2016 Genetic Testing Super WorkshopOverview of Testing

2016 Super Workshop

Mike Stahr

Food Value Chain

Seed Testing Aka Testing Seeds (1939)Neat YouTube video (discovered by John Ferrari)

Some things the same • Constant halving• Movable examination belts • Speed of testing (?)• Checking for “fruitful” seeds• …….

Some things not• Super magnification, very clear images• Electronic seed herbariums, nearly instant information• Cultivar purity by DNA• ……..

Why Test Seeds? To provide a basis for the sale of seeds To diagnose problems To determine:

how much of a bag of seeds is the correct species Mechanical Purity

if seeds are the desired variety or cultivar Cultivar Purity

if biotech (etc.) seeds are present Biotech Purity/AP Testing

The germinability of a seed lot Standard Germination

the overall vigor of a seed lot Vigor Testing

the health of a seed lot (fungi, bacteria, viruses, nematodes) Health

chemical aspects, such as protein, oil, starch, linolenic acid, …

amount of damage to surface of the seeds Fast Green, Bleach

number of seeds per pound Seed Count

moisture content Moisture

…….

Why Test Seeds?

Today’s agriculture depends on the efficient supply of quality seed at a reasonable price.

The potential of dry seeds to establish seedlings can’t be determined until the seed is germinated.

Dr. Miller McDonald – The Ohio State University

• Seeds that will cross state lines to be sold must meet requirements of the U.S. Federal Seed Act (FSA).

• Intrastate seed movement is regulated within that state with some oversight by USDA.

• The Federal Seed Act regulations are tied to the Association of Official Seed Analysts Rules for Testing Seeds.

•Testing must be done by an official lab (state, federal, some crop improvement) or at a private or seed company lab employing a Registered Seed Technologist (RST).

U.S. Seed System

U.S. Seed TagTesting for the label Germination Purity

Pure seed Inert matter Other Species

Not on the label Seed health Seed Vigor Biotech Trait Purity

(but bags are marked)

Liberty Link

Testing According to What …

• AOSA “Rules for Testing Seeds” • CFIA “Methods & Procedures” • ISTA

Association of Official Seed AnalystsCanadian Food Inspection AgencyInternational Seed Testing Association

Dates in Seed Testing

~1700 Seed production separated from crop production 1816 Standard germination test developed as a result of clover

seeds being adulterated with small stones 1869 1st seed lab in Tharandt, Germany by Frederick Nobbe

1876 1st U.S. seed lab in Connecticut by E.H. Jenkins 1891 1st samples tested at Iowa State by Dr. Pammel 1917 1st AOSA Rules for Testing Seeds published

1924 International Seed Testing Association founded 1939 Federal Seed Act becomes law ~2000 RGT accreditation starts

Rules for Seed Testing

Volume 4Seedling Evaluation

Volume 3Uniform Classification

Volume 1Principles & Procedures

Volume 3Uniform Blowing Procedure

ISTA Rules

AOSA Rules

Handbooks

SamplingEvaluationVigorTZFlowerCultivar PurityMoisturePurity (AOSA-coming)

Sampling

• Testing is only as good as how well the sample represents the seed lot.

• Seed testing begins with the sampling process.

* Volume 1, AOSA Rules for Testing Seeds

Fig 2.1. Page 2-4

• The flow scheme isn’t fixed.• Not indicating correct sampling

intensity nor size relationships between samples

• Flow will depend on test requested and lab set up.

2004 ISTA Handbook on Seed Sampling

Trait (incl. DNA lab)Mechanical Purity

Seed HealthSmall Seed Germination

Large Seed Germination

ISU Seed LabInternal Labs

Testing MethodsPurity: purity, noxious weed exam (other species – ISTA), seed

count, moisture, damage, insect/soil exam, varietal purity.

Viability: warm germ, tetrazolium (TZ).

Vigor: cold test, AA test, TZ, growth rate, EC, etc. .

Health: visual, blotter, plate, wash, ELISA, grow out, PCR.

Trait/LLP: herbicide bioassay, immunoassay, PCR, electrophoresis

Miscellaneous: fast green, hypochlorite soak, etc.

What are GMOs?

According to ISTA: Genetically modified organisms are organisms in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating or natural recombination.

It allows selected individual genes to be transferred from one organism into another.

Why Develop GM Crops?

Traditional breeding: long and difficult process. more than just the desired trait is transferred. limited to artificially crossing plants within the same,

or closely related, species.

Transgenic technology allows genes from a wide range of living sources to be inserted in a plant

Are GM Crops Bad?It is important to know what is hype and what is fact.

Only one type of Bt corn affected caterpillars. It is no longer on the market.

Monarch caterpillars feed on milkweed plants.

If pesticide is used instead of Bt to control corn borers, many types of insects would be killed.

Biotech Trait Purity vs Unintended Presence

Biotech Trait Purity Desire trait to be present at a certain level.

Tests must be quantitative (# seeds or seedlings).

Adventitious (or Unintended) Presence LLP Biotech (all or certain) traits not desired.

Qualitative testing may be sufficient.

Genetic Testing

• Biotech Trait Purity

• Adventitious Presence/ Low Level Presence

• Genetic Purity

Testing Methods

Bioassay

ImmunoassayELISA microwell plateLateral flow strips

Electrophoresis Starch, Isoelectric Focusing, etc.

PCRGel, Real-time

Basics Herbicide Bioassay

What is it? Exposing seeds or seedlings to a herbicide solution to determine tolerance to the herbicide(s) in question.

Methods: Spray (greenhouse or lab)Substrate ImbibitionSeed SoakCombination of methods

Herbicide Bioassay Non-tolerant (Susceptible) Seedlings(SCST Non-Tolerant Seedling Web Page)

SoybeansTolerant seedlings have the characteristics of normal seedlings which include sufficient roots (vigorous primary or vigorous secondary roots); hypocotyl not markedly short and thickened; no hypocotyl lesions into the central conducting tissue; 50% of healthy cotyledons present, and at least one primary leaf.

Round up® seedlingsImbibition and Seed Soak Method

Round up® seedlings Spray Method

STS® seedlingsSeed Soak Method

Non-Tolerant

Liberty Corn

GT Corn (RR)

Non-biotech

Herbicide Bioassay (Substrate Imbibition)

Tolerant Seedlings

Non-Tolerant Abnormal

TolerantNon-Tolerant

Basics - Immunoassay

ELISA Plates: Microwell plate is pre-coated with antibody of trait of interest. Protein is removed from seeds or leaves and transferred to

plate. Antigen binds to wells. After several steps of adding chemicals and washing material

from plate, color develops in wells. Plate is read visually or with spectrophotometer.

Enzyme Linked ImmunoSorbent Assay

Immunoassay Principle Immunoassay-ELISA

Basics- Immunoassay

Lateral flow strips: Seeds or leaves are ground, water or buffer added, and

material mixed. Liquid containing protein is transferred to reaction tubes. “Pre-coated” strip is placed in tube. After 3-5 minutes (typically), strip is removed from tube and

examined for development of lines.

Lateral Flow Strips Immunoassay –Lateral Flow Strips

Lateral Flow FormatQuickStixTM Strips

Lateral Flow FormatQuickStixTM Strips

Get required number of seeds.

Grind seeds Add water & mix.

Insert strip Results

Image courtesy of Agdia

Positive

Negative

What is Electrophoresis?

Migration of a charged particle through a medium (agarose, polyacrylamide, starch) under the influence of an electrical field.

Proteins and Nucleic Acid Separations Molecular Weight Electrical Charge Biological or Chemical Properties Combination of the above methods

IEF gel with 96 seeds (no parent seed)

Missing band

Genetic Purity – Protein Electrophoresis

Polymerase Chain Reaction

Fingerprint of variety tested Increases the amount of DNA being checked for

(if present, through use of primers). Expensive equipment, but test is very precise

Composition of DNA

Adenine always pairs with Thymine and Cytosine always pairs with Guanine across the two strands of the helix.

From Department of Energy’s Primer on Molecular Genetics

Three Major Steps in PCR(which are repeated for 30-40 cycles)

Denaturation (at 94o C): Paired strands separate (single strands now accessible to primers).

Annealing (at 54o C): Excess primers added, cooling allows double-strands to bind to primers.

Extension (at 72o C): Ideal working temperature for the polymerase. Primers without exact match get loose again, and don’t give an extension of the fragment.

PCR Animation

PCR Gel

From SCST Seed Technologist Training Manual

Real-Time PCR

Why Knowing Capabilities Matters

Sample Trait Added Immunoassay Bioassay*1 8 seeds NK603 positive 8 seeds +

2 none negative all negative

3 10 seeds GA21 negative 8 seeds +

4 8 seeds NK603 positive 4 seeds +

* Two reps of 100 seeds tested for bioassay

Testing vs Technology

Technology Areas: Herbicide Bioassay, Immunoassay,

Electrophoresis, PCR

Testing Areas: Genetic Purity Biotech Trait Purity Adventitious Presence/LLP

What is Appropriate for Genetic Purity Testing?

Protein Electrophoresis DNA (SNPs, RAPDs, etc.) ?

What is Appropriate for Biotech Trait Purity Testing?

Herbicide Bioassay ELISA (immunoassay) Lateral Flow Strips (immunoassay) PCR (not as often) ?

What is Appropriate for AP Testing?

ELISA (immunoassay) Lateral Flow Strips (immunoassay) PCR Herbicide Bioassay (less often) ?

Now what? Preparing for the CGT/RGT exams Qualifying for the exams Taking the exams Staying certified Moving SCST and Genetic Technology forward