Genetic Maize

As you may already know, I’m a co-blogger at Biofortified, the group blog on plant genetics and genetic engineering. Especially since winning the Ashoka Changemakers GMO Risk or Rescue contest, I’ve committed to helping to make Biofortified the best possible resource on the web for these topics. One of the unique aspects of Biofortified is the potential for discussion among diverse people. As we ramp up the number of visitors with different points of view, the discussion will get better and better. In the interests of getting those discussions going at Biofortified, I’m turning off comments at Genetic Maize. I hope you will visit Biofortified and get involved in the conversation. Any posts I write at Biofortified will also be posed here as a sort of online portfolio. If you have a specific question or comment for me, feel free to use the contact from.

The 2010 Maize Genetics Conference started with a call for maize geneticists to take on one of the greatest challenges of human history – feeding the world. Marianne Bänziger of CIMMYT presented the first plenary talk, titled Stress tolerant maize for the developing world – Challenges and prospects. Find the abstract of her talk at the end of this post.

Of all of the staple grains, maize is the most drought susceptible. Wheat is fairly drought tolerant, and rice is irrigated. Maize is sensitive to variation in rainfall, and since it is typically not irrigated, any year to year variation in rainfall will be seen as year to year variation of yield, with low rainfall years yielding less than high rainfall years. There are some drought tolerant varieties that don’t have such variation with rainfall, but they are consistently low yielding, even in high rainfall years. In order to provide enough food for growing populations, maize must be developed that can maintain reasonably high yields even in drought years.

A second major problem with maize is nitrogen. Maize reacts well to fertilizer application, providing (to a point) higher yields with higher amounts of nitrogen. However, less than 50% of applied fertilizer is used by the plant, leaving much of the nitrogen unused This unused fertilizer can be carried via surface waters to places like the Gulf of Mexico where it can contribute to hypoxic zones. Additionally, synthetic nitrogen fertilizer can be expensive to produce because it requires natural gas. Both synthetic nitrogen and non-synthetic fertilizers take fuel to distribute through fields. Maize that can use applied nitrogen more efficiently without laving so much behind must be developed both in order to provide enough food and to ensure that we are using both renewable and nonrenewable resources efficiently while protecting the environment.

CIMMYT aims to solve problems of drought and nitrogen by breeding under stress conditions. Their fields look more like a field in Africa than a field in Iowa. They simply select for stress tolerant plants that grow successfully under low water and low nitrogen conditions. They’ve found that genetic markers in typical yield selected lines and in stress selected lines are very different. CIMMYT is also looking at breeding under low phosphorus and low potassium.

While CIMMYT is primarily focused on breeding, they believe the key to meeting future food needs lies in matching breeding and transgenics. In particular, CIMMYT has partnered with seed companies to develop transgenics that will enhance productivity. The current traits on the market are protective: Bt protects the crop plants from insect damage which can reduce yield, Roundup Ready protects the crop plants from having to compete with weeds for resources, and virus resistance protects papaya from reduced yield due to virus infection. Productivity traits would directly increase yield instead of protecting it. Castigiloni showed in the 2008 paper Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions that yield could be significantly improved with a transgene.

Finding appropriate transgenes that will improve yield isn’t the end of the story. Each transgene needs to be investigated for genotype x environment interactions to see if the productivity transgenes behave differently under different environmental conditions. In addition, the transgenes may behave differently in different varieties, so each individual variety would need to be tested for yield changes with the productivity transgene. More layers of complication are added when multiple genes of similar and different traits are stacked. The combinations of transgenes may behave differently than each gene alone, and the combinations may have different interactions with each variety and environment.

CIMMYT has partnerships with Monsanto to work on water efficient maize for Africa (WEMA) and with Pioneer to work on improved maize for African soils (IMAS) which is also known as nitrogen use efficiency (NUE). These partnerships have many benefits. They can combine CIMMYT germplasm which is adapted for the farming conditions of low-income farms with the elite germplasm held by the corporations. They can ensure that the poor can aces the seed at no cost or at costs they can afford. They can also depend on the companies to provide funding to develop and deregulate the traits.

Developing and using transgenics is not without barriers, of course. In short, transgenics are expensive. Developing a transgenic trait costs $25 to $100 million dollars or more. Costs include finding a gene that does what you want it to, testing efficacy of the gene in many different varieties and environments, safety testing to ensure that the transgenic plants are substantially equivalent to their non-transgenic sister plants, and so on. For the forseeable future, the cost of transgenic traits will remain high. For the price of one commercial transgenic cultivar, CIMMYT believes they can characterize the entire genetic heritage of the two principal cereal crops, wheat and maize.

During her talk, Marianne announced the new CIMMYT program Seeds of Discovery for the first time. This exciting program will examine ancestral varieties of maize and wheat to enable breeding programs globally to use crop biodiversity in developing new lines. They aim to discover the extent of allelic variation in these varieties. They also hope to better understand how the different varieties are related in core sets. Right now, varieties are organized by geographic origin or phenotype but grouping by genotype will allow for better explanation of genetic similarities and differences. When more is known about the allelic diversity in ancestral varieties, marker assisted breeding can be used to bring those rare useful alleles into breeding programs.

In addition to ancestral varieties, CIMMYT looks at farmers’ varieties. They have partners in 14 countries that are both looking for potential lines for breeding that have traits like drought tolerance and looking into how new traits will work with the varieties farmers are currently using. They are also looking into other traits that are important to farmers in the developing world, including taste and appearance.

Greater than 80% of the required yield grain has to come from breeding. No other method, including fertilizer and transgenic traits, will be able to come close to breeding. Making these increases requires scientists from the developed world and from the developing world to both form partnerships and to work on their own areas of expertise. Marianne called upon the maize genetics community to help characterize the genes and alleles that CIMMYT finds in their Seeds of Discovery program. They plan to provide seed to scientists so they can begin to investigate the traits.

Talk Abstract:

Increasing demands for the main food staples, climate change, and increasing water, nutrient and land costs give a new urgency to developing and making available stress tolerant crops. This urgency is the greatest in the developing world where investments in research, capacity building and infrastructure development still lag far behind the developed world. The presentation gives an overview of CIMMYT’s investment in the development of stress tolerant maize which has recently gained significant leverage through stronger research collaboration with public and private partners, and now extends from native and transgenic trait discovery to large scale application of marker assisted selection approaches tailored to the improvement of highly quantitative traits such as yield under drought and low soil fertility. Many years of CIMMYT research indicate that these traits are highly polygenic, which has implications for the use of transgenics, identification of effects within association mapping studies, and the choice of appropriate marker based breeding strategies. In addition to assessing front line transgenics originating from the private sector for use in particular in Africa, current efforts focus on marker assisted recurrent selection (MARS), which is being implemented in over 40 biparental populations in Africa, Asia, and Latin America. Current MARS populations are selected on an index of 200 to 300 anonymous SNP markers, a density chosen because it is affordable with current genotyping technology. In 2010, pilot projects on the implementation of genomic selection (GS) using much higher marker densities will be initiated on new platforms based on next generation sequencing technologies, and it is expected that by 2011 genotyping costs will have dropped enough to permit their routine application across the CIMMYT maize breeding program and facilitate innovative native gene discovery and allele mining approaches. With that, CIMMYT is among the first public sector breeding programs that integrate cutting edge transgenic and molecular techniques on a large scale for germplasm development and dissemination to the tangible benefit of resource poor farmers.

Castiglioni P, Warner D, Bensen RJ, Anstrom DC, Harrison J, Stoecker M, Abad M, Kumar G, Salvador S, D’Ordine R, Navarro S, Back S, Fernandes M, Targolli J, Dasgupta S, Bonin C, Luethy MH, & Heard JE (2008). Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant physiology, 147 (2), 446-55 PMID: 18524876

That headline catches your eye, doesn’t it?

We’ve seen such claims made in popular media such as the March 2010 Fury as EU approves GM potato: Critics claim plant could spread antibiotic-resistant diseases to humans in the Independent: “Opponents fear bacteria inside the guts of animals fed the GM potato – which can cause human diseases – may develop resistance to antibiotics.” Groups that actively work against deregulation of genetically engineered crops have been making such claims for years.

We’ve also seen these claims in peer-reviewed journals (although, far less frequently than in non-peer reviewed media and reports). For example, in the February 2009 issue of Critical Reviews in Food Science and Nutrition, the review Health Risks of Genetically Modified Foods: “An area of concern focuses on the possibility that antibiotic resistance genes used as markers in transgenic crops may be horizontally transferred to pathogenic gut bacteria, thereby reducing the effectiveness of antimicrobial therapy.”

Are antibiotic marker genes in genetically engineered crops really a risk to human health? Many people have raised this question and there seems to be a lot of confusion about the issue. It’s time to look into the risks and reasons more deeply.

What are antibiotic resistance genes for?

In order to understand why these genes are used, we have to look a little at the process of genetic engineering. For some plant types, including corn and rice, immature seeds are dissected to expose the developing embryo. Pieces of carrot roots can be transformed, as can the leaves of tobacco. The desired genes are transferred into the plant cells with either a gene gun or Agrobacterium. The plant cells are then grown up into whole plants in petri dishes, with the help of plant hormones. The process is similar to other asexual plant propagation techniques, but much smaller!

Not every cell receives the gene of interest, however, so researchers need a way to find the cells that have it. Enter antibiotic resistance genes. If the cells are transformed with the gene of interest and an antibiotic resistance gene, the appropriate antibiotic can be added to the media in the petri dish so that any cells that didn’t get the genes will die. The antibiotic resistance gene is being used as a selectable marker, since it allows the researcher to select only the desired cells.

Of course, just because these genes are useful doesn’t mean that they are safe or that they should be used. What does the research tell us?

Risk: Gene transfer from plants to bacteria

Fear of antibiotic resistance markers is mainly due to fear of gene transfer from genetically modified plants to bacteria in the soil or bacteria in human or animal guts. There are at least two reasons why this fear is unwarranted. First, soil and gut bacteria naturally contain a variety of antibiotic resistance genes without any human intervention. Second, transfer of genes from a plant to a bacterium is extremely unlikely.

Natural antibiotic resistance

Life for a bacterium isn’t easy. They have to compete fiercely for resources, so it’s not surprising that some bacteria have evolved to produce poison that kills their competitors: antibiotics. The producers of these antibiotics also evolved antibiotic resistance mechanisms so they could survive their own weapons. Additionally, bacteria develop resistance to antibacterial compounds in the environment.

Often, antibiotic resistance is conferred by a single gene. Any bacteria that can find that resistance gene and use it have an advantage. Consequently, antibiotic resistance genes are widespread in natural environments. When humans intervene, using antibiotics in ways that encourage development of resistance in bacteria, that resistance is passed around even faster (no GMOs needed). For some information on how humans, check out the CDC’s pages on antibiotic resistance.

Gene swapping

Genetically modified crops: methodology, benefits, regulation and public concerns, a 2000 review in the British Medical Bulletin has a summary of the risks of horizontal gene transfer from genetically modified crops:

…horizontal transfer of a gene from ingested plant material to bacteria has never been demonstrated, and there is no indication that it has ever occurred during evolution. The probability that it could occur is, therefore, considered to be so low that it is not relevant when compared with the natural occurrence of antibiotic resistance genes.

They sound awfully confident, don’t they?

Bacteria (prokaryotes) are fairly promiscuous when it comes to genes. Many types of bacteria (not all) have the ability to take up DNA from the environment and from other bacteria and integrate it into their own genome during parts or all of their life cycle. This process is called horizontal gene transfer, and the bacteria that have this ability are called competent. Since they have this ability, it makes sense to worry about bacteria picking up antibiotic resistance genes (and other genes as well) from other organisms, including genetically modified crops.

Interestingly, eukaryotes (multicellular organisms like plants and people) also have the ability to take up DNA into their genomes. For example, the August 2007 Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes shows transfer of the entire genome from endosymbiotic bacteria into their hosts’ genomes. A more recent example appeared in January 2010 in the New York Times: Hunting Fossil Viruses in Human DNA. An entire virus genome resides in the human genome, and has been passed down from our simian ancestors.

So we know that bacteria can swap DNA and that eukaryotes can take up DNA from bacteria and viruses. Can prokaryotes take up DNA from eukaryotes?

It doesn’t look like it.

The European Food Safety Authority has the following to say about the subject in their excellent 2009 Statement of EFSA on the consolidated presentation of opinions on the use of antibiotic resistance genes as marker genes in genetically modified plants (section 2.1.1.2., edited slightly for clarity):

While many studies support the evolutionary significance of horizontal gene transfer between bacteria, eukaryotic genes in prokaryotic genomes are a rarity. There is no definitive report of DNA transfer from eukaryotes to bacteria.

As of 24 September 2008 the public genome databases included more than 750 completed prokaryotic genomes. In the first annotation of the putative genes there are frequent cases where closest matches are found with eukaryotic genes, but these preliminary results have not manifested into demonstrations of horizontal gene transfer from eukaryotes to prokaryotes, as judged by the scientific publications interpreting the genomic sequencing data. For one functional gene (phosphoglucose isomerase), phylogenetic analyses indicated that the gene might have been transferred from a eukaryote to bacteria. The transfer was estimated to have happened approximately 500 million years ago.

Multiple studies have found that bacteria can take up eukaryote DNA, but only in certain conditions, where the researchers used a sort of genetic trick called homologous recombination. In short, homologous recombination can occur when the ends of the donor DNA have sequences similar to part of the acceptor DNA. The homologous sequences can bind together, and in the next round of replication, the donor DNA can be integrated. In studies that aimed to find evidence of transfer of DNA from eukaryotes to prokaryotes without genetic tricks, none was found.

Table 1 of the EFSA Statement lists all studies prior to its publishing that examined horizontal gene transfer in bacteria (25 studies in all). Of the 18 studies that looked for gene transfer with homologous recombination, 15 found gene transfer and 3 did not. Of the 16 studies that looked for gene transfer without homologous recombination, no evidence of gene transfer was found.

In short, there are many DNA sequences that look like eukaryote genes in prokaryote genomes, but so far only one has been found that might be an actual functional gene. All evidence to date shows that gene transfer from eukaryotes to prokaryotes can only occur when homologous DNA sequences are present in donor and acceptor genomes. The lack of evidence for horizontal gene transfer in the wild suggests that there are some sort of barriers to gene transfer from eukaryotes to prokaryotes.

Barriers to gene swapping

Prokaryotic DNA and eukaryotic DNA are sort of like different computer languages. In fact, each species has slightly different ways of “personalizing” its own DNA with things like methylation and other DNA modifications, different codon preference, post translational modification of RNA, and whether or not introns are present. Eukaryotic DNA is so different from prokaryotic DNA that the bacteria just can’t take it up and use it as they would other bacterial DNA. Additionally, even if all of the barriers to gene uptake occur and all the barriers to gene expression are overcome, the likelihood that the gene will confer a positive trait for the bacterium is low. Most eukaryotic genes aren’t going to be helpful for a prokaryote, such that the few useful genes are few and far between. Even if a bacterium was able to uptake and express an antibiotic resistance gene from a genetically engineered plant, there would have to be selective pressure (i.e. an environment that included the antibiotic that the gene conferred resistance to) in order for the gene to be maintained in a bacterial colony. For more information about barriers to gene swapping, check out the EFSA Statement.

Avoiding the unlikely

Despite the fact that horizontal gene transfer from eukaryotes to prokaryotes is so unlikely (the only known example was estimated to have happened 500 million years ago), there are still precautions that can be taken to make it even more unlikely. For example, if antibiotic resistance genes are used as selectable markers in genetically modified organisms, researchers can avoid using sequences with homology to known bacterial genomes, they can be sure to only use antibiotic resistance genes that include features that make the gene unusable in bacteria, and they can avoid using promoters that are active in bacteria, just to name a few.

Alternative markers

Another option is to find alternative marker genes and alternative strategies. Herbicide resistance genes are an alternative selectable marker. Visible markers like GFP or GUS are screenable markers. Marker genes can be bred out, meaning that the final plant line will not contain the marker gene. Finally, it is possible to use no marker genes at all, but that does require far more screening of adult plants which can add expense and time to any project.

Peer-reviewed works cited

Dona A, & Arvanitoyannis I (2009). Health Risks of Genetically Modified Foods Critical Reviews in Food Science and Nutrition, 49 (2), 164-175 DOI: 10.1080/10408390701855993

Halford NG & Shewry PR (2000). Genetically modified crops: methodology, benefits, regulation and public concerns. British medical bulletin, 56 (1), 62-73 PMID: 10885105

Hotopp JC, et al. (2007). Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science (New York, N.Y.), 317 (5845), 1753-6 PMID: 17761848

Other works cited

Collins JD, et al. (2009). Statement of EFSA on the consolidated presentation of opinions on the use of antibiotic resistance genes as marker genes in genetically modified plants. European Food Safety Authority.

Hickman M & Roberts G (2010). Fury as EU approves GM potato: Critics claim plant could spread antibiotic-resistant diseases to humans. The Independent.

Zimmer, C (2010). Hunting Fossil Viruses in Human DNA. New York Times.

Note: This work was originally posted at Scientific Blogging as an entry in their 2010 Scientific Blogging Contest.

This summer will be my 4th year growing corn for my research. Every year, I’ve seen some crazy things in the transgenic and non-transgenic fields alike. For example:

On the right is “tassel ear”, where silks and kernels (female, seed producing plant parts) appear on the tassel (male, pollen producing plant parts), where they are most certainly NOT supposed to be – it’s ok for sorghum and other grasses, but not for corn! On the left, there are at least 2 ears where there should be one, and those leaves poking out between the two might be more ears. Neither of these plants are transgenic or carry heritable mutations that cause these strange phenotypes. Both transgenic and non-transgenic fields are treated with a herbicide before we plant but after that the plants are grown with no additives, chemical or otherwise.

So, what the heck is going on?

I’ve always meant to look it up, but pollination season is so busy, and then it’s harvest season which is so busy, and then we’re analyzing the seeds… you get the idea.

While looking for pictures of corn borer damage, I found an awesome site by Peter Thomison and Allen Geyer of the Horticulture and Crop Science Department of Ohio State University: Troubleshooting Abnormal Corn Ears and Related Disorders.

They say that tassel ear is due to a variety of causes, including mechanical injury due to hail, which we did have pretty badly last year. No one really knows what causes “bouquet ear” with multiple ears appearing where there should be one, but it might be due to temperature stress due to cold.

There are many other common but strange corn phenotypes explained on their site. Check it out!

James, over at James and the Giant Corn, has written a post about the long lasting tomatoes from India: Scientists at India’s NIPGR Create a Longer-Lasting Tomato (Studying The Regulation of Fruit Ripening). He does a great job of explaining cell wall chemistry, which I neglected to cover in I say tomato… I appreciate that he pointed out something that I forgot to mention (emphasis added):

I shouldn’t have to say this, but there are currently no genetically engineered tomatoes on the market. For a short time in the 1990s Calgene sold the Flavr Savr tomato in California grocery stores, but they weren’t able make a profit doing so, so they stopped. The poor taste of most tomatoes for sale in the grocery store today is purely the result of conventional breeding (my post on the subject and Mat_kinase’s).

I just submitted a poster abstract featuring Biofortified for the Maize Genetics Conference 2010. The title is: Biofortified: An educational resource for plant genetics and genetic engineering. What do you think? I’m looking forward to presenting the idea of science blogging to all of the maize geneticists and to hopefully recruiting more regular and guest bloggers. We probably should get some non-maize people on Biofortified, though. Know anyone?