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Doubled haploid technology brings promise to wheat breeders

By Jennifer M. Latzke

EMASCULATING WHEAT—Chenggen Chu, Ph.D., delicately emasculates a wheat plant, preparing it for the doubled haploid breeding process. By taking out the male portion of the wheat's genetic code and manipulating the plant into producing seed, the doubled haploid process results in a wheat seed that is genetically pure for researchers to study further—all in nine months instead of six to eight years. (Photo courtesy Heartland Plant Innovations.)

Tucked away in a few small labs and greenhouse spaces, in Throckmorton Hall on the Kansas State University campus, scientists with Heartland Plant Innovations are bringing about a revolution in wheat breeding efficiency.

For decades wheat breeders have had one overwhelming limitation to their programs--time. But, with HPI's new Wheat Doubled Haploid laboratory, time may no longer be the limitation it used to be for the discovery of new and improved wheat varieties.

Trimming time

Traditionally, wheat breeders identify two parental lines that they believe have potential and breed them for a better offspring variety. With traditional breeding, plants receive half of their chromosomes from each parent plant. Then, through inbreeding, scientists eventually get a plant that is completely homozygous. From the first cross to the finished pure new wheat variety, the traditional process can take up to 12 years. And seven of those years are devoted to just inbreeding the wheat so that breeders find a genetically pure, true breeding line to evaluate.

But, at HPI's new Wheat Doubled Haploid laboratory, a technology for corn breeders has been adapted for wheat and is drastically reducing the time from first cross to pure line--from the typical six to eight generations to one.

In doubled haploid breeding, plants are manipulated so that they have two copies of each chromosome and are essentially a clone of the gamete used. The haploid genome of the gamete, when doubled, results in a plant with a complete genome with two identical copies of every gene. Doubled haploids are homozygous at every locus--in one generation, versus six or more generations in conventional inbreeding programs. This helps wheat breeders trying to identify and isolate valuable new traits for future varieties.

"Doubled haploid breeding can speed up the discovery of new genes," said Forrest Chumley, Ph.D., president and chief executive officer of HPI. "Breeders can compare traits in lines quicker, using genetic markers and develop lines quicker."

Last fall HPI brought Chenggen Chu, Ph.D., from North Dakota to head its new Doubled Haploid lab, the key component of its Advanced Plant Breeding Services business. Chumley explained that while many companies use doubled haploid breeding techniques in their own breeding programs, HPI's doubled haploid program is the first to help public and private wheat breeders. HPI's customers include public wheat breeders from Kansas State University, and public breeding programs all over the Great Plains.

And, this June, the first seed lines to successfully undergo doubled haploid breeding were delivered to public wheat breeders.

Fooling Mother Nature

The doubled haploid process takes about nine months, from the time the doubled haploid lines are ordered to their delivery, Chu explained. HPI uses the "wheat x corn method," because it is more efficient and more successful, Chumley added. Currently, HPI is only working with winter wheats, but the method can also be used on durum wheats, sorghum and barley.

The work begins when a customer sends a batch of F1 seeds from the crossbreeding of two parent plants to HPI. The F1 seeds are grown in the greenhouse to the flowering stage, where Chu and his staff begin the process of emasculating the flowers. They cut the tips of the flowers on the head, and pull out the male anthers, which are practically microscopic. This leaves the flower with just the female portion of chromosomes, Chumley explained.

Chu then takes fresh corn pollen, which is grown in a nearby greenhouse. The alien corn pollen is shaken onto the wheat flowers a few days after emasculation. "The pollen tricks the egg into becoming an embryo," Chu said. It won't, though, pass on any corn genetics to the wheat. Since the wheat egg only has half of the chromosomes it needs at this point, if left on its own it won't develop into a viable seed. So, Chu applies a dose of 2,4-D to the wheat the next day.

The 2,4-D, Chumley explained, works as a growth stimulant in the wheat and the wheat develops a seed.

The work doesn't stop there, though. Chumley and Chu added that that seed is a haploid that doesn't have a functional endosperm, which means that it won't survive if planted in soil like a typical wheat seed. So, Chu and his workers will cut the embryos from those developing seeds about two weeks into their development. These rescued embryos are microscopic and it takes a deft touch, Chumley said. They're placed in test tubes in a growth medium and put into a dark cooler for about five days until the embryos start germinating and small roots and shoots start forming.

From there, they're moved to a warmer environment and allowed fluorescent light for two weeks. After this vernalization the plants are transferred to a soil medium for further growth. These haploid seedlings still won't make viable seeds if left on their own, so Chu has to treat them with a substance called "colchicine."

"Colchicine was developed as a cancer drug," Chumley said. It prevents cell division by stopping the spindle mechanism in the developing cell. It "pulls apart" the chromosomes and creates a mix of haploid and doubled haploid cells. After this treatment, the plants will be planted in soil, grown in a greenhouse and allowed to develop doubled haploid seeds, which will then be sent back to the wheat breeders for further study.

Wheat whisperer

The doubled haploid process is time consuming and more delicate than traditional breeding. It takes a special touch to get viable wheat, which is why HPI brought Chu to its Manhattan, Kan., facility.

Chu is known for his skill as a "wheat whisperer," Chumley said, because of his innate sense at each stage of the process. "When we emasculate about 100 flowers, we get at least 40 viable embryos," Chumley said. Most programs get a return of 30 percent, and Chumley attributes HPI's success to Chu's touch.

Despite the extra care this breeding technology takes, it will be a valuable tool for future developments in wheat, Chumley said. It also works well with the genomic selection that HPI is promoting in its wheat breeding efforts. Breeders can use a synergy of the methods, Chumley said, to integrate individual traits into wheat varieties they develop in the future--without the time of inbreeding and backcrossing.

The doubled haploid breeding is also a tidy profit generator for HPI, which is a public-private, for-profit collaboration with private investors and public funds through the Kansas Bioscience Authority. Currently, Chumley said, HPI has taken orders for 7,500 doubled haploid lines, with the hopes of expanding the number of lines they're breeding at a time to 25,000 or 50,000 per year once HPI moves into its new facility, the Kansas Wheat Innovation Center, in Manhattan.

"Right now, we are limited in how many numbers of doubled haploid lines we can develop because of our limited space," Chumley said. "Our lease with K-State runs out June 30, 2012, which is about the time we should get into the new building. KSU has been extremely helpful and supportive of HPI."

HPI contracts delivery of a minimum of 10 doubled haploid lines for every F1 seed, and a minimum of 20 seeds per doubled haploid line. At a price of $30 per line, it's just enough to cover costs and provide a return, Chumley said.

But, the extra value is in the development of wheats that will help farmers and end users, five and six years sooner than traditional breeding allows, Chumley said. Wheat is a big economic driver in Kansas, he added. As a KBA Center of Innovation, HPI has a goal to use science and technology to improve agriculture's value in the state.

And, in those small labs and greenhouses, the revolutionary work of Chu and his staff is making that goal a reality today.

Jennifer M. Latzke can be reached at 620-227-1807, or jlatzke@hpj.com.

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