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research: Biotechnology

Meet The Indian Researcher Helping To Solve The Deadly Aflatoxin Puzzle

Indian agricultural researcher Pooja Bhatnagar-Mathur, a Principal Scientist at CIMMYT, says aflatoxin, a toxin produced from soil fungus and found in groundnuts like peanuts, is a serious public health and food safety problem around the globe.

Read more: https://www.forbes.com/sites/andrewwight/2021/12/29/meet-the-indian-researcher-helping-to-solve-the-deadly-aflatoxin-puzzle/?sh=1bfada46f305

Scientific opportunities and challenges

Maize and wheat fields at the El Batån experimental station. (Photo: CIMMYT/Alfonso Cortés)
Maize and wheat fields at the El Batån experimental station. (Photo: CIMMYT/Alfonso Cortés)

The first meetings of the Accelerating Genetic Gains in Maize and Wheat for Improved Livelihoods (AGG) wheat and maize science and technical steering committees — WSC and MSC, respectively — took place virtually on 25th and 28th September.

Researchers from the International Maize and Wheat Improvement Center (CIMMYT) sit on both committees. In the WSC they are joined by wheat experts from national agricultural research systems (NARS) in Bangladesh, Ethiopia, Kenya, India, and Nepal; and from Angus Wheat Consultants, the Foreign, Commonwealth & Development Office (FCDO), HarvestPlus, Kansas State University and the Roslin Institute.

Similarly, the MSC includes maize experts from NARS in Ethiopia, Ghana, Kenya and Zambia; and from Corteva, the Foundation for Food and Agriculture Research (FFAR), the International Institute for Tropical Agriculture (IITA), SeedCo, Syngenta, the University of Queensland, and the US Agency for International Development (USAID).

During the meetings, attendees discussed scientific challenges and opportunities for AGG, and developed specific recommendations pertaining to key topics including breeding and testing scheme optimization, effective engagement with partners and capacity development in the time of COVID-19, and seed systems and gender intentionality.

Discussion groups noted, for example, the need to address family structure in yield trials, to strengthen collaboration with national partners, and to develop effective regional on-farm testing strategies. Interestingly, most of the recommendations are applicable and valuable for both crop teams, and this is a clear example of the synergies we expect from combining maize and wheat within the AGG project.

All the recommendations will be further analyzed by the AGG teams during coming months, and project activities will be adjusted or implemented as appropriate. A brief report will be submitted to the respective STSCs prior to the second meetings of these committees, likely in late March 2021.

Faster results at a lower cost

Usman Kadir and his family de-husk maize on their farm in Ethiopia. (Photo: Apollo Habtamu/ILRI)
Usman Kadir and his family de-husk maize on their farm in Ethiopia. (Photo: Apollo Habtamu/ILRI)

The current COVID-19 pandemic — and associated measures to reduce its spread — is projected to increase extreme poverty by 20%, with the largest increase in sub-Saharan Africa, where 80 million more people would join the ranks of the extreme poor. Accelerating the process of delivering high-quality, climate resilient and nutritionally enriched maize seed is now more critical than ever.However, developing these varieties is not a rapid or cheap process. Over the course of five years, researchers on the Stress Tolerant Maize for Africa (STMA) project developed a range of tools and technologies to reduce the overall cost of producing a new high yielding, stress tolerant hybrids for smallholder farmers in the region.

Maize breeding starts with crossing two parents and essentially ends after testing their great-great-great-great grandchildren in as many locations as possible. This allows plant breeders to identify the new varieties which will perform well in the conditions faced by their target beneficiaries — in the case of STMA, smallholder farmers in Africa. In other parts of the world, new tools and technologies are routinely added to breeding programs to help reduce the cost and time it takes to produce new varieties.

Scientists on the STMA project focused on testing and scaling new tools specifically for maize breeding programs in sub-Saharan Africa and began by taking a closer look at the most expensive part of the breeding process: phenotyping or collecting precise information on plant traits.

“Within a breeding program, phenotyping is the single most costly step,” explains CIMMYT molecular breeder Manje Gowda. “Molecular technologies provide opportunities to reduce this cost.” The research team tested two methods to speed up this step and make it more cost efficient: forward breeding and genomic selection.

Speeding up a long and costly process

Two important traits maize breeders look for in their plant progeny are susceptibility for two key maize diseases: maize streak virus (MSV) and maize lethal necrosis (MLN). In traditional breeding, breeders must extensively test lines in the field for their susceptibility to these diseases, and then remove them before the next round of crossing. This carries a significant cost.

Using a process called forward breeding, scientists can screen for DNA markers known to be associated with susceptibility to these diseases. This allows breeders to identify lines vulnerable to these diseases and remove them before field testing.

Scientists on the STMA project applied this approach in CIMMYT breeding programs in eastern and southern Africa over the past four years, saving an estimated $300,000 in field costs. Under the AGG project, research will now focus on applying forward breeding to identify susceptibility for another fast-spreading maize pest, fall armyworm, as well as extending use of this method in partners’ breeding programs.

A CIMMYT research associate inspects maize damaged by fall army worm at KALRO Kiboko Research Station in Kenya. (Photo: Peter Lowe/CIMMYT)
A CIMMYT research associate inspects maize damaged by fall army worm at KALRO Kiboko Research Station in Kenya. (Photo: Peter Lowe/CIMMYT)

Forward breeding is ideal for “simple” traits which are controlled by a few genes. However, other desired traits, such as tolerance to drought and low nitrogen stress, are genetically complex. Many genes control these traits, with each gene only contributing a little towards overall stress tolerance.

In this case, a technology called genomic selection can be of service. Genomic selection estimates the performance, or breeding value, of a line based largely on genetic information. Genomic selection uses more than 5,000 DNA markers, without the need for precise information about what traits these markers control. The method is ideal for complicated traits such as drought and low nitrogen stress tolerance, where hundreds of small effect genes together largely control how a plant grows under these stresses.

CIMMYT scientists used this technology to select and advance lines for drought tolerance. They then tested these lines and compared their performance in the field to lines selected conventionally. They found that the two sets of resulting hybrid varieties — those advanced using genomic selection and those advanced in the field — showed the same grain yield under drought stress. However, genomic selection only required phenotyping half the lines, achieving the same outcome with half the budget.

Innovations in the field

While DNA technology is reducing the need for extensive field phenotyping, research is also underway to reduce the cost of the remaining necessary phenotyping in the field.

Typically, many traits — such as plant height or leaf drying under drought stress — are measured by hand, using the labor of large teams of people. For example, plant and ear height is traditionally measured by a team of two using a meter stick.

Mainasarra Zaman-Allah, a CIMMYT abiotic stress phenotyping specialist based in Zimbabwe, has been developing faster, more accurate ways to measure these traits.  He implemented the use of a small laser sensor to measure plant and ear height which only requires one person. This simple yet cost effective tool has reduced the cost of measuring these traits by almost 60%. Similarly, using a UAV-based platform has reduced the cost of measuring a trait known as canopy senescence — leaf drying associated with drought susceptibility —by over 65%.

The identification of plants which are tolerant to key diseases has traditionally involved scoring the severity of disease in each plot visually, but walking through hundreds of plots daily can lead to errors in human judgement. To combat this, CIMMYT biotic stress phenotyping specialist LM Suresh collaborated with Jose Luis Araus and Shawn Kefauver, scientists at the University of Barcelona, Spain, to develop image analysis software that can quantify disease severity, thereby avoiding problems associated with unintentional human bias.

Plant breeders need uniform, or homozygous, lines for selection. With conventional plant breeding this is difficult: no matter how many times you cross a line, a small amount of DNA will remain heterozygous — having two different alleles of a particular gene — and reduce accuracy in line selection.

A technology called doubled haploid allows breeders to develop homozygous lines within two seasons. While this technology has been used in temperate maize breeding programs since the 1990s, it was not available for tropical environments until 10 years ago. In 2013, thanks to joint work with Kenyan partners at the CIMMYT Doubled Haploid facility in Kiboko, this technology was made available to African breeding programs. Now Vijay Chaikam, a CIMMYT doubled haploid specialist based in Kenya, is working towards reducing the cost of this technology as well.

The efforts begun by the STMA research team is now continuing under the Accelerating Genetic Gains in Maize and Wheat for Improved Livelihoods (AGG) project. As this work is carried forward, the next crucial step is ensuring that the next generation of African maize breeders have access to these technologies and tools.

“Improving national breeding programs will really drive success in raising maize yields in the stress prone environments faced by many farmers in our target countries,” says Mike Olsen, CIMMYT’s upstream trait pipeline coordinator. Under AGG, in collaboration with the CGIAR Excellence in Breeding Program, these tools will be scaled out.

Unique partnership to transform maize breeding and production in Africa

Scientists part of the Seed Production Technology for Africa (SPTA) and the Maize Lethal Necrosis Gene Editing projects are leveraging innovative technologies to transform seed production systems and speed up the delivery of disease resistance in elite new hybrids. This research is helping smallholder farmers in sub-Saharan Africa to access high-quality seed of new hybrids that were bred to perform under stressful low-input, drought-prone conditions, including farming regions impacted by maize lethal necrosis (MLN).

Fast delivery of MLN-tolerant varieties

The fight against maize lethal necrosis (MLN) has persisted for almost ten years now.

Collaborative efforts in diagnostics, management and systematic surveillance have limited its spread and confined the disease to the eastern Africa region. However, ongoing work is required to efficiently develop MLN-tolerant varieties for smallholders in endemic areas and prepare for the potential further movement of the disease.

“Maize lethal necrosis still exists. It has not been eradicated. Even though it has reduced in its prevalence and impact, it is still present and is a latent threat in Ethiopia, Kenya, Rwanda, Tanzania and Uganda, with potential to spread further,” said B.M. Prasanna, director of CIMMYT’s Global Maize Program and the CGIAR Research Program on Maize.

“That is why the work of the gene editing project is critical to rapidly change the genetic component of those susceptible parent lines of popular hybrids into MLN-tolerant versions,” said Prasanna. Scientists will edit the four parent lines of two popular hybrids, currently grown by farmers in Kenya and Uganda, which are susceptible to MLN. The edited MLN-tolerant lines will be used to make MLN-tolerant versions of these drought-tolerant hybrids.

Through gene editing technology, the time it takes to develop hybrids using traditional breeding methods will be cut in half. By 2025, the edited MLN-tolerant hybrids will be available for planting on approximately 40,000 hectares by about 20,000 Kenyan farmers.

A non-pollen-producing maize plant (on the left) on farm trial in Zimbabwe. (Photo: Jill Cairns/CIMMYT)
A non-pollen-producing maize plant (on the left) on farm trial in Zimbabwe. (Photo: Jill Cairns/CIMMYT)

Business as unusual

The unique seed production technology developed by Corteva Agriscience seeks to transform the seed production process in sub-Saharan Africa. This technology utilizes a dominant non-pollen producing maize gene to create female plants that are unable to produce pollen.

Seed companies that use seed production technology eliminate the need to detassel the female parent: a manual process through which tassels are removed from plants to prevent self-pollination and ensure that the intended male parent is the only source of pollen in the hybrid seed production field. Targeted small and medium-size seed companies could make significant savings to the cost of production if they were to eliminate manual detasseling. The method also helps to ensure the purity of the hybrid seed by removing the risk of unintentional self-pollination.

Hybrids produced using the seed production technology, characterized as 50 percent non-pollen producing (FNP), are unique since only half of the plants will produce pollen in the field. FNP hybrids re-allocate energy from the tassel and pollen production to grain formation, thus delivering an additional 200 kilograms per hectare yield advantage to the farmer. This represents a 10 percent productivity boost for farmers who will harvest approximately 2 tons per hectare, the average maize yield across sub-Saharan Africa. Farmers engaged in participatory research have demonstrated preference for FNP hybrids and associate the trait with higher yield and larger ear size.

As the first phase of Seed Production Technology for Africa (SPTA) wraps up, the collaborators are preparing for the next phase that will focus on commercializing, scaling up and increasing smallholders’ access to FNP. “This is among the unique partnerships funded by the foundation and I am hopeful that this incredible work will continue through the next phase,” said Gary Atlin, program officer at the Bill & Melinda Gates Foundation.

Resistant hybrid (on the right) grows beside a susceptible commercial check at the Kenya Plant Health Inspectorate Services' (KEPHIS) National Performance Trial. (Photo: CIMMYT)
Resistant hybrid (on the right) grows beside a susceptible commercial check at the Kenya Plant Health Inspectorate Services’ (KEPHIS) National Performance Trial. (Photo: CIMMYT)

A win-win collaboration

Research and development work under the SPTA and the MLN Gene Editing projects has immensely benefited from the support of public and private partners. Seed companies and national institutions have contributed to improving access to and knowledge of these technologies as well as creating a crucial link with farmers. Ongoing engagement with regulatory agencies through the different stages of the projects ensures transparency and fosters understanding.

In order to assess the progress of these two initiatives, representatives from regulatory agencies, seed trade associations, seed companies, national agricultural institutions and funders came together for a virtual meeting that was hosted on July 29, 2020.

“KALRO embraces partnerships such as those that are delivering these two projects. That synergy helps us to resolve challenges faced by farmers and other actors in various agricultural value chains,” observed Felister Makini, deputy director general of Crops at KALRO.

As the primary technology provider, Corteva Agriscience provides the seed production technology system on a royalty-free basis and grants access to key gene editing technologies, which are the foundation for the two projects. Corteva Agriscience is also actively involved in project execution through collaborative scientific support.

“We have appreciated the opportunity to work with CIMMYT, KARLO, Agricultural Research Council (ARC) of South Africa and the Bill & Melinda Gates Foundation to bring some of the technologies and tools from Corteva to address significant challenges facing smallholder farmers in Africa. We could not have done this alone, it requires the partnerships that exist here to bring forth these solutions,” said Kevin Diehl, director of the Global Seed Regulatory Platform at Corteva Agriscience.

MLN Gene Editing Project

The Maize Lethal Necrosis (MLN) Gene Editing Project uses gene editing technology to transform four elite CIMMYT maize lines which are susceptible to a devastating maize disease known as MLN. The disease first appeared in Kenya in 2011, and by 2013 it had reduced maize yields across the country by an average of 22%, resulting in loss of production worth $180 million and forcing many smallholder farmers to abandon planting maize. By 2014 it had spread to D.R. Congo, Ethiopia, Kenya, Rwanda, Tanzania and Uganda, hence posing a major threat to the food security and livelihoods of millions of Africans.

CIMMYT and its partners have responded to the problem by successfully developing MLN-tolerant hybrids through conventional backcrossing, which takes approximately 4-5 years. On the other hand, with the use of a gene editing technology known as CRISPR-Cas9, the breeding process can be accelerated, thereby reducing the time required to 2-3 years only, so that smallholders get faster access to improved maize varieties.

In partnership with Corteva Agriscience — which has significant expertise in the genome-editing field and who is the technology owner — and KALRO (Kenya Agricultural and Livestock Research Organization), CIMMYT scientists have been able to make a breakthrough via the CRISPR-Cas9 technology. The technology, Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) along with CRISPR-associated System (Cas) containing Protein 9, functions to replicate natural mutations in maize that will help strengthen its resistance to MLN. At the same time, this precisely targeted crop improvement process eliminates the transfer of many undesirable genes that would often accompany the desired ones as with the case in traditional backcrossing.

Under this project, four CIMMYT inbred lines, that are parents of two commercial hybrids in eastern Africa but susceptible to MLN, have been selected to undergo gene editing to become MLN-resistant. The edited, MLN-resistant lines will in turn be used to produce MLN-resistant hybrids which will still carry all the farmer-preferred agronomic traits including drought tolerance, similar to other elite maize hybrids developed by CIMMYT and released through partners.

CIMMYT is working in close collaboration with KALRO and other partners from the public and private sectors to increase the number of MLN-resistant Africa-adapted inbred lines and hybrids, as well as to make deployment efforts. By 2025, subject to compliance with regulatory procedures, commercial seeds of the gene-edited MLN-resistant elite maize hybrids will be available to up to 20,000 smallholder farmers for approximately 40,000 hectares of planting. In line with the CGIAR Principles on the Management of Intellectual Assets and CIMMYT’s constant endeavor to treat its improved germplasm as international public good, the MLN-resistant hybrids will be available royalty-free and seed companies entering into commercialization/licensing agreements in connection with this project will not be allowed to charge smallholder farmers higher seed cost. In this way, more farmers in MLN-affected countries in eastern and Central Africa can eventually benefit from increased supply of high-yielding, MLN-resistant and affordable maize products.

Related documents:

MLN Gene Editing project brief

MLN Gene Editing project: FAQs

Kanwarpal Dhugga awarded top honor in science

Kanwarpal S. Dhugga, a Principal Scientist at the International Maize and Wheat Improvement Center (CIMMYT) who specializes in biotechnology, has been elected a Fellow of the American Association for the Advancement of Science (AAAS), Section on Biological Sciences, in recognition of his invaluable contributions to science and technology.

Announced by AAAS on November 26, 2019, the honor acknowledges among other things Dhugga’s leading research on plant cell wall formation, with applications including their role in lodging resistance and in producing high-value industrial polymers in maize and soybean, and the assimilation, transport, and metabolism of nitrogen in plants.

“I consider this a special honor,” said Dhugga, who leads CIMMYT’s research in biotechnology with a focus on editing genes for disease resistance in maize and wheat. He has published in high-impact scientific journals including Science, the Proceedings of the National Academy of Sciences (USA), Plant Cell, Molecular Plant, Plant Biotechnology Journal, Plant Physiology and others.

AAAS Fellows are elected each year by their peers serving on the Council of AAAS, the organization’s member-run governing body. Scientists who have received this recognition include the inventor Thomas Edison (1878), anthropologist Margaret Mead (1934), and popular science author Jared Diamond (2000), as well as numerous Nobel laureates. The election of Dhugga doubles the tally of AAAS fellows at CIMMYT, the other one being Ravi P. Singh, Distinguished Scientist and Head of Global Wheat Improvement.

“Kanwarpal merits CIMMYT’s wholehearted congratulations for this prestigious recognition of his standing in science,” said Kevin Pixley, director of CIMMYT’s Genetics Resources program, to which Dhugga belongs. “I’m humbled and grateful to count him as a member of our team.”

Dhugga identified the gene for an enzyme that propels the chemical reactions to produce guar gum, a cell wall polymer that is a dominant component of the edible kernel of the coconut. (Photo: Allen Wen/CIMMYT)
Dhugga identified the gene for an enzyme that propels the chemical reactions to produce guar gum, a cell wall polymer that is a dominant component of the edible kernel of the coconut. (Photo: Allen Wen/CIMMYT)

A native of Punjab in India, Dhugga has a M.Sc. in Plant Breeding from Punjab Agricultural University and a Ph.D. in Botany (Plant Genetics) from the University of California, Riverside. He was introduced to membrane protein biochemistry and cell wall synthesis during his postdoctoral research at Stanford University in the laboratory of Peter Ray. Prior to joining CIMMYT in 2015, Dhugga worked at DuPont Pioneer (now Corteva) from 1996 to 2014.

In addition to scientific excellence, Dhugga counts among his achievements prominent international, public-private partnerships, such as the one he led between DuPont Pioneer and the Australian Centre for Plant Functional Genomics to explore new avenues to improve plant nitrogen use efficiency and reduce culm (stalk) lodging in cereals from 2004 to 2014. He continues to explore opportunities to secure funds for undertaking joint work with the collaborators from that period, thanks to the relationships fostered then. One of the scientists in his current group actually completed his Ph.D. under that collaboration.

As part of science outreach he has guided the research of many graduate students in Australia, Canada, India, and the US, a country of which he is also a citizen, and helped make high-quality education accessible to the underprivileged, including establishing a private school in his ancestral village in the state of Punjab in India.

The 2019 Fellows will receive rosette pins in gold and blue, colors symbolizing science and engineering. (Photo: AAAS)
The 2019 Fellows will receive rosette pins in gold and blue, colors symbolizing science and engineering. (Photo: AAAS)

Dhugga has also been successful as a principal or co-principal investigator in attracting significant funding for scientific research from public agencies such as the US Department of Energy, the US National Science Foundation, USAID, and the Australian Research Council. Part of his current research is supported by a grant from the Bill & Melinda Gates Foundation. At DuPont Pioneer he was the recipient of two separate, highly competitive research grants to carry out high-risk, discovery research outside of the area of the assigned company goals.

Among his research endeavors, Dhugga highlights a breakthrough he made in the area of cell wall biosynthesis under a discovery research grant from DuPont Pioneer. He identified the gene for an enzyme that propels the chemical reactions to produce guar gum, a cell wall polymer that is also used in industrial products from shampoos to ice cream and is a dominant component of the coconut kernel. The results were published in Science. On a basic level, this provided biochemical evidence for the first time for the involvement of any of the genes from the large plant cellulose synthase gene family in the formation of a cell wall polymer. Dhugga also confides that whenever he flies over coconut plantations anywhere, he gets butterflies in his stomach at the thought that he was the first one to know how simple molecules made a complex matrix that became the edible kernel of the coconut.

“That study constituted a prime example of the power of cross-disciplinary research in answering a longstanding fundamental question in plant biology,” he said. “Assaying enzymes involved in the formation of cell wall polymers is extremely difficult. The approach we used — identify a candidate gene by combining genomics with biochemistry and then express it in a related species lacking the product of the resulting enzyme to demonstrate its function — was subsequently applied by other scientists to identify genes involved in the formation of other key plant cell wall polymers.”

Dhugga will receive a pin as a token of his election as Fellow in an AAAS ceremony in Seattle, Washington, USA, on February 15, 2020.