1998 REPORT OF THE COMMITTEE OF THE USE OF BIOTECHNOLOGY RESEARCH IN ALFALFA IMPROVEMENT

1998 REPORT OF THE COMMITTEE OF THE USE OF BIOTECHNOLOGY RESEARCH IN ALFALFA IMPROVEMENT

Deborah A. Samac, E. Charles Brummer, Bryan McKersie, and Daniel Z. Skinner

Preamble

The 1998 report was compiled from responses received from inquiries regarding biotechnology research at laboratories around the world. The report is organized geographically. The names and addresses for a contact person at each location are listed at the end of the report. These individuals are identified by a * in the narrative. Although we attempted to contact all labs conducting alfalfa biotechnology research, we regret any omissions that may have occurred. Please inform the committee of any omissions so that the next report will be complete.

Canada (compiled by Bryan McKersie)

Canadian efforts over the past years in the application of biotechnology to alfalfa continue to become centralized in a few research centres.

At Agriculture and Agri-Food Canada, Sainte-Foy, Québec, Y. Castonguay, S. Laberge, P. Nadeau, R. Michaud and L.-P. Vézina are studying cold-induced changes in genetic expression in alfalfa: 1) identification and characterization of cold-induced cDNAs in alfalfa with emphasis on genes more highly or specifically expressed in cold-hardy cultivars; 2) assessment of heritability of cold-induced expression of COR genes; 3) identification and characterization of cold induced promoters in alfalfa; and 4) production of pedigree populations and phenotypic identification for a genome mapping project. S. Laberge and L.-P. Vézina are studying transgene expression in alfalfa: 1) isolation and characterization of constitutive promoters, 5' and 3' mRNA stabilizing sequences and other means of increasing the expression of foreign genes; 2) optimization of foreign protein production through subcellular targeting and protein fusion; and 3) field assessment of transgene dispersal. S. Laberge, D. Prévost, L. Barran and E. Bromfield are studying symbiotic nitrogen fixation: 1) genetic characterization of the nodulation gene content and nodulation specificity of a Canadian high arctic rhizobia adapted for growth and nitrogen fixation at low temperature; 2) nodulation specificity of the Rhizobium-legume symbiosis through nodulation genes characterization; and 3) diversity and dynamics of Rhizobium populations in soils.

At the University of Guelph, Larry Erickson is continuing his ongoing investigation of pollen-specific genes and regulatory sequences. He is also developing platform technologies for the expression of foreign proteins in alfalfa, with a special emphasis on veterinary biologicals, i.e. proteins which enhance the growth, health and development of animals. Bryan McKersie and Steve Bowley are continuing their work on the modification of winterhardiness using genetic engineering technology. The emphasis is on the modification of oxidative stress tolerance and carbohydrate metabolism in alfalfa crowns and roots. The work includes: 1) isolation of promoter and regulatory sequences for low temperatures; 2) isolation and characterization of cDNA from subtracted cDNA libraries that are associated with stress tolerance; and 3) production of transgenic plants and their evaluation in field trials.

At Agriculture and Agri-Food Canada, Saskatoon, M. Gruber is continuing her work on bloat through a collaboration with Bryan McKersie and Steve Bowley at the University of Guelph. They are attempting to alter the expression of genes associated with tannin metabolism in transgenic plants. Bruce Gossen has also been evaluating transgenic plants overexpressing superoxide dismutase for tolerance to Verticilium and bacterial wilts.

At the University of Victoria, Santosh Misra, in collaboration with Lidia Watrud at the Corvallis, OR USEPA lab, has genetically engineered alfalfa plants with a human MT gene expressed under a constitutive promoter and a root-specific promoter. The control and transgenic plants are being assessed for their ecological impact on the rhizoshere microflora.

At McGill University, Raj Dhindsa is studying the regulation of cold tolerance in alfalfa at the molecular level. He has isolated specific cold regulated genes and is studying the signaling process that regulates their enhanced expression at low temperatures.

United States (compiled by Charlie Brummer and Daniel Skinner)

At the University of Georgia, M.K. Sledge, W.A. Parrott, G. Kochert, and J.H. Bouton* are using molecular markers to assemble synthetic cultivars based on genetic dissimilarity as measured by the dissimilarity of AFLP markers. They are also working to confirm previously identified QTL for aluminum tolerance in diploid alfalfa, and to transfer these QTL from the
diploid to the tetraploid level in cultivated alfalfa.

At Iowa State University, C. E. Brummer* is using molecular markers in genetic mapping of heterosis and inbreeding depression for forage and seed yield in tetraploid alfalfa, and is using markers to help identify and develop heterotic groups.

At Kansas State University, D. Z. Skinner*, USDA-ARS, and D.E. Obert and D. L. Stuteville, are using AFLPs to define molecular regions associated with disease resistance. Skinner and J. C. Cordero are using degenerate primer PCR to clone candidate disease resistance genes. Skinner and M. M. Hetzel are using specific PCR to amplify two hypervariable chloroplast DNA regions to measure genetic distances within and between populations and cultivars. P. C. St. Amand* , and K. G. Glover are using RAPDs and AFLPs to evaluate genetic diversity among isolates ofCercospora medicaginis. St. Amand and D. Clark are using AFLPs to identify genes involved in the host-parasite interaction of alfalfa anthracnose.

Biotechnology research being conducted at the Beltsville Agricultural Research Center, USDA-ARS: T. A. Campbell* is assessing the possibility of reducing inbreeding and increasing heterosis through the use of self-incompatible (SI) parents. RAPD analyses and Anchored Microsatellite Priming (AMSP) were used to assess genetic distance. The same techniques are being used to investigate genetic relationships in collections of M.ruthnica and M. edgeworthii. Other research will entail using SSR primer sets to determine whether or not specific alfalfa loci also exist in M.ruthenica and M. edgeworthii. N. O'Neill* is using AFLP markers and rDNA sequence homologies to clarify the genetic relationships and population structure within fungal genera and species that are foliar pathogens of alfalfa (including Colletotrichum, Pleospora, Stagonospora, and Phoma) and that are not clearly distinguished by their morphology. T.C. Elden*, A.C. Smigocki, S.E. Wilhite, G.R. Bauchan*, and D.A. Samac, St. Paul, MN, are genetically engineering alfalfa to produce proteinase inhibitors which interfere with the digestive physiology of several major alfalfa insect and nematode pests. They are also cloning cysteine proteinase genes involved in the digestive physiology of the alfalfa weevil to serve as tools to express recombinant proteinases for selecting potent inhibitors from a library of novel cysteine proteinase inhibitors. Bauchan and his colleagues have utilized Giemsa banding techniques and a computerized image analysis system to identify chromosomal modifications and banding polymorphisms in diploid Medicago sativa ssp. falcata. They have also studied the 9 germplasm sources of tetraploid alfalfa using these technologies.

At the University of Minnesota, D. A. Samac * C. P. Vance, and H. Jung, USDA-ARS, are using Agrobacterium-mediated transformation to generate transgenic alfalfa plants with increased disease resistance and for new crop uses. Work is on-going to increase alfalfa tolerance to acid soil conditions, alter pectin content in alfalfa stems, and engineer alfalfa to produce value-added products. Genes involved in leaf senescence and disease resistance are being cloned and characterized.

At the University of Wisconsin, D. J. Brouwer and T. C. Osborn* are using molecular markers to identify and compare some of the genes that control winter injury, fall growth, and freezing injury in tetraploid alfalfa. Several QTL for each trait were identified. A genetic basis for using fall growth and freezing injury to predict winter injury is shown by these results, but the three traits can also be manipulated independently since some genetic factors significantly affected only one of these traits. S. Austin-Phillips, T. Zeigelhoffer, R. Strab, R. Koegel, E.T. Bingham*,and M. Cook, have been conducting a multidisciplinary feasibility study for the production of industrial enzymes in transgenic alfalfa, including developing genetically-engineered alfalfa that produces high levels of industrially-important enzymes, rapid methods for extracting and purifying these enzymes from alfalfa to provide a high value product which takes advantage of existing agricultural productivity.

Mexico (compiled by Charlie Brummer)

In Mexico, at the Nitrogen Fixation Research Center-UNAM, in Cuernavaca, Mexico, the group led by G. Hernández* is genetically modifying both alfalfa andRhizobium meliloti to improve their symbiotic capacity. In alfalfa they are
obtaining transgenic plants that overexpress or inhibit (antisense) a gene important for the symbiotic process, such as genes that code for ammonium assimilation enzymes (GS or GOGAT) or leghemoglobin genes. In R. meliloti they are working on induced DNA amplification of nif or nod genes and in the expression of a heterologous globin gene that alters the oxygen regulation of N fixation.

Europe (compiled by Deborah Samac)

In France, at the Institut des Sciences Végétales, A. Konderosi* and colleagues are conducting research aimed at the elucidation of the molecular communication between Rhizobium meliloti and its host plants, M. sativa and M. truncatula. Areas of interest include: Nod signal perception and transduction, control of root nodule organogenesis, suppression of plant defence reactions during symbiosis, and plant genetic approaches to Rhizobium-legume symbiosis. At INRA Montpellier, J. M. Prosperi and J. Ronfort* are using molecular (RAPDs and microsatellites) and biochemical (allozyme) markers to understand the population biology of M. sativa and M. truncatula. RFLPs of mitochondrial DNA have been developed to analyze the cytoplasmic diversity present within natural and cultivated populations of M. sativa.

In Hungary, at the Institute of Genetics, Szeged, G. B. Kiss* and coworkers are constructing a highly saturated genetic linkage map of alfalfa (Medicago sativa) with RFLP or PCR based markers using Medicago sativa ssp. coerulea and M. s. ssp. quasifalcata). The genetic map is being used for isolating genes by map-based cloning and to establish synteny between alfalfa and other legumes like M. truncatula and pea, and Arabidopsis thaliana. Presently, two genes involved in symbiotic nitrogen fixation conditioning a nod and fix minus phenotype, are being isolated using a BAC library constructed by D. Cook (Texas A & M University).

In Italy, at the University of Perugia, G. Barcaccia, F. Veronesi, M. Falcinelli and F. Lorenzetti* are saturating an RFLP linkage map of a M. falcata reproductive mutant (developed by S. Tavoletti) using a combination of RAPD, AFLP and SSR markers. The goal is to eventually assemble a functional apomictic system in alfalfa. The integrated genetic map is currently being used for i) targeting chromosome regions involved in diplosporic meiosis and ii) detecting progenies from somatic parthenogenesis. At the Instituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere (IRMGPF), S. Arcioni* and coworkers are conducting research to increase the transformation efficiency of alfalfa by Agrobacterium tumefaciens. Genetic transformation of alfalfa is being used to control synthesis of condensed tannins, increase sulphur amino acids and introduce resistance to pathogenic fungi. In addition, interspecific somatic hybridization is being used to transfer agronomic traits into alfalfa. A putative asymmetric somatic hybrid between M. sativa and M. littoralis is under evaluation. At the University of Ancona, S. Tavoletti* and Roberto Papa are working on the development and application of genetic linkage maps in alfalfa. Land races will be used to study genetic diversity with RFLP and AFLP markers. Genes for 2n gamete formation are also being mapped using F1 segregating populations and a half tetrad analysis project aims to position centromeres within the alfalfa genetic linkage map.

At the University of Oslo, Norway, G. Caetano-Anolles*, Department of Biology, is searching for nodulation-related genes in alfalfa by using differential display and serial analysis of gene expression. The techniques being developed will facilitate the cloning of developmental genes involved in nodule organogenesis without resorting to classical positional cloning
strategies.

Asia and Australia (compiled by Robin Groose)

In Japan, T. Takamizo and T. Komatsu* at the National Grassland Research Institute have transformed alfalfa with a CAD anti-sense gene. Transgenic plants carrying Aralia cordata CAD (Cinnamyl alcohol dehydrogenase) cDNA were produced by the Agrobacterium method. The have also somatically hybridized alfalfa and salt-tolerant Medicago ruthenica using electro-fusion
and have demonstrated various levels of ploidy among fusion products using flow-cytometry.

In Australia, at the University of Adelaide, J.W. Randles* and coworkers have demonstrated that sense and antisense coat protein genes of alfalfa mosaic virus strain N20 confer protection in transgenic tobacco plants. He continues collaborative work with Newcastle in New South Wales, R.J. Rose at the University of New Castle and with A.W.H. Lake, South Australian Research
and Development Institute, Adelaide to develop AMV resistance in annual medics and to produce Sitona weevil resistance in M. truncatula via transgenic plants.

Also in Australia, at CSIRO Plant Industry, Canberra, research in alfalfa biotechnology continues to focus on four areas. J. Watson is improving stem digestibility by the genetic modification of stem lignin by targeting genes in lignin biosynthesis which can modify the amount of lignin and alter the chemical composition of lignin polymers. C. Jenkins and W. Henderson are enhancing digestible energy content by introducing genes which enable the
accumulation of novel high molecular weight glucans and fructans. P. Chu, A. Walter, T. Richardson, and T.J. Higgins are improving the resistance to Alfalfa Mosaic Virus using various pathogen derived genes. G. Tanner, S. Abrahams and P. Larkin* are working on bloat safety by cloning and introducing genes associated with condensed tannin (proanthocyanidin) synthesis.

Also in Australia, G. Bender* of Inoculant Services, SGB Pty Ltd is genetically improving rhizobia for alfalfa and annual medics.

List of Contacts

Sergio Arcioni
Istituto Ricerche sul Miglioramento Genetico delle Piante Foraggere
VIA Madonna Alta, n. 130
06128 Perugia
ITALY
Tel: 0039 75 5005217
Fax: 0039 75 5005228
E-mail: S.Arcioni@IRMGPF.PG.CNR.IT

G. R. Bauchan USDA/ARS/PSI Soybean & Alfalfa Research Lab. Bg. 006,R. 14,10300 Baltimore Ave. Beltsville,MD 20705 - 2350 Tel:301/504-6649 Fax:301/504-5728 E-mail:gbauchan@asrr.arsusda.gov G. Bender
Inoculant Services
Seed Grain and Biotechnology Australia
RMB 221
Wymah NSW 2640
Australia
Fax: 61-60-202-001
Email: fairview@albury.net.au

E. T. Bingham 1575 Linden Dr.,Agronomy Dept. University of Wisconsin Madison,WI 53706 Tel:608/262-9579 Fax:608/262-5217 J. Bouton
Dept. of Crop and Soil Sciences
3111 Plant Sciences Building
University Of Georgia
Athens,GA 30602
Tel: (706)369-5808
Fax: (706) 542-0914
E-mail:jbouton@uga.cc.uga.edu

E. Charles Brummer
1204 Agronomy Hall
Iowa State University
Ames, IA 50010
Tel: (515) 294-1415
Fax: (515) 294-6505
E-mail: brummer@iastate.edu

Gustavo Caetano-Anolles
Plant Molecular Genetics
University of Tennessee
P.O. Box 1071
Knoxville,TN 37901 - 1071

T. Austin Campbell
USDA/ARS/PSI/SARL
Bldg. 002,Room 12
10300 Baltimore Avenue
Beltsville,MD 20705 - 2350
Tel: (301) 504-5638
Fax: (301)504-5167

Thomas C. Elden
USDA/ARS
Bldg. 467,BARC-E
10300 Baltimore Ave.
Beltsville, MD 20705 - 2350
Tel: (301)504-8392
Fax: (301) 504-8526
E-mail: telden@asrr.arsusda.gov

Georgina Hernández
Nitrogen Fixation Research Center
Ap. Postal 565-A
Cuernavaca, Mor.
MEXICO
Tel: (527) 317-4357
Fax: (527)317-5581
E-mail: gina@cifn.unam.mx

Gyorgy B. Kiss
Medicago Genetics Group, Institute of Genetics,
Biological Research Center of the Hungarian Academy of Sciences
Temesvari krt. 62, Szeged
H-6726 Hungary

T. Komatsu
Biotechnology Laboratory
National Grassland Research Institute
Nishinasuno Tochigi, 329-2793
Japan
Fax: 81-287-36-6629
Email: tkomatsu@ngri.affrc.go.jp

Adam Kondorosi
CNRS, Institute des Sciences Végétales
Avenue de la Terrasse, Bat. 23,
91198 Gif-Sur-Yvette Cedex
FRANCE
Tel: 01 69 82 36 96
Fax: 01 69 82 36 95

P. Larkin
CSIRO Plant Industry
P.O. Box 1600 Canberra 2601
Australia
Fax: 61-2-6246-5000
Tel: 61-2-6246-5060
Email: p.larkin@pi.csiro.au

Franco Lorenzetti
Instituto di miglioramento Genetico Vegetale
Universia degli Studi di Pesugia
Borgo XX Giugno, 74
06121 PERUGIA
ITALY
Tel: 39 75 5856206
Fax: 39 75 5856224

Nichole R. O'Neill
USDA/ARS,Soybean & Alf. Res. Lab.
Bldg. 009,Rm. 3-1
10300 Baltimore Avenue
Beltsville, MD 20705 - 2350
Tel: (301) 504-5331
Fax: (301) 504-5728

T. C. Osborn
Dept. of Agronomy
University of Wisconsin-Madison
1575 Linden Drive
Madison,WI 53706
Tel: (608) 262-2330
Fax: (608)262-5217
E-mail: osborn@calshp.cals.wisc.edu

J.W. Randles
Department of Crop Protection
University of Adelaide
Glen Osmond SA 5064
Australia
Fax: 61-8-8374095
Email: jrandles@waite.adelaide.edu.au

J. Ronfort
INRA
Centre de Montpellier
Domaine de Melgueil
34130 MAUGUIO
FRANCE
Tel: 04 67 23 06 37
Fax: 04 67 29 39 90

Deborah A. Samac
USDA-ARS
1991 Upper Buford Circle
495 Borlaug Hall
St. Paul, MN 55108
Tel: (612) 625-1243
Fax: (612) 649-5058
E-mail: debbys@puccini.crl.umn.edu

Daniel Z. Skinner
USDA/ARS/NPA
Agronomy Dept.,Throckmorton Hall
Kansas State University
Manhattan,KS 66506 - 5501
Tel: (785) 532-7247
Fax: (785) 532-6094
E-mail:dzolek@ksu.edu

Paul C. St. Amand
2004 Throckmorton Hall
KSU Agronomy Dept.
Manhattan,KS 66506 - 5501
Tel: (785) 532-7746
FAX: (785) 532-6094
E-mail:pst@ksu.edu

Stefano Tavoletti
Dipartmento di Biotecnologie Agratie ed Ambientali
Universita degli Studi di Ancona
Via Brecce Bianche
60131 ANCONA
ITALY
Tel: 071 2204934
Fax: 071 2204858

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