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Plant Physiol, July 2000, Vol. 123, pp. 1069-1076
Increasing Tryptophan Synthesis in a Forage Legume
Astragalus sinicus by Expressing the Tobacco
Feedback-Insensitive Anthranilate Synthase (ASA2) Gene1
Hyeon-Je
Cho,
Jeffrey E.
Brotherton,
Hee-Sook
Song,2 and
Jack M.
Widholm*
Department of Crop Sciences, University of Illinois, Edward R. Madigan Laboratory, 1201 West Gregory, Urbana, Illinois 61801
A cDNA clone that encodes a feedback-insensitive anthranilate
synthase (AS), ASA2, isolated from a 5-methyl-tryptophan
(Trp) (5MT)-resistant tobacco cell line under the control of the
constitutive cauliflower mosaic virus 35S promoter, was introduced into
the forage legume Astragalus sinicus by
Agrobacterium rhizogenes with kanamycin selection. The
35S-ASA2 gene was expressed constitutively as
demonstrated by northern-blot hybridization analyses and the presence
of feedback-insensitive AS. Hairy root lines transformed with
35S-ASA2 grew in concentrations of up to 100 µM 5MT, whereas the controls were completely inhibited by
15 µM 5MT. Expression of the feedback-insensitive ASA2
resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of
the AS activity demonstrate the Trp feedback alterations and indicate
that the ASA2  -subunit can interact with the native A.
sinicus  -subunit to form an active enzyme. The
ASA2 transcript and high free Trp were also detected in
the leaves, stems, and roots of plants regenerated from the transformed
hairy roots. Thus, we show for the first time that ASA2
can be used to transform plants of a different species to increase the
levels of the essential amino acid Trp and impart 5MT resistance.
1
This work was supported by the Consortium for
Plant Biotechnology Research, by the Illinois Soybean Program Operating
Board, by the United Soybean Board, and by the Illinois Agricultural Experiment Station.
2
Present address: BASF Plant Science, 26 Davis Drive,
Research Triangle Park, NC 27709.
*
Corresponding author; e-mail widholm{at}uiuc.edu; fax
217-333-4777.
© 2000 American Society of Plant Physiologists
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