Substrate Imbibition Rolled Towel Bioassay

Transcription

1 Substrate Imbibition Rolled Towel Bioassay Brenda Johnson 02/11/

2 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 2

3 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 3

4 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) 4

5 Sample Preparation Prepare working solution of herbicide Concentration determined during method validation Varies based on herbicide state, i.e. concentrated solution, dry chemical 5

6 Sample Preparation Prepare towels Soak towels in working solution of herbicide 6

7 Sample Preparation Plant seeds Place seed on herbicide soaked towels using planting apparatus (planting board, planting head) Roll towels to contain seeds 7

8 Sample Preparation Germinate seeds Place containers into humidity and temperature controlled chamber Allow to grow for defined period of time 8

9 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 Glufosinate Tolerant Controls Trait Negative Controls 9

10 Sample Evaluation Assess Sample Seedlings Cotton rolled-towel after germination period Corn rolled-towel after germination period 10

11 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 Trait Negative Controls 11

12 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 Abnormal because they have been affected by herbicide (an unfavorable condition), but would most likely appear Normal in the absence of herbicide 12

13 Normal Corn Seedlings Late Germinating Seedling- Has all necessary and healthy characteristics, but is slow/small 13

14 Abnormal Corn Seedlings Leaf Emerging from base of coleoptile Leaf badly shredded Leaf and coleoptile damaged 14

15 Abnormal Corn Seedlings Shoot damaged and not developing No primary root with insufficient secondaries Shoot damaged and weak roots Deep Lesion 15

16 Abnormal Corn Seedlings Decay at point of attachment to scutellum For all species, refer to AOSA Rules for Testing Seeds, Volume 4. Seedling Evaluation Hollow tips: Leaf extending less than halfway up coleoptile 16

17 Glyphosate-Corn Events: NK603, GA21, MON88017 Normal glyphosate tolerant corn seedling Normal glyphosate nontolerant corn seedlings Symptoms: Stunted, stiff root with little if any secondary roots that are unrelaxed Flattened shoot with little pigmentation 17

18 Glyphosate-Soybean Events: , MON89788 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 18

19 Glyphosate-Cotton Events: MON1445, MON88913 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 19

20 Glufosinate-Corn Events: Bt11, TC1507, DAS , T25 Normal Glufosinate non-tolerant corn seedling Normal Glufosinate tolerant corn seedlings Normal Glufosinate Non-tolerant corn seedlings Symptoms: Shortened shoot growth with necrotic mesocotyl Inhibition of root growth Little shoot pigmentation 20

21 Glufosinate-Soybean Events: A (LL55) Normal glufosinate nontolerant soybean seedlings Symptoms: Short or curled primary roots Inhibited primary leaf development Yellow pigmented cotyledons NOTE: Substrate imbibition method is not appropriate for A2704 (LL27) 21

22 ALS Inhibitors-Corn Events: Endogenous (IMI) 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 22

23 ALS Inhibitors-Soybean Events: Endogenous (STS) Normal Sulfonylurea nontolerant soybean (STS) seedlings Symptoms: Crossed leaves that are stunted in growth Roots unaffected 23

24 ALS Inhibitors-Sunflower Events: Endogenous (IMI) Normal IMI non-tolerant sunflower seedlings Symptoms: Stunted, stiff root with little if any secondary roots that are unrelaxed Shortened, thickened shoot growth 24

25 2,4-D-Soybean Events: DAS Normal 2,4-D non-tolerant soybean seedlings Symptoms: Callusing and curling of hypocotyl and radical (primary root) Leaves absent or strapped and elongated 25

26 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% ( ) This sample meets trait providers requirement of 98% 26

27 Substrate Imbibition-Q&A 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 Say, what s a mountain goat doing way up here in a cloud bank? The Far Side by Gary Larson 27