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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

9Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016

Glufosinate Tolerant Controls Trait Negative Control s

Sample Evaluation

Assess Sample Seedlings

10Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016

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

13Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016

Late Germinating Seedling -Has all necessary and healthy characteristics, but is slow/small

Abnormal Corn Seedlings

14Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016

Leaf Emerging from base of coleoptile

Leaf badlyshredded

Leaf and coleoptile damaged

Abnormal Corn Seedlings

15Document name: Substrate Imbibition Rolled Towel Bioassay File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02042016

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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