1. Springer Science+Business Media, B.V.Dear Reader,We would very much appreciate receiving your suggestions and criticisms forthe Plant Molecular Biology Manual, r d Edition, They will prove to be mosthelpful during our preparations for future supplements.Would you please answer the questions listed below, and send your commentswith any further suggestions you may have, to Drs. Gilles Jonker at theabovementioned address.Thank for your assistance!Drs. Gilles JonkerPublisherPLANT MOLECULAR BIOLOGY MANUAL1. What errors have you found? (list page numbers and describe mistakes)2. What protocols do you tind to be confusing or lacking in detail? (listchapter numbers and page numbers and describe problems)3. What protocols do you feei should be replaced in future supplements withnewer (better) methods?4. What new topics or other material would you like to see incIuded in futuresupplements?Please print or type your answers in the space below, and continue overleaf.Name: Date:Address:

2. PLANT MOLECULAR BIOLOGY MANUAL 3. PLANT MOLECULARBIOLOGY MANUALSecond editionEdited bySTANTON B. GELVINPurdue University, West LafayetteIndiana, USAROBBERT A. SCHILPEROORTLeiden State University, LeidenThe NetherlandsSpringer Science+Business Media, B.V. 4. A C.I.P. Catalogue record for this book is available from the Library of Congress.ISBN 978-94-011-7654-5 ISBN 978-94-011-0511-8 (eBook)DOI 10.1007/978-94-011-0511-8Neither Kluwer Academic Publishers nor any person acting on its behalf is responsible for the use whichmight be made of the information contained herein.Printed on acid-free pa perAll Rights Reserved 1994 Springer Science+Business Media DordrechtOriginally published by Kluwer Academic Publishers in 1994No part ofthe material protected by this copyright notice may be reproduced or utilized in anyform or by any means, electronic or mechanical, inc1uding photocopying, recording, or by anyinformation storage and retrieval system, without written permis sion from the copyright owners. 5. ContentsSECTION A: In vitro methods of gene transfer to plant cells1. PEG-mediated direct gene transfer and electroporationRoland Bilang, Andreas Kloti, Martin Schrott & Ingo Potrykus2. Gene transfer to plants via particle bombardmentPaul ChristouSECTION B: Agrobacterium-mediated gene transfer to plant cellsVII1. Agrobacterium-mediated gene transfer to plant cells: cointegrate and binaryvector systemsCindy R. Walkerpeach & 1. Velten2. Specialized vectors for gene tagging and expression studiesCsaba Koncz, Norbert Martini, Laszlo Szabados, Milan Hrouda,Andreas Bachmair & leff Schell3. Agrobacterium molecular geneticsPaul 1.1. Hooykaas & Teresa Mozo4. Genetic manipulation of Agrobacterium tumefaciens strains to improvetransformation of recalcitrant plant speciesStanton B. Gelvin & Chang-Nong Liu5. Transient expression assays using GUS constructs and fluorometricdetection for analysis of T-DNA transferLinda A. Castle & Roy O. Morris6. Agrobacterium inoculation techniques for plant tissuesNancy L. Mathis & Maud A.W. HincheeSECTION C: Selectable and screen able markers for plant transformation1. Antibiotic-resistance markers for plant transformationGeert Angenon, Willy Dillen & Marc van Montagu2. Reporter genes for plantsLuis Herrera-Estrella, Patricia Le6n, Olof Olsson & Teemu H. Teeri 6. Vlll3. Opines as screenable markers for plant transformationYves Dessaux & Annik PetitSECTION D: Nucleic acid extraction from plant tissue1. Extraction of total cellular DNA from plants, algae and fungiScott O. Rogers & Arnold J. Bendich2. Isolation and characterization of nuclear scaffoldsGerald E. Hall, Jr. & Steven Spiker3. Isolation of plant mitochondria and mitochondrial nucleic acidsSally A. Mackenzie4. Isolation of chloroplasts and chloroplast DNAC.A. Price, Noureddine Hadjeb, Lee Newman & Ellen M. Reardon5. Isolation of total, poly (A) and polysomal RNA from plant tissuesKatharina Pawlowski, Reinhard Kunze, Sacco de Vries & Ton BisselingSECTION E: Transcription and translation systems1. Assay for gene expression using run-on transcription in isolated nucleiImre E. Somssich2. Preparation of an in vitro transcription system of plant origin, with methodsand templates for assessing its fidelityYuhki Yamaguchi, Fujio Mukumoto, Hidemasa Imaseki & Ken-IchiYamazakiSECTION F: Blotting and gene detection systems1. Southern, Northern and Western blot analysisJohan Memelink, Kathleen M.M. Swords, L. Andrew Staehelin &J. Harry C. Hoge2. Screening of cDNA expression libraries with synthetic oligonucleotides forDNA-binding proteinsWolfgang Werr, Barbel Oberiacker & Bettina Klinge3. Non-radioactive nucleic acid detection systemsSusan J. Karcher 7. SECTION G: In situ hybridization and immunodetection1. RNA in situ hybridization in plantsNicholas B. DuckIX2. In situ hybridization to plant metaphase chromosomes using digoxigeninlabeled nucleic acid sequencesS. Hinnisdaels, I. Farbos, J. Del-Favero, J. Veuskens, M. Jacobs &A. MourasSECTION H: Cloning and detection of DNA sequences from large DNAmolecules1. Methods for generating plant genomic librariesMarjory A. Snead, Patricia L. Kretz & Jay M. Short2. Construction of plant yeast artificial chromosome librariesGregory B. Martin3. Preparation of high molecular weight plant DNA and analysis by pulsedfield gel electrophoresisRaymond A.J.J. van Daelen & Pim Zabel4. Random amplified polymorphic DNA (RAPD) markersAntoni Rafalski, Scott Tingey & John G.K. WilliamsSECTION I: Protein-nucleic acid interaction analyses1. Gel mobility shift assayKoji Mikami, Hisabumi Takase & Masaki Iwabuchi2. Optimization of DNase I footprinting experimentsSusan J. Martino-Catt & Steve A. Kay3. Analyses of plant chromatin and in vivo protein-DNA interactionsAnna-Lisa Paul & Robert J. Fer!4. Expression and characterization of recombinant plant trans-acting factorsLee Meisel & Eric Lam 8. xSECTION J: Subcellular targeting of proteins1. In vitro import of proteins into chloroplastsBarry D. Bruce, Sharyn Perry, 10hn Froehlich & Kenneth Keegstra2. In vitro targeting of proteins to mitochondriaMarc A. Boutry, Didier Thomas & Fran90is Chaumont3. Targeting of proteins to the vacuolelames E. Dombrowski, Luis Gomez, Maarten 1. Chrispeels & NatashaV. Raikhel4. Visualizing protein import into the plant cell nucleusVitaly CitovskySECTION K: Gene tagging using transposons1. Gene tagging by endogenous transposonsWolf-Ekkehard Lonnig & Peter Huijser2. Heterologous transposon tagging as a tool for the isolation of plant genesErik A. van der Biezen, Mark 1.1. van Haaren, Bert Overduin, H. 10hn1. Nijkamp & 1 acques Hille 9. XlList of ContributorsAuthorAngenon, G.Bachmair, A.Bendich, A.J.Bilang. R.Bisseling, T.Boutry, M.A.Bruce, B.D.Castle, L.A.Chaumont, F.Chrispeels, J.Christou, P.Citovsky, V.Del-Favero, J.Dessaux, Y.De Vries, S.Chapter AddressClB201Al051211B51213A2J4G2C305Laboratorium voor Genetica, UniversiteitGent, Ledeganckstraat 35, B-9000 Gent, BelgIUmMax-Planck Institut fUr Zuchtungsforschung,Carl-von-Linne-Weg 10, 0-50829 Kaln 30,GermanyBotany Dept. KB-15, University of Washington,Seattle WA 98195, USAInstitute of Plant Sciences, Swiss Federal Instituteof Technology, ETH-Zentrum, CH-8092 Zurich, SwitzerlandDept. of Molecular Biology, Transitarium,Agricultural University Wageningen, Dreyenlaan3, 6703 HA Wageningen, The NetherlandsUnite de Biochemie Physiologique, Universityof Louvain, Place Croix du Sud 2-20, 1348Louvain-la-Neuve, BelgiumDepartment of Botany, University ofWisconsin,Madison, WI 53706, USADept. of Plant Biology, University of California,Berkeley, CA 94720, USAUnite de Biochimie Physiologique, Universityof Louvain, Place Croix du Sud, 2-20, B-1348Louvain-la-Neuve, BelgiumDepartment of Biology, University of Calif ornia- San Diego, La Jolla, CA 92093-1116,USAAgracetus Inc., Research & Development,8520 University Green, Middleton WI 53562,USADept. of Biochemistry and Cell Biology, StateUniversity of New York, Stony Brook, NY11794, USAFree University of Brussels, Institute forMolecular Biology, Paardenstraat 65, B-I640St.-Genesius-Rode, BelgiumInstitut des Sciences Vegetales, Biltiment 23C.N.R.S., Avenue de la Terrasse, 91198 Gifsur-Yvette Cedex, FranceDepartment of Molecular Biology, Agricul- 10. XliAuthor Chapter AddressDillen, W. ClDombrowski, E. 13Duck, N. GlFarbos, I. G2F erl, R.J. 13Froehlich, J. 11Gelvin, S.B. B4Gomez, L. 13Ha~eb,N. D4Hall, G.E., Jr. D2Herrera-Estrella, L. C2Hille, J. K2Hinchee, M. B6Hinnisdaels, S. G2Hoge, J.H.C. Fltural University Wageningen, NL-6703 HAWageningen, The NetherlandsLaboratorium voor Genetica, UniversiteitGent, Ledeganckstraat 35, B-9000 Gent, BelgIUmMSU-DOE Plant Research Laboratory, MichiganState University, East Lansing, MI48824-1312, USAMonsanto Company, 700 Chesterfield VillageParkway, St. Louis MO 63198, USAUniversite de Bordeaux II, Laboratoire deBiologie Cellulaire, Av. des Facultes, F-33405Talence-Cedex, FranceDept. of Horticultural Sciences, University ofFlorida, Gainesville FL 32611, USADOE Plant Research Laboratory, MichiganState University, East Lansing, MI 48824,USADept. of Biological Sciences, Purdue University,Lilly Hall of Life Sciences, West LafayetteIN 47907, USADepartment of Biology, University of California- San Diego, La Jolla, CA 92093-1116,USAWaksman Institute, Rutgers University, Piscataway,NJ 08855-0759, USADepartment of Genetics, North CarolinaState University, Raleigh, NC 27695-7614,USADepartment of Plant Genetic Engineering,CINVESTAV del I.P.N., Unidad Irapuato,Apartado Postal 629, 36500 Irapuato, Gto,MexicoDept. of Genetics, Institute for MolecularBiological Sciences, BioCentrum Amsterdam,Free University, De Boelelaan 1007,1001 HV Amsterdam, The NetherlandsCrop Transformation, Monsanto, Plant ProtectionImprovement, 700 Chesterfield VP,St. Louis MO 63198, USAFree University of Brussels, Institute for MolecularBiology, Paardenstraat 65, B-1640 St.Genesius-Rode, BelgiumInstitute of Molecular Plant Sciences, Leiden 11. AuthorHooykaas, P.J.J.Hrouda, M.Huijser, P.Imaseki, H.Iwabuchi, M.Jacobs, M.Karcher, S.J.Kay, S.A.Keegstra, K.Klinge, B.KlOti, A.Koncz, C.Kretz, P.L.Kunze, R.Lam,E.XlllChapter AddressB3B2KlE2IiG2F31211F2AlB2HID514University, Clusius Laboratory, Wassenaarseweg64, 2333 AL Leiden, The NetherlandsInstitute of Molecular Plant Sciences, ClusiusLaboratory, Leiden University, Wassenaarseweg64, 2333 AL Leiden, The NetherlandsResearch Institute for Crop Production,Drnovska 507, Prague 6, Ruzyne, 161 06Czech RepublicMax-Planck Institut fOr Zuchtungsforschung,Carl-von-Linne-Weg 10, 5000 Koln 30, GermanyResearch Institute for Biochemical Regulation,School of Agricultural Science, N agoyaUniversity, Chikusa, Nagoya 464-01, JapanKyoto University, Faculty of Science, Dept.of Botany, Ktrashirikawa, Kyoto 606-01, JapanInstitute for Molecular Biology, Free Universityof Brussels, Paardenstraat 65, St. GenesiusRode, 1640 BelgiumDept. of Biological Sciences, Purdue University,B-315 Lilly Hall, West Lafayette, IN47907-13902, USANSF Center for Biological Timing, Dept. ofBiology, Gilmer Hall, University of Virginia,Charlottesville VA 22903, USAUniversity of Wisconsin, Dept. of Botany,430 Lincoln Drive, Madison WI 53706, USAInstitut fOr Genetik, Universitat zu Koln,Weyertal 121,50931 Koln, GermanyInstitute of Plant Sciences, Swiss Federal Instituteof Technology, ETH-Zentrum, CH-8092 Zurich, SwitzerlandMax-Planck Institut fOr Zuchtungsforschung,Carl-von-Linne-Weg 10, D-50829 Koln 30,GermanyStratagene Cloning Systems, La Jolla, CA92037, USAInstitute of Genetics, Universitat zu Koln, D-50931 Koln, GermanyRutgers University, AgroBiotech Center and 12. XIVAuthor Chapter AddressLeon, P. C2Liu, C.-N. B4Lonnig, W.E. K1MacKenzie, S. D3Martin, G.B. H2Martini, N. B2Martino-Catt, S.J. 12Mathis, N.L. B6Meisel, L. 14Memelink, J. F1Mikami, K. 11Morris, R.O. B5Mouras. A. F2Mozo, T. B3Graduate Program in Microbiology, WaksmanInstitute, Piscataway NJ 08854, USAInstituto de Biotecnologia UNAM, ApartadoPostal 510-3, Cuernavaca, Morelos, MexicoDept. of Biological Sciences, Lilly Hall of LifeSciences, Purdue University, West Lafayette,IN 47907 USAMax-Planck-Institut fOr ZOchtungsforschung,Carl-von-Linne-Weg 10, 50829 Koln, GermanyDept. of Agronomy, Lilly Hall of LifeSciences, Purdue University, West Lafayette,IN 47907, USAPurdue University, Dept. of Agronomy, 1150Lilly Hall, West Lafayette IN 47907-1150,USAMax-Planck Institut fOr ZOchtungsforschung,Carl-von-Linne-Weg 10, D-50829 Koln 30,GermanyNSF Center for Biological Timing, Departmentof Biology, University of Virginia, Charlottesville,VA 22903, USACrop Transformation, Monsanto Co., PlantProtection Improvement, 700 ChesterfieldVP, St. Louis, MO 63198, USAAgBiotech Center and Graduate Program inMicrobiology, Rutgers University, WaksmanInstitute, P.O. Box 759, Piscataway, NJ08854, USAClusius Laboratory, Molecular PlantSciences Institute, Leiden University, Wassenaarseweg64, 2333 AL Leiden, TheNetherlandsDivision of Developmental Biology, NationalInstitute for Basic Biology, Okazaki 444, J apanDept. of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USAUniversite de Bordeaux II, Laboratoire deBiologie Cellulaire, Av. des FacuItes, F-33405Talence-Cedex, FranceInstitute of Molecular Plant Sciences, Clusius 13. AuthorMukumoto, F.Newman, L.Nijkamp, H.J.J.Olsson, O.Overduin, B.Paul, A.-L.Pawlowski, K.Perry, S.Petit, A.Potrykus, I.Price, CA.Rafalski, A.Raikhel, N.Reardon, E.M.xvChapter AddressE2D4K2C2K213D511C3AlD4H413D4Laboratory, Leiden University, Wassenaarseweg64, 2333 AL Leiden, The NetherlandsResearch Institute for Biochemical Regulation,School of Agricultural Science, NagoyaUniversity, Chikusa, Nagoya 464-01, JapanWaksman Institute, Rutgers University, Piscataway,NJ 08855-0759, USADepartment of Genetics, Institute for MolecularBiological Sciences, BioCentrum Amsterdam,Vrije Universiteit, De Boelelaan 1087,1081 HV Amsterdam, The NetherlandsDepartment of Forest Genetics and PlantPhysiology, Swedish University of AgriculturalSciences, S-90187 Umea, SwedenDepartment of Genetics, Institute for MolecularBiological Sciences, BioCentrum Amsterdam,Vrije Universiteit, De Boelelaan 1087,1081 HV Amsterdam, The NetherlandsDepartment of Horticultural Sciences, Universityof Florida, Gainesville, FL 32611,USADepartment of Molecular Biology, AgriculturalUniversity Wageningen, NL-6703 HAWageningen, The NetherlandsDepartment of Botany, University ofWisconsin,Madison, WI 53706, USAInstitut des Sciences Vegetales, Batiment 23,CNRS, Avenue de la Terrasse, F-91198 Gifsur-Yvette, FranceInstitute of Plant Sciences, Swiss Federal Instituteof Technology, ETH Zentrum LFVE20,8092 Zurich, SwitzerlandWaksman Institute, Rutgers State University,Piscataway NJ 08855-0759, USADuPont Co. Agricultural Products &Biotechnology, P.O. Box 80402, WilmingtonDE 19880-0402, USAMichigan State University, MSU-DOE PlantResearch Laboratory, East Lansing MI48824-1321, USAWaksman Institute, Rutgers University, Piscataway,NJ 08855-0759, USA 14. XVIAuthorRogers, S.O.Schell, J.Schrott, M.Short, J.M.Snead, M.Sommsich, I.E.Spiker, S.Staehelin, L.A.Swords, K.M.M.Szabados, L.Takase, H.Teeri, T.Thomas, D.Tingey, S.Chapter Address01B2AlHIHIEl02FlFlB211C212H4Environmental and Forest Biology, StateUniversity of New York, College of EnvironmentalScience and Forestry, Syracuse, NY13210, USAMax-Planck Institut fOr Zuchtungsforschung,Carl-von-Linne-Weg 10, 0-50829 Kaln 30,GermanyInstitute of Plant Sciences, Swiss Federal Instituteof Technology, ETH-Zentrum, CH-8092 Zurich, SwitzerlandStratagene Cloning Systems, La Jolla, CA92037, USAStrategene Cloning Systems, La Jolla CA92037, USAMax-Planck Institut fOr Zuchtungsforschung,Carl-von-Linne-Weg 10, 5000 Kaln 30, GermanyDept. of Genetics, 3530 Gardner Hall, NorthCarolina State University, Raleigh NC27695-7614, USADepartment of Molecular, Cellular, and DevelopmentalBiology, University of Colorado,Boulder, CO 80309-0347, USADepartment of Molecular, Cellular, and DevelopmentalBiology, University of Colorado,Boulder, CO 80309-0347, USAInstitute of Plant Physiology, Biological ResearchCenter of Hungarian Academy ofSciences, Temesvari krt 62, P.O. Box 521,H-6701 Szeged, HungaryDivision of Developmental Biology, NationalInstitute for Basic Biology, Okazaki 444, JapanDept. of Genetics, Institute of Biotechnology,University of Helsinki, Arkadiankatu 7,00100 Helsinki, FinlandUnite de Biochimie Physiologique, Universityof Louvain, Place Croix du Sud, 2-20, B-1348Louvain-la-Neuve, BelgiumDuPont Co. Agricultural Products, Biotechnology,P.O. Box 80402 Wilmington, DE19880-0402, USA 15. XVIIAuthor Chapter AddressOberlacker, B. F2Van Daelen, R.A.J.J. H3Van der Biezen, E.A. K2Van Haaren, M.J.J. K2Van Montagu, M. ClVelten, J. BlVeuskens, J. G2Walkerpeach, C.R. BlWerr, W. F2Williams, J.G.K. H4Yamaguchi, Y. E2Yamazaki, K. E2Zabel, P. H3Institut fUr Genetik, Universitat zu KOln,Weyertal 121, 50931 KOln, GermanyWageningen Agricultural University, Departmentfor Molecular Biology, Dreyenlaan 3,6703 HA Wageningen, The NetherlandsDepartment of Genetics, Institute for MolecularBiological Sciences, BioCentrum Amsterdam,Vrije Universiteit, De Boelelaan 1087,1081 HV Amsterdam, The NetherlandsDepartment of Genetics, Institute for MolecularBiological Sciences, BioCentrum Amsterdam,Vrije Universiteit, De Boelelaan 1087,1081 HV Amsterdam, The NetherlandsLaboratorium voor Genetica, UniversiteitGent, Ledeganckstraat 35, 9000 Gent, BelgieUSDA-ARS, New Mexico State University,Box 3GL, Las Cruces NM 88003, USAFree University of Brussels, Institute for MolecularBiology, Paardenstraat 65, B-1640 St.Genesius-Rode, BelgiumPlant Sciences, Monsanto Co., 700 ChesterfieldVillage Parkway, St. Louis, MO63198, USAInstitut fUr Genetik, Universitat zu KOln,Weyertal 121,50931, KOln, GermanyDuPont Co. Agricultural Products, Biotechnology,P.O. Box 80402 Wilmington, DE19880-0402, USAResearch Institute for Biochemical Regulation,School of Agricultural Science, NagoyaUniversity, Chikusa, Nagoya 464-01, JapanResearch Institute for Biochemical Regulation,School of Agricultural Sciences, NagoyaUniversity, Chikusa, Nagoya, 46401, JapanDept. of Molecular Biology, AgriculturalUniversity Wageningen, Dreyenlaan 3, 6703HA Wageningen, The Netherlands 16. XIXPrefaceFive years ago, the first edition of the Plant Molecular Biology Manualappeared. At that time, the editors felt that the field of plant molecular biologyhad matured to a point that the publication of a series of protocols in plantmolecular biology was warranted. During the past five years, the field of plantmolecular biology has expanded rapidly. This expansion is, among otherthings, reflected by the presence of several journals in the plant sciences, as wellas by the increasing amount of plant sciences articles that are published in themore general journals. In 1991 approximately 3000 people attended the ThirdInternational Congress of Plant Molecular Biology in Tucson, Arizona, wheremore than 2000 posters were presented. It is also remarkable to see thatnowadays botanical and physiological meetings pay a considerable amount ofattention to plant molecular biology.Since the first edition of this manual appeared, we have published, yearly,a series of supplements to the original volume. These supplements covered newsubjects and described new methods that had been developed. With time,however, the editors realized that the original manual plus supplements hadbecome cumbersome to use, and we decided to publish a reorganized versionof the manual. This newly organized edition eliminates much of the dupli~ationof procedures found previously, and incorporates new techniques (such asspecialized transformation vectors, particle bombardment, nuclear scaffolds,in vitro transcription systems, non-radioactive detection systems, in situ hybridization,Y AC library construction, protein-nucleic interaction assays, andtransposon tagging). We again plan to publish supplements to this secondedition, to keep the work up-to-date. We hope that this manual will continueto help researchers and students in the field of plant molecular biology byclearly describing up-to-date techniques. We welcome suggestions for supplementarychapters.As before, the editors thank the authors for the speed with which theycontributed their chapters. Special thanks go to Ms. Janet Hollister (PurdueUniversity) for her secretarial assistance. 17. Plant Molecular Biology Manual AI: 1-16, 1994. 1994 Kluwer Academic Publishers. Printed in Belgium.PEG-mediated direct gene transfer and electroporationROLAND BILANG, ANDREAS KLOTI, MARTIN SCHROTT andINGO POTRYKUSInstitute of Plant Sciences. Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092Zurich, SwitzerlandIntroductiona) Transformation of protoplastsFor many years of genetic manipulation in plants, direct uptake of naked DNAby plant protoplasts has been the sole alternative to Agrobacterium tumefaciensmediatedgene transfer. The first experiments demonstrating direct gene transferincluded the delivery of isolated plasmid DNA to protoplasts of petunia andtobacco in the presence of poly-L-ornithine or polyethylene glycol (PEG)[1-4]. During the following years, protoplast transformation mediated by PEG[5] or electroporation [6] was substantially simplified and their efficiency inmodel systems was increased by several orders of magnitude (reviewed byPaszkowski et al. [7]).The production of transgenic plants via direct gene transfer to protoplastsdepends on protoplast-to-plant regeneration and on efficient selection systemsfor transgenic clones. Early gene transfer experiments focused on protoplastsof Solanaceae species that are easily regenerable, and on the use of the bacterialgene for neomycin phosphotransferase (npt II), conferring antibiotic resistanceto transformed clones (Table 1). During the past few years, protoplast-to-plantregeneration was achieved for many other plant species. Transgenic plants ofthe model plant Arabidopsis thaliana, of important crops such as J aponica andIndica rice varieties, maize, and forage grasses have been obtained (Table 1).Natural resistance of many monocotyledonous species to the antibiotic kanamycin[8,9] made the development of other selection systems necessary. Inaddition to the npt II gene, the genes for hygromycin-phosphotransferase (hpt)[ 10] and phosphinotricin-acetyltransferase (pat) [11] have proven useful forthe selection of stably transformed colonies in mono- and dicotyledonousspecies (Table 1). Other selectable markers in use are streptomycin-phosphotransferase[12], a mutant acetolactate synthase from Arabidopsis thalianaconferring resistance to sulfonylurea herbicides [13], and a mutant dihydrofolatereductase, conferring resistance to methotrexate [9].PEG- and electroporation-mediated gene transfer is simple and efficient:dozens of protoplast samples can be treated in a single experiment, andthousands of individual transgenic plants can be obtained in model systemsPMAN-AI/I 18. Table I. Stable transformation of plants via DNA-mediated direct gene transfer to protoplastsYear Plant species Trans- Type of Selectable marker Referenceformation transgenics gene 2/technique' Selecting agent1984 Nicotiana tabacum C Fertile plants npt II/kanamycin [4]1985 Lotium multiflorum C Callus npt II/G-418 [8]1985 Triticum monococcum C Callus npt II/kanamycin [44]1986 Brassica campestris C Callus npt II/kanamycin [45]1987 Petunia hybrida C Plants npt II/kanamycin [46]1987 Brassica napus E Callus npt II/kanamycin, [47]paromomycin1988 Panicum maximum E Callus dlifr/methotrexate [9]1988 Oryza sativa (Japonica) E Fertile plants hpt/hygromycin [48]1988 Dactytis glomerata C,E Plants hpt/hygromycin [49]1989 Solanum tuberosum E Fertile Plants npt II/paromomycin [50]hpt/hygromycinals/chlorosulfuron1989 Arabidopsis thaliana C Fertile plants hpt/hygromycin [51, [52]1990 Oryza sativa (Indica) C Fertile plants hpt/hygromycin [53]1992 Festuca arundinacea C Fertile plants hpt/hygromycin [54]pat/phosphinotricin1993 Zea mays C Fertile plants nptll/kanamycin [55]pat/phosphinotricin'Transformation of protoplasts was performed by (E) electroporation or by (C) chemicalmethods, i.e. treatment with PEG. 2 npt II, neomycin phosphotransferase gene; hpt, hygromycinphosphotransferase gene (both from Escherichia coli); pat, phosphinotricin acetyltransferase gene(Streptomyces ssp.); dhfr, dihydrofolate reductase gene (Mus musculus); als, mutant acetolactatesynthase (Arabidopsis thaliana).with tobacco. Manipulation of nucleic acids prior to transformation is possible,and there are no host-range limitations. These advantages allowed the developmentof a number of transient and integrative gene expression assays, whichare important tools for the investigation of the regulatory mechanisms of geneexpression.Among the most important parameters that affect the efficiency of PEGmediatedgene transfer to Nicotiana protoplasts are the concentration ofmagnesium or calcium ions in the incubation mixture, the presence of inertcarrier DNA, and the molecular weight and concentration of PEG [5]. Thephysical configuration of nucleic acids has an impact on gene transfer efficiency:linearized double-stranded plasmid DNA molecules are more efficiently expressedand integrated into the genome than are supercoiled forms [e.g. 5, 14].After delivery to protoplasts, single-stranded DNA molecules were efficientlyused as templates for in vivo duplex formation followed by genomic integration[15, 16]. mRNA molecules transferred to electroporated protoplasts of dicotyledonousand monocotyledonous species were efficiently translated [17].Multiple copy integration of the foreign DNA and rearrangements of thePMAN-Al/2 19. original sequences are observed frequently [7]. As with other transformationtechniques, integration of foreign DNA into the nuclear genome occurs predominantlyat random sites; frequencies of site-directed integration eventsobtained in tobacco and Arabidopsis mesophyll protoplasts ranged from 10 - 4to 10 - 5 [18, 19]. In contrast, PEG-mediated stable transformation of tobaccochloroplasts [20, 21] led to integration of the foreign DNA predominantly athomologous sites within the plastome.b) Transformation of intact cells by efectroporationElectroporation has been used for a long time for transient and integrativetransformation of protoplasts [22, 6] instead of or in addition to PEG-treatment(see Table 1 for examples). Only recently, electroporation conditionshave been found that deliver DNA molecules into intact plant cells stillsurrounded by a cell wall [23-28]. In most reported cases, transformability ofintact plant cells or plant tissues depends on pretreatment of the cells or tissuesto be transformed, either by mechanical wounding or by treating the cells ortissue with hypertonic or enzyme-containing solutions. D'Halluin et af. [26]regenerated transgenic plants after electroporating either maize immatureembryos briefly preincubated in a 0.3 % macerozyme solution or embryogenicmaize callus wounded mechanically by cutting. However, certain cells arecompetent for DNA-uptake by electroporation without any pretreatment, forexample cells of maize, rice and wheat immature embryos [27, 28]. Besideswounding, several relevant parameters to tissue transformation by electro porationhave been found: electric field strength, capacity, ion nature and concentrationin the electroporation buffer, preincubation time of wounded tissue inelectroporation buffer to minimize damage by released nucleases, coincubationtime with the plasmid DNA and heat shock treatment before electroporation,and orientation of the tissue in the electroporationchamber [25, 27-29].The reasons for cell competence for DNA-uptake by electroporation are stillunknown [30]. Compared to particle bombardment, the range of tissues thatcan be transformed by electroporation seems to be narrower. For tissues thatare susceptible to DNA-uptake by electroporation, this method is a simple, fastand inexpensive way for transient and stable transformation in differentiatedtissues.Proceduresa) Transient and stable transformation of tobaccoEstablishment of a sterile shoot culture of Nicotiana tabacum L.The example given is for protoplasts from N. tabacum cv. Petit Havana SR 1,a widely used genotype [31].PMAN-Al/3 20. Steps in the procedure1. Surface-sterilize tobacco seeds in a hypochlorite solution (1.4% w/vCa(CIO)2' 0.05% w/v Tween 80) for 15 min.2. Rinse 4 times in sterile distilled water.3. Plate for germination on half-strength MS medium solidified with 0.8%agar. For storage, sterilized seeds are dried in a sterile bench air flow.4. Cut shoots with 2 to 3 leaves and culture in glass jars containing MSmedium solidified with 0.8% agar. Good aeration is provided by a holein the lid of the glass jar, plugged with a Cepharen stopper.5. Culture under a 16 h photoperiod (20 ~E/m2 s; e.g. Osram L36W/21 Lumilux white tubes) at 25 C.6. Rooted shoots are subcultured at 6 weeks interval as stem cuttingsseveral times before use.Medium:- MS medium [32] (Table 2).Isolation of mesophyl/ protoplasts of Nicotiana tabacumProtoplasts are isolated following the modified protocol of Nagy and Maliga[33, 34]. For other systems, growth conditions of donor plants and/orparameters of the protoplast isolation procedure might require modifications.Steps in the procedure1. Take three fully expanded leaves 1 of a shoot culture under sterile conditionsand put them in a 9 cm petri dish. Wet the leaves thoroughly withenzyme solution and remove the mid-ribs. Cut the leaf halves into 2 to3 pieces and wound the upper epidermis with parallel cuts. Put the leafpieces bottom side down into two 9 cm Petri dishes containing 10 mlof enzyme solution each. Seal the dishes with Parafilm and incubateover-night (e.g. 14 h) at 26 C in the dark without shaking.2. Gently agitate the dishes after over-night treatment and incubate theleaves for another 30 min. Take up the protoplast suspension with a10 ml pipette with a broken-off tip and pour through a 1 00 ~m stainlesssteel mesh sieve. Add 5 ml of K4 medium to each dish and disruptremaining tissue by carefully pumping it up and down the pipette. Sievethis suspension, too.3. Agitate the protoplast suspension gently and distribute into 4 capped12 ml centrifuge tubes. Carefully overlay the suspension with 1 ml of W5PMAN-Alf4 21. solution. Centrifuge for 10 min at 80 X g. Good protoplasts will float atthe interphase.4. Collect the protoplasts with a 2 ml pipette, taking as little as possibleof the lower phase. Put the protoplasts of two tubes together into a newone.5. Gradually add 10 ml of W5 solution and resuspend the protoplasts bygentle shaking. Pellet the protoplasts (centrifuge 70 X g, 5 min). Removethe supernatant solution. Repeat this step.6. Resuspend protoplasts in a total volume of 5 ml W5 solution (the densitywill be approx. 106 protoplasts/ml) and store them for at least 30 minin a sterile Erlenmeyer flask at 4 0 C in the dark.7. Shake the suspension carefully, take 100 III and dilute in 900 III of W5solution. Count the protoplasts in a 10 Ill-hematocytometer (sporecounter, Thoma chamber).Note1. Three fully expanded tobacco leaves yield between 5 and 10 million mesophyll protoplasts.SolutionsMedium K4: K3 medium [33] (Table 2) with 0.4 M instead of 0.3 Msucrose.Enzyme solution: 1.2% w/v Cellulase 'Onozuka' R 10,0.4% w/v MacerozymeR 1 0 in K4 medium, filter sterilized.W5 solution: 154 mM NaCI, 125 mM CaCI2 , 5 mM KCI, 5 mM glucose;pH 5.8-6.0; autoclaved.PEG-mediated direct gene transfer to protoplastsThe direct gene transfer method is based on the work of Negrutiu [5]. Duringthe past years of intensive use of this protocol in our laboratory, severalsimplifications could be introduced without loss of integrative or transienttransformation efficiency in tobacco [e.g. 35, 36].Steps in the procedure1. Pellet the protoplasts (centrifuge 70 X g, 5 min), remove the supernatantsolution and resuspend the protoplasts in MMM solution to a density of2 106 protoplasts/ml. 5 105 protoplasts are needed per sample.2. Distribute aliquots of 250 III of the protoplast suspension (i.e. 5 105pps.) into 12 ml tubes, using a clipped blue tip. Add 20 III of plasmidPMAN-Al/5 22. DNA, mix by shaking. Add 250 III of PEG solution; pipet slowly becauseof the high viscosity. Shake several seconds. 13. Incubate 5 min, shake several times. Then gradually add 10 ml of W5solution. Pellet protoplasts (centrifuge 70 X g, 5 min).4. For transient gene expression experiments, remove the supernatantsolution and add 2.5 ml of K3 medium. Incubate the protoplasts for geneexpression (26C, dark, 24 h). To assay ,B-glucuronidase (GUS) activity,proceed as described in the next protocol.5. For stable transformation experiments, resuspend the protoplasts in0.5 ml of K3H medium and proceed as described in the correspondingprotocol.Note1. Sterilize plasmid DNA by precipitation and washing in 70% ethanol. Dry in sterile airflow and add H2 0 to a final concentration of 1 fl9/fll. Check the concentration withspectrophotometer measurement and on an agarose gel. The physical structure of theDNA should be super-coiled for transient and linear for stable transformation. For stabletransformation, inert carrier DNA from calf thymus, sheared to an average size of 5 to10 kb, is added to the DNA mixture to a final concentration of 2 fl9/fll.Take care neither to store the protoplasts in MMM solution for a prolonged time, norto leave a long interval between the addition of the DNA and PEG solutions to theprotoplasts. The time of the PEG-incubation is not crucial, but make sure to treat allthe samples the same way.SolutionsMMM solution: 15 mM MgCI2 , 0.1 % w/v 2[N-morpholino]ethanesulfonicacid (MESl. 0.5 mM mannitol; pH 5.8; autoclaved.PEG solution: 40% w/v PEG 4000 (Merck) in 0.4 M mannitol, 0.1 MCa(N03 )2; pH 8-9 with KOH; autoclaved. PEG is dissolved in 0.4 Mmannitol, 0.1 M Ca(N03 )2 (i.e. the final concentration of these twocomponents will be lower due to the volume of PEG). The pH takesseveral hours (e.g. overnight) to stabilize in this solution and will drop toa physiologic level (5 to 6) after autoclaving.K3 medium: [33] (Table 2).K3H: 1: 1 mixture of K3 and H [34]; modified from 5p medium [37](Table 2).PMAN-Al/6 23. Protoplast extraction, assay for transient GUS activityThe GUS-assay was described by Jefferson [38].Steps in the procedure1. Add 8 ml of W5 solution to the protoplast suspension, mix gently andpellet the protoplasts (5 min, 80 X g). Remove the supernatant solution,leave ca. 1 ml in the tube, resuspend the protoplasts. Repeat this step.2. Transfer the suspension into a 1.5 ml Eppendorf tube, pellet the protoplasts(centrifuge full speed, 30 s). Remove the supernatant solutioncompletely. Add 100 III of extraction buffer, vortex briefly.3. Shock-freeze the protoplasts in liquid N2 and vortex while thawing todisrupt the cells. Check the disruption under the microscope. Pellet celldebris (centrifuge full speed, 30 s). The supernatant solution (i.e. extract)can be stored at -70C or at +4 C, but not at -20C.4. To measure the protein concentration in the extract, take a 200-folddiluted sample and stain with Coomassie blue according to Bradford [39].5. For the GUS-assay, add 50 III of extract to 500 III freshly prepared,pre-warmed assay buffer. Incubate this assay mix at 37C in the darkfor several minutes up to days.6. To stop the assay, take a 100 III sample of the assay mix, add 900 IIIstop buffer and mix. Stopped samples can be stored at 4 C in the dark.7. Check the fluorescence of the stopped samples under UV-light. Measurethe fluorescence on a fluorimeter (365 nm excitation, 445 nm emissionwavelength). Use 10 nM to 100 llM 4-MU (4-methyl-umbelliferone) asstandards. Calculate GUS-activity to [nM 4-MU/min mg protein].SolutionsExtraction buffer: 50 mM Na2HP04 (pH 7), 10 mM Na2EDTA (pH 8)'0.1% w/v N-Iauroyl-sarcosyl, 0.1% v/v Triton X-100 (Sigma),0.07% v/v fJ-mercapto-ethanol.Assay buffer: 1 mM 4-methyl-umbelliferyl-glucuronide (MUG) in extractionbuffer.Stop buffer: 0.2 mM Na2C03 .PMAN-Al/7 24. "1:1 Table 2. The composition of the media used~ c> Z, Media component A H K3 MS MS-> morpho -Q-C-Macroelements (mg/I final concentration):KN03 [Merck] 1010 1900 2500 1900 1900NH4 N03 [Merck] 800 600 250 1650 1650CaCI2 X 2H2 O [Merck] 440 600 900 440 730MgS04 X 7H2 O [Merck] 740 300 250 370 370(NH4 )2S04 [Merck] 250KH2P04 [Merck] 136 170 170 170NaH2P04 X H2 O [Merck] 150(NH4 )Succinate [ICN] 50CaHP04 [Sigma] 50Micro elements (mg/I final concentration):Na2 EDTA [Fluka] 37.3 37.3 37.3 37.3 37.3FeS04 X 7H2 O [Merck] 27.8 27.8 27.8 27.8 27.8H3B03 [Merck] 3.0 3.0 3.0 6.2 6.2KI [Merck] 0.75 0.75 0.75 0.83 0.83MnS04 X H2 O [Merck] 10.0 10.0 10.0 16.9 16.9ZnS04 X 7H2 O [Merck] 2.0 2.0 2.0 8.6 8.6CuS04 X 5H2 O [Merck] 0.025 0.025 0.025 0.025 0.025Na2Mo04 X 2H2 O [Merck] 0.25 0.25 0.25 0.25 0.25CoCI2 X 6H2 O [Merck] 0.025 0.025 0.025 0.025 0.025Carbohydrates (g/I final concentration):D( + ) Sucrose [Roth] 30 0.125 102.69 10 30D(+)Glucose X lH2 0 [Sigma] 68.40D-Mannitol [Sigma] 50 0.125D-Sorbitol [Merck] 0.125D-Celiobiose [Serva] 0.125D ( - ) Fructose [Sigma] 0.125 25. o( +) Mannose [Merck] 0.125"'Cl L( +) Rhamnose [Fluka] 0.125 =: o(-)Ribose [Fluka] 0.125 > Z o(+)Xylose [Fluka] 0.125 0.25t myo-Inositol [Merck] 0.1 0.1 0.1 0.1 0.1--C- Hormone (mg/I final concentration):2,4-D (2,4-Dichlorophenoxyacetic acid) [Serva] 0.1 0.1NAA (1-naphthylacetic acid) [Sigma] 0.1 1.0 1.0 0.1BAP (6-benzylaminopurine) [Sigma] 1.0 0.2 0.2 1.0Vitamins (mg/I final concentration):Pyridoxine HCI [Merck] 1.0 1.0 1.0 0.5 0.1Thiamine HCI [Merck] 10.0 10.0 10.0 0.1 0.1Nicotinamide [BRL] 1.0Nicotinic acid [Merck] 1.0 1.0 0.5 0.1Folic acid [Merck] 0.2o-Ca-Pantothenate [Merck] 0.5 1.0p-Aminobenzoic acid [Sigma] 0.01Choline chloride [Sigma] 0.5Riboflavin [Sigma] 0.1L( + )Ascorbic acid [Merck] 1.0Vitamin A [Serva] 0.005Vitamin D3 [Merck] 0.005Vitamin B12 [Sigma] 0.01o-Biotin 0.005Organic acids (mg/I final concentration):Sodium pyruvate [Sigma] 5Citric acid [Sigma] 10Malic acid [Sigma] 10Fumaric acid [Fluka] 10Other organics (mg/I final concentration):Glycine [Serva] 2.0Casein hydrolysate [Fluka] 250 26. Selection of stable transformants and plant regenerationPlants are regenerated from mesophyll protoplasts using a method modifiedfrom Potrykus and Shill ito [34].Steps in the procedure1. Place 0.5 ml of the protoplast suspension (i.e. approx. 5' 105 protoplasts)in a 6 cm Falcon petri dish and add 4.5 ml of pre-warmed(40-45 C) K3H medium containing 0.6% SeaPlaque agarose. 1 Mixgently and allow to set.2. Seal the dishes with parafilm and culture the protoplasts for 24 h indarkness at 24 C followed by 6 d in continuous dim light.3. Cut the agarose containing the protoplasts into quadrants and placethese in 50 ml of A medium containing the appropriate antibiotics orherbicides for selection of stably transformed clones. 2 The culture vesselsshould have a diameter of approx. 10 cm. Incubate on a shaker with80 rpm at 24 C in continuous dim light.4. After 5 to 6 weeks, when the resistant colonies are 2 to 3 mm indiameter, they are transferred onto MS morpho medium in glass jars andkept at 24 C in 16 hid light. Normal looking shoots will spontaneouslygrow out from the protoplast-derived calli during the next 1 to 2 weeksof culture. When they reach a size of 3 to 5 cm, they can be cut off andtransferred onto MS medium, where roots will form in 1 to 3 weeks.5. Plantlets with an established root system are treated as shoot cultures(see first protocol). Alternatively, they can be transferred to soil oncethey have an established root system: the agar is gently washed awayand the plantlets potted. They require a humid atmosphere for the firstweek and can then be hardened off and grown under normal greenhouseconditions.Notes1. SeaPlaque agarose (FMC Corp., Rockland, ME) is autoclaved dry, K3 medium is addedand the agarose molten. After cooling to 45C, H medium is added.2. Selection in the agarose bead type culture system [40] has been found to be superiorto selection in other culture systems tested. This way, a nearly constant selectionpressure is maintained during the first four weeks of culture, thus suppressing anypossibility of background colonies arising due to reduced selection pressure because ofdecay of the drug. Some examples of selection schemes used in our laboratory to selectstably transformed tobacco clones: 50 mg/I kanamycin sulfate; 5 mg/I paromomycin;12 to 100 mg/I hygromycin; 20 to 100 mg/I phosphinothricin. Resistant colonies are,depending on the selection protocol, first seen 2 to 4 weeks after the start of selection.PMAN-Al/10 27. SolutionsA medium: [41] (Table 2).MS medium: [32] (Table 2).MS morpho medium: [42] (Table 2).b) Electroporation-mediated gene transfer to intact cellsThe following protocol describes gene transfer by electroporation to intactscutellum cells of wheat immature embryos (i.e. 8 to 12 days post anthesis),as determined by transient expression of fJ-glucuronidase or anthocyaninregulatory proteins [28]. We have constructed a special electroporationchamber (Fig. 1 A) in which the embryos could easily be fixed and orientated.A gene pulser apparatus with capacitance extender from Bio-Rad (Richmond,CAl was used. The electric pulses had an exponential decay waveform.Steps in the procedure1. Surface sterilize wheat inflorescences by immersing them in 70%ethanol for 5 min.2. Isolate the caryopses and place them in an empty petri dish.3. Excise the immature embryos with two needles and place them with thescutellar surface uppermost on eMS-plates containing 6% sucrose.Always keep the petri dish with the isolated embryos closed to preventthem from drying out.4. Prepare the agarose supports: place a 22 X 60 mm-Thermanox coverslip(Nunc Inc., Naperville, IL) on a ceramic plate. Above this slide forma tunnel with three microscope slides. Boil the medium and add 1 mlof this medium into the tunnel to form a 1 mm thick agar layer (Fig. 1 B).Put a filter paper into the cover of a 9 cm petri dish, wet it with 1 mlH2 0 and put a microscope slide onto it. Transfer the Thermanoxcoverslip with the polymerized agarose onto the microscope slide. Withthe top of a 1.5 ml-Eppendorf tube, cut disks of 9 mm in diameter(Fig. 1C).5. Place ten embryos on an agarose disk, add 1 ~I of alginate and movethe embryos into this drop; the scutellar surface must not get coveredby the alginate (Fig. 10). Transfer the agarose supports with theembryos onto eMS-plates with 6% sucrose. Open the cover of the petridish for 30 min to dry the embryos.PMAN-Aljll 28. +, , A pc*- anbuchca1----1 10mmtcrna BmecpH 10mmDpe81agepH 10mm 1 10mm- --ag E- "---- em1----l10mmFig. I. Electroporation of wheat embryos. A) Sectional elevation of the electroporation chamber.pc, plexiglass cover; an, anode; ch, chamber; bu, electroporation buffer; ca, cathode. B) Preparationof a I mm thick agarose layer. tc, Thermanox coverslip; ms, microscope slides; me,medium; cp, ceramic plate. C) Cutting agarose supports of 9 mm in diameter with the top of anEppendorftube. pe, petri dish; sl, Thermanox coverslip on microscope slide; ag, polymerizedagarose; ep, 1.5 ml-Eppendorf tube. D) 10 embryos mounted with 4% alginate on an agarosesupport. em, embryos; ag, polymerized agarose; ai, alginate. The scutella must not get coveredby the alginate. E) Sectional elevation of the electroporation chamber with fixed and orientatedembryos. ag, polymerized agarose; em, embryos.6. Sterilize the components of the electroporation chamber by rinsing with70% ethanol and mount the chamber. Make sure that the electrodesare connected correctly (during the delivery of the pulse, the negativelyPMAN-Al/12 29. charged DNA molecules move towards the anode, therefore the scutellahave to face the cathode).7. Fill the electroporation chamber with 140 J.l1 of electroporation-buffercontaining the plasmid DNA (50 J.lg/ml).8. Place one agarose support with 10 fixed embryos upside down ontothe electroporation chamber in a way that the embryos are immersedin the buffer (Fig. 1 E). Make sure there are no air bubbles in thechamber.9. Place the anode onto the chamber setup.10. Immediately deliver one electric pulse of 275 V/cm (0.11 kV on thereading) from the 960 J.lF-capacitor. With this setup the time of deliveryshould be in the range of 150 to 200 ms.11. Remove the agarose support with the scalpel and wash it for 1 min in10 ml of washing solution in a 9 cm-petri dish.For electroporation of the next sample add 10 to 20 J.l1 of electroporationbuffer containing plasmid DNA to the remaining buffer in thechamber, remove all the small air bubbles in the chamber with thepipette and transfer the next agarose support with the fixed embryosto the chamber.12. Carefully transfer the agarose support with the embryos to an eMSculture plate containing 3% sucrose.13. Seal the petri dish with parafilm and incubate at 26 0 C for transientgene expression.Solutionselectroporation buffer: 35 mM potassium aspartate, 35 mM potassiumglutamate, 5 mM calcium gluconate, 5 mM 2[N-morpholino]ethanesulfonicacid (MES) and 0.4 mM mannitol, pH 5.8 [29]; filter sterilized.eMS-culture plates: MS medium [32] supplemented with 500 mg/I glutamine,100 mg/I casein hydrolysate, 2.0 mg/12,4-D [43],0.8% agaroseType I and 6% or 3% sucrose, respectively.eMS-agarose supports: MS medium supplemented with 500 mg/I glUtamine,100 mg/I casein hydrolysate, 2.0 mg/I 2,4-D, 6 mM CaCI2 , 3%SeaPlaque agarose (FMC Bioproducts, Rockland ME) and 6% sucrose.alginate: 4% alginic acid, 6% sucrose, pH 5.6.washing solution: MS medium supplemented with 500 mg/I glutamine,100 mg/I casein hydrolysate, 2.0 mg/I 2,4-D, 3% sucrose.PMAN-Al/13 30. References1. Davey MR, Cocking EC, Freeman J, Pearce N, Tudor I (1980) Transformation of Petuniaprotoplasts by isolated Agrobacterium plasmids. Plant Sci Lett 18: 307-313.2. Draper J, Davey MR, Freeman JP, Cocking EC, Cox BG (1982) Ti plasmid homologoussequences present in tissue from Agrobacterium plasmid transformed Petunia protoplasts.Plant Cell Physiol 23: 451-458.3. Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation ofplant protoplasts with Ti-plasmid DNA. Nature 296: 72-74.4. Paszkowski J, Shillito RD, Saul MW, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Directgene transfer to plants. EMBO J 3: 2717-2722.5. Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis oftransformation conditions I. Setting up a simple method for direct gene transfer in plantprotoplasts. Plant Mol Bioi 8: 363-373.6. Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I (1985) High efficiency directgene transfer to plants. Bio/technology 3: 1099-1103.7. Paszkowski J, Saul MW, Potrykus I (1989) Plant gene vectors and genetic transformation:DNA-mediated direct gene transfer to plants. Cell Culture and Somatic Cell Genetics ofPlants 6: 51-68.8. Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer tocells of a graminaceous monocot. Mol Gen Genet 199: 178-182.9. Hauptmann RM, Vasil V, Ozias-Atkins P, Tabaeizadeh Z, Rogers SG, Fraley RT, HorschRB, Vasil IK (1988) Evaluation of selectable markers for obtaining stable transformants inthe Gramineae. Plant Physiol 86: 602-606.10. Gritz L and Davies J (1983) Plasmid-encoded hygromycin-B-resistance: The sequence ofhygromycin-B-phosphotransferase gene and its expression in Escherichia coli and Saccharomycescerevisiae. Gene 25: 179-188.11. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Motterman J(1987) Characterization of the herbicide gene bar from Streptomyces hygroscopicus. EMBOJ 6: 2519-2523.12. Jones JDG, Svab Z, Harper EC, Hurwitz CD, Maliga P (1987) A dominant streptomycinresistance marker for plant cell transformation. Mol Gen Genet 210: 86-91.13. Haughn WG, Smith J, Mazur B, Somerville C (1988) Transformation with a mutantArabidopsis acetol act ate synthase gene renders tobacco resistant to sulfonylurea herbicides.Mol Gen Genet 211: 266-271.14. Ballas N, Zakai N, Friedberg D, Loyter A (1988) Linear forms of plasmid DNA are superiorto supercoiled structures as active templates for gene expression in plant protoplasts. PlantMol Bioi 11: 517-527.15. Rodenburg KW, De Groot MJA, Schilperoort RA, Hooykaas PJJ (1989) Single-strandedDNA used as an efficient new vehicle for transformation of plant protoplasts. Plant Mol Bioi13: 711-719.16. Furner 11, Higgins ES, Berrington AW (1989) Single-stranded DNA transforms plant protoplasts.Mol Gen Genet 220: 65-68.17. Gallie DR, Lucas WJ, Walbot V (1989) Visualizing mRNA expression in plant protoplasts:Factors influencing efficient mRN A uptake and translation. Plant Cell 1: 301-311.18. Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021-4026.19. Halfter U, Morris P-C, Willmitzer L (1992) Gene targeting in Arabidopsis thaliana. Mol GenGenet 231: 186-193.20. Golds T, Maliga P, Koop H-U (1993) Stable plastid transformation in PEG-treated protoplastsof Nicotiana tabacum. Bio/technology 11: 95-97.21. O'Neill C, Horvath GV, Horvath E, Dix PJ, Medgeysy P (1993) Chloroplast transformationin plants: Polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolisticdelivery systems. Plant J 3: 729-738.PMAN-Al/14 31. 22. Fromm ME, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot anddicot plant cells by electroporation. Proc Natl Acad Sci USA 82: 5824-5828.23. Morikawa H, Iida A, Matsui C, Ikegami M, Yamada Y (1986) Gene transfer into intact plantcells by electroinjection through cell walls and membranes. Gene 41: 121-124.24. Lindsey K, Jones MGK (1987) Transient gene expression in electroporated protoplasts andintact cells of sugar beet. Plant Mol BioI 10: 43-52.25. Dekeyser RA, Claes B, De Rycke RMU, Habets ME, Van Montagu MC, Caplan AB (1990).Evaluation of selectable markers for rice transformation. Plant Cell 2: 591-602.26. D'Halluin K, Bonne E, Bossut M, De Beuckeleer M, Leemans J (1992) Transgenic maizeplants by tissue electroporation. Plant Cell 4: 1495-1505.27. Songstad DD, Halaka FG, DeBoer DL, Armstrong CL, Hinchee MAW, Ford-Santino CG,Brown SM, Fromm ME, Horsch RB (1993) Transient expression of GUS and anthocyaninconstructs in intact maize immature embryos following electroporation. Plant Cell TissOrgan Cult 33: 195-201.28. Kloti A, Iglesias VA, Wiinn J, Burkhardt PK, Datta SK, Potrykus I (1993) Gene transferby electroporation into intact scutellum cells of wheat embryos. Plant Cell Rep 12: 671-675.29. Tada Y, Sakamoto M, Fujimura T ( 1990) Efficient gene introduction into rice by electroporationand analysis of transgenic plants: Use of electroporation buffer lacking chloride ions.Theor Appl Genet 80: 475-480.30. Potrykus I (1990) Gene transfer to cereals: An assessment. Bio/technology 8: 535-542.31. Maliga P, Breznovitz A, Marton L (1973) Streptomycin resistant plants from callus culturesof tobacco. Nature New BioI 244: 29-30.32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobaccotissue cultures. Physiol Plant 15: 473-497.33. Nagy 11, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplastsof Nicotiana sylvestris. Z Pftanzenphysiol 78: 453-455.34. Potrykus I, Shillito RD (1986) Protoplasts: Isolation, culture, plant regeneration. MethEnzymol 118: 549-578.35. Bilang R, Iida S, Peterhans A, Potrykus I, Paszkowski J (1991) The 3' -terminal region ofthe hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotianatabacum. Gene 100: 247-250.36. Bilang R, Peterhans A, Bogucki A, Paszkowski J (1992) Single-stranded DNA as a recombinationsubstrate in plants as assessed by stable and transient recombination assays. Mol CellBioI 12: 329-336.37. Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cellsat very low population density in liquid medium. Plant a 126: 105-110.38. Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. PlantMol BioI Rep 5: 387-405.39. Bradford MM (1976) A rapid and sensitive method for the quantification of microgramquantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248-254.40. Shillito RD, Paszkowski J, Potrykus I (1983) Agarose plating and a bead-type culturetechnique enable and stimulate development of protoplast-derived colonies in a number ofplant species. Plant Cell Rep 2: 244-247.41. Caboche M (1980) Nutritional requirements of protoplast-derived haploid tobacco cellsgrown at low densities in liquid medium. Plant a 149: 7-18.42. Spangenberg G, Osusky M, Oliveira MM, Freydl E, Nagel J, Pais MS, Potrykus I (1990)Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: Morphological,genetic, and molecular characterization. Theor Appl Genet 80: 577-587.43. Vasil V, Redway F, Vasil IK (1990) Regeneration of plants from embryogenic suspensionculture protoplasts of wheat (Triticum aestivum L.) Bio/technology 8: 429-433.44. Lorz H, Baker B, Schell J (1985) Gene transfer to cereal cells mediated by protoplasttransformation. Mol Gen Genet 199: 178-182.45. PaszkowskiJ, Pisan B, Shillito RD, Hohn T, Hohn B, Potrykus I (1986 ) Genetic transformationPMAN-Al/15 32. of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaicvirus genome. Plant Mol Bioi 6: 303-312.46. Kriiger-Lebus S, Potrykus I (1987) Direct gene transfer to Petunia hybrida without electroporation.Plant Mol Bioi Rep 5: 289-294.47. Guerche P, Charbonnier M, Jouanin L, Tourneur C, Paszkowski J, Pelletier G (1987) Directgene transfer by electroporation in Brassica napus. Plant Sci 523: 111-116.48. Shimamoto K, Terada R, Izawa T, Fujimoto H ( 1988) Fertile transgenic rice plants regeneratedfrom transformed protoplasts. Nature 338: 274-276.49. Horn ME, Shillito RD, Conger BV Harms CT (1988) Transgenic plants of Orchard grass(Dactylis glomerata L.) from protoplasts. Plant Cell Rep 7: 469-472.50. Masson J, Lancelin D, Bellini C, Lecerf M, Guerche P, Pelletier G (1989) Selection ofsomatic hybrids between diploid clones of potato (Solanum tuberosum) transformed by directgene transfer. Theor Appl Genet 78: 153-159.51. Damm B, Schmidt R, Willmitzer L (1989) Efficient transformation of Arabidopsis thalianausing direct gene transfer to protoplasts. Mol Gen Genet 217: 6-12.52. Karesch H, Bilang R, Mittelsten Scheid 0, Potrykus I (1991) Direct gene transfer toprotoplasts of Arabidopsis thaliana. Plant Cell Rep 9: 571-574.53. Datta SK, Peterhans A, Datta K, Potrykus I (1990) Genetically engineered fertile indica-ricerecovered from protoplasts. Bio/technology 8: 736-740.54. Wang Z-Y, Takamizo T, Iglesias VA, Osusky M, NagelJ, Potrykus I, Spangenberg G (1992)Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transferto protoplasts. Bio/technology 10: 691-696.55. Omirulleh S, Abraham M, Golovkin M, Stefanov I, Karabaev MK, Mustardy L, MoroczS, Dudits D (1993) Activity of a chimeric promoter with doubled CaMV 35S enhnacerelement in protoplast-derived cells and transgenic plants in maize. Plant Mol Bioi 21:415-428.PMAN-Alf16 33. Plant Molecular Biology Manual A2: I-IS, 1994. 1994 Kluwer Academic Publishers. Printed in Belgium.Gene transfer to plants via particle bombardmentPAUL CHRISTOUAgracetus Inc .. 8520. University Green. Middleton. Wisconsin 53562. U.S.A.IntroductionApproximately six years ago, Klein et al. described a procedure in which highvelocity microprojectiles were utilized to deliver nucleic acids into living cells[1]. In those experiments, transient expression of exogenous RNA or DNAwas demonstrated in epidermal cells of onion (Allium cepa). Following theseexperiments, the technique of particle bombardment (otherwise known asbiolistics, microprojectile bombardment, particle acceleration etc.) has beenshown to be the most versatile and effective way for the creation of manytransgenic organisms, including microorganisms, mammalian cells, and a largenumber of plants species. Tables 1 and 2 provide a comprehensive listing ofmicroorganisms and plant species, respectively, that have been successfullyengineered using particle bombardment technology. An estimated two hundredpapers have been published on various aspects of the technique, including anumber of comprehensive reviews [2-4]. Several advantages make microprojectilebombardment the method of choice for engineering crop species:a) Transformation of organized tissue: The ability to engineer organized andpotentially regenerable tissue permits introduction offoreign genes into elitegermplasm.b) Universal delivery system: Transient gene expression has been demonstratedin numerous tissues representing many different species. In particular casesin which recovery of transgenic plants has not been reported, this deficit ismore due to the lack of a favorable tissue culture response than the DNAdelivery method.c) Transformation of recalcitrant species: Engineering of important agronomiccrops such as soybean, cotton, maize, rice, etc. has been restricted to a fewnon-commercial varieties when conventional methods are used. Particlebombardment technology allowed recovery of transgenic plants from manycommercial cultivars.d) Study of basic plant development processes: By utilizing chromogenic markersit is possible to study deVelopmental processes and also clarify the originof germline in regenerated plants.PMAN-A2/1 34. "'tI$:z> > N --NTable 1. Organelle and microorganism transformation through particle bombardmentSpecies Organism Tissue/organelle Method Gene Year ReferencetransformedSaccharomyces cerevisiae yeast mitochondria Biolistic oxil/oxi3 1988 43Chlamydomonas reinhardtii alga chloroplast Biolistic atpB compo 1988 46Saccharomyces cerevisiae yeast nucleus Biolistic Ura 3/Leu2 1990 42Saccharomyces pombe yeast nucleus Biolistic Ura 3/Leu2 1990 42Neurospora crassa fungus nucleus Biolistic qa2 1990 42Podospora anserina fungus mitochondria Biolistic sen DNA 1990 44Drosophila insect embryo Biolistic beta-gal 1990 45Chlamydomonas reinhardtii alga nucleus mechanical hmr, pAc3 1990 47Tobacco plastid chloroplast Biolistic aadA, gus 1990 50Bacillus megaterium bacterium Biolistic npt II 1991 48Escherichia coli bacterium Biolistic various 1992 49Agrobacterium tumefaciens bacterium Biolistic various 1992 49Erwinia amylovora bacterium Biolistic various 1992 49Erwinia stewartii bacterium Biolistic various 1992 49Pseudomonas syringae bacterium Biolistic various 1992 49Physcomitrella patens moss proton em a Biolistic gus 1992 52Cryptococcus neoformans yeast nucleus Biolistic ade2 1993 51Abbreviations: qa: quinic acid catabolizing gene; Ura3: Uracil catabolising gene; Leu2: Leucine catabolising gene; atpB: photosynthetic gene; b-gal: betagalactosidase; senDNA: senescence genes; pAc3: activator; oxil: mtDNA gene; oxi3: respiration gene; aadA: spectinomycin and streptomycin resistance;ade2: phosphoribosylaminoimidazole carboxylase. 35. Critical variablesA number of parameters have been identified and need to be consideredcarefully in experiments involving transformation using particle bombardment.These can be classified into three general categories:Physical parameters- Nature, chemical and physical properties of the metal particles utilized tocarry the foreign DNA.- Nature, preparation and binding of DNA onto the particles.- Target tissue.Particles should be of high enough mass in order to possess adequatemomentum to penetrate into the appropriate tissue. Suitable metal particlesinclude gold, tungsten, palladium, rhodium, platinum, iridium and possiblyother second and third row transition metals. Metals should be chemicallyinert to prevent adverse reactions with the DNA or cell components. Additionaldesirable properties for the metal include size and shape, as well asagglomeration and dispersion properties.The nature, form and concentration of the DNA need also be considered.In the process of coating the metal particles with DNA, certain additives suchas spermidine and calcium chloride appear to be useful. The nature of theDNA, ego single versus double stranded, may also be important under someconditions, even though this was shown not to be a significant variable inspecific cases.It is very important to target the appropriate cells that are competent for bothtransformation and regeneration. It is apparent that different tissues havedifferent requirements; extensive histology needs to be performed in order toascertain the origin of regenerating tissue in a particular transformation study.Depth of penetration thus becomes one of the most important variables andthe ability to tune a system to achieve particle delivery to specific cell layersmay be the difference between success and failure in recovering transgenicplants from a given tissues. In cases in which the Biolistic device has beenused, particularly with the original version of the instrument, cells near thecenter of the target are injured and cannot proliferate. This injury was attributedto physical trauma to the cells from the gas blast and acoustic shock generatedby the device. The use of baffles or mesh screens reduced cell death andincreased transformation frequency significantly [5, 6].Environmental parametersThese include such variables as temperature, photoperiod and humidity ofdonor plants, explants and bombarded tissues. These parameters have a directPMAN-A2/3 36. Table 2. Transgenic plants obtained through particle bombardment technologyPlant species Common name Explant InstrumentGlycin max soybean meristems AccellNicotiana tabacum tobacco suspension culture BiolisticZea mays com suspension culture BiolisticCarica papaya papaya embryos/hypocot. BiolisticArabidopsis thaliana arabidopsis roots pneumaticPopulus hybrids hybrid poplar nodules AccellOryza sativa rice immature embryos AccellHelianthus annuus Sunflower meristems BiolisticLiriodendron tulipifera yellow poplar suspension culture BiolisticTriticum aestivum wheat embryogenic callus BiolisticAvena sativa oat embryogenic callus BiolisticVaccinium macrocarpon cranberry stem sections AccellDendrobium orchid dendrobium protocorms BiolisticSaccharum officinarum sugarcane embryogenic callus BiolisticCucumis sativus cucumber embryogenic callus BiolisticArachis hypogaea peanut meristems AccellPhaseolus vulgaris common bean meristems AccellGossypium hirsutum cotton meristems AccellZea mays corn immature embryos BiolisticPicea glauca white spruce somatic embryos AccellHordeum vulgare barley immat embryos/embr. cal- BiolisticsIusTriticum aestivum wheat immature embryos BiolisticsAbbreviations: * Confined to systems capable of regeneration from embryogenic suspension orcallus; Biolistic: instrument developed by J. Sanford and currently marketed by Bio-Rad; Accell:Instrument developed by Agracetus Inc., based on electric discharge; BGMV: bean goldenmosaic virus; PRY: papaya rings pot virus; als: Acetolactate synthase gene; bar: phosphinothricinacetyltransferase gene; gus: beta-glucuronidase gene; npt II: aminoglycoside phosphotransferasegene; bt: Bacillus thuringiensis insecticidal protein gene; lux: firefly luciferase gene; hmr:hygromycin resistance gene (aminoglycoside phosphotransferase IV); epsps: glyphosate resistancegene (5-enolpyruvylshikimate-3-phosphate synthase). BYDVcp: barley yellow dwarf viruscoat protein.effect on the physiology of tissues and this is also an important variable. Suchfactors will influence receptiveness of target tissue to foreign DNA delivery andalso affect its susceptibility to damage and injury that may adversely affect theoutcome of the transformation process. Some explants may require a 'healing'period after bombardment under special regimens of light, temperature andhumidity.Biological parametersChoice and nature of explants and pre- and post-bombardment culture conditionsare factors that may determine whether experiments utilizing particlePMAN-A2/4 37. Genes Germline transform. Widely Transform. Referenceapplicable reportgus, npt II, cat, bar, yes yes 1988 40epsps, btgus, npt II yes yes 1988 32, 33gus, bar, lux, als yes no 1990 5, 34npt II, gus, PRY yes probably 1990 37npt II yes yes 1991 20gus, npt II, bt yes probably 1991 29gus, bar, hmr, others yes yes 1991 21,22gus, npt II not reported probably 1992 23gus, npt II yes * 1992 24gus, bar yes * 1992 25gus, bar yes * 1992 26gus, npt II, bt veget. propagated probably 1992 27npt II, PRY veget. propagated probably 1992 31gus, npt II veget. propagated yes 1992 36npt II yes * 1992 39gus, bar yes yes 1992 41gus, bar, BGMV yes probably 1993 28gus and others yes yes 1993 30bar, gus, bt yes probably 1993 35gus, npt II, bt yes * 1993 38gus, bar, BYDVcp yes probably 1994 53gus, bar, npt II yes probably 1994 54, 55bombardment are successful. In addition, explants derived from plants that areunder stress, ego infected with bacteria or fungi, over-or under-watered etc, willprovide inferior material for bombardment experiments. Considerable evidencehas been accumulating to indicate that in order to achieve high transformationfrequencies, metal particles need to be directed to the nucleus [7]. Osmoticpretreatment of target tissues have also been shown to be of importance [8, 9].In addition, experiments performed with synchronized cultured cells indicatethat transformation frequencies may also be influenced by cell cycle stage [10].Physical trauma and tungsten toxicity were found to reduce efficiency oftransformation in experiments performed with tobacco cell suspension cultures[6 ].Many investigators have over-stressed the significance of transient expressiondata. Transient expression studies should only be used as a guide todevelop systems for the stable transformation of a given species. In some casesexhaustive experiments were performed using transient expression data in anattempt to achieve complete protocol optimization for the recovery of stabletransformants. This, however, may be unwise as optimization or maximizationof transient activity does not necessarily result in optimal or any stable transformation.Therefore, studies involving numbers of transiently expressing cellsand foci per unit mass or volume of recipient cells may be meaningless and inPMAN-A2j5 38. a lot of cases irrelevant to the final outcome, particularly when the object isrecovery of transgenic plants. It is important to utilize data from stable transformationexperiments to draw conclusions pertaining to stable transformation.Of course, if no transient activity is observed following a bombardmentexperiment, the likelihood of obtaining stable transformants is practically zero.InstrumentsA number of different instruments based on various accelerating mechanismsare currently in use. These include the original gunpowder device [11], anapparatus based on electric discharge [12], a microtargeting apparatus [13],a pneumatic instrument [ 14], an instrument based on flowing helium [15, 16]and an improved version of the original gunpowder device utilizing compressedhelium [17]. Hand-held devices for both the original Biolistics device and theAccell device are also in use. The most widely-used instrument is the onecurrently marketed by Bio-Rad, Inc. (Biolistics) but Accell-based methodologyhas been particularly useful in developing variety-independent genetransfer methods for the more recalcitrant cereals and legumes. Detailed descriptionsof the various acceleration devices, principles of operation and otherdetails may be found in the primary references.Remaining problemsUntil recently the key barrier in achieving effective transformation ofagronomically-important species was the DNA delivery method. Microprojectilebombardment has had a tremendous impact on this limitation. Thechallenge now is shifting back to the biology of the explant used in bombardmentexperiments. It is apparent that the conversion frequency of transient tostable transformation events is low. This does not mean, however, that transgenicplants from most of the crops that have been engineered cannot beobtained at high enough frequencies to make the process commercially usefuland economical. More attention needs to be paid to the biology of explantsprior to, and following bombardment. We need to identify how more cells canbe induced to become competent for stable DNA uptake and regeneration.Optimization of biological interactions between physical parameters and targettissue needs to be better studied and understood. Not much is known aboutthe fate of DNA from the time particles are introduced into plant cells. Recipienttissue variation and variability due to bombardment conditions complicatethe picture even further. Additional issues such as irregular particle sizeand uniformity as well as improvements in hardware design need also beaddressed.PMAN-A2/6 39. Preparation of DNA/metal mixturesMethods for preparing DNA/metal mixtures have now been standardized. Theonly exception is transformation utilizing the microtargeting device in whichDNA is not bound onto the metal particles prior to bombardment.In a standard procedure in which gold is used as the accelerating particle,DNA is typically loaded onto 1.5-3 f.lm gold beads (Alpha Chemicals Inc.) ata rate of up to 40 f.lg DNA/mg of gold using CaCI2 and spermidine [1] toprecipitate the DNA onto the gold. The coated beads are centrifuged gentlyand re-suspended in 100% ethanol, then pipetted onto the carrier sheets(18 x 18 mm squares of 1/2 mil metalized mylar; Dupont 50 MMC). After abrief period of settling, the ethanol is drained away and the sheet dried.In typical procedures in which tungsten is used [18] 60 mg of particles arewashed extensively in a 1 ml of 70-100 % ethanol. The particles are soaked inethanol for 15 min, pelleted by a 15 min centrifugation (15,000 rpm), decanted,washed three times with sterile distilled water and brought up to a final volumeof 1 ml in a 50% (v/v) glycerol solution. The particles can be stored at roomtemperature for up to two weeks. DNA used for biolistic experiments shouldbe free of protein. Twenty-five f.ll of the tungsten suspension is transferred intomicro centrifuge tubes and vortexed continuously while removing aliquots ofthe suspension to avoid non-uniform sampling; 2.5 f.ll DNA (1 f.lg/f.li) 25 f.llCaCI2 (2.5 M) and 10 f.ll spermidine (0.1 M) are added in that order, while themicrocentrifuge tube is continuously being vortexed. The mixture is allowed toreact for several minutes with continuous vortexing. The coated particles arethen gently pelleted by pulse centrifugation. It is recommended that thetungsten/DNA complex be used as soon as it is made due to the fact that suchmixtures have been shown to be unstable. For the helium-driven system, all ofthe supernatant is removed and the pellet is washed in 70% ethanol. Theparticles are then gently pelleted and brought up in 24 f.ll of 100% ethanol. Sixmicroliters of the mixed suspension are loaded onto the carrier.For the microtargeting instrument, uniform size particles have been used[13]: To prepare 1.5 f.lm particles, place 10 ml of a 1 % aqueous solution ofgold trichloride acid trihydrate yellow (Merck, Darmstadt, Germany) in aplastic centrifuge tube, and add 200 f.ll 'Rodinal' (Agfa Gevaert). Shake themixture briefly and after 30 sec. incubation at room temperature, stop thereaction by adding 2 ml photographic fixer (e.g. 'Ilfospeed', diluted 1: 4 withdistilled water; Ilford foto AG). Centrifuge the suspension for 5 min at2,200 x g in a swing-out rotor, discard the supernatant solution, and resuspendthe pellet in 1.5 ml water. Transfer the suspension to an Eppendorf tube andwash any residual fixer salt by two further centrifugations (3 min at 10,000 x g)discarding the supernatant solution and resuspending each time in 0.5 mlwater. Autoclave the suspension in two aliquots at 120C for 30 minutes. Theautoclaved suspension contains approximately 109 particles per ml, and isstored under refrigeration. It may be resuspended by a short ultrasonic pulsejust before use. The DNA-particle mixture is prepared immediately prior to usePMAN-A2/7 40. by combining sequentially the following: 0.5 1111.0 M Tris-HC1, pH 7.0, 0.5 11110 mM Na-EDTA, 5111 plasmid DNA, 5111 particle suspension.Bombardment and culturing proceduresI. Transformation of corn using a modified gunpowder Biolistic@ instrument[5JFriable embryogenic type II callus is initiated from immature embryos excisedfrom greenhouse grown A 188 X B73 and A 188 X B84 plants, on N6medium supplemented with glycine (2 g/I) proline (2.9 g/I) casein hydrolysate(100 mg/I) dicamba (13.2 mg/I, or 2,4D (1 mg/ll and sucrose(20 g/I), and solidified with Gelgro (2 g/I; ICN Biochemicals, Cleveland, OH).Suspension cultures are initiated by placing 1 g of callus tissue into 20 mlof modified liquid MS medium containing thiamine (0.25 mg/I) L-proline(2.9 g/I) myo-inositol (100 mg/I), casein hydrolysate (200 mg/I) dicamba or2,4D (9.9 or 1 mg/ll NAA (1.6 mg/I) and sucrose (30 g/I). Suspensions arecultured for a number of months and subjected to various treatments,including cryopreservation, to establish material optimum for bombardmentexperiments. Suspension cultures used for transformation experimentsshould be growing rapidly, dispersed and heterogeneous.Steps in the procedure1. Sieve cells through a 1,000 Jlm stainless steel mesh. From the fractionof cell clusters passing through the sieve, pipette approximately 0.5 mlof packed cell volume onto 5 cm filters (Whatman No.4) and filterthrough a Buchner funnel.2. Transfer filters to petri dishes containing three 7 cm filters moistenedwith 2.5 ml of suspension culture medium. The tissue is positionedapproximately 5 cm below the macroprojectile stopping plate, and a100 Jlm mesh stainless steal screen is placed halfway between thestopping plate and the tissue to aid in dispersion of the tungsten particles.3. Bombard cells twice with 1 JlI aliquots of the DNA-tungsten mixtureunder partial vacuum (50-100 mm Hg).4. Following bombardment, culture the cells in liquid medium for 7 -14 dat which point they are plated on media containing 1-3 mg/I bialaphosfor selection of transformed colonies.PMAN-A2f8 41. 5. Transgenic plants may be regenerated by transferring embryogenic callusto MS medium containing 2,4-0 (0.25 mg/I) and BAP (10 mg/l).II. Transformation of tobacco NT cells using a Helium version of theBiolistics instrument [9, 17JCell suspension cultures of the NT 1 line of Nicotiana tabacum [19] are usedto determine the efficiency of transient and stable gene transfer. Four-dayold cell suspensions are collected onto 5.5 cm Whatman No.1 filter paperdiscs using a Buchner funnel. For transient assays the gus A gene can beused. Transient expression is measured two days after bombardment byadding x-gluc solution to each plate and counting the number of blue cellsfollowing a 4-8 h incubation at 37C. To determine rates of stable transformationthe gus gene is fused with the npt /I gene.Steps in the procedure1. After the macroprojectile (macrocarrier) and target cells are in place, thesample chamber is evacuated to O. 1 atm and the high pressure chamberis pressurized to 1 ,000 psi with helium gas.2. The membrane (rupture disk) which restrains the helium is then ruptured.The resultant shock wave of helium launches and accelerates the macroprojectilewhich is positioned 9 mm below the rupture disk. The macroprojectileis stopped by a steel screen placed 10 mm below the launchpoint. The microprojectiles continue onward to penetrate the cells whichare placed 115 mm below the macroprojectile stopping screen.3. Selection for kanamycin-resistant colonies is performed two days postbombardment on NT1 medium containing 350 mg/I kanamycin. Resistantcolonies can be observed 6-8 weeks later.III. Transformation of Arabidopsis thaliana using an Airgun apparatus[14,20JThe pneumatic particle gun used in this experiment is driven by compressedair. A polyethylene projectile with the top surface covered with DNA-coatedgold particles is used. A plunger pump is used to compress and accumulateair in the chamber (200 Kg/cm2 ). The sample to stopper distance is 10 cm,and 4 Ilg DNA/mg gold particles are used. Roots are harvested fromPMAN-A2/9 42. 4-6 week-old aseptically grown Arabidopsis thaliana plants, dissected into0.5-1.0 cm sections and cultured for a period of 3 days on solid B5 mediumcontaining 2,4-D (0.5 mg/I), kinetin (0.05 mg/I), and sucrose (3%).Steps in the procedure1. Remove root segments onto filter paper and place on target area.2. Reduce the pressure to 60 mm Hg and release the compressed airinstantaneously from the chamber to the barrel by triggering the exhaustvalve. The projectile is accelerated in the barrel and collides with thestopper sealing off the aperture. The gold particles continue their trajectorythrough the aperture of the stopper.3. Bombard a second time under the same conditions.4. Transfer sections onto culture medium (0.5-0.05 ml) and incubate for48 h at 26C.5. Transfer sections to fresh B5 medium containing 2-iP (5 mg/I). sucrose(3%) and agar (0.8 g/I) supplemented with kanamycin (50 mg/I).6. After 10 days, transfer onto the same medium with lower levels ofkanamycin (20 mg/I) and resistant callus will appear 3 weeks later.7. Transfer callus onto the same medium containing geneticin disulphate( 10 mg/I) and subculture every 4-7 d. Shoots will regenerate from thiscallus after 4 months. In order to promote elongation, shoots need to betransferred to hormone-free MS medium containing geneticin (20 mg/I)and cultured for two weeks. Rooting can be accomplished on MS mediumcontaining IBA (1 mg/I) in the presence of geneticin (20 mg/I).IV. Microtargeting device. Transformation of tobacco cells [13]Protoplast-derived microcolonies of tobacco are cultured in the dark for threeweeks and bombarded with the npt " gene for the recovery of stabletransformants. Parameters that can be varied include diameter and length ofthe restriction, pressure, working distance, particle size and density, DNAconcentration in the suspension, and the vacuum in the chamber.Steps in the procedure1. Mount the tissue on the surface of a 1.5 mm thick 2% agarose layer ofsupport. The agarose contains 10 mM CaCI2 .2. Pipette a small droplet (1-5 ~I) of autoclaved and filter-sterilized alginatePMAN-A2/10 43. onto the agarose surface. Place blotted tissue on top of the alginatedroplet and remove excess alginate. This keeps the surface to be bombardedfree of alginate. The alginate polymerizes in the presence of Ca2 +within 5-15 min, resulting in a sandwich of agarose support, the alginateand the tissue.3. Microtargeting to a given small area of tissue is achieved by placing thetarget stage under the cross wires of a stereomicroscope. The targetstage can be moved by two screws in two dimensions perpendicular tothe flight of the particles. After adjustment of the system for the firstshot, each of the following shots targets the same area.4. Layer the agarose support onto the target stage of the accelerator.5. A pressure pulse of 2 ms and up to 60 bar is provided by an air gun,supplied with carbon dioxide or nitrogen.6. After bombardment the tissue is removed from the alginate and subjectedto selection. To allow for sufficient expression of the npt /I genein the transgenic cells, the cultures need to be incubated for one weekbefore selection is applied. Transformants are selected using 5 mg/Iparomomycin for 10 d, followed by 50 g/I kanamycin. Plants are regeneratedby following standard protocols.V. Device based on flowing Helium-transient expression of gus in leaf tissueof cowpea [16JThe procedure described below was carried out utilizing the Particle InflowGun.Steps in the procedure1. Place 2 III of particle/DNA suspension in the center of the screen in adisassembled syringe filter unit. Reassemble and screw into the needleadaptor of the apparatus.2. Cowpea leaf tissue, in a petri dish, is placed on adjustable shelves atdistances between 14-23 cm from the screen in the syringe filter unit.The tissue is preferably bombarded with baffles made of nylon screens(1 mm or 500 11m) placed either directly on top or at a distance of 9 cmabove the tissue.3. Apply a vacuum of 28-30 in Hg; the particles are discharged when thehelium (at 40-80 PSI) is released following activation of a solenoid bya timer relay.PMAN-A2Jll 44. 4. Transient activity is determined 2 d after bombardment using standardprocedures.VI. Transformation of rice immature embryos using the Acce/tID instrument[22.23JThis protocol results in the recovery of clonal plants, suggesting that transformationevents are of single cell origin. No chimeric plants are recoveredin this procedure. Twelve to fifteen-day old rice immature embryos areexcised from greenhouse grown plants. These serve as target tissues fortransformation experiments.Steps in the procedure1. Load the Accell 'gun' by placing a 10111 drop of water between thepoints and cover the spark chamber with the reflecting cap.2. Place the carrier sheet over the top of the reflection chamber and put theretaining screen in place.3. Prepare and position the target in a way that will allow the desired areato be exposed as it is inverted above the retaining screen.4. Evacuate the assembly to 600 millibars before the discharge is activated.The scutellar region of the embryo is bombarded following charging thecapacitor to 10-12 kV.5. Plate bombarded tissue on regeneration media (basal medium supplementedwith 2,4-0 and appropriate selective agents, preferably hygromycinat 50 mg/I). Continuous selection of the proliferating tissue resultsin transformed embryogenic callus.6. Transfer embryogenic callus to shooting media for recovery of transgenicplantlets.ReferencesI. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for deliveringnucleic acids into living cells. Nature 327: 70-73.2. Birch RG, Franks T (1991) Development and optimization of microprojectile systems forplant genetic transformation. Aust J Plant Physiol 18: 453-469.3. Christou P (1992) Genetic transformation of crop plants using microprojectile bombardment.Plant J 2: 275-281.4. Klein TM, Arentzen R, Lewis PA, Fitzpatrick-McElligott (1992) Transformation ofmicrobes, plants and animals by particle bombardment. Bio/technology 10: 286-291.5. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'BrienPMAN-A2/12 45. JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, KauschAP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenicplants. Plant Cell 2: 603-618.6. Russell JA, Roy MK, Sanford JC (1992) Physical trauma and tungsten toxicity reduce theefficiency of biolistic transformation. Plant Physiol 98: 1050-1056.7. Yamashita T, lida A, Morikawa H (1991) Evidence that more than 90% of p..glucuronidaseexpressingcells after particle bombardment directly receive the foreign gene in their nucleus.Plant Physiol 97: 829-831.8. Vain P, McMullen MD, Finer 11 (1993) Osmotic treatment enhances particle bombardmentmediatedtransient and stable transformation of maize. Plant Cell Rep 12: 84-88.9. Russell JA, Roy MK, Sanford JC (1992) Major improvements in biolistic transformation ofsuspension-cultured tobacco cells. In Vitro Cell Dev BioI 28: 97-105.10. Iida A, Yamashida T, Yamada Y, Morikawa H (1991) Efficiency of particle bombardmentmediatedtransformation is influenced by cell cycle stage in synchronized cultured cells oftobacco. Plant Physiol 97: 1585-1587.II. Sanford JC, Klein TM, Wolf ED, Allen NJ (1987) Delivery of substances into cells andtissues using a particle bombardment process. J Part Sci Techn 6: 559-563.12. Christou P, McCabe DE, Martinell BJ, Swain WF (1990) Soybean Genetic EngineeringCommercialproduction of transgenic plants. Trends Biotech 8: 145-151.13. Sautter C, Waldner H, Neuhaus-UrI G, Galli A, Neuhaus G, Potrykus I (1991) Microtargeting:High efficiency gene transfer using a novel approach for the acceleration ofmicro-projectiles. Bio/technology 9: 1080-1085.14. lida A, Seki M, Kamada M, Yamada Y, Morikawa H (1990) Gene delivery into culturedplant cells by DNA-coated gold particles accelerated by a pneumatic particle gun. TheorAppl Genet 80: 813-816.15. Takeuchi Y, Dotson M, Keen NT (1992) Plant transformation: a simple particle bombardmentdevice based on flowing helium. Plant Mol BioI 18: 835-839.16. Finer 11, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gunfor DNA delivery to plant cells. Plant Cell Rep 11: 323-328.17. Sanford JC, Devit MJ, Russell JA, Smith FD, Harpending PR, Roy MK, Johnston SA (1991)An improved, helium driven biolistic device. Technique 3: 3-16.18. Sanford J, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biologicalapplications. Meth Enzymol 217: 83-509.19. Paszty C, Lurquin PF (1987) Improved plant protoplast plating/selection technique forquantification of transformation frequencies. BioTechniques 5: 716-718.20. Seki M, Shigemoto N, Komeda Y, Imamura J, Yamada Y, Morikawa H (1991) TransgenicArabidopsis thaliana plants obtained by particle bombardment-mediated transformation.Appl Microbio Biotech 36: 228-230.21. Christou P, Ford T, Kofron M (1991) Production of transgenic rice (Oryza sativa L.) plantsfrom agronomically important indica and japonica varieties via electric discharge particleacceleration of exogenous DNA into immature zygotic embryos. Bio/technology 9: 957-962.22. Christou P, Ford T, Kofron M (1992) The development of a variety-independent genetransfermethod for rice. Trends Biotech 10: 239-246.23. Bidney D, Scelonge C, Martich J, Burrus M, Sims L, Huffman G (1992) Microprojectilebombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.Plant Mol BioI 18: 301-313.24. Wilde HD, Meagher RB, Merkle SA (1992) Expression of foreign genes in transgenicyellow-poplar plants. Plant Physiol 98: 114-120.25. Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenicwheat plants obtained by microprojectile bombardment of regenerable embryogenic callus.Bio/technology 10: 667-674.26. Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR (1992) Fertile, transgenic oatplants. Bio/technology 10: 1589-1594.27. Serres R, Stang E, McCabe D, Russell D, Mahr D, McCown B (1992) Gene transfer usingPMAN-A2/13 46. electric discharge particle bombardment and recovery of transformed cranberry plants. JAmer Soc Hort Sci 117: 174-180.28. Russell DR, Wallace KM, Bathe JH, Martinell BJ, McCabe DE (1993) Stable transformationof Phaseolus vulgaris via electric-discharge mediated particle acceleration. Plant CellRep 12: 165-169.29. McCown BH, McCabe DE, Russell DR, Robison DJ, Barton KA, Raffa KF (1991) Stabletransformation of Populus and incorporation of pest resistance by electric discharge particleacceleration. Plant Cell Rep 9: 590-594.30. McCabe DE, Martinell BJ (1993) Transformation of elite cotton cultivars via particle bombardmentof meristems. Bio/technology 11: 596-598.31. Kuehnle AR, Sugii N (1992). Transformation of Dendrobium orchid using particle bombardmentof protocorms. Plant Cell Rep II: 484-488.32. Tomes DT, Weissinger AK, Ross M, Higgins R, Drummond BJ, Schaaf S, MaloneSchonebergJ, Staebell M, Flynn P, Anderson J, Howard J (1990) Transgenic tobacco plantsand their progeny derived from microprojectile bombardment of tobacco leaves. Plant MolBioI 14: 261-268.33. Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P (1988) Stable genetictransformation of intact Nicotiana cells by the particle bombardment process. Proc NatlAcad Sci USA 85: 8502-8505.34. Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritanceand expression of chimeric genes in the progeny of transgenic maize plants. Bio/technology8: 833-839.35. Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J,Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR,Merlin E, Rhodes R, Warren GW, Wright M, Evola SV (1993) Field performance of elitetransgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis.Bio/technology 11: 194-200.36. Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment.Plant J 2: 409-416.37. Fitch MMM, Manshardt RM, Gonsalves D, Slightom JL, Sanford JC (1990) Stable transformationof papaya via microprojectile bombardment. Plant Cell Rep 9: 189-194.38. Ellis DD, McCabe DE, Mcinnis S, Ramachandran R, Russell DR, Wallace KM, MartinellBJ, Roberts DR, McCown BH (1993) Stable transformation of Picea glauca by particleacceleration. Bio/technology 11: 84-89.39. Chee PP, Slightom JL (1992) Transformation of cucumber tissues by microprojectile bombardment:Identification of plants containing functional and non-functional transferredgenes. Gene 118: 255-260.40. Christou P, McCabe DE, Martinell BJ, Swain WF (1990) Soybean Genetic EngineeringCommercialproduction of transgenic plants. Trends Biotech 8: 145-151.41. Brar GS, Cohen BA, Vick CL (1992). Germline transformation of peanut (Arachishypogaea L.) utilizing electric discharge particle acceleration (ACCELL ) technology. ProcAmer Peanut Research Education Soc, Inc. Norfolk, Virginia. Vol 24: 21.42. Armaleo D, Ye GN, Klein TM, Shark KB, Sanford JC and Johnston SA (1990) Biolisticnuclear transformation of Saccharomyces cerevisiae and other fungi. Current Genet 17:97-103.43. Johnston SA, Anziano PQ, Shark K, Sanford JC, Butow RA (1988) Mitochondrial transformationin Yeast by bombardment with microprojectiles. Science 240: 1538-1541.44. Cummings DJ, Domenico JM, Sanford JC (1990) Mitochondrial DNA from Podosporaanserina: Transformation to senescence via particle injection of plasm ids. In: Finch CE andJohnson TE (ed) Molecular Biology of Aging, pp. 91-101. Wiley-Liss, New York45. Baldarelli RM,