Alfalfa Transformation Workshop
August 2, 1998

The Biotechnology Committee organized a pre-meeting workshop on alfalfa transformation to bring together scientists working in the area of alfalfa transformation in order to share ideas and information in an informal setting. The speakers were asked to address any of the following topics as they pertained to their research: gene promoters for expression of transgenes, selectable markers, levels of gene expression in transgenics, value-added traits for alfalfa, strategies for expressing transgenes in adapted germplasm, challenges of establishing transgenic research in industry, and field tests with transgenic alfalfa.

Deborah Samac, USDA-ARS and University of Minnesota, St. Paul, MN described development of a transformation system and its uses to study ammonia assimilation in nodules and for biotechnological applications. Critical to success of their system was combining a compatible Agrobacterium strain (LBA4404) with a highly regenerable clone of RegenSY. Cocultivation periods of 7-8 days resulted in optimal production of transgenic embryos. The transformation system has been used to introduce promoter- glucuronidase (GUS) gene constructs into alfalfa for six genes and for overexpression and antisense expression of cDNAs from three genes. While alfalfa promoters were found to be expressed as expected, two promoters tested from Arabidopsis were not expressed as expected in alfalfa. Also, many of the plants transformed with genes using the CaMV 35S promoter had low levels of gene expression. Several new viral promoters are being evaluated to increase transgene expression.

Edwin Bingham, University of Wisconsin, Madison, WI reported on transformation work carried out by a team of scientists lead by Sandra Austin-Phillips. The team has been conducting a multidisciplinary feasibility study for the production of industrial enzymes in transgenic alfalfa for the past 7 years. The goal of the program is to develop genetically engineered alfalfa that produces high levels of industrially important enzymes and to develop methods for rapidly extracting and purifying the enzymes. The transformation system uses a RegenSY and Agrobacterium LBA4404 with kanamycin as the selectable marker. Regeneration is typically 70-90% with 30-65% efficiency of transformation. Genes expressed include: GUS, ÿ-amylase, manganese dependent lignin peroxidase, phytase and cellulase. Among transformants a 10-20 fold range in expression of heterologous proteins was found. Accumulation of most enzymes was 0.1% or less of total leaf protein, however, phytase accumulates to 1-2% of total protein. Both the 35S promoter and Mac promoter were found to give good constitutive expression in alfalfa. In field tests, transgenic lines continued to express the introduced gene at comparable levels throughout the growing season and in subsequent years. They have found that introgression of the transgenic trait into cultivated germplasm by backcrossing to elite cultivars is rapid and efficient, provides many parents for production of new cultivars and removes any somaclonal variation introduced through tissue culture.

Steve Bowley, University of Guelph, Guelph, Ontario described his group's work to improve winter hardiness in alfalfa. Their transformation system utilizes alfalfa petioles and Agrobacterium mediated transformation. They have successfully utilized kanamycin, hygromycin, Basta and Pursuit resistance as selectable markers. The 35S promoter has been used successfully to express superoxide dismutase (SOD) genes. Currently they select primary transgenic plants that have SOD levels above the control plants and test primary transgenic plants in the field. They have found that greenhouse testing for cold tolerance is not predictive of field performance. Dr. Bowley discussed the additional regulations and fees Canadian researchers now face in order to field test transgenic plants. He also discussed with the audience the possible cosuppression of transgenes as plants are crossed to increase the frequency of a transgene and the need for novel production systems for transgenic alfalfa.

Nancy Paiva, Noble Foundation, Ardmore, OK described her group's work to express novel phytoalexins in alfalfa to improve disease resistance. They have used the pBI vectors with the dual 35S promoter with a tobacco etch virus leader sequence developed by Jim Carrington for better translation of mRNA. Plants containing the resveratol synthase transgene varied 50-fold in levels of expression. Expression is very high in leaves and low in roots. Dr. Paiva pointed out that no one has investigated the effect of the gene terminator on improving transgene expression and message stability. She reviewed the procedures required for field testing of transgenic alfalfa in the US. Currently alfalfa field trials only require notification for most trials, the notification can be submitted electronically, and most requests are approved rapidly. The issue of feeding transgenic alfalfa to animals was discussed with the audience members. It was felt that current restrictions will be eased in the near future.

Jay Sandman, Cal/West Seeds, West Salem, WI described the challenges of transgenic research in industry. His group has identified regenerating genotypes in germplasm with fall dormancy ratings of 2 through 9. Approximately 2-3% of plants tested would regenerate. Regeneration has been improved by crossing. Plants are being tested to select ones that transform well with Agrobacterium. He described the limited freedom to operate that a small company faces due to patent protection on most promoters, genes, selectable markers, and transformation systems. Licensing agreements must be executed carefully so that all parties will accept the use of their material in the project.

Christian Huyghe, INRA, Lusignan, France described results from their study to localize transformation events from Agrobacterium mediated transformation. In experiments using alfalfa petioles and a 35S-GUS construct, GUS expression was seen in epidermal cells and near vascular tissue after a 2 day co-cultivation period. Later, GUS positive cell clusters were observed surrounded by phenolic compounds and dead cells that may inhibit further regeneration of cultures.

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