and crops/ species to be collected should be prioritized after
thorough gap analysis based on information from different sources including database of
National Genebank/field genebank. However, for foreign explorations guidelines of the
agricultural research department /ministry in the country to be followed to develop and
finalize the mission.
The explorer should be well-versed with the nature and extent of diversity and breeding
behaviour of crop/species to be collected and plan well in advance to facilitate preparations
of proposed missions except those to be carried out under special situations like rescue
collecting.
Visit to herbaria should be made to know the range of distribution, localities, diversity
pattern and period of collection particularly for wild species. Flora of targeted area, R&D
institutions and experts in area should be consulted.
Collaborator(s) should be identified and communicated to join the mission well in advance
with details of preparations, if required, particularly in case of vegetatively propagated and
recalcitrant material.
Phytosanitary regulations should be followed in case the material is transported from foreign
country.
1.1.3. Finalization of Mission
Gathering eco-geographic information: Information on topography, climatic conditions,
vegetation, crops in cultivation and their maturity, etc. needs to be gathered to finalize the
itinerary of collecting mission. Besides, explorers should establish local contacts especially
at grass root level to seek the social, cultural, ethnic and other information of interest.
Types of survey: Coarse grid survey should be conducted in unexplored areas to capture
the overall variability, while fine grid survey is carried out to build-up more collections for
specific trait(s) known to exist in identified pockets in previously explored areas.
Multi-crop/ crop-specific explorations: Multi-crop exploration is carried out to collect the
diversity in general of a given region (also referred as region-specific exploration) or in
unexplored areas. Crop-specific exploration are undertaken to collect the variability in
particular crop and its genepool. The samples collected must be representative of the
diversity that exists within each crop/ crop groups in a given area.
Permission of collection from protected/restricted areas: Prior permission should be
obtained from the concerned authorities for exploring and collecting in protected (biosphere
reserves, sanctuaries, national parks) and restricted areas (international border) and
particularly for foreign missions.
Tour itinerary: A tentative tour itinerary should be drawn up at an early stage of the
planning, showing the main target areas (or even precise localities) to be visited within the
overall target region, the roads/tracks to be followed and the proposed timings of each visit.
The mode of transport should also be specified. Letters of introduction to local government
officials are often useful, and their preparation again will require some rough idea of the
itinerary to be followed. Maps will clearly be needed in planning the itinerary, but local
contacts are essential for advice on the feasibility of following particular routes at different
times of the year (Hawkes, 1980).
Period of collection: For germplasm collection of seed producing crops and species,
exploration should be undertaken when these are physiologically mature and ready for
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
harvest. In case of species with shattering nature, missions are executed rather earlier (7-10
days depending on crop/ species) before their maturity. Further, longer duration (2-3 weeks)
mission and repeat visits are suggested for collection of wild species. For taxonomic and
herbarium collection, the flowering season of species is best. For vegetatively propagated
crops/species, the targeted areas should be surveyed first for identification and marking of
elite types at the time of flowering/fruiting and subsequently the collections are made at
appropriate time. Period of explorations being organized within the country should be of at
least 10-15 working days (excluding journey period) and more than a month when organised
in foreign countries.
Team composition: The exploration and collecting team should be familiar with basics of
agriculture/plant genetic resources to meet the objective of the mission. Team consisting of
2-3 members including a collaborator and need-based local-aid may be formed preferably a
botanist/ breeder as team leader. Team of more than three persons is not desirable due to
practical problems and that create hesitation in farmers, villagers.
Area and route of exploration: This should be fine-tuned in consultation with the subject
experts of local bodies, staff of forest, agriculture departments, as soon as the team reaches
to the starting point keeping in view the targeted species and areas of the proposed mission.
Items and equipments required: As per the nature of the germplasm to be collected (fruit/
seed/ vegetative propagule/ in vitro/ live plants) and the area(s) to be explored, several items
and equipments are required is given in box. Herbarium press, secateur and large size poly
bags (1.5x2 m) are essential items required for herbarium specimen collection.
Domestic quarantine: All precautions including need-based domestic quarantine should be
followed for pest-free collection and its transportation.
List of items and equipments for collecting
Survey /
collecting
items
Global Positioning System (GPS), digital camera with additional memory card,
binocular, magnifying glasses, handheld microscope, digital Vernier calliper and
portable balance, Haversack/ kitbag, seed envelopes, cloth bags, polythene bags,
aluminium & tag labels, drying sheets, old newspapers, plant press, moss, rubber bands,
packing tape, sutli (thick and thin), secateurs, scissors, knife, digger, torch light,
measuring tape, passport data book, field note book, pencil, ballpoint pen and permanent
marker.
Reference
material
Regional/ national flora, digital herbarium, lap-top and accessories, list of local names
of plants, road-map, vegetation/climate map, list of rest-houses/ lodges, hotels, resting/
stay places and list of local contacts (phone, fax, e-mail).
First Aid-
Box
Anti-malaria pills, anti-allergen tablets, pain killers, anti- amoebic and anti-diarrhoeal
tablets, mosquito repellent, antifungal/ antibacterial/ antiseptic creams or lotions, cotton-
packs, band-aid, dettol, dressing gauze, water-purifying tablets, etc.
1.2 Collecting strategy and Sampling Methodology
At the actual collecting sites, there will be need to apply sampling method to ensure that the
genetic diversity of crop/species is adequately represented in the sample collected. The
following points are given to take care depending upon the situations.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
1.2.1. Sampling Sites
Inaccessible areas of valleys, isolated hills, villages at the edge of deserts, forests, mountains
and isolated coastal belts may hold rich genetic diversity, potential/ trait-specific germplasm
and wild relatives. For cultivated species, sampling sites in order of preference should be
farmers’ field, backyard/ kitchen garden, threshing yard, farm store, local village market,
etc.
Collecting mission should be started first from warm, drier tracts (vs. humid), un-irrigated
areas (vs. irrigated), valleys (vs. hills) to capture maximum available diversity in a planned
manner.
The crops often vary with ethnic diversity and different array of materials may be collected
even from contiguous belts occupied by different tribes.
Sites having stress situations viz. saline habitat, un-irrigated/ drought conditions, desert
(cold and hot), flood prone areas should be identified as target areas for collection of
respective trait-specific material. In such cases, selective sampling of promising genotypes
should be done.
For biotic stress tolerant material, hot-spot areas should be visited to collect healthy plants
in fields where severe pest damage is evident.
The frequency of sampling (number of samples per site) should be decided based on on-the-
spot observations on the variability available. In general, more sites per target area are
preferred to sample the targeted species rather than sampling from a few sites.
1.2.2. Sampling Method (Self, cross pollinated and vegetatively propagated material)
While collecting the seed, the explorer should keep in mind the required quantity of material
to be sampled for long term conservation (2000 and 4000 seeds for self and cross pollinated
crops, respectively) besides meeting the requirement of characterization, evaluation and
related studies.
The optimum sample size per site would be the number of plants required to obtain, with 95
percent certainty, all the alleles at a random locus occurring in the target population with
frequency greater than 0.05 (Hawkes, 1976; Marshall and Brown, 1975).
In case of species with extremely small-sized seeds, low seed-set, asynchronous maturity
and low seed viability, care should be taken to collect adequate sample size.
In case of extremely variable populations, one can either make larger samples (bulking), or
take as sub-samples if observed interesting variants, and be given separate collecting
numbers.
In general, random sampling should be made by collecting single spike/panicle or
fruit/berry/pod from at least 50 plants along a number of transects throughout the field
(Hawkes, 1976 & 1980) to obtain a representative and adequate sample. Plant population at
border of field should be avoided.
In a situation when wild population with few individuals occur, one should better collect
from all the plants so as to make the representative sample from that site. In case of certain
wild and semi-domesticated species occurring in small pocket with scattered populations
(treated as sampling site) having specific use/traits, the seed should be bulked. However one
should not deplete the populations of farmers’ planting stocks or wild species, or remove
significant genetic variation.
In case of large tubers, only a portion, e.g. head or proximal ends in yams, crown or tuber
in taro and other aroids should be collected. Since vegetative propagules are subject to rapid
deterioration after harvest and damage during transportation care should be taken while
sampling and in transportation.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
In case of scion collection for budding and grafting the sample size will depend upon the
number of rootstocks available but not less than ten per sample so that at least eight grafts
may survive. In case of cuttings and rooted suckers (e.g. grapes, ornamentals, passion fruits,
black pepper, beetle vine, banana, cardamom, etc.) 15-20 cuttings may be sufficient.
1.2.3. Establishing Taxonomic Identity
Material with dubious identity, unidentified material and only vernacular name should be
collected along with herbarium specimen and photographs for authentication. In case, when
herbarium specimens are not available, efforts should be made by the explorer to raise plants
to establish its correct identity.
Normally 4-5 individual plants/parts having representation of all parts especially flowers,
or fruits or both should be collected for preparing herbarium specimen. Locality, date of
collection and field notes should be clearly recorded. Characters which are lost on drying,
or which may not be represented in the herbarium specimen (plant height), flower colour,
leaflets (which may be shed on drying) should be mentioned in field notes. The detailed
guidelines for preparation and processing herbarium specimen should be followed as per
Jain and Rao (1977).
1.2.4. Type of Material
Depending on the objective of the collection mission, seed, vegetative propagule, in vitro
material and pollen are collected. For herbarium specimens, all possible parts of plants
including root, stem, leaves, buds, flowers and fruits are collected. Herbarium specimens,
in general and especially of the wild types and wild relatives should always be collected to
help in identification/ authentication. Wherever possible efforts should be made to collect
economic products of local and wider use as supportive material.
1.2.5. Transportation
In case of vegetative propagules, if required, the explorer should make prior arrangements
for the en-route transportation of collected material to the place of its establishment/
maintenance to avoid deterioration. Daily checking of collected material, change of blotting
papers of herbarium sheets, room drying of collected material after reaching at place of halt
is essential.
1.3. Recording Information
1.3.1. Passport Data
Passport data are important source for database, documentation, enhanced utilization of
PGR and studying the variation in distributional pattern with respect to ecological and socio-
economic factors. It is advisable to record information on both the essential and optional
fields in the passport data sheet or field book at the site of the collection itself by the
explorer. Sample content of passport data book of NBPGR, New Delhi is given in annexure-
I. However, in any circumstances, the explorer should not leave the information blank on
essential fields namely sample labelling (name of organization(s) and collectors, collectors’
number, date and type of material); sample identification (botanical identity, vernacular
name, its biological status); sampling information (sampling type, method and source) and
collecting site localization (state, district, village, latitude, longitude and altitude). This
information is important for herbarium specimens and recorded in field books.
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1.3.2. Related Information
Information on genetic erosion should be gathered from aged-farmers particularly on the
depletion of landraces cultivated over the time and the reasons for their loss in general and
native crops originated in particular. Indigenous traditional knowledge on plants, their use,
agricultural technologies etc. are also asked and recorded. Meaning of the name of landraces
and their properties should be asked from farmers and recorded. Observations on the
distribution pattern and frequency status of crop wild relatives, rare, endangered and
threatened species of PGR importance should be recorded for their sustainable management.
Ethnobotanical observations and new uses of plants, especially those collected from tribal
dominated tracts, are currently recorded as a database which would be available for
reference in future collections.
1.4. Post Collection Handling
1.4.1. Seed Extraction, Cleaning/Drying
The extraction and cleaning of seed should be done preferably on the same day or
immediately after completing the expedition and process for their drying under shade/ sun/
controlled conditions. The seeds with short longevity should be processed at the earliest and
care should be taken during threshing/ cleaning to avoid damage. In a situation, when delay
in processing is anticipated, all precautions should be taken to maintain its viability. The
observations on variability parameters on fruit/ pod/ seed should be recorded along with
photographs for report writing, documentation and publication.
1.4.2. Packaging and Labelling (Sharing, Accessioning, Multiplication and Conservation)
The clean and dried material should be kept in the envelopes with proper label specifying
its botanical name and collector number. One set of the material along with passport data
should be sent for accessioning, conservation (LTS/MTS) and another set be sent to the
collaborating institute for initial seed increase (if required), maintenance, characterization
and evaluation.
1.4.3. Establishment / Maintenance for Vegetatively Propagated Material
The vegetatively propagated material should be sent for establishment/ maintenance in field
genebank or at suitable site. The material for in vitro and cryo-genebank should be handed
over to the concerned curators. The elite material, if any, should be studied in detail to
generate supporting information as well as for validation of the known trait(s) for its
registration with concerned agency.
1.5. Report Writing and Publication
After completing the mission of herbarium and germplasm collection from a target area and
processing the collected material, it is important to write the comprehensive report to fulfil
the mission’s objectives. This helps in follow-up collecting(s) and also the users to know
the availability of the germplasm as well as in publications. The information on the samples
collected can be entered into database for its access to users. The report on the exploration
and collection should broadly include:
Name of the organisation(s)
Name of the scientist(s)/person(s) involved
Collaborating organisation(s)
Objectives of the collecting mission
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A description of the environment, flora, people of the target area
An account of the logistics and scientific planning
Details of the execution of the mission (timing, itinerary, sampling strategy and collecting
techniques)
A summary of the results (areas surveyed along with route maps, germplasm and herbarium
specimens collected, indigenous knowledge documented and extent and magnitude of
diversity collected with elite germplasm, if any)
Role of women in conservation of diversity
Details of sharing germplasm and information
Photographs
An account on loss of germplasm and ITK, if any
Difficulties encountered during collecting mission
Recommendations for follow-up action(s)
Acknowledgement
1.6. DO'S AND DON’TS
In addition to above guidelines for exploration, germplasm and herbarium collection, the
collector(s) should observe a well-defined code of conduct as well as take necessary
precautionary measures in its smooth execution as mentioned below:
1.6.1. Do's
Get acquainted with the International Code of Conduct for Plant Germplasm Collecting and
Transfer developed by FAO (1993).
Always keep a route map of the target area with list of important places and the distance
covered during travel to facilitate report writing.
Before entering into a forest take the help of forest guards to have forehand knowledge of
possible dangers in the target area. If needed, help of a gunman is taken during survey in
dense forest.
Explain the purpose and get consent from the farmers for collecting germplasm.
Keep important telephone numbers of concerned officers including district authorities,
hospitals, dispensaries and police station.
Keep your identity card and a certificate from Head of Organization for proposed mission.
Honour social customs of local inhabitants of the target area.
While talking and discussing with ladies, be polite and respectful to them.
After day's collection and before retiring to bed, have a glance at your equipments, passport
data and collected material for need-based updating.
1.6.2. Don’ts
Do not provide lift to strangers in your vehicle under any pretext.
Do not indulge in unnecessary discussion related to politics, religion and local beliefs with
the local people.
Do not make false promises with donors.
Do not plan the expedition during important festivals and peak election campaign in the
target area.
Do not enter any house for seed collection in absence of male members of the family.
Do not eat unknown wild fruits since some of them may be toxic or internally infected.
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Do not collect the seed in large quantities from any household if the farmers wish so.
Over-collecting of the genetic diversity with similar attributes should be avoided to save
time and energy in collection and evaluation and to save space in the genebank.
Fig. 1. Flow chart of Exploration for germplasm and herbarium collection activities
Why to collect For utilization, germplasm in danger of extinction or erosion (wild species), gap filling in existing ex-
situ collections , rescue collecting, loss of genetic diversity due to over exploitation, for future use: the
material not considered useful today may turn out to be vital tomorrow particularly in changed
climatic condition, to address issues of IPR and CBD
Exploration Aims at
Capture prevailing genetic diversity; search of novel/unique genetic material
Planning of Exploration Missions
National Exploration Plan (Five years)
Annual Exploration Plan (at national level)
Based on need of breeders, conservation for posterity, gaps in collections,
taxonomic and phylogenetic study on prioritized species
Gathering information on areas of diversity, unique traits, landraces,
availability, crop maturity, visit twice for disease/pests tolerance
Exploration Missions (Type)
Specific (crop based, trait-specific, biotic stress, quality)
Broad based (multi-crop/Region specific)
Rescue Mission (during calamity or landuse change)
Planning of exploration including logistic arrangements
Team composition, liaison and involving crop breeder/collaborator, area and route of exploration,
collection time, exploration items and equipment (transport, instruments, GPS, chemicals,
miscellaneous items, published material, medical items, other items)
Exploration and Collection Methodology
Method: Coarse grid and fine grid survey in targeted areas/fields
Sampling Techniques: Random sampling, biased sampling, bulk sampling
Plant parts collected: Based on reproductive nature of crop such as seed,
pollen, vegetative dormant bud, rhizome, tuber, stolen, etc.
Sample Size: 2000/4000 seeds of self/cross pollinated crops
Recording Passport data, ITK information
Germplasm: Processing, multiplication and conservation in genebank
Germplasm: Characterization and evaluation
Germplasm: Supply, Pre-breeding and Use
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Annexure-I
NATIONAL BUREAU OF PLANT GENETIC RESOURCES, NEW DELHI -12
PASSPORT DATA SHEET
Date.............................................Collector’s No..........................................Accession No........ ........................
Botanical Name........................................................... Common Name (English)...........................................
Crop/Vern. Name................................. Cultivar name..................................... Region Explored.......... ............
Village/Block......................................District............................State.............................. Latitude…..……………0N
Longitude…..………………0E Altitude….………..m
Temp........................................ Rainfall .........................................
COLLECTION SITE 1. Natural wild 2.Disturbed wild 3.Farmer’s field 4.Threshing yard 5.Fallow 6.Farm
store 7.Market 8.Garden 9.Institute 10..................
BIOLOGICAL STATUS 1.Wild 2.Weed 3.Landrace 4.Primitive cultivar 5. Breeder’s line
FREQUENCY 1.Abundant 2.Frequent 3.Occassional 4.Rare
MATERIAL 1. Seeds 2.Fruits 3.Inflorescence 4.Roots 5.Tubers 6. Rhizomes 7. Suckers 8.Live
plants 9.Herbarium 10…………..
BREEDING SYSTEM 1.Self-pollinated 2.Cross-pollinated 3.Vegetatively propagated
SAMPLE TYPE 1.Population 2.Pure line 3.Individual plant
SAMPLE METHOD 1.Bulk 2.Random 3.Selective (non-random)
HABITAT 1. Cultivated 2.Disturbed 3.Partly disturbed 4.Rangeland 5………………..
DISEASE SYMPTOMS 1.Susceptible 2.Mildly susceptible 3.Tolerant 4.Resistant 5.Immune
INSECT/ PEST/
NEMATODE INFECTION
1. Mild 2.Moderate 3. High
CULTURAL PRACTICE
SEASON
1. Irrigated 2.Rainfed 3.Arid 4.Wet 5...........
1.Kharif 2.Rabi 3.Spring-summer 4.Perennial type
ASSOCIATED FLORA 1. Sole 2.Mixed with………………………
SOIL COLOUR 1. Black 2.Yellow 3.Red 4.Brown 5……..
SOIL TEXTURE 1.Sandy 2.Sandy loam 3.Loam 4.Silt loam 5.Clay 6.Silt
TOPOGRAPHY 1.Swamp 2.Flood plain 3.Level 4.Undulating 5.Hilly dissected 6.Steeply dissected
7.Mountainous 8.Valley
AGRONOMIC SCORE 1.Very poor 2. Poor 3. Average 4. Good 5. Very good
ETHNOBOTANICAL USES
PART(S) 1. Stem 2. Leaf 3.Root 4. Fruit 5.Flower 6.Whole plant 7.Seed 8.Others
KIND 1.Food 2.Medicine 3.Fibre 4.Timber 5.Fodder 6.Fuel 7.Insecticide/ Pesticide 8.Others
HOW USED ................................................................................
INFORMANT(S) 1.Local Vaidya 2.Housewife 3.Old folk 4.Graziers /Shepherds 5.Others
PHOTOGRAPH 1.Colour/Video
FARMER’S/ DONOR’S NAME.......................ETHNIC GROUP............................Mobile No………………………
ADDRESS
PLANT
CHARACTERISTICS/
USES ADDL. NOTES
References
Arora RK (1981). Plant genetic resources exploration and collection: planning and logistics.
In: Plant exploration and collection (eds KL Mehra, RK Arora and SR Wadhi), NBPGR
Sci. Monograph 3, National Bureau of Plant Genetic Resources, New Delhi, pp 46-54.
Burkill IH and RS Finlow (1907) Races of jute. Agric Ledger 14: 41-137.
Castañeda Álvarez NP, HA Vincent, SP Kell, RJ Eastwood and N Maxted (2011)
Ecogeographic surveys. In: Guarino L, V Ramanatha Rao, E Goldberg (eds). Collecting
Plant Genetic Diversity: Technical Guidelines. 2011 update. Bioversity International,
Rome.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
FAO (1993). International Code of Conduct for Germplasm Collecting and Transfer. FAO,
Rome. www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en
FAO (1997). The state of the world’s plant genetic resources for food and agriculture. FAO,
Rome. PP 444. http://www.fao.org/3/a-w7324e.pdf
Frankel OH, Brown AHD (1984) Plant genetic resources today: a critical appraisal. In: Holden
JHW and JT Williams (eds.) Crop genetic resources: Conservation and evaluation. Allen
and Unwin, Winchester p 249–257.
Gautam PL, BS Dabas, U Srivastava and SS Duhoon (1998) Plant Germplasm Collecting:
Principles and Procedures. NBPGR, New Delhi, 218 p.
Greene SL and TC Hart (1999) Implementing a geographic analysis in germplasm
conservation. In: Greene SL, L Guarino (eds.) Linking genetic resources and geography:
emerging strategies for conserving and using crop biodiversity. American Society of
Agronomy; Crop Science Society of America, Madison, pp 25–38.
Guarino L, V Ramanatha Rao, R Reid (1995) A brief history of plant germplasm collecting.
In: Guarino L, Ramanatha Rao V and Reid R (eds.) Collecting plant genetic diversity.
Technical guidelines. CAB International, Wallingford, UK, pp 1-11.
Guarino L, V Ramanatha Rao and R Reid (1995). Collecting plant genetic diversity: Technical
guidelines. International Plant Genetic Resources Institute (IPGRI), Rome, Italy; Plant
Production and Protection Division, FAO, Rome, Italy; World Conservation Union
(IUCN), Gland, Switzerland; CABI, Wallingford, UK, 748 p.
Hawkes JG (1976). Manual for field collectors (Seed crops), International Board for Plant
Genetic Resources, FAO, Rome, Italy.
Hawkes JG (1980). Crop genetic resources - A field collection manual, IBPGR/EUCARPIA,
Univ. of Birmingham, UK.
Howard A and GLC Howard (1910) Wheat in India, its Production, Varieties and
Improvement. Thacker Spinn & Co., Calcutta.
Humphreys Aelys M., Rafaël Govaerts, Sarah Z. Ficinski, Eimear Nic Lughadha & Maria S.
Vorontsova (2019). Global dataset shows geography and life form predict modern plant
extinction and rediscovery. Nature Ecol. Evol. 10th June 2019, http://doi.org/gf3szp
IBPGR (1985) Ecogeogrpahic surveying and in situ conservation of crop relatives. Report of
an IBPGR task force meeting held at Washington DC. IBPGR, Rome, 33 p.
Jain S.K and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and
Tomorrow Printers and Publishers, New Delhi, p. 157.
Khoshbakht and Hammer (2007) Threatened and Rare Ornamental Plants. Journal of
Agriculture and Rural Development in the Tropics and Subtropics 108(1):19-39.
Marshall, DR and AHD Brown (1975). Optimum sampling strategies in genetic conservation.
In: Genetic resources for today and tomorrow (eds OH Frankel and JG Hawkes).
Cambridge Univ. Press, Cambridge, pp. 53-80.
Maxted N, MW van Slageren, JR Rihan (1995) Ecogeographic surveys. In: Guarino L,
Ramanatha Rao V, Reid R (eds.) Collecting Plant Genetic Diversity. CABI International,
Wallingford, UK, pp. 255–285.
Maxted N, Ford-Lloyd B V, Jury S L, Kell S P and Scholten M A. (2006). Towards a definition
of a crop wild relative. Biodiversity and Conservation 14: 1-13.
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Pal BP (1937) The search for new genes. Agriculture and Livestock 7(5): 573-578.
Shehadeh A, A Amri, N Maxted (2013) Ecogeographic survey and gap analysis of Lathyrus L.
species. Genet. Resour. Crop Evol. 60: 2101-2113.
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CONSERVATION STRATEGIES FOR AGRI-HORTICULTURAL CROPS
J. Radhamani, Vimala Devi S and Chithra Devi Pandey
Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic Resources,
New Delhi – 110 012
Introduction
Conservation of plant genetic resources is vital to combat biodiversity loss through natural and
anthropogenic factors. Many vulnerable plant groups are either endemic or threatened,
jeopardizing the future sustainable development. Many strategies such as in-situ conservation
in the form of biodiversity parks, sanctuaries and other reserved forests are in place, ex-situ
conservation is also a priority. Ex-situ conservation in any form involves huge cost, manpower
and energy consumption, which makes the decision of the strategy/process a priority. The
strategies of conservation, depends mainly on the seed.
Seed is the easiest and convenient form of not only propagation, but also the conservation
process. Large majority of the plant species produce seeds, hence conservation through seed
genebanks is the most optimal choice. In case, where seed production/propagation is a
problematic, such as in many medicinal, bulbous or plantation crops like banana, in-vitro
genebanks are the next best options.
Factors influencing selection of conservation strategies:
In case of seed storage, many factors influence the strategies to be developed/followed for
conservation such as the type of seeds, seed morphology, chemical composition of seeds, seed
maturity and seed moisture content.
Type of seed:
Seed storage may be defined as the preservation of viable seeds from the time of collection till
sowing (Holmes and Buszewicz, 1958) and successful storage depends on the seed quality in
term of viability and vigour. Any conservation strategy involves huge cost, manpower and
energy consumption. Hence, the process of conservation should be based on the nature of seeds.
A large variation in storability pattern is observed between species. Roberts (1973) classified
seeds into two broad physiological categories; (1) orthodox and (2) recalcitrant based on
storage characteristics.
Orthodox seeds generally tolerate desiccation to low moisture contents (4–10 %) on a wet
weight basis (w/w), are comparatively long lived if handled appropriately and tolerate being
stored at sub-zero temperatures. Viability is prolonged in a predictable manner by such
reduction in moisture and storage temperature. These include crops such as rice, wheat, maize,
lentil, pea, sunflower, groundnut, etc. (Fig.1). Any seed that does not behave this way is non-
orthodox category, and in fact, the seeds of a great number of tropical region are classified
under non-orthodox which may be either recalcitrant (Roberts 1973) or intermediate (Ellis et
al., 1990) according to their storage behavior.
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Fig.1. Seeds of field crops with orthodox storage behaviour
In contrast, recalcitrant seeds are desiccation sensitive, short lived and are generally intolerant
of low temperatures. Seeds of recalcitrant species maintain high moisture content at maturity
(often more than 30–60 %) and are sensitive to desiccation below 12–30 %, depending on
species. They rapidly lose viability under any kind of storage conditions. These include crops
such as cocoa, jackfruit, coconut, etc. (Fig. 2). Recalcitrant seeds are desiccation sensitive to
various levels and degrees of dehydration are tolerated depending on the species which simply
indicates that the processes or mechanisms that confer desiccation tolerance are variably
developed or expressed in the non-orthodox condition in different ways.
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Fig.2. Recalcitrant seeds of cocoa, litchi, jackfruit and coconut
Apart from these two categories, the intermediate seed species generally do not react adversely
to low temperatures, but are chilling sensitive so cannot be stored at low temperature. i.e.,
neem, coffee (Fig. 3.).
Fig. 6.3. Intermediate seeds of neem and coffee
Although the terms ‘orthodox’ and ‘recalcitrant’ are relatively well established, storage
physiology of seeds seems to cover a more or less continuous spectrum, ranging from
extremely recalcitrant, which lose viability in few days, to extremely orthodox, the viability of
which under optimal conditions counts in decades or centuries (Farrant et al., 1988). Between
these two categories further sub-divisions can be made.
Seed morphology
It plays an important role in context to the storage life of seeds. The hard seed coat in legumes
helps to maintain the level of seed metabolism by prevention of moisture and oxygen. The
softer seed coat shortens the life span of seeds as uptake of moisture is fast.
Chemical composition of seeds
In general, storage is directly related to the chemical composition of the seeds. Oily seeds do
not store as good as starchy seeds. However, exceptions are always there. For example, a corm
of black oak (oily with low carbohydrates) stores longer than the corm of white oak (high
carbohydrate with low oil).
Seed maturity
Physiologically immature seeds with insufficient accumulation of food reserves, low level of
necessary enzymes/growth regulators are prone to be damaged fast with less longevity while
the seeds at full maturity have higher survival curves.
Seed moisture content
The amount of moisture in the seed is probably the most important factor influencing how long
the seed maintains its quality remains alive. It is a function of relative humidity. The response
of seed longevity to storage at different moisture contents varies between species. This appears
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to be mainly a function of seed oil content. The longevity of oily seeds is less sensitive to a
given difference in moisture content than seed with less oil content. However, since the
potential longevity of seed with high oil content is less, it is vital that these species be dried
despite the smaller beneficial effect.
Different factors or combination of factors operate at different moisture levels causing seed
deterioration. If the seed moisture is between 40–60 % germination occurs, at 18–20 %
moisture, heating may occur; at 12–14 % mould growth may destroy the seed; at 8–9 % insects
are active and multiply; below 5% physico-chemical reactions cause deterioration of the seed.
The drier the seed, lesser the number of factors that will be operating to destroy the seed. As
the moisture increases the activity of each factor increases rapidly. Generally fungal growth is
minimum in grain seeds of 12% moisture content. At 13% moisture content a few Aspergilli
grow slowly at ideal temperature of 75–80 °F (23–27 °C), at 15% fungal growth is much faster
and at18% fungal growth is so rapid that the respiration of the fungi and the seed may cause
heating of the seed. Thus moisture content between 5–7% is ideal for long term storage, where
all deleterious factors are eliminated.
Seed storage
Based on the above factors, the orthodox seeds are mainly conserved in seed genebanks with
minimum moisture content and at minimal temperature. The period of conservation depends
on the purpose of storage.
1. Very short periods between collection and sowing– Short term storage (STS)
2. Several years (5–10) – Medium term storage (MTS) to ensure reliable supply of seed
in annual crops
3. Long period (10–50) – Long term storage (LTS) for germplasm conservation.
Short term storage
The period for such storage of seeds is between 1 year and 18 months. In the arid and cool
agro-climatic zones seeds of most cultivated species will maintain a high germination capacity
for as long as 18 months with only the basic storage conditions. However, in warm and humid
environments the viability loss is very rapid in the simple seed warehouse. Moreover, seeds of
poor storers such as soybean cotton, onion and several flower and tree species lose germination
capacity rapidly under warm humid storage environments and may deteriorate within 2 to 3
months under simple storage under warm tropical and sub-tropical climatic conditions.
Therefore, more stringent storage facilities are needed to protect the seeds from adverse
environmental conditions.
Conditions for short term storage
Storage period: 12–18 months.
Temperature: 18–20 °C
RH: 45–50 %
Storage container type: Cloth bags, paper bag, glass bottles.
Seeds can be shade dried and stored up to 2 years in sealed plastic containers, paper bags or
muslin cloth bags at 18–20 °C and 45% RH for short-term. The primary requirement is some
insulation to keep the storage units as cool as possible. One possibility is a false ceiling with
ventilation between ceiling and the roof. Heat inflow will be reduced by thick stone or brick
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wall or by a layer of insulation in walls or ceiling. Ventilation fans to bring in cool night air
can help if it is not too humid as to raise the seed to moisture contents to levels that allows
attack by storage fungi. The second requirement is to keep the seed dry. If bags are used, storage
on pallets will keep the seed from direct contact with a damp floor. Sealing the floor against
moisture penetration is also useful. Storage of seeds in steel bins with tight fitting lids or in
moisture proof bags will solve the problem of moisture penetration provided the seeds are
already dry enough for sealed storage. Additionally, fitting one or two air-conditioners
depending on the size of the storage room will help to provide a cooler as well as drier
atmosphere (20–22 °C and 45–50 % RH) where the seeds can be conveniently stored for one
to two seasons without much loss in their viability. Under such situations, the moisture content
of the seeds will equilibrate with the relative humidity of surrounding air and undergo drying
to some extent. The germplasm (seeds) are maintained under short term until they are further
processed for medium/ long term storage.
Medium term storage
The size of the accessions kept in the medium term is generally larger than those meant for
long-term storage. These working collections have a higher rate of usage as they are distributed
regularly for evaluation and breeding purposes. The accessions have to be regenerated
periodically due to depletion of stocks rather than from loss of viability. Therefore, the time
period for storage is not more than 5–10 years.
Conditions for medium term storage
Storage period: 5–10 Years
Temperature: 0–10 °C
RH: 35–40 %
Storage container type: Cloth bag, metal can, glass bottle and plastic jars (Fig. 4)
Fig. 4. Types of containers used for medium term seed storage
The active collections are stored at temperatures ranging from 0–10 °C. The seed samples in
these collections are stored in various types of containers such as cloth bags, metal cans or
glass jars. If the containers are hermetically sealed after drying the samples, then the relative
humidity of the medium term storage is not important. If unsealed containers are used, then the
relative humidity should be brought down. This shall, however, increase the running costs. The
method adopted thus shall be decided by the frequency of withdrawal of the samples. A
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temperature of 5 °C and relative humidity of 35–45 % is adequate for maintaining the viability
of most orthodox seeds for 5–20 years. If unsealed containers are used, then regular monitoring
of seed moisture content is important.
Long term storage
The base collections and the duplicate collections are stored for long-term for use in the future
crop improvement programmes. The conditions recommended by International Board for Plant
Genetic Resources (IBPGR), now known as Bioversity International for storage of base
collections are probably the most suitable and economical to be maintained by conventional
mechanical refrigeration methods which are generally followed at NBPGR. The preferred
standard for base collections is storage at 5 ± 1 % seed moisture content in hermetically sealed,
moisture proof containers preferably laminated trilayered aluminium foil packets at −18 °C to
−20 °C. A survey of monitoring data of stored accessions from several genebanks suggest that
accessions stored at −10 °C will require monitoring and regeneration twice as frequently as
those at −18 °C. Consequently, it is recommended that new facilities should adopt −18 °C or
less, because any saving on the capital and running costs of the store will be outweighed in the
long term by the increased staffing and cost required for these increased frequencies for
monitoring and regeneration.
Conditions for long term storage
Storage period: 50–100 Years
Temperature: −18–20 °C
RH: No control
Storage condition: Trilayered Laminated Aluminum foil packet, gasketed rigid plastic
container, sealed aluminum tins/cans, Polythene bags (over 700-gauge thickness) and
sealed bottles.
At NBPGR, the seed processing for long term storage is undertaken following International
Seed Testing Association (FAO/IPGRI,1994) standards. Once the seed samples are received
(germplasm collections) for long term storage, they are quarantined and then processed for
conservation. The samples which meets the minimum standards and qualify for storage i.e.,
germination percentage (>85%), sufficient seed quantity (2,000 in self-pollinated and 4,000 in
cross pollinated species), pest free, complete passport information (species, place of collection,
biological status, etc.). are issued the National identity and processed for storage. The seed
processing involves cleaning, moisture determination, drying, viability testing, packing and
labeling and arranging them in baskets. Once samples are accessioned and dried to the require
moisture levels are packed in trilayered laminated aluminum foil packets. All these packets are
labeled and arranged accession wise in baskets. Their genebank data along with the passport
data is documented along with the sample wise location in the genebank. Location is allotted
based on module number, shelf number, rack number and basket number for easy retrieval of
samples (Fig. 5).
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Fig.5: ICAR-NBPGR National Seed Genebank
Important factors for long-term storage
Long-term storage of seed aims at
1. reducing the metabolism of seeds (with minimum temperature and moisture content
possible),
2. keeping insects, fungi and other pathogens away and
3. reducing general seed ageing (stored in dark at low temperatures).
Freshly harvested seeds require drying to optimum moisture levels depending on type of
storage. All seeds are hygroscopic in nature and they tend to absorb or desorb moisture
depending on the surrounding humidity of the air. Seeds of the field crops are generally at 8–
12 % moisture content at harvest. They are dried to lower moisture levels in muslin cloth bags
over silica gel or in seed driers.
Long-term conservation help ensure the seeds to be conserved for very long period, without
losing viability of the seeds. However, there are many factors which influence the reduction of
seed viability in seed storage, such as (1) moisture content of the seed, (2) storage temperature
and (3) storage atmosphere (oxygen) all of which have influence on the rate of respiration.
Deterioration in seed leads to deterioration in viability and vigour predisposing the seed to
eventual death. The higher the moisture content and temperature conditions are, more rapidly
the seed deteriorates. Harrington (1960) proposed two thumb rules for safe storage of seeds as
follows:
• For every decrease of 1% moisture in the seed, the life of the seed is doubled
• For every decrease of 10 °F (5°C) in the storage temperature, the life of the seed is
doubled.
As an independent factor, temperature is considered next only to moisture in causing damage
to stored seeds. When the seeds are dry enough and sealed in moisture proof packets, they
remain viable for longer duration even at higher temperatures. It is important that seeds for
long term storage at sub-zero temperature are drier having a moisture content of 3–7 % only.
Seeds with higher moistures cannot be safely stored at subzero temperatures since they undergo
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freezing injury resulting in loss of viability. When moisture and temperature interact with each
other they produce a geometric effect on seed longevity. For example, onion seeds stored with
12% moisture content at 110 °F (43 °C) were found to lose complete viability within one week
whereas those with 7% moisture content stored at 50 °F (10 °C) had retained their germination
capacity even after 20 years of storage.
Apart from the moisture and temperature, the potential longevity of the seeds depends to a great
extent on the pre-storage environments. This includes the conditions experienced around the
time of harvest. The storage pattern of seed varies in different species and often the accessions
of same species differ. The seed extraction, transport and conditioning are the main factors for
extending the storage in orthodox seeds. Cracks and breaches of seed coat due to improper seed
handling which can allow the microorganisms to enter leading to seed deterioration.
Determination of seed moisture content
Moisture estimation is a critical component of germplasm processing. Seed samples are tested
for their moisture content, immediately on the receipt of the sample and also after drying of the
sample. The methods employed to determine seed moisture content are designed to reduce
oxidation, decomposition or loss of other volatile substances while ensuring the removal of as
much moisture as possible (ISTA, 2015).
Procedure
Grinding and weighing
Grinding is obligatory for the seeds larger in size to increase the surface area from which the
moisture before drying in the oven (Table 1.) for moisture estimation. About 4-5 grams of seeds
are weighed and placed in pre-weighed clean moisture bottles. The weight should be recorded
in grams to three decimal places.
Methods
Low constant temperature oven method
This method is generally used for moisture estimation of oily seeds. The seeds in moisture
bottles with their lid open are placed in an oven maintaining a temperature of 103±20C for
17±1 hours. After the prescribed period is over, the moisture bottles covered with their
respective lids are placed in a desiccator to cool for 30-45 minutes. After that, the moisture
bottles along with dry seed and lid are weighed
High constant temperature oven method
This method is generally recommended for the seeds of field crops. The procedure is same as
above except that the seeds are placed in the oven at 130±20C for 2-4 hours.
Period of seed drying
The prescribed period of seed drying shall be 17± 1 hrs at 130°C under low constant and 1 to 4 hrs at
130°-133° C under high constant temperatures. Maize seed be dried for 4 hrs, cereals and / or other
millets for 2 hrs and the remaining species for 1 hr. seeds rich in oil content or with volatile substance
be dried 17± for hrs under low constant temperature. Seed drying period begins from the time oven
returns to maintain the desired temperatures.
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Sample size
The ISTA rules recommend that two replicates, each with 4 gm of seed be used for determination of
seed moisture content. This seed sample weight may be modified to 0.2 to 0.5 gm per replicate, with
precise weighing for use in seed genebanks, to avoid unnecessary depletion of precious biological
resources.
Calculation of results
The percentage of moisture content on weight basis may be calculated by using the following
formula:
MC % = W2-W3 X 100
W2-W1
Where,
W1= Weight of moisture bottle along with lid
W2= Weight of moisture bottle along with lid and seed before drying
W3= Weight of moisture bottle along with lid and seed after drying
Table 1.Species for which the low constant temperature (103°C) oven method be used
Allium spp Linum ustatissimum
Arachis hypogeal Raphanus sativus
Brassica spp Ricinus communis
Camelina sative Sesamum indicum
Glycine max Sinapsis spp
Gossypium spp Solanum melongene
Table 2. Species for which high constant temperature (130° to 133° C)
Agrostis spp Citrullus lanatus lolium spp
Alopecurus pratensis Cucumis spp Lotus spp
Anthum graveolens Cucurbita spp Lupinus spp
Anthoxathum Cuminum cyminum Lycopersicon lycopersicum Poa spp
odratum
anthriscus spp Cynodon dactylon Medicago spp
Apium Graveolens Cynosurus cristatus Melilotus spp
Arrhenatherum Daucus carota
officinalis
Avena spp Deschampsia spp Ormithopus
sativus
flavescens
Beta vulgaris Fagopyrum esculentum
Table 3. Species for which grinding is obligatory
Amorpha fruticosa Fagopyron esculantum Lupinus spp
Arachis hypogaea Glycine max Oryza sativa
Avena spp Gossypium spp
Cicer aritinum Hordeum vulgare Pisum sativum
Citrullus lanatus Lathyrus spp Ricinus communis
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Seed drying
Seed drying is the reduction of the seed moisture content to the level recommended for its safe
conservation, at sub-zero temperature. As per international standards, 3-7% seed moisture
content is recommended for long-term conservation. There are many methods for seed drying
but gradual drying of seed by providing the environment of 10-15% relative humidity (RH)
and 150 C temperature is the most ideal and safe as recommended by the IBPGR advisory
committee on seed storage. In this condition, the seed moisture of most of the orthodox seeds
is equilibrated to the recommended range of 3-7 % in 3-4 weeks. The seeds packed in muslin
cloth bags are placed in a single layer in a closed chamber fitted with a dehumidifier and a
cooling system. The cool dry air is passed over the seeds to pick up the excess moisture released
by the seeds and then passed through the dehumidifier to remove the moisture from the air.
This continues till the seeds equilibrate to the desired moisture. In Genebanks, walk-in-drying
chambers functioning at 15% RH and 15oC is used for the drying purpose. For species with
hard seed coats and which require longer drying duration, batch dryers are used.
Seed Packaging
The best time to package seeds is immediately after the moisture content has been determined
and found to be within the required limits for safe storage. Dry seeds will reabsorb moisture
from ambient air. Therefore, seeds should be packaged into containers and hermetically sealed
without delay, soon after removal from the drying room or cabinet.
There are different kinds of containers available for long term storage of seeds. Some of the
moisture vapour proof containers are given below.
Three layered aluminium foil pouches with the specifications of (a) outer polyester
layer of 12 µ (b) middle aluminium layer of 12 µ and (c) inner layer of 250 gauge of
polyethylene.
Sealed aluminium tins, cans
Bottle with air tight lid and
Gasketed rigid plastic containers
Three layered aluminium foil pouches are the most ideal containers for storage seeds at sub
zero temperature because they need lesser space, can be cut to desired size and sealed again
and capable to withstand the temperature of -200C to 400C.
The vacuum sealer machines are used for hermetic sealing and packaging of the seed material.
References
Abdil-baki AA and Anderson JG 1972 Physiological and biochemical deterioration of seeds In:
Seed Biology Vol 2 (ed TT Kozlowski) Acad Press New York pp 283-315
Agrawal PK (ed) 1993 Handbook of seed Testing DAC Min of Agril GOI New Delhi pp 340
Agrawal PK and Dadlani M (eds) 1992 Techniques in Seed Science and Technology South Asian
Publ New Delhi pp 2007
Anonymous 1976 International rules for seed testing Annexes 1976 Seed Sci Technol 4: 51-177
Anonymous 1994 Genebank Standards FAO / IPGRI Rome pp 46
Anonymous 1999 International Rules for Seed Testing Seed Sci Technol 27 Supple pp 333+vii
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Anonymous 2004 A and Douglas JE 1967 Seed Testing Seed Sci Technol pp xiii+166+11
Bonner FT (1990) Storage of seeds: potential and limitations for germplasm conservation. Forest
Ecology and Management 35: 35–43.
Chalam GV, Singh A and Douglas JE 1967 Seed Testing Manual ICAR & USAID New Delhi pp
267
Dickie JB Linigton S and Williams JT 1984 Seed Mangement Techniques for Genebanks Proc
Workshop RBG Kew 6-9 July 1982 IBPGR Rome pp 294
Ellis RH, TD Hong and EH Roberts (1985) Handbook of seed technology for genebanks. Volume
I. Principles and methodology. Handbook for genebanks. No. 2. Rome, Italy: International
Board for Plant Genetic Resources. 210p.
Ellis RH, TD Hong and EH Roberts (1990) An intermediate category of seed storage behaviour I.
Coffee. Journal of Experimental Botany 41, 1167–1174.
Farrant JM, NW Pammenter and P Berjak (1988) Recalcitrance - a current assessment. Seed
Science and Technology 16, 155–166.
Hanson J (1985) Procedures for handling seeds in genebanks. Practical Manual for Genebanks:
No. 1. Rome, Italy: International Board for Plant Genetic Resources. 115p.
Harrington, J.F. (1960). Thumb rubs of drying seed. Crops and Soils 13:16–17.
Harrington JF (1972) Seed Storage and Longevity. In: Kozlowski, T.T. (ed.) Seed Biology. Vol.
III. pp 145–245.
Hong TD, S Linington and RH Ellis (1996) Seed storage behaviour: A Compendium: Handbooks
for Gene Bank No.4. International Plant Genetic Resources Institute, Rome, Italy.
King MW and EH Roberts (1979) The Storage of Recalcitrant Seeds: Achievements and Possible
Approaches. International Board for Plant Genetic Resources, Rome.
Holmes, G.D. and Buszewicz, G., 1958. The storage of seed of temperate forest tree species.
Commonwealth Agricultural Bureaux.
Roberts E. H. 1973. Predicting the storage life of seeds. Seed Science and Technology 1, 499–
514.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
CONSERVATION OF PLANT GENETIC RESOURCES
Neeta Singh and Sushil Pandey
Division of Germplasm Conservation, ICAR-National Bureau Plant Genetic Resources,
New Delhi-110012
There is a global recognition that biodiversity at all levels- gene pools, species and biotic
communities is important for many reasons and that it is being rapidly diminished by habitat
destruction and other damaging influences resulting from human population growth, climate
vagaries, pollution and economic expansion. Habitat destruction, genetic homogeneity in
farming systems, and alien species invasion are some of the causes of erosion. Loss of genetic
diversity has serious implications on economic and social development of any nation.
In the changing international scenario, global climatic changes, unsustainability of high input
agriculture, rehabilitation of wastelands and the search for novel genes for nutrition, biotic and
abiotic stresses, diversification, environmental security have increased the focus on the extent
of variability in genetic resources collections of crops, and the effectiveness of its utilization in
raising the productivity through widening of genetic base and mapping genes for biotic and
abiotic stresses.
Therefore, management, conservation and sustainable use of plant genetic resources are
fundamental to ecologically sustainable development and food security of a nation.
There are two broad strategies for plant genetic resources conservation
Ex-situ conservation- conservation of components of genetic material of biological diversity
outside their natural habitat. The latter includes seed gene banks, field genebank/repositories,
botanical gardens and in vitro gene banks. Three main conceptual categories of collection -
base, active and safety duplicate – are recognized as serving different purposes (see Gene bank
Standards – FAO/IPGRI 1994).
The establishment of ex situ germplasm collections has been the result of several decades of
global efforts to conserve plant biodiversity. Long term seed storage under the preferred
conditions of seed storage, viz. with 3-7% moisture content -18oC, enables plant germplasm to
be conserved cheaply and safely for seeds of orthodox nature. Since about 80% of higher plants
species show orthodox seed storage behavior ex situ biodiversity conservation by long term
seed storage is, in principle, possible for a large proportion of higher plants (World
Conservation Monitoring Centre, 1992). There are now more than 1,750 individual genebanks
worldwide holding an estimated 7.4 million accessions globally. Seed banking offers a good
compromise between ability to store intra-specific variation, applicability to wide range of
species, potential sample longevity, recovery of gene products and technical input requirement.
Modern crop seed genebanks conserve seed for either a few years as medium-term or many
decades to centuries as long-term. Short-term banks operating in form of community banks
also help in ensuring the continued availability of local varieties to resource-poor farmers.
In situ conservation-conservation of genetic resources within their ecosystem and natural
habitat. In situ conservation is concerned with maintaining species populations in the natural
habitats where they occur, whether as uncultivated plant communities or in farmer’s fields as
part of existing agro-ecosystems. In the Global Action Plan of FAO it has now been
recommended to accord priority for in situ methods of maintenance. This method of
conservation largely overlooked in the past is now being discussed widely and has achieved
some success .Several non-government organizations are also engaged in in situ conservation
of targeted species through national or external assistance or both.
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Maintenance and conservation strategies depend on the kind of targeted diversity besides other
factors, for example, ecosystem diversity with wild species is best conserved by in situ
methods, species diversity in cultivated plants in gene banks and on farm. Today these
approaches are seen as complementary and not exclusive as these methods address different
aspects of genetic resources and neither alone is sufficient to conserve the total range of genetic
resources that exist.
The Indian National Germplasm Conservation System
National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi was established in
1976 as a nodal institute for assembly of global diversity of plant genetic resources (PGR) that
are of direct or indirect value to humans. The component activities include PGR collection
through exploration, characterization, evaluation, safe conservation using both conventional
storage and biotechnological approaches for in vitro conservation and cryopreservation;
generation and conservation of genomic resources. The National Genebank Network consists
of the National Genebank at National Bureau of Plant Genetic Resources (NBPGR), New
Delhi, which is primarily responsible for conservation of germplasm on long-term basis. The
11 regional stations of NBPGR, in different agro-climatic zones of the country and the 59
National Active Germplasm Sites (NAGS) are the integral component of the network. The
NAGS are based at the premier institutes for specific crops or crop groups and are entrusted
with the responsibility of multiplication, evaluation, conservation of active collections and their
distribution to users both at national and international levels. Various other National institutes,
All India Coordinated Crop Research Projects, State Agricultural Universities and other
stakeholders are also linked to the network. International Agricultural Research Centers
involved in conservation and use of PGR are also effectively linked to the network.
The major components of the National Genebank located at New Delhi include the
seed genebank(-18°C), Cryogenebank (-150 to -196°C), In vitro Genebank (25°C) and Field
Genebanks, There are two types of seed conservation methods : those conserving accessions
for long-term and future use (referred to as base collections) and those conserving seed
accessions for immediate or short term needs (referred to as active collections). The ex situ
seed genebank at NBPGR comprises 12 long-term modules (total capacity: 1million
accessions) maintained at –18oC for housing the base collections, In addition, six modules are
also used to conserve active collections under medium-term storage conditions.. The
temperature, RH, seed moisture content, containers and distribution arrangements vary
according to the requirement of conservation period.
The active collections are distributed in 22 medium-term modules maintained at 4 oC for storing
germplasm at active sites. At present, the genebank holds approximately, 0.44 million
accessions belonging to nearly 2,000 species of different agri-horticultural crops belonging to
as the base collections and 10,235 duplicate safety samples of pulses received from ICARDA,
Syria and ICRISAT . In addition about 11,200 accessions are conserved in cryogenebank and
1,902 in the in vitro genebank. The longevity of seeds depends on the initial seed quality,
moisture content and temperature at which it is conserved. The seed samples for conservation
in genebank should meet the minimum standards for seed quality and quantity. The operational
sequence to integrate an accession into the genebank involves cleaning, seed health testing,
determination of moisture content, drying, viability testing and packaging. The germplasm
conserved as a base collection is assigned a national identity number, dried to seed-moisture of
around 5+2 percent at 15oC and 15 per cent relative humidity. The accessions meeting
international standards, (IBPGR, 1994), with seed viability more than 85 per cent and 2000-
4000 seeds are transferred to long-term storage. Base collection held in the gene banks may
have duplicates. The passport, characterization (including molecular) and evaluation data can
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be used to identify duplicates or near duplicates. Base collections are being regularly monitored
for seed viability, quantity, health etc., at an interval of 10 years (or depending on the species).
For conservation of active collections, seed moisture is brought down to 8-10 per cent. The
active collections are kept in medium-term storage maintained at 4-10oC and 35-40 per cent
relative humidity.
The field genebanks are spread across the 10 regional stations of NBPGR in various agro-
climatic zones of India and conserve about 51,000 accessions .
Long-term storage (base collections): A base collection defined as a set of accessions which
are clearly distinct and are sufficiently close to the original sample submitted in the genebank
with respect to the genetic identity is held at the Headquarters of NBPGR at New Delhi.
Normally, seeds are not distributed from base collections directly to users for the crop
improvement programme and are only used to regenerate active collections (FAO/IPGRI,
1994). Base collections are hermetically packed in tri-layered aluminum foil packets and
conserved under long-term storage conditions at sub-zero temperature at -18°C to -20°C. The
crop group wise holdings in base collection are detailed in table below.
Table 1.1 Number of accessions conserved in NGB in various crop groups (as on June 2019)
Crop Group No. of accessions
Agroforestry (136 Crops) 1,646
Cereals (9 Crops) 1,64,403
Fibre (20 Crops) 15,731
Forages (114 Crops) 7,213
Fruits & Nuts (45 Crops) 276
Grain legumes (39 Crops) 66,772
Medicinal & Aromatic plants (376 Crops) 8,071
Millets (11 Crops) 59,272
Oilseeds (27 Crops) 59,434
Ornamental (66 Crops) 661
Pseudocereals (7 Crops) 7,697
Spices, Condiments and Flavour (20 Crops) 3,163
Vegetables (70 Crops) 26,383
Duplicate safety samples 10,235
Trial material 10,771
Total 4,41,728
Medium-term storage (Active collections)
It consist of accessions which are immediately available for multiplication and distribution for
use. Because these are accessed frequently, therefore, maintained under medium-term
conditions at 4-100C temperature and 30-35% RH.
The accessions have to be regenerated periodically due to depletion of stocks rather than from
loss of viability. The seed samples in these collections are stored in various types of containers
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such as cloth bags, metal cans or glass jars. A temperature of 50C and relative humidity of 35-
45% is adequate for maintaining the viability of most orthodox seeds for 5-25 years.
The operational sequence to integrate an accession into the genebank involves cleaning, seed
health testing, determination of moisture content, drying, viability testing, labelling, packaging
and documentation. The management of seed collections requires that germplasm accessions
be maintained with a high proportion of viable seeds and this involves conservation under
appropriate conditions, periodic monitoring of seeds for viability and quantity, and
regenerating accessions as and when required according to the situation.
Seed processing includes steps necessary to ensure that seeds are brought from the field to the
genebank with minimal loss of viability and highest level of seed purity and seed health are
maintained till their conservation in the genebank. Upon receipt of the seed samples in the
genebank, seed are processed for conservation which involves the following major steps:
Seed Registration
After receiving the seeds for conservation in the genebank, the accessions are registered in
Germplasm Handling Unit (GHU) with all details of the germplasm related to the origin and
the donor.
Seed Cleaning
The purpose of cleaning is to remove impurities such as thrash, leaves, broken seeds, sand and
grit, weed seeds and those of other plant species, and immature, shriveled, unfilled and empty
spikelets. Seed can be cleaned manually or by winnowing. Seeds should be cleaned
immediately after harvest or soon after received in the genebank. Cleaning methods will vary
according to the type and size of seeds.
Seed Health Testing
Crops are frequently infected with a range of common seed-borne pathogens that may not be
visible or easily recognized during seed collection. Seed-borne inoculums reduce longevity in
genebank and cause poor germination or field establishment. Seed-borne inoculums also
promote disease in the field, reducing the value of germplasm. Exchange of infected seeds may
allow spread of diseases and pests into new regions. Genebanks should ensure that seeds
prepared for conservation are free from seed-borne diseases and pests. Seed health testing is
one of the main component of the seed quality testing and refers primarily to the presence or
absence of disease causing organisms – pathogens such as fungi, bacteria, viruses, nematodes
and insect pests.
After cleaning of the seeds, samples are sent to germplasm handling unit for critical
observation to achieve the pest-free status of the germplasm, through visual
examination and X-Ray radiography.
Wherever possible, the germplasm is salvaged without compromising the seed health
status and passed for conservation in National Genebank (NGB). However, in case of
hidden infestation, if the material cannot be salvaged, same may be fumigated and sent
to National Active Germplasm Sites (NAGS) for further multiplication of the samples.
Heavily infested seeds are incinerated.
Seed Moisture Content Determination
Moisture content of the seed is the most critical factor which controls the keeping quality of
seeds. Although, all seeds deteriorate with time, their rate of deterioration is dependent on the
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seed moisture content, storage temperature and seed species. Thus, it is essential to determine
the initial moisture content of the seeds so that the same could be dried to safe levels where
hydrolytic reactions are arrested.
For estimation of moisture content, the low constant oven drying method for oily seeds
and medicinal and aromatic species and high constant oven drying method for the other
crops is used.
In general, seeds which are larger in size should be ground to smaller coarse particles
to increase the surface area from which the moisture evaporates. In limited seed
quantity and to avoid wastage of precious germplasm, seeds moisture determinations
may be done with 0.5-1.0 g in two replications.
The seed moisture content (MC) of a sample is the loss in weight when it is dried and
is expressed as a percentage of the weight of the original sample as follows:
MC = wet weight – dry weight x 100
wet weight
Seed Drying
Drying (seed moisture equilibration) is the most important step in the processing of orthodox
seeds for conservation. Most seeds are hygroscopic and hence the water content of the seed
depends upon the relative humidity (RH) of the surrounding air at a given temperature. Drying
seeds from the moisture content levels in equilibrium with low relative humidity conditions of
~15% RH and low temperature of 150C results in several fold increase in subsequent longevity.
Seeds to be stored in long-term conservation module as base collections, are to be dried
to 3-7% MC and for active collections to be stored in medium-term conservation
module are to be dried to 8-10% MC.
Methods that minimize loss of viability during drying should be used. The most
common and safe methods used for drying are dehumidified drying and silica gel
drying.
Drying rate depends on seed size, shape, structure, composition, initial seed moisture
content, amount of seeds and layers, air movement, temperature and RH.
Large number of accessions can be dried in specially designed seed dryers maintaining
15% RH and 150C temperature is used to allow slow and safe seed drying process at a
temperature where minimum loss of seed quality occurs.
Small number of accessions can be dried in desiccators using silica gel.
Seed Viability Testing
It is very important that seeds stored in the genebank are capable of producing structurally and
functionally normal plants when sown in the field. Seeds used for conservation must have high
initial viability and maintain it during storage. Seeds with a high initial viability will also
survive longer in storage for efficient conservation. It is important to know when this decline
occurs in order to take action to regenerate the accession. before packaging, as per the
procedure prescribed by International Seed Testing Association (ISTA).
The initial germination value should exceed 85% for most seeds of cultivated field crop
species.
Exceptions may be granted for some specific accessions of horticultural crops, forestry
species, forage grasses and crop wild relatives, where quality seed production
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problems exist, by taking into consideration their reproductive biology, basic seed
characteristics, seed multiplication ratio and growth cycle.
The Indian Minimum Seed Certification Standards for viability, wherever available,
may be acceptable for long-term storage.
Seed Packaging
The best time to package seeds is immediately after MC has been determined to be within the
required limits (3-7%) for safe conservation. After drying and viability testing, the seed
samples qualifying for long term-conservation are counted or weighed, as per the prescribed
standards of minimum 2000 seeds in self-pollinated species and 4000 seeds in cross-pollinated
species, and placed moisture impervious container which is then preferably hermetically sealed
for subsequent conservation to avoid exchange of moisture and contamination from pests and
diseases.
Seeds should be packaged immediately after the desired moisture content (3-7%) has
been achieved to avoid reabsorbtion of moisture from ambient air by dry seeds.
Specially designed tri-layered aluminum foil packets (outer polyester layer: 12µm,
middle aluminum foil layer 12µm, inner polythene layer: 250 gauge), which have been
found most effective for long-term conservation of all types of seeds.
Packaging should be done in an air conditioned room where the RH is controlled
(preferably 15% RH) so that the dried seeds are exposed to the ambient air for the
shortest possible time.
Monitoring of Seed Viability
Monitoring is the regular checking of quality (viability) and quantity (number or weight) of
germplasm accessions conserved in a genebank. The objective of monitoring is to determine
whether regeneration or multiplication of an accession is required. Accessions are monitored
for two main reasons (a) the viability of seeds conserved in the genebank decreases during
storage; it is important to monitor viability of accessions to ensure that they do not lose their
capacity to produce viable plants when needed and (b) The removal of seeds for distribution
and germination testing results in a decrease of seed quantity over time. To avoid excessive
deterioration of seed quality or quantity, genebank accessions should be monitored both for
viability and seed quantity during conservation.
Genebank Standards for Seed
All material conserved in the genebank should have minimum passport data.
Untreated seeds free from pests and diseases should be conserved.
Seeds should have ≥ 85% initial viability and 3-7% moisture content.
The number of seeds to be stored will depend on the species being conserved. In case of
genetically heterogeneous materials viz., cross-pollinated crops and land races, an accession
should consist of at least 4000 seeds. For material which is genetically homogeneous viz.,
self-pollinated crops and genetic stocks, a minimum of 2000 seeds are acceptable.
Exceptions are allowed for wild and rare accessions.
Monitoring of viability should be done after 10 years of storage in case of accessions
conserved n long-term storage and after 5 years for the accessions conserved in medium-
term storage conditions, in order to assess loss in viability before it falls below the threshold
for regeneration.
The time when regeneration of the seeds in storage should be done will also depend on the
threshold viability for regeneration. FAO/ IPGRI recommend 85% of initial viability.
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However, in some species or races, the threshold can be lowered especially if the initial
viability is appreciably lower than that obtained in other species. The size of the population
for regeneration will depend on the mode of pollination because it involves the risk in
loosing genetic integrity of the regenerated germplasm on account genetic drift due to
mechanical admixture, out-crossing, selection pressure and other related factors.
In case of self-pollinated crops which are homogenous, minimum number of 100-150
randomly selected seeds should be supplied for regeneration, based on the viability and
availability of the germplasm accessions intended for regeneration so as to ensure a
minimum plant stand of 75 or more.
Similarly, in case of cross-pollinated crops which are heterogenous in nature, minimum
number of 200-250 randomly selected seeds should be supplied for regeneration, based on
the viability and availabililty of the germplasm accessions intended for regeneration so as
to ensure a minimum plant stand of 150 or more.
After the regeneration, the seed material should be processed and the germplasm accessions
that qualify as per the genebank standards should be conserved along with the original base
material.
Distribution
Germplasm is supplied in response to requests from bonafide researchers. National laws and
regulations like seed health requirement and signing of Material Transfer Agreement (MTA)
should be complied with before distribution of germplasm.
Wherever the seed stock is sufficient, a minimum of 20-50 seeds may be supplied. In case of
wild and endangered species as well as those intended only for research use, the size of sample
may be further reduced as per the availability. The indenters may be advised to multiply their
own seed stock for any further use of the distributed germplasm and also provide the multiplied
seed to the genebank, wherever the seed quantity or quality is of concern during distribution.
Documentation and Genebank Information System
Documenting the information received along with a sample is an important aspect of
seed genebanking. All data and information generated throughout the process of
acquisition, registration, storage, monitoring, regeneration and distribution should be
recorded in a suitably designed database and employed to improve conservation and
use of the germplasm.
Data management systems in genebanks are vital to tracking accessions for
management purposes and for adding value to accessions for efficient utilisation.
Data on any accession should be as complete as possible in order to identify it as a
distinct accession, although accessions without extensive data are also valuable and it
may be justified to include them in base collections.
Information documented consists of passport data providing basic information for
identification and general management of individual accessions.
The information system maintains a record of genebank operation data, including
storage location, stocks, monitoring, health tests and the distribution status.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Guidelines for sending seeds to genebanks
Clean, physiologically mature and freshly harvested free from any pest and pathogens
should be sent to the genebank. Undersized, immature and shriveled seeds should be
avoided.
Sample should contain at least 2000 seeds for self-pollinated crops and 4000 seeds for
cross pollinated crops to fully represent variability of original sample and also allow
sufficient seeds for monitoring of viability during storage and subsequent regeneration.
Minimum number of seeds for registration can be estimated from the desired plant
population for regeneration, minimum number of regeneration, the sample viability and
the expected field establishment.
Seeds should not be treated with any chemical.
All seed packets should be correctly and neatly labeled and accompanied with complete
passport data .
Seed material should be packed in strong, moisture pervious paper packets/cloth bags
to minimize damage during transit.
Germplasm Registration
Recognizing the importance of PGR with novel, unique, distinct and high heritability
traits of value that could be used in crop improvement, and to facilitate flow of
germplasm to users. ICAR-NBPGR plays a vital role in germplasm registration. More
than 900 potentially valuable germplasm of over 120 species of various crops registered
so far. To facilitate smooth registration process, a fully online system of filing
registration applications, their scrutiny, review and communications at every stage has
been developed (http://www.nbpgr.ernet.in:8080/registration/). Details of the registered
germplasm can be accessed at http://www.nbpgr.ernet.in:8080/ircg/index.htm.
Reference
Crop Genebank Knowledge Base initiative of the System-wide Genetic Resources Programme
(SGRP) of the Consultative Group on International Agricultural Research (CGIAR):
http://cropgenebank.sgrp.cgiar.org/
Draft Genebank Standards for Plant Genetic Resources for Food and Agriculture, Commission
on Genetic Resources for Food and Agriculture, Food and Agriculture Organization:
http://typo3.fao.org/fileadmin/templates/agphome/documents/PGR/ITWG/ITWG6/worki
ng_docs/CGRFA_WG_PGR_6_12_4.pdf
ISTA (2012) International Rules for Seed Testing, International Seed Testing Association,
Basserdorf, Switzerland.
Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowell D and Larinde M (2006) Manual of seed
handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome,
Italy.
Smith RD, Dickie JB, Linington SH, Pritchard HW and Probert RJ (2003)(eds). Seed
conservation: turning science into practice. London: The Royal Botanic Gardens, Kew,
1023.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
ALTERNATIVE CONSERVATION STRATEGIES FOR CLONALLY
PROPAGATED CROPS
Ruchira Pandey, Gowthami R., Vartika Srivastava, Era V. Malhotra, Neelam Sharma
and Anuradha Agrawal
Tissue Culture & Cryopreservation Unit, ICAR-NBPGR, New Delhi-110012
Introduction
Plant Genetic Resources (PGRs) conservation has gained momentum, several folds, in recent
years owing to increasing threat to biodiversity, due to various climatic and anthropogenic
factors. A prudent approach involving both in situ and ex situ methods in combination with
emerging technologies is required for saving these valuable resources. Former approach,
though more effective as it permits maintenance of genetic integrity (of wild species, forest
species and wild forms of domesticated species) under natural evolutionary process, is beset
with climatic perturbations, security
and high cost. Conservation needs
for the clonally propagated crops are
conventionally met with by
maintaining them as live plants in the
Field Gene Bank (FGB) / clonal
repositories, botanical gardens,
arboreta etc. (Fig. 1) as these species
are either seed-sterile or highly
heterozygous due to which they are
traditionally clonally propagated. Ex
situ conservation through field gene
banks/clonal repositories though not
entirely satisfactory for clonal crops
yet provides easier access to conserved germplasm for research and utilization. Seed sterility
coupled with high degree of heterozygosity makes seed gene banking a non-viable option in
these crops.
Rapid advances in biotechnological innovations have brought about a revolution in the way
clonal crops can be conserved and utilized. These advances, made in the past three decades,
are attributable to application of in vitro techniques in combination with genetics and molecular
biology. Clonal crops comprise a wide range of species including tuber and root crops (cassava,
potato, taro, yams etc.), fruits (apple, banana, citrus, pear etc.) and many others including
alliums, ginger, turmeric, vanilla, hops, sugarcane, and medicinal and aromatic plants.
Depending on the requirement of the species, in vitro techniques provide tools which can be
utilized in different ways. In vitro or tissue culture techniques possess great potential as these
enable conservation of disease-free (particularly virus-free) germplasm, in an aseptic
environment, away from environmental disturbances, preservation of elite and unique genetic
constitutions and safer international exchange besides facilitated molecular investigations. In
vitro multiplication protocols have been standardized for over thousand plant species but
conservation protocols are limited to several hundred due to lack of infrastructure, trained
human resource, limited funds, unreliable supply of electricity, pest/pathogen problems etc.
particularly in developing economies of the world.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Field Genebanks
Field genebanks represent one of the methods of ex situ conservation for perennial plant species
with recalcitrant seeds or for those which are clonally propagated. Germplasm accessions are
maintained, in the orchard or field as a permanent living collection, away from its original
location. While being conserved under semi-isolated conditions, natural evolution and
adaptation processes are either temporarily halted or altered by introducing the material to a
habitat with suppressed selection pressure. For clonal crops, FGBs represent complimentary
conservation strategy to in vitro and cryo banks.
Field genebanks have traditionally been used for perennial crops, including:
Species producing recalcitrant seeds
Species producing little or no seeds
Species that are preferably stored as clonal material and
Species that are long-lived perennials
In India, ICAR-National Bureau of Plant Genetic Resources (NBPGR) is actively involved in
collection and conservation of clonally propagated crops which are being maintained in FGBs
at its regional stations/ base centers at Akola, Bhowali, Cuttack, Hyderabad, Jodhpur, Ranchi,
Shillong, Shimla, Srinagar and Thrissur. Besides, 59 National Active Germplasm Sites
(NAGS) under ICAR, State Agricultural Universities (SAUs) and other research organizations
in India are also involved in maintaining valuable germplasm in FGBs.
Limitations
Requirement for large area for maintaining crop species
Vulnerability of species to natural disasters
Lack of pollinators
Coverage of limited genetic diversity of a species
Susceptibility to pest and pathogen attack
Cost and labour intensive
Botanical Gardens Botanic gardens are living collections of plants, in general, held for public display, education,
economic exploitation and scientific enquiry. Botanical gardens provide housing and care for
endangered species. Botanical gardens are the most widely visited ex situ conservation sites by
the public. There are about 1500 botanical gardens in the world. Botanical gardens are often
part of very stable institutions and likely to be continuously maintained by trained staff and
knowledge transformation about the importance of the species.
In Situ on Farm Conservation
In situ on-farm conservation is defined as “the continuous cultivation and management of a
diverse set of populations of crop by the farmers in the agro-ecosystems where a crop has
evolved”. It is one of the most suitable methods of ex situ conservation for sustainable
management of diversity of traditional crop varieties, associated with wild and weedy species.
With on farm conservation, farmer is encouraged to select and manage local crop populations
with impact of both natural and human selection in the production system.
Home Gardens
Home gardens are the places where living collections of different crops, trees etc. are managed
by the members of the family. In India, small to large farmers have their own home gardens to
meet their daily requirements of food, income and well being. Many progressive farmers,
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
including several custodian farmers identified, have well-tended home gardens which harbor a
rich diversity of tropical fruits. Ensuring the continued existence and maintenance of regions
with such interconnected home gardens might represent an effective way for the on-farm
conservation of a wide range of neglected and underutilized crops and fruits including their
intraspecific diversity.
Arboreta
An arboretum is a place where, an area contains collections of trees only for conservation and
scientific study. An arboretum is different from a botanical garden, as botanical gardens may
contain all the plant forms from small bushes to big trees, while an arboretum contains only
trees. One of the most common example for arboretum in India is located in Forest Research
Institute located at Dehradun (Uttarakhand) which is connected with researchers working on
different aspects of forest trees.
In Vitro Conservation
In vitro conservation refers to maintenance of germplasm in a relatively stable form, on a
defined nutrient medium, under artificial conditions in the In vitro genebank (IVGB). This
technique is especially useful for clonally propagated species which are either seed-sterile or
produce highly heterozygous seeds or produce seeds with a short viability or species with long
juvenile periods.
In vitro conservation, which involves maintenance of explants in a sterile, pathogen-free
environment, is therefore, preferentially applied to clonal crop germplasm and multiplication
of species that produce recalcitrant seeds, or do not produce seeds. It also supports safe
germplasm transfer under regulated phytosanitary control.
In vitro conservation methods vary depending on the requirement for storage duration.
Medium-term storage (MTS), aims at slowing down growth and extending the sub culture
duration whereas long-term storage (LTS) relies on cryopreservation which is storage of living
tissues at ultra-low temperature, usually that of liquid nitrogen (LN) (−196°C) for theoretically
indefinite period. Germplasm once stored in LN is protected from contamination and requires
minimal inputs. Thus, in the IVGB, tissue culture techniques are employed for medium-term
storage (MTS) [In vitro active genebank (IVAG)] and cryopreservation techniques for long-
term storage (LTS) ] In vitro base genebank (IVBG)].
In vitro active genebank
In the IVAG, cultures are maintained under sub-optimal growth conditions with an objective
to extend the subculture duration by slowing down or retarding the growth. Stepwise flow of
genetic material in the In vitro genebank (IVGB) is represented in Figure 2.
Step 1: Sample acquisition: The germplasm for in IVGB should be obtained as whole plants,
vegetative propagules or as in vitro cultures from an authentic source, with information about
correct identity of germplasm, complete passport details and characterization/evaluation data
(if available) through field explorations and importing from other countries.
Step 2: Quarantine/health testing of samples: Germplasm samples in the form of mother
plants/propagules should be always healthy and disease-free before conservation. In case of
exotic collections, samples should pass through appropriate quarantine checks along with
phytosanitary certificate. In case of live plants/cuttings, they should be established under
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
controlled conditions (screen house or in growth chamber) for proper growth and virus
indexing should be done prior to their introduction in vitro.
Step 3: Establishment of in vitro cultures
Explant selection: Type, source and physiological stage of explant is one of the
important factors in optimizing the in vitro establishment protocol. As the aim is in vitro
conservation, explants should preferably have pre-existing meristems which include
shoot tips, nodal segments, and perennating organs like suckers, tubers, bulbs, corms
or rhizomes, depending on the species.
Sterilization:The explants must be properly sterilized to remove microbial and surface
contaminants. Sterilization of explants may vary depending upon the crop and the type
of explant and it should be carried out using a disinfectant and a surfactant (type,
concentration and duration of
treatment may vary). This is
usually done by dipping the
explants in 70% ethanol for 2-5
min., followed by sterilant
treatment using mercuric chloride
or sodium hypochlorite
(concentration and duration may
vary) which will kill or remove
pathogens without injuring the
plant cells. Pre-soaking in an anti-
oxidant solution or short periods of
sonication (10-15 min in NaCIO)
may be useful for woody species.
Media standardization: Media
requirement for culture initiation
may be different from that for
optimal shoot multiplication and
rooting of shoots. Thus,
crop/genotype/accession specific
media as also the additives e.g.
plant growth regulators, activated
charcoal, amino acids, antioxidants
etc. should also be pre-determined
for optimal response.
Culture initiation: Depending on
the species, culture initiation may be carried out employing basal medium (Murashige
and Skoog 1962 or Gamborg et al., 1958) with or without growth regulators and
composition of culture medium may be determined from the published work
concerning culture of similar species or related genera. Sterilized explants are
implanted on a suitable medium in a culture tube with each tube covered with a plastic
cap and sealed with parafilm/cling film. Labelling in each tube is done for culture
number, species name, accession number, the date of inoculation etc. Culture tubes are
transferred to a growth chamber (temperature 20–25oC, suitable photoperiod and light
intensity). Screening for bacterial and fungal infection needs to be periodically
monitored. Seasonal variation, physiological stage and physico-chemical factors also
influence the response of explants in terms of culture establishment.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Virus indexing: Aseptic cultures should be indexed for specific viruses using standard
virus indexing methods based on serological (ELISA), molecular (PCR-based) and
ultrastructural (Electron microscopy) techniques.
Step 4. In vitro multiplication: Aseptic cultures established in vitro should be transferred to a
suitable multiplication medium for obtaining adequate number of explants for shoot
multiplication and subsequent conservation in IVAG. The medium required for culture
initiation may be different from that required for shoot multiplication, plantlet regeneration and
storage. Periodic monitoring for bacterial/ fungal contamination, hyperhydricity, growth
abnormalities (somaclonal variation, loss of regenerability etc.) after each subculture cycle has
to be carried out.
Step 5. In vitro conservation (slow-growth strategies): The main objective of slow growth
strategy is to enhance subculture duration without risking germplasm loss and compromising
genetic stability through stressful treatments. Consequently, there is reduction in maintenance
cost, coupled with efficient use of resources and manpower as growth is restricted using various
methods. Slow growth can be achieved employing several methods, such as using low
temperature, low-light intensity or no light, use of minimal media or osmotic agents (sucrose,
mannitol etc,), growth retardants and several others (see Table 1). Normally two or more
techniques are combined to obtain slow growth under in vitro conditions. Stored cultures need
to be scored for viability, chlorosis, defoliation, browning, tip necrosis, hyper hydricity and
contamination. Depending on the strategy adopted, cultures remain viable from 6 months to 5
years . Subculture should be carried out when 50% of the cultures are dead/dried/contaminated.
Table 1. Slow growth strategies in clonally propagated crops
I. Physical growth limitation
a) Low temperature
Temperature is the main limiting abiotic factor. The basic
principle of this method is that if in vitro plants are
maintained at a temperature below the optimum
temperature required for growth, the metabolic activities
are affected and there by the growth of plants becomes
restricted. For temperate species, storage temperature in the
range of 5-100C is suitable, whereas for tropical crops,
which are often sensitive to low temperatures, 10-150C is
beneficial.
Low temperature (200C) storage of cassava
cultures led to reduction in shoot growth by 15%
compared to those maintained under standard
growth conditions at 25-300C.
In taro (90C) and Prunus spp. (40C), subculture
duration was extended to 1-2 years.
b) Low light intensity
Reduction of light intensity or a complete darkness is often
used in combination with temperature reduction.
Subculture duration in banana could be increased
to 2 years following incubation of cultures in low
light (1000 lux) and at low temperature (150C).
c) Type of the enclosures
Type of the enclosures influence the rate of evaporation of
the water content of the media. Small culture vessels
minimize the growth and development of plants by limiting
gaseous exchange, space and nutrient supply.
Replacement of cotton plugs with polypropylene
caps has been beneficial in prolonging subculture
duration in species like Rauvolfia serpentina,
yams, sweet potato, ginger, turmeric, Allium
species and banana.
d) Size and type of culture vessels
The type of culture vessel can play a very important role
e.g. sterile, heat sealable polypropylene bags, large glass
bottles versus glass test tubes.
Storing of cassava plants in bottles (50×140 mm)
instead of culture tubes (25x150 mm) increased
the viability by decreasing the leaf fall. Use of
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
sterile heat sealable polypropylene bags for
strawberry has been very promising.
e) Reduced oxygen concentration
Growth of tissues decline when oxygen decreases and
viability of the callus cultures increase when stored under
less than 2-4ml of mineral oil (substitute for O2) at 220C.
Plantlets of Chrysanthemum and tobacco could
be stored for 6 wks at 1.3% O2.Somatic embryos
of oil palm were stored for 4 months at 1%
oxygen.
f) Osmotic adjustment
Use of osmotic regulators like sucrose, mannitol, sorbitol
etc.is recommended as these are relatively metabolically
inert and minimize the growth by imposing a level of
osmotic stress on the cultures.
Potato (60C and 4% mannitol. garlic (40C and
10% sucrose) and sweet potato (5% sorbitol) was
beneficial in prolonging the subculture duration
to1-2 years.
g) Modification of gaseous environment
Composition of gaseous environment with regard to gases
such as CO2, ethylene etc. inside the culture vessel
influences the growth rate of the cultures.
Though promising, it is expensive to maintain the
gaseous atmosphere in a large number of
individual vessels.
II. Chemical growth limitations
a) Minimal media
Lowering the mineral contents and sucrose has proved
beneficial in many species. Different genotypes may react
differently. With reduced temperature, it becomes the most
realistic method due to the synergistic effect.
Shoots of Ananas maintained at 250C with
quarter-strength salts. Replacement of sucrose by
ribose allowed the conservation of banana
plantlet for 24 months and in papaya, inclusion
of fructose in place of sucrose at 250C extended
subculture interval up to 12 months. In Allium
scorodoprasum, cultures could be stored for 5
years on minimal medium at low temperature.
b) Growth retardants
Growth retardants such as maleic hydrazide, abscisic acid,
n-dimethyl succinamic acid, phosphon-D and cycocel
reduce the overall growth rate of in vitro cultures and
enhance the subculture duration.
Use of growth retardants is generally not
preferred as it is likely to induce genetic
instability in cultures.
III. Other methods
Induction of in vitro storage organs
Useful for crops with natural storage organs (e.g. alliums,
ginger, turmeric, taro, yam, potato, sweet potato etc.).
Inclusion of high sucrose (8% or more) in the medium in
combination with light/dark conditions is conducive for in
vitro storage organ formation, which prolongs the shelf-life
of cultures
Induction of in vitro storage organs like bulblets,
micro rhizhomes, micro corms and micro tubers
has been beneficial in increasing the storage
duration up to 1 to 3 years in alliums, ginger,
turmeric, taro etc.
Step 6: Monitoring genetic stability of in vitro conserved germplasm: In vitro selection
pressure can potentially generate variants or mutants. Also some clonal genotypes have a
propensity for producing off-types and variants (due to natural chimeras). Plantlet regeneration
through pre-existing meristems (apical and axillary buds) and avoiding adventitious shoot
regeneration greatly aids in maintaining true-to-typeness of a genotype under culture
maintenance and offsets the risks of somoclonal variations.
In vitro base genebank
Cryopreservation is the non-lethal, viable, long-term storage of living tissues at ultralow
temperatures (−196 °C) usually that of liquid nitrogen (LN). At such a low temperature, plant
cell metabolism is in a suspended state of animation, eliminating the need to rejuvenate or
regenerate the plant. It is presently a supplementary tool to improve conservation of germplasm
on a long-term perspective. Cryopreservation is the most reliable method of choice for ensuring
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
the long-term storage of non-orthodox seeds, clonally propagated species and
biotechnologically important plant cell lines. Many studies have confirmed that it is
economically more viable than other conservation methods as the cost of maintaining an
accession in LN for the long-term (over 20 years) is substantially lower than that of in the field
or in vitro, particularly when dealing with a large number of accessions. Over the past 40 years,
individual scientists developed and tested a range of cryopreservation techniques for preserving
plant cells and tissues, but routine storage of plant germplasm other than seeds in LN, is gaining
momentum in recent years. Cryopreservation can be attempted using classical (freeze-induced)
or new cryopreservation (vitrification-based) techniques (Figs 3, 4, 5 &6 Table 2). Recently,
the latest method called V-cryo plate
(VCP) developed by the National
Institute of Agrobiological Sciences
(NIAS) in Japan was introduced at
IITA.
Table 2. Cryopreservation techniques
Cryopreservation
method
Explant Procedure
Pregrowth Meristems
Shoot tips/buds
Somatic embryos
Pregrowth of explants in cryoprotectants
(DMSO, PEG, Sucrose etc.)
Cryopreservation and storage in LN
Thawing and regeneration
Pregrowth-
dehydration Shoot tips/buds
Nodal segments
Somatic embryos
Zygotic embryos
Pregrowth of explants in cryoprotectants
(DMSO, PEG, Sucrose etc.)
Dehydration in a laminar airflow or over silica
gel
Cryopreservation and storage
Thawing and regeneration
Dehydration Zygotic embryos and
embryonic axes of non-
orthodox seed species
Explant dehydration using silica gel or air flow
for 60-360 min (250C)
Cryopreservation and storage in LN
Thawing and regrowth
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Vitrification Meristems
Shoot tips/buds
Nodal segments
Somatic embryos
Zygotic embryos
Cell suspensions
Preconditioning of explant source
(cryoprotectants/low temperature) (optional)
Pregrowth of explants in cryoprotectants and/or
at low temperature
Pretreatmentof explants with loading solution (
2M glycerol+ 0.4M sucrose or 10% glycerol
(optional)
Cryoprotective dehydration withcryoprotectant
mixtures (PVS2/PVS3)
Cryopreservation and storage in LN
Thawing and unloading
Regrowth
Encapsulation-
vitrification Meristems
Shoot tips/buds
Nodal segments
Somatic embryos
Zygotic embryos
Cell suspensions
Preconditioning of explant source
(cryoprotectans/low temperature) (optional)
Encapsulation of explants in sodium/ calcium
alginate (2-3%)
Cryoprotective dehydration with a cryoprotectant
mixture (PVS2)
Cryopreservation and storage in LN
Thawing and unloading
Regrowth
Encapsulation-
dehydration Meristems
Shoot tips/buds
Nodal segments
Somatic embryos
Zygotic/microspore
embryos
Cell suspensions
Preconditioning of explant source
(cryoprotectants/low temperature) (optional)
Encapsulation of explants in sodium/ calcium
alginate (2-3%)
Preculture in the medium containing high
concentration of sucrose (0.3 M - 1.2 M) for 1-7
days
Dehydration of encapsulated beads by air drying
in a laminar airflow or by exposure to silica gel
Cryopreservation and storage in LN
Thawing and regrowth
Droplet
vitrification Meristems
Shoot tips/buds
Nodal segments
Somatic embryos
Zygotic embryos
Cell suspensions
Preconditioning of explant source
(cryoprotectans/low temperature) (optional)
Pregrowth of explants in cryoprotectants and/or
at low temperature
Pretreatment of explants with loading solution (
2M glycerol+ 0.4M sucrose or 10% glycerol
(optional)
Cryopreservation (explants kept in droplets of
PVS2/PVS3 placed in aluminium foil strips) of
explants directly in LN
Storage in liquid nitrogen
Thawing and unloading
Regrowth
Dormant bud
cryopreservation
Dormant buds Collection and transportation of bud woods
Sealing cut ends with wax and packing in
polythene bags
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Excision of explants
Viability testing (TTC staining and in vitro
sprouting)
Determination of moisture and air desiccation
Storage in LN (Programmable freezing and step-
wise freezing)
Thawing and rehydration of bud
Recovery through in vitro and in vivo methods
Table 3. Status of in vitro conserved germplasm (as on March 31, 2019)
Crop group Gener
a
(no.)
Species
(no.)
Cultures
(no.)
Total
accession
s (no.)
Major collections
(no. of accessions)
Tropical
fruits
2 16 9,000 430 Musa spp. (429), Ensete glaucam (1)
Temperate
and minor
fruits
10 42 8,500 350 Actinidia spp. (6), Aeglemarmelos
(2), Artocarpouslakoocha (1),
Fragaria x ananasa (81),
Malusdomestica (30), Morus spp.
(61), Prunus spp. (13),
Pyruscommunis (73), Rubus spp.
(62), Vaccinium spp. (21)
Tuber crops
5 14 6,000 518 Alocasia indica (4), Colocasia
esculenta (90), Dioscorea spp. (153),
Ipomoea batatas (261), Xanthosoma
sagittifolium (10)
Bulbous and
other crops
4 14 3,500 171 Allium spp. (157), Dahlia sp.(6),
Gladiolus sp. (7), Cicer
microphyllum (1)
Medicinal
and aromatic
plants
25 34 4,000 172 Coleus forskohlii (14), Plumbago
zeylanica (19), Rauvolfia serpentina
(13), Tylophora indica (10),
Valeriana wallichii (16)
Spices and
industrial
crops
8 24 4,300 227 Curcuma spp. (110), Elettaria
cardamomum (5), Humulus lupulus
(8), Piper spp. (7), Simmondsia
chinensis (12), Stevia rebaudiana
(1), Vanila planifolia (4), Zingiber
spp. (80)
TOTAL 54 144 35,300 1,868
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Table 4. Status of germplasm in Cryogenebank (as on March. 31, 2019)
Categories Total no of
accessions
Recalcitrant & Intermediate 6,782
Fruits & Nuts 3,520
Spices & Condiments 152
Plantation Crops 88
Agroforestry, Industrial crops, Medicinal & Aromatic Plants 3,022
Orthodox 3,902
Dormant Buds 387
Pollen Grains 572
Genomic Resources 1,934
Total 13,577
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Engelmann F 2011. Use of biotechnologies for the conservation of plant biodiversity .In Vitro
Cell Dev Biol (Plant) 47:5–16.
Mandal BB, Chaudhury R, Engelmann F, Bhag Mal, Tao KL and Dhillon BS (eds) 2003.
Conservation Biotechnology of Plant Germplasm. NBPGR, New Delhi, India/IPGRI,
Rome, Italy/ FAO, Rome, Italy.
Normah MN, Chin HF and Reed BM 2013.Conservation of Tropical Plant Species.Springer,
New York, 538p.
Rajasekharan PE and Ramanatha Rao V (eds). 2019. Conservation and Utilization of
Horticultural Genetic Resources. Springer, Singapore, 663p.
Reed BM 2008. Plant Cryopreservation: A Practical Guide. Springer, NewYork, 511p.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
GUIDELINES FOR SENDING GERMPLASM FOR PEST FREE CONSERVATION
Veena Gupta, Sushil Pandey and Smita Lenka
Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic Resources,
New Delhi-110 012
Seed genebanks play a pivotal role in sustainable agriculture and global food security by virtue
of being rich reservoirs of potential genes. The germplasm conserved in these seed genebanks
offer genes resistance to pest and diseases and resilience to abiotic stresses, thus catering the
need of plant breeder/ conservationists/ scientists by ensuring the supply of desired germplasm
for crop improvement programmes. The proactive role of the genebank managers is to ensure
high viability and vigour of the incoming germplasm so that they can be safely conserved for
longer periods. Therefore, proper handling of the germplasm at seed genebanks is inevitable
for cost effective, safe and efficient conservation. This in turn facilitates the pest-free
conservation and subsequent distribution of germplasm from genebanks. It also helps in
preventing the risk of accidental failures in domestic quarantine measures during distribution
of germplasm from genebanks.
In order to achieve this, following steps should be strictly followed before sending the
germplasm for long term conservation at genebank.
Harvesting of seeds at their physiological maturity results in maximum viability and
longevity of the germplasm. Thus harvesting should be done at stage showing
maximum maturity of the crop.
Utmost care should be taken to avoid harvesting in rainy season as the seeds will absorb
the atmospheric moisture and will affect the moisture content of the seed and during
storage, such seeds will be susceptible to deterioration, develop diseases and become
infested with insects and pests.
Seed should be dried soon after harvest (under shade only) to around 10–12 % moisture
content. This is to be done with constant stirring of the seed to reduce the moisture
content evenly. Exposures to direct sunlight sometimes cause rapid and uneven loss of
moisture which is not advisable. If possible, dry the seeds in air-conditioned or
dehumidified rooms.
In case of regenerated/multiplied seeds, care should be taken to avoid mixing of seeds
harvested in different seasons, as the quality and durability of the samples can be
different. Batch numbers (indicating season of harvest, site or field number and
generation number) may be assigned to differentiate the seed lots.
After proper drying, seeds should be stored in a cool and dry environment with proper
ventilation to avoid spoilage due to pathogens and pests. The duration between harvest
of the seed lots and their dispatch to gene bank should be minimized to reduce the risk
of disease outbreak.
Purity should be ensured that the samples are devoid of seeds of weeds and other crop
species, debris and inert material. It should also be free from empty, immature, damaged
and infected seeds. Only mature and clean seeds free of any insect pests and fungi
should be sent to the gene bank. Undersized, shrivelled and immature seeds must be
discarded.
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Sample should contain at least 2,000 seeds for self-pollinated crops and twice that
number for cross pollinated crops (i.e., 4,000 seeds) so that it completely represents the
variability of the original sample and also permits enough seeds for monitoring of
viability during storage and subsequent regeneration. If it is freshly collected
germplasm, the explorer should work towards collecting sufficient quantities of seed to
the extent possible; otherwise the germplasm should be sent for long term conservation
after multiplication in the next season. Sample size may be reduced in case of
wild/weedy/wild relatives of crop plants/vegetable germplasm where standard seed
quantity as per genebank guidelines is difficult to achieve.
Careful scrutiny of the seed samples should be done by visual examination using hand
lens or stereoscopic microscope. Samples free of pathogens, insects, fungal growth,
bacterial and viral infections should be used for long term conservation in the genebank.
Seeds are to be packaged properly to prevent absorption of water during transit.
Accessions are kept separately to avoid mixing of samples. Seed material for gene bank
needs to be packed in either muslin cloth bags or paper bags and wrapped in polythene
bags to minimize damage during transit and to prevent contamination from pests,
insects and diseases.
Proper documentation of seed packaging is of utmost importance. So prepare label for
each packet in duplicate, put one inside the packet and affix the other one on the packet.
After labelling, prepare a list of all germplasm which is meant for dispatch and long
term conservation. Carefully enclose this list with the seed packets.
Pack all seed packets in a carton/cardboard box and seal it properly to avoid any damage
during transportation. Packing in gunny bags should be avoided.
Seeds should not be treated with any chemical or insecticide or pesticide. If necessary,
put naphthalene balls in separate muslin cloth bag and put this bag in the cardboard
box. Direct contact of naphthalene balls/powder with seed germplasm should be strictly
avoided.
Passport data proforma is a must for the allotment of IC numbers. To ensure proper
identity in the genebank all samples should be accompanied by adequate passport
information (especially cultivar name, collector number and pedigree for genetic stocks
and improved material). The minimum required passport data sheet developed by
NBPGR should be filled carefully (Annexure 1). It should be submitted to Head,
Division of Germplasm Exploration and Collection, NBPGR under intimation to Head,
Division of Germplasm Conservation.
In case of availability of characterization data for specific attributes like stress
tolerance, disease and pest resistance, those should be indicated against each and every
accession/ collection.
Germplasm processing activities at GHU
When seed samples are received at the Germplasm Handling Unit (GHU), there is a general
work plan which is being followed so that seeds are processed and entered into the genebank
with speed, accuracy and efficiently (Fig. 1).
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Fig. 1. Flowchart of activities at the Germplasm Handling Unit
As soon as the plant germplasm are received, it is ensured that all seed packets/cloth bags are
properly labelled for the accession identity. Each accession is treated separately and
documented digitally. Documentation management system will facilitate the effective and
efficient use of stored germplasm materials in the genebank.
To ensure the maximum potential of the seed endurance, high quality seeds should be stored.
Seed cleaning is crucial to maintain the seed purity and health, i.e., separating of seeds of
interest from other seeds, and inert matter or debris. Debris can consist of a wide range of plant
No
* Self-pollinated (>2000), Cross-pollinated (>4000) and wild (>500)
** Viability relaxation for wild species in Vegetable/Medicinal/Rare
endangered /forage species (50-70%)
No
Yes
No
No
No
Yes
Yes No
Yes
Are the documents complete?
Verification of Documents
i. Passport Data sheets in case of germplasm
ii. Proposals in case of release varieties
iii. Registration Proforma in case of registered germplasm
Correspond with the Donor
and get more information
Domestic Quarantine (DQ) number allotment and entry in
GHU database and check for the duplicates in the National
Genebank database
Is the sample Unique?
Is the sample with sufficient
quantity*?
Correspond with donor for
more information
Program it for
regeneration/multiplication
Is the seed sample clean?
Remove debris, infested
and broken seeds
Is the seed health satisfactory as
per standards of plant quarantine?
Yes
Send it to concerned crop curator for
viability testing
Is viability** >85%
Sent back to the
donor for
multiplication
Assign National ID (PGEC Division) and document information in database and
conserve the sample in the Genebank
Fumigated samples
Germplasm Acquisition
Fumigate or discard the
sample under intimation to
donor
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and soil-derived matter which were adhered to the seeds and not separated during harvesting
and threshing. They may comprise soil, sand, stones, chaff, plant parts, pests, etc. As soon as
the germplasm are received at GHU, the removal of weeds, impurities and debris is done by
hand cleaning.
As seed is the most important medium of spread of pests (fungi, bacteria, virus,
nematodes, weeds, insects and mites, etc.), maintenance of seed purity is essential to ensure
that the accessions stored are true to type, maximize the use of storage space and prevent
contamination by seeds of weeds and other species.
All the germplasms received by GHU are sent for seed health testing to detect and
identify the pests and make them free from pests and pathogens in Plant Quarantine Division
of NBPGR before their conservation in the National Gene Bank. With the onset of mission
mode National Agricultural Technology Project (NATP) on agro-biodiversity in 1998, seed
health testing (SHT) was initiated in 1998 at ICAR-NBPGR, New Delhi for the germplasm
collected under the project on agro-biodiversity.
Annexure-I
PASSPORT DATA FORM
Collector’s Name and Address:
Collaborating Institute: Name of Scientist(s) and Address:
Area surveyed:
S.N Collector
No.
IC.
No.
Crop
Name
Botanical
Name
Vernacular
Name
Cult./Wild/
Hybrid
Type of
Material*
Date of
Collection
Source Frequency
S.N. Collector
No.
Sample
Type
Sampling
Method
Habitat Site of Collection Latitude Longitude Altitude Remarks
Village Mandal District State
*Type of Material: Seeds, Fruits, Inflorescence, Roots, Tubers, Rhizomes, Suckers, Live Plant,
Herbarium,
The completed sheets for the allotment of IC number should be sent to:
The Head
Division of Germplasm Exploration and Collection
ICAR-National Bureau of Plant Genetic Resources
Pusa Campus, New Delhi-110 012
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
SEED HEALTH TESTING FOR PEST-FREE CONSERVATION OF PLANT
GENETIC RESOURCES
Jameel Akhtar, Kavita Gupta, Zakaullah Khan, Moolchand Singh, V Celia Chalam,
Meena Shekhar, SP Singh, T Boopathi, BH Gawade, Pardeep Kumar, Raj Kiran,
AK Maurya, DS Meena, Smita Lenka and SC Dubey
ICAR-National Bureau of Plant Genetic Resources, Division of Plant Quarantine,
NBPGR, New Delhi – 110012
ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi, India,
has one of the mandates for acquisition and management of Plant Genetic Resources (PGR)
for conservation and their utilization towards food security and sustainability. Pest-free
conservation of PGR is one of the goals of seed-health testing (SHT) and the key to target
the success in achieving ‘Food security’. No country in the world is self-sufficient in PGR
for developing new varieties of crops to overcome the various types of threats viz.
insurgence of new/ more virulent pests, weather calamities, extreme temperatures, etc.
PGR are increasingly under threat because of continuing degradation of natural habitats
and rapid replacement of locally adapted indigenous cultivars by modern high yielding
varieties. Thus, the fast shrinking genetic diversity of commercially grown crops renders
them vulnerable to widespread epidemics and pest rampage. Therefore, SHT of PGR is
carried out to detect pests associated with seeds.
About 4.41 lakh germplasm accessions of various crops belonging to > 1900 crop species
are conserved in the National Genebank (NGB), ICAR-NBPGR, New Delhi. About 1500
seed-borne fungi, ~302 bacteria (Richardson, 1990) and ~131 viruses (Power and Flecker,
2003) affect 534 crops of 109 plant families. Some of the major seed-borne pathogens
causing diseases on crops of economic importance are Drechslera oryzae, D. maydis, D.
sorokiniana, Botrytis cinerea, Colletotrichum dematium, Fusarium verticillioides (syn: F.
moniliforme), F. solani, Macrophomina phaseloina, Pyricularia grisea, Phoma betae, P.
lingam, Phomopsis vexans Puccinia helianthi, Rhizoctonia solani, Tilletia barclayana and
Xanthomonas campestris pv. campestris, Bean common mosaic virus, Cherry leaf roll
virus, Cowpea mottle virus, etc. (Richardson, 1990). Hence, seed is the most important
means of spread of pests and pathogens. Therefore, SHT of germplasm before their
conservation is utmost important step for long term conservation in pest-free state.
Processing of germplasm
About 10,000 accessions of indigenously collected/ multiplied PGR are received annually
through Division of Germplasm Conservation, ICAR-NBPGR, New Delhi, India for seed
health testing (SHT). SHT at ICAR-NBPGR was first initiated in 1998 for the germplasm
collected under mission mode National Agricultural Technology Project (NATP) on
agrobiodiversity. The Division of Plant Quarantine at ICAR-NBPGR has developed
procedures for systematic and stepwise processing for detection of pests associated with
plant genetic resources (Fig. 1). The methods used for seed-health testing are: visual
examination of dry seeds, washing test, seed soaking, incubation method using blotter test,
X-ray radiography, seed transparency. Visual and stereo-binocular examination to detect
presence of smut and bunt balls, ergot sclerotia, rust pustules, spores on the seed; washing
test for rusts and downy mildews; blotter method for seed-borne fungi and bacteria, soil
clods, weed seeds, insect eggs, adults, exuvae, etc.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Fig. 1. Seed health testing procedure for pest free conservation of indigenous crop
germplasm.
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Observation on associated pests
Seed-borne pests and pathogens may result in poor quality seed, loss in germination,
development of epiphytotics, distribution of new strains or physiological races of
pathogens along with the seeds and planting material to new geographical areas. Therefore,
critical laboratory examinations with specialized seed health testing methods are conducted
and ensure the identification of fungal, pathogens associated with seeds and other planting
materials.
Dry examination of seeds and washing test
Preliminary examination with naked eye or with the help of a magnifier to detect presence
of abnormalities such as discoloration, deformation shriveling, pigmentation,
malformation of seed with fungal growth like mycelial mats or fructifications like
chlamydospores, acervuli, pycnidia, perithecia and other impurities associated with a seed
lot such as sclerotia, smut balls, or spore masses, soil clods, plant debris, etc. and washing
test for the presence of rusts and downy mildew spores. Visual examination of seed/
washing test results in detection of economically important pests in crop germplasm. This
includes fungal/ viral pathogens, insects and weeds. Some fungal and viral disease
symptoms such as purple stain (Cercospora kikuchii) and mottling symptoms (Bean pod
mottle virus) in Glycine max; tennis ball and split seed coat in Pissum sativum; grain smut
(Sphaecelotheca sorghi) in Sorghum bicolor; smut (Ustilago crameri) in Setaria italic;
Tilletia barclayana and Ustilaginoidea virens in Oryza sativa; Karnal bunt (T. indica) and
hill bunt (T. foetida) in Triticum aestivum (Fig. 2) are very important.
Fig. 2. Symptoms of seed-borne fungal diseases in different crops; a & b) Karnal bunt and
hill bunt of wheat, c) sorghum grain smut, d) foxtail millet smut, e & f) purple stain
and mottling of soybean, g & h) split seed coat and tennis ball of pea, i & j) kernel
smut and false smut of rice.
The insect species commonly detected in crop germplasm are Rhizopertha dominica,
Sitotroga cerealella, Sitophilus zeamais, Lasioderma serricorne, etc. (Fig. 3).
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
a b c d
e f g h
Fig. 3. Detection of insect infestation in crop germplasm, a) mung bean, b) urd bean, c &
d) faba bean, e) Rhizopertha dominica, f) Sitotroga cerealella, g) Sitophilus
zeamais and h) Lasioderma serricorne in crop germplasm.
The weed species commonly detected in crop germplasm are Anagalis arvensis, Avena
fatua, Chenopodium album, Emex australis, Lathyrus aphaca, Lolium perenne, Melilotus
indica, Phalaris minor, Sorghum halpens, Vicia hirsute, Echinochloa crusgalli, E. Colona,
Dactyloctenium egyptium. Etc. (Fig. 4)
a b c d e
Fig. 4. Detection of weeds, a) Avena fatua, b) Phalaris minor, c) Sorghum halpens, d) Emex
australis and e) Lolium perenne in crop germplasm.
Seed soaking paddy bunt: For detection of bunt (Tilletia barclayana) in rice, seeds are
soaked overnight in 0.2 per cent sodium hydroxide and examined. The infected seed shows
shiny jet black discolouration. Infected seeds rupturing in a drop of water, releases a stream
of bunt spores
Incubation Method: Incubation is a simple method commonly used for detection of
mycoflora accompanied as mycelium, spores, or fruiting structures capable of growing on
the seed during incubation of seed on wet blotter or agar. Surface sterilization of the seeds
using a 4% NaOCl solution is carried out before incubation to eliminate fast growing
saprophytes if the seeds are heavily contaminated.
Blotter test: Blotter test, generally referred as the standard blotter test, is the most efficient
means of detecting a large number of seed-borne fungal pathogens.
Visually discoloured, deformed and unhealthy looking/ suspected seed are undergone for
blotter test by placing the seeds on 3 layers of moist blotter paper in plastic petriplates with
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
proper labelling and incubated at 22±20C under light in alternate cycles of 12 h light and
darkness for 7 days and examined on 8th day under stereo-binocular microscope for
presence of seed-borne fungi (Mathur and Kongsdal, 2003). The critical microscopic
examination enables the observation of pathogens as developed on their hosts in situ,
undisturbed and in a condition of natural growth. The identification is confirmed up to
species level by making slides for examining the structure, size and colour of fruiting
bodies/ conidiophores/ conidia under compound microscope at different levels of
magnification i.e. 4.0 X to 40.0 X. A critical stereo-cum-compound microscopic
examinations of seeds on 8th day after incubation results in detection of many seed-borne
pathogens. Major detection inclused Alternaria brassicae, A. brassicicola, Bipolaris
oryzae, B. sorokiniana, Botrytis cinerea, Colletotrichum capsici, Dendryphion
penicillatum, Stenocarpella maydis, Fusarium oxysporum, F. solani, F. verticillioides,
Phoma sorghina, Macrophomina phaseolina, Myrothecium roridum, Rhizoctonia solani,
Sclerotinia sclerotiorum, Verticillium albo-atrum, etc. on different crop germplasm.
a b
c d
Fig. 5. Detection of Bipolaris oryzae (a) and Phoma sorghina on rice; Pestalotia juepini
(c) and Myrothecium roridum (d) on cucurbits.
X-ray radiography and Transparency test: X-ray radiography is used to detect seeds
infested with phytophagous chalcidoids, bruchids and certain other insect groups which do
not show any external symptoms on seed surface. Based on literature survey and past
experience a list of >340 plant genera has been drawn up that are compulsorily subjected
to X-ray radiography. On developing the X-ray plates, insects if present, are hand-picked
and healthy seeds released to the indenter. Transparency method is used for detecting
infestation in small seeds and seeds of family Graminae. In case of real-time X-ray
radiography, the process is much faster and salvaging is done immediately after the image
of infested sample appears on the computer screen. The seeds are boiled in lacto-phenol
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
solution (phenol, lactic acid, distilled water and glycerin in the ratio of 2:2:2:1 respectively)
for 1-2 hours depending on the hardness of the seeds. This renders them transparent to
reveal insect infestation. Several species of bruchids viz., Callosobruchus maculatus, C.
chinensis, C. phaseoli, C. analis, Bruchus pisorum, Caryedon serratus and chalcids such
as Systole coriandri were detected using X ray radiography (Fig. 8). The detection of
important hidden infestation caused by insects was instrumental in pest free conservation
of precious germplasm collected/ regenerated by the NARS.
a b c d
Fig. 6. X –ray radiographs of seeds showing hidden infestation of bruchids (a, b & c) and
chalcids in Trifolium seed (e).
Seed soaking for detection of Phytonematodes
Soaking of seeds known/ suspected to carry seed-borne nematodes in water overnight
softens the seeds which are teased/ crushed enabling the nematodes, if present, to come out
in water (Fig. 7). Soaking of some plant material in water and when sieved through
nematological sieves (the finest sieve is of 400 mesh per linear inch) reveals nematodes
that are retained on the sieve, such as Aphelenchoides besseyi from rice seeds (Fig. 8),
Anguna tritici from wheat galls. These are recovered and examined under the compound
microscope for identification. Staining technique is used for quick detection of nematodes
in vegetative propagules where a part of the plant tissue (especially roots) is boiled in acid
fuchsin lacto-phenol solution for a few minutes and de-stained in clear lacto-phenol. The
nematodes, if present such as root knot nematodes, Meloidogyne spp. and root lesion
nematode, Pratylenchus spp, retain the red stain more deeply than the plant tissue and can
easily be detected under stereo microscope. Examination of accompanying soil shows the
presence of viable nematodes, especially, ectoparasites and cysts of cyst forming
nematodes (Heterodera and Globodera spp).
a b c d e f
Fig. 7. Processing of samples for nematode detection and identification from different plant
parts of crops and soil clods; a) Saplings, b) sapling roots, c) rice seed, d) monk
fruit seed, e) saffron bulb and f) soil clods.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Fig. 8. Detection and identification Aphelenchoides besseyi in rice seed; A) nematode
suspension, B, anterior region, C) posterior region, D, tail terminus bearing mucro
with pointed processes.
All these pests and pathogens are of economic significance as heavy crop losses have been
reported by them from different parts of the country. If infected/ infested/ contaminated
seeds are conserved and/ or distributed either for research purpose or their commercial use,
these seeds can act as a source of inoculum and the pests and pathogens will further spread
across the country which may hamper the cultivation of the crops and their wild relatives
leading to reduction in yield and quality. Therefore, seed health testing is of high
importance in conserving pest-free germplasm for quality assurance and minimizing the
risk of spreading pests in the country.
References
Bhalla S, VC Chalam, A Lal and RK Khetarpal (2009) Practical Manual on Plant
Quarantine. National Bureau of Plant Genetic Resources, New Delhi, India p. 204
+ viii.
Elling A (2013) Major emerging problems with minor Meloidogyne species.
Phytopathology 103:1092-1102.
Ferris H, KM Jetter, IA Zasada, JJ Chitambar, RC Venette, KM Klonsky and JO Becker
(2003) Risk assessment of plant-parasitic nematodes, in exotic pests and diseases:
biology and economics for biosecurity (ed. DA Sumner), Blackwell Publishing
Company, Ames, Iowa, USA. doi: 10.1002/9780470290125.ch8
Kaura, A (1959) A new transparency method for detecting internal infestation of grain.
Grain Storage News Letter, 1(1): 12.
Mathur SB and O Kongsdal (2003) Common laboratory seed health testing methods for
detecting fungi. International Seed Testing Association, Basserdorf, Switzerland.
425 p.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Richardson MJ (1990) An Annotated list of seed-borne diseases. Fourth Ed. International
seed Testing Association, Zurich, Switzerland, 387 p.
Siddiqi, M.R. 1986. Tylenchida parasites of plants and insects. Wallingford, UK, CAB
International. 645 pp.
Singh Baleshwar, PC Agarwal, Usha Dev, Indra Rani, Dinesh Rai, and RK Khetarpal
(2004) Detection of pathogenic fungi associated with indigenous germplasm during
1999-2001. Indian Journal of Plant Protection 32: 102-106.
Udaigiri, S. and Wadhi, S.R. 1982. A key to world bruchid genera. NBPGR Sci. Monogr.
No.5: 1-16.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
SEED VIABILITY TESTING: PRINCIPLES AND PRACTICES
Anjali Kak Koul and Sherry Rachel Jacob
Division of Germplasm Conservation, ICAR-NBPGR, New Delhi-110012
The primary objective of long term conservation of seeds in the genebanks is to ensure
maintenance of viability of the seed for maximum possible duration. Viability testing is
one of the primary tests that are conducted on each accession, when seeds are received for
conserving as base collection. The purpose of laboratory testing of seed germination is to
assess seed quality or viability and to predict performance of the seed and seedling in the
field. The most common and reliable method of testing seed viability is the germination
test. As per international genebank standards prescribed by Food and Agriculture
Organization of the United Nations, the initial germination value should exceed 85% for
all cultivated crop species. For wild species, where high germination percentage is
practically not attainable, lower values are accepted.
Germination is defined as the “emergence and development from the seed embryo of those
essential structures which, for the kind of seed tested indicate its ability to develop into
normal plant under favorable conditions in the soil” (Anonymous 1985). In order to bring
uniformity in the testing procedures, the International Seed Testing Association (ISTA)
formulated a set or rules. However, for gene bank purpose IBPGR Advisory Committee on
seed storage has also formulated a set of rules which are basically ISTA rules with slight
modifications. Any genetic resources laboratory should have both sets of rules to guide
them for the germination testing procedures.
General Principles
The germination test in the laboratory should always be done on pure seed fraction. A
random sample of 400 pure seeds should be taken and put for germination; in replicates of
100, 50 or 25 seeds. The seeds should be uniformly spaced on a moist substratum. Since
germplasm collected is very valuable and sometimes only limited quantity of seed is
available under such circumstances IBPGR Advisory Committee recommends that for the
initial germination test for the species where a reasonable germination technique is
available, a minimum two replicates using 200 seeds (100 seed per replicate) is acceptable,
providing that the germination is above 90%. If not a further 200 seeds should be tested as
before and the overall result for seed viability taken as mean of the two tests. The replicates
are then placed under optimal germination conditions, usually including a treatment to
break dormancy, if needed. The first count is made when the majority of seedlings have
reached the developmental stage at which proper evaluation is possible. Since the seedlings
being tested are entirely dependent for growth on the nutrients stored within the seed, they
must be evaluated before they exhaust the nutrients and begin to rot.
The normal seedlings are removed and counted. Rotten seeds and decayed seedlings are
also removed to prevent further contamination. At the time of final recording, numbers of
fresh ungerminated and hard seeds are also counted. At the end of the test if the results of
the replicates fall within the tolerance range, the average of normal seedlings of the
replicates represents the percentage germination of that seed lot/accession.
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Germination Medium
Various types of media can be used for germination testing depending of type of the seed,
its size. The media should be sufficiently porous to allow penetration of air and water and
should also permit unrestricted root and shoot growth. The growing medium can be paper,
pure sand or mixtures of organic compounds with added mineral particles.
(A) Paper: Paper, in the form of filter papers, blotters or towels, is the most commonly
used growing medium.
The basic methods of germination testing using paper medium is elaborated below-
1. Top-of-paper method: The seeds are germinated on pre-moistened, double-layered
filter papers that are placed in Petri dishes. The lids are tightly closed to prevent
evaporation. The method is generally practiced in case of small seeds.
2. Between Paper (BP) method: The seeds are germinated between two layers of
paper. Most commonly moist, rolled paper towels are used for this. The paper
towels are layered with a wax paper on the outside, to minimize evaporation and
drying. The rolls are then packed in an upright position, within plastic bags. The
method is generally used for medium and large seeds such as those of cereals,
legumes, vegetables etc.
The Petri dishes or paper towels are placed in germination incubators that are pre-
set at the required temperature. Relative humidity of the incubator is preferably
maintained at near saturation point.
(B) Sand: Sand is normally used as substrate for lager seeds such as castor, groundnut,
beans etc. Depending on their size, the seeds can either be planted on a layer of moist sand
and covered with 10-20 mm of loose sand, or planted on the top of the most sand and
pressed into it. The sand should be properly graded, sterilized and free from impurities and
toxic chemicals. In the case of sand medium, at least 90 % of the particles must pass
through a sieve with holes or meshes of 2.0 mm width.
The amount of water to be added to the sand will depend upon its characteristics
and the size of the seed to be tested. The optimum amount should be determined for the
main kind of seeds, so that a measured quantity could always be added in routine testing.
Generally for cereals except maize, sand is moistened to 60 per cent of its water holding
capacity.
The approximate quantity of water to be added to the sand can be calculated by the
formula given below.
ml of water to be added = 118.3ml sand x(20.2-8)
to every 100g of sand wt of 118.3ml of sand (in gms)
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
(C) Soil: Soil or artificial compost is commonly used instead of sand to test sample that
produce seedlings with phyto-toxic symptoms when germinated in sand or paper. The soil
or artificial compost is generally more difficult to standardize and is therefore, liable to
cause greater variation between results. But this substratum must be used to confirm the
evaluation of seedlings in doubtful cases and for testing samples which produce seedlings
with phyto toxic symptoms when germinated on paper. Water should be added until the
consistency of soil is such that the ball formed by squeezing it in the palm of the hand is
easily broken when pressed between two fingers.
Test conditions
a) Moisture and aeration
The moisture requirements of the seed will vary according to its kind. Large seeded species
require more water than the small seeded species. It is essential that the substratum must
be kept moist throughout the germination period. Care need to be taken that the sub-stratum
should not be, too moist. The excessive moisture will restrict the aeration and may cause
the rotting of the seedlings or development of watery seedlings. Except the situations where
a high moisture level is recommended (e.g. Paddy and jute), the substratum should not be
so wet that a film of water forms around the seeds. In situations where low level of moisture
is recommended, the moist substratum should be pressed against the dry blotters or towel
paper, to remove excess moisture. The water used for moistening the substratum should
have pH in the range of .5-7.5. In order to reduce the need for additional watering during
the germination period, the relative humidity of the air surrounding the seeds should be
kept at 90-95 % to prevent loss of water by evaporation.
Special measures for aeration are not usually necessary in case of top of paper (TP)
tests. However, in case of ‘Roll towel’ test (BP) care should be taken that the rolls should
be loose enough to allow the presence of sufficient air around the seeds. In case of sand
media, the sand should not be compressed while covering the seeds.
b) Temperature
The temperature is one of 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. This can vary within the species and with the age of seeds. At very low or high
temperatures, the germination is prevented to a larger extent. The temperature should be
uniform through out the germinator and the germination period. The variation in
temperature inside the germinator should not be more than 1°C. The prescribed temperature
for germination of agricultural, vegetable or horticultural seeds, provided in the Rules for
Seed Testing can be broadly is classified into two groups, viz. constant temperatures and
alternate temperatures.
Constant temperature
Wherever, the constant temperatures are prescribed or recommended for the germination
tests, the tests must be held at the specific temperature during the entire germination period.
Alternate temperature
Wherever, the alternating- temperatures are prescribed, the lower temperature should be
maintained for 16 hours and the higher for 8 hours; a gradual changeover lasting 3 hours
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
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.
c) Light
Seed of most of the species can germinate, in light or darkness. It is always better to
illuminate the tests 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 that any heating from the source that an even intensity is obtained over
the entire substrate, and that any heating from the source does not affect the prescribed
temperature.
Seed 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 alternating” temperatures together with light, the tests should
be illuminated during high temperature period.
Duration of testing
The duration of the test is determined by the time prescribed for the, final count (ISTA
Seed Testing Rules, Table 2) but the chilling, periods before or during the test, which is
required to break dormancy, is not included in the test may be extended for an additional
period up to 7 days. A test may be terminated prior to the test 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. The First
count may be delayed to permit the development of root hairs in order to be certain that
root development is normal, or may be omitted. Intermediate counts may be at the
discretion of the analyst to remove seedlings, which have reached a sufficient state of
development for evaluation, to prevent them becoming entangled. But the number of
intermediate counts should be kept to a mini-mum to reduce the risk of damaging any
seedlings that are not sufficiently developed.
Seedlings may have to be removed and counted at more frequent intervals during the
prescribed period of the test when a sample contains is infected with ‘fungi or bacteria.
Seeds that are obviously dead and decayed, and may, therefore, be a source of
contamination for healthy seedlings, should be removed at each count and the number
recorded.
Evaluation of germination test
The germination tests need to be evaluated on the expiry of the germination period, which
varies according to the kind of seed. 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 test.
The first and subsequent counts, only normal and dead seed (which are source of infection)
are removed and recorded. In evaluating the, germination test, the, seedlings and seeds are
categorized into normal seedlings, abnormal seedlings, dead seeds, fresh ungerminated
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
and hard seeds. The fresh ungerminated or hard seeds and abnormal seedlings should be
evaluated at the end of germination.
1. Normal Seedlings: Seedlings which have the capacity for continued development into
normal plant when grown in favourable conditions of soil, water, temperature and light.
Characters of normal seedlings:
(a) A well-developed root system with primary root-, except in certain species of
Gramineae which normally producing seminal roots or secondary root.
(b) A well-developed shoot axis consisting of intact hypocotyls in seedlings with
epigeal germination.
(c) A well developed epicotyle in seedlings of hypogeal germination .
(d) One cotyledon for seedlings of monocotyledons and two cotyledons and seedlings
of dicotyledons.
(e) A well developed coleoptiles in graminae containing green leaf
(f) A well developed plumule in dicots
1. Abnormal seedlings
Seedlings which do not show capacity for continued development into normal plants
when grown under favorable conditions of light temperature and water.
Seedling with following defects can be classified as abnormal seedlings.
(a) Damaged seedlings:Seedling with any one of essential structures missing or
badly damaged so that the balanced growth is not expected . Seedslings with no
cotyledons with splits cracks and lesions of essential structures and without
primary roots in those species where primary root is essential
(b) Deformed seedlings: Weak or unbalanced development such as spirally twisted
or stunted plumules, hypocotoyls and epicotyl, swollen shoots,stunted roots,split
plumules,empty coleoptile watery and glassy seedlings etc.
(c) Decayed Seedlings: Decay in essential structures resulting from seed borne
infection.
2. Hard Seeds: Seeds, which remain hard and dormant at the end of the prescribed test
period because they have not absorbed water due to an impermeable seed coat, are
classified as hard seeds.
3. Fresh ungerminated seeds: Seeds, other than hard seeds, which remain firm and
apparently viable.
4. Dead seeds: Seeds which at the end of the test period are neither hard nor fresh and
have not produced seedling are classified as dead seeds. Often dead seeds collapse and
milky white exudate comes out when pressed at the end of the test.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Calculation and expression of result
The seedlings are assessed when they have reached a stage where the essential seedling
structures can be clearly examined. The day for first and final examination has been
standardized by ISTA for each species. The result of the germination test is expressed as
percentage by number of normal seedlings. The percentage should be rounded to the
nearest whole number. If there are ungerminated seeds on the final count day, these seeds
should be cut and examined, to verify whether they are dead or dormant. If the dormant
seeds are significantly large in number, the seeds should be retested, after applying
dormancy breaking treatments that are recommended for the species.
The ISTA recommended medium, temperature and days of first / final counts, for major
agricultural and horticultural crops, are listed in Table 1.
Essential equipment required for germination in laboratory
a) 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
b) Counting devices
The counting devices include the counting boards, automatic seed counter and vacuum
seed counter. These devices are required to aid germination testing by minimizing the time
spent on plating 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 the absence of counting devices, the work
may be accomplished manually.
c) Other requirements
The other equipments required for routine germination testing include refrigerators,
scarifier, hot water bath, incubator, germination boxes, Petri dishes, forceps, plastic trays,
oven for sterilization of glassware, sand/soil germination paper, beaker, measuring
cylinders, scales etc. Certain chemicals like Gibbrelic acid, potassium nitrate. Thiourea,
Sulphuric acid, Tetrazolium chloride, distilled water etc. are also required for specific
purposes.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Fig. 1. Germination testing for some agricultural and horticultural seeds using
different substrata for germination
Table 1. Germination standards for some agricultural and horticultural crops as
recommended by ISTA (ISTA rules, 2015).
Species Substrata Tempoc First
count
Final
Count
Abelmoschus esculentus (Okra) TP;BP 20-30 4 21
Arachis hypogaea (Groundnut) BP;S 20-30;25 5 10
Allium cepa (Onion) BP;TP 20;15 6 12
Avena sativa (Oat) BP;S 20 5 10
Beta vulgaris (Sugar beet) TP;BP;S 20-30;15-25 4 14
Brassica juncea (Sarson) TP 20-30;20 5 7
Brassica juncea (Rapa) TP 20-30;20 5 7
Brassica oleracea (Cabbage, Cauliflower) TP 20-30;20 5 10
Cajanus cajan (Red gram) BP;S 20-30;25 4 10
Capsicum sp.(Chilly) TP;BP 20-30 7 14
Cicer arietinum (Bengal gram) BP;S 20-30;20 5 8
Corchorus sp.(jute) TP;BP 30 3 5
Crotalaria juncea (Sunhemp) BP;S 20-30 4 10
Cucumis melo (Muskmelon) BP;S 20-30;25 4 8
Cucumis sativus (cucumber) TP;BP;S 20-30;25 4 8
Cucurbita maxima (Winter squash) BP;S 20-30;25 4 8
Cucurbita moschata (Pumpkin) BP;S 20-30;25 4 8
Cucurbita pepo (Summer squash) TP;BP;S 20-30;20 7 14
Daucus carota (Carrot) TP;BP;S 20-30;20 7 14
Glycine max (Soybean) BP;S 20-30;25 5 8
Fossypium Sp. (Cotton) BP;S 20-30;25 4 12
Helianthus annus (Sunflower) BP;S 20-30;25;
20
4 10
Hordeum vulgare (Barley) BP;S 20 4 7
Lactuca sativa (Lettuce) TP;BP 20 4 7
Lens culinaris (Lentil) BP;S 20 5 10
Lium usitatissimum (Linseed) TP;BP 20-30;20 3 7
Lycopersicon lycopersicum (Tomato) TP;BP 20-30 5 14
Medicago sativa (Alfa alfa) TP;BP 20 4 10
Nicotiana tabacum (Tobacco) TP 20-30 7 16
Oryza sativa (Paddy) TP;BP;S 20-30;25 5 14
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Pennisetum typhoides (Pearlmillet) TP;BP; 20-30 3 7
Pisum sativum (pea) BP;S 20 5 8
Ricinus communis (Castor) BP;S 20-30 7 14
Secale cereal (Rye) TP;BP;S 20 4 7
Sesamum indicum (Sesame) TP 20-30 3
Solanum melongena (Brinjal) TP;BP 20-30 7 14
Sorghum vulgare (Jowar) TP;BP 20-30 7 14
Triticum aestivum (Wheat) TP;BP;S 20 4 8
Triticum durum (Wheat) TP;BP;S 20 4 8
Vicia faba (Broad bean) BP;S 20 4 14
Vigna mungo (Black gram) BP;S 20-30;25; 20 4 7
Vigna radiate (Green gram) BP;S 20-30;25 5 7
Vigna unguiculata (Copea) BP;S 20-30;25 5 8
Zea mays (Maize) BP;S 20-30;25; 20 4 7
Abbreviations used have the following meaning:
TP: Top of paper
BP: Between paper (including rolled towels and pleated paper)
S: Sand
References
Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowell D and Larinde M. 2006. Manual of seed
handling in genebanks. Handbooks for Genebanks No. 8. Biodiversity
International, Room, Italy.
Agrawal PK and Dadlani M. 1987. Germination test under controlled conditions and its
Evaluation. In :Agrawal and Dadlani (eds) Techniques in Seed Science and
Technology (second edition) pp.61-83.Mehra Offset Press, Chandni Mahal,
Dariyaganj, New Delhi,India.
Anonymous (1985). International rules for seed testing. Seed Sci. & Technol. 13(2), 307-
520
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
QUICK VIABILITY TEST USING TETRAZOLIUM SALT
AD Sharma and Veena Gupta
Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic
Resources, New Delhi
Viability is generally assessed through standard germination test in the laboratory prior to
sowing in the field or storing in the genebank. It is important to ensure that the germplasm
stored in the genebank over the years when sown in the field should be capable to produce
a healthy plant. A seed with high initial viability can be stored for a longer period though
initially the viability declines slowly and then rapidly when the seed age.
There are many methods to determine the seed viability which depend on the crop species.
Standard germination test using various substrata is the most accurate and reliable method
for determining seed viability but it is lengthy and time consuming. Other than the standard
germination tests there are quicker biochemical tests. These are not as accurate as the
standard germination tests. These are not recommended for routine tests and need more
expertise as compared to general tests.
Tetrazolium (TZ) assay, one of the biochemical test, is the fast evaluation for seed viability
and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955).
The living cells converts the TZ salt (2,3,5 triphenyl tetrazolium chloride) to a carmine red
coloured water-insoluble formazan (red staining on seeds) by hydrogen transfer reaction
catalysed by the cellular dehydrogenases while the dead tissues remain unstained due to
absence of respiration.
The objects of quick viability tests are
To determine quickly the viability of seeds of species which normally germinate
slowly or show dormancy under the normal germination methods.
To determine the viability of samples which at the end of the germination test reveal
a high percentage of fresh ungerminated or hard seeds.
Material and Method
1. Seeds (Monocots / Dicots)
2. 1% Tetrazolium (TZ) solution (2,3,5 triphenyl tetrazolium chloride)
3. Distilled water
4. Sunlit filter paper
5. Incubator
6. Shaker
7. Weighing balance
8. pH meter
9. Magnifying glass
10. Sharp blade
11. Needle
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Procedure
Soak 25 seeds in water for 16 hours and place in the incubator chamber at 250 C. Take out
the seeds and cut the monocot seeds longitudinally into two equal halves using sharp blade.
Remove the seed coat of dicots using needle without damaging the cotyledon. Immerse the
seeds in 1% Tetrazolium (TZ) salt solution (2,3,5 triphenyl tetrazolium chloride) prepared
in distilled water or in 0.067 M phosphate buffer of pH 7 for 2 hours at 370 C for staining.
A concentrated (1.0%) solution can be used for legumes, cotton and grasses that are not
bisected through the embryo. Dilute (0.25% or 0.50%) solution for grasses and cereals that
are bisected through the embryo. Wash the seeds with distilled water several times, place
on filter paper and examine under magnifying glass for staining pattern. According to
Verma et al., 2013, viable seeds will be with bright red staining while the seeds which may
grow either normal or abnormal seedlings shall be partially stained. Dead tissues will be
indicated by greyish red stain. However, non-viable seeds will remain completely
unstained.
Precautions (Porter et al. (1947) and Wharton (1955):
1. The pH of the TZ staining solution should be 7. Solution with pH > 8 or pH < 4
would result in either intense staining or would not stain even viable seed tissues.
If water is out of neutral range then use phosphate buffer with pH 7 to dissolve TZ.
2. TZ assay can be used for seeds of legume, cotton and grasses. The incubation time
varies with seed type and morphology. Remove the seed coats of larger seeds (like
legume seeds) before examination.
3. The dicot seeds can be germinated further as the stained seeds are not damaged.
4. When performed appropriately, the percentage of viable seeds obtained by
tetrazolium assay is very close to the percentage of seed germination expected
under most favourable conditions.
Evaluation Sheet of the test
Appearance Category Interpretation
Some unstained areas, but the ‘essential’ areas
missing, flaccid, unhealthy, unstained or
uncharacteristically coloured.
Whole seed (or, if appropriate, embryo) unstained.
Empty seeds,
Underdeveloped,
shriveled or damaged
Completely unstained
Non-viable
Seed appears intact, firm, fresh, healthy and fully
stained the characteristic rich, formazan red
according to the ISTA staining patterns of each
species.
Completely stained Viable
Some unstained tissues, but the‘ essential’
areasintact, firm, fresh, healthy and fully stained
with the characteristic rich, formazan red, or an
acceptable plum or pink colour.
Specific stained areas Viable
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Advantages/disadvantages of different germination/viability tests
Test Advantage Disadvantage
Germination Direct measure of germination Duration may exceed time available
Duration may exceed longevity of
some recalcitrant fruits/seeds
Cut Quick result (hours)
Cheap equipment
Especially useful for checking maturity
and quality before collection and during
processing, also ungerminated seeds at
end of a germination test
Indirect measure of germination
Subjective interpretation
X-ray Quick result (hours)
Non-destructive
Permanent record (photograph)
Especially suited to many wild species
which habitually produce of empty, insect-
damaged
and poorly formed fruits and seeds
Indirect measure of germination
Expensive equipment
Only reveals missing/damaged tissues
Does not reveal whether tissues are
dead or alive (unless combined with
contrast agents e.g., heavy metal ions)
Two dimensional representation of 3 D material
Tetrazolium Quick result (1–3 d)
Only method of assessing some hard
coated, deeply-dormant woody fruits seeds
(e.g., Cornus, Euonymus, Juglans Rosa,
Viburnum) where germination tests are
often precluded due to pretreatment
durations, and it is impossible/ impractical
to excise an intact embryo for EE testing
Especially suited to most other deeply
dormant species requiring > 6 weeks to
pretreat and germinate
Indirect measure of germination
Very labour intensive
Fairly dextrous surgical skills required Skilled
interpretation of staining patterns, colours and
intensities necessary
Unable to detect phytotoxic effects of some seed
dressings (MacKay, 1972)
Unable to detect abnormal germinants (Schubert,
1961)
Unable to differentiate between dormant and
non-dormant
Not suited to very small seeds
Not suited to some fruits with inhibitors which
prevent enzyme reaction e.g., Quercus
Excised
embryo
Quick result (7–14 d)
Especially suited to most deeply dormant
species requiring > 6 weeks to pretreat and
germinate
Indirect measure of germination Exceptionally
labour intensive Exceptionally dextrous surgical
skills
Fairly skilled interpretation necessary
Not suited to very small seeds
Not suited to some fruits with a very tough fruit
case and convoluted seed preventing extraction
of intact embryo (e.g., Juglans)
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Standard methodology for conducting TZ test in few crops
Crop Moistening
Method time (h)
Preparation before staining Staining at 30° C
(conc of TTC)
(duration in
hours)
Barley W/BP 6-18 Bisect longitudinally through embryo and ¾ endosperm 1.0 3-24
Maize W/BP 18 Bisect longitudinally almost full depth through the midsection and spread the cut surfaces
slightly apart
1.0 2-24
Oat W/BP 6-18 Bisect longitudinally through embryo and ¾ endosperm 1.0 2-24
Paddy W/BP 18 Bisect longitudinally through embryo and ¾ endosperm remove or severe lemma 0.5 6-24
Pearl millet W/BP 6-18 Bisect completely through the midsection of distal half 0.5 6-24
Ragi W/BP 6-18 Bisect completely through the midsection of distal half 0.5 6-24
Sorghum W/BP 6-18 Bisect completely through the midsection of distal half 0.5 6-24
Wheat W/BP 6-18 Bisect longitudinally through embryo and ¾ endosperm 0.5 3-24
Chickpea
Cowpea
W 18 Remove seed coat 0.5 6-24
BP 18 Remove seed coat, or cut through the coats near the midsection 0.5 6-24
Pea W/BP 18-24 Cut through the coats near the midsection 0.5 6-24
Soybean BP 18 Remove seed coat 0.5 6-24
Oilseeds castor W 18 Cut through the coats, entire length, near the midsection 0.5 6-24
Groundnut BP 18 Remove seed coat. One cotyledon may be severed 0.5 6-24
Sunflower W 6-18 Bisect entirely through the midsection of the distal half 0.5 6-24
cotton BP 18 Bisect entirely through the midsection of the distal half 0.5 6-24
Jute W/BP 18 Bisect entirely through the midsection of the distal half, expose the embryos by spreading
the cut surfaces
0.5 24-48
Alfa-alfa W 6-18 No seed coat preparation is generally needed can be cut through the coats near the mid-
section of the distal half
0.5 6-24
Berseem
(Trifolium)
W 18 Cut through the coats near the midsection of the entire cotyledon length 0.5 6-24
Beet root W 18 Remove the seed coat, cut/puncture/remove the inner coat 0.5 24-48
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Bittergourd W 6-18 Cut completely through the midsection of the distal half. Expose embryos by spreading
the cut surfaces
0.5 6-24
Brinjal W/BP 18 Puncture the seed coat near the centre, cut longitudinally or laterally between the radicle
and the cotyledon
0.5 6-24
Chilli W/ BP 18 Puncture the seed coat near the centre, cut longitudinally or laterally between the radicle
and the cotyledon
0.5 6-24
Cucumber W/BP 6-18 Bisect longitudinally through the midsection of the half and expose the embryo by
spreading the cut surfaces
0.5 6-24
Tomato W/BP 18 Puncture the seed coat near the centre and cut longitudinally the entire almost full depth
in the midsection cutting towards the radicle and cotyledon tips
0.5 18-24
Citrus W 18 Remove the slipperiness of the seed coat by drying or wiping with a cloth/paper. Cut,
puncture and remove the seed coat
0.5 6-24
Coffee W 18 Cut the seed longitudinally entire length and almost full depth starting in the crease 0.5 24-48
Rubber W 18 Bisect the seed off-centre through the coats and nutritive tissues to expose the embryo
outline
0.5 2(40°C)
Tobacco
W/BP 24 Cut longitudinally near the midsection 0.5 24-48
Baliospermum** W 2 Cut longitudinally near the midsection 0.1% 17
Cassia** W 2 Remove seed coat and cut longitudinally near the midsection 0.1% 4
Jatropha** W 17 Remove seed coat and cut longitudinally near the midsection 0.1% 6
*BP= Between paper; W=Soaked in water at room temperature ** developed by Author at GCD, ICAR-NBPGR
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Reference
Porter, R., Durrell, M. and Romm, H. (1947). The use of 2, 3, 5-triphenyl-tetrazoliumchloride
as a measure of seed germinability. Plant Physiol 22(2): 149
Verma, P., Kaur, H., Petla, B. P., Rao, V., Saxena, S. C. and Majee, M. (2013). Protein L-
isoaspartyl methyltransferase2 is differentially expressed in chickpea and enhances
seed vigor and longevity by reducing abnormal isoaspartyl accumulation
predominantly in seed nuclear proteins. Plant Physiol 161(3): 1141-1157.
Wharton, M. J. (1955). The use of tetrazolium test for determining the viability of seeds of the
genus Brassica. Proc Int Seed Test Assoc 20: 81-88.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
MODELLING AND MONITORING OF SEED LONGEVITY IN CONSERVED
GERMPLASM
J Aravind, Chithra Devi Pandey, Neeta Singh and Anjali Kak Koul
Division of Germplasm Conservation, ICAR-NBPGR, New Delhi-110012
Introduction
A mathematical model describes a system, event or real-life scenario in terms of mathematical
concepts and language. This enables us to describe their characteristics in simple terms as well
as in prediction and forecasting. In seed biology experiments particularly in genebanks, two
kinds of data are generated (Fig. 1).
a) Counts of germinating seeds at different periods of time from the same sample of seeds
known as cumulative germination counts and
b) Total germination of independent samples of seeds stored under the same set of
conditions for different periods of time known as the seed viability data.
Fig. 1. a) Seed germination progress and b) Seed viability loss data
Based on the nature of the data, distinct approaches need to be taken to model seed germination
progress versus seed viability loss (Hay, et al., 2014). In this write up, a brief introduction to
the concepts underlying these approaches will be outlined. In addition, a brief tutorial is
presented on how to use two software packages - ‘germinationmetrics’ (Aravind et al., 2018a)
and ‘viabilitymetrics’ (Aravind et al., 2018b) where these have been implemented. Both these
packages are developed as add-on packages to the free and open-source statistical
programming language ‘R’.
Modelling seed germination progress
Emergence of seedling from the seed after a particular length of time from sowing is recorded
as seed germination. However, this does not encompass the complex processes preceding the
protrusion of embryo such as imbibition and metabolic activation. This is further confounded
by the fact, for a single seed the germination is recorded as a qualitative binomial trait while
for the seed lot, it is recorded as a quantitative percentage trait. Hence in addition to the end
point germination, cumulative germination progress count data taken at regular intervals can
be used to describe the entire process. Initially, several single-value germination indices were
proposed to describe the features of the germination progress curve such as capacity, time, rate,
uniformity, synchrony etc. A brief outline of these indices is given by Brown and Mayer
(1988); and Ranal and Santana (2006) Table 1. An overview is also available in the
‘germinationmetrics’ documentation.
2 4 6 8 10 12 14
01
02
03
04
0
int
y
2 4 6 8 10 12 14
02
04
06
08
01
00
years
y
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
Table 1. Features of seed germination progress described by the various single-value seed
germination indices.
Germination Indices Capacity Time Rate Uniformity Synchrony
Germination percentage ✓
t50
(Median germination time) ✓
Mean germination time ✓
Mean germination rate ✓
Germination Index (AOSA) ✓ ✓ ✓
Timson's index ✓ ✓
Coefficient of uniformity of
germination
✓
Uncertainty of the germination
process (U)
✓
Synchrony of germination (Z
index)
✓
Peak value ✓
Germination value ✓ ✓ ✓
However, as these failed to describe the entire process accurately, non-linear functions such as
Weibull, Richard’s, logistic etc. were employed (Brown and Mayer, 1988). Among these, the
four parameter-hill function (El-Kassaby et al., 2008) is implemented in ‘germinationmetrics’
(Fig. 2).
𝑦 = 𝑓(𝑥) = 𝑦0 +𝑎𝑥𝑏
𝑐𝑏 + 𝑥𝑏
Where, 𝑦 is the cumulative germination percentage at time 𝑥, 𝑦0 is the intercept on the y axis,
𝑎 is the asymptote, 𝑏 is a mathematical parameter controlling the shape and steepness of the
germination curve and 𝑐 is the “half-maximal activation level” (Table 2).
Fig. 2. Seed germination progress modelled by the four-parameter hill function (FPHF) curve.
(TMGR: Time to maximum germination rate; MGT: Mean germination time; RoG: Rate of
germination; t50: Time to 50% germination; U: Uniformity)
Table 2. Features of seed germination progress described by the parameters from the four-
parameter hill function
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Parameter Capacity Time Rate Uniformity Synchrony
a (asymptote, or maximum
cumulative germination
percentage)
✓
b (shape and steepness of
curve)
✓
c (half-maximal activation
level or time for 50% of viable
seeds to germinate)
✓
t50 (Time to 50% germination) ✓
Uniformity
✓
TMGR (Time to maximum
germination rate)
✓
AUC (Area under the curve) ✓ ✓ ✓
MGT (Mean germination
time)
✓
Skewness of MGT ✓
One of the basic assumptions of such non-linear model is the independence of the observations
recorded. As the germination counts are taken from the same sample after set intervals of time,
this is clearly violated. Hence a class of models known as survival, time-to-event, failure-time
or reliability models have been proposed (McNair et al., 2012). They are based on the
distribution of germination times of individual seeds rather than on cumulative germination in
case of non-linear models. Such models can be fitted to the cumulative germination data using
the ‘R’ package ‘survival’ (Therneau, 2015).
Modelling seed viability loss
Orthodox seeds, which can be conserved over extended periods of time under reduced moisture
and temperature conditions form the bulk of the collections held in ex situ genebanks
worldwide including in India. In such species, the relationship between seed longevity and seed
storage conditions are described by seed viability equations (Ellis and Roberts, 1980). It is the
driving force behind the development of seed storage facilities and the genebank standards
particularly for seed viability monitoring and regeneration. The seed death or survival over
time follows a normal distribution (Fig. 3a) and hence the cumulative seed death over time
follows a negative cumulative normal distribution curve or a negative sigmoidal curve. Probit
transformation of the seed survival data transforms the sigmoidal curve to a straight line. The
slope of this curve gives the longevity (σ) or the time to lose one probit viability or the standard
deviation of the normal distribution of seed deaths for the seed lot (Ellis and Roberts, 1980;
Pritchard and Dickie, 2003).
𝑣 = 𝐾𝑖 – (1/𝜎) 𝑝 (1)
Where 𝑣 is probit viability, 𝑝 is storage period and 𝐾𝑖 is viability (in probits) before
storage. The 𝐾𝑖 value is seed lot dependant, while 𝜎 remains constant between seed lots for the
same species under the same storage conditions.
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Fig. 3. a) Normal distribution of seed survival over time, b) negative cumulative normal
distribution of the seed viability loss over time and c) transformation of seed viability to
corresponding probits. (Figure adapted from Pritchard and Dickie (2003))
So the seed longevity (𝜎) in turn depends on the seed storage conditions (moisture and
temperature) and the species. This relationship has been worked out as follows.
𝑙𝑜𝑔 𝜎 = 𝐾𝐸 – 𝐶𝑊 log 𝑚 – 𝐶𝐻𝑡 – 𝐶𝑄𝑡2 (2)
Combining (1) and (2), we get the improved seed viablity equation as follows.
𝑣 = 𝐾𝑖 – (1
10𝐾𝐸 – 𝐶𝑊 log 𝑚 – 𝐶𝐻𝑡 – 𝐶𝑄𝑡2) 𝑝
Where 𝑣 is probit viability, 𝑝 is storage period, 𝐾𝑖 is viability (in probits) before storage, 𝑚 is
the seed moisture content, 𝑡 the seed storage temperature, 𝐾𝐸 is the species constant, 𝐶𝑊 is the
moisture content constant; and 𝐶𝐻 and 𝐶𝑄 are the temperature constants. 𝐾𝐸, 𝐶𝑊, 𝐶𝐻 and 𝐶𝑄
are species specific. If these constants are known, then for any seed lot, several predictions can
be made. For example the seed viability after a period of storage under a particular set of storage
conditions for a seed lot on the basis of the initial viability. Similarly the storage conditions
required to store a seed lot for a particular period without significant loss of viability can be
predicted. These predictions are however bounded by the limits established for moisture
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content (2-6.25% to 15-28%) and temperature (-13 to 90 °C) within which the assumptions of
the viability equation holds well (Pritchard and Dickie, 2003). The ‘viabilitymetrics’ package
includes functions for such predictions.
These constants can be empirically determined for each species from factorial storage
experiments at different combinations of storage temperatures and moisture contents.
However, if the longevity of a large number of species is to be established, the comparative
longevity protocol can be employed. Here accelerated ageing test at a single environment for a
number of species is conducted and compared with the species included in the experiment with
known seed longevity (Newton et al., 2014). The ‘viabilitymetrics’ package includes functions
for fitting seed viability equations to factorial seed storage equations as well as conversions for
comparative longevity testing.
Monitoring of seed viability in Genebank
Checking of quality (seed viability/germination) and quantity (number or weight) of
germplasm accessions during storage in a genebank is known as monitoring. The germination
ability of seeds conserved in genebank decreases over time during storage and before it reaches
unacceptable levels seeds should be regenerated. Similarly, retrieval of conserved seeds for
research/utilization and germination testing results in a decrease of seed quantity.
Determination of seed vigour, in addition to germination percentage, could provide the
genebank curator with early indications of a decrease in viability.
Need for monitoring
• Even under optimal ex situ storage conditions viability declines.
• Reduction in viability results in loss of both genes and genotypes.
• Monitoring of viability is necessary to take decisions on timely regeneration and should
be a priority activity of all gene banks.
• The cost of storing germplasm is high therefore seed should not be wasted and a balance
should be made between monitoring too frequently and not monitoring at the
appropriate time.
Hence, Monitoring is an important activity in genebank management as it provides
information on accessions that are being depleted in seed numbers and those that require
regeneration (Ellis et al., 1980; Ellis et al., 1985a).
Monitoring viability
The viability of many different crop species conserved in a genebank is tested mainly using a
standard germination test following the International Seed Testing Association (ISTA)
guidelines (Ellis et al., 1985b) or in specific cases using quick viability test. However, different
genebanks use different strategies based on their past results/ experience. One of the constraints
is the insufficent data on the extent of loss of seed viability of different species over time
(described by the shape of survival curve). Accessions of the same species and even seed lots
within the same accession may differ in storability. The monitoring procedure and interval
should be in such way that the seed viability does not deteriorate beyond the permissible level.
For most cultivated crop species, initial germination value should be equal to or more than
85%. For wild/weedy/endangered/ and forest species, a lower percentage can be accepted. The
monitoring interval depends on the species, initial viability conditions of storage or in the
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previous test and conditions of storage. The first monitoring test should normally be conducted
after 10 years for seeds with high initial germination percentage and those conserved in long-
term storage. Species known to have inherently short storage life or accessions of poor initial
quality or those conserved in medium-term storage should be tested after 5 years. The interval
between later tests should be based on experience and could in many cases be greater than the
recommended 10 or 5years.Suggested monitoring intervals for non-oil rich and oil rich seeds
are given in Table 1
Table 1. Monitoring intervals for species with non-oil rich and oil rich seeds.
Active collections of most crops and base collections of oil rich seeds with initial
viability > 95% are monitored every 10 years. Accessions with initial viability between
85% and 95% are monitored every eight years and those with < 85% every five years.
Base collections of non-oily crops with >95% viability are monitored every 20 years,
those with viability between 85 and 95% every 15 years and accessions with viability
<85% every 10 years.
Active collections of oily crops with >95% viability can be monitored every 8 years,
accession with 85-95% viability every 5 years and those with <85% viability every 3
years. However, these are suggested monitoring intervals which should be adjusted
according to the data received from germination tests. Monitoring intervals should be
shortened when significant decline in viability is detected. This will better help to
predict the time to reach the viability standards.
Viability is monitored (Fig.1) by conducting germination test on a fixed sample size
or sequential germination test which require substantially less seed per test as
described below:
1. Identify and list the accessions, which require viability testing based on initial values
and year of storage using genebank data.
2. Locate the containers in storage from database.
3. Take out the containers from storage and leave them to warm up till it reaches room
temperature.
4. Open the container in a controlled environment atmosphere to take out a sample of seed
needed for the test and close the containers.
5. Update the seed quantity in database, deducting the number of seeds drawn.
6. Conduct the germination tests (fixed sample or sequential).
7. Specific germination information and test recommendations should be followed.
Germination
(%)
Monitoring interval (years)
Active collection (4°C) Base collection (−20°C )
Non-oil rich seeds Oil rich seeds Non-oil rich seeds Oil rich
seeds
<85 5 3 10 5
85-95 8 5 15 8
>95 10 8 20 10
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8 Update the germination data in the database and mark those requiring regeneration
based on the genebank standard of maintaining at least 85% of the original viability
(Table 2).
Table 2. Threshold germination percentages for regeneration of accessions
Initial germination percentage Regenerate if percentage germination after
monitoring is below
100 85
99 84
98 83
97 82
96 82
95 81
94 80
93 79
92 78
91 77
90 77
89 76
88 75
87 74
86 73
85 72
Sample size for germination test
• For the fixed sample, size germination test use at least 200 seeds (two replications of 100
seeds each) or if seeds are limited 50 seeds per replicate.
• If the percentage germination is above 85% of the initial germination percentage, the
accession is continued in storage.
• Depending on the current percentage germination date for next test is fixed.
• In case the germination is below 85% of the initial germination percentage, the accession
is marked for regeneration.
Sequential germination test
The sequential germination test uses on an average fewer seeds per replicate than the standard
germination test for a similar degree of accuracy. Otherwise, the methods and conditions for
germination are the same as described for the fixed sample size germination test.(Ellis et al.,
1980). Sequential germination test is a series of discrete seed tests in which the decision to
further test seeds or stop the test depends upon the cumulative result. This test is only necessary
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when seed quantity is limited. The test is open ended and there is an intermediate zone of
results, where no decision can be taken.
• It is recommended to use 40 seeds per replicate although lesser number can also be
used.
• Ensure that the same number of seeds are used when repeating the test so that the
different samples can be treated as replicates.
• Repeat the test if the cumulative germination percentage is less than the acceptable
level.
• The test is continued until a decision can be made to regenerate or continue storage or
until it is repeated 10 times (Table.3).
Table 3. Sequential germination test plan for 85 per cent regeneration standard for group of 40
seeds (Ellis et al., 1980)
No. of
Seeds
tested
Regenerate if the number
of Seeds number
germinated is less
than/equal to
Repeat test if
number of
germinated seeds
is in the range of
Store if number of
Seeds germinated
more than /equal to
40 29 30-40 -
80 64 65-75 76
120 100 101-110 111
160 135 136-145 146
200 170 171-180 181
240 205 206-215 216
280 240 241-250 251
320 275 276-285 286
360 310 311-320 321
When 400 seeds have been tested, the test can be terminated because enough tests have been
conducted for an informed decision to be made.
Monitoring for seed quantity
Seed quantity is recorded in a computerized genebank management database.
The weight/ number of seeds initially conserved in the genebank as well as all
subsequent seed withdrawals for distribution, regeneration and germination testing are
recorded to update seed stock accounting all seed withdrawals.
Seeds are not withdrawn from accessions having less seed numbers ( less than that
required for at least one regeneration cycle)
To know more about standards for viability monitoring in genebanks refer to the
Genebank Standards (FAO, 2014) and for procedures for viability testing the IPGRI handbooks
on seed technology for genebanks as well as Ellis et al.,( 1985a, 1985b).
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References
Aravind J, S Vimala Devi, J Radhamani, SR Jacob and Kalyani Srinivasan (2018)(a)
Germinationmetrics: Seed Germination Indices and Curve Fitting. 2018.
https://aravind-j.github.io/germinationmetrics/.
Aravind J, S Vimala Devi, J Radhamani, SR Jacob and Kalyani Srinivasan (2018)(b)
Viabilitymetrics: Seed Viability Calculations and Curve Fitting. 2018.
https://aravind-j.github.io/viabilitymetrics/.
Brown RF and DG Mayer (1988) Representing Cumulative Germination. 2. The Use of the
Weibull Function and Other Empirically Derived Curves. Ann. Bot. 61: 127–38.
https://doi.org/10/gfpgjj.
El-Kassaby YA, I Moss, D Kolotelo and M Stoehr (2008) Seed Germination: Mathematical
Representation and Parameters Extraction. For. Sci. 54: 220–227.
https://doi.org/10.1093/forestscience/54.2.220.
Ellis R and E Roberts (1980) Improved Equations for the Prediction of Seed Longevity.
Ann. Bot. 45: 13–30.
Hay FR, A Mead and M Bloomberg (2014) Modelling Seed Germination in Response to
Continuous Variables: Use and Limitations of Probit Analysis and Alternative
Approaches. Seed Sci. Res. 24: 165–86.
https://doi.org/10.1017/S096025851400021X.
McNair JN, A Sunkara and D Frobish (2012) How to Analyse Seed Germination Data
Using Statistical Time-to-Event Analysis: Non-Parametric and Semi-Parametric
Methods. Seed Sci. Res. 22: 77–95. https://doi.org/10/f3x44z.
Newton R, F Hay and R Probert (2014) Protocol for Comparative Seed Longevity Testing.
Millennium Seed Bank Partnership, Royal Botanic Gardens, Kew.
Pritchard HW and JB Dickie (2003) Predicting Seed Longevity: The Use and Abuse of
Seed Viability Equations. RD Smith, JB Dickie, SH Linington, HW Pritchard, and
RJ Probert (eds.) In: Seed Conserv. Turn. Sci. Pract. Kew, UK, Royal Botanic
Gardens, pp 653–721.
Ranal MA and DG de Santana (2006) How and Why to Measure the Germination Process?.
Braz. J. Bot. 29: 1–11. https://doi.org/10.1590/S0100-84042006000100002.
Therneau TM (2015) A Package for Survival Analysis in S. https://CRAN.R-
project.org/package=survival.
Ellis RH, EH Roberts and J Whitehead (1980) A new more economic and accurate
approach to monitoring the viability of accessions during storage in seed banks.
Plant Genetic Resources Newsletter 41:3-18.
Ellis RH, TD Hong and EH Roberts (1985a) Handbook of Seed Technology for Genebanks
Vol. 1 :Principles and Methodology. IBPGR, Rome, 210 p.
Ellis RH, TD Hong and EH Roberts (1985b) Handbook of Seed Technology for Genebanks
Vol. 2: Compendium of Specific Germination Information and Test
Recommendations. IBPGR, Rome, 456 p.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
FAO (2014) Genebank Standards for Plant Genetic Resources for Food and Agriculture.
Rev. ed. Food and Agricultural Organization, Rome, Italy, 166 p.
Kameswara Rao, N., and Paula J Bramel (eds.). 2000. Manual of Genebank Operations and
Procedures. Technical Manual no. 6. Patancheru 502 324, Andhra Pradesh, India:
International Crops Research Institute for the Semi-Arid Tropics.
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OPERATION AND MAINTENANCE OF SEED DRYERS/DE-HUMIDIFIER AND
SEED GERMINATOR
Satya Prakash and Lal Singh
Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic Resources,
New Delhi-110012
Survival of seed for a longer period of time is possible by storing them in a controlled
environmental conditions. The process of eliminating of moisture from the seed is called seed
drying. The rate of seed drying is depending on initial seed moisture.
Seed dryer
Seed dryer is a machine which provided/reduced the seed moisture as per requirement of seed
by controlling the relative humidity and temperature for gradual drying of seed material without
losing its viability.
Principle
Seed dryer works on the principle of physical adsorption, for removal the moisture from the
seed.
The moisture is adsorbed in the dehumidification sector by the slowly rotating fluted, metal
silicate desiccant synthesised rotor and is exhausted in the reactivation sector by a stream of
hot air in a counter flow. Following the reactivation process, the adsorption sector is again
ready to adsorb the moisture. Thus, the two processes of “moisture adsorption” and
“reactivation” takes place with separate airflows continuously and simultaneously. Positive
sealing between chambers prevents mixing of the process and reactivation air stream.
Operation/ Functioning
For achieving the controlled air, relative humidity and temperature, seed dryer should have a
combination of De-humidifier section to controlled relative humidity and a refrigeration system
for controlling the required air temperature to the specified level.
The seed dryer essentially consists of two parts the first is air light chamber with perforated
trays where the seed is to be placed for drying, second parts is dehumidifying dryer section
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which located on top and continuously supply controlled dry air into the tray chamber. When
the de-humidified air passed over the seed loaded on perforated tray in air tight chamber, it
picks up the moisture from the seeds.
The moisture loaded air is again passes through the de-humidifier to remove its moisture. This
process will continue till the desired moisture content of the seed is achieved. The above
process is completed in a closed loop re-circulating system and a unique air distribution pattern
through the trays to achieve the required drying without losing the viability of the seeds.
Maintenance
Even though Seed dryers and Dehumidifiers require very little maintenance, this section shall
provide as an easy and quick reference to effective maintenance/ service requirement of Seed
dryers and Dehumidifiers.
Filter
Seed dryer has filters for both, process and reactivation air flow. Each filters made of multiple
layers of expended aluminium in a metal frame.
The maintenance interval for filters depends directly on the cleanliness of the air entering in
the dryers. Filters should be cleaned properly every fortnightly. It should not be clogged in any
condition. Filter can be clean with compressed air or wash with warm soap solution if required.
Desiccant Rotor
Check the desiccant rotor for smooth rotation. There should be no sign of discoloration, pits,
cracks due to dirt, dust or other foreign materials. It should be cleaned with soft brush within
the interval of every three months. To clean rotor use vacuum cleaner with dry vacuum and
dusting brush with the attachment of soft Bristol brush. Vacuum both surface of the rotor.
Reactivation Heater and Fan Motors with alignments
The reactivation heaters are located near the intake of reactivation air. To check the heater
element, turn the power of and check the resistance across each element and clean them
quarterly.
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Both blower motors have permanent lubricated ball bearings and require no additional
lubrication. Fan wheals require periodic inspection for accumulation of dust and dirt, If
cleaning become necessary, clean then.
Misalignments can cause overheating, wear of seals, bearing failure and unbalance of rotating
parts. Periodic check to ensure alignments and smooth running of various support/ bearing
surfaces is vital.
Hardware, Air Flow, Access Panels/gaskets, Refrigeration system
Check tightness of all bolts, screws and electrical connections.
Ensure that airflows in and out of the ducts are free from obstructions.
All service panel cover gasket should be observed during inspection and servicing to
ensure a good seal. Any leaks must be sealed for proper dehumidification operations.
Refrigeration system should be check quarterly with checking of suction and discharge
pressures, noise of compressor, condenser and motors etc.
Seed Germinator
To germinate a seed, it is essentially required an optimum environmental condition and a
photoperiodic cycle.
Seed Germinator is a machine which provided the controlled temperature, relative humidity
(RH) and photoperiodic cycle which is optimum for germination of seeds.
Seed Germinator has the following systems.
Temperature control system
Humidity control system
Photo photoperiodic cycle control system
Temperature control system
In seed germinator, temperature is being controlling by a digital, dual temperature controller
cum indicator. Temperature control system has two parts.
1. Electric air heater for rise the temperature with the help of hot air circulating fan.
2. Refrigeration system to cool down the temperature with a cool air circulating fan.
Humidity control system
In seed germinator relative humidity (RH) is being control by a digital humidity controller cum
indicator.
The required relative can be achieved by injector the water vapours in the form of spray into
the chamber the uniformity of RH in the chamber can be achieved by effectively circulating
the chamber air.
Photo periodic cycle/ Illumination cycle system
The photo periodic cycle can be achieved by illuminating the tube lights which may be switch
ON/OFF either manually or automatically by means of a 0-24 hours’ cyclic timer for
controlling the day and night temperature.
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Common type of faults and their remedies
No heating
Causes:
i) There is some disconnection or breakage of connecting leads or wires.
ii) Switch or relay coil has become permanently open.
iii) Heating element has become open.
The fault can be removed/isolated using a series lamp or multi meter. Once the fault is isolated
the needful can be done.
Equipment giving shock
Either the heating element or connecting leads is leaking the current.
Using a series lamp, all the components of the circuit need to be checked one by one and the
fault to be isolated.
No cooling
o Go for detailed check up from the relay or controller to cooling relay. If any fault
detected remove it. If the supply is available upto cooling relay, then check for over
load protector. If it is open, check for compressor temperature. It must be excessively
hot. Allow it to cool. Check for over load. If it starts functioning OK
o Compressor might be over worked if it trips again. Consult a refrigeration expert.
Compressor is working but no cooling effect. Check the condenser for its being hot. It
its not hot check for condenser fan motor. Fan may be loose on the motor shaft or the
motor may be jammed. Do the needful.
o If the compressor is working, condenser fan is working but the desired cooling is not
achieved i.e. cooling is less, it must be due to lesser quantity of gas. If the problem
again erupts after a few days, it must be due to a leakage in the system which needs to
be detected and remedial action for plugging the same to be taken.
Problems in the control circuitry
Most of the time, loose connections are the culprit. These need to be tightened.
Identify the problem area. Isolate the component which is faulty. Replace the same.
Sometimes a particular component which is replaced becomes faulty due to some other
malfunctioning. For this type of fault, the inter connected components also need to be
checked.
Humidity is not achieved
Burning of the heating element in the tank, jamming of water circulation pump, clogging of
water line or no water in the tank, relay mal functioning, humidity controller man functioning.
Do the needful.
Air circulation systems
The motors which prime the air circulation fans, need to be oiled regularly as these are most of
the time bush based. So, to prevent the bushes from cutting/wearing out due to friction, these
needs to be oiled regularly.
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International Training Manual on "Management of PGR" (for officials of MoA, IRAQ) 15-20 July, 2019
INFORMATION MANAGEMENT SYSTEM FOR PLANT GENETIC RESOURCES
Sunil Archak, Rajeev Gambhir and Nirmala Dabral
AKMU, ICAR-National Bureau of Plant Genetic Resource, New Delhi-110012
Ever-increasing significance of conservation and utilization of plant genetic resources (PGR)
on one hand and advancements in computer technology for digitization and management of
data on the other have catapulted PGR Informatics into limelight.
What is PGR Informatics
PGR Informatics is the management (creation, storage, retrieval and presentation) and analyses
(discovery, exploration and extraction) of diverse information (facts, figures, statistics,
knowledge and news). PGR Informatics has assumed significance because of the following
factors:
(i) Increased awareness about PGRFA
(ii) Various international agreements (CBD, GPA, ITPGRFA) coming into force
(iii) Availability of information in text, images, maps, videos, etc.
(iv) Technologies to record, link and archive such diverse types of information
(v) Growing power (and falling costs) of computers and internet to facilitate access and
retrieval
Fundamental merit of an organized digital information system is that it provides fair and just
opportunity for all to access. On-line portals, as a consequence of PGR Informatics, enable
non-exclusive access to PGR information to a large number of users involved in overlapping
research areas on PGR management.
Typically information is collected on details of multitude of Passport data including taxonomy,
biogeography, and ethnobotany of the germplasm acquisitions (domestic collections and exotic
introductions), their Seed Health, multiplication for Supply and Conservation, Regeneration,
experimental data on Characterization and Evaluation leading to Utilization. In addition to
field data, it also includes biochemical and genomic data as well as publications. Once the
information is digitized and stored, computer technologies allow management and analysis
irrespective of the scale and types of data leading better visualization and predictions.
Biodiversity informatics as a discipline started with the construction of the first taxonomic
coding system by researchers at the Virginia Institute of Marine Science for the Biota of
Chesapeake Bay in 1972. This work led to development of a number of other taxonomic
databases specializing in particular groups of organisms culminating into the "Catalogue of
Life" in 2001 as well as into "Biodiversity Information Projects of the World."
Encyclopedia of Life, Consortium for the Barcode of Life (CBOL), TreeBASE, Species 2000,
Global Biodiversity Information Forum (GBIF), Inter-American Biodiversity Information
Network (IABIN), World Biodiversity Information Network (REMIB), Indian Bioresources
Information Network (IBIN) inter alia have been the torchbearers of biodiversity informatics
(Agrawal et al. 2012).
Relevance of PGR informatics
The need for countries to develop, maintain and exchange information "from all publicly
available sources, relevant to conservation and sustainable use of biological diversity"
including "results of technical, scientific and socioeconomic research" has been recognized in
the Convention on Biological Diversity (CBD, Articles 7d, 17), and the Global Plan of Action
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(GPA, priority activities 17 and 18). Information of this nature is imperative for planning and
implementing activities; sustainable use and sharing of benefits accrued from its use.
Global assessment indicates that many of the world’s PGR are insufficiently and poorly
documented. The passport information and characterization and evaluation data on genebank
accessions conserved in genebanks are either lacking or poorly recorded or scattered at
different places, such as passport data sheets, reports of collection and exploration missions,
crop catalogues, published articles, etc. In addition, there exist informal or non-coded
knowledge held by traditional farmers and indigenous people. To use this information
efficiently and effectively, the valuable information need to be collected, collated, maintained
and exchanged with the help of PGR Informatics.
Global initiatives on PGR informatics
These mainly include database systems and online portals associated with genebanks (Table 1).
(i) Germplasm Resources Information Network (GRIN): supports the national germplasm
collections important to food and agriculture, collectively called the National Genetic
Resources Program of United States Department of Agriculture. GRIN provides
genebank personnel and germplasm users with access to databases that maintain
passport, characterization, evaluation, inventory, and distribution data important for the
effective management and utilization of national germplasm collections.
(ii) European Search Catalogue for Plant Genetic Resources (EURISCO) is a search
catalogue providing information about ex situ plant collections maintained mainly in
Europe. It is based on a network of National Inventories of 43 member countries and
400 institutes providing information on ~2 million accessions.
(iii) The Japanese Genebank of National Agriculture and Food Research Organization
(NARO), manages databases that include information on passport data, evaluation as
well as more general information on genetic resources.
(iv) GENESYS is a global portal to information about PGR, from which information on
germplasm accessions from genebanks around the world can be found. GENESYS
resulted from collaboration between Bioversity International on behalf of System-wide
Genetic Resources Programme of the CGIAR, the Global Crop Diversity Trust and the
International Treaty on the Plant Genetic Resources for Food and Agriculture. In
addition to passport data, GENESYS provides access to over 11 million records of
characterization and evaluation data.
(v) PGR Portal: It is a gateway to information on PGR conserved in the Indian National
Genebank housed at ICAR-NBPGR, New Delhi, with information on about 400,000
accessions.
Important PGR Informatics applications developed and maintained at NBPGR
1. PGR Portal pgrportal.nbpgr.ernet.in
2. Import Permit and EC Data Search exchange.nbpgr.ernet.in
3. Genebank Dashboard genebank.nbpgr.ernet.in
4. PGR Map pgrinformatics.nbpgr.ernet.in/pgrmap
5. National Herbarium of Crop Plants pgrinformatics.nbpgr.ernet.in/nhcp
6. Biosystematics Portal pgrinformatics.nbpgr.ernet.in/cwr
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7. PGR Climate pgrinformatics.nbpgr.ernet.in/pgrclim
8. PGR and IPRs http://pgrinformatics.nbpgr.ernet.in/ip-pgr/
Recent advances in PGR Informatics in India
NBPGR has been striving to establish PGR information set up since 2002 (Archak and
Agrawal, 2012). Development of mobile apps in PGR Informatics facilitates Enhanced access
to PGR information can lead to enhanced utilization. NBPGR has developed two mobile apps
“Genebank” and “PGR Map”. Both the apps are first of their kind for any genebank in the
world. The apps have been developed for both Android and iOS. No other ICAR app is
available for iPhone. Licenses were purchased and the apps have been hosted on Google Play
and App Store.
Genebank app provides a dashboard view of indigenous collections (state-
wise), exotic collections (country-wise), addition of accessions to genebank,
etc. The app also helps generate routine genebank reports. The app uses
databases live on the backend and hence always gives updated information.
PGR Map app offers three benefits: “What’s around me” helps user to obtain
quickly the accessions that have been collected and conserved in the genebank
from a particular location in India where the user is located at the moment;
“Search the map” helps user to list the accessions that have been collected and
conserved in the genebank from any selected location in India; “Search for
species” helps user to map the collection sites of a crop species.
Acknowledgments
The author acknowledges the contributions made by colleagues at NBPGR particularly Mr.
Rajeev Gambhir and Mr. Vijay Kumar Mandal. Author is supported by ICAR-National
Fellowship.
References
RC Agrawal, S Archak, RK Tyagi (2012). An overview of biodiversity informatics with special
reference to plant genetic resources. Computers and electronics in agriculture, 84: 92-
99.
S Archak and RC Agrawal (2012). PGR informatics at the National Bureau of Plant Genetic
Resources: status, challenges and future In: A road map for implementing the
multilateral system of access and benefit-sharing in India. (Eds. Halewood et al.).
ICAR-NBPGR and Bioversity International, Rome.
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Table 1. PGR Informatics databases, portals and websites
Information resource Web address
Japanese genebank portal www.gene.affrc.go.jp/databases_en.php
European genebanks portal eurisco.ipk-gatersleben.de
Genesys portal www.genesys-pgr.org
Indian genebank portal pgrportal.nbpgr.ernet.in
Barcode of Life www.barcodeoflife.org
Convention on Biological Diversity www.cbd.int
Encyclopedia of Life www.eol.org
Global Biodiversity Information Forum www.gbif.org
Indian Bio-resources Information Network www.ibin.gov.in
International Legume Database www.ildis.org
International variety protection database www.upov.int
National Plant Germplasm System of USDA www.ars-grin.gov/npgs
Species 2000 www.sp2000.org
World Information and Early Warning System www.fao.org/wiews/en
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CHARACTERIZATION OF PLANT GENETIC RESOURCES
K K Gangopadhyay, Kuldeep Tripathi and S K Kaushik
Division of Germplasm Evaluation, ICAR-NBPGR, New Delhi-110012
Plant Genetic Resources (PGR) is the key component of any agricultural production system-
indeed of any ecosystem. The “germplasm” or “genetic diversity” refers to the plant genetic
resources with actual or potential value that exists among individuals or group of individuals
belonging to a species. The full spectrum of PGR consists of diverse type of collections such
as those derived from the centres of diversity, centres of domestication and from breeding
programs. Characterization and evaluation including regeneration of germplasm is an integral
component of PGR management and is the key to accelerate utilization in crop improvement
programme by exposing the actual value of germplasm. The characterization of germplasm
deals with recording of highly heritable characters that can be seen easily by the eye, and
equally expressed in all environments where as evaluation deals with the attributes related to
agronomic, biotic and abiotic stresses, and quality traits.
Need for Characterization and Evaluation
• Estimate the extent of variation in the Genebank collections
• Botanical identification and establish diagnostic keys for identifying/distinguishing
• Categorize accessions into different groups as per requirement
• To know the accessions and its actual and potential value
• Assess inter-relationships among accessions, traits and different geographic groups
• Identify and remove duplicates present in the existing collection
Principles of Germplasm Characterization, Evaluation and Maintenance
Germplasm characterization and evaluation are primarily the description of a particular
accession. It covers the whole range of activities starting from the receipt of the new samples
by the curator and growing these for seed increase, characterization and preliminary
evaluation, and also for further detailed evaluation and documentation. There is a need
for its systematic evaluation in order to know its various morphological, physiological and
developmental characters including some special features, such as stress tolerance,
insect pest and disease resistance. Newly explored collections, trait specific exotic
introductions for location specific character expression, repatriated germplasm accessions
conserved in Genebank of other countries/ international organizations and the accessions
redrawn from Genebank after long interval form the basic material for characterization and
evaluation. The germplasm accessions are usually evaluated in augmented block design
(ABD) with atleast one local checks and national checks for two consecutive years for
documentation and preparation of crop catalogue. For effective evaluation of germplasm, a
close collaboration between curator and breeder is necessary in the context of breeding
objective vis-a-vis evaluation programme.
Characterization
Characterization should provide a standardized record of readily assessable plant
characters which, together with passport data, go a long way to identify an accession.
Characterization descriptors include spike/panicle shape, seed shape and colour, and
other characters which are generally more of taxonomic type. Their recording along with
the passport data provides an overall picture of the range of diversity in the collections,
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so badly required by the users. Various techniques used for characterization and
evaluation depending upon the need is given below:
• Agro-morphological Characterization It is easier and usually based on visual observations. This technique can’t be ignored even
it is highly influenced by environment. The field experiment should be conducted with
statistically sound experimental design depending upon the quantity of seed and number
of germplasm accessions.
• Biochemical Characterization
Biochemical analysis is based on the separation of proteins into specific banding patterns
but only a limited number of enzymes are available and thus, the resolution of diversity is
limited.
• Molecular Characterization The DNA based markers or molecular markers are also gaining importance because of no
environmental influence on these molecular makers. The highly reproducible molecular
Markers used for characterization are Amplified Fragment Length Polymorphisms
(AFLPs), Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs),
etc.
Seed Increase
This is usually the first step and done either with characterization or separately if quantity of
seed is very low. This needs care as it involves the risk of losing a particular accession due to
poor adaptation, disease and pest damage, introducing admixtures through contamination or
error and altering the genetic composition of the original genetic make up through conscious
(human) or unconscious (natural) selection. During initial seed increase, data on agro-
morphological traits and other traits of interest are recorded. Duplicate accessions are also
identified at this stage and promising ones are identified for intensive evaluation.
Evaluation
There is need for its systematic evaluation in order to know the potential of germplasm after
collection of genetic resources and characterization. The following steps are followed for
germplasm evaluation:
Preliminary evaluation
It consists of recording a limited number of agronomic traits thought desirable by users of the
particular crop in addition to characterization descriptors. Characterization of physiological
characters by curators can be of considerable help to the breeders through providing baseline
data which would help to narrow the selection of potential breeding stocks. Most important
characterization and preliminary evaluation descriptors and descriptor states to be used are site
data, leaf, floral, seeds and fruits characters.
Detailed evaluation
Detailed evaluation consist of recording potential characters viz. stress tolerance, disease and
pest resistance and quality characters. Detailed evaluation of large collections requires
multidisciplinary approach and specific testing conditions. Such systematic evaluation
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operations, though expensive and time consuming, are of great value. The principal goal in
exploiting useful genes from germplasm collections vary greatly among crops and for different
ecological zones within a crop. In general, characterization and preliminary evaluation is done
by the curator/germplasm scientists; further evaluation or detailed evaluation is mostly done
by the breeders for taking additional information. However, no hard and fast rule prevails and
the detailed evaluation can also be done by the curator in collaboration with breeders,
pathologists, entomologists, agronomists and biochemists as per needs.
Agronomic Abiotic stresses
Biotic Stresses Quality
Focused Identification of Germplasm Strategy (FIGS) is a revolutionary and rapid tool
which facilitates gene bank managers and agricultural researchers to screen large collections
of PGR more accurately than done earlier using traditional methods. FIGS grew out of early
work done at ICARDA in 1980s searching for boron-tolerant wheat for Australian farmers.
Accessions that had been collected from Mediterranean sites with soils of marine origin-soils
that commonly contain toxic levels of boron were evaluated and found to have all the genetic
variation needed to develop boron-tolerant cultivars. This helps to improve the effectiveness
of crop improvement programs. The global genebanks hold more than 7.5 million accessions
of crops and their wild relatives – a vital source of novel genes that can improve drought
tolerance, disease resistance, and other traits. But the sheer number of accessions makes it
difficult for breeders to identify those that might have useful traits. FIGS combines agro-
ecological information with data on plant traits and characteristics to narrow down the search-
identifying sets of plant genotypes with a higher probability of containing specific ‘target’
traits. FIGS has been used successfully to identify sources of resistance to powdery mildew,
Sunn pest, Russian wheat aphid, and stem rust (Puccinia graminis Pers.) in wheat, net blotch
in barley, and drought stress in faba bean.
Descriptors
Crop curators with their own experience, technical inputs from the Germplasm Advisory
Committee (GAC) and experts from relevant fields like chemistry, pathology, entomology etc.
develop descriptors’ list for each crop. ICAR-NBPGR, Bioversity International and CGIAR
institutions developed descriptors for characterization and evaluation of targeted crops. The
use of uniform descriptors and descriptor states facilitate the utilization of germplasm by
different research workers. The different kinds of descriptors are as follows:
a. Passport descriptors
These descriptors are recorded at the time of collection of germplasm.
b. Environmental and site descriptors
These describe the environmental and site-specific parameters that are important when the
characterization and evaluation trials are held.
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c. Characterization descriptors
These kinds of descriptor expressed in all kind of environments.
d. Evaluation descriptors
Evaluation descriptors are mostly quantitative characters used for the agronomic
performance, quality parameters.
Besides these descriptors, Minimal Descriptors on Agri-horticultural Crops (Mahajan et al.,
2000) is widely used for characterization and evaluation for PGR.
Core collection
Frankel (1984) termed ‘core collection’ which would represent ‘with a minimum
repetitiveness, the genetic diversity of a crop species and its wild relatives. Remaining
accessions of entire collection is defined as reserve collections. It does not replace the existing
collection from which it is obtained. Brown (1989b) suggested that it should not be more than
10% of the whole collection and always less than 2000 entries. They are distinct from each
other genetically and ecologically. Most of the developed core collections were 5–20% of the
size of the collection from which they were established. A general procedure for the selection
of a core collection can be divided into five steps, which are described in the following sections.
i) Identify the material (collection) that will be represented ii) Decide on the size of the core
collection iii) Divide the set of material used into distinct groups iv) Decide on the number of
entries per group v) Choose the entries from each group that will be included in the core vi)
Validation testing
Function of core collection:
• Provide a reference set for comparing the novel material
• Provide set for priority handling when needed
• Provide appropriate set of accessions for monitoring in genebanks by routine seed
testing
• Act as a priority group for safety duplication, for further distribution to regional or
international genebanks or for maintenance in different conditions (e.g. as DNA
libraries, in field banks or in vitro)
• Provide test material of choice for possible improved maintenance procedures (e.g.
ultra-dry seeds, in vitro and cryopreservation)
• Provide benchmark standard for documentation and allow stratification of whole
collection to be recorded
• Preferred material for developing authentic and accurate list of descriptors
• Allow selection of optimal material for studies of trait inheritance and estimation of
general combining ability
Medium Term storage (MTS)
Loss of crop diversity poses serious threat to agriculture and livelihood of millions of people.
Realizing the emerging importance of PGR for food security through conservation and use,
Medium Term storage was established at ICAR-NBPGR and National Active Germplasm Sites
(NAGS) for distribution and regular use of ‘Active Collections’ holistically, where the seeds
are stored in modules at 5oC and the relative humidity of 35-40 percent in various containers
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such as cloth-bags, metal cans or glass jars. MTS reduces the post-harvest losses and maintain
the seed viability and other associated standards.
Germplasm Field days
Germplasm field day is the primary activity of ICAR-NBPGR where diverse germplasm is
displayed with distinct traits. Breeders and crop experts are invited from all parts of country
representing whole National Agricultural Research System (NARS) for on-spot selection of
the promising germplasm to accelerate the crop improvement programmes in India.
Registration of Germplasm
Indian Council of Agricultural Research (ICAR) made NBPGR a nodal institute to register the
trait-specific germplasm developed/ identified by researchers in India. Germplasm or Genetic
stock of agricultural, horticultural and other economic crops, including agro-forestry species,
spices, medicinal and aromatic plants, ornamental plants, which is unique and has potential
attributes of academic, scientific or commercial value can be registered. Registered germplasm
is ready material which can be utilized as donors for targeted breeding programmes in India.
References:
Brown, A.H.D. 1989b. The case for core sets. p. 136–155. In A.H.D. Brown, O.H. Frankel,
D.R. Marshall and J.T. Williams (ed.) The use of plant genetic resources. Cambridge
Univ. Press, Cambridge, England.
Frankel, O.H. 1984. Genetic perspective of germplasm conservation. p. 161–170. In W. Arber,
K. Llimensee, W.J. Peacock, and P. Starlinger (ed.) Genetic manipulations: Impact on
man and society. Univ. Press, Cambridge, England.
Mahajan RK, RL Sapra, U Srivastava, M Singh and GD Sharma. 2000. Minimal Descriptors
for Characterization and Evaluation of Agri-horticultural Crops (Part I). National
Bureau of Plant Genetic Resources, New Delhi, pp 230.
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ENHANCING UTILIZATION OF CONSERVED PLANT GENETIC RESOURCES
Jyoti Kumari, Sherry Rachel Jacob and Ashok Kumar
Division of Germplasm Evaluation, ICAR-National Bureau of Plant Genetic Resources,
New Delhi-110012
Introduction
Plant genetic resources is the basic foundation block of sustainable agriculture on which food
and nutritional security rely. Plant genetic diversity provides farmers and plant breeders with
options to develop, through selection and breeding, new and more productive crops, that are
resistant to virulent pests and diseases and adapted to changing environments. Human
population is increasing at an alarming rate and is expected to increase from 6.9 billion to 9
billion by 2050. To feed the world population, we need to increase the food production by 60%
up to 2050 with the limited land and water resources (FAO, 2012) and plant genetic resources
will play a major role in boosting crop production by utilizing trait specific germplasm through
introgression into elite lines, allele mining, wide crossing, molecular marker assisted breeding
etc.
The value of conserved germplasm can be assessed for the useful traits in plant breeding and
the economic impact on germplasm utilization in crop production and productivity. Utilization
of germplasm in agriculture system for crop improvement are of two types: One is direct use
of the germplasm resources as crop cultivars and another is indirect use of germplasm resources
as parents in crop improvement. Direct use of germplasm contribution to crop improvement is
not expected to increased crop yield to higher level. However, indirect use to crop improvement
is more important than direct use and will contribute more in the future. Categorization of
Germplasm resources are based on: Germplasm of indigenous origin and germplasm
introduced from other countries (exogenous). Crop germplasm resources include five major
types, i.e. landraces, varieties (obsolete and in use), genetic stocks, wild relatives and breeding
lines.
Status of PGR Conservation
Genebanks, living seed collections serve as repositories of genetic variation present in the
entire gamut of germplasm comprising primitive varieties, landraces, wild relatives of crop
species and modern varieties etc. Currently, more than 1,750 individual gene banks are in place
across the globe, and about 130 of which hold more than 10,000 accessions each. Besides, there
are also substantial ex situ collections in botanical gardens of which there are over 2,500 around
the world. The total number of accessions conserved by ex situ methods worldwide has
increased by approximately 20 per cent since 1996, reaching 7.4 million, out of which only 25
- 30 per cent of the total holdings (1.9-2.2 million accessions) are distinct, with the remainder
being duplicates held either in the same or, more frequently, a different collection (FAO, 2010).
A total of 2,802,770 accessions are being conserved world-wide by 446 organizations
represented in Genesys. National Genebank of India at NBPGR, New Delhi conserves more
than 4 lakhs accessions comprising cultivars, wild relatives and land races of about 2000 crop
species.
Characterization and Evaluation
The accessibility of collections depends largely on the information available on them. Accurate
passport and characterization data are the first requirements, but users of plant genetic
resources, particularly plant breeders, have also emphasized the need for detailed evaluation of
accessions. Evaluation is a complex process and there is serious backlog in most collections.
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There are often very large numbers of accessions involved (frequently many thousands) and a
number of the traits (e.g. resistances to biotic and abiotic stresses) are difficult to measure and
subject to significant variation according to the environment in which they are measured.
Improved evaluation procedures are needed and the use of augmented plot designs (Narain,
1990) provides one way of assessing large numbers of accessions in a single replicate with
control plots that produces statistically satisfactory data.
Recent Efforts at National Level
Recently, In India, ICAR-National Bureau of Plant Genetic Resources (NBPGR) along with
national partners has taken up initiatives to characterize gene bank material for prioritized crops
for various economically important traits and also for identification and isolation of novel
genes/alleles under various programs like National Initiative on Climate Resilient Agriculture
and Consortium Research Platform. A mega trial was conducted by ICAR-NBPGR scientists
to characterize the entire set of around 22,000 wheat accessions for 34 agro-morphological
traits, terminal heat tolerance using 18 morpho-physiological traits like canopy temperature,
leaf rolling, heat susceptibility index etc. and biotic stresses including leaf, stem and stripe rusts
and spot blotch diseases so that useful germplasm lines can be identified and used in national
crop improvement program. Similarly, around about 16,000 chickpea accessions were
characterized for agronomic traits and core sets have been developed in both the crops (wheat-
2226 acc. and chickpea-~1100 acc.). Also, wheat minicore of 224 accessions was developed
using Powercore.
Under multi-location evaluation programme, ICAR-NBPGR along with its national
collaborators has identified trait specific germplasm in prioritized crops rice, wheat, maize,
chickpea, pigeonpea, brassica, okra. The information on identification of trait specific
germplasm is available on NBPGR website. Also link of PGR portal comprising all the passport
databases as well as characterization data are available for the researchers.
Strategies for Enhancing PGR Utilization
The major bottleneck for limited use of germplasms in crop improvement programme is the
large size of germplasm collections. Further, lack of infrastructure facility and manpower to
carry out high throughput phenotyping in controlled environments are other important issues
posing hindrance in PGR utilization. Documentation and easy access to characterization and
evaluation data of germplasm is also very important for awareness of the users. Knowledge of
gene pool and breeding methodologies are required for utilization of germplasm, through
varietal development, marker assisted utilization etc. Some of the strategies for germplasm
utilization are mentioned below.
Core and Minicore Collection
Surprisingly, in spite of large collections available across the world, only a few germplasm
accessions (<1%) have been utilized in crop improvement programs such as in wheat, maize,
spring barley, soybean and other grain legumes (Sharma et al., 2013). The major factor
responsible for low utilization of plant genetic resources worldwide is the unavailability of
evaluation and characterization data as the primary thrust so far has been mainly on
characterization and regeneration of gene bank material and the value of PGR was not known.
Characterization and evaluation of plant genetic resources for different morpho-physiological,
biotic and abiotic stress and quality related traits etc. is essentially required for their efficient
utilization in breeding programmes. Further, the major bottleneck for limited use of
germplasms in crop improvement programme is the large size of germplasm collections.
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Therefore a core collection is intended to contain, with a minimum repetitiveness, the genetic
diversity of a crop species and its wild relatives (Frankel and Brown, 1984; Brown, 1989). It
was envisaged that such collections, which would contain approximately 10% of the collection,
or 2000–3000 accessions, whichever is the smaller, would provide the starting material for
breeders in search of new variation or specific characters and research workers investigating
diversity. Core collections have been developed by different countries and organizations for
wheat, rice, maize, barley, beans, cassava, sweet potato, soybean, oats, brinjal etc. and we need
to extend it further in other crops. Further for extensive study of complex traits a bigger core
set is difficult to study and replicate, hence minicore was proposed as ~10 % of core collection
and ~1 % of entire collection representing entire diversity with minimum repetitiveness.
The concept of the core collection appears to offer a number of potential benefits to users of
genetic resources. Plant breeders would have a manageable number of accessions to use in the
search for new characters or character combinations and a structured way to evaluate whole
collections. Other research workers would be able to concentrate studies on inheritance or test
new technologies on a defined subset on which a substantial amount of data would be collected.
More practically, genebanks with limited resources would be able to maintain the core
collection, a rationally chosen set of accessions of crop species at relatively low cost. There
remain important issues to be addressed in ensuring that optimum procedures are used for
developing core collections. These include the extent to which ecogeographic data can provide
an adequate basis for the development of a core, the sampling strategy to be adopted (so that
interesting traits with low frequency will be represented), the importance of the genetic
structure of the crop or species concerned, and the ways in which procedures should be
modified for crops with different breeding systems and for clonally propagated ones.
Alternative Search for Genes: FIGS
Many plant genotypes are potential sources of novel genes that can improve drought tolerance,
disease resistance and other traits. Until now, breeders have not succeeded in combing through
huge gene bank collections to identify useful traits. Since 1990s, geographic information
system (GIS) has been specifically applied to the genetic resources conservation which is a
database management system that can simultaneously handle digital spatial data and attached
non-spatial attribute data (Gepts, 2006). Focused Identification of Germplasm Strategy (FIGS)
combines agro-ecological information with data on plant traits and characteristics. It is a new
tool which was developed jointly by ICARDA, the Vavilov Institute of Plant Industry in
Russia, and the Grains Research and Development Corporation in Australia. FIGS datasets
identify sets of plant genotypes from large number of collections with a higher probability of
containing specific ‘target’ traits. This strategy allows gene bank managers and agricultural
researchers worldwide to screen large plant genetic resource collections more rapidly and
accurately than was previously possible using traditional methods. It has helped identify
sources of resistance to biotic stresses in wheat such as powdery mildew, Russian wheat aphid,
stem rust, net blotch disease resistance in barley and abiotic stress tolerance such as drought
tolerance in Vicia faba (Gopal Krishnan and A K Singh, 2015).
Pre-breeding Approach
Pre-breeding refers to all activities designed to identify desirable characteristics and/or genes
from unadapted materials. Pre-breeding is a vital step to link conservation and use of plant
genetic resources especially in breeding programs. It aims to reduce genetic uniformity in crops
through the introduction of a wider base of diversity, as well as to increase yields, resistance to
pests and diseases, and other quality traits. Pre-breeding aims to provide breeders with
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enhanced germplasm materials which have specific traits of interest as well as a means to
broaden the diversity of improved germplasm.
Wild species are the reservoir of many useful genes/alleles as they have evolved under natural
selection to survive climate extremes. In pulses, wild species of Cicer, Cajanus, and Arachis
have been extensively screened and several of them were reported to have very high level of
resistance/tolerance to various stresses. Among wild Cicer species, C. bijugum, C. judaicum,
and C. pinnatifidum are the most important sources having the highest levels of
resistance/tolerance to multiple stresses (Sharma et al., 2013). The dwarfing genes in wheat
Rht1 and Rht2 were derived from a Japanese land race “Shiro Daruma” and in rice sd1 was
from “Dee-gee-woogen”. Some other successful examples of germplasm utilization include,
the only source of resistance to grassy stunt virus in rice, Oryza nivara (Plunkett, 1987),
chickpea variety Pusa 1103 using Cicer reticulatum.The emerging threat to global crop
production is climate variability, leading to frequent droughts as a result of erratic rainfall,
prevalence of high temperature, water-logging, increased soil salinity, and emergence of new
insect-pests and diseases. Due to climate change, several areas are now becoming unsuitable
for cultivation of traditional crops. To cope with this situation, there is a need to breed new
crop cultivars with a broad genetic base capable of withstanding frequent climatic fluctuations
and wider adaptability due to co-adapted gene complex.
During the past decade, pre-breeding was recognized as an important tool to broaden the
genetic base of the crops in Brazil, Cuba, Tajakistan, Ethiopia and The Russian Federation.
However the major bottlenecks in using wild species are the compatibility in wide crossing and
the linkage drag associated with the utilization of crop wild relatives. Further breaking the
undesirable linkage drag through pre-breeding makes the breeding program more time taking
and cumbersome (Sharma et al., 2013). In wheat, the discovery of the Ph1/ph1 locus which
regulates pairing and recombination between homoeologous (as opposed to homologous)
chromosomes in wheat has been a very important finding (Riley and Chapman 1958; Riley et
al., 1959). Ph1 has been used widely and successfully in wheat to induce homoeologous
recombination and the introgressed genome segments can be trimmed repeatedly to eliminate
most of the linked undesirable alleles and/or genes.
Genomics Assisted Utilization
The potential impact of molecular genetics on plant breeding is enormous and not so surprising
given the explosion of new molecular technology and applications developed during the last
decade. Progress in DNA markers became particularly important with the development of
reliable polymerized chain reaction (PCR) based markers, such as microsatellites and amplified
fragment length polymorphism (AFLP), Single Nucleotide Polymorphism (SNP), Genotype
based sequencing (GBS) etc. Genotyping by sequencing, or next-generation sequencing, an
ultimate MAS tool and a cost-effective technique, has been successfully used in implementing
genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis,
molecular marker discovery and genomic selection under a large scale of plant breeding
programs (He et al., 2014). Furthermore, DNA markers are now used extensively to
characterize germplasm (fingerprinting), to evaluate the genetic distance among accessions
(genetic diversity) and to provide important supportive information to the fields of ecology,
population genetics and also evolution. Molecular markers are also used for gene mapping and
tagging using biparental and association mapping. Enormous progress has been made in the
last 15 years in depositing an exponential amount of sequence information into GeneBank.
Based on gene and genome sequences, polymerase chain reaction (PCR) strategies are devised
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to isolate useful alleles of genes from a wide range of species. This capability enables direct
access to key alleles conferring resistance to biotic and abiotic stresses, greater nutrient use
efficiency, enhanced yield and improved quality. Using novel genomic tools, similar alleles
responsible for a given trait and their variants in other genotypes can be identified through
‘allele mining’. Identification of allelic variants from germplasm collections not only provides
new germplasm for delivering novel alleles to targeted trait improvement but also categorizes
the germplasm entries for their conservation.
Fig 1. Utilizing Exotic Germplasm through novel tools [Source: Trends in Plant Science
(2017). 10.1016/ j. tplants.2017.04.002.]
Exotic germplasm such as landraces and wild relatives possess high levels of genetic diversity
for valuable traits, including adaptation to stressful environments and more efficient nutrient
utilization. The advent of affordable high-throughput genotyping and phenotyping
technologies, together with omics-based systematic genetic technologies and emerging
statistical genomic methods, provide new avenues for efficient management, characterization,
and utilization of exotic germplasm (Fig 1). Novel biotechnologies such as genome editing
allow direct transfer of beneficial genes or gene complexes into an elite genetic background or
manipulation of existing genes in a very efficient way to obtain expected phenotypes, without
lengthy backcrossing. Genomic selection (GS) can be used to identify pre-breeding materials
with beneficial genetic variation for complex traits.
Varietal Development
Development of superior variety by accumulation of beneficial alleles from vast plant genetic
resources is a major challenge. However Indian plant breeding programme has successfully
utilized the germplasm of some major crops in India in varietal development either through
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direct use or indirectly through conventional breeding programme. The case of utilization of
germplasm in varietal development in rice and wheat are mentioned below for example.
Rice: During 1911 to 1956, about 400 cultivars were released through pure line selection of the
traditional cultivars. These improved local types were virtually the cream of the traditional rice
germplasm of India and made 10 to 20 percent increase in yield over the traditional types under
local agronomic practices and ecological conditions. They have continued to play a significant
role in the varietal improvement of rice even to the present day by providing a well-adapted
genetic background for incorporating other desirable characters (Sharma et al., 1988). Many
of these cultivars were adapted to and selected for upland and/or drought conditions (N 22, Lal
Nakanda 41, Jhona 349, MTU 17, CO 31, PTB 28), deep water and/or flood conditions (HBJ
1, HBJ 2, HBJ 3, HBJ 4, AR 1, EB 1, EB 2, FR 13A, BR 15, BR 41, BR 46) and saline soils
(Kumargone, Patnal 23, Getu, Damodar, SR 26B).
Scientists in India have made effective use of the indigenous genepool which provides
resistance to pests or tolerance to abiotic stresses. The drought-resistant N 22 was used in
breeding Bala. TKM 6, which has multiple resistance to insects and diseases, became a parent
of Ratna, Saket 4, Parijat, CR 44-1 and other improved varieties. The Tungro virus-resistant
PTB 10 has been bred into improved varieties such as Aswini, Bharathi, Jyothi, Rohini, Sabari
and Triveni. Similarly, PTB 18, possessing multiple resistance has been widely used in India
(Anon. 1980) and at IRRI (Khush, 1977; 1980). Some of the promising introductions which
have been utilised in the breeding programmes include Taichung Native 1, IR 8, Mahsuri, Leb
Mue Nahng and China 1039. Taichung Native 1 and IR 8 were the principal source of semi-
dwarfism during the mid-1960s. Mahsuri of Malaysia and Leb Mue Nahng of Thailand were
used to develop photoperiod-sensitive varieties. Rajendra Dhan 20 and Pusa 4-1-11 derived
their disease resistance from Tadukan of Philippines (Chaudhary, 1979). Indian rice germplasm
has also provided resistance source to many improved cultivars developed at IRRI, viz.
Pankhari-203 for tungro virus; many accessions from the Assam rice collection and TKM-6
and BJ-1 to bacterial leaf blight; Oryza nivara germplasm for grassy stunt virus resistance;
Assam rice collection for sheath blight; TKM-6, CO-13, Patna-6 and PTB-10 for stem borer;
and PTB-18 and PTB-21 for gall midge.
Wheat: During 1960’s, there has been a mass scale introduction of improved germplasm,
carrying the Norm-dwarfing genes from international organizations (mainly CIMMYT,
ICARDA and USDA), procured through the NBPGR. The systematic screening of indigenous
wheat germplasm was also initiated at Punjab Agricultural University, Ludhiana for various
diseases, and a number of resistant types have been identified to one or more diseases. IC35119
and IC35127 from Karnataka in Triticum durum; IC36706 and IC36729 from Himachal
Pradesh, and IC47490 from Karnataka in T. aestivum; and IC47453 from Karnataka in T.
dicoccum, showed high level of resistance to rust diseases under epiphytotic conditions. IC
28594, a durum collection from Gujarat, observed to be highly resistant to brown and yellow
rusts, also showed resistance to loose smut and powdery mildew. Wheat germplasm was also
screened for salinity tolerance at the Central Soil Salinity Research Institute, Karnal, and some
promising germplasm were identified with tolerance to saline/alkaline soils, such as IC 28609,
IC 28674, Kharchia 65, KRL 2-22, KRL-4-1, KRL 4-2, KRL 4-3, K-7435, HD 2177, BHP 10,
BHP-31, CSW 538, CSW 540 and Rata wheat.
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Conclusion
The very basis of any crop improvement programme is the extent of variability available for
different economically important traits in the germplasm. The germplasm exploration and
collection have resulted in accumulation of enormous genetic diversity of crop plants in the
gene banks. Therefore, concerted efforts need to be focussed for identification of valuable
alleles in the germplasm especially from unexplored/‘exotic’ germplasm using high throughput
phenotyping, multi-location evaluation and modern genomic tools. Once identified, these
alleles can be effectively utilized in crop improvement programmes through targeted
introgression using molecular marker assisted/ genomic assisted breeding. Further,
development of core and minicore sets along with the reference sets for trait of interest, focused
identification of germplasm strategy, pre-breeding, gene prospecting and allele mining are
essentially required for effective utilization of genetic resources.
References
Brown AHD. (1989). Core collections: A practical approach to genetic resources management.
Genome. 31: 818–824.
FAO. (2012). www.fao.org/statistics/en/
FAO (2010). The second report on the state of the world’s plant genetic resources for food and
agriculture. Rome. 399p.
Gepts P. (2006). Plant genetic resources conservation and utilization: the accomplishments and
future of a societal insurance policy. Crop Sci. 46: 2278–2292.
Gopala Krishnan S and AK Singh. 2015. Strategies for Enhancing the Utilization of Plant
Genetic Resources. Jacob Sherry R et al. (eds.) Management of Plant Genetic
Resources, National Bureau of Plant Genetic Resources, New Delhi, 323 p.
He J, Zhao X, Laroche A, Lu Z-X, Liu H and Li Z. (2014). Genotyping-by-sequencing (GBS),
an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding. Front.
Plant Sci. 5:484. doi: 10.3389/fpls.2014.00484
Plucknett DL. (1987). Gene Banks and the World’s Food. Princeton Univ. Press, 247p.
Riley R and Chapman V. (1958). Genetic control of the cytologically diploid behaviour of
hexaploid wheat. Nature 182: 713–715.
Riley R, Chapman V and Kimber G. (1959). Genetic control of chromosome pairing in
intergeneric hybrids with wheat. Nature 183: 1244–1246.
Sharma S, Upadhyaya HD, Varshney RK and Gowda CLL. (2013). Pre-breeding for
diversification of primary gene pool and genetic enhancement of grain legumes.
Frontiers in Plant Science 4:309.
Wang, C., Songlin H., Candice G., and Thomas L. "Emerging Avenues for Utilization of Exotic
Germplasm." Trends in Plant Science (2017). 10.1016/ j.tplants.2017.04.002
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OPERATIONS AND MAINTENANCE OF GENEBANK FACILITY
Rajvir Singh and Anjali
Division of Germplasm Conservation, ICAR-NBPGR, New Delhi-110012
In general refrigeration is defined as a process of heat removal. More specifically refrigeration
is defined as the branch of science that deals with the process of reducing and maintaining the
temperature of a space or material below the temperature of the surroundings.
In a refrigerator heat is being virtually pumped from the lower level to the higher level of
temperature and rejected at the higher level of temperature. This process according to second
law of thermodynamics can only be performed by the aid of external work. Hence, supply of
power from an external source is required to operate a refrigerating machine. The total quantity
of heat which is rejected to outside body is made up of two part, one part is the heat which has
been extracted at the low level of temperature from the body that is being kept cold and the
second part is the heat which is equivalent to the mechanical work which has been spent in
extracting it (work spent in driving the machine).
Theoretically, a reversed heat engine will act as refrigerator when run in the reversed direction
by means of external power. Such an engine will become a heat pump, which will pump heat
from a cold body and will deliver heat to a hot body. Thus, mechanical refrigerator operates on
the reversed heat engine cycle. The physical idea about employing the reversed heat engine as
a refrigerator can be conceived by comparing the arrangements of elements of the power plant
cycle and refrigeration cycle. A schematic diagram of the refrigeration system installed in the
Field Medium Term Storage (FMTS) is shown in fig. A and B.
The direction of flow of the working fluid at the power plants is clockwise and their cycle
follows the processes of evaporation, expansion, condensation and compression in turn in the
components condenser and feed pump.
If the direction of flow of working fluid is reversed and made anticlockwise and the order of
operations reversed such that, starting with evaporation then compression, condensation and
expansion. It is seen that the components are required to be interchanged. Evaporator
exchanged with condenser and compressor exchanged with expansion valve. Thus, it can be
said that by reversing the cycle completely in all respects, cycle of refrigeration can be evolved
which can truly be said as a reversed cycle. It may be noted that the working also requires to
be changed to a refrigerating agent (refrigerant) to make the cycle practicable.
BASIC COMPONENTS OF REFRIGERATION SYSTEM
The refrigeration system for the FMTS is assembled and supplied by MIS Heat Craft
(USA) a world famous company for designing the refrigeration systems. The major
components of the system are compressor, condenser, expansion valve and evaporator.
Their functions, operations and types are explained below:
a) Compressor: The type of compressor may be either reciprocating, rotary or centrifugal.
The compressors installed in the FMTS are Copeland Compressor. Its function is to
receive refrigerant at a particular temperature and pressure and to deliver it after
compression at higher temperature and pressure. The temperature of the refrigerant
delivered will be higher than the temperature of the cooling fluids used; so that heat will
flow from the refrigerant to the cooling fluid which is at higher temperature then that of
the refrigerant space.
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b) Condenser: The high pressure and high temperature compressed vapor is discharged
into the condenser where heat is transferred to the cooling fluid which is normally water
or air. The vapor cools and then condenses. at saturation temperature, which corresponds
to the pressure in the condenser. The vapor after condensing is passed into the liquid
storage vessel.
c) Expansion Valve: This is a throttling valve in which throttling process is carried over.
It is a valve with narrow passage through which high-pressure liquid passes and expands
from high pressure to low pressure at constant enthalpy. This is transferred to the receiver
from where it passes to the evaporator through a control valve.
d) Evaporator: In the evaporator liquid vapor absorbs heat from the space to be
cooled for its vaporization. The evaporator is in the form of coil or bare pipe or tubes, as
the case may be, through which liquid vapor flows. The evaporated vapor is sucked by
the compressor from the evaporator and delivered to the condenser. Thus the cycle is
completed.
ELECTRICAL CONTROLS
The working of the refrigeration systems are electrically controlled it is essential to have a clear
understanding of these systems also in order to efficiently maintain and operate the FMTS. The
important components of the electrical circuit controls of the compressor and the control panel
in the refrigeration system in the FMTS. Based on the experience of the maintenance at the
various sites a list for the troubleshooting of the FMTS are presented below for ready reference
of the users.
TROUBLESHOOTING CHART FOR MAINTAINANCE OF MTS AND LTS
Symptom possible cause Solution
Chamber will not
start
Disconnect switch in off position Check disconnect switch
No power from stabilizer Check stabilizer and its cause of tripping
stabilizer trips
Control panel –
No
Temp/humidity
indication
Control circuit breaker tripped check heater/recorder circuit
Incandescent door circuit
UPS
Breaker OK but still no indicator
check
UPS output or bypass UPS
24V AC at output of transformer
5823A-refer manual
24V AC not available, locate the
fault as per circuit diagram
5823A-refer manual or call BSL
engineer.
One of the temp/humidity control
not responding properly
Replace the controller
Check its sensor is storage room
Incandescent does
not lighting
Light circuit breaker Check for short circuit
Incandescent Switch Check Switch position
Lamp holder Replace defective lamp holder / bulb
Emergency alarm circuit breaker Check for short circuit
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Audible
alarm/personnel
emergency
Transformer Check input / output voltage of transformer
Switch Switch personnel emergency
Recorder Transformer Check input / output voltage
Recorder Calibrator Do the calibration of recorder
Check its sensor only temp
Evaporator Fan
motors
Check circuit breaker Check circuit breaker, if required replace
Defect in motor / Electrical
Connection
Check voltage at input of motors, if required
replace the motor
Dehumidifier Drier – Circuit breaker Check circuit breaker
Defect in dehumidifier Check indicator on the Dehumidifier if it is
not ON check dehumidifier circuit (refer
manual Check fuses in the Dehumidifier. If
defect replace. Check M.C.B in
dehumidifier.
Compressor will
not run
1. Compressor circuit
2. Disconnect switch OFF
3. Fuse Blown
4. Thermal over load
5. Defective contractor coil
6. System shut down by safety/No
cooling
7. Liquid line solenoid will not
open
8. Motor electrical trouble
9. Loose wiring
1. Check circuit breaker in control panel
2. Close switch
3. Check electrical circuit for short /
overloading from defective parts.
4. If overload is automatically reset, check
unit closely when unit comes on
5. Repair or replace.
6. Determine and correct the cause
7. Check the circuit if required replace coil.
8. Check motor for open / short.
9. Check all wire junctions.
High discharge
pressure
1. Condenser fan not running
2. System over charged
3. Head Pressure control setting
4. Dirty Condenser coil
1. Check condenser fan motor and its
electrical circuit
2. Remove excess
3. Adjust / or may be faulty set
4. Clean with water & liquid soap
Low suction
pressure
1. Lack of refrigerant
2. Evaporation dirty or iced
3. Clogged liquid filter drier
4. Expansion value
1. Check for leak repair / charge refrigerant
2. Clean evaporator coil Check fan motor
3. Replace drier
4. Check and adjust Super heat, replace
expansion value
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Figure: A
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Figure: B
Preventive Maintenance Schedule for MTS and LTS
To ensure proper operation and maintenance of equipments of Genebank, it is highly
recommended that the following list of checks and maintenance items be performed as
scheduled.
Each Day:
Refrigeration site glass-green colour, no bubbling
Recorder charts-replace where required.
Lamps- replace where necessary.
Temperature operating as programmed.
Humidity operating as programmed.
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Each Week:
All items shown under “ Each Day”, plus;
High/Low temperature limits-check operation.
Evaporator circulating fans-check to make sure operating.
Each Month
All items shown under “Each Week”, plus;
Chart recorder- check calibration (at 25° C).
Emergency lighting- ensure system activates by shutting off main lighting circuit
breaker.
Panic alarm –ensure alarm system is operational.
Smoke detector-check battery, check operation.
Redundant refrigeration systems-switch over to other system to ensure operation.
Remote condenser-clean units with whisk brush and vacuum cleaner or use
compressed air. This is very important to good chamber performance.
Chemical Dryer air filters (process and reactivation)- inspect and clean as necessary.
Chemical Dryer Homey Combe wheel-check for rotational binding.
Chemical Dryer Honey Combe wheel- should not be plugged with dirt.
Chemical Dryer upper and lower air seals- check for excessive wear.
Chemical Dryer reactivation outlet temperature –should be 120° F/40° C ± 12-5%.
Every Three Months
Oil condenser fan motors if required (per manufacturer’s instructions)
Every Six Month
All items mentioned previously, plus;
Door hinges and latches- adjustment if necessary.
Drain traps-clean to avoid blockage.
Refrigeration system- have a refrigeration technician check unit for small faults and
leaks before they develop into major breakdown. Check the pressure controls and
operating pressures
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INTELLECTUAL PROPERTY RIGHTS (IPR) ISSUES RELATED TO PLANT
GENETIC RESOURCES
Vandana Tyagi, Pragya and Pratibha Brahmi
Division of Germplasm Exchange and Policy Unit, ICAR-National Bureau of Plant Genetic
Resources, New Delhi-110012
Abstract
Plant Genetic Resources (PGR) include plants of potential value for food and agriculture
including their wild and weedy relatives and play a vital role in crop improvement
programmes worldwide. Currently access to PGR is regulated by various national and
international instruments related to bio-diversity and agriculture. Under the provisions of
Convention on Biological Diversity (CBD), which entered into force in 1993, access to
biological resources is based on the principle of ‘sovereign rights of nations’. It provides for
access to genetic resources and transfer of relevant technologies on mutually agreed terms
(MAT) and subject to prior informed consent (PIC). In response to CBD, India enacted the
Biological Diversity Act (BDA), 2002 and established the National Biodiversity Authority
(NBA) in 2003. Access to PGR from India is therefore regulated by BDA, 2002. ICAR-
National Bureau of Plant Genetic Resources (NBPGR) is recognized in India as the nodal
organization facilitating the exchange of PGR for research purposes, to users in India and in
other countries. As per the provisions of the International Treaty on Plant Genetic Resources
for Food and Agriculture (ITPGRFA), 2001, facilitated access to plant genetic resources for
food and agriculture to all member countries, is provided for the crops listed in Annex 1 of
ITPGRFA. The access as per the ITPGRFA is solely for utilization and conservation for
research, breeding and training. The Treaty has established a multilateral system (MLS) of
exchange of PGRFA. All exchange under the provisions of the Treaty is done after signing
the Standard Material Transfer Agreement (SMTA), ensuring that the material accessed
under treaty shall be freely available to others for use in research, breeding and training
provided the third and subsequent parties are bound by the same conditions of the SMTA.
IPRs cannot be claimed by the recipients on the material received from the MLS and if any
commercial utilization is done, the benefits would be deposited in a trust fund of the Treaty.
The Nagoya Protocol entered into force from October 2014 further defines the international
regime within the framework of CBD to promote and safeguard the fair and equitable sharing
of benefits arising from the utilization of genetic resources. In relation to PGR the IPRs are
granted in the forms of protection through registration of germplasm, as variety under
PPVFRA and Geographical indications.
Keywords: Access, BDA, ITPGRFA, MTA, PGR
Introduction:
Plant Genetic Resources (PGR) comprise of crop plants and their wild/weedy related species
of actual or potential use. The development of improved types used today and those that
would be cultivated in future is based on the effective utilization of PGR. These have helped
in broadening the genetic base of crop plants within species and among species and also in
diversification of cropping and farming systems through stability and sustainability. There is
continuous search for newer resources to meet the future demands that arise with the
emergence of climate change, new diseases, and enhanced demands food and nutritional
security (De Jonge 2009). PGRs are exchanged and searched continuously for specific traits
to improve crops in terms of yield and nutritional value. All countries are therefore,
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interdependent on each other for sources of PGR, since many crops cultivated in a country
have not originated there. Even biodiversity rich regions depend for more than 30% of their
food production on crops originating from other countries (Cooper, 1994) and this
interdependence plays a very important role in international collection and exchange of
germplasm. Every nation is concerned with acquisition of diverse and superior germplasm
for conservation and utilization.
India is very rich in terms of plant genetic resources and was considered to be one of the eight
‘centres of origin’ of crop plants and had two sub centers of crop diversity as described by
Vavilov (1926). It was described as one of the twelve mega centres of crop diversity (Zeven
and de wet, 1982). Endless diversity of useful genes and traits had been utilized for crop
improvement from Indian plant genetic resources. About 166 cultivated species are reported
to be native to this region along with 320 wild relatives distributed in different agro-ecological
zones in India (Arora, 1991). PGR have been collected, used and improved for centuries, but
the formal concern regarding their conservation has been voiced only since 1930s mainly
following voyages of Vavilov who described the concept of centres of origin of crop plants.
Short statured, lodging resistant, input responsive, high yielding introductions of wheat and
rice played a pivotal role in ushering in the era of Green Revolution; and those carrying
cytoplasmic-nuclear male sterility and fertility restoration genes brought in the era of hybrid
breeding in crops like sorghum, pearl millet and rice that enabled the exploitation of heterosis.
Further, it was the introduced germplasm that enabled soybean and sunflower to become
major field crops in India and among horticultural crops some major crops introduced into
the country are apple, kiwi fruit, peach, sea buckthorn, French bean, pepper mint, and sugar
beet. Introductions therefore have played a pivotal role in the establishment of large number
of crops and development of improved varieties in India (Singh, 2003). The exchange of
germplasm especially from ex situ collection of CGIAR centres has also helped most
countries including India to strengthen their crop improvement programmes. As agriculture
progressed, a number of species that were not native were introduced from different parts of
the world. Thus exchange of PGRs offers enormous opportunity for sustainable agriculture
as there is continuous need of PGRs for utilization in various crop improvement programs.
For sustainable agriculture, food and nutritional security, the planners and policy makers at
national and international level decided to strengthen the activities related to plant genetic
resources management. In India the Indian Council of Agricultural Research (ICAR) created
a new organization in 1976 and named it National Bureau of Plant Introduction (NBPI),
renamed as National Bureau of Plant Genetic Resources (NBPGR) in 1977. NBPGR after its
creation in 1976 has developed a very strong Indian Plant Germplasm Management System
which operates in a collaborative and partnership mode with other organizations. The system
has contributed immensely towards safeguarding the indigenous crop genetic resources and
regulating access of PGR for enhancing the agricultural production and productivity in the
country. India being one of the gene-rich countries of the world faces a unique challenge of
protecting its natural heritage and evolving suitable mutually beneficial strategies for
germplasm exchange with other countries. Since its inception NBPGR is working towards
achieving collection, conservation, characterization of PGR addressing to and compliance of
national and international regulations related to exchange of plant genetic resources. As per
TRIPS Agreement the term 'intellectual property' refers to all categories of intellectual
property that are the subject of Sections under the Act. These sections deal with copyrights
and related rights, trademarks, geographical indications, industrial designs, layout designs of
integrated circuits, patents and the protection of undisclosed information (trade secret). As
per Section 5 of the TRIPS Agreement, there is need to provide for the protection of plant
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varieties by patents or by an effective sui generis system or by any combination thereof. It
follows that, like patents and all the other rights the sui generis system also has to be an IPR.
In relation to PGR the forms of IPRs are either through registration or protection as
Geographical indications. As per the definition of Intellectual Property Rights, anything
occurring in nature cannot be granted a patent however GI protection and protection of plant
varieties is provided under the PPVFRA, 2001 and GI Act. Further, with the objective of
providing due credit to the scientists and developers who identify unique and promising
research material registration certificate is provide for such unique accessions which are in
public domain and available for research programmes.
Protection of Plant Varieties &Farmers Right Act (PPVFRA)
The Indian PPV& FR Act provides for effective system for protection of plant varieties, and
protects rights of farmers and breeders. The Act recognize the farmer as a conserver, provider
of genetic resources, breeder and as a producer and consumer of seed. PPVFRA is effective
from January 2005 and addresses the rights of plant breeders and farmers. With regard to
developing or selecting varieties, the Act refers to the value added by farmers to wild species
or traditional varieties/ landraces through selection and identification for their economic
traits. PPVFR Authority awards farming communities who are engaged in conservation and
improvement of PGR (economic plants and wild relatives).
As per the Act, Farmers’ variety is defined as a variety which has been traditionally
cultivated and evolved by the farmers in their fields, or is a wild relative or land race of
variety about which the farmers possess the common knowledge under the PPVFRA and
qualify for protection as per the Law.
The relationship between IPRs and benefit sharing is especially complex with relation to
access to PGR. The access to PGR and equitable sharing of benefits policies and programmes
are now well placed and regulated under different Acts and Treaties.
Geographical Indications (GIs):
The Geographical Indications of Goods (Registration and Protection) Act, 1999 under sui
generis system provide protection as GI at national level. The Government of India has
established the 'Geographical Indications Registry' with all-India jurisdiction at Chennai,
where the GIs can be registered. The Controller General of Patents, Designs and Trademarks,
is the Registrar of Geographical Indication of India for administering the GI Act. Protection
of goods with unique quality or reputation by registering them as GIs does not only provide
them legal protection but would also help to build a reputation in international market for
good economic returns. Like trademarks or commercial names, GIs are distinctive signs
which permit the identification of products in the market.
Since GI is a community right granted to the producers of concerned good in the defined
region, for registration of GI, any association of persons or producers or any organization or
authority established by or under the law for the time being in force can file an application
for registration of said good under the GI Act 1999 on behalf of producer of concerned goods.
The grant of GI registration would be the date of making of the application. Upon registration,
the registrar issues to the applicant and its authorized users, a certificate of registration sealed
with the seal of GI Registry. The registration of GI is valid for a period of ten years but may
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be renewed from time to time in accordance with the provisions given in the Act. Some of
the agricultural produce which is specific to a geographical area regarding its quality and
specific traits can be protected by the communities/ producers in the specific geographic
region examples Darjeeling tea, hail banana of Karnataka and Guntur chillies.
Convention on Biological Diversity (CBD)
The exchange scenario has changed fast during the last decade, most obviously due to the
trends of globalization and privatization. As a result, a paradigm policy shift was witnessed
in the international policy environment from “heritage of mankind” to “sovereign rights of a
nation”. The major events which led to this shift was the Convention of Biological Diversity
(CBD) which came into force in 1993, adopted during the Rio Earth Summit of the United
Nations. It was the first legally binding institutional mechanism, providing for conservation
and sustainable use of all biological diversity and intends to establish the process of the
equitable sharing of benefits arising out of the use of biodiversity. The CBD reaffirmed
national sovereignty over genetic resources and stressed that the authority to determine access
to genetic resources rests with the national governments and is subject to national legislations.
It provides for a bilateral approach to access/exchange between countries on prior informed
consent (PIC) and mutually agreed terms (MAT). Prior Informed Consent (PIC) is an
instrument that user has received informed consent from provider prior to accessing a
particular resource. MAT is an element that needs to be negotiated between users and
providers of genetic resources and associated traditional knowledge.
CBD is UN agreement in the background of increased threat to PGR by the developments in
biotechnology. Accordingly, patents on genetic material need to be consistent with the CBD
and resources are acquired legally. Thus country of origin and proof of PIC together known
as disclosure issues are needed to be indicated in patent applications. CBD has therefore,
regulated the flow of germplasm between the nations. The objectives of CBD are to conserve
biological diversity; to use biological diversity in a sustainable manner and to share the
benefits of biological diversity fairly and equitably.
The Biological Diversity Act (BDA), 2002
In response to CBD, Government of India enacted legislation called Biological Diversity Act
(BDA), 2002 and notified the Biological Diversity Rules, 2004. The objectives of the
Biological Diversity Act are to provide for conservation of biological diversity, sustainable
use of its components and fair and equitable sharing of benefits arising out of the use of
biological resources. As per the provisions of the Act, National Biodiversity Authority
(NBA) is established which regulates conservation and access to biological diversity for
sustainable utilization and equitable sharing of benefits arising out of the utilization of
biological resources.
Section 3 (2) of the Act, defines the non- Indian entity and describes that non-Indian entity
cannot access any biological resource occurring in India without the prior approval of NBA.
However, Section 5 of BDA, 2002, provides for exemption from Section 3 and 4 for
exchange of PGR/ germplasm for research which are agreed under the collaborative research
project between Government sponsored institution and confirming to the policy guidelines
issued by Ministry of Environment, Forests and Climate Change. As per the Act, the non-
India entity is defined as a person who is not a citizen of India; a citizen of India, who is a
non-resident as defined in clause (30) of section 2 of the Income-tax Act, 1961; a body
corporate, association or organization- (i) not incorporated or registered in India; or
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incorporated or registered in India under any law for the time being in force which has any
non-Indian participation in its share capital or management.
Section 6 of BDA, 2002 also mentions no person (Indian or non-Indian) shall apply for any
IPR by whatever name called, in or outside India for any invention based on any research or
information on a biological resources obtained from India without obtaining the previous
approval of the NBA before making such application.
Nagoya Protocol
India has ratified the Nagoya Protocol (NP) which is an international agreement which aims
at sharing the benefits arising from the utilization of genetic resources in a fair and equitable
way, including by appropriate access to genetic resources and by appropriate transfer of
relevant technologies, taking into account all rights over those resources and to technologies,
and by appropriate funding. NP entered into force on 12 October 2014.The regulatory
mechanism for access to biological resources under BDA, 2002 ensures compliance to
Convention and NP.
Nagoya Protocol provides a strong base for legal certainty and transparency for both providers
and users of genetic resources. As nonmonetary benefit, NP provides for the provision of
transfer of knowledge and technology that make use of genetic resources, including
biotechnology, or that are relevant to the conservation and sustainable utilization of biological
diversity to the provider of PGR under fair and most favorable terms, including on
concessional and preferential terms where agreed. It recognizes the importance of promoting
equity and fairness in negotiation between providers and users.
International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA)
CBD does not categorize the nature of biological resources and do not differentiate the
treatment of plant genetic resources for food and agriculture (PGRFA). The special nature of
PGRFA, their interdependence, was thus felt to have a special treatment which is very
essential and crucial for the sustainable utilization and food security, and on the universally
made principle that PGR is heritage of mankind and consequently should be available without
restriction. FAO in 1983 adopted an International Undertaking on Plant Genetic Resources
(IUPGR) with the objective to ensure that PGR are of economic and / or social interest
particularly for agriculture, will be explored, preserved, evaluated and made available for
plant breeding and research purposes. FAO Commission on Genetic Resources for Food &
Agriculture (CGRFA) monitored the implementation of IUPGR. The revised text of IUPGR
was adopted as the International Treaty on Plant Genetic Resources for Food and Agriculture
(ITPGRFA) on 3 November, 2001 (FAO, 2002). Legally binding ITPGRFA was thus
negotiated as a direct response to CBD in 2001, came into force in 2004 to facilitate access
to PGRFA in harmony with CBD, through an efficient mutually agreed system of access and
benefit sharing. Access here is only for research, breeding and training and not for chemical,
pharmaceutical or nonfood/feed industrial use. No IPRs can be claimed on PGRFA in the
form received from the multilateral system that limits the facilitated access to PGRFA/genetic
parts or components. Its centerpiece is a multilateral system of facilitated access and benefit
sharing that directly supports the work of breeders and farmers everywhere. Its objectives are
conservation and sustainable use of PGRFA and the fair and equitable sharing of benefits
derived from their use, in harmony with the CBD for sustainable agriculture and food security.
It covers all PGR relevant to food and agriculture. Each ratifying government agreed to ensure
the conformity of its laws, regulations and procedures with its obligations under the treaty.
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The Governments of the countries that ratified the treaty form its governing body. Currently
144 countries are the contracting parties including India who had ratified the treaty.
The treaty provides for facilitated access to a specified list of PGRFA, balanced by benefit-
sharing in the areas of information exchange, technology transfer, capacity building and
commercial benefit-sharing. Presently, the multilateral system applies to a list of over 64 plant
genera, including 35 crop and 29 forage plants, agreed on the basis of interdependence and
food security and are referred to as Annex 1 crops. The list may be revised or expanded
based on the criteria of food security and interdependence. The conditions for access and
benefit sharing are set out in a ‘Standard Material Transfer Agreement’ (SMTA), adopted by
the Governing Body of the Treaty.
An important point is equitable sharing of the benefits arising from the commercialization of
a product that uses PGR from the multilateral system except when the product is available
without restriction for further research and breeding. The treaty also recognizes the enormous
contribution that farmers and farming communities have made and continue to make to the
conservation and development of PGR, and puts the responsibility for realizing farmers’
rights on national governments.
Conclusions
The regulations for access to PGR are in place and every researcher in the country need to
know the rules for exchange of germplasm. Categories of germplasm need to be treated
differently for exchange as per these rules. The CBD encourages bilateral exchange based on
mutually agreed terms and ensures equitable benefit sharing. However PGRFA which are
important for present and future food and nutritional security need to be handled differently
as provided under the ITPGRFA. The SMTA of the ITPGRFA has further taken the debate
to other genetic resources and the need for an internationally recognized frame work for
access and benefit sharing was recognized. This has culminated in the adoption of the Nagoya
Protocol negotiated under the CBD, which entered into force in October 2014.
References:
De, Jonge., Plants, Genes and Justice: An enquiry into fair and equitable benefit-sharing.
Unpublished Ph D dissertation, 2009, Wageningen: Wageningen University
Vavilov, N. I.,Studies on the origin of cultivated plants. Bulletin of Applied Botany, 1926,
26: 1–248.
Zeven, A. C. and J. M. J. de wet, Dictionary of cultivated plants and their regions of diversity
:excluding most ornamentals, forest trees and lower plants, 2nd rev. ed., Wageningen
: Pudoc, Centre for Agricultural Publishing and Documentation, 1982, 263 p.
Arora, R. K., Plant diversity in Indian Gene Centre, Plant Genetic Resources: Conservation
and Management (eds. R. S. Paroda and R. K. Arora), IBPGR, Regional Office, New
Delhi, India, 1991, pp.25-54.
Cooper D., J. Engels and E. Frison, A Multilateral System for plant genetic resources:
imperatives, achievements and challenges, Rep.2, 1994, Int. Plant Genetic Resources
Institute, Rome, Italy
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Singh, R.V., Chand, D., Tyagi, V., Verma, N., Singh, S. P. and Dhillon, B. S., Important crop
germplasm introduced into India during 2001, Indian J. Plant Genet. Resour., 2003,16
(2):87-90.
Dhillon, B.S. and Anuradha Agarwal, Plant Genetic Resources: Ownership, Access and
Intellectual Property Rights In: Plant Gentic Resources: Oilseed and Cash Crops (eds.
Dhillon BS, R K Tyagi, S Saxena and Anuradha Agarwal) 2005, Narosa Publication,
pp. 1-20.
FAO, The International Treaty on Plant Genetic Resources for Food and Agriculture, 2002,
Rome, Italy
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LIST OF THE PARTICIPANTS
Name : Asaad Abdul Rassoul
Designation : Chief of Agronomist
Department : Plant Genetic Resources
Organization : Directorate of Seed Testing and Certification
E-mail ID : [email protected]
Phone No. : 07713236036
Name : Qasim Mohammed
Designation : Senior Agronomist
Department : Plant Genetic Resources
Organization : Directorate of Seed Testing and Certification
E-mail ID : [email protected]
Phone No. : 77118276663
Name : Osamah Sami Noori
Designation : Senior Agronomist
Department : Plant Genetic Resources
Organization : Directorate of Seed Testing and Certification
E-mail ID : [email protected]
Phone No. : 009647700124020
Name : Hadeel Sabri Nasser
Designation : Agronomist
Department : Plant Genetic Resources
Organization : Directorate of Seed Testing and Certification
E-mail ID : [email protected]
Phone No. : 07705009275
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LIST OF FACUALTY AND THEIR CONTACT DETAILS
Dr. Kuldeep Singh Director
ICAR-NBPGR
Dr. Veena Gupta Principal Scientist and Head (Acting)
Division of Germplasm Conservation
Dr. SP Ahlawat Principal Scientist and Head
Division of Plant Exploration and
Germplasm Collection
Dr. Ruchira Pandey Principal Scientist
Tissue Culture and Cryopreservation Unit
Dr. Neeta Singh Principal Scientist
Division of Germplasm Conservation
Dr. J. Radhamani Principal Scientist
Division of Germplasm Conservation
Dr. Anjali Kak Koul Principal Scientist
Division of Germplasm Conservation
Dr. Chitra Pandey Principal Scientist
Division of Germplasm Conservation
Dr. Sushil Pandey Principal Scientist
Division of Germplasm Conservation
Dr. Vimla Devi S. Senior Scientist
Division of Germplasm Conservation
Dr. Sherry R Jacob Senior Scientist
Division of Germplasm Conservation
Dr. Sunil Archak ICAR- National Fellow &
Officer-In-Charge
Agriculture Knowledge Management Unit
Dr. Era Malhotra Scientist
Tissue Culture and Cryopreservation Unit
Dr. J. Aravind Scientist
Division of Germplasm Conservation
Dr. Jameel Akhtar Principal Scientist
Division of Plant Quarantine
Dr. Jyoti Kumari Principal Scientist
Division of Germplasm Evaluation
Dr. Kavita Gupta Principal Scientist
Division of Plant Quarantine
Dr. KK Gangopadhyay Principal Scientist(Hort.)
Division of Germplasm Evaluation
krishna.gangopadhyay(@icar.gov.in
Dr. Vandana Tyagi Principal Scientist
Germplasm Exchange and Policy Unit
Dr. Vartika Srivastava Scientist
Tissue Culture and Cryopreservation Unit
Dr. Gowthami R Scientist
Tissue Culture and Cryopreservation Unit
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Dr. Axma Dutta Sharma Assistant Chief Technical Officer
Division of Germplasm Conservation
Ms. Smita Lenka Jain Assistant Chief Technical Officer
Division of Germplasm Conservation
Mr. Rajeev Gambhir Assistant Chief Technical Officer
Agriculture Knowledge Management Unit
Ms. Nirmala Dabral Technical Officer
Division of Germplasm Conservation
Mr. Rajvir Singh Assistant Chief Technical Officer
Division of Germplasm Conservation
Mr. Satyaprakash Technical Officer
Division of Germplasm Conservation
Mr. Lal Singh Technical Officer
Division of Germplasm Conservation
Ms. Anjali Technical Assistant
Division of Germplasm Conservation
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