New technique speeds up transgenic crop design - applications in bioenergy

Scientists have developed a new method of constructing artificial plant chromosomes from small rings of naturally occurring plant DNA which can be used to transport multiple genes at once into embryonic plants where they are expressed, duplicated as plant cells divide, and passed on to the next generation - a long-term goal for those interested in improving agricultural productivity. The technique is set to revolutionize the design of robust energy crops, which speeds up the transition to third and fourth generation biofuels (earlier post). In a first stage, researchers want to apply the method to maize, sugarcane, switchgrass and other biofuel plants.

In an open access article in the current issue of PLoS-Genetics, the team of academic and commercial researchers report that their 'maize mini-chromosomes' (MMC) can introduce an entire 'cassette' of novel genes into a plant in a way that is structurally stable and functional. Early results, the study authors say, suggest that the MMC could be maintained indefinitely.

Daphne Preuss, PhD, professor of molecular genetics and cell biology at the University of Chicago, says the technique has ready applications for energy crops and biofuel production:

This appears be the tool that agricultural scientists, and farmers, have long dreamed of. This technology could be used to increase the hardiness, yield and nutritional content of crops. It could improve the production of ethanol or other biofuels. It could enable plants to make complex biochemicals, such as medicines, at very little expense. - Professor Daphne Preuss

Preuss is chief scientific officer and president of Chromatin, Inc., the makers of the MMCs, which cut one to two years out of any new transgenic project. Preuss, who is taking a leave of absence from the University to bring this technology into the marketplace, says the technique allows researchers to get a better product faster, which saves time, reduces costs, and frees up resources.

The production of transgenic plants, including maize, has historically relied on techniques that integrate DNA fragments into a host chromosome. This can disrupt important native genes or lead to limited or unregulated expression of the added gene.

Currently, to add a single gene, plant scientists create hundreds of transgenic plants in which the new gene is randomly inserted into a plant chromosome. Then they screen the gene-altered plants to find the few that might be suitable for commercial use. If they want to add two genes, they create twice as many new plants, screen for single-gene successes, then cross breed them to get both new genes, a slow and laborious process.

Instead, Preuss and colleagues have constructed MMCs that contain DNA sequences found in maize centromeres, the chromosomal regions needed for inheritance. Rather than inserting the new genes randomly into a plant's natural chromosomes, these mini-chromosomes remain separate:
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As a result, the new genes can be arranged in a defined sequence, with each gene surrounded by the desired regulatory mechanisms. This results in more consistent and controlled expression. The whole cassette of genes is passed on as a group during cell division as well as to the next generation.

In their PLoS paper, the researchers characterized the behavior of the maize mini-chromosome through four generations. Using a gene for red color as a marker, they showed that the added genes are expressed "in nearly every leaf cell, indicating stability through mitosis" - the process in which a cell duplicates its chromosomes to generate two identical daughter cells.

They also show that the MMC is efficiently passed on through meiosis, the creation of gametes, to the next generation, at ratios 'approaching Mendelian inheritance.'

Taken together, the authors conclude, the maize mini-chromosome, once introduced, behaves much like an ordinary chromosome. It remains distinct from the other chromosomes. Its gene cassette is structurally stable from generation to generation. The genes it carries are expressed and it is transmitted through mitosis and meiosis.

This development has not gone unnoticed. Six years ago, Preuss and two of her post-doctoral students at the University, Gregory Copenhaver and Kevin Keith, started Chromatin to refine and apply this technology. On October 10, 2006, the United States Patent and Trademark Office issued Chromatin patent No. 7,119,250, which extends the exclusive right to use these mini-chromosomes to all plants. This includes "a crop plant," the patent states, "a commercial crop plant, a vegetable crop plant, a fruit and vine crop plant, a field crop plant."

On May 22, 2007, biotech giant Monsanto Company purchased non-exclusive rights to use Chromatin's mini-chromosome stacking technology in corn, cotton, soybeans, and canola. Chromatin is in discussions to license this technology to other companies, potentially capturing most of the US corn market.

The timing looks ideal. The US, in order to limit oil imports and reduce greenhouse gasses, hopes to double its use of ethanol in fuels by 2012 and to double that twice over by 2022. Because of increased demand, corn prices rose this summer by about 50 percent over last year.

Preuss and colleagues hope to apply the technology to other plants, including sugar cane and switch grass, which could also serve as biofuel sources. They are also looking at other applications and expanding the gene carrying capacity of their mini-chromosomes. They have successfully delivered mini-chromosomes about six times the size of MMC1, suggesting that this platform can carry 'a large number of genes.'

Image: the autonomous minichromosomes (arrowheads); integrated constructs appear as pairs of FISH signals (arrows); size bar, 5 μm.

References:
Carlson SR, Rudgers GW, Zieler H, Mach JM, Luo S, Eric Grunden, Cheryl Krol, Gregory P. Copenhaver, Daphne Preusset, "Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome", PLoS Genetics, Vol. 3, No. 10, e179 doi:10.1371/journal.pgen.0030179

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