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