0:05
Yep.

0:05
Thank you very much, Chris.

0:06
By the invitation by the opportunity to share our work with you guys.

0:11
My name is Rubens.

0:12
I work at the Department of Bacteriology in the lab of Jean Michel Anna.

0:17
Today I’m going to talk a few about our projects involving involving biological nitrogen fixation in corn.

0:27
I, I split my presentation in three parts.

0:30
Firstly, I’m I’m going to give one introduction to the biological nitrogen fixation.

0:36
If so, then I’m going to talk about our scientific approach, that is the genetic regulation involved in the formation of our roots.

0:45
And then finally in the our technological approach that is our results involving our maze breeding program for biological nitrogen fixation.

0:56
So let’s start talking about the nitrogen.

1:00
As you may know, nitrogen is a significant cost for farmers that grow corn.

1:07
It’s about 10 to 12% of the costs.

1:11
This costs and the rates of nitrogen depends of several things like soil management and soil morphogenesis.

1:20
It also important, as Lindsay mentioned, like the rotation that the, the crops that we plant before corn.

1:28
The level of technology of the farmers is also very important.

1:32
And here I’m not talking about only about machines, but mainly about seeds.

1:38
As you know, great hybrids.

1:40
They, they request more, more a better fertilization.

1:45
And also it’s important the youth that the farmer aims to produce.

1:51
If I’m making a simple math, let’s say that we’re expecting something about 200 bushel acres.

1:58
That means that we need like 180, five, 190 N points.

2:03
And multiply by the price of the the point of North, that’s about $0.06.

2:09
And the area that you have in USA with corn, that’s about 95 millions of acres.

2:15
We see that we spend USA something about 10:50 billions of dollar every year with nitrogen.

2:25
But that’s the the economical problem is not the only problem as we see, as we saw in the presentation of fleets, there are a lot of problems involving the nitrogen requiring the environment like the acidification of habitats.

2:43
One good example is the otification in the Mississippi River Delta.

2:50
And also we have like programs involve the health, especially the production of particular matter that caused like significant cardiovascular and respiratory disease.

3:01
If some colleagues from the University of Minnesota, they published in our work a couple years ago that they estimated that this costs involving ammonia in like health and environmental costs, it’s about 21 billions of dollars.

3:20
So it’s a lot of money that you spend in this issue.

3:27
So how could we use the the environment in our favour?

3:32
Probably you guys have seen this graph several times in in your life.

3:37
So we are liking deep in the ocean of nitrogen, right About 78% of the atmosphere it’s make of nitrogen.

3:46
But unfortunately the plant cannot assess this nitrogen from the air.

3:51
The plants can only assess the nitrogen that we are, we are put in the fertilizer.

3:56
So which kind of strategies could you make to could you adopt to make quite fixed nitrogen from the air?

4:05
Basically we could like transferring regulatory pathways involved in nodulation from legumes.

4:12
Here is a picture from Med cargo.

4:14
That’s one legume that produce nodulus.

4:18
So we can like teach the plant, the coin plants to produce nodulus, nodulus.

4:25
But it’s a, it’s evolves like a very hard genetic engineer.

4:29
There are a couple scientists work in in that issue, but it’s not something that we’re going to achieve in a couple years.

4:35
Probably we’ll speed decades to to have any success in this approach.

4:42
We also could try to introduce in the nitrogenise, nitrogenise enzyme from bacteria in corn cells.

4:51
But again, it’s a not another hard strategy that demands a big genetically engineered process.

5:00
We could also engineer the bacteria that the azotref that they are associated with with corn.

5:09
Just to mention diazotref is the name of bacteria that are able to fix nitrogen from there.

5:16
So we could engineer there, there then to make them more efficient.

5:22
But also it’s a genetic engineer process.

5:25
It’d be faster than any genetic engineer process in plants.

5:30
But we know that there are a lot of regulations involving the adoption of of this kind of bacteria, engineered bacteria in the field.

5:40
So the approach that make that seems to be most doable for us at that point is to explore corn varieties that they produce this natural, this mucilage, this mucilage, this gel that I’m going to explain to you how it’s useful for an biological nitrogen fixation.

6:03
We use some some corn from the state of Oaxaca in Mexico, that state, this part of Mexico.

6:13
It’s a very particular environment.

6:15
It’s like a high altitude place.

6:20
It’s a mountain.

6:21
It’s there are several villages in the mountain.

6:23
They have a lot of fog.

6:25
So it’s a very it’s super humid the the the ambient there.

6:30
And they are like one, they have like people that are, it’s a very rich in terms of culture.

6:38
They use this kind of corn.

6:40
You’ll see here that they produce a lot of iron roots and they create this this kind of mucilage.

6:47
And they use this mucilage for rituals and several things.

6:52
But for us, our main interest is to because in this mucilage in the bacteria that lives here, we can take that nitrogen from the air transform into ammonium and then the plant can use this this nitrogen.

7:08
So how how does the nitrogen fixation process occur?

7:13
OK, here we have the plant with this air roots and secreting mucillage.

7:19
Let’s imagine that this green illustration, it’s their root that’s secrete the mucilage.

7:26
The mucilage will have like the solve the dinitrogen gas that is the the the nitrogen from there the atmospheric nitrogen then after here it’s where the bacteria acts like the nitrogen is the enzyme from diazotraph.

7:47
They break this nitrogen gas into ammonia molecules like this is a very hard reaction.

7:55
It requests a lot of energy and just this kind of bacteria they can do that also that we have protonation.

8:04
So ammonia becomes ammonia and then it comes inside to the coin tissues through the ammonia transporters.

8:14
Here in blue you can follow the nitrogen atoms.

8:18
So after that this nitrogen can can go into the glutamine synthetase and the glutamate synthase pathway.

8:28
So here you can see the blue balls, the nitrogen here the nitrogen is fixed and can be used for corn for diverse metabolism activities, metabolic activities.

8:41
So that’s how it works.

8:45
So, but let’s think a little bit a little bit deeper.

8:49
If our goal at the end of the day it’s have high yield and low cost corn, we know that it’s a a function of three things, environment, plant and bacteria.

9:01
We know that it’s very hard to not say impossible to modify our environment.

9:06
So we need to work in plant and bacteria sites.

9:10
Here we have in Jean Michel and a lab, we have a big team of scientists that work.

9:17
Some of the scientists work more in the bacteria site and others like me work more in the plant side and we have other scientists that work in the interaction between there.

9:30
From my side, I’m more interested in corn, so I want to discover how it it happens.

9:39
So we have two basic questions here.

9:42
Why do some corn cultivars produce arrowroots and others don’t and how it’s mucilage produced and discreted?

9:51
To the second question, my colleague Rafael Vonado that’s now in Summit, he has published some good results about that.

10:00
His paper is impressed.

10:03
And so today I’m going to to focus more in the production in the formation of the IR roots.

10:10
OK, now I’m going to the second part of the presentation and, and we can talk about the genetic regulation involved in the formation of this arrow roots twisted that that we started with like some double haploid populations for those that are not used about that.

10:32
We we we firstly requested seeds from this Oaxaca land races from Simit and USDA and then we crossed with a Midwest adapted in the red line and then we have the F1 hybrid and then we back crossed with the Midwest adapted in red line to make easier the the evaluation in field.

10:56
Because of this Oaxaca land races, they hate the rest like they don’t like when we plant them coin belt, they have a totally different phonological cycle.

11:08
OK.

11:08
After that we crossed the BC1 hybrid with the applied industry line and then select the the applied seats grow the applied City made the cautious in treatment and then self pollination.

11:26
The almost goes the eight line and then we have a population of the 8 lines.

11:32
We did that in partnership with Limagran.

11:35
That’s the French company.

11:37
We did it 8 times.

11:39
So we have 8 different population of the 8 lines.

11:43
So Limogram also helped us in doing the genotyping of these plants.

11:50
What mean genotyping at that point?

11:52
I would like you guys think for those that are not to use this kind of technology, think about the genome, the DNA of a plant like an address, like we can find each gene, each region of the genome of the plant has its own address.

12:12
So OK, here’s the genotype activity and then we need, it’s going to be over in about 6 minutes.

12:22
Yeah, yeah, then we can.

12:27
OK.

12:27
And then after making the genotyping, we need to do the phenotyping of these plants of just eight different populations.

12:36
We have do it, we have done it in West Madison, that’s our field this year.

12:41
We have done it also in 2023 and 2024.

12:48
We assess several IR route related traits like arrow route diameter, the number of of of nodes with arrow routes.

12:57
That’s one’s the most important.

13:00
The stock diameter, flaglift height and flowering time, flowering time, flaglift height and stock diameter we use as a cover rate.

13:10
In 2025 here in West Madison, we have like about 3000 plots.

13:16
We evaluated like 6 plants per plot.

13:20
I mean all these traits.

13:21
So we have collected about one more than 100,000 data.

13:25
We are go actually we are going to finish it hopefully next week.

13:31
And we have a big team of about 20 students that helped us a lot.

13:37
OK, after crossing this genotyping data with the phenotyping data, we have a graph like this.

13:47
Do you remember that I talked about the DNA, thinking about the DNA as a map, OK, here we have the 10 chromosomes of corn, right?

13:59
And so each regional here has like different genes.

14:05
So here, how to interpret a graph like that.

14:08
Here we have the logarithm of the score that it’s the the parameter that we use to make the quantitative trait loci mapping.

14:19
It means that if we have some peaks like this, it it says that in this interval.

14:27
Here we have genus, here they are likely to be involved in this trait.

14:34
In the skies of trait.

14:35
They are the format the number of nodes with R roots.

14:39
So if we want to discover the genes that are involved in this trait, that’s the first step.

14:47
But we we can increase the, the looking, the search for this kind of genes, the important genes for our root formation.

14:56
We can use it like put data from different populations.

15:01
So in blue, it’s from one population, in red we have data from another population.

15:07
We can add one more.

15:10
We love when we find something like this, like overlap between two populations.

15:15
It means that the the probability of some gene here it evolved in the in the Strait, in the number of nodes of our roots, it’s super high.

15:26
But we also can improve this mapping, I mean the search for the key genes.

15:32
We can.

15:33
We adopt modern algorithms.

15:35
Right now we are using one algorithm called the inclusive composite interval mapping.

15:41
We use covariates to reduce residual variation, especially days to enthesis flowering time.

15:48
It’s a super important covariate for us that help us to to have better results.

15:54
We evaluate like multiparent populations, like nested associate mapping populations.

16:02
That’s a different approach.

16:04
We compare these results with no gene annotation.

16:07
So we use a lot the data in the maze GDB.

16:11
We look for information on related species like sugar cane or SORG especially.

16:18
And right now we are not, not right now.

16:20
We are going to start pretty soon the genome assembly of one of these land ratios also to try to improve the to look for gene variations.

16:34
Yeah, that’s we we also have another fine, fine mapping, fine tiny mapping approach, but we can talk about it later.

16:44
But now in the third section, I want to talk about the breeding program, our breeding program to introduce this trait.

16:52
I mean there are root related traits into Midwest adopted in Brett Alliance.

16:57
So how did our breeding started?

17:00
We started cross again Oregon seeds from Simit and West DA.

17:05
There are calendar races and they cross the 2 Midwest adapted embedded lines.

17:09
So we have the F1 again self pollinated and then we have F2 and then we conducted with several generations of self cross.

17:19
Then we have like some reels recombination recombinated embedded lines.

17:26
We also conducted the the breeding program through self to back crosses trying to like increase the adaptability of the plant to the environment in the corner belt.

17:42
So we have different populations and combinant inbred lines that came from the crosses between Midwest adapted inbred line and Oaxaca and ladder races.

17:53
So in that case in the breeding we evaluated all these higher root related traits, but also we evaluate agronomic traits because you know, we don’t want to plant their propensity logic or they have like small ear size or they are like resistant to some kind of disease.

18:13
So we have much more things to evaluate in the breeding program.

18:17
At the end of the day, we want to plant 1 inbred line with six to nine nodes, 9 will be a dream, 6 is much achievable forming this higher roots and secreting this mucilage and also exhibiting good agronomic traits.

18:36
We are at that point now we are splitting our breeding programs to have plants with different flowering times that we hope it can be used in different latitudes in US.

18:50
You know, we are, we are dealing about with one polygenic trait, right, with moderate editability.

19:00
So it’s a kind of hard to make breeding in that condition.

19:03
So we need a different points of the breeding program.

19:07
We need to make top classes to help to, to increase the deficits of selection.

19:13
And our, our, our youth trials are conducted in Hancock.

19:19
There we have like more than 100 hybrid.

19:23
We evaluate that in two environments with standard nitrogen and with like about 40% off like like very low nitrogen.

19:35
And also we have irrigation each three days because we need like moisture to, to get better results.

19:44
And then we assess all these route related to it, chlorophyll content and field.

19:51
One important step of our our experiment in Hancock is to inoculate the diazotref.

19:59
As I mentioned, we have like a lot of students that help that help us to make the inoculation.

20:05
Of course, in the future, we hope that the stock in this technology, the bacteria, could be inoculated in the seed.

20:13
But here by for experimental purpose, we are spraying the bacteria in the plants to make sure that the bacteria is there.

20:23
So we we did not harvest yet our our fielding hand clock this year, but we have like very nice results.

20:32
Last year we compared hybrids with different with similar backgrounds, sorry, but one hybrid made with an inbred line that has their root related trait and another hybrid that not, doesn’t have this interest trait.

20:51
So and this results are are about in the very low nitrogen place.

20:59
I mean like a lack of 80 points of nitrogen per acre.

21:03
So here you have that you see that we don’t have any any good change in terms of chlorophyll content, but some for some hybrids that that has like more our roots.

21:19
We see that the the chlorophyll content increases very much and you know that the chlorophyll content it’s related with the nitrogen status of the plant.

21:31
But let’s see the result in terms of yields that we know.

21:35
At the end of the day, yield is the most important trade for farmers.

21:39
Again, we compared the hybrids with similar backgrounds, but the difference that the one of the parent has the trait that’s blue and another one doesn’t have.

21:53
So here we don’t have, we don’t see too much difference.

21:57
But when we go to the our best hybrids, you’ll see that there are a big increase in the youth under environment with very low nitrogen.

22:09
And we assume that this this increase is due to the bacteria association, this associate the symbiotic association.

22:19
When we plot the data about the number of nodes with our roots with the yield, we see a very super high correlation.

22:27
What that was that yes, when the plant has more nodes and of course they have the bacteria inoculated, it tends to have like a bigger yield to be to, to make sure about our data, we need to validate that in laboratory.

22:47
So we make some experiments involve the bacteria.

22:53
Here we have the different bacterias and here the the level of acetone reduction activity.

23:01
Basically by accessing the level the of conversion of Acetylene to ethylene, we can quantify ductive of nitrogenase enzyme enzyme from the bacteria.

23:15
So these three bacteria Crypsilo michiganensis, Crypsilo Varicola and Azotobacter vinyl and are the bacteria that spray in the field.

23:24
Here we have the controls that as you can see they don’t fix nitrogen, but also we make another experiment in greenhouses that we put a type of nitrogen, A fertilizer called the 50 N nitrogen, 50 N fertilizer.

23:44
That means that this fertilizer has a different nitrogen, it’s a label is isotope.

23:52
So you can track this nitrogen in the plant.

23:57
What means that you can also after you you, you take the tissues of the the plants and ground this mute this you can see how the percentage of the nitrogen that comes from fertilizer and then the percentage of nitrogen that comes from the air.

24:20
So here the results are telling us that we have about 50% of the nitrogen that the plant use it, it’s from the air, which is great.

24:33
In Oaxaca in Mexico, these plants fix about 82%.

24:38
Here in greenhouse we, we, we got like 50%.

24:44
We will be super happy in the field in the future.

24:47
If farmers can reach something between 25 and 30%, that means something about 3 billions of dollars saved in fertilizers every year.

24:58
So at the end of the day, we, we want to like, give another option for the farmers to save money and protect the environment at the same time.

25:08
So I would like to talk my team, especially our Pi, Jean Michelani, our Copis.

25:15
We have a, a partner in Georgia.

25:18
We, we do the same experiments in Georgia to have two locations and especially all the undergrads from our current team and the, and the workers from from the agricultural research Station, especially Janet and her team in West Madison and Paul and his team in Hancock.

25:40
Yeah, that’s our our.

25:42
I would like to highlight that our project, it’s funded by USDA and I’ll be happy to reply and answer any doubt that you guys have.

25:53
Thank you very much, Chris.

25:55
Yeah.

25:56
Thank you.

25:56
Rubens.

25:57
One question I had listening to your presentation was you definitely noted that the the nitrifying corn in those low nitrogen systems, you know the the yield got higher.

26:10
So where is growing natively in Mexico?

26:13
Are they following like the same nitrogen practices like in the US where we really put a lot of nitrogen on?

26:23
Or is this better in a scenario where maybe it’s difficult to fertilize or you have restrictions and you can’t fertilize?

26:32
Is this going to be a better corn to grow in the future?

26:36
Yeah, that, that that’s the goal.

26:38
We use that especially in like let’s say poor fields where we don’t like, I mean for farmers that don’t have condition to, to put like super high inputs.

26:51
We have some, we are starting to have some partnerships in Africa and India.

26:56
We bet that it to be very useful in this place.

27:00
But yeah, talking about Oaxaca, the big difference in Oaxaca is, well, this plant has a huge like phenological circle that takes forever to flower.

27:12
So they have more time to produce this roots.

27:18
And also they, they are very sensitive.

27:20
I, I, I do not have time to, to include more data, but we have some interesting data that plants in the edge of our plots, they produce more, they have more roots and there’s a big influence of the environment.

27:35
We realized that it’s not about the nitrogen availability in the soil and that it’s not the moisture of the lower part.

27:44
So our big bet now it’s that could be light could be a trick.

27:49
And in in, you know, a hacker, they don’t plant like USA in several roles.

27:54
They are individual.

27:55
It plants.

27:56
So each plant it’s an edge.

27:59
So for sure it it has a role in the Strait.

28:05
Yeah.

28:06
So we are trying to figure out this, this, like, light influence.

28:11
Yeah, there are, like, too much things to consider.

28:14
I was gonna say one question leads to another question.

28:17
Yeah.

28:17
Yeah, that’s the point.

28:18
Love it.

28:18
Well, thank you for bringing an update to our crowd and our webinar attendees.