in Placerville, California. Library of Congress Cataloging-in-Publication Data. NATO Advanced Research Workshop on Woody Plant Biotechnology 11989.
Woody Plant Biotechnology Edited by
M. R. Ahuja Federal Research Centre for Forestry and Forest Products Institute of Forest Genetics and Forest Tree Breeding Grosshandsdorf, Germany
Plenum Press New York and London Published in cooperation with NATO Scientific Affairs Division
Proceedings of a NATO Advanced Research Workshop on Woody Plant Biotechnology, held October 15-19, 1989, in Placerville, California
Library of Congress Cataloging-in-Publication Data
NATO Advanced Research Workshop on Woody Plant Biotechnology 11989 Placerville, Calif.) Woody plant biotechnology I edited by M.R. Ahuja. p. em. -- !NATO ASI series. Series A, Life sciences ; v. 210) "Proceedings of a NATO Advanced Research Workshop on Woody Plant Biotechnology, held October 15-19, 1989, in Placerville, Calif."-T.p. verso. Includes bibliographical references and index. ISBN 0-306-44019-9 1. Forestry biotechnology--Congresses. 2. Trees--Biotechnology-Congresses. 3. Woody plants--Biotechnology--Congresses. I. AhuJa, M. R., 1933II. North Atlantic Treaty Organization. Scientific Affairs Division. III. Title. IV. Series. SD387.B55N37 1989 634.9--dc20 91-24161 CIP
ISBN 0-306-44019-9
© 1991 Plenum Press, New York A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Printed in the United States of America
TRANSFORMATION OF POPULUS - FROM SYSTEM DEVELOPMENT TO FIELD PLANTINGS
N. B. Klopfenstein, R. W. Thornburg, H. S. McNabb, Jr., R. B. Hall, E. R. Hart, Y. W. Chun, A. Kernan and N.-Q. Shi Depts. Forestry, Plant Pathology, Entomology, and Biochemistry and Biophysics Iowa State University Ames, IA 50011 USA Populus hybrids are being used for woody plant transfor~ation in attempts to increase insect and disease resistance. Refined in vitro - - L. - -X P. micropropagation systems were the initial basis for Populus alba grandidentata Michx. transformation system development (Chun~al., 198Sb). An optimum concentration of N6 -benzyladenine (1 mg/1) in combination with alpha-naphthaleneacetic acid (0.1 mg/1) was determined for regenerating adventitious shoots from leaves. An Agrobacterium tumefaciens host range study established that strain A281 (containing pTiBo542) was capable of forming galls on the Populus hybrids (Chun et al., 1988a). Kanamycin sensitivity studies determined that 10 mg/1 kanamycin sulfate inhibited adventitious shoot regeneration from leaves and inhibited root formation from explanted shoot segments (Chun et al., 1988a). This allo>ved the use of Neomycin Phosphotransferase II (NPT II) as a selectable marker gene. A binary vector plasmid, pRT45, was constructed containing 2 chimeric gene constucts: 1)Nopaline synthase (nos) promoter-NPT II-nos terminator and 2) potato Proteinase Inhibitor II (pin2) promoter-Chloramphenicol Acetyltransferase (CAT)-pin2 terminator for studying wound-inducible gene expression. The binary vector was transferred to A. tumefaciens strain A281 by triparental mating (Thornburg et al., 1987). Excised leaves of in vitro cultured poplar were co-cultured for 2 days with the Agrobacterium binary vector system. Following 10 days of preselective culture, co-cultured leaves were transferred to selective (40 mg/1 kanamycin) shoot regeneration medium. After at least 24 days, kanamycin-resistant shoots developed to a stage that allowed transfer and secondary selection on root-inducing medium containing kanamycin (20 mg/1). Root initiation was observed after 10 days. Kanamycin-resistant, rooted plantlets were subsequently adapted to soil conditions and greenhouse grown. NPT II expression in transgenic poplars was confirmed with NPT assays of leaf protein extracts. Southern hybridization was used to confirm CAT gene insertion into the hybrid poplar genome. Assays of wound-inducible CAT expression are hampered by a compound interfering with CAT assays that is wound inducible in the hybrid poplar leaves. Northern hybridization does demonstrate, however, that the wound-inducible promoter of pin2 functions in Woody Plant Biotechnology, Edited by M.R. Ahuja Plenum Press, New Yolk, 1991
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poplar by increasing levels of CAT mRNA. Attempts to further characterize wound-inducible CAT expression by Western blotting are in progress. A 4-year field test planting of transgenic poplar, Tr15, was established 28 July 1989 in accordance with guidelines set by USDA/APHIS Biotechnology Permit Unit (permit number 89-109-03). This field test is designed to determine the effects of the transgenes on poplar growth and development, as well as characterizing transgene expression in tissue developed under field conditions. REFERENCES Chun, Y. W., Klopfenstein, N. B., McNabb, H. S., Jr., and Hall, R. B., 1988a, Transformation of Populus species by an Agrobacterium binary vector system, J. Korean For. Soc., 77:199. Chun, Y. W., Klopfenstein, N. B., McNabb, H. S., Jr., and Hall, R. B., 1988b, Biotechnological applications in Populus species, J. Korean For. Soc., 77:467. Thornburg, R. W., An, G., Cleveland, T. E., Johnson, R., and Ryan, C. A., 1987, Wound-inducible expression of a potato inhibitor IT-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants, Proc. Natl. Acad. Sci. USA, 84:744.
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