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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 situEx situ Conservation of Seeds 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
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
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 techniquesƒ Number of plants„ Security of site… Replication/duplication† Adaptation‡ Size of the plotˆ Pests and diseases/natural calamities‰ IsolationŠ Ease of access
Conservation of Plants – FGB (contd.)
Conservation of Tissues/Cells
Difficult-to-conserve species Recalcitrant seeds Vegetatively propagated Large seeds
For some species the only option
Cryopreservation Rapid progress madeGreat potential for
conservation In vitro conservation: security, availability & cost
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
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
Conservation of Tissues/Cells (contd.)
Essential requirements• Capacity of plants to regenerate in vitro • Establish in the filed Genes for regeneration ability?
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
Conservation of Tissues/Cells (contd.)
CryopreservationCryopreservation
• Theoretically ideal• Quality preservation possible • Relatively new method• Costs involved require repeated
evaluation
Conservation of Tissues/Cells (contd.)
AdvantagesPhysical & genetic stability/ Relatively
economicalEase 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
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 SeedsSynthetic 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 genebanksIn 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
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 Tissues/Cells (contd.)
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 genesAdditional research is needed
Conservation of Pollen
DNA or Gene BanksDNA or Gene BanksProgress 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
DNA Storage
Botanical Gardens> 1500 BGs worldwideObjectives 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 practiceBiological 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
• 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
Next steps (Contd.)
Concluding Remarks
EX situ conservation forest biodiversity is feasible Is a component CCSMany countries in APO conserving Forest GD In situ approach - main current focus Need to exploit advantages of ex situ approachTogether they can help to conserve & maximum crop GDNeed research on seed storage behaviour Need research on cost-effective conservation as plants