WSU CAHNRS

College of Agricultural, Human, and Natural Resource Sciences

Regenerating Pixie is Important Step in Grasping Grape Genetics

Understanding the grape genome in all its vast variety will translate into sustainable viticulture practices and a deeper understanding of wine quality. Wine grape growers have been plagued by an economically devastating pest, phylloxera, which has necessitated the replacement of almost all vines with new ones grown on pest-resistant rootstocks. Fungal diseases are not only an economic threat but an environmental one as well, since heavy fungicide treatments are required to beat back the spread of powdery mildew.

Graduate student Kathie Nicholson holds regenerated Pixie grape plants. Photo by Brian Charles Clark/WSU.

Graduate student Kathie Nicholson holds regenerated Pixie grape plants. Photo by Brian Charles Clark/WSU.

Getting a grip on grape genetics requires not just the sequencing of entire genomes but detailed pictures of which genes do what. To get that information, scientists need a way to quickly grow sample plants that have been genetically transformed. Transformation means that a particular gene is silenced or added to the organism in order to learn what effect the change has on the plant.

“We need regeneration in order to do transformation,” said Kathie Nicholson, a doctoral student working in horticultural genomicist Amit Dhingra’s lab. “But it’s been hard to find a reliable method of regenerating grape in the lab.” Regeneration is a carefully controlled laboratory technique in which whole plants are grown from a few cells.

Nathan Tarlyn, one of Nicholson's co-authors, working on a plant tissue culturing project. Photo by Brian Charles Clark/WSU.

Nathan Tarlyn, one of Nicholson’s co-authors, working on a plant tissue culturing project. Photo by Brian Charles Clark/WSU.

Nicholson is the lead author of a paper that outlines a grape regeneration technique she and her colleagues developed using Pixie, a dwarf variety that flowers continuously and reaches maturity in just a few months. Only available since 2006, Pixie appears to be an ideal candidate as a grape “lab rat”: it grows quickly, does well in the greenhouse, takes up very little space and, now, can be regenerated reliably. In other words, Nicholson and her team’s work is an important step forward in the development of a tool that can be used to understand grape genetics.

“Technically, traditional breeding is genetic transformation,” Nicholson said, but traditional breeding is like taking a slow boat to an uncertain shore: you don’t know when you’re going to get there and you can never be sure where you are going. With the systematic exploration of gene function, Nicholson said, researchers will be able to zero in on the precise factors that give a plant resistance to certain diseases, the ability to withstand stresses like cold and drought, and the ability to produce the combination of complex chemicals that are so prized by winemakers.

Granted, Nicholson said, Pixie may not be an ideal model for studying wine grape genetics (although Pixie was developed from Pinot Meunier, an important grape used in the production of champagne.) “That’s why we are trying the same regeneration protocol on Chardonnay and Merlot,” she said.

Plants have the ability to regenerate themselves from a single cell. This ability, called totipotency, is sometimes compared to the ability of animal stem cells (which are pluripotent) to differentiate themselves into the wide variety of cells in an organism. A plant cell can divide, differentiate, and become the cells of shoots, roots, and leaves. This, anyway, is the theory: getting a few plant cells to differentiate and grow into mature plants in the lab is much more difficult.

Nicholson and her colleagues experimented with a variety of mixes of plant growth hormones and regimes of light and dark in order to find the sweet spot that encouraged the regeneration of Pixie. Starting with a tiny piece of leaf from the very top of a plant, she and her team found that a particular combination of stress-easing dark, a particular ratio of hormones, and a nutrient medium coaxed the cells to regrow into new Pixie plants.

Nicholson did the regeneration work as part of her Master’s degree program. Now, as a doctoral student, she is continuing her investigation of regeneration techniques, while also working to explore the genetic differences between wine grape varieties.

Brian Clark

This article is based in part on a paper by Nicholson et al. in Plant Cell, Tissue and Organ Culture and available online at http://bit.ly/Q5JyZ6.

Read more about Pixie in Voice of the Vine, WSU’s wine science e-newsletter for wine lovers: June 24, 2010 and Oct. 30. 2008.

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