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

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

Ex situ conservation Main justification  Semi-static method 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

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 500-1500 individuals or more The different numbers are based on population genetic considerations

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

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

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

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?

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

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

 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

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

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

 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

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

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

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

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

Thank you!