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Agrobacter ium rhizogenesMediatedTransformation of the ForageLegumes Medicago sativaand Onobrychisviciifolia

1. T. J. GOLDS,2. J. Y. LEE,3. T. HUSNAIN,4. T. K. GHOSEand5. M. R. DAVEY1

-Author Affiliations1. Plant Genetic Manipulation Group, Department of Botany, University of NottinghamUniversity Park, Nottingham NG7 2RD, UK

1. 1To whom correspondence should be addressed

Received November 2, 1990. Accepted March 22, 1991.

Abstract

Three cultivars ofM. sativa and one cultivar of O. viciifolia were evaluated for their response to inoculation with A. rhizogenes strain A4T(containing pRiA4b). A cultivar-dependent response was observed inM. sativa with 94%, 25%, and 4% of infected stem explants producing

transformed roots in the cultivars Vertus, Regen-S, and Rangelander, respectively. In O. viciifolia cv. Hampshire Giant, an explant-

dependent response was observed with 78% and 50% of seedling cotyledon and hypocotyl explants responding, respectively. Leaf explantsfailed to produce transformed roots. Transformed roots showed plagiotropic and negatively geotropic growth on hormone-free agar MS

medium. Production of transgenic shoots from O. viciifolia root cultures occurred spontaneously. Recovery of transgenic plants fromM.

saliva cv. Rangelander was achieved by transfer of callus (induced on UM medium containing 20mg dm3 2,4-D and 025 mg dm3 kinetin)to MS medium containing 05 ing dm3 BAP and 005 mg dm3 NAA. Cultured roots of both species synthesized opines (agropine and

mannopine). Extensive morphological variation was observed in plants ofM. sativa (clone Al) and O. viciifolia (clone A4Tl) established in

the glasshouse. DNA sequences homologous to TL-DNA and TR-DNA were present in root clones and regenerated plants.

http://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-

f34a20787e2e

Transformation ofMedicago sat ivaL. Using a Ti Plasmid Derived Vector

1. M. Pezzotti,

2. F. Pupilli,

3. F. Damiani,

4. S. Arcioni

Article first published online: 28 APR 2006

DOI: 10.1111/j.1439-0523.1991.tb00477.x

Keywords:

Medicago sativa;

Agrobacterium tumefaciens;

transformation-binary-vector;

kanamycin resistance

Abstract

A cultivar ofMedicago sativa was transformed withAgrobacterium tumefaciens strain At81 binary-vector carrying the plasmid

pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem

http://jxb.oxfordjournals.org/search?author1=T.+J.+GOLDS&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+J.+GOLDS&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=J.+Y.+LEE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=J.+Y.+LEE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+HUSNAIN&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+HUSNAIN&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+K.+GHOSE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+K.+GHOSE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=M.+R.+DAVEY&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=M.+R.+DAVEY&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=M.+R.+DAVEY&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/content/42/9/1147.abstract?sid=47183503-9775-4516-b518-f34a20787e2ehttp://jxb.oxfordjournals.org/search?author1=M.+R.+DAVEY&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=M.+R.+DAVEY&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+K.+GHOSE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+HUSNAIN&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=J.+Y.+LEE&sortspec=date&submit=Submithttp://jxb.oxfordjournals.org/search?author1=T.+J.+GOLDS&sortspec=date&submit=Submit

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segments were infected with the bacterium and kanamycin resistant calli were obtained; plant regeneration via somatic

embryogenesis and polyembryogenesis was achieved only in media devoid of the antibiotic. Genetic transformation was

confirmed by the presence of the structural gene through DNA-DNA hybridization and the enzymatic assay showed its functional

expression. Mesophyll protoplasts, leaf calli and S1 progeny of transformed plants grew in the presence of kanamycin (100

gml-1). Results are discussed in relation to the use of kanamycin-resistant plants in somatic hybridization in the

genus Medicago.

http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0523.1991.tb00477.x/abstract

Efficient Transformation of Alfalfa Protoplasts by the IntranuclearMicroinjection of Ti PlasmidsT. J. Reich1, V. N. Iyer1 & B. L. Miki2

Nature Biotechnology4, 1001 - 1004 (1986)

doi:10.1038/nbt1186-1001

Abstract

Intranuclear microinjection of alfalfa (Medicago sativa L.) protoplasts yielded transformationfrequencies of 1526%. Over 70 transformed callus lines were recovered without selection bymicroinjecting a variety of plasmids. Analyses of several lines transformed with pTiC58 showed thatintegration did not occur by the TDNA mechanism typical for crown gall tissues. The presence of afunctional TDNA right border on smaller plasmids or the coinjection of a functional virregion on aseparate plasmid did not increase the transformation frequencies. The novelty of this approach tothe genetic transformation of plants is that selection systems are not required and restrictions on

the host range ofAgrobacterium tumefaciens may be circumvented.

http://www.nature.com/nbt/journal/v4/n11/abs/nbt1186-1001.html

http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0523.1991.tb00477.x/abstracthttp://onlinelibrary.wiley.com/doi/10.1111/j.1439-0523.1991.tb00477.x/abstracthttp://www.nature.com/nbt/journal/v4/n11/abs/nbt1186-1001.htmlhttp://www.nature.com/nbt/journal/v4/n11/abs/nbt1186-1001.htmlhttp://www.nature.com/nbt/journal/v4/n11/abs/nbt1186-1001.htmlhttp://onlinelibrary.wiley.com/doi/10.1111/j.1439-0523.1991.tb00477.x/abstract