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Making fusarium-resistant plants from pollen

Corn embryos transferred to solid medium begin to develop shoots. All photos courtesy of Dr. Laima Kott, University of Guelph.

University of Guelph researchers are developing a new technique to produce corn plants with specific traits from individual pollen grains. The technique, called microspore culture, involves a step to induce small genetic mutations, followed by the selection of individuals that are likely to exhibit the desired new trait. The end product is inbred plants that can be used in corn breeding programs. The first trait being pursued is fusarium tolerance.

Microspore culture is not exactly a new concept. It was originally developed for canola in the mid-1980s by Dr. Laima Kott, researcher and adjunct professor in the Department of Plant Agriculture at the University of Guelph.  Although similar culture systems have since been established for barley and rice, corn has been notoriously difficult to manipulate in such a system. However, Kott and laboratory technician Ecaterina Simion, who have a combined total of about 50 years’ experience with microspore culture, are getting close to completing a functional microspore culture system for corn after almost three years of work. Hyland Seeds and Thompsons Ltd., the project collaborator, provided hybrid corn lines and some funding; Ontario Pork, the Ontario Corn Producers Association, and the Ontario Ministry of Agriculture, Food, and Rural Affairs provided additional funding for the research. 

Selecting beneficial mutations
A number of steps are involved in producing a plant from a pollen grain. To begin, microspores
(pollen grains) are obtained from the mature plant. “We extract the pollen grains out of the anthers so they’re loose, and then we get rid of the debris and put these pollen grains into liquid medium in a Petri dish, where they can start growing,” says Kott. This can yield hundreds to millions of tiny pollen grains per dish.

Before the pollen grains start to grow, they are exposed to ultraviolet (UV) light for a specific amount of time. This creates small mutations, called point mutations, in which only a small number of the DNA building blocks are altered. Many of these mutations will be lethal, but some will not. “Because we have a million or so pollen grains per plate, there’s no problem with how much tissue we have to work with; it’s totally abundant,” explains Kott. “So, if we kill half of them by this UV treatment, that’s okay. By killing half of them, we know that the remaining living half has probably been altered.” 

The next step is to take the surviving, altered pollen grains that are beginning to grow into embryos and pick out the ones that have the desired trait. To do this, the researchers use a chemical selection agent that is specific to the trait being sought. The chemical kills most of the embryos, leaving alive the ones that are able to survive in the chemical. “So what we’ve done is basically killed everything that didn’t have the right mutation and kept the ones that have the mutation that we prefer,” says Kott.

Small corn plants are transferred to a culture box for root and shoot development.
These plants began as pollen grains and survived the mutagenesis and selection steps to eventually produce normal tassel and silk.

As a simple example, she explains selection for glyphosate tolerance, which was used in canola. “We’ve got our plate of isolated pollen grains floating around in the medium, and we’ve stuck it on a UV light and done the mutation. As the embryos grow to a certain stage, we want to produce glyphosate-tolerant plants. So we add glyphosate and let them continue growing. Generally, 99 percent of them will die, but the mutation that actually disables the glyphosate or breaks it down or does something to it to make it not be toxic, those embryos will survive.”
Selecting for something like disease tolerance is a bit trickier because the researchers must first figure out which chemicals to use as selection agents. For fusarium tolerance, they are using three selection agents that they hope will kill off the fusarium-intolerant embryos and
leave behind those that have tolerance. One is the deoxynivalenol (DON) toxin produced by the fungus, which is harmful both to the plant and to animals that consume the toxin. The other two selection agents are specific acids that are produced by the fungus to aid its invasion of the plant tissue. If the embryos can survive in these selection agents, they likely have some way of breaking them down or making them ineffective. The resultant plants may then be resistant to fungal invasion.

Each selection agent alone kills 99 percent or more of the embryos, so they are not combined in the screening tests, says Kott. “We don’t know which one’s going to work best. If we get three or four kinds of mutations, it’s easy for the breeders to cross them and stack them.” 

The surviving embryos are removed and grown to maturity in the lab. In canola, the embryos, which only have one set of chromosomes from the original pollen grain, must undergo another treatment to duplicate the chromosomes and produce a normal plant. However, in most of the corn embryos, the single set of chromosomes from the pollen grain doubles spontaneously, so no extra steps are required. Each new corn plant is then self-pollinated to maintain the genetic purity of the new trait and to multiply the seed to produce inbred stock. 

Testing new varieties
Once the materials have been mutagenized, selected, and grown into plants, they are returned to Hyland Seeds for further testing. This involves exposing the plants to fusarium and evaluating their disease tolerance. “At this stage (July 2009), we have our first corn plants that went through the entire process. They’re just finishing silking in the growth room,” says Henry Olechowski, research director at Hyland Seeds in Blenheim, Ontario. “As we get more of these, we will either be screening them with the actual disease in the greenhouse this winter or in the field in 2010, depending on the numbers of plants we get.” 

The inbred plants identified through the screening process as fusarium-tolerant can then be included in the parental lineup to begin crossbreeding and yield testing. If the project is successful, new varieties could be rolled out. “By the time you’d be confident about the variety, you’re looking at 10 generations, or five years down the road,” says Olechowski.

Establishing the microspore technique for corn provides several benefits. First, tremendously large numbers of pollen grains can be manipulated, increasing the chance of creating and finding a successful mutation. Second, the final plant is not regarded as transgenic because no new genes have been inserted. This avoids some of the regulatory analyses required for transgenic crops in variety registration, says Olechowski. Finally, the technique can be used to develop many different traits, simply by changing the selection agent. For example, traits that have been developed or are in development in canola using microspore culture include sclerotinia resistance, clubroot resistance, winter hardiness, low saturated oil and reduced glucosinolate content in seed.

“Fusarium is just one trait; there are other things we can screen for,” says Kott. “Once we have the microspore system working, we just have to develop a new screening system for whatever trait we’re after. It’s a different selection agent for every trait.”

Olechowski says that if the microspore culture system is successful, they may be developing other traits for corn in the future. “We’ve got a few ideas, but we haven’t made any decisions at this time. We’re just thrilled that the microspore technique is actually working, and we’re excited that we’ve got a fusarium screen. We’ll have to see what happens down the road.”

Fusarium-resistant wheat
Fusarium is a major disease problem in Canada and the northern United States,” says Henry Olechowski, research director at Hyland Seeds in Blenheim, Ontario. “It has a tremendous impact on farm feeding for hog producers, as well as yield losses for farmers. To date, we don’t have a silver bullet to control fusarium in wheat or corn.”

For this reason, Hyland Seeds, Dr. Laima Kott and technician Ecaterina Simion at the Department of Plant Agriculture, University of Guelph, are also exploring microspore culture in wheat. “Right now, we’re just developing the method. It should probably be pretty much done by February of 2010,” says Olechowski. “Then we will be looking at fusarium tolerance.”