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United States Department of Agriculture

Agricultural Research Service

Virtual Tour of NCGRP
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NCGRP’s state-of-art facilities combine an unusual mix of laboratories designed to process diverse germplasm for storage, measure germplasm quality and viability and evaluate genetic diversity of collections.
 
Facilities, procedures and instrumentation vary according to each CRIS program’s major focus. 

   World map indicating where our accessions come from.

Virtual Tour - Plant Genetic Resources Preservation Program
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Come and see how we handle and store the valuable materials when they arrive. Take a look inside the vaults to see both conventional and liquid nitrogen (cryogenic) storage.

 Seed and Plant Material Donors/Sources

NCGRP receives seed and plant materials from all over the United States and the world.  NCGRP stores the base collection of the National Plant Germplasm System. NCGRP also works with international organizations such as the International Maize and Wheat Improvement Center (CIMMYT - Mexico), the International Rice Research Institute (IRRI - Philippines) and the International Plant Genetic Resources Institute (Bioversity International - Rome), as well as the Centers for Plant Conservation (CPC), the Crop Science Society of America for Crop Science Registration (CSR), and the Plant Variety Protection Office (PVPO).

Quarantine Seed Quarantine is a government program to monitor and control the importation of plant and animal materials. Samples that arrive at NCGRP under quarantine restrictions are handled in the quarantine laboratory. Samples are inspected and prepared for storage in a laminar flow biological safety cabinet. Material being prepared for increase is surface sterilized and treated with a fungicide prior to shipment. All germplasm is increased and inspected for disease and/or insect infestations by APHIS inspectors on the island of St. Croix (a substation for the Tropical Agriculture Research Station in Mayagüez) or other approved sites prior to release into the National Plant Germplasm System.

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Seed Quality Evaluation Laboratory

Seed Receiving

Upon receipt of seed samples, information is verified with GRIN, NCGRP serial numbers are assigned, germination cards and bar code labels are printed. After unpacking, samples are placed in an equilibration room with 23% relative humidity at 5° C. Samples are equilibrated for a few weeks to achieve the optimum moisture content for storage.   Moisture tests are conducted to verify that appropriate seed moisture contents have been reached.

 seed equilibration room moisture oven

Cleaning

After optimal moisture content is achieved, seed analysts remove inert material, empty seeds and contaminating seeds (i.e. weed seeds). Seed blowers such as this one remove all light material and chaff, leaving only the heavy seed. Seed may also be cleaned through the use of sieves and/or rubbing boards.

 

screens and blower

Viability Assessment

Viability assessment is a means of testing a sample to determine how many seeds are alive and may produce a normal plant.  Initial tests are conducted to determine viability before samples are stored. Analysts then may assess viability using germination and tetrazolium tests.  Subsequently, samples are tested periodically to monitor seed viability during storage.

seedlings on paper towels

Amaranthus seedlings on blotter paper

germination cart

Analysts select appropriate conditions and plant a subsample of the pure seed on standard germination blotters or towels. The number of seeds tested is dependent on the size of the sample. All samples scheduled for cryogenic storage in liquid nitrogen have paired tests performed to check for damage caused by the liquid nitrogen (LN2).

small liquid nitrogen container

amaranthus seedlings     The LN2 test exposes some of the seed for 24 hours to the vapor phase of LN2 (approximately -160° C, or -320° F). Seedling evaluation of the germinated seeds follows the Association of Official Seed Analysts' Seedling Evaluation Handbook. Abnormalities and pathogen problems are assessed. Moisture content, viability, cleanout, and seed counts are tracked and results uploaded to the Germplasm Resource Information Network (GRIN).


Storage

NCGRP stores samples in conventional storage at -18° C and cryogenic storage using liquid nitrogen at -196° C (-160° C in the vapor phase). Storage method is determined by the type of seed, the size of the seed, the number of seeds in the sample, the viability of the sample, and sometimes the instructions from the donor.

Samples stored by conventional methods are transferred to heat sealable, moisture-proof, foil laminated bags. Locations are assigned and barcodes for the location and serial numbers are placed inside and on the outside of the bags. The bags are then heat sealed and placed in trays in the cold storage vault.

Samples stored by cryogenic methods are transferred to clear polyolefin tubes, barcoded, and crimped closed. Filled tubes are then placed in metal boxes, labeled with serial numbers and locations, and stored in the LN2 tanks.

heat sealing bags

Vaults

The  vault area at NCGRP is a separately constructed portion of the building, self-contained with its own air handling unit and electrical generator in case of emergency.  It is able to withstand possible floods from nearby Horsetooth Reservoir, tornadoes, and the dropping of a 2500 pound object traveling at 125 miles per hour. The vaults have the capacity to store 1.5 million samples, 50% in conventional storage, and 50% in liquid nitrogen.

 -18 C cold vault

Conventional Vault Area

The individual storage units have walls with six inches of insulation and are metal covered. Temperature is kept at -18 C. This vault is 5000 square feet. The samples are stored in plastic trays on shelves which are part of a moveable rack system, allowing a 60% greater room capacity over the old multiple aisle system. As each carriage is moved, the light from the previous aisle is turned off and the light above the newly opened aisle is turned on. The life expectancy of a seed sample stored in this vault is from 20 to 50 years.  Because of the variability of seed longevity, periodic retesting is done to identify regeneration needs.

Cryogenic Vault Area

Two floors of the vault area are designed for cryogenic storage. One is in current use, and the other is designed for expansion. Each floor can hold 112 storage tanks, and has double-walled stainless steel piping for filling from the 13,000 gallon tanks located outside the building.

Cryotanks are like giant thermos jugs with vacuum jacketed double walled insulation.  Each tank will hold 330 metal containers (metal boxes containing tubes of  seed samples) on a lazy susan which holds the samples above the liquid nitrogen and allows easy access to each section. A tank holding wheat could contain  2640 samples, whereas a tank of tobacco (a very tiny seed) would contain 26,400 samples. We try to have a minimum of 3000 seeds per sample for cross pollinated crops and 1500 seeds for self-pollinated crops.   

liquid nitrogen tank

Tanks are filled once a week, with about 56 liters of liquid nitrogen.     
Air circulation is very important in the vault since nitrogen gas is constantly boiling from the tanks. Oxygen monitors are placed around the vault for detection of oxygen content of the air. Emergency fans are triggered by the oxygen monitors or emergency buttons. During an emergency, liquid nitrogen is shut off and fans provide 14 air exchanges per hour.

All of these elaborate precautions are to ensure that our valuable germplasm lives as long as possible, and is available for plant breeders and researchers to produce the food and fiber products we all depend upon.

Tissue culture and Dormant Buds

 

Plants that are propagated clonally are preserved as meristematic shoots or dormant  winter buds. The meristematic shoots are excised from tissue cultures, under a dissecting microscope and cryopreserved according to a procedure that is genus or species specific. When needed, the shoots are regenerated to whole plants in culture vessels, and planted in the field.

Preservation of dormant winter buds also follows an established protocol but after storage in liquid nitrogen, the buds are grafted to a rootstock or in rare exceptions, the segments with the cryopreserved dormant buds are rooted (e.g. willow) and whole plants are established. Examples of crops that are cryopreserved as vegetative propagules are apples, banana, blueberries, fruit and nut trees, garlic, potato, strawberries, sweet potato and willow. (vegetatively-propagated germplasm)  

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Virtual Tour - National Animal Germplasm Program
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Samples are collected from animals from all over the U.S.

Shorthorn cows in Nebraska.  Shorthorn cows in Nebraska.

 Navajo women with a churro sheep.  Navajo Churro sheep in Arizona.

Upon arrival, sample viability is evaluated using computer assisted sperm analysis (CASA).  Evaluating sample viability in the laboratory.

Semen samples are cooled to 5°C and cryoprotectants are added.  Samples are then packaged with an automated straw filler and labeler.

 Filled, sealed, and labeled semen straws. Example of filled, sealed and labeled straws ready to be cryopreserved.

 Straws in a programmed cooling apparatus.

Straws are cooled to -140°C using a programmed cooling apparatus, and then they are plunged into LN2.

 

Once samples have received proper cooling treatments, they are placed in cryovats for long term storage. >

 Straws being placed in cryovats.

 Animal-GRIN database form. Information about sample provenance and semen storage location is entered into the Animal-GRIN database.

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Virtual Tour - Plant Germplasm Preservation Research
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The six laboratories of the PGPR are divided by discipline.

The Biochemistry lab houses analytical instrumentation to measure metabolic capacity of cells and cellular constituents such as small molecular weight carbohydrates, DNA and protein concentrations, lipid composition, and volatile compounds produced during storage.

Gas chromatography instrumentation measures volatiles produced during seed storage.

 Gas is sampled from the headspace in vials.Seeds stored in vials produce volatiles that can be identified using gas chromatography.

Trace showing volatile production.

 

The Genomics/Population Genetics lab houses DNA extraction and sequencing instrumentation required for high throughput genotyping.

 Undergraduate student pouring a gel.  Sequencing gel is loaded.  Sequence information differentiates samples.

 Differential scanning calorimetry instrumentation.

Abronia ammophila fruits that sometimes do not contain seeds.

The Seed Quality/Biophysics lab is equipped with instrumentation to evaluate development and viability of seeds from diverse species and measure thermal and mechanical  properties of seeds and cellular constituents as development progresses.

Crimson clover seeds stored for 40 years at 4, 7 and 10% water as part of the Louis Bass sealed can experiment. Seeds in the most humid treatment are darkest because of the aging reactions that occurred.

 Extraction of embryos from recalcitrant seeds.

Microscopy and Imaging is equipped with instrumentation to evaluate viability and how cell structures change during cryopreservation treatments.

 Cross section of recovering shoot tip.

Viability staining in pecan pollen.

Fluorescein staining staining for viability in suspension cells.

Cuphea embryos successfully preserved.

 Collection of imaging data.

The Gene Expression lab uses molecular tools to identify changes in gene product levels during acclimation to cold and cryoprotective treatments and upon recovery from storage.

Preparation of samples for PCR.Gel electrophoresis equipment.

 Sample extraction.

Cryobiology and Tissue Culture is structured as a suite of laboratory space, cell transfer, autoclave and growth rooms that provide ideal workspace for plant microculture and cryoprotection treatments.

Ancillary space provides room for incubators, programmable freezers, cryogenic facilities, growth chambers and other instrumentation required to understand structural, biochemical, and genetic traits of cells that affect response to genebanking.

 Tissue culture growth facility.  Fast cooling procedures using sub-cooled liquid nitrogen slush.  Slow cooling apparatus for dormant buds.

 Cryopreserved dormant apple bud succussfully grafted onto rootstock.

Grafting of dormant buds.

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Last Modified: 5/3/2013
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