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Sugarbeet molecular biology--News GLYPHOSATE TOLERANT SUGARBEETS

Novartis Seeds and Monsanto have petitioned for deregulation of a sugar beet line designated as GTSB77, which has been genetically engineered for tolerance to the herbicide glyphosate. GTSB77 has been genetically engineered to express an enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme derived from Agrobacterium sp. strain CP4, and the ß-D-glucuronidase (GUS) protein from Escherichia coli. The CP4 EPSPS protein confers tolerance to the herbicide glyphosate, and the GUS protein serves as a marker in the plant transformation process. The subject sugar beet line also expresses a novel protein known as 34550, which has no known biological activity, and was apparently created when a truncated glyphosate oxidoreductase gene fused to sugar beet DNA. The Agrobacterium tumefaciens method was used to transfer the added genes into the parental sugar beet proprietary line A1012, and expression of the added genes is controlled in part by gene sequences derived from the plant pathogens figwort mosaic virus and cauliflower mosaic virus. The sugar beet line has been field tested since 1996 under APHIS permits and notifications.

As noted above, FIFRA requires that all pesticides, including herbicides, be registered prior to distribution or sale, unless exempt by EPA regulation. In cases in which genetically modified plants allow for a new use of an herbicide or involve a different use pattern for the herbicide, EPA must approve the new or different use. Accordingly, a submission has been made to EPA for registration of the herbicide glyphosate for use on sugar beet.

When the use of the herbicide on the genetically modified plant would result in an increase in the residues of the herbicide in a food or feed crop for which the herbicide is currently registered, or in new residues in a crop for which the herbicide is not currently registered, establishment of a new tolerance or a revision of the existing tolerance would be required. Residue tolerances for pesticides are established by EPA under the FFDCA and enforced by the FDA. Novartis and Monsanto have begun consultation with FDA on the subject sugar beet line.

For further information contact Dr. James White, Biotechnology and Biological Analysis, PPQ, APHIS; 301-734-5940. Comments on this petition must be received on or before October 19, 1998. Please state that your comments refer to Docket No. 98-079-1.

Plant Research News CELL TRACKING SYSTEM BOOSTS TRANSFORMATION SUCCESS

Plant biotechnology in the late 70's and early 80's was primarily manipulation at the cellular level and gene transfer methods utilizing single cells or protoplasts. It was believed that the techniques of cell fusion and gene transfer could contribute significantly to overcoming barriers to crossing in conventional breeding. Both technologies required a good protoplast system and development of protocols to induce regeneration in amorphous cell clusters (calluses). With the advent of simpler techniques using Agrobacterium and the leaf disc method, transformation of protoplasts was no longer the method of choice for many species. The value of protoplasts, however, is that they can be isolated in vast numbers and can subsequently be used for studying numerous cellular processes, such as cytoskeleton formation, metabolism, and physiology.

Now with the help of a computer-controlled cell finder system, parameters that determine competence for regeneration from a single cell into a multicellular organism are being investigated by scientists at the Department of Cell Biology, CPRO-DLO (Centre for Plant Breeding and Reproduction Research, Wageningen, the Netherlands). The cell tracking system combines step-motors for microscope stage movement, fixed reference points for calibration of immobilized target-cell positions, a video recording system, and specially designed software.

The system allows specific cells within a sample to be identified, either beforehand or in retrospect, and their locations fixed. Subsequent development of the individual cells can be monitored daily using computer assisted relocation. In this way, a specific cell type capable of sustained division and regeneration has been identified in what has been the low-efficiency protoplast system of a recalcitrant species, sugarbeet.

The regeneration competent sugarbeet cells were seen to have originated from stomatal guard cells. Isolation and purification procedures were then optimized to yield millions of guard cell protoplasts (GCPs). Studies using transient expression vectors and polyethylene glycol mediated gene transfer proved that GCPs were amenable for transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure enables one person to generate 600 individual transgenic plants per year. This number allows for the selection of plants having optimal transgene expression, efficacy, and field performance. GCP technology is presently being investigated for application to other recalcitrant crops such as onion.

The costs of this technology, particularly those of the equipment and the input needed for software maintenance and updating, might present limitations for its widespread use. Yet spin-offs of the research performed at CPRO-DLO have already appeared in other laboratories. The cell finder system is being used to examine other protoplast systems, including cell-suspension derived cultures, for the occurrence of particular cell types with specific competencies, for example regeneration and/or gene uptake.

In addition, the tracking system can also be envisaged to play a role in more fundamental research areas, such as understanding the process of foreign DNA integration into recipient plant genomes. This investigation could lead to enhanced control over the process, increasing both the efficiency and the accuracy of this critical step in the production of transgenic crops.

References

1. F.A. Krens, H.A. Verhoeven, A.J. Van Tunen, and R.D. Hall. 1998. The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation. In Vitro Plant Cellular & Developmental Biology 34:81-86.

2. Hall, R.D., H.A. Verhoeven, F.A. Krens. 1995. Computer assisted identification of protoplasts responsible for rare division events reveals guard cell totipotency. Plant Physiol 107:1379-1396.

3. Toonen, M.A.J., S.C. de Vries. 1997. Use of video cell tracking to identify embryogenic cultured cells. In: K.J. Lindsey, ed. Plant Tissue Culture Manual. Kluwer Academic Publisher, Dordrecht, Netherlands.

Nature/Biotechnology HIGH LEVEL FRUCTAN ACCUMULATION IN A TRANSGENIC BEET Dutch researchers have successfully transferred a gene from Jerusalem artichoke into sugarbeet that causes sugarbeets to store fructose polymers (fructans) rather than sucrose. Fructans currently are used in several food products and may have wide use in food industry applications. Becaue humans cannot digest fructans, lower molecular weight polymers act as low calorie sweeteners. Longer chain fructose polymers can substitute for fats in baked goods and other food products. Beneficial bacterial species in the digestional tract use them, and these bacteria are reputed to have positive health benefits connected to cholesterol reduction. The growth and development of the transformed sugarbeets was similar to non-transformed individuals under laboratory and greenhouse conditions. The majority of storage products in the roots were fructans, with very little sucrose being present. This transformation suggests that sugarbeets might be successfully used for the production of other useful storage compounds as well.

References

Smeekens, S. (1998). A convert to fructans in sugar beets. Nature/Biotechnology 16:822-823.

Sevnier, r., Hall,R.D., van der Meer, I.M., Hakkert, H.J. C., van Thunen, A.J., and Koops, AJ. (1998). High level fructan accumulation in a transgenic beet. 16: 843-846.