1. Ex situ conservation of forest genetic resources: Principles and practices Ramanatha Rao

2. Genetic resources Conservation Genotypes or populations Cultivars
Genetic stocks, &
Related wild & weedy species
Conservation
 Species level
 Genepool level or
 Ecosystem level

3. Ex situ conservation Main justification  Semi-static method
Capturing current genetic diversity/species diversity
Readily available for use and research
Complements in situ conservation

4. 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

5. 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

6. 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

7. Additional methods for seed storage
 Mainly for recalcitrant seeds
Imbibed storage
Storage in LN2
Ultradry seed storage

8. Managing the FGR genebanks
Collecting
Monitoring storage conditions
Viability monitoring
Regeneration and multiplication,
Characterization and evaluation
Documentation and information
Distribution for use

9. 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

10. Seed collection  At least 150 trees if their relationship is unknown
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 individuals or more
The different numbers are based on population genetic considerations

11. Important considerations
Conservation of Plants – FGB (contd.)
Important considerations
 Establishment of seedlings
‚ Sampling techniques
ƒ Number of plants
„ Security of site
… Replication/duplication
† Adaptation
‡ Size of the plot
ˆ Pests and diseases/natural calamities
‰ Isolation
Š Ease of access

12. 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

13. For the long‑term conservation of shoot cultures
Conservation of Tissues/Cells (contd.)
For the long‑term conservation of shoot cultures
 Cryopreservation in LN2 becoming available
Other roles
Distribution of germplasm
Collecting of samples from the field
Need to keep culture growth to
 Minimum or arrested

14. Ways to achieve slow growth
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?

15. Much research needed in
Conservation of Tissues/Cells (contd.)
Status
Protocols for many species
Work in progress on many others
Work on somaclonal variation
Genetic uniformity of the material
Much research needed in
Monitoring genetic stability &
Ways & means of conserving GD through in vitro

16. Quality preservation possible Relatively new method
Conservation of Tissues/Cells (contd.)
Cryopreservation
Theoretically ideal
Quality preservation possible
Relatively new method
Costs involved require repeated evaluation

17.  Various stages in the process are:
Conservation of Tissues/Cells (contd.)
 Advantages
Physical & genetic stability/ Relatively economical
Ease of access to material
 Various stages in the process are:
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

18. Vitrification Vitrification appears to be promising
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

19. Another promising method for Clonally propagated or
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

20.  Discussed only different components  Need align all the components
Conservation of Tissues/Cells (contd.)
In vitro genebanks
 Discussed only different components
 Need align all the components
Protocols for tissue culture
Successful regeneration
Transfer to soil
Genetic stability
Cryopreservation of cultured material
Vitrification or encapsulation
 For viable long term conservation strategy

21. Day to day management of in vitro genebanks
Conservation of Tissues/Cells (contd.)
Day to day management of in vitro genebanks
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

22. 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

23. 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

24. 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)

25. 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

26. Create/use existing institutional framework Assess & Locate diversity
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

27. 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

28. 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

29. Thank you!