Double-Haploid Induction Speeds Up Plant-Breeding Process

Syngenta scientists have solved the mystery behind an abnormal corn line responsible for revolutionizing corn breeding. Discovered in 1959 by University of Missouri Professor Edward H. Coe, Ph.D., the line produces haploid plants that contain just half the DNA of normal corn.

The ability to use this line to speed up breeding caught the attention of the corn-breeding industry. Today, all corn-breeding companies use haploids to shorten the time required to produce parent lines by several years. Reduced time and increased efficiencies for scientists to develop new hybrids have the potential to bring about higher-yielding, better-adapted seed options for growers at a faster pace.

But the reason why this odd and naturally evolved line produces haploids was never understood until recently. In 2007, Syngenta scientists began a quest to locate the genes responsible for haploid production. They found their answer by 2013 and followed up with gene editing to verify the discovery in 2015.

Solving this mystery will help Syngenta improve how scientists use haploids in current breeding systems and may lead to applying the technology in other crops. It also shows how new biotechnology can find solutions located deep in genetic coding.

Doubling Haploids

Some basic corn biology helps explain why haploids are so important to corn breeding. Corn is a diploid, meaning it has two copies of every chromosome in every cell. That’s 10 chromosomes that come from the female parent and 10 from the male parent. A haploid occurs when there is only one copy of every chromosome coming from one of the parents, while the copies from the other parent are gone.

Haploids become valuable when scientists double them and use them to produce homozygous breeding lines. In homozygous lines, all genes on each pair of chromosomes in every cell of the plant are identical. These homozygous lines are 100-percent inbred lines, which otherwise would have to be produced by repeated forced self-pollinations. The haploid method lets breeders produce inbred lines within just two generations, while traditional breeding takes 10 generations.

“We had this one huge-effect gene, the big gun. It was exciting that it was a major gene contributing so much of the trait.”

“It speeds up parent line development for hybrid crops by several years,” says Michiel van Lookeren Campagne, Ph.D., head of Syngenta Seeds Research. “The way we do this is by regenerating new plants out of pollen or egg cells, which each have only one set of chromosomes, and then doubling the chromosomes of these plants through a chemical treatment. The end result of this process is a doubled-haploid plant.”

The most efficient way to produce doubled haploids in corn is through haploid induction, he adds. “It can be done cheaply in the field and is broadly applicable across all genetic starting material.”

Haploid induction requires taking pollen from a haploid-inducer plant and putting it on any female ear of corn. The result will be an odd-looking ear that’s populated with about 13 percent haploid kernels.

The Search for Answers

The discovery of corn haploids has been around since 1959, but its use really took off in the 1990s, as scientists learned how to effectively double the haploids and breeders efficiently used them in their breeding programs, says Brent Delzer, Ph.D., Syngenta corn breeder. Delzer was part of the team that searched for the gene source of the haploid induction.

“As scientists, we have inquiring minds and want to know what the genetic basis is that is contributing to haploid induction,” he says. In 2007, Syngenta made the first crosses of haploid inducers with non-inducers, while also developing a mapping population to search for the chromosome position of the genetic trait.

“We initiated that work in our nursery in Hawaii,” Delzer says. “We were able to get several generations a year and set up the breeding population so we could map the gene.”

In the summer of 2008, Delzer planted some of the first crosses at his location near Janesville, Wisconsin. The next winter, his colleagues in Hawaii grew fields with crosses for evaluation. The team was looking for the chromosome region containing genes contributing to the haploid-induction trait.

“Chromosomes are rather big with a lot of genes on each one,” Delzer says. “So after we mapped a spot on the chromosome, we had to do fine mapping.” Syngenta geneticist Satya Chintamanani, Ph.D., became involved with the search and during 2009 and 2010 helped hone in on a small region of a chromosome.

The team was able to identify six different genes in the region. Using gene sequencing, they found one of the genes had a mutation that produced haploids. They baptized it as the MATRILINEAL (MATL) gene.

The Big Gun

The results surprised the team. The MATL gene was responsible for nearly 70 percent of the haploid-induction trait. “Corn has as many genes as people do—about 30,000—and almost all traits are controlled by many, many genes,” Delzer says. “But we had this one huge-effect gene, the big gun. It was exciting that it was a major gene contributing so much of the trait.”

There was another surprise ahead, which came during the verification process. Tim Kelliher, Ph.D., principal scientist for reproduction biology at Syngenta, led the verification to prove the gene was the correct one. The team used gene editing to recreate the small mutation in a normal inbred. By doing the minor edit, the plant produced a working haploid inducer, just like the one found decades ago in Missouri.

During the process, the team discovered the gene’s unusual type. “The gene produces a protein that modifies pollen fats or lipids,” Kelliher says. “Lipids are an important but poorly understood part of cellular biology. Now we are looking at the lipid composition of pollen grains and how they change to figure out ways to make haploids without editing genes.”

Future Work

The team’s work on the haploid mystery is not done, Kelliher says. Syngenta will continue to study the MATL gene and also identify the other minor genes involved in haploid production.

The value of this long-term research for Syngenta is two-fold, according to van Lookeren Campagne. The first comes with “making existing haploid-induction systems more efficient and thereby saving costs.”

The second is “deploying the technology to other crops that do not have any doubled-haploid production system,” he says. “That is where the real value would be, as it could really make a breakthrough in the breeding of these crops.”

Corn is a prime example of what can happen when scientists use the doubled haploid. “The line that Professor Coe found, the haploid inducer, has really underpinned the success of corn as a crop in the marketplace,” Kelliher says. “Corn is king, and a lot of it is due to this line.”

For Syngenta scientists, helping another crop achieve similar success and sustainably feeding the world are high on their list of priorities.