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Protein derived from cottonseed for human nutrition is one step closer to reality

22Oct

By: Kay Ledbetter
Contact: Dr. Keerti Rathore – rathore@tamu.edu

Cottonseed ground into flour to deliver protein to millions of people, a project to which Dr. Keerti Rathore has devoted more than half his professional career, is one step closer to reality.

Dr. Keerti Rathore looking at immature cotton boll

Dr. Keerti Rathore, a Texas A&M AgriLife Research plant biotechnologist in College Station, received word that Texas A&M’s “Petition for Determination of Non-regulated Status for Ultra-Low Gossypol Cottonseed (ULGCS) TAM66274” has been approved by the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service, or APHIS. (Texas A&M photo by Lacy Roberts)

Rathore, a Texas A&M AgriLife Research plant biotechnologist in College Station, received word that Texas A&M’s “Petition for Determination of Non-regulated Status for Ultra-Low Gossypol Cottonseed (ULGCS) TAM66274” has been approved by the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service, or APHIS.

Texas A&M University Chancellor John Sharp, who oversees Texas A&M AgriLife Research along with 11 universities and seven state agencies, said Rathore’s work will have a dramatic effect across the world.

“The work and dedication of Dr. Rathore has paid off,” Sharp said. “He and his team exemplify the values of the Texas A&M System, and because of them, more than half a billion people across the world may have access to a new form of protein, and our farmers will be able to earn a much better living.”

Through a project funded by Cotton Incorporated, Rathore and the Texas A&M team have developed a transgenic cotton plant – TAM66274 – with ultra-low gossypol levels in the seed, while maintaining normal plant-protecting gossypol levels in the rest of the plant.

Dr. Kater Hake, vice president of agricultural and environmental research at Cotton Incorporated, said it has been a decades-long journey.

“Gossypol suppression in cottonseed has been part of our funded research portfolio for over 30 years,” Hake said.

Tom Wedegaertner, director of cottonseed research and marketing at Cotton Inc., underscores the potential of the breakthrough and the journey through the regulatory process.

cottonseeds cut in half to show many black specks inside seed

Seeds containing gossypol have glands showing up as black specks. (Texas A&M AgriLife photo by Dr. Devendra Pandeya)

“Gossypol in the leaves and stalks of the cotton plant serve as a pest deterrent, but its presence in the seed serves no purpose,” Wedegaertner said. “The more widespread use of cottonseed as a livestock feed and even for human consumption has been stymied by the natural levels of gossypol in the seed. As we progress through the regulatory review, the ability to utilize the protein potential in the seed gets that much closer.”

The recent USDA action confirms that TAM66274 and any cotton lines derived from crosses between TAM66274 and conventional cotton or biotechnology-derived cotton granted non-regulated status by APHIS are no longer considered federally regulated articles, he said.

For the past 23 years, Rathore has been determined to create cotton plants that produce seeds containing gossypol well below what the U.S. Food and Drug Administration considers safe levels while maintaining normal levels of gossypol and related chemicals in the foliage, floral parts, boll rind and roots.

cottonseed halves showing very few black specks.

Glands are still present, but are much lighter, reflecting the very low levels of gossypol in the deregulated cottonseed. (Texas A&M AgriLife photo by Dr. Devendra Pandeya)

Gossypol, while toxic to humans and monogastric animals such as pigs, birds, fish and rodents, is useful to cotton plants for defense against insects and pathogens. Therefore, cottonseed containing gossypol is currently used mainly as ruminant animal feed, either as whole seed or cottonseed meal after oil extraction.

“Biotechnology tools that made the ULGCS technology successful had just become available when I started looking at the potential to make this new source of protein available to hundreds of millions of people,” Rathore said.

“I also realized the value to cotton farmers everywhere of removing gossypol from the cottonseed because such a product is likely to improve their income without any extra effort on their part or additional input,” he said. “Such a product can also be important from the standpoint of sustainability because farmers will produce fiber, feed and food from the same crop.”

Cotton-producing countries with a limited supply of feed protein can realize great benefits by utilizing this seed-derived protein as a feed for poultry, swine or aquaculture species, Rathore said.

These animals are significantly more efficient in converting plant protein into high-quality meat protein, he said. Egg and broiler production could become the most efficient use of any available feed protein source, including the ULGCS.

Despite the obstacles, failures and lack of funding at times, Rathore said it was the dedication and loyalty of his team and supporters such as the late Dr. Norman Borlaug, who was known as the “father of the Green Revolution,” that kept him going on this project.

“Dr. Borlaug was the biggest supporter of this project. During the lean times when I was struggling to get funding and after the failed attempts – there were many, it was his words of encouragement that provided the inspiration to continue,” Rathore said.

While there were many team members over the years working on the project, he said key contributors to its advancement were Dr. Devendra Pandeya, LeAnne Campbell, Dr. Sreenath Palle and Dr. Sunilkumar Ganesan, all who worked in his laboratory at Texas A&M, as well as by Dr. Robert Stipanovic and associates with USDA-Agricultural Research Service who conducted biochemical analysis of gossypol levels in the ULGCS lines.

Dr. Rathore and two others in a greenhouse with cotton plants

Dr. Keerti Rathore discusses the ultra low gossypol cotton with his team, Dr. Devendra Pandeya and LeAnne Campbell. (Texas A&M photo by Beth Luedeker)

“It feels good to have come this far as Texas A&M AgriLife is only the fourth public institution to have accomplished such a feat as deregulation of an engineered crop.”

Rathore has been granted several U.S. patents. In 2006, he published in the Proceedings of the National Academy of Sciences announcing the cotton plants had been successfully altered in the lab to “silence” gossypol in the seed. In 2009, field trials verified the lab and greenhouse studies indicating the crop could become a source of protein.

The cottonseed from these plants met World Health Organization and FDA standards for food consumption, he said, thus opening the potential to make the new source of high-protein food available to hundreds of millions of people a year.

Rathore said cottonseed, with about 23 percent protein content, can play an important role in human nutrition with the gossypol eliminated, especially in countries where cereal/tuber-based diets provide most of the calories but are low in protein content.

“Growing up in rural India as the son of a doctor, I had seen the effects of malnutrition firsthand in my father’s patients,” he said. “Many of their health issues were due to inadequate food and nutrition.”

Rathore said for every pound of cotton fiber, the plant produces about 1.6 pounds of seed. The annual global cottonseed production equals about 48.5 million tons.

“The kernels from the safe seed could be ground into a flour-like powder after oil extraction and used as a protein additive in food preparations or perhaps roasted and seasoned as a nutritious snack,” he said.

Rathore said cotton will continue to be grown as a source of natural fiber, but the adoption of the ultra-low gossypol varieties by farmers has the potential to make the seed just as valuable as the lint.

“Our approach, based on the removal of a naturally occurring, toxic compound from the cottonseed, not only improves its safety but also provides a novel means to meet the nutritional requirements of the burgeoning world population,” he said.

Aside from the human aspect, Rathore said the potential of ultra-low gossypol cottonseed as a fish meal replacement in the diets of shrimp and southern flounder has been demonstrated. Additional aquaculture and poultry feeding studies are planned to fully evaluate the nutritional value of the unique cottonseed.

Even after this deregulation hurdle has been jumped, the team knows the work is not done.

“The next major effort will be aimed at activities to demonstrate the value-added potential of this technology,” Wedegaertner said. “The first step will be to produce enough ULGCS seed for a commercial-scale production run at a cottonseed oil mill. This will take a couple of years.”

Rathore said development of ULGCS involved several patented technologies, so additional steps must be taken to secure agreements with the patent holders, then to find a seed company willing to market the ULGCS trait and make it available to cotton farmers worldwide.

Rathore said as a scientist who has conceived and developed this technology, “My personal preference as we move forward would be to follow the ‘Golden Rice’ example in terms of its use for humanitarian purposes.”

Engineered cotton uses weed-suppression chemical as nutrient

16Jul

Writer: Kay Ledbetter, 806-677- 5608, skledbetter@ag.tamu.edu
Dr. Keerti Rathore, 979-862-4795, rathore@tamu.edu

 

COLLEGE STATION – A newly developed fertilizer system will provide nutrition to engineered cotton crops worldwide and a deadly dose to weeds that are increasingly herbicide resistant, according to a Texas A&M AgriLife Research study.

Keerti Rathore, Devendra Pandeya and LeAnne Campbell in greenhouse

Dr. Keerti Rathore examines the health of ptxD-cotton plants being grown in the greenhouse for seed increase for a field trial with Dr. Devendra Pandeya and LeAnne Campbell. (Texas A&M AgriLife photo by Beth Luedeker)

The new system applies phosphite to cotton crops engineered to express a certain gene — a gene that makes cotton able to process the phosphite into nutrition while the same compound suppresses weeds that are unable to use it, researchers said.

“Our researchers here at Texas A&M AgriLife have addressed an issue that costs producers billions of dollars,” said Dr. Patrick Stover, vice chancellor of agriculture and life sciences at Texas A&M in College Station and AgriLife Research acting director. “This is an economical, envrionmentally safe and sustainable solution.

Stover said this is an exciting and timely discovery in the movement to get ahead of the ongoing problem of weeds evolving faster than the chemicals and other methods developed to control them.

“We believe the ptxD/phosphite system we have developed is one of the most promising technologies of recent times that can help solve many of the biotechnological, agricultural and environmental problems we encounter,” said Dr. Keerti Rathore, an AgriLife Research plant biotechnologist in College Station.

pigweed in cotton field

Palmer amaranth, more commonly known as pigweed, infests a High Plains cotton field. (Texas A&M AgriLife photo)

“Selective fertilization with phosphite allows unhindered growth of cotton plants expressing the ptxD gene while suppressing weeds” is the title of a Proceedings of the National Academy of Sciences of the United States of America journal article to be released the week of June 4. The article will be found at: https://tinyurl.com/ptxDcottonphosphite.

Phosphorus is a major element required by all living beings – life is not possible without it. Most organisms can only utilize phosphorus in the form of orthophosphate.

“We have determined ptxD-expressing cotton plants can utilize phosphite as a sole source of phosphorus while weeds cannot, thus making it effective at suppressing weed growth,” Rathore said.

The transgenic plants expressing the bacterial ptxD gene gain an ability to convert phosphite into orthophosphate, he said. Such plants allow for a selective fertilization scheme, based on phosphite as the sole source of phosphorous for the crop, while offering an effective alternative to suppress the growth of weeds that are unable to utilize this form of phosphorus.”

The international research team led by Rathore consists of Dr. Devendra Pandeya, Dr. Madhusudhana Janga, Dr. Muthu Bagavathiannan and LeAnne Campbell, all with Texas A&M AgriLife in College Station. Others are Dr. Damar Lopez-Arredondo and Dr. Priscila Estrella-Hernandez at StelaGenomics Inc. and Dr. Luis Herrera-Estrella at the Center for Research and Advanced Studies of the National Polytechnic Institute, all in Irapuato, Mexico.

LeAnne Campbell and Keerti Rathore looking at tiny plants sealed in small jars

LeAnne Campbell and Dr. Keerti Rathore examine the quality of engineered transgenic cotton plants regenerated from tissue cultures. (Texas A&M AgriLife photo by Beth Luedeker)

This research was funded in part by Cotton Inc. Weed herbicide resistance and weed control are the No. 2 and No. 3 concerns of U.S. cotton farmers after input costs.

“We can and will deliver for our cotton producers in Texas and beyond, in collaboration with Cotton Inc. and partners,” said Dr. Bill McCutchen, executive associate director of AgriLife Research in College Station.

Weeds typically are managed manually, mechanically or chemically. However, he said, chemical control options are rapidly shrinking due to an increasing number of herbicide-resistant weeds in crop fields, with few alternatives on the horizon.

“Over the years, it has become abundantly clear that new strategies are needed for weed control to sustain agriculture production while reducing our dependence on herbicides,” Herrera-Estrella said. “There is an urgent need for alternative weed suppression systems to sustain crop productivity, while reducing our dependence on herbicides and tillage.”

Rathore, who has been researching genetic improvement of cotton for more than 20 years, said herbicide-resistance in weeds in not just a U.S. problem, but rather a global challenge for producers of cotton, corn and soybeans.

Such a development will also relieve some of the negative perceptions associated with the use of herbicide-resistance genes and heavy dependence on herbicides, he said.

Rathore has also developed cotton plants that produce very low levels of gossypol in the seeds to improve the safety and nutrition aspects of the cotton seed, but simultaneously maintain normal levels  of this chemical in the foliage, floral parts, boll rind and roots for protection against insects and pathogens.

He previously published a report identifying ptxD as a selectable marker gene to produce transgenic cotton plants. The ptxD gene derived from Pseudomonas stutzeri WM88 encodes an enzyme that changes phosphite into orthophosphate, a metabolizable form of phosphorus, when expressed in transgenic plants.

Importantly, the ptxD/phosphite system proved highly effective in inhibiting growth of glyphosate-resistant Palmer amaranth, Rathore said. Resistance to current technologies in this highly noxious weed started showing up in fields about 10-15 years ago.

tiny cotton plantlets in covered petri dishes

A cultured somatic embryo developing into a normal cotton plantlet following introduction of a transgene into cotton cells some 10 months previously. (Texas A&M AgriLife photo by Beth Luedeker)

“The results presented in our paper clearly demonstrate the ptxD/phosphite system can serve as a highly effective means to suppress weeds under natural, low-phosphorus soils, including those resistant to the herbicide glyphosate, while allowing better growth of the ptxD-expressing cotton plants due to lesser competition from the debilitated weeds,” Rathore said.

Unlike weeds acquiring resistance to herbicides, he said it is highly unlikely weeds will gain the ability to use phosphite as a source of phosphorus.

“In order for a weed to acquire the ability to utilize phosphite, one of its dehydrogenase genes will have to undergo a complex array of multiple mutations in its DNA sequence – that’s unlikely to happen by random mutations that occur in all organisms,” Rathore said.

Another important point, he said, is compared to phosphate, phosphite has higher solubility and a lower tendency to bind soil components. So, if it is applied in proper formulation to prevent leaching, lower quantities can be used without sacrificing the crop yields.

“Even if some phosphite ends up in streams and rivers and eventually in lakes and the sea, the algal species will be incapable of using it as a source of phosphorus, thus preventing toxic algal blooms that kill fish and other creatures in water bodies,” Herrera-Estrella said.

Future studies will focus on testing ptxD-transformants in the fields that are low in phosphorus as well as evaluating the utility of phosphite as an over-the-top ‘herbicide,’ Rathore said. Also, long-term impact of the use of phosphite as a source of phosphorus on the soil microflora under field conditions needs to be investigated.

Genetic discovery another tool in battle against wheat pests

17Nov

Writer: Kay Ledbetter, 806-677-5608, skledbetter@ag.tamu.edu
Contact: Dr. Shuyu Liu, 806-677-5600, SLiu@ag.tamu.edu

AMARILLO – Greenbug and Hessian fly infestations can significantly reduce wheat yield and quality in Texas and worldwide. Breeding for resistance to these two pests using marker-assisted selection just got a new tool from a Texas A&M AgriLife Research study.

Because genetics is the most economical strategy to minimize losses, AgriLife Research wheat geneticist Dr. Shuyu Liu began two years ago searching for breeder-friendly markers for those two insects. This step is a continuation of ongoing genetic work on insect resistance.

greenbugs

Greenbug cluster on wheat. (Texas A&M AgriLife photo)

Through the years, a number of greenbug resistance genes have been identified in wheat and its relatives based on their differential reactions to different biotypes, which range from A through K. There are also 18 Hessian fly biotypes, and because it has the ability to overcome resistance genes deployed in wheat cultivars through mutations, it is necessary to identify and utilize resistance genes from diverse sources for wheat breeding.

Scientists use genetic markers to identify regions where specific genes can be found on a particular plant. Liu has identified the neighborhoods or markers for a gene offering greenbug resistance, Gb7, and a gene that provides Hessian fly resistance, H32, in wheat.

Liu’s work was recently published in the Theoretical and Applied Genetics Journal of Plant Breeding Research, detailing the development of the Kompetitive Allele Specific Polymerase Chain Reaction or KASP assays for both genes.

hessian fly

Hessian fly adult. (Texas A&M AgriLife photo)

Joining Liu on the publication were AgriLife Research wheat team members Drs. Jackie Rudd, Amarillo, and Amir Ibrahim, College Station, both wheat breeders; Dr. Qingwu Xue, crop stress physiologist; Dr. Chor Tee Tan, an associate research scientist; as well as other students and staff in Amarillo.

Both genes were identified through previous research, and linked markers for them were mapped, but the detection methods were not well suited for marker-assisted selection for evaluating thousands of plants, Liu said.

He said knowing an address doesn’t mean someone knows where in the city to start looking for it. But by developing single nucleotide polymorphism, or SNPs, which include flanking markers closely linked and located on chromosomes, geneticists are able to give breeders the neighborhood to search.

SNPs are then converted into KASP assays, which are considered breeder-friendly because they are easier to use, faster and more accurate, he said.

Effective molecular markers closely linked to the target genes are the key for the success of marker-assisted selection on traits such as greenbug and Hessian fly resistance, Liu said. For instance, a breeder will typically screen 1,000s of breeding lines, and the KASP acts as a flag to say the necessary genes for a particular trait exists in a particular line.

Through Liu’s work, both genes can now be easily transferred into a new wheat line through marker-assisted selection.

Dr. Shuyu Liu

Dr. Shuyu Liu, a Texas A&M AgriLife Research geneticist in Amarillo, looks at the results from a KASP assay for insect resistant wheat lines. Clustered in blue are the resistant lines, the susceptible lines are clustered in red, and green indicates lines not pure resistant or susceptible. (Texas A&M AgriLife photo by Kay Ledbetter)

Liu said the Gb7 and H32 are both found in a synthetic wheat, W7984, which is a parental line for a mapping population that wheat researchers are using worldwide. Synthetic wheats are man-made crosses between Durum or pasta-type wheats and Aegilops tauschii. These initial crosses provide access to genes of the wild relatives of wheat, thus increasing usable genetic diversity for breeders to improve winter wheat varieties.

The mapping population was developed more than 10 years ago by the International Triticum Mapping Initiative, but neither of these genes has been used for resistance in breeding programs to this point, he said.

“The reason I think they were not being used is they were in a synthetic line and it required more effort to transfer them into adaptive wheat lines,” he said. “What we have done with the KASP marker is make them easier to find and utilize.”

For example, TAM 114, a newer, increasingly popular variety of Texas A&M wheat, does not have greenbug resistance and only has limited Hessian fly resistance, Liu said.
“But with this new knowledge, breeders can cross with TAM 114 and keep its superior end-use quality and improve it with the Gb7 and H32 genes,” he said. “This will make the new line more adaptable to the regions where Hessian fly is a problem.”

By crossing wheat lines with the identified KASP markers, the process to develop the pure line with selected properties can be much more accurate, Liu said.

Liu said he began searching for these markers because the TAM breeding program has made heavy use of synthetic germplasm so the markers will quickly be implemented.

To get to this point, Liu utilized genotype-by-sequencing markers developed by other research groups, and ultimately the KASP markers were validated using the set of synthetic wheat lines. Each line of that mapping population was screened for reactions by greenbug and Hessian fly by two U.S. Department of Agriculture Agricultural Research Service centers.

“We’ve determined they are very effective under many genetic backgrounds,” he said. “Genetic diversity and genetic gains are always important to wheat breeders.”

New wheat streak mosaic virus resistance genetic markers developed

6Feb

Advancement made in battle of major disease in the Great Plains

Writer: Kay Ledbetter, 806-677-5608, skledbetter@ag.tamu.edu
Contact: Dr. Shuyu Liu, 806-677-5600, SLiu@ag.tamu.edu
Dr. Chor Tee Tan, 806-677-5600, chor_tee.tan@ag.tamu.edu

AMARILLO – The Wsm2 gene is located on chromosome 3BS in wheat and most recently eight tightly linked flanking markers have been identified and mapped.

Dr. Chor Tee Tan, a Texas A&M AgriLife Research associate research scientist in Amarillo, prepares KASP assays. (Texas A&M AgriLife photo by Kay Ledbetter .

To most, that means very little. To Texas A&M AgriLife Research geneticists and breeders, it’s the key to battling one of the most important biotic stresses affecting wheat.

Dr. Shuyu Liu, AgriLife Research small grains geneticist in Amarillo, and his team recently published two articles, “Saturated Genetic Mapping of Wheat Streak Mosaic Virus Resistance Gene Wsm2 in Wheat” and “Development and Validation of KASP Markers for Wheat Streak Mosaic Virus Resistance Gene Wsm2,” in the Crop Science journal.

In both articles, Liu and his collaborators outlined how wheat streak mosaic virus, also known as WSMV, is a major threat to wheat production in the Great Plains, one of the largest wheat regions in the nation, Liu said. While the threat may not be as high as it is in the U.S., the virus has been found in all major wheat-growing regions of the world.

There is no effective chemical treatment available for the disease, he said. Host resistance is the most cost-effective and environmentally safe approach for combating this disease.

“WSMV resistance is an important target trait for our wheat breeding program,” said Dr. Jackie Rudd, AgriLife Research wheat breeder in Amarillo. “We have evaluated many diverse sources of genetic resistance over the years, including a wheat breeding line from Colorado State University, CO960293-2, known to have good resistance.”

Rudd and the AgriLife Research wheat team began crossing with the Colorado line in 2005 and by 2011 had identified and mapped WSMV resistance on chromosome 3BS and named the gene Wsm2.

Liu said wheat breeding programs worldwide now rely on two primary sources of resistance, Wsm1 – identified in the early 1990s from a wheatgrass, and the Wsm2 from a bread wheat, for development of WSMV-resistant wheat cultivars.

Effective molecular markers closely linked to the target genes are the key for the success of marker-assisted selection on traits such as WSMV resistance, he said. Among the available markers, single nucleotide polymorphisms or SNPs are routinely used in plant breeding programs to distinguish potentially superior genotypes with genetic merit for traits of interest.

Two clusters from the KASP assay shows wheat lines exhibiting resistance with Wsm2, blue, and susceptibility, red, to WSMV.

His genetic team has identified eight SNPs flanking Wsm2, Liu said. This helps increase the efficiency in selection for the resistance needed to battle the virus.

“A single marker linked to target genes may not be sufficient to screen across diverse genetic backgrounds,” Liu said. “Therefore, a set of tightly linked markers on each side of the gene is the best predictor for Wsm2 with higher accuracy.”

These tightly linked SNPs will be useful for marker-assisted selection for WSMV resistance, he said.

Dr. Chor Tee Tan, an associate research scientist in Amarillo, has been able to carry the AgriLife Research work one step further. Working in the wheat genetic program for almost four years, he led the effort to develop breeder-friendly Kompetitive Allele Specific Polymerase Chain Reaction or KASP assays for those SNPs tightly linked to Wsm2 and validated them in multiple breeding populations.

If a breeder is screening 1,000s of breeding lines, as is typical, the KASP acts as a flag to say the necessary sequence for wheat streak mosaic virus resistance exists in a particular line of wheat, Tan said.

“These KASP SNPs were tested on a panel of nine wheat breeding and mapping populations with diverse genetic backgrounds and were found to be very effective in differentiating resistant and susceptible genotypes for WSMV,” he said.

“We have many lines with Wsm2 in our breeding pipeline now and these new markers will help us get to the finish line,” Rudd said. “Marker technology has greatly improved, and these latest findings are easy to use and very predictable.”

Liu said the joint effort from breeders at AgriLife Research, Kansas State University-Hays and Colorado State University, has “made great progress to fight this disease that is not easy to screen by infection.”

Research for these studies was supported in part by the Texas Wheat Producer Board, Monsanto’s Beachell-Borlaug International Scholars, AgriLife Research and a U.S. Department of Agriculture National Institute of Food and Agriculture grant to the Triticeae-CAP project.

Both journal articles can be accessed at http://bit.ly/2kSxobG.

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