r.k. tyagi and anuradha agrawal - nbpgr · seed vault in vitro repository . seed propagated...
TRANSCRIPT
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
ITR
O C
ON
SE
RV
AT
ION
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
eban
k
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