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

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

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

3. Ex situ conservation  Semi-static method Main justification • 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. 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

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

17. 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. 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. 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. 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. 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. 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!