Introduction to Glyphosate Mode of Action and Bioassay Calculations

Introduction to Glyphosate 

Mode of Action and 

Bioassay Calculations 

Michael Lovelace, Ph.D. 

Agronomist, USDA, AMS 

Seed Regulatory and 

Testing Branch 

Gastonia, NC 

http://www.ams.usda.gov/lsg/seed.htm 

Agricultural Marketing Service 

Marketing and Regulatory Programs

The Seed Regulatory 

and Testing Branch 

AMS Lab and Office Complex, Gastonia, NC 

• Enforces interstate commerce provisions of 

the Federal Seed Act (FSA). 

• Provides the global seed community with 

services that promote the truthful marketing of 

seed. Agricultural Marketing Service 


The Seed Regulatory 

and Testing Branch 

AMS Lab and Office Complex, Gastonia, NC 

• ISTA accredited 

• ASL accreditation pending 

• ISO 9001 compliant 



SRTB Tests Performed 

• Purity 

• Germination 

• Noxious weed seed 

• Moisture / Conductivity 

• Species identification 

• Plant pathology 

• Variety verification 

– Varietal purity / Presence of 

biotech derived seed 

• Bioassay 

• Protein based 

• DNA based 


Marketing and Regulatory Programs 

New Test Development

New Test Development 

• Where do we begin? 

• Important things to know: 

– Previous literature / studies 

– Background on the crop 

– Background on the herbicide 

– Knowledge of experimental design / 

statistics 



Biochemistry Review 



• What are: 

– Amino Acids? 

– Proteins? 

– Enzymes?

Amino Acids 

• An amino acid is any molecule 

containing amino and carboxylic 

acid functional groups. 

• AAs are the basic structural 

building units of proteins. 

• AA form short polymer chains 

called peptides or polypeptides, 

which in turn form proteins. 

• The coding sequences of genes 

determines the AA sequences of 

almost all naturally occuring 

proteins though transcription 

and translation. 



Proteins 

• Protein comes from the Greek word 

"protos“, meaning "of primary 

importance". Proteins are essential to 

the structure and function of all living 

cells. 

• Many proteins are enzymes or subunits 

of enzymes. Others are important for 

structural or mechanical roles, 

signalling functions, immune response, 

storage, and transport. 

• In nutrition, proteins are broken down 

through digestion to provide amino 

acids for an organism, including those 

the organism may not be able to 

synthesise itself. 



Enzymes 

• An enzyme is a protein that catalyzes, or 

speeds up, a chemical reaction. 

• Enzymes are essential to life because 

most chemical reactions in biological 

cells occur too slowly, or lead to 

different products, without enzymes. 

• Enzymes lower the reaction activation 

energy, thus speeding up the reaction. 

They remain unaltered by the reaction 

and, therefore, can continue function. 

• Enzyme activity can be affected by other 

molecules. Inhibitors decrease or 

abolish enzyme activity, while activators 

increase activity. Aspirin inhibits the 

COX enzymes that produce the 

inflamation messenger prostaglandin, 

thus suppressing pain and inflammation. 



H 

Amino Acid Synthesis 

NH 3 + 

R 

COO - 

• Carbon skeletons come from 

intermediates of glycolysis, the 

pentose phosphate pathway, or 

citric acid cycle. These 

skeletons contain carboxylic 

acid groups. 

• Nitrogen is assimilated into 

amino acids by way of glutamate 

and glutamine. 

• Synthesize using five different 

precursors via various 

pathways. 



Alanine (Ala) Arginine (Arg) 

Aspargine (Asn) Aspartate (Asp) 

Cysteine (Cys) 

Glutamate (Glu) Glutamine (Gln) 

Methionine (Met) 

Branched Chain 

Amino Acids 

Aromatic 

Amino Acids 

Isoleucine (Ile) 

Amino Acids 

Proline (Pro) 

Glycine (Gly) 

Leucine (Leu) 

Histidine (His) 

Serine (Ser) Threonine (Thr) 

Valine (Val) 

Phenylalanine (Phe) Tyrosine (Tyr) Tryptophan (Trp) 

Lysine (Lys) 

Inhibited by 

ALS Inhibiting 

Herbicides 

Inhibited by 

Glyphosate

D-Erythrose-4-Phosphate 

H 2 O 

P i 

7-Phospho-2-dehydro- 

3-deoxy-Darabinoheptulosonate 

NAD + 

NADH, 

H + , P i 

Shikimate Pathway 

Production of Aromatic Amino Acids 

3-Dehydroquinate 

Phosphoenolpyruvate 

O 

O - 

Enzyme 

complex 

O 

O- O- P 

O 

Co++ 


synthase 


dehydratase 

H 2 O 

3-Dehydroshikimate 

Chorismate 

Shikimate 

dehydrogenase 

NADPH, 

H + 

NADP + 

3-Phospho-5-enoylpyruvylshikimate 

Chorismate 

synthase 

P i 

Shikimate 

3-Phospho-5enoylpyruvylshikimate 

synthase 

Mg++ 

Shikimate 

kinase 

3-Phosphoshikimate 

(S3P) 

ATP 

ADP 

P i 

PEP

Chorismate 

Precursor for Aromatic Amino Acids 

Chorismate 

Prephenate Anthranilate 

Phenylalanine Tyrosine Tryptophan

Influence of Glyphosate in Plants 

Glyphosate molecules 

in a spray droplet

Chloroplast 

O 

O - 

O 

O- O- P 

O 

Chorismate 

3-Phospho-5enoylpyruvylshikimate 

synthase 

Shikimate 

x

O - 

O - 

O 

P 

O 

O - 

P 

O - 

O 

PEP 

N 

Competitive Inhibition 

COO - 

Glyphosate 

COO - 

EPSPS 

open conformation closed conformation

How does resistance work? 

Site of herbicide action 

in a conventional plant 

O - 

O 

P 

O - 

N 


Interaction results in a conformational change that will not allow PEP to bind. 

Site of herbicide action 

in a transgenic plant 

O - 

O 

P 

O - 

N 

COO - 


Changing a base pair that codes for an amino acid in the site of herbicide action 

changes the localized charges of the enzyme, thus inhibiting glyphosate binding. 

This permits PEP to bind and the shikimate pathway to function normally. 

COO -

How does this knowledge relate 

to bioassays? 

• Glyphosate activity occurs in 

the chloroplast. A light cycle 

must be present for 

glyphosate to have an affect. 

• Seeds do not have 

chloroplasts, so a certain 

level of plant development is 

necessary in order for the 

bioassay to work. 

• This is the reason we have 

some growth of the plants 

before growth is inhibited. 



Summary 

• Glyphosate translocates into 

the chlorophyll. 

• In the chlorophyll, glyphosate 

inhibits the EPSPS enzyme, 

thus inhibiting production of 

aromatic amino acids. 

• Plants die due to starvation. 

• In transgenic plants, a mutated 

EPSPS enzyme is insensitive to 

glyphosate, thus allowing PEP 

to bind normally. 



Time to Change Gears!! 



Bioassay Setup 

• Acquire sample seeds from purity 

test and count out the number of 

seeds needed to conduct the test. 

– Sample in question 

• Roundup Treatment 

• Water Check 

– Know susceptible sample 



– Known resistant sample 



• Place the seeds in a petri-dishes and 

correctly label the seeds for each 

treatment.

Prepare Working Solution 

• A working solution is the herbicide 

mixture used for seed soaking or 

wetting the towels. 

• Determine the concentration of the 

working solution from the test 

directions and gather information from 

pesticide label. 



Preparation of Herbicide 

Working Solutions 

Prepare calculations for 

your herbicide working 

solution. 

Calculate concentration in 

PPM (Parts Per Million). 

or 

Calculate concentration in 

molarity (g / L). 



Short Quiz 

• What is a PPM? 

• How do you calculate PPM? 

• What are the units of PPM? 

• Why is PPM unit-less?

Gathering Pertinent Information 

• Locate the area on the 

container that contains the 

pertinent information 

• Identify the active ingredients 

and corresponding 

concentrations. 

• Know what information is 

useful. 



How much glyphosate is in 1 

Liter of Roundup? 

• Container indicates 50.2% 

glyphosate. 

• 50% of 1 liter is 500 ml, thus 

there are 500 ml of 

glyphosate in 1 L. 

• The container indicates there 

are 600 ml of glyphosate in 1 

L. 

• Where did we go wrong? 



Why Not Calculate 

Based on Percentages? 

• Existing bioassay methods 

indicate concentration 

calculations should be 

based on percentage. 

• This can lead to problems 

because the label usually 

does not specify if 

percentages are based on 

weight or volume. 

• Why does it matter? 



Importance of Percentage Type 

50% V / V (volumetric) 

Solvent 

1.4 g / cm 3 

glyphosate 

1.7 g / cm 3 

If the 

solution is 

mixed on a 

volume 

basis, 

there will 

be about 

10% more 

glyphosate 

on a weight 

basis. 

50% g / g (gravimetric) 

Solvent 

1.4 g / cm 3 

glyphosate 

1.7 g / cm 3 

If the 

solution is 

mixed on a 

weight 

basis, 

there will 

be about 

10% more 

solvent on 

a volume 

basis. 

If we assume that the percentage of glyphosate is 50% by volume 

and glyphosate is actually 50% by weight, we would be adding 10% 

less roundup to our working solution than we would anticipate.

Why Not Calculate 

Based on Percentages? 

• So, is it volumetric or 

gravimetric?? 

• We don’t always know!! 

• Instead, use g ai / Liter 

or lb ai / gal 



Calculating Working Solutions 

Determine the concentration of 

your working solution and how 

much you will need for the 

experiment. 

Examine herbicide container to 

establish concentration of stock 

solution. 

Calculate the dilution of your 

stock solution to make your 

desired working solution. 



1000 PPM; 

1 Liter 

600 g / Liter 

???

Calculating Working Solutions in PPM 

1,000 parts 

999,000 parts 

1 part 

999 parts 

Roundup Ultra Max = 600 g ai 

1000 ml 

600 g ai 

1000 ml 

= 1 g ai 

cross multiply 

X 

Water 1 ml = 1 g 

1 g 

999 g 

= = 

= 

X = 

1 ml 

999 ml 

Must dilute roundup to acquire 

desired concentration. 

1 g ai X 1000 ml 

600 g ai 

X = 1.67 ml 

Add the 1.67 ml of herbicide solution to 998.33 ml of water to make a 

1000 PPM working solution. 

Are we done?? Not so fast!! There is still more to consider!! 




• Lets revisit the label. 

• The label indicates glyphosate is in 

the form of isopropylamine salt. 

• Why is this important? 

• The isopropylamine salt portion of 

the formulation has no herbicidal 

activity. Furthermore, different 

formulations of glyphosate have 

different salts. 

• In order to avoid any confusion, 

our calculations need reflect the 

amount of glyphosate free acid 

(portion without the salt) in the 

solution.

Formulation Comparisons 

HO 

O 

O 

N P 

O - 

Glyphosate Free Acid (169 g / mole) 

+ H3 N 

Isopropylamine Salt (59 g / mole) 

• Glyphosate free acid = 169 g / mole 

• IPA salt of glyphosate = 228 g / mole 

• DAM salt of glyphosate = 201 g / mole 

• TMS salt of glyphosate = 244 g / mole


1,000 parts 

999,000 parts 

1 part 

999 parts 

1 g 

999 g 

= = 

= 

Roundup = 600 g IPA salt of glyphosate 

Ultra Max 1000 ml herbicide solution 

Roundup = 0.445 g glyphosate free acid 

Ultra Max ml herbicide solution 

0.445 g ai 

1 ml herb 

= 1 g ai 

X 

cross multiply 

Water 1 ml = 1 g 

X = 

1 g ai X 1 ml 

0.445 g ai 

1 ml 

999 ml 

X = 2.25 ml herb 

Due to the different densities of water and herbicide solution, it may not 

be appropriate to add 2.25 ml of herbicide solution to 997.75 ml of water. 

Instead, it would be more appropriate to tare a beaker on a balance. 

Add about half the quantity of water desired. Add your herbicide 

solution. Add water to the beaker until the scale reads 1000 grams. 

X 

169 g glyphosate free acid 

228 g IPA salt of glyphosate 

Herb solution 1 ml ≠ 1 g

Use of Molarity 

• Use g ai / Liter or lb ai / gal 

• Use molecular weight (169.07) 

of the glyphosate molecule. 

• Percentage, density of 

solution, and makeup of inert 

ingredients is irrelevant in 

calculation. 

• Calculations are accepted SI 

units, whereas PPM is not. 



Conclusions 

• Things are not always as 

easy as they seem. 

• We need to rethink the way 

we do our calculations. 

• A common worksheet may 

standardize our herbicide 

solutions. 

• Molarity is a great 

alternative because there is 

no confusion with units.

Questions or Comments

Introduction to Glyphosate Mode of Action and Bioassay Calculations

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