Agriculture Biotechnology- A Tool for Functional Genomics and Complementing Plant Breeding Programs
Thomas Clemente, Eugene W. Price Distinguished Professor of Biotechnology, Dept of Agronomy & Horticulture, University of Nebraska-Lincoln
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05/15/2025
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Clemente received a BS degree in Biology with a minor in Chemistry from the Indiana University of Pennsylvania in 1985. He earned an MS degree in Plant Pathology from Oklahoma State University in1989 and a Ph.D. in Plant Pathology from North Carolina State University in 1993. Following his doctorate program, he did postdoctoral training at Monsanto Company from 1993 to 1996. For the past 29years he served as the Director of the University of Nebraska-Lincoln’s Plant Transformation Core Research Facility. Upon retirement in June of 2025 Clemente plans on writing a memoir of his career in science entitled, ‘Clowns to the left of me, jokers to the right, here I am stuck in the middle with you’
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- [00:00:00.240]The following presentation is part of the Agronomy and Horticulture Seminar Series at the University of Nebraska-Lincoln.
- [00:00:07.720]Good morning, I'm Preston Stevenson. I'd like to welcome you all to our Spring 2025 Seminar Series.
- [00:00:13.920]It's my pleasure to introduce Dr. Tom Clemente, the Eugene W. Price Distinguished Professor of Biotechnology here in the Department of Agronomy and Horticulture.
- [00:00:23.540]Dr. Clemente received a Bachelor of Science degree in Biology with a minor in Chemistry from Indiana University of Pennsylvania in 1985,
- [00:00:31.500]earned his Master's degree in Plant Pathology from Oklahoma State University in 1989,
- [00:00:37.320]and a PhD in Plant Pathology from North Carolina State University in 1993.
- [00:00:42.720]Following his doctorate program, he did postdoctoral training at Monsanto Company from 1993 to 1996,
- [00:00:50.900]and for the past 29 years,
- [00:00:53.120]he's served as a professor of biology and horticulture at the University of Pennsylvania.
- [00:00:53.520]He's also served as the director of the University of Nebraska-Lincoln's Plant Transformation Core Research Facility.
- [00:00:58.720]Upon retirement in June of this year, Dr. Clemente plans on writing a memoir of his career in science,
- [00:01:06.340]entitled, with no apologies whatsoever, To Stealer's Wheel.
- [00:01:09.680]Clowns to the left of me, jokers to the right, here I am stuck in the middle with you.
- [00:01:15.160]I note that I am on your left.
- [00:01:17.420]Probably accurate.
- [00:01:21.500]His presentation is,
- [00:01:23.500]titled Agricultural Biotechnology, a Tool for Functional Genomics and Complementing Plant Breeding Programs.
- [00:01:29.520]Dr. Clemente.
- [00:01:30.280]All right.
- [00:01:30.740]Well, thank you very much.
- [00:01:31.780]I appreciate the seminar committee for the invitation.
- [00:01:34.840]And I'm grateful for all you folks attending here today and whoever's online, anybody online.
- [00:01:40.120]So I'll jump right into this thing.
- [00:01:42.380]Budget cuts.
- [00:01:45.740]This doesn't work, huh?
- [00:01:53.480]There you go.
- [00:01:54.460]So this is the outline of my presentation.
- [00:01:56.500]I'm going to first going to give you an overview of what we've been doing in the lab, the activities over the last seven years.
- [00:02:02.460]First, two little short stories on soybeans.
- [00:02:05.080]I'm going to describe the outcomes of editing the G model that underlines the major QTL found on chromosome 20.
- [00:02:11.960]And it's paralog on chromosome 10.
- [00:02:15.220]This QTL has a high impact on impacting total protein in the seed.
- [00:02:21.100]And then I'm going to describe the outcomes of it.
- [00:02:23.480]Enhancing expression of the microtubule-associated proteinase called MAP65,
- [00:02:28.400]one of the members of that MAP65 family called MAP65-1.
- [00:02:33.060]And then I'm going to give you a couple little vignettes around sorghum,
- [00:02:36.860]and it's going to be collectively trying to design an idiot type sorghum feedstock for the bioeconomy.
- [00:02:42.520]And I misspelled that right there.
- [00:02:43.540]That's okay.
- [00:02:43.940]And then last, if anybody gets insulted easy, you may want to leave,
- [00:02:48.220]but I'm going to describe what's known as the Clemente commentary.
- [00:02:53.440]Now, throughout my entire career,
- [00:02:56.260]we've been doing this so-called build, test, learn approach,
- [00:02:59.080]where we design genetic constructs,
- [00:03:01.500]introduce them to the major crops pertinent to the state of Nebraska,
- [00:03:04.700]wheat, soybean, maize, a little bit of sorghum, and so forth.
- [00:03:09.560]And then we learn from that, you know, with some help from our friends,
- [00:03:14.820]what's going on, most of the stuff we don't know what we're doing,
- [00:03:17.600]and we start to cycle back up again.
- [00:03:19.420]And so what we've built over the years, what we refer to as the agricultural,
- [00:03:23.420]wild technology pipeline, there's a lot of public sector institutions
- [00:03:26.520]that have the capacity to introduce novel genetic variation into higher plants,
- [00:03:30.400]but they don't have the capacity to really get them out of the greenhouse
- [00:03:34.040]in a stewardship fashion.
- [00:03:35.880]And so what we have is we built all the capacity to introduce the genetic variation
- [00:03:40.560]into model plant species and commodity crops and then get them out to the field.
- [00:03:45.540]We have dedicated acreage space, 25 acres in the eastern part of the state,
- [00:03:49.560]another 10 acres in the western part of the state,
- [00:03:53.400]and it's all with stewardship in mind.
- [00:03:55.460]And so this is the facility that was started in 2005.
- [00:04:00.220]We're part of the USDA's APHIS BQMS program, which may be cut now, I have no idea,
- [00:04:05.680]but this is a compliance assistance program that helps you make sure you're in line
- [00:04:11.580]and in accordance with all the rules and regulations that govern the movement
- [00:04:14.680]and release of regulated materials.
- [00:04:16.920]And this is the infrastructure that really sets out the University of Nebraska
- [00:04:20.920]than other public sector institutions.
- [00:04:23.380]Okay, so that's a little background.
- [00:04:25.820]And now, the outcomes, the first little short story on soybeans.
- [00:04:29.180]The outcomes of editing the G model that underlines this major QTL found on chromosome 20
- [00:04:34.820]and its paralog found on chromosome 10 that has a strong impact on total seed protein at harvest.
- [00:04:41.760]Now, typically soybeans, about 36% protein, 90% oil, 30% carbohydrates and fiber and ash,
- [00:04:48.460]what a bean looks like, right?
- [00:04:50.520]And about 30 years ago, a guy named Brian D.
- [00:04:53.360]while walking the field with a soybean breeder, Walt Fair,
- [00:04:57.820]identified this QTL on chromosome 20, as I said.
- [00:05:02.180]And the underlying gene model that it took him 30 years to map-based fine clone this thing
- [00:05:07.340]is this number you see right here.
- [00:05:09.540]And this is confirmed by a number of independent groups that this is the gene model, okay?
- [00:05:14.820]And so it took 30 years to get this thing cloned,
- [00:05:17.640]but we still don't know the mechanism by which it influences seed reserves.
- [00:05:21.520]And that's very important for it.
- [00:05:23.340]It's dropped like soybean in all the major commodities.
- [00:05:25.240]So what they found out was that in this chromosome 20,
- [00:05:29.780]there is a small indel, about 300, not small, decent size,
- [00:05:33.720]insertion of 321 base pairs.
- [00:05:36.240]And it's right here on this exon right here, number 4 exon.
- [00:05:40.140]And what that does is it interrupts this little what's known as a CTT motif.
- [00:05:44.500]And if this indel is present there and interrupts this motif, you get a low protein.
- [00:05:50.180]If it's not there, you get a higher protein, all right?
- [00:05:53.320]And so when you know this, then you want to come out and ask the question, if you up-regulated
- [00:05:58.240]or down-regulated, what's the outcome there?
- [00:06:00.140]So you start gaining insight of what's going on there.
- [00:06:02.220]And that's exactly, oh, so let me just step back.
- [00:06:04.300]The CT domains are named, they're first described in the Rabidopsis, and this is where the name
- [00:06:10.100]comes from, because these are the gene families that they all have these little motifs.
- [00:06:13.500]It's about a 43 stretch of amino acids, as I mentioned, is found in the Rabidopsis.
- [00:06:18.000]And what they do is they kind of act like this is a recruiting aspect.
- [00:06:22.480]So they land upstream of a gene model, in this case, in terms of flowering.
- [00:06:29.100]And then they impact when this gene is expressed and not.
- [00:06:32.560]It's kind of what other players involved to either more expression or low expression.
- [00:06:36.900]So all these genes that have this CTT motif, this is how they function in a way.
- [00:06:42.760]And so what we did is then we designed an editing reagent that resides in this construct, 1576.
- [00:06:52.360]And then we had a fourplex of guide RNAs.
- [00:06:56.080]And of these guides, they had two of them will interrupt exon 2 of the main gene model on chromosome 20.
- [00:07:03.000]And then two of them will target the 5' UTR.
- [00:07:05.460]And one of the guides, this guide 1, will also hit the exon 2 of the paralog in chromosome 10.
- [00:07:11.540]And one will also hit the 5' UTR.
- [00:07:15.240]So the four gene models, all four will target the main gene model on chromosome 20 that has that indel.
- [00:07:22.260]And the other one that still has an intact CTT domain, it will only have one on exon 2 and one on exon 3.
- [00:07:28.560]We introduced this editing reagents into soybean.
- [00:07:32.240]We generated 18 independent events.
- [00:07:34.600]And then what we did is we looked at the next generation from a subset of this.
- [00:07:39.380]You do editing, it's a hell of a lot of work.
- [00:07:41.360]All right, to introduce the thing in there and get the edits, but then find it, weed it through, get it down to homozygosity,
- [00:07:46.600]a hell of a lot more work than actually working with the transgene.
- [00:07:49.520]But nonetheless, what you see, and this work was done,
- [00:07:52.160]mostly headed up by Ming Hua in our lab,
- [00:07:54.920]and everything you see with the blue,
- [00:07:56.560]so here's the wild type up here, what it looks like,
- [00:07:59.160]where the guides come in.
- [00:08:01.300]Everything you see in here is where the insert, a deletion,
- [00:08:04.880]and you go through the whole aspect or the 5' UTR.
- [00:08:08.260]And you can see, we see a whole array of different edits in line with this.
- [00:08:15.480]So we're making these various mutations that we couldn't follow all of them on,
- [00:08:18.360]so we took a subset of those.
- [00:08:20.140]And the subset of the edited lines, and this is,
- [00:08:22.180]T1 generation and T2 generation, we followed something like this.
- [00:08:25.640]And we have one line that we're following that's only edited in the chromosome 20 allele,
- [00:08:31.060]and that paralog is intact.
- [00:08:32.500]And the other line we're following, we're calling a dual edit,
- [00:08:35.560]where we call GM10 and DM20, where both the exon 2s have indels or insertions.
- [00:08:42.060]So this is a complete null of both the main gene model and its paralog,
- [00:08:46.680]or just the one on chromosome 20.
- [00:08:48.300]You're with me on that?
- [00:08:49.340]All right?
- [00:08:50.140]And so we then took these to the field.
- [00:08:52.920]You're not with me after you've laughed.
- [00:08:54.340]You're not listening.
- [00:08:54.820]You don't know what the hell I'm talking about.
- [00:08:55.900]That's okay.
- [00:08:56.580]I'll keep trying to do it better.
- [00:08:57.840]So here we took these to the field,
- [00:08:59.660]and then we have either the single interruption of the chromosome 20
- [00:09:04.180]or the dual insert, and this is in 2022.
- [00:09:06.360]And this is a more vegetative state.
- [00:09:08.440]They look pretty much the same.
- [00:09:09.540]But then at harvest, there was a drastic reduction in maturity.
- [00:09:13.260]All right?
- [00:09:14.000]Delayed, and here's the wild type.
- [00:09:15.240]These are just the border rows, border rows,
- [00:09:17.120]and then either the single mutation or the W mutation.
- [00:09:20.060]And then days of maturity, you could see,
- [00:09:22.160]oh, it's done.
- [00:09:22.620]So the single mutation, slight increase,
- [00:09:25.240]but the doubling is a major increase in maturity.
- [00:09:28.440]All right?
- [00:09:29.680]But the interesting thing was we saw a drastic reduction
- [00:09:33.520]in protein.
- [00:09:34.100]The single insert, slight reduction, but not significant.
- [00:09:37.000]I don't have this one.
- [00:09:38.060]This one was significant.
- [00:09:38.900]This one was not.
- [00:09:39.580]Okay?
- [00:09:40.080]So from about just under 30%, but it's under 40%,
- [00:09:44.080]down to about 30% at harvest.
- [00:09:46.080]Oil wasn't much changed.
- [00:09:47.960]Crude fiber, drastically reduced.
- [00:09:52.160]Reduced.
- [00:09:52.760]And then starch increases.
- [00:09:54.320]So now we have this soybean that has a drastic reduction in protein.
- [00:09:58.800]Elevation in starch.
- [00:10:00.460]Oil stays pretty much the same.
- [00:10:01.920]I think we're getting closer to a P.
- [00:10:03.880]So now we have about 60 pounds of this stuff,
- [00:10:06.500]and we're going to try just to test in this stuff for a direct food application.
- [00:10:09.460]So some of the things when you're doing this kind of discovery
- [00:10:12.360]with how a functionality of a gene works, you could come up,
- [00:10:15.100]can we make a product of that as you're going along the way?
- [00:10:17.620]Okay?
- [00:10:17.860]So what did we do then?
- [00:10:19.800]We had that outcome in here in Nebraska,
- [00:10:22.160]we sent the seed to Missouri, Georgia, South Carolina,
- [00:10:25.720]and we also sent it up to Minnesota,
- [00:10:27.560]but they didn't do a field trial.
- [00:10:28.740]So we did it in the field trials in these four areas in 2023,
- [00:10:32.480]and it was the same exact outcome.
- [00:10:34.580]And either the double edit or the single edit,
- [00:10:38.600]down seed reserve of protein keeps coming down at each location,
- [00:10:44.420]oil raised a little bit, but it was pretty much the same,
- [00:10:48.600]and then the starch went up.
- [00:10:50.460]So it wasn't that.
- [00:10:52.160]Regardless of the environment, same outcome.
- [00:10:54.700]Then we did a growth chamber study with both the dual edit,
- [00:10:59.500]single edit, and wild type under a short day or long day.
- [00:11:02.580]And it was only the short day where we saw this drastic increase in days
- [00:11:07.020]to maturity compared with the wild type.
- [00:11:09.920]And again, total protein went down, oil went up a little bit this time,
- [00:11:14.900]and starch also went up.
- [00:11:16.940]So now what are we doing?
- [00:11:19.500]Once you have these edits, you come back with stone
- [00:11:22.160]what's known as a complementation study.
- [00:11:23.840]We introduced all these four cassettes.
- [00:11:26.780]We took the 2KB upstream region of the glymacol on chromosome 20,
- [00:11:33.220]put it in front of the wild type coding sequence with the indel,
- [00:11:38.020]or without that transposon, still has that CTT domain in it.
- [00:11:43.020]And so we did that under the endogenous promoter here and here,
- [00:11:47.620]or the seed-specific promoter, beta-conglycinate.
- [00:11:50.500]Now, Charles,
- [00:11:52.160]at the Danforth Center essentially introduced equivalent to this one here,
- [00:12:00.580]this construct in this wild-type soybean, Maverick and Williams 82,
- [00:12:03.800]and saw a significant increase in total protein.
- [00:12:06.380]So we're expecting that to be the same outcome.
- [00:12:08.800]But here's the number of events we have in soybeans T2 population.
- [00:12:12.320]These will be in the field this year.
- [00:12:14.360]And so now we have this in wild-type,
- [00:12:18.120]all 40s in wild-type background, and a subset of those,
- [00:12:22.160]also in each one of those mutants.
- [00:12:24.060]So this is going to be, at the end of the day,
- [00:12:27.020]we want all combinations.
- [00:12:28.280]Mostly going to have to do that by breeding.
- [00:12:30.120]But this collective of all these biologicals
- [00:12:32.600]is going to be a very powerful resource
- [00:12:34.660]to allow us to understand how that gene model
- [00:12:37.680]and this paralog influence total protein seed reserves at harvest.
- [00:12:42.920]Now I'm going to jump ship.
- [00:12:45.260]All right, a little different, a little story.
- [00:12:47.320]All right, and this one is
- [00:12:49.260]Translational Outcomes Gaining Insight on
- [00:12:52.160]Understanding Host-Microbe Interactions.
- [00:12:54.060]We have Agrobacterium Tumor Fossians,
- [00:12:56.480]a vehicle to introduce novel genetic variation
- [00:12:59.500]for over 45 years in higher plants.
- [00:13:01.360]We knew how to do that from Understanding Host-Microbe Interactions.
- [00:13:04.740]We have these TALENs, genome editing reagents.
- [00:13:08.140]We understand how to do that from Understanding Host-Microbe Interaction.
- [00:13:12.140]And then we have CRISPR, right, coming on around 2012.
- [00:13:16.340]We understand how to do that, Understanding Host-Microbe Interactions.
- [00:13:20.320]This time it's a bacterium phage.
- [00:13:22.580]So sometimes you want to go through, as you're going through your life and career in science,
- [00:13:26.540]and you think you're doing this nebulous stuff, but at the outcome, it's very impactful translational biology.
- [00:13:32.540]In this case, all under the same space of the people in this world of Host-Microbe Interaction,
- [00:13:38.380]what's known as plant pathology and agriculture.
- [00:13:40.660]All right?
- [00:13:42.100]Yeah.
- [00:13:44.040]All right.
- [00:13:44.800]So here's a simplistic view of Host-Microbe Interaction.
- [00:13:47.920]When a plant cell, or even you, are challenged with an organism,
- [00:13:52.160]that organism either has molecular patterns around its outside
- [00:13:56.500]that that cell can recognize and put up a defense.
- [00:13:59.560]All right?
- [00:14:00.360]These are called PAMPs or MAMPs.
- [00:14:02.280]All right?
- [00:14:02.860]And that's the first line of defense.
- [00:14:04.480]But mostly microbes can overcome that.
- [00:14:07.020]They overcome that, then they can inject these so-called effector proteins.
- [00:14:11.440]And these effector proteins target cellular proteins
- [00:14:14.860]to perturb the cellular dynamics, if you will,
- [00:14:18.820]to make it more susceptible for disease.
- [00:14:22.160]And then the plant's trying to counteract that
- [00:14:24.720]with coming back with genes to overcome those effectors.
- [00:14:27.420]And so if you understand what targets these effectors are going after,
- [00:14:31.400]you can then understand, well, this is how this bacteria
- [00:14:34.660]or this organism wants to induce disease.
- [00:14:36.840]If we can understand that,
- [00:14:39.040]then we can understand how to protect against disease.
- [00:14:41.600]And one of these so-called effectors was cloned by,
- [00:14:45.220]this is led by Ming-Hua and Panya Kim,
- [00:14:47.840]where this one effector from this organism called Pseudomonas syringa,
- [00:14:52.160]Favard DC3000, introduces into the plant cell this effector called HOPPY1.
- [00:14:58.560]And this HOPPY1 targets a kinase called MAP65-1.
- [00:15:03.940]And these MAP65 family members are known to stabilize
- [00:15:09.120]and enhance stability of microtubules.
- [00:15:11.260]So now this effector is going in there, targeting MAP65-1,
- [00:15:16.880]disrupting the stability of the cellular matrix of these microtubules,
- [00:15:22.160]and in this way, making the plant more susceptible to disease.
- [00:15:26.300]So then we asked the question, what if we go in,
- [00:15:29.360]drop like soybean, and enhance expression of this MAP65,
- [00:15:33.400]in turn theoretically should more stabilize this microtubule matrix,
- [00:15:39.120]what will the outcome of that be?
- [00:15:41.060]And that's exactly what we did.
- [00:15:42.440]We introduced into soybeans two constructs called 6015 and 1613.
- [00:15:48.940]And we took the MAP65-1 from a rabbit option
- [00:15:52.160]or the soybean MAP65-1
- [00:15:55.020]and then constituently expressed them under the 35S promoter
- [00:15:58.340]and put those both in the soybeans.
- [00:16:00.140]And then just showing you the soybean transformation protocol,
- [00:16:02.980]all the major commodities.
- [00:16:04.520]It's about six months from the greenhouse,
- [00:16:07.400]from that laboratory to get a plant in a greenhouse,
- [00:16:09.460]so it's not a short turnaround time.
- [00:16:11.140]And here's the outcome of that.
- [00:16:13.020]We took in soybeans, here's the resistance to Pseudomonas glycine,
- [00:16:16.800]the Pseudomonas syringae, the strain that impacts soybeans.
- [00:16:20.440]And so this is the back.
- [00:16:22.160]The bacterial growth after you do that, the wild type is in blue,
- [00:16:24.600]and then the red is the soybeans that carry the expression
- [00:16:29.260]of the Arabidopsis version or ectopic expression of the soybean version.
- [00:16:33.720]So at day zero, no change.
- [00:16:35.860]This is a, orizolin is a herbicide that when you apply it,
- [00:16:39.940]destabilizes, its mode of action is destabilizing microtubules.
- [00:16:43.440]So when you do that, you see a slight reduction in the growth
- [00:16:47.340]of both these, either with the Arabidopsis or the soybean one.
- [00:16:50.720]But now when you add...
- [00:16:52.160]When you add the Orizolin, which will destabilize the microtubules
- [00:16:55.860]and then come back with the challenge of this bacterial pathogen,
- [00:17:00.480]you see a more significant reduction in growth.
- [00:17:06.660]So enhancing the stability with this overexpression of these MAP65 kinases,
- [00:17:13.060]either from Arabidopsis or soybeans, had an outcome of an enhanced
- [00:17:17.380]disease resistance towards this bacterial pathogen.
- [00:17:20.740]Then we looked at another...
- [00:17:22.160]Another pathogen, Phytophthora soji, again, in these lines,
- [00:17:25.860]either in thorn or with the Arabidopsis version or the soybean version.
- [00:17:31.020]And in each case, we saw a slight protection against this pathogen,
- [00:17:35.580]which you see here.
- [00:17:36.440]We did two strains.
- [00:17:37.640]This one strain over here was not even a pathogen on soybeans,
- [00:17:40.340]but this one was.
- [00:17:41.460]And here we saw a significant reduction.
- [00:17:43.980]In this case, what you're looking at is you're looking at the gene,
- [00:17:47.720]a constitutive housekeeping gene, if you will, from the pathogen.
- [00:17:50.660]And so let's...
- [00:17:52.160]The recovery of that amplification of that housekeeping gene
- [00:17:57.580]is significant, as it reflects on the growth of that pathogen,
- [00:18:00.300]just like we did with the bacterial one.
- [00:18:02.280]But in this case, we saw a significant reduction
- [00:18:04.900]in the ability of this pathogen to colonize soybeans
- [00:18:09.820]when they are expressing these two gene models.
- [00:18:12.680]Okay?
- [00:18:13.500]Lastly, as I mentioned, this herbicidal rhizoline
- [00:18:17.760]falls under the class, its mode of action of destabilizing microtubules.
- [00:18:22.160]That's how it acts as herbicidal activity.
- [00:18:24.140]And so logically, we think if we enhance the stability of that,
- [00:18:27.300]then we should see some herbicide-tolerant outcome.
- [00:18:30.720]And indeed, we did.
- [00:18:31.900]So when you challenge the soybeans that carry these two alleles,
- [00:18:36.740]transgenic alleles, either in tissue culture or spraying outcome,
- [00:18:40.900]we saw a herbicide-tolerant outcome.
- [00:18:43.520]So the take-home message of this second little story here is simply this.
- [00:18:49.320]Understanding host-microbe interaction,
- [00:18:52.160]allows you to gain insight on how that pathogen can induce in disease,
- [00:18:55.960]and that knowledge gained from that can help you for translational outcomes
- [00:19:00.160]that can benefit agriculture and disease resistance,
- [00:19:02.940]in this case, herbicide tolerance.
- [00:19:04.440]Now, jumping ship.
- [00:19:08.340]Now we're moving along here.
- [00:19:10.240]Still with me?
- [00:19:10.740]All right.
- [00:19:11.680]Now we're talking about, oh, the bioeconomy and stuff like this.
- [00:19:15.100]And so here we're trying to design, I dismissed it,
- [00:19:17.920]designing an idiot, that should be an E, I don't believe in this,
- [00:19:22.160]all that, designing an idiot-type sorghum feedstock for the bioeconomy.
- [00:19:25.300]Now, what I want to remind folks in this, in the United States of America,
- [00:19:30.140]we go through 20 million barrels of oil per day.
- [00:19:37.060]Let me say that again, 20 million barrels of oil per day.
- [00:19:42.740]And if you think you're going to replace that amount of energy in agriculture,
- [00:19:47.800]that's a huge hill to climb.
- [00:19:50.680]But we have a lot of people.
- [00:19:52.160]We have a lot of people out there saying, we're going to do this,
- [00:19:54.080]the 50 billion ton harvest.
- [00:19:56.000]We're going to make all this ethanol.
- [00:19:57.680]Ethanol is made for drinking, not driving.
- [00:19:59.700]All right?
- [00:20:00.700]But nonetheless, if there's a battle of oil,
- [00:20:03.860]and this is like every barrel is like 42 gallons, I think.
- [00:20:06.500]And in this case, most of it's for gasoline, distillates, and some others.
- [00:20:09.920]But there's some of these high-value products that we can do in agriculture
- [00:20:13.700]and displace a portion of that barrel of oil if you can
- [00:20:17.080]and create jobs in the interim.
- [00:20:19.440]All right?
- [00:20:20.100]And so that's the focus.
- [00:20:22.160]That always reminds me of the line that Dr. Speck likes to use.
- [00:20:26.040]Keep the height-to-reality ratio in check,
- [00:20:28.620]because sometimes that denominator is approaching zero.
- [00:20:31.340]All right?
- [00:20:32.120]Now, and so what we did here to design this ediotype of sorghum feedstock
- [00:20:37.200]for the bioeconomy, we looked at – again, that should be an E.
- [00:20:41.380]I didn't track the – I always do that.
- [00:20:44.080]All right?
- [00:20:44.680]That bothers the shit out of me.
- [00:20:46.640]All right?
- [00:20:47.160]So we looked at two input traits.
- [00:20:50.080]All right?
- [00:20:50.780]And we tried to make the smart –
- [00:20:52.160]canopy, leaf angle, right, like this,
- [00:20:55.680]try to increase the leaf just so it goes up.
- [00:20:58.100]We tried to reduce the stomatal numbers on the leaf surface
- [00:21:02.040]to improve water use efficiency.
- [00:21:03.580]And lastly, looking at an output trait.
- [00:21:06.820]Here we're trying to make vegetative lipids
- [00:21:08.960]in the vegetative portion of the plant.
- [00:21:11.000]Right?
- [00:21:11.700]And so you do this where people talk about sustainability,
- [00:21:15.060]and they always think about sustainability as,
- [00:21:17.320]oh, the impact on the environment,
- [00:21:18.500]the impact on society,
- [00:21:19.980]but they always ignore the third,
- [00:21:22.140]the third prong of sustainability,
- [00:21:24.060]that's economic sustainability.
- [00:21:25.800]For producing a ton of biomass,
- [00:21:27.900]you've got, a producer has to say,
- [00:21:30.040]am I going to get that money back?
- [00:21:31.540]And you're not going to really get it
- [00:21:32.980]if you're only going to have this biomass.
- [00:21:34.340]But if you can accumulate oil, right,
- [00:21:38.380]to feed the renewable diesel aspect,
- [00:21:40.500]that has value.
- [00:21:41.740]So the farm gate value of that ton is now increased,
- [00:21:45.400]and they won't have to rely on government subsidies
- [00:21:48.000]if they can do this.
- [00:21:49.560]All right?
- [00:21:50.180]Economic sustainability,
- [00:21:51.620]should be the first of the prong,
- [00:21:53.780]the other two fall below,
- [00:21:54.980]because if you don't have the economics,
- [00:21:56.460]you're going to have to have government support somewhere.
- [00:21:58.960]Sounded like Trump, huh?
- [00:22:01.560]Oh.
- [00:22:01.840]So we have this reliable platform
- [00:22:08.920]we've been doing since about 2005
- [00:22:10.820]to add novel genetic variation to sorghum,
- [00:22:13.140]and this is like soybeans.
- [00:22:14.120]It takes about, you know,
- [00:22:15.640]six months to get it from start
- [00:22:17.400]to get a plant in a greenhouse.
- [00:22:18.560]These are the folks that have been the workforce for us
- [00:22:21.140]over the last at least 10 years or so.
- [00:22:23.460]Cheryl's been with me for longer than that.
- [00:22:24.960]Sometimes she may regret it.
- [00:22:26.400]And covering all this,
- [00:22:28.440]it's an agrobacterium media transformation.
- [00:22:31.080]Time frame is six to eight months,
- [00:22:32.600]and we can do this in a grain genotype,
- [00:22:34.680]and so far we've done it in a couple of sweet genotypes,
- [00:22:36.980]Ramada and Rio, right?
- [00:22:38.960]And so first let me just talk about the,
- [00:22:40.940]I'm going to slam James Schnabel here a little bit.
- [00:22:44.060]So first let me talk about leaf angle, all right?
- [00:22:48.620]And so these mappers,
- [00:22:50.660]three different GWAS studies, three of them, right?
- [00:22:53.240]I have them listed here.
- [00:22:54.300]They all target this dwarf 3 allele.
- [00:22:57.780]Come up here in chromosome 7.
- [00:22:59.760]That's significant input on the leaf angle.
- [00:23:02.460]It has to be dwarf 3.
- [00:23:03.820]Look it.
- [00:23:04.460]Dwarf 3 is one of the dwarfing alleles,
- [00:23:08.820]and it's recessive, and it carries,
- [00:23:10.820]this was reduced to practice by a guy at Purdue
- [00:23:13.780]about 15 years ago.
- [00:23:15.300]There's also a little 882 base pair duplication
- [00:23:18.240]in exon 5 of the dwarf 3,
- [00:23:20.180]gene that interrupts it,
- [00:23:21.900]kind of like involved in growth regulatory transport.
- [00:23:24.540]But nonetheless, so they say, oh, look at here.
- [00:23:28.160]Here's one that's homozygous for the recessive allele,
- [00:23:30.800]laser up.
- [00:23:31.800]Here's one that's not, wild type, laser out.
- [00:23:34.480]I knew there was something wrong with this
- [00:23:36.920]because the genotype we are workhorse for transformation
- [00:23:39.400]is Texas 430.
- [00:23:40.420]That's also homozygous recessive for DW3.
- [00:23:44.820]It's laser like this.
- [00:23:46.560]So I didn't have to go through all these GWASs saying,
- [00:23:49.700]I don't think it's 430.
- [00:23:50.900]Maybe something related to it, but I don't know.
- [00:23:53.480]So what I did then is I think these sorghum people
- [00:23:56.360]are full of shit.
- [00:23:57.040]And then what I went back to do was rely on the maize folks.
- [00:24:00.000]The maize people know what they're doing.
- [00:24:01.560]And then the sorghum people say, oh, well,
- [00:24:03.420]I'll do what maize is doing.
- [00:24:04.240]And so we target ligulus.
- [00:24:06.980]And ligulus is right there, right
- [00:24:09.340]at the base of the blade and the stem proper.
- [00:24:12.520]And so what we did is took the two sorghum maize alleles
- [00:24:16.760]for ligulus 1 and ligulus 2, blasted
- [00:24:19.220]that to the phytosome in the sorghum,
- [00:24:22.000]and then came up with those homologs.
- [00:24:23.600]And then what we did is made a hairpin.
- [00:24:25.680]And we wanted to downregulate both ligulus 2 and ligulus 1.
- [00:24:29.780]We targeted the 3-prime UTR of each one of these gene models.
- [00:24:33.060]And this was work done with Nikhil.
- [00:24:35.120]He was at the University of Illinois under Steve Long,
- [00:24:38.040]and now he's currently at Illinois State.
- [00:24:39.700]And this vector was called 1355,
- [00:24:41.860]and we constitutionally expressed this hairpin
- [00:24:44.080]to dual downregulate these ligulus alleles.
- [00:24:48.740]And we kind of expect we knew what the outcome based what's
- [00:24:51.160]known in MACE.
- [00:24:52.680]And sure enough, this is really a southern blot analysis.
- [00:24:55.460]So when we probe with that, do those two, that hairpin allele,
- [00:24:58.980]these are the two endogenous things.
- [00:25:00.560]And when you do a southern blot like this,
- [00:25:02.040]it's really informative.
- [00:25:02.900]I like southern blots.
- [00:25:03.800]Nobody wants to do them anymore.
- [00:25:04.940]But here's what you can see in one shot.
- [00:25:06.660]These are the endogenous alleles,
- [00:25:07.980]and then all these other bands are the transgenic alleles,
- [00:25:10.780]all right?
- [00:25:11.380]But the outcome here, here's the copies of each one
- [00:25:14.040]of those endogenous alleles, and this is tissue taken
- [00:25:18.260]right from the dejuncture of the stem
- [00:25:20.400]and the leaf blade proper.
- [00:25:21.840]Here's wild type, expression of those copies
- [00:25:24.740]and nanograms of RNA, and then here's
- [00:25:27.300]three independent events, significantly down-regulated
- [00:25:30.060]in both of those gene models, all right?
- [00:25:32.600]And here's what it looks like, all right?
- [00:25:34.300]Here's a transgenic that carries it.
- [00:25:37.120]You can see even the head's got a bullet to it,
- [00:25:39.260]and then here's the wild type.
- [00:25:40.560]Here's a progeny from one that doesn't carry that hairpin,
- [00:25:44.340]and here's the one that does, all right?
- [00:25:46.360]Both of this.
- [00:25:47.780]And the value of doing this, you get planting density,
- [00:25:50.140]aeration through the lower part of the canopy,
- [00:25:52.760]and photosynthesis through lower part of the canopy,
- [00:25:55.760]because normally if you've got a field that looks like this,
- [00:25:59.840]all the sunlight's hitting the top,
- [00:26:01.680]and down bottom's nothing but shade, all right?
- [00:26:04.080]And so here's just a closer picture of this.
- [00:26:05.980]This is led by Ming Hua who's in charge of all this.
- [00:26:08.480]You can see the ligule here, and then it's gone into transgenics, all right?
- [00:26:12.820]And again, I showed you this picture.
- [00:26:14.100]So under the greenhouse, a significant reduction,
- [00:26:17.300]and leaf angle, and also under the field,
- [00:26:19.460]significant reduction in leaf angle,
- [00:26:21.740]and this is showing you some of the pictures of what that looks like, all right?
- [00:26:24.860]And so with this allowed to, we did a field trial.
- [00:26:28.040]Now, Nikhil did that, and this was done in Illinois.
- [00:26:30.600]And so what you're seeing is light availability through the canopy
- [00:26:34.340]at basically noon, much greater with the ones that are as a vertical leaf,
- [00:26:39.440]vertical plant architecture.
- [00:26:42.020]And then photosynthesis, canopy plot-wise photosynthesis
- [00:26:46.820]significantly increased because you're getting more photosynthesis
- [00:26:50.300]through the canopy, all right?
- [00:26:51.940]And so the outcome of that was we saw a significant increase in biomass,
- [00:26:57.080]some slight boost in the yield, and then no compromise in soil moisture.
- [00:27:02.860]So we had all these positive agronomic outcomes
- [00:27:05.980]with no change in water you'd need, all right?
- [00:27:09.900]It's a plus.
- [00:27:10.880]Now, I'll step back.
- [00:27:14.040]We don't know what a smart canopy looks like.
- [00:27:16.580]Right now, that's too much of a bullet.
- [00:27:17.900]We think if you could come up with a promoter
- [00:27:20.220]that's only on around that, comes on later in,
- [00:27:23.400]like maybe V5 or later, and then not control that hairpin,
- [00:27:27.660]that can give you this, we want upright up top
- [00:27:30.920]and out long down the bottom of the canopy, all right?
- [00:27:34.980]Folks have done this.
- [00:27:36.080]Freddie Allpeter did this in sorghum using genome edits,
- [00:27:39.180]and you can even get a more bullet, you know,
- [00:27:41.520]with a ligulus.
- [00:27:42.100]So it's probably not the ideal canopy.
- [00:27:44.620]A smart canopy, not sure.
- [00:27:46.500]What that quite looks like.
- [00:27:47.540]But I think with the hairpin model approach strategy
- [00:27:50.460]would be better than going with the gene editing model
- [00:27:52.960]because then you can get that upright up top of the canopy
- [00:27:55.780]and then slower leaf like this.
- [00:27:57.700]All right.
- [00:27:58.240]Now let me jump ship.
- [00:27:59.260]This was done with trying to alter a number of stomates
- [00:28:04.080]about the leaf surface.
- [00:28:05.280]This was done with John Ferguson,
- [00:28:06.580]who then was a postdoc with Andrew Leakey
- [00:28:08.960]at the University of Illinois.
- [00:28:10.200]And at that case, what we did is started about,
- [00:28:12.940]oh, in 2017, we just went back to a rabbit ops
- [00:28:16.360]assessment.
- [00:28:16.480]And took all these gene models, all these genes
- [00:28:19.060]involved, known involved in stomate organization.
- [00:28:23.020]Inducer of CBF, too many mouths to feed,
- [00:28:27.820]epidermal patterning factors, and so forth.
- [00:28:30.260]And we made designs for this, the overexpressed
- [00:28:33.900]or down regulation.
- [00:28:34.860]Hell of a lot of work.
- [00:28:36.220]And the only two that have been characterized so far,
- [00:28:38.760]these two I haven't read, and that's
- [00:28:39.860]why I'm going to give you some of the outcome results
- [00:28:42.480]from here.
- [00:28:43.660]But the point of showing you all this work is, yes,
- [00:28:46.340]there's a bottleneck in plant transformation.
- [00:28:48.100]But characterizing commodity crops
- [00:28:50.720]takes a lot of work and effort, too.
- [00:28:52.360]And so there's a lot to do.
- [00:28:55.320]And yes, you could speed up plant transformation.
- [00:28:57.180]But you know what that's going to do?
- [00:28:58.400]Just add to this list here that nothing's been characterized.
- [00:29:00.780]So what we did, and basically, and I'm
- [00:29:05.840]just showing you some of the models of how this thing works,
- [00:29:08.420]very complicated.
- [00:29:09.080]But nonetheless, when you change this thing,
- [00:29:11.400]it's been known in model plant species
- [00:29:13.680]that you can get a significant reduction in
- [00:29:16.200]the number of stomates that lay upon the epidermal factor
- [00:29:19.240]at least surface.
- [00:29:21.260]So then what we did is we had this constitutive promoter
- [00:29:24.500]ectopically expressing a synthetic version of EPF2
- [00:29:29.120]and stomagen, two of them.
- [00:29:31.380]It's a fusion protein we made, and I won't go into details
- [00:29:33.620]of why I selected that.
- [00:29:34.520]But that's what we did.
- [00:29:35.900]And then we also compared that to EPF1.
- [00:29:38.960]EPF1 was previously shown, and I should have
- [00:29:41.560]showed you a slide of that, to be able to significantly
- [00:29:44.540]reduce the number of stomates in wheat.
- [00:29:46.180]A C3 crop, and now we're working C4.
- [00:29:48.680]But when we did that, EPF1 overexpression,
- [00:29:52.180]we saw some reduction in some.
- [00:29:54.480]These are all independent events in sorghum,
- [00:29:56.520]carrying the ectopic expression of the sorghum EPF1.
- [00:30:00.540]Most of the lines, we didn't see much change.
- [00:30:03.400]A few we saw a reduction in stomata.
- [00:30:05.880]But remember, I just told you, in wheat,
- [00:30:08.320]this was a drastic reduction in stomates.
- [00:30:10.880]So a C3 outcome, ectopically expression
- [00:30:13.480]of wheat homologous, worked very well.
- [00:30:16.280]But a C4, we didn't see much.
- [00:30:18.160]However, when we did this synthetic version,
- [00:30:20.720]which was a fusion of EPF9, also known as stomagen,
- [00:30:24.100]or in EPF2, a fusion of those two,
- [00:30:27.040]I won't go into details, as I said,
- [00:30:28.580]but here's the wild type.
- [00:30:29.920]We saw significant reductions in the amount of stomates.
- [00:30:32.920]And so you can't test all of these.
- [00:30:35.080]It's a lot of work to do this.
- [00:30:36.640]So we just took two of these events here.
- [00:30:38.940]It's called 12B and 13A.
- [00:30:40.720]You see the numbers, about 30% reduction
- [00:30:43.540]in number of stomates, and did deeper characterization.
- [00:30:46.140]And that's what you're seeing here.
- [00:30:47.960]And so this line here, the counting of stomates
- [00:30:52.020]was greatly facilitated by a guy named Edgar Spalding.
- [00:30:55.400]He had a guy collaborating with him
- [00:30:57.400]named Nathan Miller from University of Wisconsin,
- [00:31:00.360]worked with Andrew Leakey to come up
- [00:31:02.200]with an imaging platform where once you take a picture,
- [00:31:04.600]the computer can tell you what's a stomate and it's not,
- [00:31:06.760]so it's easy to count.
- [00:31:07.680]But whether it was on the adaxial side of the leaf
- [00:31:10.440]or the abaxial side of the leaf,
- [00:31:12.280]I just reversed those, but it's fine.
- [00:31:14.200]You saw still the reduction
- [00:31:16.120]in the number of stomates was maintained
- [00:31:18.020]approximately 30%.
- [00:31:19.400]And so we did some greenhouse studies
- [00:31:22.220]with these materials, and then what we did
- [00:31:24.320]is kind of a drought mimic sort of thing.
- [00:31:26.440]And so here's the regular soil
- [00:31:28.080]moisture level over days
- [00:31:30.080]once the drought mimic was basically stopped watering.
- [00:31:32.480]And what you could see is
- [00:31:33.960]saving of water compared to the
- [00:31:36.320]wild type that has full number of stomates,
- [00:31:38.260]stomatal conductance maintained
- [00:31:42.540]in the ones that reduced
- [00:31:44.080]number of stomates relative to wild type
- [00:31:47.000]and photosynthesis being maintained in the ones
- [00:31:50.660]with the reduced stomatal numbers
- [00:31:53.700]across relative to wild type. This was published recently
- [00:31:56.540]in the Journal of Experimental Botany.
- [00:31:57.920]And then what we also done is drive down this picture of that
- [00:32:02.680]at harvest. You can see under
- [00:32:05.300]sufficient water or water withdrawal
- [00:32:08.020]we did pretty good. And so these look
- [00:32:11.600]good. But the outcome is a constitutive expressor
- [00:32:14.740]had some pleiotrophic effects. Very few
- [00:32:17.740]seed set. The plants kind of look spindly
- [00:32:20.980]and so forth like that. So what we've done over the last two
- [00:32:23.820]years is put different promoters in front
- [00:32:26.720]of that synthetic version of the EPF2
- [00:32:29.760]and stomagen that are only on more
- [00:32:32.640]in epidermal cells rather than the whole plant.
- [00:32:35.800]And these are the one
- [00:32:37.780]of them looks pretty good
- [00:32:38.660]and rather promising
- [00:32:39.480]and folks in Illinois are moving forward with that.
- [00:32:41.880]And so now I'm going to jump ship again.
- [00:32:45.320]We did these two input traits
- [00:32:47.240]to try to make this ediotype feedstock
- [00:32:49.740]for the bioeconomy.
- [00:32:51.640]Leaf angle, water use efficiency.
- [00:32:55.160]Now we're targeting the value added trait
- [00:32:59.040]vegetative lipids.
- [00:33:00.100]And so what I just told you about this,
- [00:33:02.500]we want to increase the farm gate value
- [00:33:04.980]of this harvest, the biomass of this
- [00:33:07.540]so this person is going to make a profit
- [00:33:10.200]from that rather than losing money.
- [00:33:12.420]All right.
- [00:33:13.100]And so we work with Vijay Singh,
- [00:33:15.280]who does this part of this conversion,
- [00:33:17.340]sending biomass down to Illinois.
- [00:33:19.640]And then we work with folks
- [00:33:21.440]in Brookhaven National Lab
- [00:33:23.600]to try to say when we're going through
- [00:33:25.240]this build test, learn to try to make
- [00:33:26.820]these vegetative lipids.
- [00:33:27.960]How can we make more?
- [00:33:29.460]What are we missing?
- [00:33:30.200]And then he's going to be telling us
- [00:33:32.140]what our target is.
- [00:33:33.520]And our target needs to be
- [00:33:35.140]something more than 5% tag.
- [00:33:37.300]If you get anything over 10% tag
- [00:33:38.960]in the vegetative lipids,
- [00:33:39.900]you got a product.
- [00:33:40.860]All right.
- [00:33:41.540]But nonetheless,
- [00:33:42.380]what we're trying to do is
- [00:33:44.520]feed this renewable diesel production.
- [00:33:47.000]All right.
- [00:33:47.740]Why soybean prices are going up?
- [00:33:49.800]Historically, it was driven by protein.
- [00:33:51.680]Now, if the protein and oil
- [00:33:53.960]are 50-50% of the harvest cost,
- [00:33:56.700]priced value,
- [00:33:58.060]because of renewable diesel.
- [00:33:59.900]All it is much better than biodiesel.
- [00:34:02.460]All right.
- [00:34:02.720]That's the driver is this.
- [00:34:04.180]So can we make a feedstock,
- [00:34:06.540]C4,
- [00:34:07.060]feedstock that we can harvest,
- [00:34:08.500]extract the oil and feed this industry.
- [00:34:11.400]Right.
- [00:34:12.240]And that was based off of this work that was done almost about 11 years ago in Australia,
- [00:34:16.240]where they put what they call the push-pull-protect model,
- [00:34:20.180]where they have,
- [00:34:21.400]they introduce that transcription factor called wrinkled one.
- [00:34:24.140]That's basically a global regulator of fatty acid biosynthesis.
- [00:34:27.600]They hooked it up to a DGAT,
- [00:34:30.360]which one of the final steps in lipid biosynthesis to make triacylglycerols and then protect by expressing,
- [00:34:36.820]an oleosin gene that protects that lipid droplet from being broken down.
- [00:34:40.380]Those are the core modules you need to make vegetative lipids.
- [00:34:43.660]In this case,
- [00:34:44.100]they did it in tobacco and they produced that tobacco that has approximately 16% oil in the leaves tag.
- [00:34:51.500]Soybean is 20%.
- [00:34:53.320]Okay.
- [00:34:53.980]So if you can translate that to a C4 feedstock,
- [00:34:57.640]you've got something.
- [00:34:58.560]And that's what we tried to do.
- [00:35:00.200]And I'm not going to go through all the iterations we did over the years.
- [00:35:02.900]Ed Cahoon's group is the major driver of this one.
- [00:35:06.080]And so,
- [00:35:06.820]this was the one that,
- [00:35:08.780]the two vectors that really gave us the best outcome.
- [00:35:11.400]And we probably did before this,
- [00:35:12.700]and I won't show you the data,
- [00:35:13.420]at least 50.
- [00:35:14.320]That was really a strikeout.
- [00:35:15.420]All right?
- [00:35:15.980]But this construct had those,
- [00:35:17.600]what you see in blue here,
- [00:35:19.100]was those core elements.
- [00:35:20.460]The transcription factor,
- [00:35:21.860]the DGAT,
- [00:35:22.880]and the oleosin.
- [00:35:23.900]And then we changed a few over here to try to,
- [00:35:26.340]based on what we were learning from Brookhaven,
- [00:35:28.700]can we change those over here?
- [00:35:30.540]All right?
- [00:35:31.120]And the outcome of this,
- [00:35:32.680]but now,
- [00:35:33.800]let me just step back.
- [00:35:34.680]These were designed to make median,
- [00:35:36.820]chain fatty acids.
- [00:35:37.800]In a testing platform of the vectors,
- [00:35:39.860]we do it in tobacco.
- [00:35:40.700]And so when you put these two vectors into tobacco,
- [00:35:43.660]you make medium chain fatty acids.
- [00:35:45.520]You put them in sorghum,
- [00:35:46.860]you don't make medium chain fatty acids,
- [00:35:49.300]but we saw a significant increase in total fatty acids.
- [00:35:52.640]So the take home message there is,
- [00:35:54.960]when you want to have a model species to give you quick data,
- [00:35:57.560]it better be predictive of the organism that you're trying to target.
- [00:36:00.560]In this case,
- [00:36:01.300]sorghum.
- [00:36:01.620]So tobacco is not a reliable predictor.
- [00:36:04.900]In this case,
- [00:36:06.120]for sorghum.
- [00:36:07.480]All right.
- [00:36:08.120]But nonetheless,
- [00:36:08.800]what you see here is a significant increase.
- [00:36:11.060]That's tag.
- [00:36:11.700]There's that little blob you see here is a soybean oil droplet.
- [00:36:14.440]And that's this wild type.
- [00:36:16.140]And you see the accumulation of tag.
- [00:36:17.860]You can measure it,
- [00:36:19.960]visually measure this.
- [00:36:20.840]There's total fatty acids,
- [00:36:22.060]everything,
- [00:36:22.600]and the amount of tag you see in this little orange color here.
- [00:36:25.400]Under the greenhouse conditions,
- [00:36:26.400]the primary event,
- [00:36:28.140]or in a grain sorghum,
- [00:36:31.840]or in a sweet sorghum.
- [00:36:33.020]So we got about maybe tag one and a half,
- [00:36:36.060]to two and a half percent.
- [00:36:37.160]Not bad.
- [00:36:37.900]All right.
- [00:36:38.500]But our goal is five to 10%.
- [00:36:40.680]All right.
- [00:36:41.360]So then what we did,
- [00:36:43.720]again,
- [00:36:44.040]looked at the next generation.
- [00:36:45.240]Is this inherited?
- [00:36:46.280]And indeed it was.
- [00:36:47.500]We're still getting about a little over 2% tag,
- [00:36:50.180]increase in total fatty acids.
- [00:36:52.140]All right.
- [00:36:52.820]Again,
- [00:36:53.640]in both a sweet genotype,
- [00:36:55.240]or in a grain genotype.
- [00:36:57.400]So this is pretty good.
- [00:36:58.420]And this was just recently communicated
- [00:36:59.880]in a plant biotechnology journal.
- [00:37:01.740]We took these to the field.
- [00:37:04.220]That's coyote.
- [00:37:04.960]Well,
- [00:37:05.460]you're seeing,
- [00:37:05.820]I won't go through that,
- [00:37:06.920]but this is the grain genotype.
- [00:37:08.360]When you take stuff to the field
- [00:37:09.880]and they're regulated,
- [00:37:11.400]it's a hell of a lot of work.
- [00:37:12.980]You got to bag the heads during flowering,
- [00:37:14.720]and then you got to bag the heads afterwards
- [00:37:16.720]to protect from birds feeding them.
- [00:37:18.120]But nonetheless,
- [00:37:18.920]this is done in 2020, 2021.
- [00:37:21.100]The oil sorghum events,
- [00:37:23.140]that's coyote there.
- [00:37:24.200]What you see there,
- [00:37:25.020]see that line there?
- [00:37:26.040]These are all Texas 430s.
- [00:37:27.340]That's where that dwarf three allele breaks.
- [00:37:29.380]And it goes right through the roof.
- [00:37:31.260]So if you ever see a field of sorghum,
- [00:37:32.660]it's not uniform, right?
- [00:37:34.260]It go like this, like this.
- [00:37:35.260]Some people start a company when they observe that.
- [00:37:37.540]That's an inside joke.
- [00:37:39.940]You know, that's an inside joke.
- [00:37:41.220]How many people know that?
- [00:37:42.140]So this is from the field in 2022.
- [00:37:47.440]And basically, I won't go through all this,
- [00:37:50.280]but it's holding up both in the green
- [00:37:52.500]and in the Ramada genotype.
- [00:37:54.620]The outcomes we saw in the greenhouse
- [00:37:56.540]is translating to the field.
- [00:37:58.100]About 2.5% tag.
- [00:37:59.920]We really want to at least double that.
- [00:38:01.920]My guess is you're going to want to get at least a 10%
- [00:38:04.700]because when you go through the processing outcome,
- [00:38:07.260]the less amount of oil you'll get,
- [00:38:08.820]the least efficient it is.
- [00:38:09.880]The more oil, the better efficiency that comes out.
- [00:38:12.000]We learned from that.
- [00:38:15.640]We had stuff going on.
- [00:38:17.300]From what we learned from those,
- [00:38:19.740]there was another vector.
- [00:38:20.680]I won't go into that.
- [00:38:21.460]What we learned from this,
- [00:38:23.060]the vectors are lead vectors
- [00:38:24.780]that gave us that 2, 2.5% tag in the stems
- [00:38:27.940]and a little bit higher in the leaves.
- [00:38:29.720]We tried to build a series of other vectors.
- [00:38:32.360]Try to build some more.
- [00:38:34.640]And we generated over 300 transgenic events
- [00:38:37.500]and nothing happened.
- [00:38:38.880]So I tell you this because people in my world
- [00:38:42.520]who go through all this work
- [00:38:43.880]and, oh, man, it could be two years later
- [00:38:46.280]and you got nothing.
- [00:38:46.940]Well, what have you been doing, Clemente?
- [00:38:49.400]Well, Elon Musk wants to know what you did last week.
- [00:38:51.900]I don't know, man.
- [00:38:52.560]I struck out, you know?
- [00:38:53.980]But here's what the people would help us learn.
- [00:38:56.380]All right, the group headed up by Ed.
- [00:38:58.260]Look younger there, thinner there.
- [00:38:59.920]That was before the Noom diet hit in.
- [00:39:01.540]All right.
- [00:39:02.400]But one of the things we did learn is that
- [00:39:04.580]the correlation between TAG expression
- [00:39:06.560]and accumulation or expression of these genes
- [00:39:10.560]was the DGAT and the cis-oleosin.
- [00:39:12.740]Strong correlation between TAG and that.
- [00:39:14.880]So that was a learning experience.
- [00:39:16.280]All right.
- [00:39:17.660]And so if this does come to fruition,
- [00:39:19.800]here's what we're going to be trying to do.
- [00:39:21.340]What would an oil sorghum cropping system look like?
- [00:39:24.000]We envision coming in in October
- [00:39:26.040]with something like camelina, canola,
- [00:39:28.300]winter canola, or pennycress.
- [00:39:29.680]So you can still get some oil, feed it constantly.
- [00:39:32.360]All right.
- [00:39:33.080]Harvest that.
- [00:39:34.520]Sometime in July.
- [00:39:35.660]And then we'll sow the seeds for the sorghum
- [00:39:38.260]that carry this lipid alleles, if you will,
- [00:39:41.320]if we get that final product level 10% in July.
- [00:39:43.960]Because we don't want seeds.
- [00:39:45.180]We only want biomass.
- [00:39:46.660]And what this allows you to do
- [00:39:48.480]is bypass the developmental stage
- [00:39:51.240]where you get a potential to outcrossing
- [00:39:52.780]the shatter cane and johnson grass.
- [00:39:54.200]And then you harvest in September.
- [00:39:56.800]And so now, this way, we can have a constant supply.
- [00:40:00.960]Not a constant, but a more uniform supply
- [00:40:03.760]to these processes.
- [00:40:04.460]Processing facilities to feed the renewable diesel.
- [00:40:06.860]If this all comes to fruition, this
- [00:40:08.860]is how we envision it would play out, if that makes sense.
- [00:40:11.040]All right?
- [00:40:12.320]Now, these are the folks I got to thank.
- [00:40:14.120]As I told you, these are the ones that
- [00:40:15.600]are workhorse doing the transformations.
- [00:40:17.020]These are the two now doing the phenotype vector assembly.
- [00:40:19.960]As I just told you, these are the folks
- [00:40:23.220]that have done a lot of the work for the lipid
- [00:40:25.600]and more characterization.
- [00:40:27.160]And these are the miscellaneous folks
- [00:40:29.140]that I've interacted with over the past seven years.
- [00:40:31.680]And I just have them lifted there.
- [00:40:34.400]And these are the organizations that have funded me
- [00:40:38.400]over the last couple of years.
- [00:40:39.540]I'm very grateful for that, primarily the soybean boards,
- [00:40:42.260]NSF, Department of Energy.
- [00:40:44.760]And we do it all for this.
- [00:40:46.120]And I keep telling us, I've been shown
- [00:40:48.320]the same image for decades.
- [00:40:50.240]You know, one of the greatest national security assets
- [00:40:53.000]the United States of America has is our ability
- [00:40:55.480]to produce a plentiful and safe food supply.
- [00:40:58.540]And we cannot leave anything on the table, right,
- [00:41:01.740]including biotechnology, and that brings me
- [00:41:04.340]to this.
- [00:41:05.100]This is the Clemente commentary, all right?
- [00:41:08.080]I don't know how many people know who that is,
- [00:41:10.180]but he had a famous quote, when things get weird,
- [00:41:12.740]when the going gets weird, the weird turn pro, all right?
- [00:41:16.940]I'm a pro, all right?
- [00:41:20.160]And so when somebody says to me, synthetic biology,
- [00:41:24.340]this is what pops into mind.
- [00:41:26.380]In microbes, we can cut and paste where you want.
- [00:41:29.820]You want to delete something, add a whole freaking pathway
- [00:41:33.140]wherever you want.
- [00:41:34.280]You can do it.
- [00:41:35.000]That's true synthetic biology.
- [00:41:37.240]We are not there in higher plants.
- [00:41:39.920]But people will keep saying we are here and I'm,
- [00:41:42.540]we got a chassis here, we're forward thinking loophole here
- [00:41:45.920]and blah, blah, blah, right?
- [00:41:47.660]We're going to get there, but we're not there yet.
- [00:41:50.500]In this case, this paper was published over 15 years ago
- [00:41:53.180]and they introduced into E. coli a pathway
- [00:41:56.220]that can allow E. coli to use glycerol as a substrate,
- [00:41:59.600]a very cheap substrate from biodiesel, renewable
- [00:42:04.220]diesel aspect, and make 1,2-propanediol
- [00:42:07.460]that's used in clothing,
- [00:42:09.920]that's used in paints and all this stuff,
- [00:42:12.960]a very high-value molecule.
- [00:42:14.360]Now, we are not there yet, but we will get there
- [00:42:18.880]and hire plants, true synthetic biology,
- [00:42:21.200]within the next decade, easily within the next decade.
- [00:42:24.500]It's going to happen. We're very close, and it's going to happen.
- [00:42:27.580]But what's going to be the roadblock when that does happen?
- [00:42:31.300]Genetically modified,
- [00:42:34.160]organism, GMO.
- [00:42:35.600]GMO is not a science term, an acronym.
- [00:42:40.260]It is a legal acronym.
- [00:42:41.900]It first came out probably in the New York Times, I think it was,
- [00:42:45.840]in 1980. Scientists don't really use this
- [00:42:49.060]because everything is genetically modified. You grow a plant in the field,
- [00:42:51.820]it's constantly getting hit by UV lights. There is freaking
- [00:42:54.820]mutations happen all the time. All the time.
- [00:42:57.340]Mutations happen in you all the time.
- [00:43:00.400]This has drastically impeded
- [00:43:04.100]products getting to the market.
- [00:43:06.560]This non-GMO project, I applaud them.
- [00:43:09.940]They've been very good because they exploited the ignorance
- [00:43:13.200]of the public. I've been showing these two examples
- [00:43:15.940]for over 20 years. In the late 90s, in the UK,
- [00:43:18.940]a survey was done where 40% of respondents
- [00:43:22.000]conducted understood that non-GMO
- [00:43:24.860]tomatoes contain genes. That means 60%
- [00:43:27.820]of the people didn't realize they're eating DNA.
- [00:43:29.760]In 2015, a survey was done at Oklahoma State University
- [00:43:34.040]where 80% of respondents wanted a label
- [00:43:36.920]on their food if it contained DNA.
- [00:43:38.440]This non-GMO means nothing to these people,
- [00:43:42.580]but you can exploit it in advertising
- [00:43:45.300]as a marketing scheme. Here we have non-GMO salt.
- [00:43:49.020]Here we have non-GMO freaking vodka.
- [00:43:52.220]Over here, we have non-GMO cannabis.
- [00:43:55.180]The last thing you want to do is get stoned
- [00:43:58.500]smoking the 35S promoter.
- [00:44:03.980]Here's the non-GMO great nuts.
- [00:44:06.980]If you ever see my talk, I give it to the public
- [00:44:09.400]because I've shown this slide before.
- [00:44:10.580]Here's the GMO great nuts.
- [00:44:12.200]You can see in the non-GMO, significant reduction in nutrition,
- [00:44:16.220]either in vitamin A, vitamin D, riboflavin, and vitamin D12.
- [00:44:19.760]Why is that? Because these vitamins come by engineered organisms
- [00:44:23.620]that are fermented. They can't add them in the quote non-GMO.
- [00:44:26.640]You're paying the same amount of money and you're getting ripped off.
- [00:44:29.840]You don't get less nutrition. You're stuck on stupor.
- [00:44:33.920]And then, here's the pet peeve of mine.
- [00:44:36.620]The allergenicity. Oh, you're going to introduce an allergenicity.
- [00:44:40.600]Allergen is a problem. There's no doubt about that.
- [00:44:44.020]In the U.S., you can't see this, but in the U.S.,
- [00:44:46.840]it's about 5% of the people have some type of food allergen.
- [00:44:49.420]And every year in the United States, approximately 200 people
- [00:44:52.800]die from food allergies. But most of those
- [00:44:55.960]deaths occur when they eat outside the house because they have no idea
- [00:44:58.520]what they're eating. Now, I feel sorry if you have a major food allergen,
- [00:45:01.200]but you have to learn how to cook and know how to eat labor
- [00:45:03.860]labels. But what happens here is this publication came on from a group
- [00:45:09.560]here in Lincoln, right over here, and they identified that a Brazil nut
- [00:45:14.140]allergen in transgenic soybeans was an allergen. So Pioneer Hybrid
- [00:45:17.800]introduced this seed reserve from Brazil nut into soybeans to increase
- [00:45:23.900]the total methionine content in a way to help you have to add
- [00:45:27.820]methionine when you're doing some feed applications. I'm not sure it was
- [00:45:31.760]a wise project to move forward. Sometimes those folks
- [00:45:33.800]are pioneer, not the brightest group. But nonetheless, what they did
- [00:45:37.880]here is saying, hey, we know that's a food allergen. What these folks did here is
- [00:45:41.560]they took those soybeans, purified that allergen, and
- [00:45:45.840]then did skin pricks. And sure enough, they selected folks that were
- [00:45:49.800]known allergic to Brazil nut, and they
- [00:45:53.860]got a little spot. And this set off a wave, like, oh my
- [00:45:57.940]God, biotechnology is increasing the allergenicity of all our plants.
- [00:46:01.400]And this set off a whole other realm
- [00:46:03.740]of more regulations that had to be done to make, say,
- [00:46:07.700]substantial equivalence between the wild type and the transgenic
- [00:46:11.800]regardless of the trait. The herbicide trait, the fangled
- [00:46:15.400]trait, is it the same? Is it more allergenic? And the problem is, why
- [00:46:19.560]this bothered me so much is, we try to work with those folks at Food Science and make
- [00:46:23.660]a hyperallergenic soybeans by putting in ERA2 and ERA6
- [00:46:27.900]from peanut. Much more significant food allergen than this.
- [00:46:31.640]We couldn't do it to work in a
- [00:46:33.680]mouse model because the best you can do in curtain, unless you do some
- [00:46:39.020]tricks, is about a half a percent of the total seed protein of that foreign
- [00:46:43.220]allele. That's about what we saw in the Brazil nut because this was done in
- [00:46:46.720]1996. I'm very confident if we purified this and did the skin test
- [00:46:52.220]prick, you would still have a spot of somebody who is known allergic to
- [00:46:56.720]peanuts. What I'm saying and what I'm showing here is
- [00:47:00.780]historically what they used to do, they don't do it anymore, is they had
- [00:47:03.620]once this all came up, you had to do this and this is what was done years ago
- [00:47:06.620]at Monsanto Company when they're trying to get
- [00:47:08.180]deregulation of Roundup Ready One. And here's the Roundup Ready One protein
- [00:47:11.780]when you do put it in simulated gastric fluid, within 15 seconds it's broken down.
- [00:47:16.760]One of the hallmarks of a food allergen, it could withstand your food,
- [00:47:19.860]your stomach gastric fluid because now it's being presented.
- [00:47:22.980]So you've got to ask, now most soybeans, we got five million acres in the state
- [00:47:28.760]of Nebraska, they're all going to feed. There is some
- [00:47:31.420]going to, you know, food products,
- [00:47:33.560]most of those it's going to be diluted down, you find it in your Snickers bar or
- [00:47:36.760]something like that. So if you only have a 5.5 percent,
- [00:47:39.960]and normal, so this particular allergen Brazil nut
- [00:47:42.860]is over five percent of the total protein, here it's tenfold less.
- [00:47:46.560]So now you can have the probability of an individual that has a food
- [00:47:51.520]allergen in the Brazil nut to be exposed to this and die,
- [00:47:54.620]was severely low, but this set off a storm
- [00:47:58.200]and drastically increased the cost of getting something deregulated and proved
- [00:48:03.060]for the
- [00:48:03.500]market is my take home message. And so the added to this is another pet peeve of mine is in the United States,
- [00:48:10.500]we have no ability to identity preserve materials. Everything's still sold as a market. Put it on a train, move it somewhere.
- [00:48:17.380]If we had the infrastructure to identity preserve something from the field to the feedlot,
- [00:48:23.280]we'd be much better off. And that's where investment should be made.
- [00:48:26.740]What's the problem with all the regulations? The regulations, the global regulatory agencies,
- [00:48:33.440]regulate the process by which you add genetic variation. They should be looking at the outcome
- [00:48:38.720]of that process. And this is a perfect example. Hyaluronic acid soybean, which should be the
- [00:48:43.220]commodity bean, and it's not. We know how to do this since 1992, John Browse's group.
- [00:48:46.980]You also have to do for soybeans is knock out this Delta-12 bisacterase. This was done through
- [00:48:51.580]mutagenesis, EMS mutagenesis. A small company, now bankrupt, did this for Talens at Genome Editing.
- [00:48:58.320]And then these two companies, Monsanto, then Monsanto, and Pioneer, did it through
- [00:49:03.380]hairpin technology. This regulated everywhere, this regulated nowhere, Canada may have some
- [00:49:09.760]questions, this regulated somewhere. Three different processes, same outcome, a soybean
- [00:49:15.480]oil that looks like olive oil. And so if the global regulatory agencies exchange one simple
- [00:49:20.340]thing, regulate the trade, not the process by which that trade is made. Huge breakthrough,
- [00:49:25.740]huge, not going to happen. And so here's another example, golden rice. Basically, it's beta
- [00:49:33.320]carotene. You're eating carrots. We were able to do this. Tricus did this in 2000. It was a
- [00:49:38.660]cover of time. Still not going off the rug. Maybe an allergen. Oh, you never know. Bullshit,
- [00:49:43.920]right? It's a damn shame. So we've been 45 years since we reduced the practice to add
- [00:49:51.340]higher novel genetic variation to higher plants. Here's the group that's probably
- [00:49:56.260]responsible for that. 45 years. How much progress have we made that now have the regular
- [00:50:03.260]regulatory, science-based regulatory governing the movement, development, and marketing of
- [00:50:09.060]these technologies? How much progress have we made in the past 45 years? Holy shit. It's
- [00:50:15.620]the big one, Elizabeth. Now we got a guy that's going to take, he's going to reverse 80 years
- [00:50:20.600]of poisonous farm policy. RFK Jr. That's where we're at. And this is called merit-based hiring.
- [00:50:27.440]Here's where we're at. This guy's going to do it. We're all good. Huh?
- [00:50:33.200]And what he wants to do is no synthetic fertilizers, no chemicals, no biotechnology.
- [00:50:39.420]It's the organic experiment. Have we done the organic experiment before? Damn right we did.
- [00:50:45.920]And it led to chaos. And here's an individual that commands $100,000 speaking engagement.
- [00:50:52.660]I got stale cookies. Remember, yield impacted by the genetics and the agronomics
- [00:51:03.140]we can't leave any technology on the table. And we have been leaving a major technology,
- [00:51:09.840]aisle technology, on the table for 45 years. It's enough.
- [00:51:15.700]Thank you. And this is the mic drop for the Tom and Shirley's Transgene R Us.
- [00:51:25.840]And always remember, if you were born with wings, why would you prefer to crawl?
- [00:51:33.080]And lastly, if you ever find yourself walking along the bike trail on the Rock Island bike
- [00:51:39.000]trail and around the Country Club neighborhood off at 33rd and Lake Street, you could take a
- [00:51:43.400]load off on our bench Shirley and I put up in memory of our four dogs that enjoyed walking
- [00:51:47.760]this trail. Thank you.
- [00:51:49.260]Questions?
- [00:52:03.020]Oh, we're trying to figure it out.
- [00:52:22.560]It's for the people online, sir.
- [00:52:32.960]With your biomass sorghum, what are you going to do with
- [00:52:36.800]the biomass that's left after you extract it?
- [00:52:40.380]That is still a question. Vijay Singh is trying to do
- [00:52:45.320]some other anthocyanins. Can you do more of that?
- [00:52:48.440]If it could be used for feed or something, I don't know, but you can't throw it away.
- [00:52:52.900]That's for sure. I don't have a good answer for that,
- [00:52:56.760]but you can't throw it away. I don't know.
- [00:53:02.900]You can't throw it away because that gets back to the economic sustainability.
- [00:53:07.040]Absolutely. David.
- [00:53:09.700]I have a question about the vertical leaf sorghum.
- [00:53:13.720]If we imagine a maize leaf or a sorghum leaf, for that matter,
- [00:53:18.560]with its blade and its sheath separated by the ligule,
- [00:53:22.920]you have to imagine that that has some effect on the structural
- [00:53:26.740]integrity. The sheath wraps around the stem
- [00:53:30.080]and forms part of that.
- [00:53:32.840]Mechanical rigidity.
- [00:53:34.060]Have you thought about any negative effects of getting rid of the ligule
- [00:53:40.620]in terms of lodging or that kind of thing?
- [00:53:42.920]I haven't thought about that, but what we have thought about is
- [00:53:46.820]that the ligus alleles are also involved in hormone movement
- [00:53:51.980]and so forth like that, and some disease pressure.
- [00:53:54.560]That's why the constitutive one may have other off-target effects
- [00:54:00.000]that you don't want besides what you're trying to articulate
- [00:54:02.780]and that's why I think if we had a promoter
- [00:54:04.740]that was coming on just about the ligulus
- [00:54:06.960]right around that junction there, it would be much better.
- [00:54:10.200]I don't think about lodging per se.
- [00:54:13.360]It's mostly the tall plants that get lodged very easily,
- [00:54:16.840]so like that ramada is huge.
- [00:54:18.280]If you get something with dwarfin, they get the thick stem
- [00:54:21.000]that they're usually okay.
- [00:54:22.200]I'd be more concerned of diseases and other things
- [00:54:25.300]that you don't think of because of down-regulating
- [00:54:27.340]those ligulus things.
- [00:54:28.180]They have those hormonal changes
- [00:54:30.180]that could impact signal transduction
- [00:54:32.720]when challenged with the pathogen.
- [00:54:33.980]That would be my guess.
- [00:54:36.180]Be something, an off-type, if you will.
- [00:54:38.520]Tom, could you elucidate on your comment
- [00:54:43.180]that you can't throw it away?
- [00:54:44.900]The reason why I ask that is in wheat,
- [00:54:47.180]Bob Klein, who's one of our best agronomists,
- [00:54:50.140]basically says if you want to get straw,
- [00:54:52.480]buy it from your neighbor
- [00:54:53.960]because the residue is so valuable
- [00:54:56.000]for maintaining soil health,
- [00:54:58.360]trapping moisture and the like.
- [00:54:59.600]So is there a reason why you can't throw it away?
- [00:55:02.660]Well, I'm just saying for price, Steve.
- [00:55:04.580]If somehow the producer can get that money back
- [00:55:07.280]by doing what you just said,
- [00:55:08.900]that would be great.
- [00:55:09.820]But again, when I hear people like you go to meetings now,
- [00:55:12.860]you hear that term sustainability.
- [00:55:14.180]When I was in college,
- [00:55:15.200]they used to have the Bob Newhart show.
- [00:55:17.360]Every time you said Bob, you had to do a shot.
- [00:55:19.040]So when if now every time you go to a conference
- [00:55:23.480]and somebody says sustainability,
- [00:55:24.360]you have to do a shot,
- [00:55:25.260]you'd be bombed by the first time.
- [00:55:26.880]So my thing is it's a pet peeve of mine
- [00:55:30.320]that everyone ignores economic sustainability
- [00:55:32.600]and that's what I was trying to get at.
- [00:55:34.120]Whether that could satisfy that, I don't know.
- [00:55:36.400]Comment?
- [00:55:41.760]Clemente, don't tell me.
- [00:55:43.060]It's somebody from food science.
- [00:55:43.980]He has no idea how bad the allergies are.
- [00:55:45.860]He has no idea this is very important.
- [00:55:47.800]BT may kill you.
- [00:55:49.160]I know.
- [00:55:50.440]I heard it all before.
- [00:55:51.840]Go ahead.
- [00:55:52.420]I would like to have one question about
- [00:55:57.060]when you try to make the,
- [00:55:59.160]try to insert the tDNA into the
- [00:56:02.540]QRT called 5-prime tDNA
- [00:56:06.840]in the chromosome 20 in soybean,
- [00:56:09.460]is it on the slide that you tried to insert it
- [00:56:13.880]in 5-prime UTI and exome 2 maybe?
- [00:56:16.240]Right.
- [00:56:16.740]My question is,
- [00:56:17.600]why do you do it in the 5-prime UTI and exome 2?
- [00:56:21.300]Is that because you're based on some other research?
- [00:56:23.680]Or is it randomly chosen?
- [00:56:25.720]Right.
- [00:56:26.220]So the idea was if you hit the 5-prime UTR,
- [00:56:28.780]you wouldn't get a total null.
- [00:56:30.020]It could just be lower expression,
- [00:56:31.340]almost like a hairpin.
- [00:56:32.480]And if you hit the exon, that was a null.
- [00:56:34.100]We just decided to go with the two exons
- [00:56:37.160]and said that's all we did.
- [00:56:38.360]There's a whole other vectors we made
- [00:56:41.720]that we just did in context promoter bashing with these,
- [00:56:44.080]and we didn't even look at that
- [00:56:45.040]because we didn't have enough money to do it.
- [00:56:46.560]But the reason why we targeted the 5-prime UTR
- [00:56:48.720]is that's a way to just get some down regulation
- [00:56:51.340]rather than a total null, not null.
- [00:56:53.360]And the second thing is after making knockdown,
- [00:56:55.980]you have the Y-type plan.
- [00:56:57.660]The mutant plan could increase the amount of stage
- [00:57:01.300]but lower amount.
- [00:57:02.420]So if you have a protein and a little bit increased amount
- [00:57:04.200]of oil, do you, but the purpose of that,
- [00:57:07.980]as that is in the soybean, and as I know that in soybean,
- [00:57:10.820]normally we expect with high amount of protein,
- [00:57:12.940]but in here when you knockdown, the protein amounts reduce.
- [00:57:16.180]Right.
- [00:57:16.740]So what we're trying to do there is just trying
- [00:57:18.920]to understand the function of this gene to see
- [00:57:20.700]how can we use it to have a designer bean,
- [00:57:23.000]we want this much oil, this much protein.
- [00:57:24.720]In this case, if we have a bean,
- [00:57:26.880]and now it's starting to look like a pea,
- [00:57:28.080]a pea is about 25% starch.
- [00:57:29.680]And so Ed and Ozan are going to try to look
- [00:57:32.360]at some of this.
- [00:57:32.960]Is there some direct food applications
- [00:57:34.580]that we can do with this bean?
- [00:57:35.640]It has a little bit starch, lower protein,
- [00:57:37.980]same amount of oil.
- [00:57:38.760]And you have to remember,
- [00:57:39.660]we can engineer the oil in soybean very easily.
- [00:57:41.820]We have an oil in soybean that is like a high solids,
- [00:57:44.320]high stearic and high oleic.
- [00:57:45.960]You know, where do you combine that
- [00:57:47.660]with a bean that looks like that?
- [00:57:48.760]Can you make a food product with that?
- [00:57:50.120]And so your answer is yes.
- [00:57:52.120]You don't want low protein
- [00:57:53.200]when you're trying to go for feed, right?
- [00:57:54.880]But it can have other uses,
- [00:57:56.320]and that's what we're trying to do.
- [00:57:57.620]All right.
- [00:57:57.940]Explore whether it's not.
- [00:57:59.280]But the main thrust of this project
- [00:58:01.000]is to understand the function
- [00:58:02.300]of this gene
- [00:58:03.060]and then learn from that
- [00:58:04.280]and then give that information
- [00:58:05.500]to the breeder
- [00:58:06.160]and allow the breeder to say,
- [00:58:07.400]all right, in the future,
- [00:58:08.480]you know, do I want,
- [00:58:09.620]it's not that way now
- [00:58:10.520]because beans are sold by weight.
- [00:58:12.280]You want a high, you know,
- [00:58:13.760]high oil bean,
- [00:58:14.600]you want a high protein bean this year,
- [00:58:16.440]you can do it.
- [00:58:17.320]But that's not going to be a benefit
- [00:58:18.680]until we get the infrastructure
- [00:58:20.140]to allow for identity preservation
- [00:58:22.180]from the farm to the processing.
- [00:58:23.700]We don't have that infrastructure
- [00:58:24.920]in the United States.
- [00:58:25.600]And the third question is,
- [00:58:27.180]you try to make the whole plan
- [00:58:29.860]with the leaf up, right?
- [00:58:32.460]But my question is whether
- [00:58:33.380]we can use two different lines
- [00:58:35.340]grown together.
- [00:58:36.360]One line is for the leaf straight up
- [00:58:40.140]and another line next
- [00:58:41.100]is a little bit shorter plan,
- [00:58:42.520]but the leaf is always open.
- [00:58:44.060]So if it's a shorter plan,
- [00:58:45.740]you're going to get a shading effect.
- [00:58:46.900]You don't want to have that.
- [00:58:47.760]You want everything the same height.
- [00:58:48.920]You wouldn't want to dual crop something
- [00:58:50.940]that's one tall, one small.
- [00:58:52.240]That's the shading's
- [00:58:53.380]going to impact you there.
- [00:58:54.260]And then if you had one wide
- [00:58:55.980]and one this,
- [00:58:56.560]that's going to,
- [00:58:57.220]you want it,
- [00:58:57.780]one of the values
- [00:58:58.480]of the upright canopy
- [00:59:00.400]is planting density.
- [00:59:01.680]You can get more
- [00:59:02.180]plants per hectare or acre.
- [00:59:03.800]So that would,
- [00:59:04.820]you would deplete
- [00:59:05.880]that aspect of it
- [00:59:06.760]if you did that, right?
- [00:59:08.120]All right.
- [00:59:08.640]Thank you.
- [00:59:10.180]All right.
- [00:59:10.640]James.
- [00:59:11.920]He's going to tell me
- [00:59:15.460]it's dwarf three.
- [00:59:15.960]I'm full of kids.
- [00:59:16.540]I knew that.
- [00:59:17.720]I completely agree with you.
- [00:59:19.180]One of the big limitations
- [00:59:20.160]we face is how hard
- [00:59:21.220]it is to identity
- [00:59:21.860]preserve anything
- [00:59:22.460]and it just adds so much
- [00:59:23.460]to your cost right away.
- [00:59:24.560]So I am curious,
- [00:59:25.980]what are the things
- [00:59:27.780]that you think
- [00:59:28.240]would make the most sense?
- [00:59:29.200]You know,
- [00:59:29.480]we're going to get rid
- [00:59:30.700]of the FDA, USDA,
- [00:59:31.480]you can put whatever
- [00:59:32.120]you want into a soybean.
- [00:59:32.940]What are the things
- [00:59:34.400]that economically
- [00:59:35.000]would make sense
- [00:59:35.820]right now to be putting
- [00:59:37.220]in a soybean
- [00:59:38.060]that would justify that cost?
- [00:59:39.580]Oh, for soybean,
- [00:59:40.960]it would be the aquaculture.
- [00:59:43.220]Oh, the soybean.
- [00:59:44.600]Yeah, not just taurine,
- [00:59:46.860]just be the carotenoids,
- [00:59:49.020]astaxanthin, and omega-3s.
- [00:59:50.560]We can make a soybean
- [00:59:52.740]that has all the ingredients,
- [00:59:54.440]terrestrial-based ingredients
- [00:59:55.680]for aquaculture.
- [00:59:56.440]That would be,
- [00:59:57.580]that's no,
- [00:59:58.940]hands down,
- [00:59:59.880]that's what you want to do.
- [01:00:00.740]Okay.
- [01:00:01.100]Yeah, no doubt.
- [01:00:02.060]Aquaculture, yeah.
- [01:00:02.840]Yeah.
- [01:00:03.200]Aquaculture.
- [01:00:04.060]Thank you.
- [01:00:04.680]Any other questions?
- [01:00:11.560]This is kind of a sayonara
- [01:00:19.520]for a long-term,
- [01:00:21.780]very good record here.
- [01:00:23.000]Do you have any
- [01:00:24.040]particular publication
- [01:00:26.280]that you have
- [01:00:27.760]that you thought
- [01:00:28.700]you ought to be,
- [01:00:30.500]that you really liked
- [01:00:32.000]and really got cited a lot
- [01:00:34.140]or something
- [01:00:34.680]that you thought was your best?
- [01:00:36.380]I know this kind of hits you
- [01:00:38.180]kind of early here,
- [01:00:39.480]but if you had anything
- [01:00:40.400]that you wanted to tell us.
- [01:00:41.360]I could just tell you,
- [01:00:42.780]I got this thing here.
- [01:00:43.480]I could just tell you that,
- [01:00:44.660]you know,
- [01:00:45.000]publications never really
- [01:00:46.460]turned me on.
- [01:00:47.180]That wasn't my thing.
- [01:00:48.340]I wanted products.
- [01:00:49.160]And I only was able
- [01:00:50.280]to do intellectual contribution
- [01:00:51.420]to one product
- [01:00:52.040]to make it on the market,
- [01:00:52.880]and that was
- [01:00:53.620]the dicamba tolerance.
- [01:00:54.880]You know,
- [01:00:55.580]the big impact on that,
- [01:00:56.680]that was great.
- [01:00:57.220]But the biggest frustration
- [01:00:58.740]in my life,
- [01:00:59.480]I don't give a shit
- [01:01:00.280]about publications.
- [01:01:00.900]I really,
- [01:01:01.260]it was great.
- [01:01:01.940]I realize it's a metric
- [01:01:03.240]and stuff,
- [01:01:03.600]but it's getting products
- [01:01:04.620]on the market.
- [01:01:05.140]And it's because
- [01:01:06.120]of these regulatory processes
- [01:01:07.420]that hindered that.
- [01:01:08.840]It's been the most regretful
- [01:01:10.520]thing of my career.
- [01:01:11.460]I should have stayed
- [01:01:12.520]on the garbage trucks
- [01:01:13.520]because it was just a shame
- [01:01:15.260]that we're still here
- [01:01:16.060]after 45 years.
- [01:01:17.260]That's my biggest frustration
- [01:01:19.820]is not,
- [01:01:20.700]there's products
- [01:01:21.760]that remain on the shelf
- [01:01:22.880]across this globe
- [01:01:24.060]that can help
- [01:01:25.040]the massive farmer,
- [01:01:26.160]small stakeholder farmer,
- [01:01:27.640]but it's not because
- [01:01:29.160]of these non-science
- [01:01:30.160]based regulations
- [01:01:30.980]and we,
- [01:01:31.880]this taken
- [01:01:32.320]with this latest round
- [01:01:33.360]of hiring,
- [01:01:33.940]merit-based hiring,
- [01:01:35.420]that it just took us
- [01:01:37.520]50 years back,
- [01:01:38.700]50 years back.
- [01:01:39.880]All right,
- [01:01:42.540]we have time
- [01:01:43.040]for maybe one more question.
- [01:01:44.400]I know I'm slow.
- [01:01:50.160]So my question is that
- [01:01:52.800]when we talking about
- [01:01:54.140]the increasing oil content
- [01:01:55.940]and basically this is that
- [01:01:57.800]if you have higher protein
- [01:01:59.600]and oil content go down.
- [01:02:01.820]So in your career,
- [01:02:03.180]long career,
- [01:02:04.560]did you ever think about
- [01:02:06.000]to knock out the pathway
- [01:02:08.460]that is going to be
- [01:02:09.700]increased the protein
- [01:02:10.740]means like amino acids
- [01:02:12.100]to increase the oil content?
- [01:02:13.920]So there's an indirect,
- [01:02:15.980]you know, as I think
- [01:02:16.940]you're alluding to there,
- [01:02:17.660]there's an indirect relationship
- [01:02:18.780]with the protein and oil
- [01:02:19.680]and the more protein
- [01:02:20.940]you tend to get less yield.
- [01:02:23.220]And so there's a penalty that way.
- [01:02:25.040]And as I mentioned,
- [01:02:25.800]historically soybeans
- [01:02:26.840]was driven by protein.
- [01:02:28.600]Oil was just like icing
- [01:02:30.020]on the cake that sold,
- [01:02:30.780]but now it's 50-50
- [01:02:31.760]and so there's going to be,
- [01:02:33.360]you know, there may be
- [01:02:34.240]the need now to go,
- [01:02:35.320]you know, get a bean.
- [01:02:36.620]I think the best you can do
- [01:02:38.020]in soybeans, 26, 20% oil.
- [01:02:40.400]You know, if you get a bean
- [01:02:41.800]that could get in the mid 30s,
- [01:02:43.440]upper 30s,
- [01:02:44.100]I think that would have value.
- [01:02:45.480]But as you go to the elevator now,
- [01:02:47.340]it's not based on the constituents.
- [01:02:49.280]It's just based on weight.
- [01:02:50.440]And that, until that metric changes,
- [01:02:52.880]people are just going
- [01:02:53.580]to be going for yield,
- [01:02:54.400]yield, yield, yield,
- [01:02:55.740]and then that's it.
- [01:03:01.700]One more chance.
- [01:03:04.820]All right.
- [01:03:06.300]Thank you, Dr. Kemp.
- [01:03:07.120]All right.
- [01:03:07.380]Well, thank you.
- [01:03:07.860]Thank you.
- [01:03:14.300]Thank you.
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