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‹‹‹ Contents page for this issue     |     Summer 2009

Strawberry fields forever?

By Barry Whyte

Related story: The strawberry genome

The search for a better strawberry is benefiting from new research tools under development at the Virginia Bioinformatics Institute and the Department of Horticulture at Virginia Tech. Scientists want to improve some of the everyday crops and fruits we eat in the hope of advancing human health and nutrition. Now that a research consortium has cracked the genetic code of the strawberry, the scientists are building a portfolio of resources aimed at crop improvement. Virginia Tech research partnerships are also helping plant breeders improve other related crops, like apple, peach, and pear — high-value nutritional foods of considerable economic importance across the globe.

Fruit is an essential part of the human diet, rich in many nutrients that benefit health. Strawberries, for instance, are an excellent source of vitamin C and other nutrients. The California Strawberry Commission, a state agency that represents strawberry growers, calculates that a serving of eight medium-sized strawberries provides more vitamin C than one medium-sized orange. As an antioxidant, vitamin C helps protect the body against the damaging effects of free radicals, molecules generated by metabolism that can overwhelm healthy cells in the body in times of stress. Damage caused by excessive free radicals has been linked to many diseases, including cardiovascular disease, certain cancers, and other conditions associated with aging.

Scientists believe that we have only scratched the surface of exploiting the nutritional value of strawberries. But before further improvements can be made, researchers need access to the entire genetic code (collectively known as the genome) of strawberries, as well as research tools that will allow them to engineer beneficial health traits into strawberry crops. Scientists at Virginia Tech and other universities around the globe are working as part of a research consortium to deliver the information and tools that will help make these improvements a reality.

A model plant for a diverse family

The Rosaceae family, named after one of its most familiar members, the fragrant garden rose, also includes many fruit species, from tasty apples, plums, peaches, and pears to appetizing raspberries, sour cherries, sweet cherries, and strawberries. In total, this economically significant family of plants comprises more than 3,000 species, many of which provide high-value nutritious fruits and nuts.

Modern plant breeding programs have produced many varieties of Rosaceae plants that play a significant role in commercial agricultural markets worldwide. Some estimates place the annual global value of strawberry production at approximately $7 billion. In recent years, an international community of scientists has come together to use the very latest techniques in molecular biology to investigate ways to improve fruit of the Rosaceae family. (Learn more in the article: “Multiple models for Rosaceae genomics,” by Shulaev et al in Plant Physiology 147(3): 985-1003, 2008.)

Vladimir Shulaev, associate professor in the Department of Horticulture and the Virginia Bioinformatics Institute (VBI), is one of the participants in the Rosaceae Genomics Consortium. “Some of the leading practitioners in the application of genomics technologies to crops like apple, pear, and strawberry are participating in large-scale collaborative efforts that target the development of improved fruit crops,” he says. “Genomics and bioinformatic advances over the past decade have provided new opportunities to identify useful compounds in plants and to investigate the genes responsible for their production. In the not-too-distant future, these research initiatives promise to bring significant economic benefits to plant growers around the globe and open up ways to advance human health and nutrition.”

Some of the shared resources that are becoming widely available and making Rosaceae crops attractive plants for functional genomic studies include the identification of sets of genes that are active at a particular time in the development of an organism (expressed sequence tags); a method to divide a large genome into smaller pieces to make it more easily studied (bacterial artificial chromosome libraries); physical and genetic maps that display the order of genes on a chromosome and distances between them; tags for genes that can be selected and mapped (molecular markers); methods to introduce alien DNA into plants (genetic transformation protocols); and a growing portfolio of bioinformatics tools. Highly efficient sequencing technologies, methods for analysis of quantitative gene expression (where many genes control a single trait), and novel phenotyping platforms for documenting the appearance of a plant in order to easily score any abnormalities are also impacting the quest for improved plant traits.

Schuyler Korban, professor of molecular genetics and biotechnology at the University of Illinois, is also helping the consortium meet its objectives. “We are working with researchers all over the country, including collaborators at Virginia Tech, to apply the very latest accumulated knowledge and resources toward genomics advances that will contribute to crop improvement,” he says.

Korban’s group focuses on genome-wide functional analysis and characterization of profiles of genes associated with important horticultural characteristics, such as fruit quality and disease resistance. They have generated the largest expressed sequence tag collection, which consists of 180,000 sequences, for the apple, and have created a large apple microarray for functional genomics studies in Rosaceae. “We have developed a genome-wide physical map for the apple that consists of an ordered arrangement of large contiguous sequences aligned to each of the chromosomes in apple,” says Korban. “Additionally, we are pursuing comparative genomic studies among the different members of the Rosaceae family so that we can extrapolate knowledge from one member of this family to another and learn about how genes operate in different genetic systems.”

Korban says, “We are very much interested in working with our colleagues at Virginia Tech in elucidating those genes that are involved in fruit quality and abiotic and biotic stress using the strawberry as an experimental tool.”

The Illinois researchers are supporting the Virginia Tech effort to sequence the whole genome of strawberry by providing expertise in annotating the strawberry genome. “We are making the tools that our research has generated directly available to the wider scientific community. This approach reflects the fundamental strategy of the Rosaceae research community — to develop family-wide resources and leverage the most appropriate system that maximizes research and development opportunities. Over the years, this strategy has been very effective for other plant and microbial research communities,” says Korban.

Tools of the trade

Scientists at VBI, the Department of Horticulture in the College of Agriculture and Life Sciences at Virginia Tech, the Institute for Sustainable and Renewable Resources at the Institute for Advanced Learning and Research in Danville, Va., and researchers at other institutions are developing new procedures for the efficient transfer of specific DNA sequences into the strawberry. In work funded by a Virginia Tech ASPIRES grant (A Support Program for Innovative Research Strategies), the scientists have used Agrobacterium tumefaciens, a plant pathogen that has been disarmed and used as a tool for genetic engineering of plants, to introduce DNA into a common field plant known as the woodland or alpine strawberry, Fragaria vesca.

“The woodland strawberry is emerging as a highly attractive system for genetic studies,” Shulaev says. “It has enabled us to develop high-efficiency gene transformation methods that greatly facilitate genetic research on this commercially grown crop.”

The gene transfer method takes advantage of Agrobacterium’s circular DNA molecule (known as T-DNA) to deliver DNA to the plant. By helping researchers establish the function of native strawberry genes, this method will be extremely useful in enhancing the nutritional value of these plants as well as the amount of health-enhancing antioxidants that they contain.

“One of the keys to success in this project was the selection of the right type of strawberry, one that was amenable to the transfer of DNA to many plants on a large scale,” says Shulaev. “Due to the small size of its genome, short reproductive cycle, and small plant size, F. vesca is an ideal candidate for genomic studies relevant to the commercial strawberry.”

Herb Aldwinckle, another member of the Rosaceae Genomics Consortium and professor of plant pathology at Cornell University, says, “The commercial strawberry familiar to most consumers is octoploid, which means that it contains eight sets of chromosomes. By using a close relative that has two sets of chromosomes and a significantly smaller genome, researchers have found a particular type of alpine strawberry that is very amenable to transformation.” Aldwinckle’s research group has developed similar technology for highly efficient genetic transformation of apple.

Richard Veilleux, professor of horticulture at Virginia Tech, has been using the DNA transformation method to study the function of genes in strawberries. He explains that to study the function of strawberry genes the researchers needed a way to generate many modified plants (See ‘ Strawberries that glow green by design’). “Our system allows scientists to investigate the function of many genes in strawberry. This is a major step in developing a way to allow scientists to identify commercially important genes, like those that convey much-sought-after health benefits.”

DNA that codes for alien genes is introduced at random into the strawberry genome to disrupt the function of a native gene. This disruption leads to changes that give clues to the function of the disrupted gene. The DNA that is introduced does not carry a gene of interest that would endow the transformed plant with some new trait but only what scientists refer to as a reporter gene, one that will help select the altered plants from unmodified plants further down the line. “Insertional mutagenesis has been a key approach to understanding gene function in many organisms, from mouse and zebrafish in animals to maize and tomato in plants. We are now witnessing the benefits of this genetic approach for strawberry,” says Veilleux.

The researchers found that leaves of 6- to 7-week-old woodland strawberry seedlings could be easily transformed using Agrobacterium. The reporter gene codes for a protein known as the green fluorescent protein. The transformed strawberry plants can be easily identified by visual inspection under a fluorescent microscope because they express an unnatural bright green glow due to the green fluorescent protein. Scientists can therefore see that the transformation has been successful by looking for the green glow. Because insertion of the green fluorescent protein gene by Agrobacterium into the millions of bases that compose the strawberry genome is random, the next challenge is to determine exactly which native strawberry gene has been interrupted in each of the new plants.

“We are currently examining several hundreds of plants generated in this way,” says Veilleux. “Some of the plants produced by this approach are shedding light on genes that impact leaf shape, the development of colored plant pigments like anthocyanin, and the accumulation of nutrients, as well as flower form and structure.” By using the modern tools of genomics, the introduction of foreign DNA from another organism (transgenics), molecular marker analysis, and tissue culture, Veilleux says he hopes to bolster a wide variety of crops, including potato, eggplant, and strawberry.

The search for a better strawberry is also benefiting from collaborative research projects underway at the Institute for Sustainable and Renewable Resources (ISRR), one of several research groups located at the Institute for Advanced Learning and Research (IALR) in Danville, Va. In addition to support for the genome sequencing effort (See ‘The Strawberry Genome’ on page 32), ISRR researchers have been contributing to the strawberry gene transformation project by generating novel strawberry plants and characterizing them. Yinghui Dan, research assistant professor; Barry Flinn, ISRR director; and Sarah Holt, a resident doctoral student at IALR, have been working to enhance the efficiency of Agrobacterium-mediated strawberry transformation; generate additional, novel strawberry plants; and help characterize some of the plants.

In addition to ASPIRES, the work is funded by the United States Department of Agriculture and the Virginia Tobacco Indemnification and Community Revitalization Commission. The Tobacco Commission became interested for two reasons. Strawberries are becoming an increasingly popular crop for Virginia and might be a new crop for Southside Virginia. And the tissue culture work required to prepare enhanced plants might be a new skill set for Southsiders, making the labor force attractive to agricultural biotechnology businesses. Eventually, tobacco greenhouses might be used to launch commercial undertakings for different kinds of crops.

Shulaev comments, “The development of this transformation protocol for strawberry represents a key milestone for researchers interested in improving strawberry and other fruit crops through genomics. We are now in a position to generate a collection of plants that will serve as an invaluable tool not only for discovering new genes in the Rosaceae family but also for establishing the functions of these genes through high-throughput screening methods.”

New horizons

Scientists hope to use their knowledge of traditional plant breeding methods to modify fruit crops in beneficial ways. The input of plant breeders and growers is used to design new approaches to crop improvement employing the very latest genomics technologies. To generate, maintain, and distribute stocks of large collections of fruit crops is an enormous task, but one that promises to shed light on how the many different genes contribute to the make-up of plants and fruit.

An extensive research toolkit and DNA sequence information are only the start of work to enhance strawberry and other fruits. The availability of these powerful genomic technologies means that scientists are one step closer to improving the health and nutritional value of fruit. Higher quality fruit should become available to consumers in forms that place less strain on the environment. If successful, this might just lead to a better, healthier berry and more sustainable agricultural practice.


Illustration by Aimee Drysdale.

Vladimir Shulaev, associate professor at the Virginia Bioinformatics Institute and the horticulture department at Virginia Tech. Photograph by John McCormick.

“Genomics and bioinformatic advances over the past decade have provided new opportunities to identify useful compounds in plants and investigate the genes responsible for their production.” — Vladimir Shulaev, associate professor, Virginia Bioinformatics Institute

Professor Richard Veilleux with Virginia Tech horticulture doctoral candidate Sarah Holt. Photo by John McCormick.

The Rosaceae family, named after one of its most familiar members, the fragrant garden rose, also includes many fruit species, from tasty apples, plums, peaches, and pears to appetizing raspberries, sour cherries, sweet cherries, and strawberries. In total, this economically significant family of plants comprises more than 3,000 species, many of which provide high-value nutritious fruits and nuts. Photo by John McCormick.

Yinghui Dan, research assistant professor at the Institute for Sustainable and Renewable Resources, Institute for Advanced Learning and Research in Danville, and collaborators are using their expertise in plant tissue culture and plant molecular biology to contribute to the development and characterization of novel strawberry plant collections. Photo by John McCormick.

Strawberries that glow green by design: Green fluorescent strawberries help scientists establish gene function. The 2008 Nobel Prize for Chemistry was awarded to Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien for their discovery and development of the important biological tracking device, green fluorescent protein. Photo by John McCormick.