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

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

Preamble

The 2000 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 an * 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 Stephen Bowley)

At Agriculture and Agri-Food Canada, Sainte-Foy, Québec, Yves Castonguay*, Serge Laberge, Paul Nadeau, and Réal Michaud have a number of projects on biotechnology and genomic research. Activities include: 1) High throughput analysis of Expressed Sequence Tags (ESTs) isolated from cDNA libraries of cold acclimated alfalfa and functional analysis of their expression on high density grids; 2) Search of genetic markers associated to cold tolerance using DNA-based molecular markers to construct linkage maps and to identify Quantitative Trait Loci (QTL) associated with freezing tolerance in alfalfa; 3) Assessment of the adaptative value of molecular changes such as cryoprotective sugar accumulation and their potential use as markers for phenotypic selection; 4) Development of an integrated perspective on the effects of environmental parameters and management practices such as soil humidity, ice encasement or fall cutting schedule on molecular and genetic changes associated with the acquisition of freezing tolerance in alfalfa; and 5) Genetic characterization of symbiotic gene content in artic rhizobia strains; 6) Isolation and sequencing of nodulation genes and genes induced by nod inducers in arctic rhizobia.

At McGill University, Raj Dhindsa* is studying the regulation of cold tolerance at the molecular level. His research has focussed on 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 to characterize the function of a pollen-specific protein in alfalfa using immunolocalization and antisense approaches. Their major activity is the development and application of platform technologies 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 date include swine viral antigens for oral vaccines, porcine epidermal growth factor and defensins. Steve Bowley* and Bryan McKersie (now located at BASF Plant Science, NC) are continuing their work on the modification of winterhardiness, growth attributes, and quality using genetic engineering technology combined with field breeding methodologies. The emphasis is on modification of environmental stress tolerance systems, carbohydrate metabolism, and promoter analysis. The work includes: 1) Isolation of promoters 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. In conjunction with Czarnecka-Verner at the University of Florida and Suzanne Cunningham and Jeff Volenec at Purdue University, they are studying the alfalfa heat shock response, characterizing an alfalfa heat shock factor and its relationship to stress tolerance.

At the University of Manitoba, Rob Hill* and Christos Doras are are attempting to transform alfalfa with the sense and antisense barley hemoglobin gene using Agrobacterium tumefaciens and A. rhizogenesas as vectors. The objective of this study is to determine the effect of nonsymbiotic hemoglobins on the physiology of the whole plant and more specifically on the tolerance to flooding.

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

At the University of Victoria, Santosh Misra* in collaboration with Lidia Watrud US EPA are examining the effects of expression of a human metallothionein gene in alfalfa on accumulation of copper and on root-associated microbial communities. Shoots of transgenic plants expressing the transgene accumulated higher amounts of copper when treated with CuSO4. No differences in arbuscular mycorrhizal infection were found between transgenic and control plants. These plants will be tested for bioremediation of contaminated soils.


United States (compiled by Charlie Brummer and Daniel Skinner)

Biotechnology research being conducted at the Beltsville Agricultural Research Center, USDA-ARS: T. A. Campbell* is using anchored microsatellite priming, RAPD, and SSR analyses of genomic DNA to compute intra- and inter-specific genetic distances for alfalfa, Medicago edgeworthii, and M. ruthenica. The co dominant SSR analyses also allows comparisons of allelic states among clones. Diversity in these species is currently under investigation in mitochondrial and chloroplast DNA. N. O'Neill* is determining evolutionary and genetic relationships, and population structure in fungal foliar pathogens of alfalfa by sequence analysis of conserved gene loci and by AFLP. Pathogens include species of Stemphylium, Phoma, and Stagonospora. G. Bauchan* and his colleagues have utilized Giemsa banding techniques and a computerized image analysis system to study the 9 germplasm sources of tetraploid alfalfa focusing on non-dormant types. These technologies can be used to identify individual chromosomes and discriminate between non-dormant and dormant types of alfalfa.

At the University of Minnesota, D. A. Samac* C. P. Vance, and H. Jung, USDA-ARS, are using Agrobacterium-mediated transformation to generate alfalfa plants with increased disease resistance and for new crop uses. Work is ongoing to increase alfalfa tolerance to aluminum in acidic soils, alter cell wall components in alfalfa stems, and engineer alfalfa to produce a biodegradable plastic polymer, polyhydroxybutyrate, as a value-added product. Genetic and pathogenic diversity in Minnesota populations of Phoma medicaginis is being investigated by rDNA sequencing and AFLP analyses. Expressed sequence tags (ESTs) associated with plant microbe interactions and T-DNA insertion mutants are being developed with Medicago truncatula.

At Iowa State University, E. C. Brummer* and colleagues are studying heterosis and winter hardiness in alfalfa using a combination of traditional breeding, genetic mapping, and genomics. They are constructing genetic maps in diploid and tetraploid populations using a combination of RFLP and AFLP markers. Forage yield and quality, morphological traits, winter survival, and autumn growth are being mapped as quantitative trait loci (QTL). Genes associated with photoperiod and temperature induced dormancy are being isolated using several genomics techniques and placed on the maps. Finally, a new project is beginning to map physiological and metabolic components of roots in these populations to help develop a framework genomic architecture of dormancy and winter hardiness.

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

At Kansas State University, P.C. St. Amand* and D.C. Clark in the Agronomy Department are currently involved in research on marker assisted selection (MAS) in autopolyploid alfalfa for resistance to 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 pre penetration morphogenesis in the fungal phytopathogen Colletotrichum trifolii, causal agent of alfalfa
anthracnose. These genes are are primarily involved in signal transduction and include kinases, small G-proteins and calcium regulated proteins. One of these genes (LAPK) is involved in appressorium 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, in Cuernavaca, Mexico, the group led by G. Hernández* is genetically modifying both alfalfa and Rhizobium 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

Europe (compiled by Deborah Samac)


In Bulgaria, at the Institute of Genetic Engineering in Kostinbrod Mariana Vlahova* and colleagues are investigating the cellular and molecular basis of direct somatic embryogenesis in alfalfa. They have found a low level of expression of the cyclin-dependent protein kinase cdc2 in non-dividing cells which are competent for cell division and expression of cyclins only in dividing cells. They are also developing herbicide resistant transgenic alfalfa in an effort to improve control of Cuscuta spp. Constructs with genes for Basta resistance and Glean resistance have been introduced into alfalfa and characterization of putative transformants is in progress. A transgenic approach is also being taken to improve alfalfa digestibility. The poplar gene encoding caffeoyl CoA 3-O methyltransferase from the phenylpropanoid pathway has been introduced in the sense and antisense orientations under the control of the double 35S promoter. All transgenic plants had a normal phenotype and fertility. Four plants had 20-22% less lignin content than the control. Analysis of lignin composition and further molecular analyses are in progress.

In France, at INRA in Lusignan, Bernadette Julier-Koubaiti* and Christian Huyghe are involved in alfalfa genetic mapping and transformation. They have established an alfalfa F1 mapping population from 2 parental plants chosen for their differences in several agronomic traits, Verticillium wilt and anthracnose resistance, lodging, fall dormancy, digestibility and are currently identifying AFLP markers on 180 F1 plants and will use micro-satellite markers originating from M. truncatula. F1 plants were planted in the field, each plant surrounded by plants from the variety Europe. Offspring of F1 plants will be sown in 2001 for agronomic evaluation and QTL for different traits will be identified. The alfalfa transformation program aims to modify the isoprenoid pathway, involved in the synthesis of several growth hormones. In tobacco, the over-expression of the gene encoding the mevalonate 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 will be tested, in spaced plants and under competition.


In Hungary, at the Institute of Genetics, Szeged, G. B. Kiss* and coworkers have constructed a highly saturated genetic linkage map of Medicago sativa with approximately 2,000 RFLP and 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. A BAC contig containing a gene involved in symbiotic nitrogen fixation conditioning a nod minus phenotype, has been sequenced and found to contain many candidate genes. A high degree of macro- and microsynteny was found between M. sativa and M. truncatula while some degree of synteny between Medicago and 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 have been used for mapping genes for 2n egg and pollen production in diploid alfalfa and characterizing Medicago species and landraces. A paper on the development and use of retrotransposon-based molecular markers in Medicago species will soon be submitted. A project involving characterization of mutants in sporogenesis will identify genes controlling 2n egg and pollen production, and female sterility. Male sterile alfalfa plants were obtained at Plant Genetic Systems, Gent, Belgium, by genetic transformation with a construct containing the Bacillus amyloliquefaciens RNAse (Barnase) gene under the control of pTA29, a tobacco anther tapetum specific promoter. F1 plants exhibiting higher sterility than the primary transformants were observed, indicating that it should be possible to obtain good male sterile plants by backcrossing this trait into different genetic backgrounds.

At the Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere of Italian Research Council (CNR) S. Arcioni* and colleagues are applying biotechnology tools in different aspects of forage breeding. F. Damiani and F. Paolocci are identifying and isolating Lotus corniculatus genes involved in the synthesis of condensed tannins with the long term goal of producing non bloating alfalfa plants. M. Bellucci is introducing maize genes encoding beta and gamma zeins in order to increase the level of sulphur amino acids in alfalfa and Lotus corniculatus. F. Pupilli is using molecular markers to differentiate populations, ecotypes and varieties of alfalfa and turf grass. In addition, with the final objective of obtaining apomictic alfalfa plants, F. Pupilli is using chromosome walking and other strategies to identify genes responsible for apomixis in Paspalum simplex. A. Mariani is analyzing micro- and macrosporogenesis in alfalfa plants at different level of inbreeding in relation to the presence of reproductive system alterations.

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

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



Asia and Australia (compiled by Robin Groose)

At the CSIRO Division of Plant Industry in Canberra ACT, CSIRO Plant Industry, Canberra, Australia, Sharon Abrahams, Tony Ashton, Paul Chu, Kathy Francki, Colin Jenkins, Phil Larkin*, Terese Richardson, Greg Tanner, and John Watson are improving the digestibility and nutritive value of alfalfa stems through the transgenic expression of bacterial genes for fructans and transgenic modification of lignins. Genes are also being cloned involved in the biosynthesis of condensed tannins with a view to modifying 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 is studying signalling in the induction of somatic embryogenesis in M.truncatula; nodule development in M. truncatula; gene transfer technologies in M.sativa, and M.truncatula; and chloroplast DNA in M.sativa and M.truncatula.


At the Waite Agricultural Reseach Institute at the University of Adelaide, John Randlels*, has demonstrated resistance and immunity to Alfalfa Mosaic Virus in two lines of Medicago truncatula cv Jemalong transformed with the coat protein gene of an Australian isolate of AMV. Resistance was demonstrated with both homologous and heterologous strains of the virus.

John Irwin* and Joanne Musial at the University of Queensland have begun a project to develop and apply molecular marker technology in lucerne to facilitate delivery of improved cultivars to industry. More specifically, the project aims to generate markers linked to resistance genes for Colletotrichum, Phytophthora, Stemphylium, Leptosphaerulina and lucerne aphids. A genetic diversity study will also be conducted to determine the level of variation in Australian grown cultivars.


List of Contacts

Sergio Arcioni
Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere CNR
(IRMGPF)
via Madonna Alta 130 06128 Perugia
ITALY
Tel: (39) 75 5005217
Fax: (39) 75 5005228
E-mail:S.Arcioni@irmgpf.pg.cnr.it

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
USDA/ARS/PSI/SARL
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
RUSSIA
Tel: 383/235-6135
Fax: 383/235-6558
E-mail:deineko@bionet.nsc.ru

Marty Dickman
Department of Plant Pathology
University of Nebraska
Lincoln NE 68583
Tel:402/472-2849
Fax:402/472-2853
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.
MEXICO
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
AUSTRALIA
Tel: 61-7-3652790
FAX: 61-7-3654771
Email: J.Irwin@botany.uq.edu.au

Bernadette Julier-Koubaiti
INRA
Unite de Genetique et d'Amelioration des Plantes Fourrageres
86600 Lusignan
FRANCE
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
HUNGARY
Tel:36-62-462-232
Fax:36-62-433-503

Phillip J. Larkin
CSIRO Plant Industry
P.O. Box 1600
Canberra ACT 2601
AUSTRALIA
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
NSW 2308 AUSTRALIA
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
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
ITALY
Tel: 037/131-838
Fax: 037/131-853
E-mail: iscfbiol@telware.it

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

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.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

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







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