R.K. Tyagi

and

Anuradha Agrawal

National Bureau of Plant Genetic Resources Pusa Campus, New Delhi -110 012

http://www.nbpgr.ernet.in

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Genetic resources conservation Ex situ conservation strategies

• PGR comprise diversity among and within plant species, that are of direct or indirect value to humans.

• Most valuable raw material in crop improvement, food and nutritional security.

• Comprises landraces, farmers’ varieties, obsolete cultivars, modern cultivars, breeding lines and genetic stocks, wild relatives, weedy races, potential domesticates or other wild species.

• PGR diversity eroded due to introduction of mono-culture of improved varieties/hybrids (narrow genetic base) and changes in agro-ecosystems, industrialisation and other developmental process (genetic erosion, extinction).

• PGR constitute national heritage that needs efficient management and conservation for posterity. “Germplasm” refers to reproductive or vegetative propagating material of plants, such as, seeds, tissues, cells, pollen, DNA molecule etc. containing the functional unit of heredity that can be utilised in crop improvement programme.

Why conserve Plant Genetic Resources (PGR)?

Management of Plant Genetic Resources

Main Activities • Acquisition

- Collection, Exchange and Quarantine

• Conservation

• Evaluation

• Utilization

Multidisciplinary, cut across various sectors of

science and society

Central topic of debate at national and

international fora

• Scientific issues - conservation and sustainable

utilization

• Social and ethical issues – Ownership, benefit

sharing

Field

Genebank

Cro

p

gen

eb

an

k

Seed Genebank

In Situ

(in original habitat,

dynamic conservation)

Ex Situ

(outside habitat,

Static conservation)

National

Parks

Biosphere

reserves

Gene

Sanctuaries

On-farm

Wild species

Wild relatives

Landraces

Traditional

varieties Arb

ore

tum

Herb

al g

ard

en

Bo

tan

ical g

ard

en

In Vitro

Genebank

Cryobank

DNA bank

Strategies for Conservation

Both static (seed banks, culture collections etc.) and dynamic (conservation of populations in

evolving natural environments) ways are needed.

How to select a suitable

conservation method?

•Biology of the species

•Size, population, genetics

•Nature of propagule

•Nature of threat

•Infrastructural and technical expertise

•Economic considerations

•Utilization

•Research

•Breeding

•Exchange

Natural Habitat

Herbal garden

Seed vault

In Vitro Repository

Seed propagated Clonally/vegetatively

propagated

How to select the best method?

• Knowledge of the seed storage behaviour of a target species is required in order to determine whether or not seed storage is suitable as a method of genetic conservation, and how to handle seeds during collection and germplasm exchange.

• If it is feasible, long-term seed storage is generally considered the safest, most inexpensive and most convenient method of genetic conservation.

• Most plant genetic resources are conserved by this means.

• The duration of storage depends upon the objectives of the particular conservation effort and the species concerned.

• The goal of genebanks is essentially the maintenance of seed viability in a very wide range of species for indefinite but considerable periods, a task which requires both specific facilities, expertise and information.

Seed Storage Physiology

Three main categories of seed storage behavior are currently recognized*: • ORTHODOX : Seeds that survive long-term dry storage • INTERMEDIATE : Seeds that can withstand dehydration to a certain extent but have reduced longevity • RECALCITRANT : Seeds that cannot withstand dehydration. *(Roberts EH, 1973, Predicting the storage life of seeds. Seed Sci.Tecnol. 1:499-514 Ellis, RH et al, 1990 An intermediate category of seed storage behavior? I. Coffee J. Exp. Bot. 41: 1167-1174.)

Orthodox Seeds Intermediate Seeds Recalcitrant Seeds

Undergo maturation

drying, shed in a relatively

dry condition (<20%mc)

Absence of maturation drying, shed at moisture contents >50%.

Absence of maturation drying, shed at moisture contents >50%.

Can be dried to low

moisture contents (<7%)

without losing viability

Survive drying to

moderately low moisture

contents (7-18%)

Continuously metabolically active, damaged on drying (below 20%) and are sensitive to desiccation and low temperatures

They can be stored for

many years at

ambient, low and ultra-low

temperatures

Store at sub-zero

temperatures.

Can be only stored at ambient temp. for short period. Cannot be stored at low temperature, only at LN temperatures.

Small seeded annual crops

eg. wheat, rice, maize,

legumes,

Eg., Neem, coffee, citrus,

papaya, star fruit

Large seeded, tree and

aquatic species, eg. tea,

mango, jackfruit, litchi,

jamun, cocoa, rambutan,

rubber as well as forest and

horticultural species

Determination of seed moisture content and viability

Seed Genebank - Procedures

• Seed samples are registered and added to the collection if they meet the minimum standards for germination, seed quantity and accompanying passport information.

Evaluation of seed viability and vigour

Seed Genebank - Procedures

• The operational sequence to integrate an accession into the genebank involves cleaning, moisture determination, drying, viability testing and packing.

Seed drying in chamber (15% RH & 15oC)

Hermatic sealing

Seed Genebank - Procedures The management of seed collections requires that germplasm accessions be maintained with a high proportion of viable seeds. This involves storage under optimal conditions, periodic monitoring of seeds for viability and quantity, and regenerating them when the situation warrants.

Retrieval and monitoring of seed germplasm from Genebank

Seed Genebank - Procedures • Germplasm regeneration

is done in post-rainy season to get quality seeds.

• Adequate number of plants are grown and sampled equally to minimize genetic drifts.

• During regeneration, the genetic integrity of cross-pollinating crops such as sorghum, pearl millet and pigeon pea is maintained by pollination control.

Bagging in pearl millet for controlled pollination

SEED BANK STANDARDS

• Untreated seeds

• High quality seed with maximum viability >85%

• Minimum 2,000 in self-pollinated and 4,000 in cross-pollinated crops

• Seed moisture content 3-8%

• Seed storage temperature -180C for long-term and +40C for medium-term

• Monitoring of viability- After 10 years in long-term and 5 year in medium-term storage

• Regeneration - 100 or more plants for regeneration to avoid the loss of alleles.

Candidates for In Vitro Conservation and Cryopreservation

Conservation of germplasm

• Vegetatively propagated species (root and

tubers, ornamental, fruit trees)

• Recalcitrant seed species (rubber, coconut,

coffee)

• Rare and endangered species

Conservation of tissue with specific

characteristics

• Medicinal and alcohol producing cell lines

• Genetically transformed tissues

• Transformation competent tissues (embryogenic

cell suspensions)

Techniques

Plant Tissue Culture Is the culture and maintenance of plant cells, tissues or organs (explants) in sterile, nutritionally (synthetic media) and environmentally (controlled) supportive conditions (in vitro)

Cryopreservation Is a process where cells or whole tissues are preserved by cooling to ultra-low temperatures (-150 °C to -196°C) usually using liquid nitrogen

IN V

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Definition

Crops

Conservation of germplasm under defined nutrient conditions in an artificial environment in form of in vitro cultures

Culture Forms

Culture systems may be in form of shoots, meristems, embryos, plantlets, callus or cell suspension

Type Conservation may be achieved through normal or slow growth (active collection) or suspended growth (base collection)

Vegetatively propagated crops – banana and plantains, yams, taro, sweet potato, cassava, potato ginger, turmeric, garlic, apple, pear, strawberry etc. Recalcitrant seeded species – oil palm, rubber, coffee, tea, jackfruit, almond etc. Rare/endangered species – medicinal plants Elite lines

In Vitro Conservation – How to Proceed?

Identify the authentic source of species from which suitable

propagules can be used for in vitro culture establishment

Test for viruses and bacteria Micropropagation

Slow growth conservation method

Germplasm distribution (for utilization)

Hardening and field transfer

Test for genetic

stability

Database

Management

Musa Culture Establishment

Explant

Suckers/shoot tip Male/female flower buds In vitro shoot culture

Suckers selected from a plant without symptoms of any disease

Suckers are trimmed to remove soil, roots and any other extraneous

material, leaving part of the central corm containing the meristem and about 10 cm above it. The overall

dimension of the block of tissue is about 20 cm high and 10-15 cm in

diameter

The material is sent to an appropriate tissue culture laboratory, if located in the same place as

field plants or

Blocks are air-dried and wrapped in newspaper and dispatched in a cardboard box

to the laboratory. No plastic should be used for wrapping

Protocol for establishing in vitro cultures of Musa for conservation

Shoot tips are excised, disinfected and cultured

Cultures are established in 4-12 weeks, depending on genotype,

often requiring repeated disinfection

Test for endogenous bacteria

Base of shoot explant

streaked on nutrient

agar medium,

observed after 1, 6

weeks

Cultures responding

positively discarded/

disinfected

Bacteria-free explant is

cloned

Tissue samples are taken from the three youngest expanded leaves

and indexed for viruses

If all tests are negative, the cultures derived from the

remaining subclones may be further propagated and

conserved in vitro

Test for viruses

ELISA

PCR Based techniques

Electron Microscopy

Culture establishment

1. Plant multiplication 2. Keeping it alive and

healthy 3. Making it available as and

when needed

Objectives of In

Vitro Conservation

• Genetic diversity requires diverse protocols

• Published protocols to be studied

• Optimization required – Explant type and conditions

– Containers

– Growth medium

– In Vitro conservation strategy

– Growth room/storage conditions

Determining Suitable Protocol and Conditions for Conservation

Advantages of In Vitro Genebanks

•Rapid multiplication

•Reduce space for storage

•Germplasm loss due to pest/pathogen and

climatic disorders can be avoided

•Facilitate exchange of germplasm

Management of large collections is problematic even if the subculture intervals are greatly extended – Genetic instability due to somaclonal variation

– Lack of effective conservation methods for all species

– Loss of regeneration potential of cultures

– Operational management difficulties

• Mislabelling

• Infection from due to mites and fungi

• Lack of skilled workers

Potential Solutions • Prioritization of collections to be maintained in vitro

• Identify and eliminate duplicates to reduce the collections to a manageable size

• Use of techniques which require minimal maintenance and space. Cryopreservation is the obvious choice.

Limitations of in vitro conservation

CRYOPRESERVATION OF PLANT GERMPLASM

Definition Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as −196 °C (the boiling point of liquid nitrogen). It can be employed for storage of germplasm under suspended growth for very long periods of time.

Advantages •No need of subculturing •Long-term conservation of germplasm •Maintains genetic stability •Storage of disease -free stock •Requires less space and energy input

What tissues can be cryopreserved ?

• Seeds and Pollen

• Zygotic embryos/embryonic

axes

• Winter buds

• Embryogenic cell suspensions

• Somatic embryos

• Meristem/shoot tip cultures

Steps for cryopreservation

Pretreatment (low temp., high sucrose, high cytokinin)

Desiccation/dehydration (high sucrose, air, DMSO)

Freezing/ Cooling (controlled/rapid)

Cryopreservation in LN

Rewarming (rapid/slow)

Post-treatment

Testing of viability and reculture

Techniques for Cryopreservation

Vitrification-Based Techniques

• Air Desiccation/ Dehydration

• Pregrowth

• Pregrowth-desiccation

• Encapsulation-dehydration

• Vitrification

• Encapsulation-vitrification

• Droplet freezing

Classical Slow Freezing

Techniques

• Chemical cryoprotectants + freeze induced dehydration

Factors important for seed cryopreservation

• Seed moisture content

– Most critical factor

– Optimum moisture content must be attained

– High or low create problems

• Developmental Stage

– Maturity level

– Desiccation tolerance

• Desiccation and Freezing Rates

– Fast vs Slow drying

– Fast vs Slow freezing

• Recovery Growth

DNA BANKS Three kind of collections:

1. Total genomic DNA

2. DNA libraries

3. Individual cloned DNA fragments (probes, satellites etc.)

Advantages:

• Convenient experimental material

• Easy to exchange and ready material for further manipulations

• Function as “time capsule”

Disadvantages:

• In vitro regeneration (only cloned fragment replicated to precision)

• Documentation

• Require high level of skills for exchange and use

• Problem of ownership and control of samples

• Total genomic samples are non-renewable

• Allow recovery of single gene not of genomes

Seed

Gen

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Cryo Genebank In Vitro Genebank

National Genebank Network

(NBPGR responsible for PGR activities at national level)

Long-term modules at-200C (12)

Medium-term modules at +4 to 80C

(6)

In Vitro Genebank at 25+20C (6 culture

rooms)

Cryogenebank at (-180 to1960C (6 cryotanks)

NBPGR, New Delhi

NBPGR, Regional Stations (10)

Medium-term modules at

+4 to 80C (18)

National Active Germplasm Sites(59 crop based ICAR

institutes)

Field Genebanks

Crop Group Accessions

Cereals 1,49,289

Millets and Forages 54,639

Pseudo cereals 6,656

Grain legumes 57,111

Oilseeds 55,798

Fiber Crops 11,512

Vegetables 24,196

Fruits 530

Medicinal and aromatic plants

6,390

Spices & Condiments 2,831

Agro-Forestry 2,433

Safety Duplicate 10,235

Cereals 39.12%

Millets and forages 14.32%

Pseudo cereals 1.74%

Grain legumes 14.96%

Oilseeds 14.62%

Fibre crops 3.02%

Vegetables 6.34%

Medicinal &

Aromatic Plants 1.67%

Safety duplicates

2.69%

Others 1.52%

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ts PGR Conservation : Seed Genebank

Accessions : 3,89,295

Species : 1,500

PGR Conservation : Cryogenebank

Category Accessions

Recalcitrant/Intermediate seed 5,969

Orthodox seed 3,182

Dormant buds 373

Pollen grains 345

Accessions : 9,869

Species : 729

Orthodox seeds

32.86%

Dormant buds 3.55%

Pollen grains 3.64%

Recalcitrant/ Intermediate seeds 59.95%

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PGR Conservation : In Vitro Genebank

Accessions : 2,075

Species : 132

Tropical fruits

20.25%

Temperate & minor

fruits 15.46%

Tuber crops

28.80%

Bulbous crops 8.45%

Medicinal & Aromatic

plants 8.35%

Spices 18.69%

Crop Group Accessions

Fruits (banana, strawberry, pear, apple, blackberry)

743

Tuber crops (sweet potato, yams, taro)

611

Bulbous crops (garlic, dahlia)

171

Medicinal & aromatic plants (Bacopa, Rauvolfia, Tylophora spp.)

170

Spices (ginger, turmeric, pepper, cardamom)

380

National Genebank at NBPGR

• Total 4,01,239 accessions conserved:

– 3,89,295 accessions as orthodox seeds (-200C) in

Seed Genebank

– 9,869 accessions as orthodox, recalcitrant seeds, pollen, dormant buds (-1960C) in Cryogenebank

– 2,075 accessions (35,000 in vitro cultures) of vegetatively propagated crops (250C) in In Vitro Genebank

– Large number of accessions of live plants in Field

Genebanks

• About 1,60,000 accessions are distinct on the basis of passport information

PGR Conserved at NBPGR Regional Stations

Akola Bhowali Cuttack Jodhpur Shillong

Germplasm Maintenance (Field Gene Bank)

84 1,724 884 750 445

MTS Holding 19,424 12,206 - 31,700 1,025

Hyderabad Srinagar Ranchi Shimla Thrissur

Germplasm Maintenance (Field Gene Bank)

45 39 1,550 1,153 1,817

MTS Holding 66,523 - 130 10,155 7,795

Thank you