Substrate Imbibition Rolled Towel Bioassay · Substrate Imbibition-Overview I. Purpose II. Sample...
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Substrate ImbibitionRolled Towel Bioassay
Brenda Johnson
02/11/2016
Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Substrate Imbibition-Overview
I. Purpose
II. Sample Preparation
• Prepare Herbicide Solution
• Wet Towels
• Plant Seed
• Germinate
III. Sample Evaluation
• Terms
• Assess control seedlings
• Assess sample seedlings
• Calculate trait purity
2Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Purpose
What is a bioassay?
• Assessment of the biological activity of a substance (herbicide) by testing its effect on an organism (seed) and comparing the result to an agreed upon standard (control)
What are we detecting/measuring?
• The reaction of the seed/seedling to the presence of the herbicide of interest to determine trait purity
3Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Purpose
Why Conduct an Herbicide Bioassay?
• Trait providers require that trait purity meet standards as indicated in licensing agreements
• To determine if plants demonstrate resistance to herbicide(s) to allow for weed control by herbicide application as required
%Trait Purity = # Normal Tolerants
(# Normal Tolerants + # Normal Non-Tolerants)
4Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Sample Preparation
Prepare working solution of herbicide
• Concentration determined during method validation
• Varies based on herbicide state, i.e. concentrated solution, dry chemical
5Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Sample Preparation
Prepare towels
• Soak towels in working solution of herbicide
6Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Sample Preparation
Plant seeds
• Place seed on herbicide soaked towels using planting apparatus (planting board, planting head)
• Roll towels to contain seeds
7Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Sample Preparation
Germinate seeds
• Place containers into humidity and temperature controlled chamber
• Allow to grow for defined period of time
8Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Sample Evaluation
Assess Control Seedlings
• Confirm that trait positive controls demonstrate the expected phenotype for herbicide tolerance
• Confirm that trait negative controls demonstrate the expected phenotype for herbicide non-tolerance
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Glufosinate Tolerant Controls Trait Negative Control s
Sample Evaluation
Assess Sample Seedlings
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Cotton rolled-towel after germination period
Corn rolled-towel after germination period
Sample Evaluation
Assess Sample Seedlings
• Each seedling is determined to be normal, abnormal, or dead
• Normal seedlings only are determined to be tolerant or non-tolerant to the herbicide
11Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Trait Negative Controls
Sample Evaluation
Normal (AOSA Rules for Testing Seedlings) -Seedling possessing the essential structures that are indicative of their ability to produce plants under favorable conditions
Abnormal Seedling (AOSA Rules for Testing Seedlings) -Seedling that cannot be classified as normal
Tolerant Seedling- Seedling that demonstrates the presence of a genetic trait providing tolerance to the herbicide in question
aka: Trait-containing, Trait positive
Non-tolerant Seedling- Seedling that does not demonstrate the presence of a genetic trait that confers resistance (or tolerance) to the herbicide
aka: Non-trait, Negative, Susceptible
Cautionary Note: Non-tolerant seedlings appear “Abn ormal” because they have been affected by herbicide (an unfavorabl e condition), but would most likely appear “Normal” in the absenc e of herbicide
12Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal Corn Seedlings
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Late Germinating Seedling -Has all necessary and healthy characteristics, but is slow/small
Abnormal Corn Seedlings
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Leaf Emerging from base of coleoptile
Leaf badlyshredded
Leaf and coleoptile damaged
Abnormal Corn Seedlings
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No primary root with insufficient
secondaries
Deep Lesion
Shoot damaged and not developing
Shoot damaged and weak roots
Decay at point of attachment to scutellum
Abnormal Corn Seedlings
16Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
“Hollow tips:”Leaf extending
less than halfway up coleoptile
For all species, refer to AOSA Rules for Testing Seeds,Volume 4. Seedling Evaluation
Glyphosate-Corn
Events: NK603, GA21, MON88017
17Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal glyphosate
tolerant corn seedling Normal glyphosate non-
tolerant corn seedlings
Symptoms: • Stunted, stiff root with
little if any secondary roots that are “unrelaxed”
• Flattened shoot with little pigmentation
Glyphosate-Soybean
Events: 40-3-2, MON89788
18Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal glyphosate non-tolerant soybean seedlings
Symptoms: • Stunted, stiff, off-color root with
little if any secondary roots that are “unrelaxed”
• Shortened hypocotyl
ISU Photo
Glyphosate-Cotton
Events: MON1445, MON88913
19Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal glyphosate non-tolerant cotton seedling (left)
Symptoms: • Stunted, stiff, off-color root with
little if any secondary roots that are “unrelaxed”
• Shortened, darkened “staining of” hypocotyl
Glufosinate-Corn
Events: Bt11, TC1507, DAS59122-7, T25
20Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal Glufosinate non-tolerant corn seedling
Symptoms: • Shortened shoot growth with
necrotic mesocotyl• Inhibition of root growth• Little shoot pigmentation
Normal Glufosinate Non-tolerant corn seedlings
Normal Glufosinate tolerant corn seedlings
Glufosinate-Soybean
Events: A5547-127 (LL55)
NOTE: Substrate imbibition method is not appropria te for A2704 (LL27)
21Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal glufosinate non-tolerant soybean seedlings
Symptoms: • Short or curled primary
roots• Inhibited primary leaf
development• Yellow pigmented
cotyledons
ALS Inhibitors-Corn
Events: Endogenous (IMI)
22Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal IMI
tolerant corn seedlings
Normal IMI non-tolerant corn seedlings
Symptoms: • Stunted, stiff root with
little if any secondary roots that are “unrelaxed”
• Shortened, thickened shoot growth
Events: Endogenous (STS)
ALS Inhibitors-Soybean
23Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal Sulfonylurea non-tolerant soybean (STS) seedlings
Symptoms: • Crossed leaves that are
stunted in growth• Roots unaffected
Events: Endogenous (IMI)
ALS Inhibitors-Sunflower
24Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal IMI non-tolerant sunflower seedlings
Symptoms:
• Stunted, stiff root with little if any secondary roots that are “unrelaxed”
• Shortened, thickened shoot growth
Events: DAS68416-4
2,4-D-Soybean
25Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
Normal 2,4-D non-tolerant soybean seedlings
Symptoms:
• Callusing and curling of hypocotyl and radical (primary root)
• Leaves absent or strapped and elongated
Herbicide Bioassay Data Analysis
Calculating Trait Purity from a Bioassay
%Trait Purity = # Normal Tolerants(# Normal Tolerants + # Normal Non-Tolerants)
Example: Standard 400 seed bioassay
400 seeds are planted-2 are dead, 8 are abnormal, 7 are normal non-tolerant, 383 are normal tolerant
%Trait Purity = 383 = 98.21%(383 + 7)
This sample meets trait providers requirement of ≥98%
26Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
I. Purpose
II. Sample Preparation
• Prepare Herbicide Solution
• Wet Towels
• Plant Seed
• Germinate
III. Sample Evaluation
• Terms
• Assess control seedlings
• Assess sample seedlings
• Calculate trait purity
Substrate Imbibition-Q&A
27Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016
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