training manual - nbpgr

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



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


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


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


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


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


11:30 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


11:30 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


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


11:30 AM - 01:30 PM Plant Genetic Resources -Indian Perspective Dr Kuldeep Singh, Director


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

1

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.

2


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.

3


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.

4


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

5


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.

6


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.

7


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.

8


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

9


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.

10


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

11


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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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.

http://www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en

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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.

14


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.

15


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.

16


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

17


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

18


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

19


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).

20


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

21


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.

22


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

23


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

24


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.

25


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.

26


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

27


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

28


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

29


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

30


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.

31


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.

32


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.

http://www.sgrp.cgiar.org/

http://www.cgiar.org/

http://cropgenebank.sgrp.cgiar.org/

http://typo3.fao.org/fileadmin/templates/agphome/documents/PGR/ITWG/ITWG6/working_docs/CGRFA_WG_PGR_6_12_4.pdf

http://typo3.fao.org/fileadmin/templates/agphome/documents/PGR/ITWG/ITWG6/working_docs/CGRFA_WG_PGR_6_12_4.pdf

33


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.

34


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,

35


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

36


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.

37


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

38


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

39


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)


Thawing and regrowth

40


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)


Thawing and unloading

Regrowth

Encapsulation-

vitrification Meristems

Shoot tips/buds

Nodal segments

Somatic embryos

Zygotic embryos

Cell suspensions


(cryoprotectans/low temperature) (optional)

Encapsulation of explants in sodium/ calcium

alginate (2-3%)

Cryoprotective dehydration with a cryoprotectant

mixture (PVS2)



Regrowth

Encapsulation-

dehydration Meristems

Shoot tips/buds

Nodal segments

Somatic embryos

Zygotic/microspore

embryos

Cell suspensions


(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


Thawing and regrowth

Droplet

vitrification Meristems

Shoot tips/buds

Nodal segments

Somatic embryos

Zygotic embryos

Cell suspensions


(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


Regrowth

Dormant bud

cryopreservation

Dormant buds Collection and transportation of bud woods

Sealing cut ends with wax and packing in

polythene bags

41


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

42


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

43


44


Reference

Benson EE 2008 Cryopreservation of phytodiversity: a critical appraisal of theory and practice.

Crit Rev Plant Sci 27:141–219.

Benson EE, Harding K, Debouck D, Dumet D, Escobar R, Mafla G, Panis B, Panta A, Tay D,

Van den houwI and Roux N 2011. Refinement and standardization of storage

procedures for clonal crops - Global Public Goods Phase 2: Part II. Status of in vitro

conservation technologies for: Andean root and tuber crops, cassava, Musa, potato,

sweet potato and yam. System-wide Genetic Resources Programme, Rome, Italy.

Benson EE, Harding K, Debouck D, DumetD, Escobar R, Mafla G, Panis B, Panta A, Tay D,

Van den houwI and Roux 2011. Refinement and standardization of storage procedures

for clonal crops - Global Public Goods Phase 2: Part III. Multi-crop guidelines for

developing in vitro conservation best practices for clonal crops. System-wide Genetic

Resources Programme, Rome, Italy.

Engelmann F 2000.Importance of cryopreservation for the conservation of plant

geneticresources.In: Engelmann F and Takagi H (eds) Cryopreservation of

TropicalPlant Germplasm Current Research Progress and Application, JIRCAS/

IPGRI pp.8-20.

Engelmann F 2004. Plant cryopreservation: progress and prospects. In Vitro Cell Dev Biol

Plant 40:427–433

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.

45


GUIDELINES FOR SENDING GERMPLASM FOR PEST FREE CONSERVATION

Veena Gupta, Sushil Pandey and Smita Lenka


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.

46


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).

47


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

48


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



49


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.

50


Fig. 1. Seed health testing procedure for pest free conservation of indigenous crop

germplasm.

51


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).

52


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

53


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

54


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.

55


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.

56


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.

57


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.

58


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)

59


(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

60


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

61


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.

62


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.

63


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

64


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

65


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

66


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

67


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


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


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)

68


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

69


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

70


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.

http://www.ncbi.nlm.nih.gov/pubmed/16654086






71


MODELLING AND MONITORING OF SEED LONGEVITY IN CONSERVED

GERMPLASM

J Aravind, Chithra Devi Pandey, Neeta Singh and Anjali Kak Koul


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

72


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

73


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.

74


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

75


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

76


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

77


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

78


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).

79


80


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.

81


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.

82


OPERATION AND MAINTENANCE OF SEED DRYERS/DE-HUMIDIFIER AND

SEED GERMINATOR

Satya Prakash and Lal Singh


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

83


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.

86


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

87


(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

88


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.

89


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

http://www.genesys-pgr.org/

http://www.ildis.org/

http://www.upov.int/

http://www.sp2000.org/

90


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,

91


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.

95


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

http://www.fao.org/statistics/en/

http://dx.doi.org/10.3389/fpls.2014.00484

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OPERATIONS AND MAINTENANCE OF GENEBANK FACILITY

Rajvir Singh and Anjali


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

111


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.

113


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




Phone No. : 77118276663

Name : Osamah Sami Noori

Designation : Senior Agronomist




Phone No. : 009647700124020

Name : Hadeel Sabri Nasser

Designation : Agronomist




Phone No. : 07705009275

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LIST OF FACUALTY AND THEIR CONTACT DETAILS

Dr. Kuldeep Singh Director

ICAR-NBPGR

[email protected]

Dr. Veena Gupta Principal Scientist and Head (Acting)


[email protected]

Dr. SP Ahlawat Principal Scientist and Head

Division of Plant Exploration and

Germplasm Collection

[email protected]

Dr. Ruchira Pandey Principal Scientist

Tissue Culture and Cryopreservation Unit

[email protected]

Dr. Neeta Singh Principal Scientist


[email protected]

Dr. J. Radhamani Principal Scientist


[email protected]

Dr. Anjali Kak Koul Principal Scientist


[email protected]

Dr. Chitra Pandey Principal Scientist


[email protected]

Dr. Sushil Pandey Principal Scientist


[email protected]

Dr. Vimla Devi S. Senior Scientist


[email protected]

Dr. Sherry R Jacob Senior Scientist


[email protected]

Dr. Sunil Archak ICAR- National Fellow &

Officer-In-Charge

Agriculture Knowledge Management Unit

[email protected]

Dr. Era Malhotra Scientist


[email protected]

Dr. J. Aravind Scientist


[email protected]

Dr. Jameel Akhtar Principal Scientist

Division of Plant Quarantine

[email protected]

Dr. Jyoti Kumari Principal Scientist

Division of Germplasm Evaluation

[email protected]

Dr. Kavita Gupta Principal Scientist

Division of Plant Quarantine

[email protected]

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

[email protected]

Dr. Vartika Srivastava Scientist


[email protected]

Dr. Gowthami R Scientist


[email protected]

mailto:[email protected]




117


Dr. Axma Dutta Sharma Assistant Chief Technical Officer


[email protected]

Ms. Smita Lenka Jain Assistant Chief Technical Officer


[email protected]

Mr. Rajeev Gambhir Assistant Chief Technical Officer

Agriculture Knowledge Management Unit

[email protected]

Ms. Nirmala Dabral Technical Officer


[email protected]

Mr. Rajvir Singh Assistant Chief Technical Officer


[email protected]

Mr. Satyaprakash Technical Officer


[email protected]

Mr. Lal Singh Technical Officer


[email protected]

Ms. Anjali Technical Assistant


[email protected]


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119
