Seed Genebank Conservation

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Seed Genebank Conservation

• Susan Stieve
• HCS 830
• October 28, 2003

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Seed Genebank Conservation

• Definitions
• Management of Collections
• Acquisition
• Characterization and evaluation
• Regeneration
• Storage
• Viability monitoring
• Data management
• Exchange
• The Cost of Conservation

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Definitions

• Germplasm-all the genes making up the hereditary
  information of a plant or collection of plants
• Accession-group of related plants
• Ex situ-maintenance of germplasm away from
  original location
• The most convenient and widely used method of
  conservation
• Seeds adapted for storage, compact, survive
  naturally in soil
• Tubers, tissue explants, pollen, DNA
• Base collections- long-term (NCGRP, Ft. Collins,
  CO)
• Active collections- medium-term working
  collection
• Used for distribution, regeneration (OPGC)

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Management of Collections-Acquisition

• Sources
• Exchange
• Other genebanks
• Botanic gardens
• Plant societies
• Private collectors
• Purchase
• Collecting expeditions

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Management of Collections-Acquisition

• Collecting expeditions
• Knowledge and preparation are critical
• The amount of genetic variation within and
  between populations
• Breeding system (in, out-breeding)
• Taxonomy
• Ecogeographic distribution
• These data may not be available

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Management of Collections-Acquisition

• Collection sites-centers of origin, diversity
• Field sampling considerations
• Distribution of sites within target area
• Number of sites sampled
• Delineation of a site
• Distribution of plants sampled at a site
• Random interval along a transect
• Selection for visible characteristics
• Number of plants sampled per site
• Number and type of sample per plant
• Documentation critical

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Management of Collections-Acquisition

• Theoretically, collect at least one sample of
  each different allele in a population
• In practical terms, aim to sample the number of
  plants that will contain 95 of the alleles at a
  random locus occurring in the target population
  with a frequency greater than 0.05 (Marshall and
  Brown, 1975)
• Typical goal bulk a random sample of 50 plants
  per population, 50 seeds per plant

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Management of Collections-Acquisition

• Other germplasm sources when wild material is not
  available at center of origin
• Farmers fields
• Farmers stores
• Kitchen gardens
• Markets
• Local researchers

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Management of Collections-Characterization and
Evaluation

• Descriptors are used to score morphological and
  cultural characters
• Many collections poorly evaluated and
  consequently under-utilized (Frankel and Brown,
  1984)
• Development of core collection concept
• Include variants, reduce redundancy

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Management of Collections-Characterization and
Evaluation

• Identification of duplicates, collection gaps
• Estimate as few as 35 of accessions in world
  collections are actually distinct (Plucknett et
  al., 1986)
• Facilitate preliminary selection of germplasm by
  end-users
• Insect and disease resistance
• Tolerance to environmental stresses
• Genetic diversity studies, taxonomic
  relationships through molecular studies

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Management of Collections-Data Management

• USDA/NPGS uses Germplasm Resources Information
  Network (GRIN) to record all information
• Inventory
• Origin, collector
• Quantity, viability
• Characterization, evaluation data
• Order processing for distribution
• Generate reports
• Facilitates selection based on criteria
• Select information made available to public
• www.ars-grin.gov

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Management of Collections-Regeneration

• Growing out seed to supply fresh seed for further
  storage
• A costly operation!
• Required when
• Viability falls below set point
• Crop dependent, based on initial viability,
  75-90
• Quantity falls below set point through
  distribution
• For example, database notification when less than
  1000 seeds
• Care must be taken to preserve original genetic
  attributes

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Management of Collections-Regeneration

• Regeneration tactics are a balance and vary
  according to
• Crop species, reproductive system (in,
  out-breeding)
• Available resources including labor, space, time
• The most efficient and cost effective way of
  maintaining genetic integrity is to keep the
  frequency of regeneration to an absolute minimum

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Management of Collections-Regeneration

• Hazards to avoid
• Human error
• Contamination from foreign pollen during
  fertilization
• Contamination through seed adulteration during
  harvesting, cleaning and packaging
• Changes due to gene mutation
• Genetic drift due to random loss of alleles,
  especially when regenerating from small numbers
  of individuals
• Bottlenecking
• Genetic shift due to unconscious natural or
  artificial selection

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Management of Collections-Regeneration

• Steps
• Identification of accessions
• Germination of seeds
• Optimal number of parental plants
• 30-100 (ICRISAT 1995)
• 100 or more (FAO/IPGRI 1994)
• 150-220 (Crossa et al. 1993)
• Plant culture to flowering
• Perennials may take several years

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Management of Collections-Regeneration

• Steps, continued
• Seed production
• May require pollinator, isolation cage
• Honey bee nucleus hive 50, unhappy confined
• Bumblebee domicile 100, okay in confinement
• Others-Blue Bottle Fly, Alfalfa Leaf Cutter,
  Osmia
• Harvesting-how much seed is enough?
• Inbreeders 1,500-6,000
• Outbreeders 4,000-50,000
• Cleaning to eliminate poor quality, off-types
• Minimize physical damage, loss of good seeds

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Management of Collections-Regeneration

• Steps, continued
• Final drying
• Necessary if storing in air-tight container
• One week at 15 RH, 15o C
• Viability testing
• Packaging
• Storage
• Working collection for distribution
• Base collection for long-term storage

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Management of Collections-Storage

• Seed Classification
• Protocol for determination-Hong and Ellis, 1996
• Orthodox
• Can be stored longer cool, dry (5 MC or less)
• Generally can be frozen if 15 moisture content
  or less
• Cereals, floriculture crops
• Small seed
• Most common
• Recalcitrant
• Lose viability if moisture content less than
  12-13
• Many tropical and temperate tree species
• Large seeds
• Least common
• Store in vitro, in field gene banks,
  cryopreservation, etc.
• Intermediate

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Management of Collections-Storage

• Orthodox Seeds
• Storage potential influenced by inherent and
  external factors
• Leguminosae-typically genetically hard seeded,
  long-lived
• High oil seeds typically short-lived
• Variation at cultivar level
• Difficult to account for seed maturity
• Seed age may vary by days, weeks
• Standardization of collecting an issue
• Fully mature seeds generally store better than
  immature
• Maternal (and paternal) plant environment can
  affect storability
• temperature, moisture, nutrition, light

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Management of Collections-Storage

• Orthodox Seeds
• Harringtons Rule of Thumb
• Longevity is doubled for every
• 5o C decrease in temperature (above freezing)
• 1 decrease in moisture content
• Less than 5 MC may cause damage
• Typical Storage dry, cold
• Refrigerator- medium-term storage
• oF RH ? 100 (or 75)
• At OPGC 40o F 30 RH 70
• Freezer- long-term storage
• Dry to 5-6 MC, place in sealed container, store
  below 18 oC
• Extreme storage-
• Less than 5 MC
• Liquid nitrogen (196 oC)

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Management of Collections-Storage

• Minimum standard
• Seeds should be stored under conditions which
  ensure the accessions viability remains above at
  least 65 for 10-20 years (FAO/IPGRI, 1994)

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Management of Collections-Storage

• Storage of vegetative material
• Bulbs-periods of dormancy, varying environmental
  conditions for flower initiation
• In vitro-storage of plants in tissue culture
• Regeneration from undifferentiated cells can
  result in somaclonal variation, therefore use
  large explants and avoid callus formation
• Normal versus minimal growth
• Cryopreservation in liquid nitrogen (196 oC)
• Glycerol, DMSO, and other cryoprotectants used to
  prevent membrane damage
• Meristems, DNA, pollen
• Cryopreservability of pollen not related to seed
  type
• Field genebanks
• Orchards
• Botanic gardens and arboreta

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Management of Collections-Storage

• Genetic change can occur during storage
• Chromosome damage is a general result of the
  ageing phenomenon in seeds, result of loss of
  ability to undergo normal DNA repair
• Increasing number mutant phenotypes from old
  seeds stored in improper conditions
• Loss of viability closely linked to genetic
  damage (Roberts et al., 1967)
• More frequent regeneration presents problems in
  terms of maintenance of genes within collections
  these may outweigh those imposed by accumulation
  of mutations

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Management of Collections-Viability

• Viability Monitoring of Orthodox Seeds
• Even under ideal storage conditions seed
  viability will decrease over time
• Stored seed should be monitored with regular
  germination testing
• Estimating the loss of viability theoretically
  possible but complex (Hong and Ellis, 1996)

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Management of CollectionsExchange

• USDA/NPGS distributes free to researchers around
  the world
• www.ars-grin.gov
• Import permits
• Phytosanitary inspections
• Shipping costs

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The Cost of Conservation

• Estimates vary, an expensive activity!
• Collecting one accession from a different country
  597
• Incorporating one accession into the genebank
  (germination testing, cleaning, packaging,
  verification) 273
• Annual curation costs per sample 5
• Distribution on request from user 15
• (Smith and Linington, 1997)

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The Cost of Conservation

• 1.50-12.00 per year, depending on location and
  plant part being conserved
• (IPGRI Newsletter for the Americas, July 2003)
• International Maize and Wheat Improvement Center,
  Mexico (CIMMYT)
• Corn
• 0.93 to conserve existing accession for one year
• 30.24 to store an accession for 40 years
  (genebank life)
• 58.85 to store in perpetuity
• Wheat
• 0.19 to conserve existing accession for one year
• 7.19 to store an accession for 40 years
• 10.26 to store in perpetuity

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Literature Cited

• FAO/ IPGRI. 1994. Code of conduct for plant
  germplasm collecting and transfer. FAO, Rome
• Frankel, O. H. and A. H. D. Brown. 1984.
  Current plant genetic resources-a critical
  appraisal. In Genetics New Frontiers Vol.
  IV, New Delhi, Oxford and IBH Publishing.
• Hawkes, J .G. N. Maxted, and B. V. Ford-lloyd
  (eds). 2000. The ex situ conservation of plant
  genetic resources. Kluwer, Boston
• Hong, T. D. and R. H. Ellis. 1996. A protocol
  to determine seed storage behaviour. IPGRI
  Technical Bulletin, IPGRI, Rome
• Plucknett, D. L., N. J. H. Smith, J. T.
  Williams, N. M. Anishetty. 1986. Gene banks and
  the worlds food. Princeton University Press,
  New Jersey.
• Sackville Hamilton, N. R. and K. H. Chorlton.
  1997. Handbook for Genebanks No. 5, Regeneration
  of accessions in seed collections a decision
  guide. IPGRI, Rome.
• Smith, R.D. and S. Linington. 1997. The
  management of the Kew Seed Bank for the
  conservation of arid land and U.K. Wild species.
  Bocconea, 273-280.