Plant Cell, Tissue and Organ Culture 69: 251–258, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
251
Agrobacterium tumefaciens-mediated transformation of the legume
Astragalus sinicus using kanamycin resistance selection and green
fluorescent protein expression
Hyeon-Je Cho
1
& Jack M. Widholm
∗
Department of Crop Sciences, University of Illinois, Edward R. Madigan Laboratory, 1201 W. Gregory Dr.,
Urbana, IL 61801, USA; (
1
Present address: MAXYGEN, 515 Galveston Drive, Redwood City, CA 94063, USA;
∗
requests for offprints: Fax: +217-333-4582; E-mail: widholm@uiuc.edu)
Received 14 June 2001; accepted in revised form 16 November 2001
Key words: Agrobacterium tumefaciens, Astragalus sinicus, green fluorescent protein, transgenic plant
Abstract
To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch),
cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector
pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance
gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l
−1
α-
naphthaleneacetic acid (NAA) and 1.0 mg l
−1
thidiazuron (TDZ). Putative transformed shoots were selected on
SR medium containing 75
µ
gml
−1
Km, 200
µ
gml
−1
Timentin, and transformation was monitored by observation
of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-
expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic
cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants
have set seed. GFP expression segregated in the T
1
seedlings of the two lines tested in a 3 – 1 ratio. In addition to
the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern
and Western blot analyses.
Abbreviations: GFP – green fluorescent protein; Km – kanamycin monosulfate; MS – Murashige and Skoog (1962)
medium; NAA – α-naphthaleneacetic acid; nptII – neomycin phosphotransferase; SR – shoot regeneration medium;
TDZ – thidiazuron
Introduction
The Leguminosae contains many economically im-
portant forage crops and there is general interest in
developing transformation systems for these species.
Astragalus sinicus (Chinese milk vetch) is a winter-
growing green manure legume that has been widely
used in rice fields for fertilizing the soil in the southern
parts of China, Japan and Korea. In China alone, it
is cultivated over an area of nearly 5 million ha (Cho
et al., 1995). The plant has a symbiotic relationship
with the soil bacterium Rhizobium huakuii (Chen et
al., 1991) or R. hualuii bv. renge B3 (Murooka et
al., 1993), which results in the formation of nitrogen
fixing root nodules. Besides being used as a green
manure, A. sinicus can also be used as fodder for an-
imals and as a source of nectar for honey bees (Yasue,
1985). Besides being an important crop plant, A. sini-
cus might also become a model laboratory leguminous
plant in addition to Lotus japonicus (Stiller et al.,
1997) and Medicago truncatula (Barker et al., 1990) if
the needed genetic and transformation techniques are
developed.
Green fluorescent protein (GFP) from the biolu-
minescent jellyfish, Aequorea victoria, is a novel
genetic reporter system (Kitts et al., 1995) that can be
very effective in plants (Cho et al., 2000b). Haseloff
et al., (1997) modified the green fluorescent protein