In This Issue:
Welcome and Introduction to New Readers
What's In The Pipeline?
Towards High Provitamin-A Rice
Now A Vitamin Enhanced Tomato, Too!
New 'Hit and Run' Strategy Solves Selectable Marker Problems
Method for Parasite-Derived Resistance Is Patented

NEWS AND NOTES

WELCOME AND INTRODUCTION TO NEW READERS
Last week, Information Systems for Biotechnology (ISB) reached a highwater mark - the ISB/NBIAP News Report is now sent to more than one thousand email subscribers, and over five hundred print copies are mailed out each month. We want to welcome our many new readers and introduce the ISB program to those who are not sure just where this newsletter comes from.

In 1988, the U.S. Department of Agriculture established the National Biological Impact Assessment Program (NBIAP) under the Cooperative State Research Service, which was reorganized in 1995 as the Cooperative State Research, Education, and Extension Service (CSREES). The program consisted of two main project areas: the Biotechnology Risk Assessment Research Grants Program (BRARG), and ISB, a publicly accessible resource for information about regulatory, environmental, and research and development issues in agricutural biotechnology.

Since its inception, ISB has operated through a USDA special grant awarded to the Agricultural Experiment Station at Virginia Tech. ISB came online as a dial-up Bulletin Board Service just as the first wave of agbiotech research was moving beyond the lab and greenhouse and the number of field tests was increasing rapidly. Since then, the computer-based information system has evolved into an interactive World Wide Web site (http://www.nbiap. vt.edu) with online searchable databases, documents, and resource information.

To serve the growing community of scientists affected by USDA and EPA regulations, ISB developed and distributed the Permit Application System and the Termination Report System, computer programs designed to assist researchers in fulfilling the regulatory requirements for field testing genetically modified organisms. Due to changes primarily in USDA procedures, both of these programs are now obsolete and are no longer supported.

The current focus of ISB activities is to provide information and training that supports the environmentally responsible use of agricultural biotechnology products. The website now carries risk assessment research reports as well as a computer-based version of the Performance Standards for Safely Conducting Research with Genetically Modified Fish and Shellfish, a risk assessment tool developed by and for aquatic biotechnology researchers. Workshops on the biosafety review process for conducting field tests of transgenic crops, and on regulatory awareness and compliance, are available by request. All technical and scientific activities are divided between two full-time employees. Doug King maintains the website, databases, and listserver. Pat Traynor conducts training, responds to scientific inquiries, and is editor of this News Report. The USDA/CSREES grant administrator is Dr. Richard Frahm.

ISB grant funding appears as a line item in the federal budget under the NBIAP name; it must be renewed annually. The risk assessment research grants program, now administered through USDA's Competitive Grants Program, continues to be funded by a one percent set-aside of USDA biotechnology research grant funds. Over the past few years, budget cuts have severely curtailed most of the non-regulatory federal programs concerned with agbiotech information and biosafety issues. The EPA Risk Assessment Grants Program, the USDA Office of Agricultural Biotechnology and Agricultural Biotechnology Research Advisory Committee, and the Biotechnology Information Center at the National Agricultural Library have either been eliminated or are in the process of being dismantled. The recently adopted federal budget agreement, however, retains level funding for NBIAP in 1998. Barring unforeseen changes, ISB will continue to provide information services to the agbiotech community.

Pat Traynor and Doug King
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
nbiap@vt.edu

WHAT'S IN THE PIPELINE?
In May of this year, USDA/APHIS Biotechnology and Scientific Services (formerly Biotechnology, Biologics and Environmental Protection, or BBEP) expanded the notification procedure for conducting field tests. Almost all genetically engineered crop species, with the exception of plant species known to be noxious weeds, can now be tested under notification as long as the release meets all other eligibility requirements established in the original notification procedure of 1993. As a result, the number of field tests requiring a permit is expected to drop sharply. Among the handful submitted since April are:

beet with an EPSPS gene for glyphosate tolerance (American Crystal Sugar)

Cephalosporium gramineum modified with a GUS gene as a visual marker (Washington State University)

grape modified for resistance to a virus deemed Confidential Business Information (GenApps) or with a visual marker (DNA Plant Technology)

lettuce carrying genes for bacterial and fungal resistance (Harris Moran)

melon engineered for resistance to zucchini yellow mosaic virus (Michigan State University) or with altered fruit ripening (Agritope)

Pelargonium carrying genes for resistance to Botrytis cinerea and a fungus (Sanford Scientific)

potato modified for resistance to potato virus Y by virtue of the viral nuclear inclusion protein b (Cornell)

rice engineered with a gene considered Confidential Business Information (Monsanto)

sunflower carrying a marker gene (VanderHave)

sweet potatoes with a modified storage protein or engineered for fungal resistance (Tuskegee University)

Xanthomonas modified for reduced virulence (Auburn University)

Pat Traynor
Information Systems for Biotechnology

PLANT RESEARCH NEWS

TOWARDS HIGH PROVITAMIN-A RICE
Vitamin A is highly essential for the human body, and widespread dietary deficiency of this vitamin in rice-eating Asian countries has tragic undertones: five million children in South East Asia develop an eye disease called xerophthalmia every year, and 250,000 of them eventually become blind. Improved vitamin A nutrition would alleviate this serious health problem and, according to UNICEF, could also prevent up to two million infant deaths because vitamin A deficiency predisposes them to diarrhea diseases and measles.

Flowers and fruits owe their dazzling colors to carotenoid pigments. Beta-carotene, the best-known carotenoid which gives carrots and sweetpotatoes their orange color, is a precursor to vitamin A. Both vitamin A and beta-carotene (provitamin A) are antioxidants which neutralize cancer-causing compounds known as free radicals, destroying their ability to damage cells. These nutrients are also known to boost the immune system and reduce the incidence of cataracts, arthritis, heart and lung diseases. Two recent reports from Europe illustrate how the wizardry of genetic engineering is being employed to boost the provitamin A content in food.

Rice is a staple that feeds nearly half the world's population, but milled rice does not contain any beta-carotene or its carotenoid precursors. A research team led by Peter Burkhardt and Ingo Potrykus of the Swiss Federal Institute of Technology in Zurich, in collaboration with scientists from the University of Freiburg in Germany, discovered, however, that rice contains geranyl geranyl diphosphate (GGPP), a 20-carbon isoprenoid molecule. Condensation of two such molecules produces a 40-carbon molecule called phytoene, the first carotenoid precursor in the biosynthetic pathway leading to the production of beta-carotene.

Burkhardt and colleagues used microprojectile bombardment to engineer rice with a gene from daffodil (Narcissus) that codes for phytoene synthase (psy), the enzyme that synthesizes phytoene from GGPP. Rice plants transformed with the daffodil gene produced phytoene in the immature endosperm; non-transgenic control plants did not have this carotenoid. This major breakthrough shows that an important step in provitamin A synthesis can be engineered in a non-green plant part that normally does not contain carotenoid pigments.

Burkhardt informed the ISB News Report that his team has just developed transgenic rice plants carrying bacterial phytoene desaturase and daffodil lycopene cyclase genes, thus providing all enzymes necessary to produce the beta-carotene in rice. These plants are now being subjected to biochemical tests, and Burkhardt feels confident that the enzymes will be active in the rice endosperm. He says "This work is just one example how the tools of modern biotechnology can be used to benefit people in less developed countries, as the results of our work will be distributed freely through the International Rice Research Institute in Manila, The Philippines".

The success of this work could have significant implications for alleviating vitamin A deficiency in the developing world. Who would have thought that daffodils, the flowers that have inspired poets for centuries, may in the future also make the world healthier?

Reference

(1) P. K. Burkhardt. 1997. The Plant Journal. 11:1071-1078.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu

AND NOW A VITAMIN-ENHANCED TOMATO TOO!
Tomatoes, the most popular vegetable in the world, have a jump on the competition when it comes to vitamin A. Tomatoes contain high amounts of carotenoid pigments, the majority of which is lycopene. This pigment, which is responsible for the tomato's red color, is an immediate precursor to beta-carotene, the provitamin that is readily converted in our bodies to vitamin A. A story carried by Reuters and the Associated Press reports that Peter Bramley and colleagues at Royal Holloway College, University of London in Egham, England have developed tomato lines that have either four times the normal levels of beta-carotene or twice the normal levels of lycopene. The research, which has not yet been published, is being reported to the European Commission which funded the study.

The gene for beta-carotene synthesis was placed under the control of tomato fruit-specific promoters so that the provitamin A would be produced only in the ripening fruit. According to the Reuters report, similar work is also underway in France with peppers. Bramley, who has eaten these tomatoes, says that they do not taste any different but look a little redder or more orange.

According to Prof. Bramley, "The studies we are undertaking are designed to increase the levels of carotenoid pigments in ripe fruit in order to provide greater amounts in the typical diet. The reason for this is that carotenoid pigments have been implicated in the reduction of certain cancers and coronary heart disease. In particular, high amounts of tomato in the diet have been shown to reduce prostate cancer and the implication is that lycopene is the cause of this beneficial effect. Beta carotene does not seem to be as effective in this respect."

High lycopene and high beta-carotene tomatoes are just two more examples of how biotechnology can be used to improve the quality of foods, which can have direct benefits on the health and well-being of the world's population.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu

NEW "HIT AND RUN" STRATEGY SOLVES SELECTABLE MARKER PROBLEMS
As plant genetic engineering becomes increasingly commonplace and new beneficial genes are identified, we may expect to see major crops carrying not one, but many foreign gene constructs. While this is fine in principle, in practice the very act of introducing a gene into a plant has some subtle, but important repercussions. To understand this it is useful to review some of the mechanics of how a novel gene is put into a plant.

Each gene inserted into a recipient plant cell carries not only the gene of interest (i.e. a gene providing resistance to a disease or tolerance to an herbicide) but also extra DNA sequences that serve the essential functions of (a) allowing the gene of interest to integrate itself into the plant genome and (b) subsequently allowing identification of the few transformed cells among the vast number of untransformed cells. For this latter purpose, the introduced DNA must carry a selectable marker gene so that only those cells containing the gene of interest will be regenerated into whole plants. The selectable marker is usually a gene that enables the transformed cell to survive while growing in the presence of a selection agent, usually an antibiotic or herbicide. The principle is that when all potentially transformed cells are placed on a medium containing the selection agent (the antibiotic kanamycin, for example), the only cells able to survive and grow will be those that contain the introduced DNA with its kanamycin-resistance marker gene. Those cells will probably have the gene of interest, too, since it is physically connected to the marker gene. In this way the researcher can easily identify, and work to regenerate, only those few cells that now carry the introduced gene. Unfortunately, this process presents some potential problems:

The selectable marker gene is unnecessary once the process of genetic engineering has been completed. If retained and expressed in the engineered crop, it may result in wasteful metabolism and loss of productivity. It may also produce a protein that requires additional testing and regulatory clearance to ensure that food from the crop is safe for consumption. Thus the presence of marker genes can result in increased costs associated with commercialization of genetically engineered crops. Furthermore, the environmental consequences of plants carrying such marker genes is unknown. For example, it is not clear what impact a field of corn plants expressing kanamycin resistance might have on natural populations of bacteria.

A plant that has been engineered once cannot be engineered a second time using the same selectable marker. Although this is only an inconvenience for crops that can be crossed sexually, it presents a problem for vegetatively propagated crops such as potato, cassava, strawberries, or apple. These crops must be sequentially engineered, rather than crossed, to combine multiple new genes into a single line. Thus, in order to add a gene conferring insect resistance to a potato variety that has already been engineered to be resistant to an herbicide, a different selectable marker must be used. The number of effective selectable marker genes currently is quite limited, yet the number of cloned agronomically useful genes is increasing rapidly. Simple mathematics dictates that sequential engineering of certain crops will be problematic.

Selectable marker genes can sometimes cause growth abnormalities. Most currently used markers are selective by virtue of their ability to allow transformed plant cells to grow in the presence of a toxin. Incomplete detoxification in the transformed plant cells can lead to physiological or morphological problems in regenerated plants that require additional time and expense to eliminate before the transgenic crop is commercially acceptable. In order to avoid these problems, the choice of the most appropriate selectable marker for the plant system in question must be made carefully.

This can further reduce the number of markers available for use in a given crop.

A new selection system that offers hope for eliminating all of these problems was recently described by Ebinuma and co-workers in a paper in the Proceedings of the National Academy of Science (USA) (1). They devised a transformation vector with what they called a "hit and run" selectable marker system. This system includes two unique elements, the first being a selectable marker that is visible as an unusual morphology in regenerating plantlets, and the second being that the selectable marker gene itself is inserted into a sequence of DNA that has a predisposition to eliminate itself from the plant genome. The result is a transgenic plant in which the gene of agronomic interest has been retained, but the selectable marker (and its associated problems) has been lost.

The first component of the system is the use of a bacterial isopentenyl transferase (ipt) gene as a selectable marker. This gene catalyzes an important step in the synthesis of the plant hormone cytokinin. High levels of cytokinin stimulate shoot growth, so transformed plantlets containing the ipt gene exhibit unusually prolific shoot growth and can be easily identified from tissue that does not posses this gene. Obviously, this would be a serious drawback for the regenerated plant since a crop with many shoots but little root growth would be an agronomic disaster, except that the vector in this case is designed so that the ipt gene can be eliminated and a normal plant results.

The second element of the system is taken from the corn transposable element gene (Ac). This gene has a tendency to excise itself from the genome and reinsert at a different location. This attribute has proven to be very useful in corn genetics and has been well characterized. The Ac element has even been found to have the same ability to move when inserted into the genomes of certain other crops. For the purpose of the "hit and run" transformation strategy, the authors have taken advantage of the fact that about 10% of the time the transposable element does not successfully reinsert into the genome and is lost completely. By placing the ipt gene within the transposable element in such a way that excision of Ac will also take the ipt gene, a mechanism was created by which the marker gene may be removed without additional effort by the scientist. A transformed cell in which this occurs would begin regenerating with a very shooty appearance, but then start to grow normally if the Ac element containing the ipt marker gene is lost. This is exactly what was observed.

Ebinuma and colleagues used this system to transform tobacco and aspen with some well-characterized marker genes in the place of the gene of interest. They showed that they were able to regenerate very shooty tobacco plants that still contained the ipt gene, but that 4.8% of the transgenic plants lost this gene, but retained the gene of interest, within six months of the time of transformation. Similar results were observed in aspen where 20 shooty plantlets were identified following transformation, of which three reverted to the normal phenotype within eight months. Analysis of plant chromosomal DNA confirmed that the Ac/ipt marker had indeed been eliminated from these lines.

Cloning strategies such as this one provide promise for the sequential insertion of multiple, agronomically beneficial genes, and may prove especially useful for crops such as fruit trees that normally require long time periods for making sexual crosses. The ability to eliminate the marker gene should also reduce the possibility of adverse environmental impact from transgenic plants, while increasing their vigor, and the acceptability of transgenic plants by a public leery of genetically engineered food products.

Reference

1. Ebinuma, H., K. Sugita, E. Matsunaga, and M. Yamakado. 1997. Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc. Natl. Acad. Sci. USA 94:2117-2121.

Jim Westwood
International Research and Development
Virginia Tech
westwood@vt.edu

INDUSTRY NEWS

METHOD FOR PARASITE-DERIVED RESISTANCE IS PATENTED
A patent for parasite-derived resistance (also known as pathogen-derived resistance) was recently issued to Stephen Johnston and John Sanford of Cornell Research Foundation, Inc. (CRF). This patent (Patent Number 5580716) claims a unique method for obtaining resistance genes. Rather than identifying resistance genes in the host organism or a related species, genes or gene fragments from the parasite itself are inserted into the host genome to confer resistance. The implications of this technique are broad and will likely impact development of agricultural crops resistant to insects and many types of microorganisms.

The patent describes how a gene taken from a virus can be used as a defense against the virus by inserting it into the host organism, such as a bacterium. The viral gene is expressed in the bacterial host and disrupts normal function of the virus parasite by competing with, or interfering with, expression of the native viral gene. Although the patent describes the use of a virus replicase gene in a bacterium, the technology can be applied to any host, including other microorganisms such as industrial yeast or bacteria, plants, or even animals including mammals. The technology also applies to any parasite including fungi, bacteria, protozoans and insects, in addition to viruses.

(Details of the patent are available at http://patent.womplex.ibm.com)

Alternative approaches for incorporating resistance genes into plant hosts are available, but may have limitations. For example, genes isolated from a resistant strain of the host species or a closely related species can be inserted into a susceptible variety. However, these genes may be difficult to identify. Also, such resistance may be polygenic requiring transfer of several genes which can greatly increase the difficulty of gene transfer. In some cases, resistance genes may not exist at all. In other cases, the parasite may have strains which have evolved virulence genes to overcome the host resistance. These genes can then spread quickly in the parasite population to overcome the resistance in the host.

Another approach is to insert genes from organisms unrelated to the host or the parasite, but which happen to possess effects detrimental to the parasite. One well known example is the insertion of Bacillus thuringiensis (Bt) genes into plants to confer resistance to specific insect pests. This approach is useful, but does not have a broad application to multiple pests or parasites.

Patent 5580716 indicates that parasite-derived resistance genes are easier to identify and easier to isolate than genes from the host organism. Usually the host genome is more complex than the parasite's genome. In addition, parasite derived genes should confer resistance to multiple parasites. For example, use of a viral replicase gene will not only make the host resistant to the particular virus from which it was isolated, but also to all viruses that have similar replicase genes.

It will be interesting to see how large seed or agricultural companies with products using parasite-derived resistance either on the market or in development, are affected by this patent. Seminis Vegetable Seeds has a patent for a virus resistant squash which contains a virus coat-protein gene. According to officials at Seminis, they are in the process of evaluating patent 5580716 and its implications, and are unable to comment until they know more about it.

Marc Law, Research Director at Novartis, believes the licensing strategy of CRF will dictate how widely parasite-derived resistance technology will be applied. "The (commercial) impact of this patent depends on how the claims are interpreted and how vigorously the patent is enforced." According to Dr. Law, if CRF issues non-exclusive, low cost licenses, the technology could have broad implications. To date, most seed companies have shown limited interest in virus resistance compared to other pest problems. The commercial impact, therefore, may not be as great until fungal, bacterial and insect resistance has been demonstrated by this technique.

Cynthia J. Sollod
Institute for Biotechnology Information, LLC
Research Triangle Park, NC
http://www.biotechinfo.com

The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. P.L. Traynor, Editor.

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