DANFORTH PLANT SCIENCE CENTER RESEARCHER DISCOVERS BREAKTHROUGH IN DROUGHT TOLERANCE

Source: Donald Danforth Plant Science Center 

Drought is one of the greatest threats to agricultural systems, resulting in unpredictable crop yields, declines in farm revenue, and an increase in disease outbreaks. In the United States alone, drought has cost the nation $249 billion since the 1980s.

One potential solution to enhancing crop resilience is the inoculation of seed with bacteria, aka. plant 'probiotics' that are known to improve a plant's drought tolerance. While scientists have identified many microbes that show promise in the lab, replicating their efficacy in agricultural field studies proves much more difficult, largely due to complex environmental variation in the real world.

New research spearheaded by Rebecca Bart, PhD, Associate Member, Donald Danforth Plant Science Center, and her colleagues tackled the challenge of bridging the gap between lab and field studies related to crop-microbial interactions and their influence on drought tolerance. Their work has the potential to accelerate crop adaptation to drought conditions and streamlines findings from the lab for farmers in the field.

Their seminal research, Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbe comparisons and Increased signal-to-noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components, was recently published in ISME Journal and eLife, respectively.

The authors took a systems-level approach to identify microbes that affected drought response in sorghum, work that spanned "sterile, controlled environments" in the lab, to field experiments chock full of complex soil properties, uneven topography, and nonuniform accumulation of water moisture.

The team found that at least six microbes that caused root developmental defects in the lab - stunting the height of sorghum seedlings - were also negatively affecting sorghum growth in the field. "The big advance here," said corresponding author Bart, "is that we observed similar patterns in a controlled environment and in the field. That result tells us that our lab observations are real and relevant to agriculture." Strikingly, the research team also identified a new microbe that promoted root growth, a critical characteristic to improve crop resilience to drought.

The research, which took place over the course of the last five years, was not without its own challenges. "Environmental variation makes the real world a noisy place to conduct science," wrote first author and Danforth Center Senior Data Scientist Jeffrey Berry. The authors needed to develop a model to account for confounding biological variables in field experiments - factors like soil pH and phosphate content, which can vary wildly across a field site.

By combining giant, multivariate datasets from collaborators across several institutions, including at University of Nebraska-Lincoln, Iowa State University, Washington State University, University of North Carolina-Chapel Hill, Colorado State University, and the Joint Genome Institute, Berry was able to use sophisticated computational models to understand and overcome variation in the field.

The result was a first-of-its-kind statistical model that accounted for soil properties that influenced traits in both crops and microbes. The authors could now compare their results between the lab and field without worrying about how environmental variation might be altering their field observations. "Jeff figured out how to connect some really complicated puzzle pieces," concluded Bart.

In addition to tackling complicated statistics and collaborating with scientists across the country, part of the teams' success was having access to the Danforth Center's unparalleled research infrastructure. For example, the authors used The Bellwether Foundation Phenotyping Facility to visualize and quantify how drought and microbe treatments affected sorghum growth and development as part of their controlled lab experiments.

The team is beginning to replicate their methodology in other crops systems like maize, and future research plans for this work will be housed out of the Danforth Center's new Subterranean Influences on Nitrogen and Carbon (SINC) Center, co-directed by Bart and three other Danforth Center members.

SINC was established to better understand the symbiotic relationships between plants and microbes and their potential to reduce chemical nitrogen fertilizer used in agriculture. SINC's cross-disciplinary approach to creating real solutions for agricultural challenges is the ideal facility to continue the multi-scale and multi-system work for Bart and her collaborators.

This work was supported by funding from the United States Department of Energy, National Science Foundation, Howard Hughes Medical Institute, and Iowa State University. 

USDA REPORTS ORGANIC TRADE REACHES $3.4 BILLION IN 2021

USDA release

The U.S. Department of Commerce actively tracks organic food in 37 export and 57 import categories. Tracked exports and imports of organic products in the United States reached $3.4 billion in 2021.

Since 2011, there has been an uptick in the total value of imported organic products, partially because more products are being tracked and partially because more high-value organic products, such as blueberries and squash, are being imported into the United States.

The United States also exports organic food, and those exports have been steadily rising since 2011, reaching $0.7 billion in 2021. For example, the United States exported 2.4 thousand metric tons of organic fresh cultivated blueberries, with more than 90 percent headed to Canada in 2021. In the same year, the United States imported 41.5 thousand metric tons of organic fresh cultivated blueberries primarily from Peru (40 percent of the total imports), Chile (32 percent), and Mexico (25 percent).

Importers of organic products must either be USDA-certified or belong to a trading partner with an organic recognition agreement with the United States, which allows foreign Governments to accredit certifying agents to USDA organic standards. Countries with such agreements include Canada, the European Union, Japan, South Korea, Switzerland, Taiwan, and the United Kingdom.

Editor's Note: What the release does not tell you is that organics constituted almost exactly 1% of the total U.S. food imports and exports by dollar value. One percent. And much less than 1% if measured by relative nutritive value.  

While regular readers of the Weekly Update are fully informed on this matter;  the last week has seen a plethora of news articles detailing the absurdity of the administration's unprecedented subsidies for the organic sector. Most of the recent articles cite food prices, up by 13.5% over the last 12 months, and food shortages predicted in the near future, seem to be inconsistent with incentivizing lower levels of food production at higher prices. Publications as diverse as the Wall Street Journal to the Washington Post have questioned the "wisdom" of subsidizing organic agriculture particularly at such a time.

We openly questioned these subsidies and organic incentives beginning four weeks ago when the USDA announced them. Further evidence of how out-of-touch the executive branch of our federal government is just now but consistent with depleting the federal petroleum reserve while curtailing offshore oil leases, etc.

Organic is marketing, pure and simple, nothing more and nothing less. As a seed producer when was the last time the USDA offered you direct payments, cash incentives, and subsidies to assist you with your marketing?  

Overcoming a Major Technical Hurdle for Gene Editing in Growing Produce

By Kenong Xu in Growing Produce

The Nobel Prize-winning CRISPR (clustered regularly interspaced short palindromic repeats)-CAS (CRISPR-associated protein) genome-editing technology has been rapidly adapted across all forms of life ever since its discovery in 2012. In plants, successful genome editing using CRISPR-CAS has been reported in many food and feeding crops, such as corn, rice, and tomato.

Recently, the first CRISPR-CAS-edited tomato fruit that has a drastically increased content of γ-aminobutyric acid (GABA), a beneficial health compound, has officially entered the marketplace for consumption in Japan.

This is an important milestone in using the CRISPR-CAS technology to develop value-added fleshy fruit for consumers. It is almost certain commercial production of many other CRISPR-CAS-edited food crops will follow soon.

However, the advance of CRISPR-CAS genome editing in perennial woody fruit crops, such as apple and grape, remains slow. Following is a major underlying hurdle and a promising approach that may overcome it.

TECHNICAL HURDLE 

One of the major advantages of CRISPR-CAS-edited crops over the classic genetically modified (GM) crops is that the former does not contain any foreign DNA, making them indistinguishable from those developed by conventional plant breeding.

In annual crops, such as rice and tomato, the CRISPR-CAS gene-editing components (considered foreign DNA) that were introduced into their genome can be quickly removed though genetic segregation in two life cycles, taking only one or two years.

However, this strategy could not be adapted in perennial woody fruit crops, causing a major technical hurdle due to the following two reasons:

The first is their long juvenile phase before flowering. In apple, for example, the juvenile phase usually requires five or more years. This implicates that once the CRISPR-CAS genome-editing components were introduced into the apple genome in a cell, it would take at least five years for the apple cell to develop into a fruit-bearing tree to produce fruit and seeds. Assuming some of the seedlings (segregants) be gene-edited and free of foreign DNA, they will need another five or more years to set fruit for performance analysis.

The second is their highly heterozygous genomes. This makes it impossible to reproduce the mother trees from their own seeds. Similarly, any foreign DNA-free segregants with desired gene edits will be different from their mother trees, inevitably causing uncertainties in their performance. Given the importance of brand and variety recognition among consumers, such loss of identity is undesirable.

PROMISING APPROACH

To overcome the hurdle, a number of approaches have been proposed and tested to enable CRISPR-CAS editing without genome integration in plants.

What looks promising is an approach that uses a DNA base editor (a modified version of CRISPR-CAS) capable of editing single DNA bases, the Agrobacterium-based DNA delivery system, and one or more herbicide-resistance genes in plants.

Agrobacterium is a plant tumor causing bacterium due to its ability to transfer DNA into plant cells, which has been used widely for developing conventional GM crops.

You might ask why this approach is promising when Agrobacterium is involved.

Well, a little less-known fact about Agrobacterium-based DNA delivery is that most plant cells that were transferred with a DNA molecule by Agrobacterium will not integrate it into their genome.

It is this fact that makes Agrobacterium a viable option to express the CRISPR-CAS gene-editing machinery without genome integration.

However, once a cell is edited in a gene of interest, the next task is to find it from a mix of millions of cells. This is essentially searching for a needle in a haystack.

To facilitate the search, an herbicide-resistance gene in the plant, such as the gene encoding an acetolactate synthase (ALS), is exploited. This is because most plants, including woody tree fruits, are susceptible to herbicide sulfonylurea (SU). If the ALS gene is edited properly, plants will become resistant to SU.

The idea here is to co-edit the ALS gene alongside a gene of interest so that such co-edited plants can be selected based on their resistance to SU, as application of SU would eliminate all plants without an ALS edit, making identification of plants with a desirable edit feasible.

Obviously, this approach will also make the edited plants resistant to sulfonylurea-like herbicides, providing an attractive arsenal for weed management.

So far, the ALS gene has been proven an effective selectable marker in apple, pear, citrus, and other woody fruit crops. Moreover, ALS gene-edited citrus plants free of foreign DNA also have been developed.

The progress shows that the Agrobacterium-mediated ALS gene co-editing approach is promising for CRISPR-CAS editing in woody fruit crops. As such, gene-edited woody fruit crops that are free of foreign DNA and have a significant improvement over their mother trees are currently underway.

Editor's Note: We understand this article has little to do, directly, with seed our members produce or sell.  However the technological aspects seemed quite impressive and we felt it worth a look.  

Purple Tomato Is First Genetically Engineered Plant To Be Deregulated Through USDA’S New Regulatory Status Review Process

By: Liz Freeman Rosenzweig in MoFo Life Sciences

On September 7, 2022, the United States Department of Agriculture’s Animal and Plant Health Inspection Service (USDA-APHIS) announced the completion of the first Regulatory Status Review (RSR) of a genetically engineered plant under the SECURE rule. APHIS concluded that a new genetically engineered tomato produced by Norfolk Plant Sciences is unlikely to pose an increased plant pest risk compared to a conventional tomato, and is therefore not subject to regulation under the SECURE rule. This means that these tomato plants, which have been engineered to produce deep purple tomatoes with enhanced nutritional quality, may be legally imported, moved interstate, or “released” into the environment (including, for example, in a field trial) in the United States without a permit from APHIS.

Notably, this finding also means that subsequent genetic transformation events involving the same combination of plant species, trait, and mechanism of action (“PTMOA”) as Norfolk Plant Sciences’ purple tomatoes are also no longer regulated under the SECURE rule. Thus, other subspecies and varieties of Solanum lycopersicum that have been modified to produce the same trait by the same mechanism of action—even if by different transgenic events—are now exempt under § 340.1(c) of the SECURE rule. More information on this so-called PTMOA exemption is available in APHIS’s “Guide for Requesting a Confirmation of Exemption from Regulations under 7 CFR part 340” (published August 31, 2022; document ID BRS-GD-2020-0001). This approach is different from the “event-by-event” regulation that was previously required, and represents the first time that APHIS has made an RSR determination under its new rules.

Additional information about the contours of the SECURE rule and the genetic engineering that Norfolk Plant Sciences used to produce their purple tomato is provided below.

About the SECURE Rule

The SECURE rule (7 CFR part 340) governs how APHIS regulates certain organisms developed using genetic engineering, with the goal of protecting U.S. agriculture from plant pest risks under the Plant Protection Act of 2000 (7 U.S.C. §§ 7701 et seq.). It replaced the previous version of 7 CFR part 340, which had been in place largely unchanged since APHIS’s biotechnology regulations were established in 1987, in phases between May 18, 2020 and October 1, 2021.

The new regulations completely overhauled and streamlined the regulatory process for assessing the plant pest risk of organisms developed using genetic engineering, taking into account advances in scientific understanding, and focusing more on the properties of the engineered organism and less on the method(s) used to produce it.

The revised regulations exempt certain types of modifications from regulation; such exemptions are self-determined, though developers may voluntarily request confirmation from APHIS that a given exemption applies. This exemption/confirmation process replaced the previous “Am I Regulated?” process on June 17, 2020, and APHIS has since issued 15 confirmation letters as of this writing, with the earliest in April 2021.

However, no plant had made it through the new RSR process until now. The RSR process is an option for instances in which no SECURE rule exemptions apply to a given engineered plant, but the developer feels that the plant nonetheless does not pose an increased plant pest risk and should therefore not be regulated by the SECURE rule. The RSR process replaced the previous “petition” process for requesting deregulation from 7 CFR part 340 due to low likelihood of posing a plant pest risk.

The RSR process became available for corn, soybean, cotton, potato, tomato, and alfalfa on April 5, 2021, and for all other plant species on October 1, 2021. APHIS received Norfolk Plant Sciences’ RSR request on August 4, 2021 and responded on September 6, 2022 (both the request and the response documents are available here, under RSR number 21-166-01rsr). As of this writing, Norfolk Plant Sciences’ tomato is the only RSR request publicly available on APHIS’s website.

Under the RSR process, APHIS reviews “the biological properties of the plant; and the trait (or new characteristic); and the mechanism of action (or how the genetic modification causes the new trait to occur)” in order to evaluate plant pest risk. There are two potential steps to this process, depending on what APHIS determines during the first step. In Step 1, APHIS identifies whether the engineered plant poses a plausible pathway to increased plant pest risk compared to a “comparator” plant. If APHIS finds no such pathway, the RSR process concludes, and APHIS notifies the requestor that the plant in question is not subject to regulation under the SECURE rule. This was the outcome for Norfolk Plant Sciences’ tomato.

On the other hand, if APHIS does determine that the engineered plant may plausibly pose an increased plant pest risk, there are several potential next steps. First, the developer may accept that the plant is regulated under the SECURE rule, and then either request a permit before moving or releasing the plant, or take no further action and not move or release the plant.

Alternatively or additionally, the developer may request that APHIS proceed to Step 2 of the RSR process, which entails a more involved review, subsequent publication in the Federal Register, and solicitation and review of public comments before a final determination. As of this writing, no plant has gone through this second step of the RSR process.

About the Purple Tomato

As described in its RSR request, Norfolk Plant Sciences created its purple tomato plant by Agrobacterium-mediated insertional mutagenesis of the “MicroTom” tomato variety, and subsequent crossing into other tomato varieties. The plants are engineered to increase expression of their natural anthocyanin pigments, which is what causes the fruits to have a deep purple color and also enhances their nutritional value.

Specifically, the inserted DNA contains two transcription factors from the snapdragon plant (Antirrhinum majus), which serve to activate production of the tomato’s native anthocyanin biosynthesis pathway, causing increased anthocyanin production. Each of these two transcription factor genes, called Del and Ros1, is expressed from the T-DNA under a native tomato promotor that causes fruit-specific expression. The T-DNA also includes the nptII selectable marker with a promotor and terminator from Agrobacterium tumefaciens, which have a decades-long history of safe use and consumption.

Complete genome sequencing revealed that the T-DNA was inserted at a single site in chromosome 4, accompanied by several small deletions. Phenotypic evaluation of the transformed plants revealed that they grew effectively the same as non-transgenic tomatoes, except that they produce deep purple fruit with significantly higher anthocyanin content. Photos of the plants and fruit are available in the published RSR request.

APHIS considered the information disclosed in Norfolk Plant Sciences’ RSR request, alongside “publicly available resources, and APHIS’ familiarity with tomato and knowledge of the trait, phenotype, and mechanism of action” and “did not identify any plausible pathway by which [the] modified tomato, or any of its sexually compatible relatives, would pose an increased plant pest risk relative to a comparator tomato” (21-116-01 RSR Response, page 1). As such, APHIS concluded that these purple tomatoes are not subject to regulation under the SECURE rule.

Other Regulatory Agencies

It is important to note that deregulation from APHIS’s SECURE rule does not mean that the plant is wholly removed from U.S. federal regulatory oversight. For example, regulations implemented by the Food and Drug Administration (FDA), Environmental Protection Agency (EPA), and/or other arms of USDA (such as Plant Protection and Quarantine (PPQ) import and export regulations, and/or Agricultural Marketing Service (AMS) labeling requirements) may still apply. Along those lines, Norfolk Plant Sciences’ RSR request states that Norfolk Plant Sciences submitted a food and feed safety and nutritional assessment of the Purple Tomato to FDA under the voluntary Biotechnology Notification Consultation program, which was received as BNF number 178. As of this writing, FDA has not yet published a completed consultation for Norfolk Plant Sciences’ purple tomato.

Conclusion

This regulatory review is an important milestone for regulation of genetically engineered plants in the United States. It is the first public test of the SECURE rule’s RSR process since its implementation more than a year ago, when it became one of the most scientifically progressive such review processes in the world, at least on paper. The deregulation of Norfolk Plant Sciences’ purple tomatoes shows that USDA-APHIS is embracing its new product-focused regulations. Although the review took more than a year—significantly longer than the 180 days promised by APHIS for Step 1—the process will likely become more efficient as the agency and developers become more familiar and comfortable with the new system. It will be interesting to see how the exemption and review processes grow and possibly become more streamlined with additional use.