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Ex situ conservation of forest
genetic resources: Principles
and practices
Ramanatha Rao
Genetic resources
 Genotypes or
populations
 Cultivars
 Genetic stocks, &
 Related wild & weedy
species
Conservation
 Species level
 Genepool level or
 Ecosystem level
Ex situ conservation
 Semi-static method
Main justification
• Capturing current genetic
diversity/species diversity
• Readily available for use
and research
• Complements in situ
conservation
Why ex Situ Conservation?
• To meet the needs of plant
improvement
• Generate knowledge base
needed for many aspects of
FBD conservation & use
including in situ conservation
For ex situ conservation we need
– Exploration
– Collecting of diversity
– Introduction of FGR
 Assemblage of accessions
Ex Situ Conservation has to take into
account
Biological factors of the
species
• Breeding systems
• Production of seeds
• Nature of seeds
• Seed size and volume
Ex situ Conservation of Seeds
Orthodox seeds
Seeds be maintained under conditions
 in which the life processes minimized
• Low MC/Low temperature
Stored safely for a number of years
• Little loss in GD, genetic integrity and
viability
• Storage facilities extend viability of seeds
• Efficient & reproducible technique for
orthodox seed species
 Work progress on alternative
methods of storage
Additional methods for
seed storage
 Mainly for recalcitrant
seeds
 Imbibed storage
 Storage in LN2
 Ultradry seed storage
Managing the FGR genebanks
• Collecting
• Monitoring storage conditions
• Viability monitoring
• Regeneration and multiplication,
• Characterization and evaluation
• Documentation and information
• Distribution for use
Conservation of Plants
Field genebanks (FGB)/Arboreta/Ex Situ
stands
Some problems
 Damaged by natural calamities
 Infection
 Neglect or abuse
 Substantial no. of individual genotypes
 Require more space
 Relatively expensive to maintain
However
 Provide easy & ready access
Alternative methods are yet to be developed
Major role for perennial species
Conservation of Plants – FGB (contd.)
Seed collection
 At least 150 trees if their relationship is
unknown
•Seed should be collected from at least 25
randomly chosen and supposedly unrelated
individuals
•Aim for final stand size of 500-1500 individuals or
more
•The different numbers are based on population
genetic considerations
Conservation of Plants – FGB (contd.)
Important considerations
•
Establishment of seedlings
‚Sampling techniques
ƒNumber of plants
„Security of site
…
Replication/duplication
†Adaptation
‡Size of the plot
ˆPests and diseases/natural
calamities
‰
Isolation
ŠEase of access
Conservation of Tissues/Cells
Difficult-to-conserve species
 Recalcitrant seeds
 Vegetatively propagated
 Large seeds
 For some species the only option
Cryopreservation
 Rapid progress made
Great potential for conservation
In vitro conservation: security,
availability & cost
Conservation of Tissues/Cells (contd.)
For the long-term conservation of
shoot cultures
 Cryopreservation in LN2
becoming available
Other roles
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Distribution of germplasm
Collecting of samples from the
field
Need to keep culture growth to
 Minimum or arrested
Conservation of Tissues/Cells (contd.)
Ways to achieve slow growth
• Use of immature zygotic embryos (not
for vegetatively propagated species)
• Addition of inhibitors or retardants
• Manipulating storage temperature
• Mineral oil overlay
• Reduced oxygen tension
• Defoliation of shoots
Essential requirements
• Capacity of plants to regenerate in vitro
• Establish in the filed
 Genes for regeneration ability?
Conservation of Tissues/Cells (contd.)
Status
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Protocols for many species
Work in progress on many others
Work on somaclonal variation
Genetic uniformity of the material
Much research needed in
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Monitoring genetic stability &
Ways & means of conserving GD through in
vitro
Conservation of Tissues/Cells (contd.)
Cryopreservation
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Theoretically ideal
Quality preservation possible
Relatively new method
Costs involved require repeated evaluation
Conservation of Tissues/Cells (contd.)
 Advantages
Physical & genetic stability/ Relatively
economical
Ease of access to material
 Various stages in the process are:
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Selection
Excision of plant tissues or organs
Culture of source material
Select healthy cultures
Apply cryoprotectants
Pregrowth treatments
Cooling/freezing
Storage
Warming & thawing
Recovery growth
Viability testing
Post-thawing
Conservation of Tissues/Cells (contd.)
Vitrification
• Vitrification appears to be
promising
• Specially for differentiated
cultures
• Technically simple method
• Requires suitable cryoprotectants
• Rapid cooling rates
 More studies undertaken in many
labs
Conservation of Tissues/Cells (contd.)
Synthetic Seeds
Another promising method for
• Clonally propagated or
• Species with recalcitrant seed
Encapsulation of shoot-tips & somatic embryos
In semi-solid material
 ‘Beads'
With current pace of development
 Production & storage of artificial seeds may be a
routine practice soon
Conservation of Tissues/Cells (contd.)
In vitro genebanks
 Discussed only different
components
 Need align all the components
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Protocols for tissue culture
Successful regeneration
Transfer to soil
Genetic stability
Cryopreservation of cultured
material
• Vitrification or encapsulation
 For viable long term
conservation strategy
Conservation of Tissues/Cells (contd.)
Day to day management of in vitro
genebanks
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Receiving vegetative material from field
genebank/ex situ stands/collecting mission
Processing of the material for disease
indexing
Therapy and quarantine, if needed
Healthy and clean material for culturing
Cryopreserved base genebank (long term)
and/or
In vitro active genebank in slow growth
Conservation of Pollen
Mainly developed as a tool
For controlled pollination of
Asynchronous flowering genotypes
 Better utilisation of available PGR
 Fewer quarantine problems
 Pollen dried down to 4-5% MC (silica gel)
 Stored under vacuum
 Viable for
Cryopreservation
 Additional technique for GR
 Conservation of genes
Additional research is needed
DNA Storage
DNA or Gene Banks
Progress in biotechnology
Breaking down species barriers
Progress in this field has led
• DNA libraries
• Single useful genes
Suggestions to store in DNA libraries
 Total genomic information of
germplasm
Despite the problems
 DNA Storage - an additional option
for
• Improves the access
• May allow recovery of genes from
extinct taxa
Botanical Gardens
> 1500 BGs worldwide
Objectives
 Maintain ecological and life
support systems
 Preserve GD
 Sustainable use of species &
ecosystem
Play limited role in conservation
Greater role in
 PA
 Education
 Research
Good management + Planning 
May emphasise conservation of
some groups of species (like FGB)
Translating research results into practice
Biological research questions & practices
What provenances are needed to recover target tree
species?
 Determine both extent of local genetic adaptation &
potential risks of introducing foreign genotypes, including
founder effects, genetic swamping and inbreeding
depression
 Determine appropriate seed transfer zones in restoration
planning for native plant populations.
Where, how and when do we collect material for ex situ
conservation?
 Access to seed sources with known origins that are
properly characterized from a genetic point of view
 Adopt/Develop protocols for seed collecting, handling,
storage, germination & bulking to be applied
Next steps)
 Create/use existing institutional framework
 Assess & Locate diversity
 Understand value of GD
 Collect/assemble diversity
 Determine seed storage behaviour
 Develop appropriate strategy for ex situ
conservation
 Link problems with new opportunities
 Link FGR conservation with national biodiversity
conservation strategy to benefit from work in other
areas, including PGR conservation
Next steps (Contd.)
• National partners to spend some
time to work out above steps for
specific target genetic diversity
/species
• Discuss & develop process of ex situ
conservation for target genetic
diversity/species
• Identify resources
• Follow up as needed
Concluding Remarks
EX situ conservation forest biodiversity is feasible
 Is a component CCS
Many countries in APO conserving Forest GD
 In situ approach - main current focus
 Need to exploit advantages of ex situ approach
Together they can help to conserve & maximum
crop GD
Need research on seed storage behaviour
Need research on cost-effective conservation as
plants