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UF research shows promise in finding cure for citrus greening

Fred Gmitter, a professor of citrus breeding and genetics with the University of Florida, peels a piece of fruit on Oct. 6 at the Lake Alfred Citrus Research and Education Center.
Fred Gmitter, a professor of citrus breeding and genetics with the University of Florida, peels a piece of fruit on Oct. 6 at the Lake Alfred Citrus Research and Education Center.
Published Oct. 17, 2016


The researchers' tools were chemistry class standards — glass flasks, test tubes and pipettes — but the task performed may be part of a revolutionary cure for the scourge of the state's orange industry: citrus greening.

The test tube in a tiny lab at the Lake Alfred Citrus Research and Education Center held dissolved citrus tissue. The fluid dripping into it from a pipette carried an enzyme that can snip genes like scissors, along with strands of RNA to guide the enzyme to precise genetic targets.

They are part of a gene-editing tool called CRISPR, which is so efficient it has been loosely compared to the "find and replace" function of a word-processing program.

Nian Wang, the University of Florida professor leading this CRISPR research team at Lake Alfred, said the tool could cut decades from reaching the holy grail of citrus research — producing varieties immune from the disease that has devastated Florida's most iconic industry.

"Come back in two or three years, and I'll show you a tree," Wang said.

The benefits of CRISPR are not just that it can eliminate genes that make citrus vulnerable to greening. It can replace them with other genes from the same plant, rather than from jellyfish or spinach, as has been the case with some previous genetically modified organisms.

Mostly because CRISPR products don't borrow genes from other plants or animals, the U.S. Department of Agriculture ruled last spring that they won't face the same regulation as earlier generations of GMOs.

"If there is any kind of regulatory approval needed, it's going to be much shorter and much cheaper," said Dr. Michael Rogers, director of the UF-run research center east of Lakeland.

The method could also provide growers fighting to hang on using a variety of expensive, lower-tech tools — including the regular replanting of groves — with a product more palatable to the public.

"To knock out a specific gene without introducing a new gene would solve the challenge facing growers and would be more acceptable for consumers," said Larry Black, a grower from Fort Meade.

The impact of greening, a bacterial disease carried by small insects called psyllids, can be seen in the many groves of dead or dying trees surrounding the research center on the northern edge of the Central Florida citrus belt.

The annual production of oranges in Florida, 242 million boxes in the 2003-2004 season, dropped to 81.6 million boxes last season. And on Wednesday, the USDA released a dire prediction for the coming harvest, 70 million boxes — though some of that predicted decline is blamed on heavy rains last spring that stripped blossoms from trees before they were able to bear fruit.

CRISPR is based on a virus-fighting system in bacteria that uses DNA, the building blocks of chromosomes, and ribonucleic acid, which transmits genetic messages.

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In the 1980s, researchers noticed that bacteria stockpile what seemed to be stray bits of DNA called "clustered regularly interspaced short palindromic repeats," or CRISPR.

They later found that these strands of genetic material match DNA of their viral enemies — and that bacteria used them as models to create RNA that takes enzymes on seek-and-destroy missions against the virus's DNA.

In recent years, scientists learned to duplicate this process and apply it to other organisms.

"All biologists have to do is feed (the enzymes) the right sequence, called a guide RNA, and boom, you can cut and paste bits of DNA sequence into the genome wherever you want," the tech website, Gizmodo, reported in 2015

The process has been touted as a possible cure for a wide range of genetic maladies in humans, and Wang has already shown it can work in citrus, producing a variety of grapefruit resistant to an earlier menace of the industry, citrus canker.

Proof of the industry's confidence in Wang's research is an initial $500,000 grant from the grower-funded Citrus Development and Research Foundation. This was followed by a $1 million line item included in the 2015-16 state budget by the Florida Legislature, which pledged to support his work for three years.

But Wang also said his project is more time-consuming than the hype over CRISPR might suggest, requiring a team of six researchers and tools far more sophisticated than test tubes and flasks, including sequencing equipment and "gene guns" that shoot DNA into cells.

The vast array of genes in the citrus genome means that some are very similar, which in turn means that the RNA might lock on to a beneficial gene rather than one the researchers are targeting.

"You have to analyze the genome very carefully to make sure you only target the bad guys and not the good guys," Wang said.

Then there's the laborious process of identifying which genes are, in fact, the "bad guys" — the ones that make the trees susceptible to greening.

This process is further complicated by the systemic nature of the disease, said Fred Gmitter, another UF professor at Lake Alfred who is conducting CRISPR research, and who earlier helped map the genome of the sweet orange.

"It's not like you have a single gene for susceptibility and you just knock that out and everything is fine," Gmitter said.

"Anything you can imagine metabolically happening in the plant, there are changes brought about by this disease."

And even after all these "susceptibility genes" are identified and deactivated, Wang said, his team must surmount the obstacle that always faces researchers of slow-growing fruit trees: A wait of years to determine whether the disease-resistant tree has retained all the qualities — especially the ability to produce sweet, marketable fruit — that growers demand.

But even that process is hastened by CRISPR, because the process gives researchers detailed knowledge of the modified tree's genetics before it goes into the ground.

"I think this is one of the most promising technologies out there," Rogers said.

Contact Dan DeWitt at Follow @ddewitttimes.


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