Tag Archives: citrus greening

“transforming a plant is still an art” even with CRISPR

“Plus ça change, plus c’est la même chose (the more things change, the more things stay the same), is an old French expression that came to mind when I stumbled across two stories about genetic manipulation of food-producing plants.

The first story involves CRISPR (clustered regularly interspersed short palindromic repeats) gene editing and the second involves more ancient ways to manipulate plant genetics.

Getting ‘CRISPR’d’ plant cells to grow into plants

Plants often don’t grow from cells after researchers alter their genomes. Using a new technology, a team coaxed wheat (above) and other crops to more readily produce genome-edited healthy adult plants. Credit: Juan Debernardi

An October 13, 2020 news item on phys.org announces research about getting better results after a plant’s genome has been altered,

Researchers know how to make precise genetic changes within the genomes of crops, but the transformed cells often refuse to grow into plants. One team has devised a new solution.

Scientists who want to improve crops face a dilemma: it can be difficult to grow plants from cells after you’ve tweaked their genomes.

A new tool helps ease this process by coaxing the transformed cells, including those modified with the gene-editing system CRISPR-Cas9, to regenerate new plants. Howard Hughes Medical Institute Research Specialist Juan M. Debernardi and Investigator Jorge Dubcovsky, together with David Tricoli at the University of California, Davis [UC Davis] Plant Transformation Facility, Javier Palatnik from Argentina, and colleagues at the John Innes Center [UK], collaborated on the work. The team reports the technology, developed in wheat and tested in other crops, October 12, 2020, in the journal Nature Biotechnology.

An October 12, 2020 Howard Hughes Medical Institute (HHMI) news release, which originated the news item, provides more detail,

“The problem is that transforming a plant is still an art [emphasis mine],” Dubcovsky says. The success rate is often low – depending on the crop being modified, 100 attempts may yield only a handful of green shoots that can turn into full-grown plants. The rest fail to produce new plants and die. Now, however, “we have reduced this barrier,” says Dubcovsky, a plant geneticist at UC Davis. Using two genes that already control development in many plants, his team dramatically increased the formation of shoots in modified wheat, rice, citrus, and other crops.

Although UC Davis has a pending patent for commercial applications, Dubcovsky says the technique is available to any researcher who wants to use it for research, at no charge. A number of plant breeding companies have also expressed interested in licensing it. “Now people are trying it in multiple crops,” he says.

Humans have worked to improve plants since the dawn of agriculture, selecting wild grasses to produce cultivated maize and wheat, for example. Nowadays, though, CRISPR has given researchers the ability to make changes to the genome with surgical precision. They have used it to create wheat plants with larger grains, generate resistance to fungal infection, design novel tomato plant architectures, and engineer other traits in new plant varieties.

But the process isn’t easy. Scientists start out with plant cells or pieces of tissue, into which they introduce the CRISPR machinery and a small guide to the specific genes they’d like to edit. They must then entice the modified cells into forming a young plant. Most don’t sprout – a problem scientists are still working to understand.

They have tried to find work-arounds, including boosting the expression of certain genes that control early stages of plant development. While this approach has had some success, it can lead to twisted, stunted, sterile plants if not managed properly.Dubcovsky and his colleagues looked at two other growth-promoting genes, GRF and GIF, that work together in young tissues or organs of plants ranging from moss to fruit trees. The team put these genes side-by-side, like a couple holding hands, before adding them to plant cells. “If you go to a dance, you need to find your partner,” Dubcovsky says. “Here, you are tied with a rope to your partner.”

Dubcovsky’s team found that genetically altered wheat, rice, hybrid orange, and other crops produced many more shoots if those experiments included the linked GRF and GIF genes. In experiments with one variety of wheat, the appearance of shoots increased nearly eight-fold. The number of shoots in rice and the hybrid orange, meanwhile, more than doubled and quadrupled, respectively. What’s more, these shoots grew into healthy plants capable of reproducing on their own, with none of the defects that can result when scientists boost other development-controlling genes. That’s because one of the genes is naturally degraded in adult tissues, Dubcovsky says.

Caroline Roper, a plant pathologist at University of California, Riverside who was not involved in the work, plans to use the new technology to study citrus greening, a bacterial disease that kills trees and renders oranges hard and bitter.

To understand how citrus trees can protect themselves, she needs to see how removing certain genes alters their susceptibility to the bacterium — information that could lead to ways to fight the disease. With conventional techniques, it could take at least two years to generate the gene-edited plants she needs. She hopes Dubcovsky’s tool will shorten that timeline.  

“Time is of the essence. The growers, they wanted an answer yesterday, because they’re at the brink of having to abandon cultivating citrus,” she says.

For anyone who noticed the reference to citrus greening in the last paragraphs of this news release, I have more information aboutthe disease and efforts to it in an August 6, 2020 posting.

As for the latest in gene editing and regeneration, here’s a link to and a citation for the paper,

A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants by Juan M. Debernardi, David M. Tricoli, Maria F. Ercoli, Sadiye Hayta, Pamela Ronald, Javier F. Palatnik & Jorge Dubcovsky. Nature Biotechnology volume 38, pages 1274–1279(2020) DOI: https://doi.org/10.1038/s41587-020-0703-0 First Published Online: 12 October 2020 Journal Issue Date: November 2020

This paper is behind a paywall.

Ancient farming techniques for engineering crops

I stumbled on this story by Gabriela Serrato Marks for Massive Science almost three years late (it’s a Dec. 5, 2017 article),

There are more than 50 strains of maize, called landraces, grown in Mexico. A landrace is similar to a dog breed: Corgis and Huskies are both dogs, but they were bred to have different traits. Maize domestication worked the same way.

Some landraces of maize can grow in really dry conditions; others grow best in wetter soils. Early maize farmers selectively bred maize landraces that were well-adapted to the conditions on their land, a practice that still continues today in rural areas of Mexico.

If you think this sounds like an early version of genetic engineering, you’d be correct. But nowadays, modern agriculture is moving away from locally adapted strains and traditional farming techniques and toward active gene manipulation. The goal of both traditional landrace development and modern genetic modification has been to create productive, valuable crops, so these two techniques are not necessarily at odds.

But as more farmers converge on similar strains of (potentially genetically modified) seeds instead of developing locally adapted landraces, there are two potential risks: one is losing the cultural legacy of traditional agricultural techniques that have been passed on in families for centuries or even millennia, and another is decreasing crop resilience even as climate variability is increasing.

Mexico is the main importer of US-grown corn, but that imported corn is primarily used to feed livestock. The corn that people eat or use to make tortillas is grown almost entirely in Mexico, which is where landraces come in.

It is a common practice to grow multiple landraces with different traits as an insurance policy against poor growth conditions. The wide range of landraces contains a huge amount of genetic diversity, making it less likely that one adverse event, such as a drought or pest infestation, will wipe out an entire crop. If farmers only grow one type of corn, the whole crop is vulnerable to the same event.

Landraces are also different from most commercially available hybrid strains of corn because they are open pollinating, which means that farmers can save seeds and replant them the next year, saving money and preserving the strain. If a landrace is not grown anymore, its contribution to maize’s genetic diversity is permanently lost.

This diversity was cultivated over generations from maize’s wild cousin, teosinte, by 60 groups of indigenous people in Mexico. Teosinte looks like a skinny, hairier version of maize. It still grows wild in some parts of Central America, but its close relatives have been found, domesticated, at archaeological sites in the region over 9,000 years old. These early maize cobs could easily fit in the palm of your hand – not big enough to be a staple crop that early farmers could depend upon for sustenance. Genetically, they were more similar to wild teosinte than to modern maize.

[] archaeologists also found that the cobs in Honduras, which is outside the natural range of teosinte, were larger than cobs of the same age from the original domestication region in southern Mexico. The scientists think that people in Honduras were able to develop more productive maize landraces because their crops were isolated from wild teosinte.

The size and shape of the ancient cobs from Honduras show that early farmers engineered the maize crop [emphasis mine] to make it more productive. They developed unique landraces that were well adapted to local conditions and successfully cultivated enough maize to support their communities. In many ways, they were early geneticists. [emphasis mine] …

We have a lot to learn from the indigenous farmers who were growing maize 4,000 years ago. Their history provides examples of both environmentally sound genetic modification and effective adaptation to climate variability. [emphases mine] …

Plus ça change …, eh?

Effective treatment for citrus-destroying disease?

Citrus greening is a worldwide problem. A particularly virulent disease that destroys citrus fruit, it’s a problem that is worsening. Before getting to the research from the University of California at Riverside (UCR), here’s more about the disease and how it’s developing from the UCR Huanglongbing, (HLB, Citrus Greening webpage,

The Situation: Citrus huanglongbing (HLB), previously called citrus greening disease, is one of the most destructive diseases of citrus worldwide.  Originally thought to be caused by a virus, it is now known to be caused by unculturable phloem-limited bacteria.  There are three forms of greening that have been described.  The African form produces symptoms only under cool conditions and is transmitted by the African citrus psyllid Trioza erytreae, while the Asian form prefers warmer conditions and is transmitted by the Asian citrus psyllid Diaphorina citri.  Recently a third American form transmitted by the Asian citrus psyllid was discovered in Brazil.  This American form of the disease apparently originated in China.  In North America, the psyllid vector, Diaphorina citri, of HLB is found in Florida, Louisiana, Georgia, South Carolina, Texas and Hawaii, and recently arrived in Southern California from Mexico. HLB is known to occur in Florida Lousiana, South Carolina, Georgia, Cuba, Belize and the Eastern Yucatan of Mexico.  A federal quarantine restricts all movement of citrus and other plants in the family Rutaceae from Asian Citrus Psyllid or HLB-infested areas into California in order to prevent introduction of the disease.

 Damage:  The HLB bacteria can infect most citrus cultivars, species and hybrids and even some citrus relatives.  Leaves of newly infected trees develop a blotchy mottle appearance.  On chronically infected trees, the leaves are small and exhibit asymmetrical blotchy mottling (in contrast to Zinc deficiency that causes symmetrical blotching).  Fruit from HLB-infected trees are small, lopsided, poorly colored, and contain aborted seeds. The juice from affected fruit is low in soluble solids, high in acids and abnormally bitter.  The fruit retains its green color at the navel end when mature, which is the reason for the common name “citrus greening disease.”  This fruit is of no value because of poor size and quality.  There is no cure for the disease and rapid tree removal is critical for prevention of spread.

Economic Impact: HLB is one of the most devastating diseases of citrus and since its discovery in Florida in 2005, citrus acreage in that state has declined significantly.  If the disease were to establish in California, the nursery industry would be required to move all of their production under screenhouses, pesticide treatments for the vector would be instituted resulting in greatly increased pesticide costs (3-6 treatments per year) and indirect costs due to pesticide-induced disruption of integrated pest management programs for other citrus pests.  A costly eradication program would need to be instituted to remove infected trees in order to protect the citrus industry.

Distribution of HLB: In April 2012, after about a week of testing, the California Department of Food and Agriculture (CDFA) removed a pumelo tree with a lemon graft from Hacienda Heights in Los Angeles County after the tree and an Asian citrus psyllid found on the tree both tested positive for Huanglongbing. In 2005, HLB was also found in Florida and it is now known to occur in Louisiana, Georgia, South Carolina, Cuba, Belze and Eastern Mexico.  Worldwide, HLB is also present in China, eastern and southern Africa, the Indian subcontinent, Mauritius, Reunion, the Saudi Arabian peninsula, and southeast Asia.

Research:  Research is focusing on characterization of the bacteria, development of detection methods, and control of the disease and the psyllid.  To date, control of the disease is based on planting HLB-free citrus germplasm, eradication of infected citrus plants, and control of the vector with systemic insecticides.  Countries with HLB learn to manage the disease so that they can still produce citrus.  In California, the best strategy is to keep this disease out. This goal is supported by both federal and state quarantine regulations and the University of California’s Citrus Clonal Protection Program, which provides a mechanism for the safe introduction of citrus germplasm into California.

A July 7, 2020 news item on phys.org announces what researchers hope can be used commercially as a new treatment for citrus greening disease from researchers University of California at Riverside (UCR), Note: Links have been removed,

UC Riverside scientists have found the first substance capable of controlling Citrus Greening Disease, which has devastated citrus farms in Florida and also threatens California.

The new treatment effectively kills the bacterium causing the disease with a naturally occurring molecule found in wild citrus relatives. This molecule, an antimicrobial peptide, offers numerous advantages over the antibiotics currently used to treat the disease.

UCR geneticist Hailing Jin, who discovered the cure after a five-year search, explained that unlike antibiotic sprays, the peptide is stable even when used outdoors in high heat, easy to manufacture, and safe for humans.

A July 7, 2020 UCR news release (also on EurekAlert) by Jules Bernstein, which originated the news item, provides technical detail and information about plans to commercialize the product,

“This peptide is found in the fruit of Australian finger limes, which can naturally tolerate Citrus Greening bacteria and has been consumed for hundreds of years,” Jin said. “It is much safer to use this natural plant product on agricultural crops than other synthetic chemicals.”

Currently, some growers in Florida are spraying antibiotics and pesticides in an attempt to save trees from the CLas bacterium that causes citrus greening, also known as Huanglongbing or HLB.

“Most antibiotics are temperature sensitive, so their effects are largely reduced when applied in the hot weather,” Jin said. “By contrast, this peptide is stable even when used in 130-degree heat.”

Jin found the peptide by examining plants such as the Australian finger lime known to possess natural tolerance for the bacteria that causes Citrus Greening Disease, and she isolated the genes that contribute to this innate immunity. One of these genes produces the peptide, which she then tested over the course of two years. Improvement was soon visible.

“You can see the bacteria drastically reduced, and the leaves appear healthy again only a few months after treatment,” Jin said.

Because the peptide only needs to be reapplied a few times per year, it is highly cost effective for growers. This peptide can also be developed into a vaccine to protect young healthy plants from infection, as it is able to induce the plant’s innate immunity to the bacteria.

Jin’s peptide can be applied by injection or foliage spray, and it moves systemically through plants and remains stable, which makes the effect of the treatment stronger.

The treatment will be further enhanced with proprietary injection technology made by Invaio Sciences. UC Riverside has entered into an exclusive, worldwide license agreement with Invaio, ensuring this new treatment goes exactly where it’s needed in plants.

“Invaio is enthusiastic to partner with UC Riverside and advance this innovative technology for combating the disease known as Citrus Greening or Huanglongbing,” said Invaio Chief Science Officer Gerardo Ramos. “The prospect of addressing this previously incurable and devastating crop disease, helping agricultural communities and improving the environmental impact of production is exciting and rewarding,” he said. “This is crop protection in harmony with nature.”

The need for an HLB cure is a global problem, but hits especially close to home as California produces 80 percent of all the fresh citrus in the United States, said Brian Suh, director of technology commercialization in UCR’s Office of Technology Partnerships, which helps bring university technology to market for the benefit of society through licenses, partnerships, and startup companies.

“This license to Invaio opens up the opportunity for a product to get to market faster,” Suh said. “Cutting edge research from UCR, like the peptide identified by Dr. Jin, has a tremendous amount of commercial potential and can transform the trajectory of real-world problems with these innovative solutions.”

You can find out more about Invaio Sciences here.

Citrus greening has been featured here before in an April 7, 2015 posting titled, Citrus canker, Florida, and Zinkicide. There doesn’t seem to have been much progress made with this Florida solution for citrus greening. This 2018 document on nano.gov was the most recent I could find, ZinkicideTM- a systemic nano-ZnO based bactericide/fungicide for crop protection by Swadeshmukul Santra.

US Dept. of Agriculture awards $3.8M for nanotechnology research grants

I wonder just how much funding the US Dept. of Agriculture (USDA) is devoting to nanotechnology this year (2015). I first came across an announcement of $23M in the body of a news item about Zinkicide (my April 7, 2015 posting),

Found in Florida orchards in 2005, a citrus canker, citrus greening, poses a serious threat to the US state’s fruit industry. An April 2, 2105 news item on phys.org describes a possible solution to the problem,

Since it was discovered in South Florida in 2005, the plague of citrus greening has spread to nearly every grove in the state, stoking fears among growers that the $10.7 billion-a-year industry may someday disappear.

Now the U.S. Department of Agriculture has awarded the University of Florida a $4.6 million grant aimed at testing a potential new weapon in the fight against citrus greening: Zinkicide, a bactericide invented by a nanoparticle researcher at the University of Central Florida.

An April 29, 2015 article by Diego Flammini for Farm.com describes the latest USDA nanotechnology funding announcement,

In an effort to increase America’s food security, nutrition, food safety and environmental protection, the United States Department of Agriculture’s (USDA) National Institute of Food and Agriculture (NIFA) announced $3.8 million in nanotechnology research grants.

Flammini lists three of the eight recipients,

University of Georgia
With $496,192, the research team will develop different sensors that are able to detect fungal pathogens in crops. The project will also develop a smartphone app for farmers to have so they can access their information whenever necessary.

Rutgers University
The school will use its $450,000 to conduct a nationwide survey about nanotechnology and gauge consumer beliefs about it and its relationship to health. Among the specifics it will touch on is the use of visuals to communicate nanotechnology.

University of Massachusetts
The researchers will concentrate their $444,200 on developing a platform to detect pathogens in food that is better than the current methods.

A full list of the recipients can be found in the April 27, 2015 USDA news release featuring the $3.8M in awards,

  • The University of Georgia, Athens, Ga., $496,192
  • University of Iowa, Iowa City, Iowa., $496,180
  • University of Kentucky Research Foundation, Lexington, Ky., $450,000
  • University of Massachusetts, Amherst, Mass., $444,200
  • North Dakota State University, Fargo, N.D., $149,714
  • Rutgers University, New Brunswick. N.J., $450,000
  • Pennsylvania State University, University Park, University Park, Pa., $447,788
  • West Virginia University, Morgantown, W. Va., $496,168
  • University of Wisconsin-Madison, Madison, Wis., $450,100

You can find more details about the awards in this leaflet featuring the USDA project descriptions for the eight recipients.

Citrus canker, Florida, and Zinkicide

Found in Florida orchards in 2005, a citrus canker, citrus greening, poses a serious threat to the US state’s fruit industry. An April 2, 2105 news item on phys.org describes a possible solution to the problem,

Since it was discovered in South Florida in 2005, the plague of citrus greening has spread to nearly every grove in the state, stoking fears among growers that the $10.7 billion-a-year industry may someday disappear.

Now the U.S. Department of Agriculture has awarded the University of Florida a $4.6 million grant aimed at testing a potential new weapon in the fight against citrus greening: Zinkicide, a bactericide invented by a nanoparticle researcher at the University of Central Florida.

An April 2, 2015 University of Central Florida news release by Mark Schlueb (also on EurekAlert), which originated the news item, describes the problem and the solution (Zinkicide),

Citrus greening – also known by its Chinese name, Huanglongbing, or HLB – causes orange, grapefruit and other citrus trees to produce small, bitter fruit that drop prematurely and is unsuitable for sale or juice. Eventually, infected trees die. Florida has lost tens of thousands of acres to the disease.

“It’s a hundred-year-old disease, but to date there is no cure. It’s a killer, a true killer for the citrus industry,” said Swadeshmukul Santra, associate professor in the NanoScience Technology Center at UCF.

The bacteria that causes HLB is carried by the Asian citrus psyllid, a tiny insect that  feeds on leaves and stems of infected citrus trees, then carries the bacteria to healthy trees.

Zinkicide, developed by Santra, is designed to kill the bacteria.

The $4.6 million grant is the largest of five totaling $23 million that were recently announced by the USDA’s National Institute of Food and Agriculture.

The evaluation of Zinkicide is a multi-institute project involving 13 investigators from six institutions. Evan Johnson of UF’s [University of Florida] Citrus Research and Education Center at Lake Alfred is the project director, and there are a dozen co-principal investigators from UF, UCF, Oak Ridge National Laboratory (ORNL), Auburn University, New Mexico State University and The Ohio State University.

”Managing systemic diseases like HLB is a difficult challenge that has faced plant pathologists for many years,” said Johnson “It is a privilege to work with an excellent team of researchers from many different disciplines with the goal of developing new tools that are both effective and safe.”

A portion of the grant money, $1.4 million, flows to UCF, where Santra leads a team that also includes Andre Gesquiere, Laurene Tetard and the Oak Ridge National Laboratory collaborator, Loukas Petridis.

HLB control is difficult because current bactericidal sprays, such as copper, simply leave a protective film on the outside of a plant. The insect-transmitted bacteria bypasses that barrier and lives inside a tree’s fruit, stems and roots, in the vascular tissue known as the phloem. There, it deprives the tree of carbohydrate and nutrients, causing root loss and ultimately death. For a bactericide to be effective against HLB, it must be able to move within the plant, too.

Zinkicide is a nanoparticle smaller than a single microscopic cell, and researchers are cautiously optimistic it will be able to move systemically from cell to cell to kill the bacteria that cause HLB.

“The bacteria hide inside the plant in the phloem region,” Santra said. “If you spray and your compound doesn’t travel to the phloem region, then you cannot treat HLB.”

Zinkicide is derived from ingredients which are found in plants, and is designed to break down and be metabolized after its job is done. [emphasis mine]

It’s the first step in a years-long process to bring a treatment to market. UF will lead five years of greenhouse and field trials on grapefruit and sweet orange to determine the effectiveness of Zinkicide and the best method and timing of application.

The project also includes research to study where the nanoparticles travel within the plant, understand how they interact with plant tissue and how long they remain before breaking down. [emphasis mine]

If effective, the bactericide could have a substantial role in combatting HLB in Florida, and in other citrus-producing states and countries. It would also likely be useful for control of other bacterial pathogens infecting other crops.

The Zinkicide project builds as a spinoff from previous collaborations between Santra and UF’s Jim Graham, at the Citrus Research and Education Center to develop alternatives to copper for citrus canker control.

The previous Citrus Research and Education Foundation (CRDF)-funded Zinkicide project has issued three reports, for June 30, 2014, Sept. 30, 2014, and Dec. 31, 2014. This project’s completion date is May 2015. The reports which are remarkably succinct, consisting of two paragraphs, can be found here.

Oddly, the UCF news release doesn’t mention that Zinkicide (although it can be inferred) is a zinc particulate (I’m guessing they mean zinc nanoparticle) as noted on the CRDF project webpage. Happily, they are researching what happens after the bactericide has done its work on the infection. It’s good to see a life cycle approach to this research.