Doubled Haploid Wheat Breeding Accelerates Process

Some potential new wheat varieties for Colorado are taking a side trip to Kansas.

A portion of the wheat crosses developed by Dr. Scott Haley and his research team at Colorado State University (CSU) are making a trip to Heartland Plant Innovations (HPI) in Manhattan, Kansas to undergo a doubled haploid breeding process which may lead to the release of varieties much more quickly than traditional breeding methods.

“Using doubled haploids we can shorten the breeding process from about 10-12 years to about 6-7 years,” said Dr. Scott Haley, CSU wheat breeder.

Update: The CSU wheat breeding team is now making doubled haploids on site. A doubled haploid lab is under construction. A detailed story will be available in the December Colorado Wheat Farmer.

The most time-consuming part of plant breeding is inbreeding several generations after the initial cross by self-fertilization to obtain a pure line – or at least pure enough for the seed certification process. Because of genetic variation, plants following the parent generation may or may not display the desired traits the breeder was seeking.

“Selfing,” where the plant is self-fertilized with its own pollen, can take up to seven years in a traditional breeding program. Through this process of inbreeding, wheat breeders select the plants with desirable traits, such as yield, quality, and pest resistance. These traits are typically selected through observation, field trials and DNA marker analysis. Selfing ensures the variety is uniform and will not change through further testing or when farmers save their seed.

By using the doubled haploid breeding method, seeds developed from an initial cross between two parents essentially represent clones of themselves, eliminating genetic variation that remains within a breeding line through conventional self-fertilization.

How does this process work? Wheat seeds that have resulted from a cross of two promising parents are sent to HPI. They are grown to the stage where they are developing pollen within the male part of the plant, which is easily seen as the anthers that protrude from the head at flowering time. At this stage, the plants are emasculated, with the glumes being clipped and the tiny anthers removed with tweezers. After a few days, the wheat ovaries are pollinated with fresh corn pollen, which is grown in another greenhouse at HPI. The corn pollen stimulates wheat embryo development, but because the wheat and corn are so different, no corn genetic material remains in the wheat embryo after the first few cell divisions. At this point, 2,4-D and a key plant hormone called gibberellic acid are applied to the developing embryo. These act as growth stimulants to encourage the immature embryo to continue to develop.

Dr. Chenggen Chu, Director of Advanced Plant Breeding Services at HPI, fertilizes wheat with corn pollen. Photo courtesy HPI.

Dr. Chenggen Chu, Director of Advanced Plant Breeding Services at HPI, fertilizes wheat with corn pollen. Photo courtesy HPI.

At this point, the embryo is not viable since it only has the single copy of chromosomes from the wheat plant. This embryo would not develop into a seed if allowed to continue development on its own. So the embryos are removed from the plants using tweezers and a microscope, and are cultured in a growth medium. This process is known as “embryo rescue.” After the embryos grow in this medium they may regenerate into whole plants that are “haploids,” meaning they only contain one copy of the chromosomes of a normal wheat plant. Following early seedling development, the haploid plants are treated with colchicine, which serves to duplicate the chromosomes in the cells. The plants are then raised in a greenhouse, and the seeds harvested and returned to the plant breeder. At each stage in this overall process there are bottlenecks which reduce the efficiency of the doubled haploid development procedure.

Rescued embryos: On the left, normal wheat embryos, on the right, haploid embryos.  Photo courtesy HPI.

Rescued embryos: On the left, normal wheat embryos, on the right, haploid embryos. Photo courtesy HPI.

The time saved by using the doubled haploid process is great, but CSU is not using doubled haploids for all of its crosses. CSU does well over 1,000 new conventional crosses each year, and using the doubled haploid process for all of them wouldn’t be possible.

“First of all, it is really quite expensive, especially compared to the conventional breeding approaches we are using,” said Haley, “Also, because of the expense you tend to target crosses you think are going to have the best chance of leading to good lines. It is possible that I would not have used the cross that led to the variety Byrd for generating doubled haploids. With the large number of crosses we do in the traditional manner, we get surprises, and sometimes those surprises can turn out to be something like the next Byrd.”

Doubled haploid wheat breeding produces instant homozygous wheat lines.

Doubled haploid wheat breeding produces instant homozygous wheat lines.

With funding from the Colorado Wheat Administrative Committee (CWAC) and the Colorado Wheat Research Foundation (CWRF), CSU is also using another advanced breeding technique known as “genomic selection” to make more rapid progress in wheat breeding. With conventional means, it takes roughly seven years from the time a cross is made until a superior experimental line from that cross has proven itself worthy to use in crossing. Using genomic selection, together with doubled haploid breeding, this time can potentially be reduced to two years.

Haley said, “We have averaged about one percent per year yield improvement over the last few decades. We need to dramatically increase this in order to feed a growing world population. Genomic selection using doubled haploids is the most promising technology on the horizon at this time to meet this challenge.” As part of this project, the breeding program is also exploring whether the same techniques used for genomic selection may also be useful to predict which crosses should be made and used for doubled haploid generation. “If this is successful, it will revolutionize wheat breeding,” Haley said.

Doubled haploid breed saves up to eight years compared to conventional breeding.

Doubled haploid breed saves up to eight years compared to conventional breeding.

In September 2011, CSU received about 1,000 doubled haploid lines from HPI, which were created from crosses made in the greenhouse in spring 2010. They were grown in Yuma, Arizona, over the winter, and in May, Haley selected 328 lines to be planted in yield trials at seven locations in Colorado. Those lines were planted this fall and will be harvested next summer. Thus, the breeding program has gone from crossing to multi-locational field trials in a little over two years, at least three years less than with conventional means. A second (and larger) set of doubled haploids from HPI was recently generated and will be grown for seed increase in Yuma, Arizona, during winter 2012-13. Haley said it is possible that the first CSU variety developed using doubled haploids will be released three or four years from now.

“It is no big secret that doubled haploid breeding is also something that private industry is doing. In order to keep public wheat breeding programs viable, we need to adapt and use all available technologies to help us develop better wheat varieties,” Haley said.

The use of the doubled haploid technique for CSU wheat lines is made possible by funding from CWRF royalties from the sale of PlainsGold certified seed varieties, and additional support from ConAgra.

As part of its continuing support for the CSU wheat breeding program, ConAgra contributes funding for additional doubled haploid crosses beyond what is funded with CWRF royalties.

Mike Veal, ConAgra vice president of marketing said, “We appreciate the value the CSU wheat breeding program not only provides to farmers and bakers, but also to consumers who enjoy the tastes and nutrition of wheat-based foods on a daily basis. By investing in the wheat breeding program, and in doubled haploids in particular, we hope to help Dr. Haley and his team develop new varieties faster that benefit farmers, end-users and the environment. Like farmers and consumers, we are interested in improved nutrition, better yields, reduced inputs, higher performance, and improved quality in hard red wheat varieties as well as hard white wheat varieties.”

Doubled haploids are part of the Wheat Research Initiatives adopted by the CWAC board of directors in November 2010. The initiative document states that the purpose of the initiatives are to: Implement the Colorado Wheat Strategic Plan which includes the following goals: to protect and position the CSU wheat breeding program and CSU germplasm; preserve and strengthen the CSU wheat breeding program; and support development of wheat research collaborations between CSU and biotech companies that strengthen the CSU wheat breeding program.

Specifically, the initiatives state, as a strategy: Implement breeding strategies to release wheat varieties more rapidly using advanced single seed descent techniques, doubled haploids (under contract via HPI) and other new breeding technologies.

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