NICO TI AN A PROCEDURES FOR EXPERIMENTAL USE isagji^- Mil *^yr ^^^ UNITED STATES TECHNICAL PREPARED BY [(ÜjJ) DEPARTMENT OF BULLETIN SCIENCE AND ^^ AGRICULTURE NUMBER 1586 EDUCATION ADMINISTRATION Contributors L. G. Burk T. Murashige SEA, USDA Department of Plant Sciences Tobacco Research Laboratory University of California Oxford, N.C. 27565 Riverside, Calif. 92502 J. F. Chaplin G. Schaeffer SEA, USDA SEA, USDA Tobacco Research Laboratory Cell Culture and Nitrogen Fixation Laboratory Oxford, N.C. 27565 Beltsville, Md. 20705 G. B. Collins L. Sequeira Department of Agronomy Department of Plant Pathology University of Kentucky University of Wisconsin Lexington, Ky. 40506 Madison, Wis. 53706 R. D. Durbin M. Shabde-Moses SEA, USDA Department of Plant Sciences Department of Plant Pathology University of California University of Wisconsin Riverside, Calif. 92502 Madison, Wis. 53706 T. A. Shalla R. W. Fulton Department of Plant Pathology Department of Plant Pathology University of California University of Wisconsin Davis, Calif. 95616 Madison, Wis. 53706 H. H. Smith J. P. Helgeson Department of Biology SEA, USDA Brookhaven National Laboratory Department of Plant Pathology Upton, N.Y. 11973 University of Wisconsin Madison, Wis. 53706 J. R. Stavely SEA, USDA M. J. Kasperbauer Tobacco Laboratory SEA, USDA Beltsville, Md. 20705 Department of Agronomy University of Kentucky H. M. Wilson Lexington, Ky. 40506 Department of Plant Pathology University of Wisconsin L A. Mastrangelo Madison, Wis. 53706 Biology Department New York University New York, N.Y. 10003 Abstract R. D. Durbin, ed. Nicoiiana: Procedures for Experimental Use, U.S. Department of Agri- culture, Technical Bulletin 1586. Background information and procedures for using the genus Nicoiiana as experimental subjects are presented. Each chapter first surveys a topic, then, in detail, presents the associated experimental techniques. The topics include plant propagating and hybridizing; cytogenetical techniques; organ, tissue, cell and protoplast culture; protoplast fusion and organelle transfer; selection of biochemical cell varients; virology; disease resistance; and bacterial hyper- sensitivity. In addition, a genetical overview is given of the genus. Keywords: Nicoiiana, tobacco, tissue culture, protoplast, disease resistance, haploids, protoplast and organelle fusion, organ culture, and bacterial hyper- sensitivity. Trade names and the names of commercial companies are used in this publication solely to provide specific information. Mention of a tr^de name or manufacturer does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement by the Department over other products not men- tioned. 449575 Foreword Advances in botanical research have often depended upon the proper choice of an experimental plant. In this regard, the genus Nicotiana has been pre- eminently successful. A few such advances in which it has played a crucial role include photoperiodism, whose study has contributed much to our knowl- edge of flowering and other aspects of plant growth, and which was first elucidated in Maryland Mammoth tobacco by Garner and Allard (Journal Agricultural Re- search 18:553) ; the discovery of cytokinins as a class of plant growth substances depended upon tobacco tissue cultures ; and quantitative virus assays were revo- lutionized by Holmes (Phytopathology 28:553) when he used leaves of N, glu- tinosa as a local lesion indicator for tobacco mosaic virus. Pioneering research in haploid plant production, quantitative genetics, incompatibility systems, and parasexual hybridization also have used tobacco as the experimental plant of choice. Because of its many advantages, the prospect for the continued and even expanded employment of Nicotiana in theoretical biology seems assured. How- ever, the necessary background information and procedures for taking full ad- vantage of its attributes have never been brought together and, in some cases, are not published. Hence, the reason for this volume. Our hopes in presenting this information are that it will benefit experimenters in various disciplines and that they will continue to find Nicotiana useful. Suppliers of the specialized materials mentioned (plants, chemicals and ap- paratus) are listed in the Appendix. The Index contains all references to Nicotiana spp. in the text, but it does not include those mentioned in figures or tables. R. D. Durbin Science and Education Administration USD A; University of Wisconsin, Madison, Wis. Ill Contents Foreword iv R. D. Durbin 1 The genus as a genetic resource 1 H. H. Smith 2 Cytogenetic techniques 17 G. B. Collins 3 Hybridization 23 L. G. Burk and J. F, Chaplin 4 Plant propagation 28 J. F. Chaplin and L. G. Burk 5 Haploid plant production and use 33 M. J. Kasperbauer and H. M. Wilson 6 Organ culture 40 M. Shabde-Moses and T. Murashige 7 Tissue and cell suspension culture 52 J. P. Helgeson 8 Protoplast isolation and culture 60 T. A. Shalla 9 Protoplast fusion and organelle transfer 65 I. A. Mastrangelo 10 Selection of biochemical variants from cell culture 74 G. Schaef f er 11 Nicotianas as experimental virus hosts 79 R. W. Fulton 12 Disease resistance 87 J. R. Stavely 13 Bacterial hypersensitivity Ill L. Sequeira Appendix-Suppliers 121 Species index 122 Issued January 1979 IV THE GENUS AS A GENETIC RESOURCE H. H. Smithi Introduction 1 The species 2 Polyploidy, aneuploidy and the origin of Nicotiana tahacum .. 3 Biometrical studies 6 Biochemical genetics 6 Alkaloids 6 Isozymes 7 Genetic tumors 7 Evidence for genetic control 7 Physiological characteristics 8 Some unique advantages of Nicotiana for future genetic research 9 Built-in selective system at the cell level 9 Fraction I protein : a molecular genetic marker 9 Consequences of interspecific gene transfer 10 References 12 Introduction The genus Nicotiana has been used widely in genetic postulating the origin of N, tahacum, establishing a series research, and in related botanical disciplines, largely be- of monosomes, interspecific transferring of genes for disease cause of the great variation and different stages of evolu- resistance, and clarifying cytogenetic and phylogenetic re- tionary divergence that species offer. Within a species, lations among species. Reviews on the cytogenetics of the genetic studies are aided by the simplicity of controlled genus Nicotiana have been published by East {42), Kos- pollination and the abundant yield of seeds and progeny toff {76), Goodspeed {53) and Smith {133, 139). (ch. 3). Between species, hybridizing has revealed dis- Throughout the 1950^s and 1960's, parallel to the study tinctive, cytogenetic relationships, and hereditary char- of tobacco cytogenetics, a separate discipline of single cell actetristics such as genetic tumors, differences in bio- culture was being developed, which made extensive use of chemical traits of alkaloids and isozymes, markers for Nicotiana spp. (ch. 7). Several investigators clearly dem- chloroplast deoxyribonucleic acid (DNA), and certain so- onstrated that whole tobacco plants could be differentiated matic instabilities that are used to demonstrate the genetic from single somatic cells {157). In the late 1960*s fungal control of gene expression. The applications that have cellulases, hemicellulases, and pectinases became commer- been made in the past suggest that, as new techniques and cially available from Japan. Techniques for their wide- concepts evolve, Nicotiana spp. will continue to find use scale use in isolating protoplasts rapidly advanced (ch. 8). in and to be applied to arising problems. In 1969-70 Takebe and his associates {106, 156) isolated The favorableness of Nicotiana spp. for experimental re- protoplasts from tobacco leaves that underwent mitotic search did not escape the notice of the pre-Mendelian hy- division and regenerated into whole plants {152). bridizers. Kolreuter, the first to undertake systematic, sci- With the consolidation of the fields of cell culture and entific hybridizing in plants, succeeded in obtaining the plant genetics using such model systems as Nicotiana cross N. rustica X N. paniculata, describing it in 1761 {138), the significance and potential of somatic cell plant more than 100 years before MendePs results were pub- genetics became recognized. Research with tobacco is in a lished {109, 151),'' Throughout the first half of the 20th better position than with any other crop plant to take ad- century, Nicotiana spp. were used in many studies aimed vantage of the new developments in genetic engineering at establishing the basis of classical genetics. These ad- {141). Not only is this because the cells and tissues of to- vances in genetics included forming the multiple factor bacco are inherently relatively easy to culture, but the hypothesis, producing new species through amphiploidy and techniques themselves have been developed to a consider- able extent by using species of the genus Nicotiana. Also, ^Department of Biology, Brookhaven National Laboratory, Upton, N.Y. 11973. as noted above, the phylogenetic structure of the genus ^Italic numbers in parentheses in each chapter refer to "References" provides a rich reservoir of genetic resources for experi- listed at the end of each chapter. ments. 1 TECHNICAL BULLETIN 1586, U.S. DEPARTMENT OF AGRICULTURE The Species A taxonomic monograph of the genus Nicotiana is in- chromosome number of each. N. sanderae is not included cluded in Goodspeed's book (53), It is classified into 3 in the table because it is a horticultural species