• Overview of Biotech Traits • Specific Procedures, Demos, Hands-On • Trait Purity • Herbicide Bioassay • ELISA Tyler Tunning • Adventitious Presence (AP) Trait Testing Research Associate • Lateral Flow Strips Iowa State University Seed Testing Laboratory • PCR
Native • According to ISTA: Genetically modified › Traits naturally present in germplasm › Bred into other populations via crossing and organisms (GMOs) are organisms in which phenotypic selection › Examples include increased seed yield, cold the genetic material (DNA) has been altered tolerance, drought resistance (water optimization), in a way that does not occur naturally by some disease resistance, etc. Biotech mating or natural recombination. › Traits introduced into germplasm from a different organism › Inserted into crops using plant transformation • It allows selected individual genes to be methods, then crossing and trait detection › Examples include herbicide tolerance, insect transferred from one organism into another. tolerance, some disease resistance, transgenic yield improvement, etc.
Commercialized Corn and Soy Varieties Trade Name Event Name Proteins Corn Single traits, double/triple stacks Syngenta Agrisure™ CB/LL Bt11 (Cry1Ab + PAT) Syngenta Agrisure® GT/CB/LL Bt11+GA21 (Cry1Ab + PAT) + cp4EPSPS
Molecular stacks, breeding stacks Agrisure Duracade™ E-Z Bt11 X MIR162 X MIR604 X (Cry1Ab + PAT) X Vip3A X Refuge™ 5222 TC1507 X 5307 X GA21 mCry3A X (Cry1F + PAT) X Herbicide tolerance, insect tolerances Cry3A-Cry1Ab (eCry3.1Ab) X cp4EPSPS Biotech and plant novel Monsanto Roundup Ready® NK603 cp4EPSPS Corn 2 Monsanto Genuity™ VT Mon89034+NK603 (Cry1A.105 + Cry2Ab2) + Double PRO™ cp4EPSPS
Monsanto Genuity™ Mon88017+Mon89034+TC1507 (Cry1F + PAT) + Cry1Ab + SmartStax™ DowAgrosciences +DAS59122-7 (Cry34/35Ab1 + PAT) + SmartStax™ cp4EPSPS Soy Roundup Ready® Soybean GTS 40-3-2 cp4EPSPS
Monsanto Genuity™ Roundup MON89788 cp4EPSPS Ready2 Yield Bayer CropScience A2704-12 PAT LibertyLink® Soybean
http://www.isaaa.org/gmapproval database/default.asp • Particle bombardment Gene Gun • Uses a ‘gene gun’ to force DNA into plant • Can introduce multiple copies of gene • Simple/fast technology, high success rate • Agrobacterium Induced Transformation • Uses agrobacteria to introduce gene via infection • More likely to result in a single gene copy • Time-consuming • ZFN, TALEN, & Crispr-Cas based Transformation • Site specific genome editing, transgene insertions
http://www.gmo-safety.eu/glossary/776.biolistic-gun-particle.html
Agrobacterium Method: The desired gene sequence replaces part of the bacteria genome and when the bacteria “infects” the host plant’s cells, DNA is transferred and reproduced.
• Marker gene: allows for selection of plants or bacteria containing the transgene • Promoter: allows transgene to “express” in plant • Transgene: codes for the protein conferring herbicide tolerance, insect resistance, etc. • Terminator: stops the protein translation so that the transgene codes the correct protein sequence and structure
This material is derived from the curriculum Fields of Genes: Making Sense of Biotechnology in Agriculture, Ó1997 National 4-H Council. (National 4-H Council can be found on the Web at www.fourhcouncil.edu/). Image A-D from NIH.gov.
Determining % trait purity • Bioassay › Testing methods approved by trait provider • Test seeds/plants with chemical or insect to › Ensure seed lot meets minimum % required by trait provider determine level of tolerance › Common methods: bioassays, ELISAs, PCR • Immunoassay › Tests must be quantitative • Test seeds/plants for presence of trait protein Determining Adventitious Presence/Low- • ELISA micro-well plate Level Presence › Biotech (all or certain) traits not desired • PCR › Qualitative testing may be sufficient • Test seeds/plants for the presence of DNA › Common methods: qPCR, lateral-flow strips sequence for trait Must observe required detection levels and limits of testing methods
Spray Method: Seedlings are grown in a growth chamber or greenhouse. When plants reach a desired height or maturity they • Exposing seeds or seedlings to an are sprayed with an herbicide concentration similar to the field herbicide solution to determine rate. After 5-7 days, evaluate for symptoms of susceptibility. tolerance to the herbicide(s) in question.
• Methods: Spray (greenhouse or lab) Substrate Imbibition Seed Soak Combination of methods
Substrate Imbibition: Moisten substrate (typically towels, blotters, or crepe cellulose paper) with herbicide. Plant seeds and “cover”. After 5-7 days, evaluate for symptoms of susceptibility.
Tolerant
Susceptible Canadian Food Inspection Agency
Formula: Non-tolerant vs Tolerant
# of Normal Tolerant => Percent Tolerant Non-tolerant vs Abnormal # NT + # Susceptible
400-10(abn)-10(dead)-10(susc) = 370 = 97.4% Variation of symptoms in 370 + 10 380 different germplasm Rounding: 97.45% = 97.5%, not 98.0%
• Bioassay • Microwell plate is pre-coated with specific antibody of trait • Test seeds/plants with chemical or insect to of interest. determine level of tolerance • Protein is removed from seeds or leaves and transferred to • Immunoassay plate. Antigen binds to wells. • Test seeds/plants for presence of trait protein • After several steps of adding chemicals and washing • ELISA micro-well plate material from plate, color develops in wells. • PCR • Plate is read visually or with spectrophotometer. • Test seeds/plants for the presence of DNA sequence for trait
Enzyme-Linked ImmunoSorbent Assay
Immunoassay Principle
• Check + and – control wells. • Calculate % of seedlings containing the protein. • (85 positive/90 seedlings) x 100% = 94.4% Images courtesy of Envirologix
Determining % trait purity › Testing methods approved by trait provider › Ensure seed lot meets minimum % required by trait • Immunoassay provider • Test seeds/plants for presence of trait protein › Common methods: bioassays, ELISAs, PCR • Lateral flow strips › Tests must be quantitative
Determining Adventitious Presence/Low- • PCR Level Presence • Test seeds/plants for the presence of DNA › Biotech (all or certain) traits not desired sequence for trait, promoters or terminators › Qualitative testing may be sufficient › Common methods: qPCR, lateral-flow strips
Must observe required detection levels and limits of testing methods
Lateral Flow Format QuickStixTM Strips
• Seeds or leaves are ground, water or buffer added, and material mixed.
• Liquid containing protein is transferred to reaction tube.
• “Pre-coated” strip is placed in tube.
• After 3-5 minutes (typically), strip is removed from tube and examined for development of lines.
Slide courtesy of Envirologix Mass required Add water & Grind seeds. number of seeds. mix.
• Immunoassay • Test seeds/plants for presence of trait protein • Lateral flow strips
Insert strip Results • PCR • Negative Test seeds/plants for the presence of DNA sequence for trait, promoters or terminators
Positive
Image courtesy of Agdia
• Immunoassays detect the presence of biotech proteins. Bioassays detect tolerance to an herbicide or herbicides.
• PCR uses DNA to indicate the presence of biotech “traits” without the need for protein or phenotypic evaluation. It is a method of making multiple copies of a DNA sequence, involving repeated reactions with an enzyme (DNA polymerase)
http://www.molecularstation.com/pcr/
• All biotech traits (GMO, GEO) have promoter and terminator sequences added to the gene that allows it to be “expressed” in the plant.
• For AP PCR testing, we screen for these promoters and terminator sequences in addition to the specific biotech trait sequence. http://www.youtube.com/watch?v=2KoLnIwoZKU There are several types of PCR: • Prepare samples for DNA extraction o Carefully divide out seed pools and 1) Qualitative - detected or not detected (+/-) grind seed o Check tissue quality and that samples 2) Semi-Quantitative - estimate % GMO* in plate match records • Extract DNA • Run PCR through standard thermocycler 3) Quantitative – “actual” GMO content. or real time machine • Run agarose gel* • Evaluate results *Based on results from pools being plugged into statistical program such as SeedCalc. * For gel-based system
Sample to Pools to Subsamples
Count seed, determine sample size
Seed samples
Grinding Ground powder
Sub sample From SCST Seed Technologist Training Manual
39
# seeds tested % GM Estimates Matrix 3000 [A useful tool to help practitioners choose pool sizes that give the desired % GM seeds estimation resolution when using a qualitative assay] # of per pools pool 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 4.0024 25 26 27 28 29 30 1 3000 0 ---- 3.50 30 pools 2 1500 0 0.05 ----
3 1000 0 0.04 0.11 ---- 3.00 25 pools 4 750 0 0.04 0.09 0.18 ---- 20 pools 5 600 0 2.50 0.04 0.09 0.15 0.27 ---- d= number of deviant pools (0 to 30) 15 pools 6 500 0 0.04 0.08 0.14 0.22 0.36 ---- n= number of pools (1 to 30) 2.00 7 428 0 0.04 0.08 0.13 0.20 0.29 0.45 ---- 10 pools 8 375 0 0.04 0.08 0.13 0.18 0.26 0.37 0.55 ---- m= number of seeds per pool (rounded 1.50 9 333 0 0.04 0.08 0.12 0.18 0.24 0.33 0.45 0.66 ---- to integer) 1.00 10 300 0 0.04 0.07 0.12 0.17 0.23 0.30 0.40 0.54 0.76 ---- 11 272 0 0.04 0.07 0.12 0.17 0.22 0.29 0.37 0.48 0.62 0.88 ---- estimate GM % 0.50 12 250 0 0.03 0.07 0.12 0.16 0.22 0.28 0.35 0.44 0.55 0.71 0.99 ---- 13 230 0 0.03 0.07 0.11 0.16 0.21 0.27 0.34 0.41 0.51 0.64 0.81 1.11 ---- 0.00 14 214 0 0.03 0.07 0.11 0.16 0.21 0.26 0.32 0.40 0.48 0.58 0.72 0.91 1.23 ---- 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 15 200 0 0.03 0.07 0.11 0.15 0.20 0.26 0.31 0.38 0.46 0.55 0.66 0.80 1.00 1.34 ---- number of positive pools 16 187 0 0.03 0.07 0.11 0.15 0.20 0.25 0.31 0.37 0.44 0.52 0.62 0.74 0.89 1.11 1.47 ---- 17 176 0 0.03 0.07 0.11 0.15 0.20 0.25 0.30 0.36 0.43 0.50 0.59 0.69 0.82 0.98 1.21 1.60 ---- Enter the total number or 18 166 0 0.03 0.07 0.11 0.15 0.20 0.24 0.30 0.35 0.42 0.49 0.57 0.66 0.77 0.90 1.07 1.31 1.73 ---- seeds to be tested and then 19 157 0 0.03 0.07 0.11 0.15 0.19 0.24 0.29 0.35 0.41 0.47 0.55 0.63 0.73 0.85 0.99 1.17 1.42 1.86 ---- 20 150 0 0.03 0.07 0.11 0.15 0.19 0.24 0.29 0.34 0.40 0.46 0.53 0.61 0.70 0.80 0.92 1.07 1.26 1.52 1.98 ---- the % GM estimates are 21 142 0 0.03 0.07 0.11 0.15 0.19 0.24 0.29 0.34 0.39 0.45 0.52 0.59 0.68 0.77 0.88 1.01 1.16 1.36 1.64 2.12 ---- calculated in the matrix for all 22 136 0 0.03 0.07 0.11 0.15 0.19 0.23 0.28 0.33 0.39 0.44 0.51 0.58 0.66 0.74 0.84 0.95 1.08 1.25 1.45 1.75 2.25 ---- possible combinations of d, n 23 130 0 0.03 0.07 0.11 0.15 0.19 0.23 0.28 0.33 0.38 0.44 0.50 0.57 0.64 0.72 0.81 0.91 1.03 1.17 1.34 1.55 1.86 2.38 ---- and m. Note: This matrix of 24 125 0 0.03 0.07 0.11 0.15 0.19 0.23 0.28 0.32 0.38 0.43 0.49 0.55 0.62 0.70 0.78 0.88 0.98 1.10 1.25 1.42 1.65 1.97 2.51 ---- Samples are compared to known 25 120 0 0.03 0.07 0.11 0.15 0.19 0.23 0.27 0.32 0.37 0.42 0.48 0.54 0.61 0.68 0.76 0.85 0.95 1.06 1.18 1.33 1.52 1.75 2.08 2.65 ---- estimates is appropriate for 26 115 0 0.03 0.07 0.11 0.15 0.19 0.23 0.27 0.32 0.37 0.42 0.48 0.54 0.60 0.67 0.75 0.83 0.92 1.02 1.13 1.27 1.42 1.61 1.86 2.21 2.79 ---- routine tests. This matrix is a 27 111 0 0.03 0.07 0.11 0.14 0.18 0.23 0.27 0.32 0.36 0.42 0.47 0.53 0.59 0.66 0.73 0.81 0.89 0.98 1.09 1.21 1.35 1.51 1.71 1.96 2.32 2.93 ---- good approximation of standards to determine the quantity of 28 107 0 0.03 0.07 0.11 0.14 0.18 0.23 0.27 0.31 0.36 0.41 0.47 0.52 0.58 0.65 0.71 0.79 0.87 0.96 1.06 1.16 1.29 1.43 1.60 1.80 2.07 2.44 3.07 ---- proficency test sample 29 103 0 0.03 0.07 0.11 0.14 0.18 0.22 0.27 0.31 0.36 0.41 0.46 0.52 0.58 0.64 0.70 0.78 0.85 0.94 1.03 1.13 1.24 1.37 1.52 1.69 1.90 2.18 2.56 3.22 ---- estimates when the proficency 30 100 0 0.03 0.07 0.11 0.14 0.18 0.22 0.27 0.31 0.36 0.40 0.46 0.51 0.57 0.63 0.69 0.76 0.83 0.91 1.00 1.09 1.20 1.31 1.44 1.60 1.78 1.99 2.28 2.67 3.34 ---- GMO in a sample. (0%, 0.1%, 1.0%, 10%) n m 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 test samples are large (e.g., 3000 kernels). Tyler Tunning [email protected]
Determine abnormal seedlings, deads, hard Herbicide bioassays for two crops: › Corn RR seeds using same rules as for germination › Soybean RR tests Each tray has 200 seeds planted of each Determine non-tolerant seedlings using crop spiked with non-tolerant seed. controls and symptoms described Controls are on each edge (positive and Use lateral flow strips to confirm tolerance or negative). Use these to help determine non-tolerance of questionable seedlings symptoms of non-tolerance. Calculate % tolerance
Normal Corn Abnormal Corn Seedling Seedling Shoot › Missing Slight Damage Vigorous roots › No leaf › primary and Root seminal › None Leaf greater than › Weak, stubby or half the coleoptile missing primary root Leaf not badly with weak seminal roots. shredded
› Primary and seminal
Normal (leaf curled due to test conditions) Abnormal
Normal Soybean Seedling Abnormal Soybean Seedling Roots Root › None › Vigorous primary or two › Weak, stubby, or missing primary root with weak secondary or adventitious roots or more secondary Hypocotyl roots › Deep, open cracks extending into the conducting tissue › Malformed, such as markedly shortened, curled or thickened Hypocotyl Cotyledons › Not markedly › Less than half of the original cotyledon tissue remaining attached shortened or thickened › Less than half of the original cotyledon tissue free of necrosis or decay Epicotyl Cotyledons › Missing › Less than one primary leaf › One or two halves › Deep, open cracks (>50%) › Terminal bud damaged, missing or decayed Epicotyl Seedling › One or more essential structures impaired as a result of decay from primary infection › At least one primary › Albino leaf; terminal bud present
Normal
Phomopsis – ? dead seed Damaged point of attachment
Normal Negative Decayed geotrophism cotyledons –
A. flavus Insufficent roots + epicotyl showing Descriptions and drawings are from • Volume 4 of the AOSA Rules for Testing Normal Abnormal Seeds - Seedling Evaluation (corn and soybean) Normal • Seedling Evaluation, ISU Seed Laboratory (soybean)
Susceptible Note: Not all of the drawings in the corn and soybean section of Volume 4 have been used in this presentation.