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Doubled Haploids: Breeding’s Future?

Sunday, November 1, 2009
filed under: Optimizing Plant Development/Yields

Is the term “doubled haploid” part of your everyday vocabulary? No? Well, you’re not alone. Most of us have little or no understanding of this term’s meaning; nor do we need to understand it in detail. But what is important is for those individuals who work to develop new sunflower hybrids to understand — and to able to exploit the benefits of — doubled haploids.

Doubled-haploid breeding is used in numerous crops — everything from wheat and barley to corn and rice, canola and sunflower. Here’s a thumbnail explanation of how it applies to sunflower specifically:

• Sunflower has 17 pairs of chromosomes (compared to 10 in corn). One chromosome in each pair comes from the male parent, the other from the female parent. The genes found on the left chromosome of a given pair may — or may not — be identical to the genes on the right chromosome.

• During the hybrid development process, a central goal of the sunflower breeder is to develop inbred parent lines with the same genes on both chromosomes of the pair. Doing so results in what’s termed a “homozygous inbred.”

• Traditionally, this objective has been achieved through a multi-year self-pollination, backcrossing and evaluation process that can involve seven, eight or even more generations. And, even upon successful inbred development, the genetic purity of that inbred still may be no more than 90%.

• Doubled-haploid breeding is a much faster way of developing an elite inbred line. Plus, it results in 100% genetic purity. In other words, the inbred’s genetic traits are completely fixed — “set in stone.”

• How does doubled-haploid breeding occur? The parent lines are pollinated with a haploid inducer, resulting in a haploid plant possessing just one chromosome — not two — from each pair. (In a haploid plant, each cell contains one set of chromosomes.) The haploids then are subjected to a special treatment that causes the single chromosome to double — i.e., producing a duplicate, with the duplicate chromosome being genetically identical to the original.

• So the haploid — which previously had 17 single chromosomes — now has 17 double chromosomes. The sunflower plant that is grown from this seed is referred to as a “doubled-haploid” inbred. The inbred can be used as a hybrid parent year after year — all the while remaining genetically pure (so long as no seed mixtures or outcrossing with other sunflower plants occur during the increase or use of the line).

Again, the bottom-line benefits of doubled haploids for a sunflower breeding program — and, eventually, for its farmer-customers — are that (1) desired inbreds can be developed much faster than through conventional methods, and (2) these inbreds possess 100% genetic purity.

The doubled-haploid approach is not yet being utilized by sunflower breeding programs. Virtually all companies are very interested in it; but the expensive high-tech laboratory facilities and skilled personnel needed for this technology do make it more feasible for some. Others, looking at the required resources believe focusing on marker-assisted breeding should rate as the top priority. (Which is not to say that the two — doubled-haploid breeding and marker-assisted selection — are exclusive of each other. They are not. Marker-assisted selection has been used, and will continue to be used, in all kinds of breeding, conventional or doubled haploid.)

Florin Stoenescu is the Fargo, N.D.-based sunflower breeder for Advanta USA, Inc., which develops hybrids that are licensed for sale by other seed companies. Stoenescu considers doubled-haploid breeding to be a “very important tool that enables the refinement of breeding of most crops.”

Stoenescu, whose experience with doubled haploids (in canola) dates back to 1989, says that using doubled haploids in concert with a molecular marker approach leads to improved selection efficiency and gene mapping. The uniformity of doubled-haploid-bred lines also reduces the cost of parental line seed production, he notes.

The road to doubled-haploid sunflower is not without some potholes, however. Stoenescu references the previously noted need for costly laboratory and growth-room facilities, as well as highly skilled technicians. Also, he says, the method works better with some sunflower genotypes than with others. Plus, since they have not yet gone through a selection and evaluation process, inbreds produced via a doubled-haploid approach must be field tested.

Still, Stoenescu is definitely an advocate of the expanded use of doubled haploids in sunflower breeding — in large part because he believes it will help sunflower be more competitive with other crops that are further advanced in its use — corn and canola being a couple prime examples.

Longtime sunflower breeder Gerhardt (Gary) Fick certainly understands the benefits of the doubled-haploid approach in sunflower, but likewise says it carries some caveats. He agrees it’s a good way to make “instant inbreds,” but also believes the approach is presently more viable for the larger companies that already have sophisticated laboratories in place.

“It’s fairly easy to develop a lot of new inbred lines using a traditional plant breeding system,” says Fick, whose sunflower career began with the USDA in the early 1970s and continues today with Seeds 2000, which he helped establish and serves as vice president/director. “We don’t have trouble developing a lot of lines. The trouble comes in finding out what’s in them — finding whether they carry the genes for disease resistance, herbicide tolerance or high-oleic acid, for example.” That’s where marker-assisted breeding can be very helpful, Fick suggests.

Fick and the sunflower breeding community in general believe that the 100% genetic purity component of doubled haploids can be particularly important when trying to add a single-gene trait (e.g., herbicide tolerance) to materials that already possess a superior set of agronomic characteristics, such as yield capacity, oil percentage and disease resistance. But anytime one seeks to incorporate a desired trait from a less-refined population (e.g., a wild species), a significant amount of backcrossing is required to eliminate unwanted traits. In those cases, utilizing the doubled-haploid approach would be premature — as it also would be in a conventional breeding approach prior to extensive selection and backcrossing.

As does Advanta’s Stoenescu, Fick points out that regardless of breeding methodology, a lot of field analysis will still be required of any new inbred lines. “The disadvantage of the doubled haploids is you don’t have the opportunity to [carry out those evaluations] on the ‘way up,’ ” Fick explains. “Even though [the inbreds] can be made very quickly, you still have to evaluate them.”

That said, Fick by no means denies the benefits that doubled-haploid breeding offers to sunflower research programs. “I think it could be very helpful for a program that already has a good marker-assisted breeding program set up, with markers to identify [desired] traits,” he observes, “and if breeders are using backcrossing to develop new inbred lines or convert inbred lines to [include traits like] herbicide tolerance or disease resistance. For that, I think it would be a nice supplement.” But, Fick suggests, if a company is not already using marker-assisted breeding, jumping into doubled haploids first could be a case of “putting the cart before the horse.”

Overall, the sunflower breeding community appears ready to embrace the use of doubled haploids. During a meeting at the National Sunflower Association’s 2009 Summer Seminar, several scientists — both private and public — spoke to the increased speed of inbred development and how doubled-haploid breeding can help sunflower compete with other crops.

Toward that objective, the National Sunflower Association, through financial commitments from the hybrid seed industry, has recently approved the investment of nearly $250,000 over a three-year period to aid in the development of an efficient protocol for doubled-haploid production in sunflower. Once developed, this protocol can be adopted by any interested seed companies — including those with more-modest laboratory facilities and limited personnel. The NSA funds will go to the USDA-ARS Sunflower Research Unit at Fargo, N.D., and used to support a postdoctoral scientist. This scientist will work under the supervision of ARS cytogeneticist C.C. Jan, molecular geneticist Lili Qi and research geneticist Brent Hulke. Jan is the project’s principal investigator (PI), with Qi and Hulke serving as co-PIs.

Once the postdoc scientist has been hired, he/she will work on two approaches simultaneously in order to evaluate which provides a more-efficient protocol for doubled-haploid sunflower production:

• Anther Culture — Here, the goal is to develop a protocol for the extraction of anthers from sunflower florets at an optimum stage of development; then, to determine optimum tissue culture media and environmental conditions for the induction of embryos, germination to plantlets, chromosome doubling, confirmation of haploid production — and growth to a mature, homozygous plant. Jan will supervise this effort.

• Foreign Pollen — In this approach, the postdoctoral scientist — supervised by Qi and Hulke — will explore the potential for sunflower haploid production by fertilization of sunflower ovules with foreign pollen. (An existing example is where wheat has been fertilized with corn pollen. After the wheat ovule has been fertilized, the corn chromosomes are eliminated, leaving a haploid wheat embryo that can then be doubled.) The ARS scientists will identify potential sources of pollen coming from the family Compositae — but outside sunflower’s genus, Helianthus — and then test them for their ability to induce haploid production in sunflower.

The ARS unit has an environmental growth chamber that can provide the precise conditions necessary for embryo induction and development, and the unit’s scientists feel quite confident they will be able to successfully develop an efficient doubled-haploid protocol that can then be used by interested commercial companies.

Steve Kent, president of Seeds 2000 and the current chairman of the NSA Research Committee, says he is very pleased with the response from the private hybrid seed sector. “We know that GMO sunflower is not an option in the present regulatory environment,” Kent remarks. “Developing a doubled-haploid breeding system is likely the next best step. Having everyone working together to get this system in place is a real tribute to the overall sunflower industry.”

— Don Lilleboe

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