Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

1. 2. 3. 4. 5. T. J. GOLDS, J. Y. LEE, T. HUSNAIN, T. K. GHOSE and M. R. DAVEY1

-Author

Affiliations

1.

1. Plant Genetic Manipulation Group, Department of Botany, University of NottinghamUniversity Park, Nottingham NG7 2RD, UK 1 To whom correspondence should be addressed

Abstract

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

Three cultivars of M. 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 in M. 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 explantdependent response was observed with 78% and 50% of seedling cotyledon and hypocotyl explants responding, respectively. Leaf explants failed 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 from M. saliva cv. Rangelander was achieved by transfer of callus (induced on UM medium containing 20mg dm 3 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 of M. 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-b518f34a20787e2e

Transformation of Medicago sativa L. Using a Ti Plasmid Derived Vector

1. 2. 3. 4. M. Pezzotti, F. Pupilli, F. Damiani, 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 of Medicago sativa was transformed with Agrobacterium tumefaciens strain At81 binary-vector carrying the plasmid pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem

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 Intranuclear Microinjection of Ti Plasmids

T. J. Reich1, V. N. Iyer1 & B. L. Miki2
Nature Biotechnology 4, 1001 - 1004 (1986) doi:10.1038/nbt1186-1001

Abstract
Intranuclear microinjection of alfalfa (Medicago sativa L.) protoplasts yielded transformation frequencies of 1526%. Over 70 transformed callus lines were recovered without selection by microinjecting a variety of plasmids. Analyses of several lines transformed with pTiC58 showed that integration did not occur by the TDNA mechanism typical for crown gall tissues. The presence of a functional TDNA right border on smaller plasmids or the coinjection of a functional vir region on a separate plasmid did not increase the transformation frequencies. The novelty of this approach to the 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