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June 1997 |
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NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
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June 1997 |
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NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
In This Issue:
APHIS Explains New Rule to Customers
Plants Pay a Price in Fitness for Herbicide Resistance
Forever Flowers and Fruits
Conference on Transgenic Animals in Agriculture
Upcoming Meetings
The new rule, which was published May 2 and became effective June 2, greatly expands the crops and gene modifications eligible under the notification procedure such that about 99% of field tests are expected to qualify. When notification first went into effect in 1993, it applied to only six crops - potato, corn, tobacco, soybean, cotton, and tomato. Now, any species not on a Federal Noxious Weed List and not considered a weed in the area of a proposed release is eligible. Restrictions on the nature of virus-derived transgenes have also been eased, so that only constructs encoding a functional virus movement protein are ineligible. Permits are still required for field testing microbes, plants expressing pharmaceutical proteins, unknown genes, functional virus movement proteins, and genes from human or animal pathogens.
A number of minor technical and procedural points were clarified. States will continue to be notified of proposed field tests, but now they may choose whether or not to respond. After a notification has been acknowledged, only a phone call is needed if the investigator wants to increase the acreage or change which lines will be tested (though all must fit the original description). If tests are to be conducted at additional sites, new collaborators have become involved, or additional gene constructs are to be tested, the investigator must file a new notification. It was emphasized that companies should make sure that cooperators at field test sites are fully informed about what procedures are in place to meet the performance standards written into the original notification rule.
Anticipating an ever increasing number of petitions, APHIS amended the petition process to include a mechanism for extending previous deregulatory actions to highly similar products ready for commercial sale. The petitioner must establish that the analysis done for the 'antecedent organism' adequately addresses all regulatory issues raised by the newer product. Examples would include transformation of similar cultivars with the same DNA construct as used previously; use of a different transformation method to introduce the same DNA into the same cultivar; and use of a vector differing only in noncoding regulatory sequences, as long as the introduced genes are not expressed in tissues other than those described for the antecedent.
In another effort to assist their customers, APHIS has prepared a Petition Checklist that itemizes the technical information petitioners should provide. Based on past experience, having an incomplete molecular characterization lacking in sufficient detail is the most common reason a petition is not accepted. Reviewers want to know what DNA has been put into the plant and what is expressed. Petitions lacking a complete description of all inserted sequences, or accompanied by maps or blots that are illegible, inaccurate, unlabeled or incomplete will be returned. In other words, data submitted in support of petitions should meet accepted standards for publication in the scientific literature. And far too many petitions are sent back because of glitches that never should have happened - pages not numbered, cover letter not signed, no table of contents, and so on.
The meeting concluded with a forward-looking discussion of what further changes could be made to improve the regulatory process. Part of the answer is greater use of technology. For example, USDA's Veterinary Services branch is able to accept electronic signatures; the Biotechnology and Scientific Services branch is working to follow suit. Encryption methods to protect Confidential Business Information would increase the number of electronic submissions, saving time and money for everyone. Improvements could also come through discontinuing some practices, such as requiring a notification or permit for simply moving transgenic materials between contained facilities. APHIS would like to hear more from their biotechnology "customers".
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
With respect to herbicide resistance, it has long been known that certain gene mutations result in reduced fitness for the plant. For example, weeds that have target-site resistance to the triazine herbicides (photosynthesis inhibitors) exhibit decreased electron transport through photosystem II. In the presence of the herbicide, the resistance trait confers a huge selective advantage over non-resistant competitors because it spares the life of the resistant plant. But in the absence of herbicide selection pressure, photosynthesis in the resistant plants is less efficient than that of wild-type counterparts, thus resistant plants are at a selective disadvantage due to their slower growth rate. The fitness costs of resistance to other herbicides is less clear. Weeds with mutations in the acetolactate synthase gene (the target of herbicides such as chlorsulfuron) have been thought to be equally competitive with their non-resistant counterparts in the absence of herbicide selection pressure, but the heterogeneity of weed populations makes this difficult to prove.
In the case of genetically engineered herbicide resistance, the gene mutations and genetic background of the transgenic crop are much more controlled than in resistant weeds. Nevertheless, it is not a simple task to determine the impact of a new gene on the overall fitness of a plant because it is difficult to discriminate between changes in fitness caused by the introduced gene itself and other disruptions arising during the process of engineering the transgenic plant. For example, unintended changes to the plant genome can be caused by the insertion of too many copies of the new gene, its position of insertion into the genome, or somatic mutations generated during plant transformation and regeneration.
Two recent papers in the Proceedings of the Royal Society of London addressed the costs of herbicide resistance in transgenic crucifers. Crawley and coworkers (1) compared seed survival over winter in three lines of rapeseed (Brassica napus). One line was not transformed, while the others were transformed with genes that conferred resistance to either the antibiotic kanamycin (representing the plasmid alone), or kanamycin plus the herbicide glufosinate. They found that while 2% of the non-transgenic plant seeds survived one winter, less than 0.3% of transgenic seeds survived. (The level of dormancy, and thus overwintering, is low in domesticated rapeseed.) They did not find a significant difference between the two transformed lines, suggesting that simply inserting the plasmid, which carries the kanamycin- resistance selectable marker, caused the same reduction in fitness as the plasmid with the glufosinate resistance gene.
Such was not the result in the study by López-Gutiérrez and colleagues (2), who took several extra steps to ensure that their transgenic lines of Arabidopsis thaliana were only altered by the addition of a gene for chlorsulfuron resistance. Plants with this herbicide-resistance gene were back-crossed two times to the parental line to remove any unwanted mutations induced during transformation. These were then selfed to produce two sets of plants, with one set being homozygous for the transgene and the second set homozygous for the absence of the transgene (controls).
As with the rapeseed study, separate lines were developed for plants transformed with plasmid alone (kanamycin resistant) and plasmid plus the chlorsulfuron resistance gene. After measuring several growth parameters, a 34% reduction in seed production was observed in the herbicide-resistant plants as compared to the non-transgenic controls. Plants containing only kanamycin resistance did not show this reduction, so the decrease in seed production could only be attributed to the presence of the herbicide resistance gene. Other factors such as changes in gene dosage or mutations during regeneration were also ruled out.
These studies measured two important aspects of plant survival; seed production, and the ability to overwinter. Both studies found that transgenic plants were less fit than their non- transgenic counterparts, and in the absence of herbicide selection pressure would not be expected to survive over the long term as well as non-transgenic plants. The reason for the decreased fitness was not clear.
It should be noted that both cases involve herbicide resistance genes that are expressed continuously, which may represent wasteful carbon metabolism and energy drain over the lifetime of the plant. More research is required to explain the decreased fitness, and it is likely that fitness costs will have to be determined on an individual basis for each new gene used in genetic engineering. In the mean time, these studies seem to support the notion that in genetic engineering, as in life, nothing worthwhile is without its price.
References:
1. Hails, R. S., M. Rees, D. D. Kohn, and M. J. Crawley. 1997.
Burial and seed survival in Brassica napus subsp. oleifera and
Sinapis arvensis including a comparison of transgenic and
nontransgenic lines of the crop. Proc. Royal Soc. Lond. B 264:1-
7.
2. Bergelson, J., C. B. Purrington, C. J. Palm, and J.-C. López-
Gutiérrez. 1996. Costs of resistance: a test using transgenic
Arabidopsis thaliana. Proc. Royal Soc. Lond. B 263:1659-1663.
Jim Westwood
International Research and Development
Virgina Tech
westwood@vt.edu
A recent report describes a new solution to this problem that entails the use of a hormone receptor gene from Arabidopsis which confers ethylene insensitivity (1). Tomato fruits ripened very slowly on plants engineered with this gene, while petunia flowers from transgenic plants remained fresh longer than their nontransgenic counterparts. The dominant mutant etr1-1 gene, cloned from Arabidopsis by Elliot Meyerowitz and colleagues at CalTech, encodes a protein that alters the perception of ethylene by plant cells and thus makes the plant unresponsive to the hormone (2).
A team led by Harry Klee, who initiated the work while at Ceregen Technology (Monsanto Company) and continued it at the University of Florida, introduced this gene into tomato and petunia using Agrobacterium vectors. Transgenic tomato plants exposed to ethylene exhibited a dramatically delayed fruit ripening and senescence compared with those on untransformed plants. Harvested tomato fruits retained their original golden yellow color even when stored for 100 days while the regular tomato fruits soon "turned red, became soft and started to rot". Similarly, petunia flowers with the 'ethylene- insensitive' gene senesced slowly and remained longer on the plant. When exposed to ethylene, the transgenic flowers stayed fresh for nine days in the vase while the untransformed flowers wilted within just three days.
According to the researchers, the ethylene insensitive gene from Arabidopsis may have to be weakened by molecular alterations to ensure its broad application, because fruits and vegetables eventually must respond to ethylene for ripening to proceed. The use of appropriate promoters may also permit targeted ripening. Monsanto scientists anticipate that the immediate beneficiary of their finding will be the floriculture business, a multibillion dollar industry worldwide.
Many chemicals that affect ethylene synthesis or its action, which are currently in use to extend the shelf life of flowers, are being banned because of environmental concerns (3). The floriculture industry thus may gain substantially from the use of the 'ethylene-insensitive' gene by making their colorful blooms last longer either on plants or in vases. Arabidopsis may never be considered pretty enough to be taken seriously by nurserymen but the Nature Biotechnology study clearly underscores one of the potential pay-offs to agriculture from the investment in research on this humble weed.
References:
1. J. Q. Wilkinson et al. 1997. Nature Biotechnology 15:
444-447.
2. C. Chang et al. 1993. Science 262: 539-544.
3. M. Bouzayen & J. Pech. 1997. Nature Biotechnology 15: 418
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
Proceedings of the meeting will be published by CAB International; a copy will be included in the cost of registration. For further information contact Martina McGloughlin, UC Davis Biotechnology Program (tel: 916-752-3260, e-mail: mmmcgloughlin@ucdavis.edu) or connect to http://pubweb.ucdavis.edu/Documents/BIOTECH/biotech1.htm.
Sept 15-19: Meeting of International Program on Rice Biotechnology, Riviera Bay Resort in Malacca, Malaysia. (Contact: The Rockefeller Foundation, 420 Fifth Avenue, New York, NY 10018-2702).
Nov 30-Dec 3: The National Research Council of Canada's VIIIth Industrial Biotechnology Conference, Toronto, Ontario. (Contact: Pierre Lamoureux, Conference Services, National Research Council of Canada, M19, Montreal Road, Ottawa, Canada K1A 0R6; tel: 613-993-9431; fax: 613-993-7250; e-mail: biotech97@nrc.ca).
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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