Deborah A. Samac, E. Charles Brummer, Steve Bowley, and Daniel Z.Skinner


The 2000 report was compiled from responses received frominquiries regarding biotechnology research at laboratories aroundthe world. The report is organized geographically. The names andaddresses for a contact person at each location are listed at theend of the report. These individuals are identified by an * inthe narrative. Although we attempted to contact all labsconducting alfalfa biotechnology research, we regret any omissionsthat may have occurred. Please inform the committee of anyomissions so that the next report will be complete.

Canada (compiled by Stephen Bowley)

At Agriculture and Agri-Food Canada, Sainte-Foy, Québec, YvesCastonguay*, Serge Laberge, Paul Nadeau, and Réal Michaud have anumber of projects on biotechnology and genomic research.Activities include: 1) High throughput analysis of ExpressedSequence Tags (ESTs) isolated from cDNA libraries of coldacclimated alfalfa and functional analysis of their expression onhigh density grids; 2) Search of genetic markers associated tocold tolerance using DNA-based molecular markers to constructlinkage maps and to identify Quantitative Trait Loci (QTL)associated with freezing tolerance in alfalfa; 3) Assessment ofthe adaptative value of molecular changes such as cryoprotectivesugar accumulation and their potential use as markers forphenotypic selection; 4) Development of an integrated perspectiveon the effects of environmental parameters and managementpractices such as soil humidity, ice encasement or fall cuttingschedule on molecular and genetic changes associated with theacquisition of freezing tolerance in alfalfa; and 5) Geneticcharacterization of symbiotic gene content in artic rhizobiastrains; 6) Isolation and sequencing of nodulation genes andgenes induced by nod inducers in arctic rhizobia.

At McGill University, Raj Dhindsa* is studying the regulation ofcold tolerance at the molecular level. His research has focussedon 1) Isolation and characterization of cold acclimation-specific(cas) genes; 2) Study of the regulation and function of cas genes;and 3) Low-temperature signal transduction in plants.

At the University of Guelph, Larry Erickson* is continuing tocharacterize the function of a pollen-specific protein in alfalfausing immunolocalization and antisense approaches. Their majoractivity is the development and application of platformtechnologies for the expression of bioactive peptides in alfalfa.This includes the development of tissue-specific and inducible
promoters and use of fusion proteins. Some genes expressed to dateinclude swine viral antigens for oral vaccines, porcine epidermalgrowth factor and defensins. Steve Bowley* and Bryan McKersie (nowlocated at BASF Plant Science, NC) are continuing their work onthe modification of winterhardiness, growth attributes, andquality using genetic engineering technology combined with fieldbreeding methodologies. The emphasis is on modification ofenvironmental stress tolerance systems, carbohydrate metabolism,and promoter analysis. The work includes: 1) Isolation ofpromoters and regulatory elements associated with cold tolerance;2) Production of transgenic plants and field evaluation; 3)Pyramiding transgenes genes using conventional breeding; and 4)Contractual transformation and evaluation of proprietary genes. Inconjunction with Czarnecka-Verner at the University of Florida andSuzanne Cunningham and Jeff Volenec at Purdue University, they arestudying the alfalfa heat shock response, characterizing analfalfa heat shock factor and its relationship to stresstolerance.

At the University of Manitoba, Rob Hill* and Christos Doras areare attempting to transform alfalfa with the sense and antisensebarley hemoglobin gene using Agrobacterium tumefaciens and A.rhizogenesas as vectors. The objective of this study is todetermine the effect of nonsymbiotic hemoglobins on the physiologyof the whole plant and more specifically on the tolerance toflooding.

At Agriculture and Agri-Food Canada, Saskatoon, M. Gruber* iscontinuing her work on reduction of bloat incidence. Their groupis attempting to alter the expression of genes associated withsecondary metabolism (tannins) in transgenic plants.

At the University of Victoria, Santosh Misra* in collaborationwith Lidia Watrud US EPA are examining the effects of expressionof a human metallothionein gene in alfalfa on accumulation ofcopper and on root-associated microbial communities. Shoots oftransgenic plants expressing the transgene accumulated higheramounts of copper when treated with CuSO4. No differences inarbuscular mycorrhizal infection were found between transgenic andcontrol plants. These plants will be tested for bioremediation ofcontaminated soils.

United States (compiled by Charlie Brummer and Daniel Skinner)

Biotechnology research being conducted at the BeltsvilleAgricultural Research Center, USDA-ARS: T. A. Campbell* is usinganchored microsatellite priming, RAPD, and SSR analyses ofgenomic DNA to compute intra- and inter-specific genetic distancesfor alfalfa, Medicago edgeworthii, and M. ruthenica. The codominant SSR analyses also allows comparisons of allelic statesamong clones. Diversity in these species is currently underinvestigation in mitochondrial and chloroplast DNA. N. O'Neill*is determining evolutionary and genetic relationships, andpopulation structure in fungal foliar pathogens of alfalfa bysequence analysis of conserved gene loci and by AFLP. Pathogensinclude species of Stemphylium, Phoma, and Stagonospora. G.Bauchan* and his colleagues have utilized Giemsa bandingtechniques and a computerized image analysis system to study the 9germplasm sources of tetraploid alfalfa focusing on non-dormanttypes. These technologies can be used to identify individualchromosomes and discriminate between non-dormant and dormant typesof alfalfa.

At the University of Minnesota, D. A. Samac* C. P. Vance, and H.Jung, USDA-ARS, are using Agrobacterium-mediated transformation togenerate alfalfa plants with increased disease resistance and fornew crop uses. Work is ongoing to increase alfalfa tolerance toaluminum in acidic soils, alter cell wall components in alfalfastems, and engineer alfalfa to produce a biodegradable plasticpolymer, polyhydroxybutyrate, as a value-added product. Geneticand pathogenic diversity in Minnesota populations of Phomamedicaginis is being investigated by rDNA sequencing and AFLPanalyses. Expressed sequence tags (ESTs) associated with plantmicrobe interactions and T-DNA insertion mutants are beingdeveloped with Medicago truncatula.

At Iowa State University, E. C. Brummer* and colleagues arestudying heterosis and winter hardiness in alfalfa using acombination of traditional breeding, genetic mapping, andgenomics. They are constructing genetic maps in diploid andtetraploid populations using a combination of RFLP and AFLPmarkers. Forage yield and quality, morphological traits, wintersurvival, and autumn growth are being mapped as quantitative traitloci (QTL). Genes associated with photoperiod and temperatureinduced dormancy are being isolated using several genomicstechniques and placed on the maps. Finally, a new project isbeginning to map physiological and metabolic components of rootsin these populations to help develop a framework genomicarchitecture of dormancy and winter hardiness.

At the USDA-ARS in Manhattan, KS, D. Z. Skinner* and D. B. Haysare developing an EST library from glandular-haired alfalfa plantssubtracted from eglandular sibs. Skinner and Brian Bellinger aredeveloping alfalfa cultivar fingerprinting techniques based ongene intron markers. Skinner and Kwang Baek are investigating geneexpression in pea aphids feeding on alfalfa vs. other host plants.

At Kansas State University, P.C. St. Amand* and D.C. Clark in theAgronomy Department are currently involved in research on markerassisted selection (MAS) in autopolyploid alfalfa for resistanceto anthracnose and finding yield and disease related QTL markers.

At the University of Nebraska, work in the laboratory of M.B.Dickman* has identified a number of genes involved in prepenetration morphogenesis in the fungal phytopathogenColletotrichum trifolii, causal agent of alfalfa
anthracnose. These genes are are primarily involved in signaltransduction and include kinases, small G-proteins and calciumregulated proteins. One of these genes (LAPK) is involved inappressorium development; when inactivated by gene replacement,appressoria do not form and the fungus is non-pathogenic.

Mexico (compiled by Charlie Brummer)

In Mexico, at the Nitrogen Fixation Research Center-UNAM, inCuernavaca, Mexico, the group led by G. Hernández* is geneticallymodifying both alfalfa and Rhizobium meliloti to improve theirsymbiotic capacity. In alfalfa they are obtaining transgenicplants that overexpress or inhibit (antisense) a gene importantfor the symbiotic process, such as genes that code for ammonium

Europe (compiled by Deborah Samac)

In Bulgaria, at the Institute of Genetic Engineering in KostinbrodMariana Vlahova* and colleagues are investigating the cellular andmolecular basis of direct somatic embryogenesis in alfalfa. Theyhave found a low level of expression of the cyclin-dependentprotein kinase cdc2 in non-dividing cells which are competent forcell division and expression of cyclins only in dividing cells. They are also developing herbicide resistant transgenic alfalfa inan effort to improve control of Cuscuta spp. Constructs with genesfor Basta resistance and Glean resistance have been introducedinto alfalfa and characterization of putative transformants is inprogress. A transgenic approach is also being taken to improvealfalfa digestibility. The poplar gene encoding caffeoyl CoA 3-Omethyltransferase from the phenylpropanoid pathway has beenintroduced in the sense and antisense orientations under thecontrol of the double 35S promoter. All transgenic plants had anormal phenotype and fertility. Four plants had 20-22% lesslignin content than the control. Analysis of lignin compositionand further molecular analyses are in progress.

In France, at INRA in Lusignan, Bernadette Julier-Koubaiti* andChristian Huyghe are involved in alfalfa genetic mapping andtransformation. They have established an alfalfa F1 mappingpopulation from 2 parental plants chosen for their differences inseveral agronomic traits, Verticillium wilt and anthracnoseresistance, lodging, fall dormancy, digestibility and are currently identifying AFLP markers on 180 F1 plants and will usemicro-satellite markers originating from M. truncatula. F1 plantswere planted in the field, each plant surrounded by plants fromthe variety Europe. Offspring of F1 plants will be sown in 2001for agronomic evaluation and QTL for different traits will beidentified. The alfalfa transformation program aims to modify theisoprenoid pathway, involved in the synthesis of several growthhormones. In tobacco, the over-expression of the gene encoding themevalonate kinase, an enzyme at the beginning of the pathway,increased the rate of phenological development and size of leaves.The effect of this gene on alfalfa growth and forage quality willbe tested, in spaced plants and under competition.

In Hungary, at the Institute of Genetics, Szeged, G. B. Kiss* andcoworkers have constructed a highly saturated genetic linkage mapof Medicago sativa with approximately 2,000 RFLP and PCR basedmarkers using Medicago sativa ssp. coerulea and M. s. ssp.quasifalcata. The genetic map is being used for isolating genesby map-based cloning. A BAC contig containing a gene involved insymbiotic nitrogen fixation conditioning a nod minus phenotype,has been sequenced and found to contain many candidate genes. Ahigh degree of macro- and microsynteny was found between M. sativaand M. truncatula while some degree of synteny between Medicagoand Arabidopsis was detected.

In Italy, at the University of Perugia, the work of F. Veronesi,F. Lorenzetti, and D. Rosellini* focuses on alfalfa breeding,genetics, and use of molecular markers. Molecular markers havebeen used for mapping genes for 2n egg and pollen production indiploid alfalfa and characterizing Medicago species and landraces.A paper on the development and use of retrotransposon-basedmolecular markers in Medicago species will soon be submitted. Aproject involving characterization of mutants in sporogenesis willidentify genes controlling 2n egg and pollen production, andfemale sterility. Male sterile alfalfa plants were obtained atPlant Genetic Systems, Gent, Belgium, by genetic transformationwith a construct containing the Bacillus amyloliquefaciens RNAse(Barnase) gene under the control of pTA29, a tobacco anthertapetum specific promoter. F1 plants exhibiting higher sterilitythan the primary transformants were observed, indicating that itshould be possible to obtain good male sterile plants bybackcrossing this trait into different genetic backgrounds.

At the Istituto di Ricerche sul Miglioramento Genetico dellePiante Foraggere of Italian Research Council (CNR) S. Arcioni* andcolleagues are applying biotechnology tools in different aspectsof forage breeding. F. Damiani and F. Paolocci are identifyingand isolating Lotus corniculatus genes involved in the synthesisof condensed tannins with the long term goal of producing nonbloating alfalfa plants. M. Bellucci is introducing maize genesencoding beta and gamma zeins in order to increase the level ofsulphur amino acids in alfalfa and Lotus corniculatus. F. Pupilliis using molecular markers to differentiate populations, ecotypesand varieties of alfalfa and turf grass. In addition, with thefinal objective of obtaining apomictic alfalfa plants, F. Pupilliis using chromosome walking and other strategies to identify genesresponsible for apomixis in Paspalum simplex. A. Mariani isanalyzing micro- and macrosporogenesis in alfalfa plants atdifferent level of inbreeding in relation to the presence ofreproductive system alterations.

At the Istituto Sperimentale Colture Foraggere, Lodi Institute,Pietro Rotili* and colleagues are using molecular markers inalfalfa breeding. RFLP markers have been used to estimateheterozygosity in families and individuals under selfing to selectvigorous individuals with low heterozygosity as parental plantswith improved genetic value. In cooperation with S. Arcioni inPerugia, RFLP and SSR makers are being used to estimate geneticdistance in parents of double hybrids and octuple hybrids. Incooperation with S. Arcioni and M. Bazzicalupo (DBAG-University ofFlorence), RFLP, RAPD, SSR, and AFLP markers are being used toevaluate Italian alfalfa ecotypes for their use in distinction,uniformity and stability tests.

In Russia, at the Institute of Cytology and Genetics inNovosibirsk, E. V. Deineko* and colleagues are working on geneexpression and gene manipulation in transgenic alfalfa plants foruse as edible vaccines. They have produced transgenic alfalfaadopted to local regions, expressing esat6 and mpt64 transgenes toafford protection against Mycobacterium bovis and M. tuberculosis.

Asia and Australia (compiled by Robin Groose)

At the CSIRO Division of Plant Industry in Canberra ACT, CSIROPlant Industry, Canberra, Australia, Sharon Abrahams, Tony Ashton,Paul Chu, Kathy Francki, Colin Jenkins, Phil Larkin*, TereseRichardson, Greg Tanner, and John Watson are improving thedigestibility and nutritive value of alfalfa stems through thetransgenic expression of bacterial genes for fructans andtransgenic modification of lignins. Genes are also being clonedinvolved in the biosynthesis of condensed tannins with a view tomodifying alfalfa for bloat-safety and rumenal protein protection. Recently, Alfalfa Mosaic Virus immunity has been achieved.

R.J. Rose* at the University of Newcastle in New South Wales isstudying signalling in the induction of somatic embryogenesis inM.truncatula; nodule development in M. truncatula; gene transfertechnologies in M.sativa, and M.truncatula; and chloroplast DNA inM.sativa and M.truncatula.

At the Waite Agricultural Reseach Institute at the University ofAdelaide, John Randlels*, has demonstrated resistance and immunityto Alfalfa Mosaic Virus in two lines of Medicago truncatula cvJemalong transformed with the coat protein gene of an Australianisolate of AMV. Resistance was demonstrated with both homologousand heterologous strains of the virus.

John Irwin* and Joanne Musial at the University of Queensland havebegun a project to develop and apply molecular marker technologyin lucerne to facilitate delivery of improved cultivars toindustry. More specifically, the project aims to generate markerslinked to resistance genes for Colletotrichum, Phytophthora,Stemphylium, Leptosphaerulina and lucerne aphids. A geneticdiversity study will also be conducted to determine the level ofvariation in Australian grown cultivars.

List of Contacts

Sergio Arcioni
Istituto di Ricerche sul Miglioramento Genetico delle PianteForaggere CNR
via Madonna Alta 130 06128 Perugia
Tel: (39) 75 5005217
Fax: (39) 75 5005228

Stephen Bowley
Department of Plant Agriculture
Crop Science Building
University of Guelph
Guelph, Ontario
N1G 2W1
Tel: 519-824-4120 Ext. 8704
Fax: 519-763-8933
Email: sbowley@uoguelph.ca

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

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

Yves Castonguay
Soils and Crops Research and Development Centre
Agriculture and Agri-Food Canada
2560 Hochelaga Blvd.
Ste-Foy, Québec
KG1V 2J3
Tel: 418-657-7980
Fax: 418-648-2402
Email: castonguayy@em.agr.ca

Elena Deineko
Institute of Cytology & Genetics
Lavrentiev Str.10
Novosibirsk 630090
Tel: 383/235-6135
Fax: 383/235-6558

Marty Dickman
Department of Plant Pathology
University of Nebraska
Lincoln NE 68583
Email: mbd@unlinfo.unl.edu

Larry Erickson
Department of Plant Agriculture
Crop Science Building
University of Guelph
Guelph, Ontario
N1G 2W1
Tel: 519-824-4120 Ext. 3398
Fax: 519-763-8933
Email: erickson@plant.uoguelph.ca

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

Robert Hill
Department of Plant Science
Faculty of Agriculture and Food Sciences
University of Manitoba
Winnipeg, Manitoba
R3T 2N2
Tel: (204)-474-6087
Email: rob_hill@umanitoba.ca

J.A.G. Irwin
Botany Department
Univ. of Queensland
Brisbane QLD 4072
Tel: 61-7-3652790
FAX: 61-7-3654771
Email: J.Irwin@botany.uq.edu.au

Bernadette Julier-Koubaiti
Unite de Genetique et d'Amelioration des Plantes Fourrageres
86600 Lusignan
Tel: 33 (0)5 49 55 60 38
Fax: 33 (0)5 49 55 60 44
E-mail: julier@lusignan.inra.fr

Gyorgy Kiss
Hungarian Acedemy of Sciences
Institute of Genetics
P.O.B. 521
Szeged H-6701

Phillip J. Larkin
CSIRO Plant Industry
P.O. Box 1600
Canberra ACT 2601
Tel: 61-2-624665060
Fax: 61-2-62465000
E-mail: p.larkin@pi.csiro.au

Santosh Misra
Biochemistry & Microbiology
University of Victoria
Victoria, BC
V8W 3P6
Tel: 604-721-8928
Email: smisra@uvvm.uvic.ca

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

J.W. Randles
Department of Crop Protection
University of Adelaide
Glen Osmond SA 5064
Fax: 61-8-374095
E-Mail: jrandles@waite.adelaide.edu.au

Ray J. Rose
Department of Biological Sciences
University of Newcastle
University Drive
Callaghan, Newcastle
Tel: 61-49-216143
Fax: 61-49-216923
E-Mail: BIRJR@cc.newcastle.edu.au

Daniele Rosellini
Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali
Università degli Studi di Perugia
Borgo XX giugno, 74
06121, Perugia, Italy
Tel: 39 075 5856211
Fax: 39 075 5856224
E-mail roselli@unipg.it

Pietro Rotili
Istituto Sperimentale Colture
Foraggere,Viale Piacenza 29
Lodi 20075
Tel: 037/131-838
Fax: 037/131-853
E-mail: iscfbiol@telware.it

Deborah A. Samac
1991 Upper Buford Circle
495 Borlaug Hall
St. Paul, MN 55108
Tel: (612) 625-1243
Fax: (651) 649-5058

Daniel Z. Skinner
Agronomy Dept.,Throckmorton Hall
Kansas State University
Manhattan,KS 66506 - 5501
Tel: (785) 532-7247
Fax: (785) 532-6094

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

E Mariana Vlahova
Institute of Genetic Engineering
Kostinbrod - 2 90001-970
Tel: 359/729-2552
Fax: 359/729-7985
E-mail: geneng@mtel.net

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