Advances in CELL CULTURE Edited by KARL MARAMOROSCH Robert L Starkey Professor of Microbiology Department of Entomology Rutgers University New Brunswick, New Jersey GORDON H SATO W Alton Jones Cell Science Center Lake Placid, New York VOLUME ACADEMIC PRESS, INC Harcourt Brace Jovanovich, Publishers San Diego London New York Berkeley Boston Sydney Tokyo Toronto This book is printed on acid-free paper @ COPYRIGHT © 1989 BY ACADEMIC PRESS, INC All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher ACADEMIC PRESS, INC San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW1 7DX ISBN 0-12-007907-0 (alk paper) ISSN 0275-6358 This publication is not a periodical and is not subject to copying under CONTU guidelines PRINTED IN THE UNITED STATES OF AMERICA 89 90 91 92 CONTRIBUTORS TO VOLUME Numbers in parentheses indicate the pages on which the authors' contributions begin D BRODY, Bodega Marine Laboratory, University of California, Bodega Bay, California 94923 (19) ERNEST S CHANG, Bodega Marine Laboratory, University of California, Bodega Bay, California 94923 (19) TOOHYON CHO, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853 (261) ELIZABETH W DAVIDSON, Department of Zoology, Arizona State University, Tempe, Arizona 85287 (125) MICHAEL ROBERT R GRAN ADOS, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853 (261) TADAAKI HIBI, National Institute of Agrobiological Resources, Tsukuba Science City, Kannondai, Ibaraki 305, Japan (147) BRUCE L NICHOLSON, Department ofMicrobiology, and Center for Marine Studies, University of Maine, Orono, Maine 04469 (1) LOWELL D OWENS, Plant Molecular Biology Laboratory, Agriculture Research Service, U.S Department of Agriculture, Beltsville, Maryland 20705 (183) GIDEON W SCHAEFFER, Plant Molecular Biology Laboratory, Agricultural Research Service, U.S Department of Agriculture, Beltsville, Maryland 20705 (161) MICHAEL L SHULER, School of Chemical Engineering, Cornell University, Ithaca, New York 14853 (261) ANN C SMIGOCKI, Plant Molecular Biology Laboratory, Agriculture Research Service, U.S Department of Agriculture, Beltsville, Maryland 20705 (183) VIRGINIA K WALKER, Department of Biology, Queen s University, Kingston, Ontario, K7L 3N6, Canada (87) M N ZAPROMETOV, K A Timiriazev Institute of Plant Physiology, Academy of Sciences of the U.S.S.R., Moscow, U.S.S.R (201) ix PREFACE The seventh volume of this landmark series provides a timely update of advances in the culturing of cells, and presents new biotechnological applications of in vitro techniques, reviewing both the advantages and disadvantages of the methods The contributions cover the cultivation of fish and crustacean cells, gene transfer in insect cells, the study of bacterial protein toxins using vertebrate cells, fusion of plant protoplasts by electromanipulation, anther and microspore cultivation, gene transfer to induce morphogenesis, formation of phenolic compounds in cells and tissues in vitro, and new media and culture systems for the large-scale production of insect cells A biographical sketch has been devoted to the 1986 Nobel Laureate Rita LeviMontalcini, the developmental biologist who identified the nerve growth factor (NGF), a protein that is necessary for the growth, development, and maintenance of nerve cells in the peripheral nervous system and, apparently, also in the brain Photograph of Dr Levi-Montalcini courtesy of Massimo Vergari A.G.F KARL MARAMOROSCH GORDON H SATO xi RITA LEVI-MONTALCINI RITALEVI-MONTALCINI Rita Levi-Montalcini was born in Turin, Italy on April 22, 1909 Her scientific career started in the early 1930s, when she became a medical student and "intern' ' in the Institute of Anatomy at the University of Turin There she was a colleague and friend of Renato Dulbecco and Salvador E Luria It is of special interest to note that years later all three were awarded Nobel Prizes In 1952, during a summer spent at Cold Spring Harbor, I learned from Professor Luria that his decision to become a research scientist rather than a practicing physician was due to the influence of his anatomy professor at Turin, Giuseppe Levi, and that not only he, but several of his colleagues, among whom were Renato Dulbecco, Rita Levi-Montalcini, and Guido Pontecorvo, came under the ' spell ' ' of Professor Levi It was probably Rita's good luck that Professor Levi realized her strong inclination to basic research and, instead of steering her to histology, encouraged her to culture different cell types in vitro The "master-pupil association" which started so early lasted till the death of Giuseppe Levi thirty-three years later In her biographical essay (1988) Rita vividly describes the influence Professor Levi had on her scientific career The topic of her doctoral dissertation was the formation of collagen reticular tissue by connective, muscular, and epithelial tissues She became passionate about this research and succeeded in demonstrating that the formation of reticular fibers is a combined property of all three types of tissue The experience gained in studying nervous and other tissues in vitro later prompted her to use the same techniques that led to the milestone discovery of the nerve growth factor (NGF) and to the penetrating analysis of its action in vivo and in vitro The study of nerve cells in vitro had been undertaken by her teacher Giuseppe Levi in 1928, when he followed the early work of Ross Granville Harrison (Maramorosch, 1981) Professor Levi was one of the first to realize the possibilities that tissue culture offered for the analysis of cell growth and differentiation in vitro He and his co-worker Hertha Meyer, who worked earlier with Emil Fischer in Germany, published several articles jointly on this subject before Dr Meyer left Italy for Rio de Janeiro in 1939 to work in the Institute of Biophysics directed by Carlos Chagas Rita graduated from medical school in 1936, having specialized in neurology and psychiatry Two years later, in 1938, Mussolini issued his anti-Jewish manifest which barred all academic and professional careers for "non-Aryan" citizens In 1939 Rita accepted an invitation from a neurological institute in Brussels and moved there, but returned to Turin when the German invasion of Belgium became imminent She then decided to build a small laboratory in her bedroom where she xiii XIV RITA LEVI-MONTALCINI assembled a microscope, a microtome, and an incubator Sewing needles, sharpened by hand on a stone, provided the microinstruments for her work on chick embryos During the period 1940-1943, when the Germans advanced all over Europe, crushing everything Rita cherished and valued, and when the anti-Semitic campaign reached its peak, Rita tried to ignore the abuses and threats and, joined by Professor Levi, became completely absorbed in her work In 1942, after a heavy bombing of Turin, she and her family were forced to move to a small country home where she rebuilt her laboratory and, despite poor conditions involving, among others, a shortage of eggs and frequent interruptions of electric current, she completed her study on acousticovestibular centers of the chick embryo, the results of which were published many years later (1949) In September 1943 Italy was occupied by Nazi Germany, and Italian Jews became the object of mass killings and deportation to the gas chambers at Auschwitz Rita was able to escape to Florence where she hid with false identity cards till the end of the war In 1945 she returned to Turin where she became an assistant to Professor Levi, who had resumed his work as anatomy professor A year later, in 1947, she and Renato Dulbecco sailed from Genoa on the Polish ship Sobieski to the United States Rita accepted an invitation from Victor Hamburger to work in his laboratory at Washington University in St Louis, Missouri and to reinvestigate the mechanism of action between nervous and non-nervous structures Quoting her own words: "If the beginning of my career was under the spell of Giuseppe Levi, the second period was under the influence of Victor Hamburger, who had already played a key role in channeling my interest toward problems of growth and differentiation of nerve cells" (Levi-Montalcini, 1975) In the spring of 1951 Rita and Dr Hamburger discovered that a growth factor of unknown nature was being released from neoplastic cells, which differed from the effect of that of normal embryonic tissues Rita then grafted fragments of chick sarcomas 180 or 37 onto chorioallantoic membranes of 4- to 6-day-old chick embryos so that the tumor and the embryo shared circulation, but were not in direct contact, and demonstrated the effect of the same mysterious growth factor (Levi-Montalcini, 1952) A few years later, at one of the first symposia of the Society for Growth and Development, I met Rita, who impressed me greatly with her scientific presentation as well as with her charming personality At that time she described the "NGF halo," which she discovered while on a Rockefeller Foundation travel grant in Dr Chagas's laboratory in Rio in 1952 This halo represents a reliable index of the NGF that permitted final identification of the factor by Rita and Stanley Cohen twenty years later, which earned them the Nobel Prize The tissue culture experiments started by Rita in 1952 opened a new sector of study in developmental biology The experiments revealed the capacity of growth factors to stimulate processes of proliferation and differentiation in cells of various RITA LEVI-MONTALCINI XV origin When Rita used tumors that had been transplanted into embryos, she found that they stimulated fiber growth far beyond her expectations After returning to St Louis from Rio, Rita set up an in vitro culture unit, and confirmed that only extracts of the mouse tumors, first transplanted into chick embryos and then excised, induced the formation of the fibrillar halo around sensory ganglia in vitro The chemical characterization of NGF was carried out by Stanley Cohen (who joined Rita and Hamburger for six years) who found the growth factor in large quantities in snake venom This permitted work on its identification as a protein of molecular weight 20,000 Complete characterization of NGF was carried out by Piero Angeletti who joined Rita in St Louis after Dr Cohen left the laboratory In 1961 he moved with Rita to a new study center in Rome This center remained in close contact with the laboratory in St Louis Currently Rita spends all her time working in the laboratory in Rome In vitro experiments revealed that NGF markedly stimulated all anabolic processes in embryonic sensory and sympathetic cells Actinomycin D does not entirely prevent the formation of the halo effect, whereas puromycin and cyclohexamine block the outgrowth of nerve fibers from explanted ganglia (Angeletti, Levi-Montalcini, and Calissano, 1968; Levi-Montalcini, 1964; Levi-Montalcini and Angeletti, 1971) Fine structure studies demonstrated the marked effect of NGF on neurofilament and neurotubule formation (Levi-Montalcini et al., 1968) Later studies by Rita were devoted to "chemical sympathectomy," a new method to suppress the sympathetic nerve system in newborn animals (Angeletti and Levi-Montalcini, 1970) The work on NGF is not yet finished I would like to quote Rita's last paragraph from her ' 'An Uncharted Route' ' ( 1975): "Now that the NGF has come of age and the most picturesque and adventurous phase of its life is over, the biographer, who has had some part in the chase, entrusts it, with love, to younger and more skillful hands." Rita's tissue culture activity was not devoted solely to the study of NGF, but also, and to a rather large extent, to the study of Periplaneta americana, explored in whole mount cultures as well as dissociated nerve cells Regretfully, she had to discontinue this work in view of the length of time the cultures required, which made it impossible for her to pursue at the same time her studies of NGF in vivo and in vitro KARL MARAMOROSCH XVI RITA LEVI-MONTALCINI REFERENCES Angeletti, P U., and Levi-Montalcini, R (1972) Growth inhibition of sympathetic cells by some adrenergic blocking agents Proc Natl Acad Sei U.S.A 69, 86-88 Angeletti, P U., Levi-Montalcini, R., and Calissano, P (1968) The nerve growth factor (NGF): Chemical properties and metabolic effects Adv Enzymol 31, 51-75 Levi-Montalcini, R (1949) The development of the acoustico-vestibular centers in the chick embryo in the absence of the afferent root fibers and of descending fiber tracts J Comp Neurol 91, 209-241 Levi-Montalcini, R (1952) Effects of mouse tumor transplantation on the nervous system Ann N.Y Acad Sei 55, 330-343 Levi-Montalcini, R (1964) Growth control of nerve cells by a protein factor and its antiserum Science 143, 105-110 Levi-Montalcini, R (1975) An uncharted route In "The Neurosciences: Paths of Discovery" (F G Worden, J P Swazey, and G Adelman, eds.) MIT Press, Cambridge, Massachusetts Levi-Montalcini, R (1988) "In Praise of Imperfection-My Life and Work," 220 pp Basic Books, New York Levi-Montalcini, R., and Angeletti, P U (1971) Ultrastructure and metabolic studies on sensor and sympathetic nerve cells treated with the nerve growth factor and its antiserum In "Hormones in Development" (M Hamburgh and E J W Barrington, eds.), pp 719-730 Appleton-CenturyCrofts, New York Levi-Montalcini, R., Caramia, F., Luse, S A., and Angeletti, P U (1968) In vitro effects of the nerve growth factor on the fine structure of the sensory nerve cells Brain Res 8, 347-362 Maramorosch, K (1981) Ross Granville Harrison: 1870-1959 Adv Cell Culture 1, xiii-xvi SELECTED PUBLICATIONS Levi-Montalcini, R., Meyer, H., and Hamburger, V (1954) In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo Cancer Res 14, 49-57 Cohen, S., Levi-Montalcini, R., and Hamburger, V (1954) A nerve growth-stimulating factor isolated from sarcomas 37 and 180 Proc Natl Acad Sei U.S.A 40, 1014-1018 Levi-Montalcini, R., and Angeletti, P U (1963) Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro Dev Biol 7, 653-659 Levi-Montalcini, R (1964) Growth control of nerve cells by a protein factor and its antiserum Science 143, 105-110 Levi-Montalcini, R (1965) Growth regulation of sympathetic nerve cells Arch Ital Biol 103, 832-846 Levi-Montalcini, R., Caramia, F., Luse, S A., and Angeletti, P U (1968) In vitro effects of the nerve growth factor on the fine structure of the sensory nerve cells Brain Res 8, 347-362 Chen, J H., and Levi-Montalcini, R (1969) Axonal outgrowth and cell migration in vitro from nervous system of cockroach embryos Science 166, 631-632 Levi-Montalcini, R., and Chen, J S (1969) In vitro studies of the insect embryonic nervous system Symp Int Soc Cell Biol 8, 277-298 Chen, J S., and Levi-Montalcini, R ( 1970) Axonal growth from insect neurons in glia-free cultures Proc Natl Acad Sei U.S.A 66, 32-39 RITA LEVI-MONTALCINI XVll Chen, J S., and Levi-Montalcini, R (1970) Long term cultures of dissociated nerve cells from the embryonic nervous system of the cockroach Periplaneta americana Arch Ital Biol 108, 503-537 Levi-Montalcini, R (1971) In vitro analysis of the insect nervous system Boll Zool 38, 385-399 Levi-Montalcini, R., and Chen, J S (1971) Selective outgrowth of nerve fibers in vitro from embryonic ganglia of Periplaneta americana Arch Ital Biol 109, 307-337 Seshan, K R., and Levi-Montalcini, R (1971) In vitro analysis of corpora cardiaca and corpora allata from nymphal and adult specimens of Periplaneta americana Arch Ital Biol 109, 81-109 Aloe, L., and Levi-Montalcini, R (1972) In vitro analysis of the frontal and inglucial ganglia from nymphal specimens of the cockroach Periplaneta americana Brain Res 44, 147-163 Aloe, L., and Levi-Montalcini, R (1972) Interrelation and dynamic activity of visceral muscle and nerve cells from insect embryos in long-term cultures J Neurobiol 3, 3-23 Levi-Montalcini, R., and Aloe, L (1972) Neuronal nets and nerve cell interactions in vitro in insect systems In Vitro 8, 178 Levi-Montalcini, R., and Seshan, K R (1973) Long-term cultures of embryonic and mature insect nervous and neuroendocrine systems In "Tissue Culture of the Nervous System" (G Sato, ed.), pp 1-33 Plenum Press Seshan, K R., and Levi-Montalcini, R (1973) Neuronal properties of nymphal and adult insect neurosecretory cells in vitro Science 182, 291-293 Seshan, K R., Provine, R R., and Levi-Montalcini, R (1974) Structural and electrophysiological properties of nymphal and adult insect medial neurosecretory cells: An in vitro analysis Brain Res 78, 359-376 TOOHYON CHO ET AL 274 Air Filter Hh Air Water for Air Humidification 1.5 cm CellColumn Water Bath, 29°C Air Flowmeter FIG A schematic diagram of the packed-bed insect cell bioreactor and airlift for oxygen supplementation For details see Shuler et al (1989) the further downstream purification process The nonionic surfactant pluronic F-68 was used to protect insect cells from the adverse effects of sparging From the preliminary experiments in a laboratory-scale prototype, glass-bead, packed-bed bioreactor, we have demonstrated t h a t the attachment-dependent T ni 5B14 cells can be effectively cultured and, when the cells were infected with recombinant AcNPV containing the ß-galactosidase gene from Escherichia coli, they produced < 3 % of the total protein as ß-galactosidase (Shuler et al., 1989) The T ni 5B14 cells did not detach from the surface even 88 hours after infection, and nearly all of the cells maintained some viability These data showed t h a t the growth of cells on glass spheres of mm diameter, with aeration in the external loop, was a promising technique for large-scale cell production to produce recombinant proteins V Summary Traditionally, serum-free media and cell culture systems have been developed primarily for the production of viral pesticides The recent interest in the use of baculoviruses as expression vectors has speeded NEW MEDIA FOR PRODUCTION OF INSECT CELLS 275 up progress on the development of new serum-free media and cell culture systems A serum-free medium that contains the pluronic F-68 and liquid mixture of cod liver oil, Tween 80, otocopherol acetate, and cholesterol instead of FBS was successfully used in a 21-liter volume in an airlift fermentor for large-scale production of suspended SF9 cells to produce recombinant M-CSF from recombinant AcNPV The glass bead-packedbed bioreactor with an external-loop airlift column developed in our laboratory, along with attachment-dependent T ni 5B14 cells isolated originally in our laboratory, looks very promising for large-scale production of insect cells It has already been demonstrated that this culture system and cell line could produce up to 33% of the total protein as recombinant ß-galactosidase from a recombinant AcNPV Based on the remarkable progress made in the last few years, new improved media formulations and novel bioreactors will soon be available for commercial large-scale insect cell production ACKNOWLEDGMENTS This work was sponsored in part by the National Science Foundation under Grant EET-8807089 The U S government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright notation hereon REFERENCES Agathos, S N., Jeong, Y H., Fallon, A M., and Venkatasubramanian, K (1987) Abstr Annu Am Inst Chem Eng Meet, New York, November 15-20 (Session 161) Billimoria, S L., and Carpenter, W M (1983) In Vitro 19 870-874 Brooks, M A., Ifcang, K R., and Freeman, F A (1980) In I n Vitro" (E Kurstak, K Maramorosch, and Dubendorfer, eds.) Elsevier-North Holland, Amsterdam Brown, M., and Faulkner, P (1975) J Invertebr Pathol 26, 251-257 Corsaro, B G., and Fraser, M J (1987) In Vitro Cell Dev Biol 23, 855-862 Dwyer, D G., Webb, S E., Shelton, A M., and Granados, R R (1988) J Invertebr Pathol 52, 268-274 Gardiner, G R., and Stockdale, H (1975) J Invertebr Pathol 25, 363-370 Goldschmidt, R (1915) Proc Natl Acad Sei 1, 220-222 Goodwin, R H., and Adams, J R (1980) In I n Vitro" (E Kurstak, K Maramorosch, and Dubendorfer, eds.) Elsevier-North Holland, Amsterdam Goodwin, R H., Tbmpkins, G J., and McCawley, P (1978) In Vitro 14, 485-495 Grace, T D C (1962) Nature (London) 195, 788-789 Granados, R R., and Hashimoto, Y (1989) In "Invertebrate Cell System Applications," Vol 2, Chapters 1, 3-13 (J Mitsuhashi, ed.) CRC Press, Boca Raton, Florida Granados, R R., Derksen, A C G., and Dwyer, D G (1986) Virology 151, 472-476 276 TOOHYON CHO ET AL Granados, R R., Dwyer, K G., and Derksen, A C C (1987) In "Biotechnology in Invertebrate Pathology and Cell Culture" (K Maramorosch, ed.), pp 167-181 Academic Press, San Diego Groner, A., and Roder, A (1982) In "Invertebrate Pathology and Microbial Control," Progr Abstr Offered Pap, 15th Annu Meet Soc Invertebr PathoL, p 167, September 6-10, Univ of Sussex, Brighton, England Hink, W F (1982) In "Microbial and Viral Pesticides" (E Kurstak, ed.), p 493 Marcel Dekker, New York Hink, W F , and Strauss, E M (1976) In "Invertebrate Tissue Culture: Applications in Medicine, Biology, and Agriculture" (E Kurstak and K Maramorosch, eds.) Academic Press, New York Hink, W F , and Bezanson, D R (1985) In "Techniques in the Life Sciences." Vol C l , "Techniques in Setting up and Maintenance of Tissue and Cell Cultures" (E Kurstak, ed.), pp C l l l / l - C l l l / Elsevier, New York Hink, W F , Strauss, E M., and Lynn, D E (1977) Annu Meet Abstr In Vitro 13, 177 Hink, W F , Ralph, D A., and Joplin, K N (1985) In "Comprehensive Insect Physiology, Biochemistry, and Pharmacology" (G A Kerkut and L I Gilbert, eds.), Vol 10, pp 547-570 Pergamon, New York Inlow, D., Harano, D., and Matorella, B (1987) Abstr Am Chem Soc Natl Meet, Sess on "Strategies in Cell-Culture Scale-up,'' September 1, New Orleans Landureau, J C , and Jolies, P (1969) Exp Cell Res 54, 391-398 Luckow, V A., and Summers, M D (1987) Biotechnology 6, 47-55 Luckow, V A., and Summers, M D (1988) Virology 167, - Lynn, D E., Dougherty, E M., McClintock, J T, and Loeb, M (1988) In "Invertebrate and Fish Tissue Culture" (Y Kuroda, E Kurstak, and K Maramorosch, eds.), pp 239-242 Springer-Verlag, Berlin Maiorella, B., Inlow, D., Shauges, A., and Harano, D (1988) Biotechnology 6, 1406-1410 Martignoni, M E., and Scallion, R J (1961) Biol Bull 121, 507-520 Miltenburger, H G., and David, P (1980) Dev Biol Stand 46, 83-186 Miltenburger, H G., Naser, W L., and Harvey, J P (1984) Z Naturfbrsch (C) 39,993-1002 Miltenburger, H G., Naser, W L., and Schliermann, M G (1985) In Vitro Cell Dev Biol 21, 433-438 Mitsuhashi, J (1982) Appl Entomol Zool 17, 575-581 Mitsuhashi, J (1987) In "Biotechnology in Invertebrate Pathology and Cell Culture" (K Maramorosch, ed.), Chapter 24, pp 387-400 Academic Press, San Diego Mitsuhashi, J., and Maramorosch, K (1964) Contrib Boyce Thompson Inst 22, 435-460 Mitsuhashi, J., Nakasone, S., and Horie, Y (1983) Cell Biol Int Rep 7, (12), 1057-1062 Murhammer, D W., and Goochee, C F (1988) Biotechnology 6, 1411-1418 Pollard, R., and Khosrovi, B (1978) Process Biochem 13(5), 31-37 Roberts, P L (1984) Biotechnol Lett 6(10), 633-638 Roder, A (1982) Naturwissenschaften 69, 92 Roder, A., and Groner, A (1982) In "Invertebrate Pathology and Microbial Control," Progr Abstr Offered Pap 15th Annu Meet Soc Invertebr PathoL, p 166, September 6-10, Univ of Sussex, Brighton, England Shuler, M L., Cho, T., Wickham, T., Ogonah, O., Kool, M., Hammer, D A., Granados, R R., and Wood, H A (1989) Ann NY Acad Sei (in press) Spier, R E (1980) In "Advances in Biochemical Engineering" (A Fiechter, ed.), Vol 14, pp 119-162 Springer-Verlag, Berlin Stockdale, H., and Priston, R A J (1981) In "Microbial Control of Pests and Plant Diseases, 1970-1980" (H D Burges, ed.), pp 313-328 Academic Press, London NEW MEDIA FOR PRODUCTION OF INSECT CELLS 277 Street, D A., and Hink, W F (1978) J Invertebr Pathol 32, 112-113 Tramper, J., and Vlak, J M (1986) Ann N.Y Acad Sei 469, 279-288 Tramper, J., Williams, J B., and Joustra, D (1986) Enzyme Microb Technol 8, 33-36 Tramper, J., Joustra, D, and Vlak, J M (1987) In "Plant and Animal Cells." 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Vol 2, "Practical Application for Insect Control" (R R Granados and B A Federici, eds.), pp 64-87 CRC Press, Boca Raton, Florida Weiss, S A., and Vaughn, J L (1987) In "The Biology of Baculoviruses" (R R Granados and B A Federici, eds.), Vol 2, App 63-67 CRC Press, Boca Raton, Florida Weiss, S A., Smith, G C, Kalter, S S., and Vaughn, J L (1981) In Vitro, 495-502 Wilkie, G E I., Stockdale, H., and Pirt, S V (1980) Dev Biol Stand 46, 29-37 Wyatt, G R., Loughheed, T C, and Wyatt, S S (1956) J Gen Physiol 17, 835-868 INDEX A Acer pseudoplatanus cell suspension cultures, 222, 246 cultured cells and tissue, 205, 209, 235, 240, 241, 243, 244, 251 Aedes aegypti, 139 cells, 135-136,137,139, 266 larvae and cells, 140 toxins from crystals, 136 Aedes albopictus cells, 90,139-140, 168-169 auxotrophic, 100 larvae, 140 mutant cell line, 99 Aedes dorsalis cells, 140 Agrobacterium rhizogenes, 184,188-190 Agrobacterium tumefaciens, plasmid from, 184-185,193 T-region mutants, 187-188 Anastrepha suspensa, 111 Anchusa officinalis, cultured cells, 206 Androgenic gland and testis, crustacean, 56-58 Anopheles gambi, 140, 266 eggs, 116 elements isolated from, 111 Anopheles stephensi, 140 Anther culture, role in germplasm development applications, 175-179 deviation from parents, 175-176 biochemical selection pressure, 177-178 future research, 178-179 spontaneous variation, 176-177 basic studies gametes as target for plant modifications, 175 probes and markers, 174-175 concepts, 161-163 cytogenetic aspects, 165-167 genetic instabilities somaclonal variation, 172-174 history, 163-164 methodologies of genotype, 167-169 media and plantlet regeneration, 171-172 microspore stage, 169, 170 temperature shock, 169,171 problems, 175-179 rice, use, 179-180 status, 164-165 Antheria eucalypti, ovarian tissues from, 263 Anthocynins, 223-224 Anthraquinones, 212-214 biogenesis, 217-219 Anticoagulation techniques in crustacean cell culture, 66 Arachis hypogaea callus cells, 247 cell suspension culture, 232 Aspergillus niger, autoclaved, 248 Astacus fluviatilus, - Astacus leptodactylous, 61 Attachment-dependent cell lines, for production of insect cells, 273-274 Autographa californica nuclear polyhedrosis baculovirus (AcNVP), 100-101 Avena sativa, 183 B Bacillus sphaericus, insect toxicity studies activity, 141-142 commonality with Bacillus thuringiensis, 125-127 strains, 139-141 Bacillus thuringiensis endotoxin, insect toxicity studies binding, studies, 131-132 commonality with Bacillus sphaericus, 125-127 dipteran toxins, 133-138 lepidopteran larvae toxins, 127-133 Bacterial protein toxins, insect cell culture studies, 125-127 279 280 INDEX Bacterial protein toxins {continued) Bacillus thuringiensis-endotoxin dipteran toxins, 133-138 lepidopteran larvae toxins, 127-133 Bacillus sphaericus, 139-142 value of, 143 Balanus amphitrite, 61 Biochemical selection pressure with S-AEC, 177-178 selective recovery of phenotypes, 177 Biophalaria glabrata continuous cell culture, 5, Birnaviridae virus family, Black beetle virus (BBV), 102 Black flies, activity of dipteran toxins towards, 134 Blue light, responses of plant cells to, 237-238 Bombyx mori, 263 chorion genes, 117 larvae, 115 Brain tissue and cell culture from fish, 11 C Calcium and neurohormone, crustacean, 21,52 Callistephus chinensis callus tissue, 242, 243 leaves and stems, 224 Calrose 76 cultivar, 176-177 Camellia sinensis callus tissue of, 209, 227, 235, 236, 239, 240, 242, 244, 247 cell suspension culture, 245 cultured cells, 245 mature shoots, 222 young leaves and stems, 234, 244 Canavalia ensiformis, callus tissue of, 227, 249 Cardisoma carnifex, 61 Carcinogens, value offish cell cultures for evaluating and testing effects, 12-13 Carcinus maenas, 56 Carpocapsa pomonella, 264 Carrot cultured cells, 229 Carrot root protoplast, and tobacco mesophyll protoplast, diheterokaryons between, 149-150, 154, 157 Cassia fistula, callus tissue, 241, 243, 246 Cassia tora, 241 Castanea sativa, 205 Catalpa ouata, 215 Catharanthus roseus callus tissue, 224 cell suspension culture, 247 Cell production, insects, see Insect cells, progress in serum-free media use in large-scale culture systems Chatetomium globosum, 248 Chalcones, 220 Channel catfish virus (CCV), Chenopodium rubrum, 205 Chorismate mutase, 203-204 Choristneura fumiferana, 127 Chromosome structure and function, aspects, localization of functions, 166-167 meiosis and genetic fidelity, 166 Chymomyza proncemis, 111 Cicer arietinum cell suspension culture, 227, 232 cells, 249 cultured cells of, 231 root tips, 220, Cinchona ledgeriana, 214 Citrus auranthium, 225 Citrus paradisi, 232 Coleus blumei, cultured cells, 206, 246 Contaminants, environmental, genotoxicity, 12 Continuous fish cell culture, preparation of, 3, see also Fish cell culture Continuous-flow electrofuser, 151-153 principles, 152-153 procedures, 153 revised method, 153, 155 scheme, 152 Corydalis sempevirens, 203 Crab, see Crustacean organ and cell culture techniques Crataegus monogyna callus tissue, 222, 242, 245 cell suspension cultures, 222 Crayfish, see Crustacean organ and cell culture techniques Crotolaria juncea, 226 Crustacean hyperglycémie hormone (CHH), 62 281 INDEX Crustacean organ and cell culture techniques conclusions, 78 hemocyte, 62 encapsulation, 64 nonself recognition factors, - serum factors affecting phagocytosis, 63 temperature effects, 63 literature, 19-20, 2 - methods anticoagulation techniques, 66 attachment factors, 67, 69 cell dispersion, 69 cell separation, 66-67, 68 media, - 6 surface sterilization and dissection, 64-65 temperature, - organ and tissue culture blood glucose, 62 metabolism, 55 molting, - culture of x-organ-sinus gland complex, 61 ecdysteroid effects in vitro, 61—62 molting-hormone secretion, - regulation of Y-organ, - nerve, muscle and neuromuscular preparations, - , - calcium and neurohormone, 21 resting and action potentials, 52 volume regulation, 54 osmoregulation, - 5 gill, 54 gut and urinary bladder, - 5 pigmentation, 62 reproduction, 5 - mandibular organ, 58 ovary, 5 - testis and androgenic gland, - responses and viability dispersed hepatocyte, 70 embryonic and ovarian cells, - muscle cells, - neuromuscular synapses, 71 neurons, - testicular and hematopoietic cells, 73-78, 79 Cryptochrome, effect on plant cells, 237 Cryptomeriajaponica callus tissue, 242 cultures, 203, 222 Culex quinquefasciatus, cell culture system utilizing, 136,137,139-142 Cucumis sativus, transformed, shoot organogenesis, 193-195 Culex quinquefasciatus, 139, 140, 141-142 Culture vessels, standard, for fish cell culture, Cydia pomonella, embryonic tissues from, 262 Cytogenic markers, 166-167 Cytogenetics, aspects in fish cell culture, 9-10 Cytokinin gene fused with strong promoter, phenotypic effects cucumber, shoot organogenesis, 195 Nicotiana, shoot organogenesis, 193-195 promoter fusions, 192-193 RNA transcript and zeatin levels, 195-196 D DAHP-synthase enzyme, activity of, 203 Datura metel, 246 Daucus carota callus cultures, 209, 235, 243, 244-245 cell suspension culture, 248 Derris elliptica, 227 Dielectrophoretic cell alignment process by high-frequency AC, 148-149 Digitalis lanata, callus tissue, 212 Diheterokaryon between tobacco mesophyll and carrot root protoplast, 153,154,157 Dimorphotheca auriculata, callus tissue, 223 Dipteran toxins, 133-138 activity toward mosquitoes and black flies, 133-138 comparison with lepidopteran, 138 strains, 134-137 Direct-pulse current (DC), cell fusion process, 148-149 DNA-mediated transfection stable transformation, 94-100 transient expression, - uptake, 8 - 282 INDEX DNA viruses, double-stranded, 102 Drosophila hydei, cell lines, 91, 99 Drosophila embryos, transformation of using three different techniques, 102-104 Drosophila melanogaster, transfer of DNA to alternative transposable elements in, 109 cell lines derived from, 91,128,136 DNA-mediated transfection, 8 - history, 87-88 P elements and, 105-109 stable transformation, 95-100 transient expression, - Drosophila melanogaster larvae, 136 E Eagle's Minimum Essential Medium (MEM), for fish cell culture, Echium lycopsis, 217 Electrical conditions for electrofusion, varying, 156-158 Electrode chamber for electrofusion, 151 Electromanipulation, technique for plant protoplast fusion applications, 158-159 apparatus for continuous-flow electrofuser, 151-153 electrode chamber, 151 generator, 150-151 frequency, factors affecting biological conditions, 154-155 experimental conditions, 155-158 electrical conditions, 156-158 fusion buffer, 155-156 protoplast density, 156 temperature, 156 history, 147-148 methods, 149-150 principles, 148-149 prospects, 158-159 Elicitors, effect of on phenolic compounds, 248-250 Embryonic cells, crustacean, - Embryos, insects, somatic transformation, 112-115 Erythrocytic necrosis virus (ENV), Escherichia coli gene from, 274 transformed, 111 Exogenous DNA, microinjection of, 175 F Fetal bovine serum (FBS), for fish cell culture, Finfish cell culture media, 6, see also Fish cell culture Fish cell culture applications, 7-14 carcinogenesis, 12-13 cellular differentiation, 10-12 cellular physiology, 10-12 cytogenetics, 9-10 education, 14 immunology, 13-14 toxicology, 12 virology, - continuous cell cultures, maintenance, - culture vessels, growth media, growth temperature, - long-term storage, - preparation of primary cell cultures for, 2-3 shellfish cell culture, - Flavonoids, natural plant, 219 anthocynins, 223-224 biogenesis, 227-232 advances in, 227-228 carrot cells, 229 enzyme operation and, 232 chalcones, 220 flavan-3-ols, 221-223 flavan-3,4 diols, 221-223 flavone and flavonol, 225-226 flavonoid-specific O-methyltransferase, 232 history, 228 parsley cells, 229 usefulness, 230-231 and 3-hydroxyflavonones, 220-221 isoflavone, 227 stilbenes, 226-227 Foeniculum vulgäre, 242 283 INDEX Freezer, ultracold, for storage offish cell cultures, - Fusion buffer, composition of, 155-156 G Galium mollugo, culture of, 214, 247 Gametic tissue for genetic engineering, 174-175 for plant modifications, 175 microinjection, 175 Gene transfer in insects conclusions, 115 considerations, 116-119 history, - 8 other methods, 102 somatic cell fusion, 100-101 somatic transformation of embryos, 112-115 transfer into germ lines alternative transposable elements in Drosophila melanogaster, 109 early techniques, 102-105 of other insects, 109-111 P elements and Drosophila melanogaster, 105-109 viral vectors, 101-102 Generator, for electrofusion, 150-151 Genetic instability, and anther culture chromosomal rearrangements, 173 multigene changes, 173-174 phenotypes, 174 point mutations, 173 somoclomal variation, 172 Genotoxicity of environmental contaminants to aquatic and marine species, testing, 12 Genotype, as important determinant in anther culture, 167-169 Germ lines, see Gene transfer in insects Ginkgo biloba callus cells of, 230 cell suspension cultures of, 221, 222, 223 Glycine max, 184 cell suspension cultures, 210, 225, 237 early history of cell culture, 184-185 tissue cultures, 203 Glycyrrhiza echinata, cultured cells, 227, 249-250,251 Goldfish cells (CAR), toxicology studies on, 12, see also Fish cell culture Gonadal fish cell and organ cultures, 11-12 Growth regulators and phytohormone, effect of on phenolic compound, 240-245 H Haploid cell doubled, application to plant breeding, 175-176 recovery from excised anthers, 164 Haplopappus gracilis callus tissue, 205, 224, 226, 238 cell suspension culture, 229-230, 241 cultured cells, 228, 242 Helianthus tuberosus, 223 tubers, 244 Heliothis zea, clone, 262, 264 Hematopoietic cells, crustacean, 73, 77-78, 79 Hemocyte, in vitro studies, - encapsulation, 64 nonself recognition factors, - serum factors affecting phagocytosis, 63 temperature effects, 63 Hepatocyte, dispersed, 70 Homarus americanus, 73, 75, 77, 79 Hordeum vulgäre, 183 Hydrangea macrophyla, 210 Hydrophora cecropia, 263 Hydrobenzoic acid in plant cell cultures data on occurrence, 209-210 enzymology, 210 Hydroxycinnamic acid and derivatives biogenesis, 206-209 history of literature, 205-206 Hydroxycoumarin, production by cultured cells, 210-212 3-Hydroxyflavonones, 2 - 2 IAA levels of tumor lines, 189,191-192 Idothea wosnesenskii, 61 Immunology, fish, 13-14 284 INDEX Impatiens balsamina, 238 Infectious hematopoietic necrosis virus (IHNV), Infectious pancreatic necrosis virus (IPNV), 7, Insect and insect cells, gene transfer in, see Gene transfer in insects Insect cells, progress in serum-free media use in large-scale culture systems insect cell lines or strains, 262-263 large scale cell production attached cell culture, 273-274 general considerations, 269-272 media costs, 272 oxygen, 270 shear stress, 270-271 temperature and pH, 271-272 suspension systems, 272-273 media formulations, 263-268 summary, 274-275 Insect cell cultures in study of bacterial protein toxins, see Bacterial protein toxins, insect cell culture studies Interferon (IFN), h u m a n fibroblast, 101 Interleukin (IL-1), 13 Isoflavones, 227 Israelensis strain 1884,134-136,137 J Juglans major, 215 Juglans microcarpa, 215 Juniperus communis, callus tissue, 222, 233,235 K Kalanchoe tubiflorae, 192 Kidney tissue from fish cultured, 10 natural killer cells from, 13-14 Kurstaki toxin activity, inhibition of against CF-1 cells, 138,142 L Lanicera proliféra, 235 Large-scale cell production, see also Insect cells, progress in serum-free media use in culture systems attached cell culture, 273-274 general considerations, 268-272 suspension systems, 272-273 Lactuca sativa, cultured in vitro, 245 Leibowitz Medium L-15, for fish cell cultures, Lepidopteran cell lines, new, 262, see also Insect cells Lepidopteran larvae toxins binding studies, 131-132 cell lines, 128-130 sensitive cell lines, 129 comparison with dipteran, 138 mode of action, 132-133 specificity of for mammalian cells, 130-131 Leucania separate cells, 269 Light, effect of in phenolic compound formation in plant cell and tissue cultures, 236-240 chloroplast, 238-239 dependence, 237-238 effect on formation of shikonin, 238 nonphotosynthesis, 236 photosynthesis, 236 phytochrome system, 237 Lignin formation, 234-235 structure in cultured cells, 236 Liquid nitrogen, use of for long-term storage offish cell culture, - Linum usitatissimum, 223 cotyledons of, 244 Lithospermum erythrorhizon callus tissue, 241 dark-grown, 217 cell suspension culture, 238 culture, 206, 211, 216, 219 Liver tissue from fish, cultured, 10 Lobster, see Crustacean organ and cell culture techniques spiny, 7, see also Fish cell culture Locusta migratoria, gene transfer in, 108, 110,114 embryos, 115 Lymantria dispar, embryonic tissues from, 262, 265 M Macrobrachium rosenbergii, 58 Malacosoma disstria, 127 285 INDEX Malus pumila, 223 Mamestra brassicae, cell lines, 266, 268-269 Mandibular organ, crustacean, 58 Media for crustacean cells and tissues, 65-66 Media formulations for insect tissue culture, 263-269, see also Insect cells, progress in serum-free media use in large-scale culture systems costs, 272 history, 263-265 low protein, serum-free, 273 nutritional requirements for viral replication, 265-266 protein-free insect tissue culture media, 268, 269 shear stress formed in, 270 Wilkie's chemically defined, 266, 267 Meiosis, and genetic fidelity, 166 Microinjection, of exogenous DNA, 175 Microspore development in anther culture, 169,170 Microspore, role in advancing technology, see Anther culture, use in germplasm development Mollusks, bivalve, primary cell culture, 6, see also Fish cell culture Molting, 58-62 culture of x-organ-sinus gland complex, 61 ecdysteroid effects in vitro, - molting-hormone secretion, - regulation of Y-organ, - Morinda citrifolia, cell suspension culture, 213, 217, 240,248 cultured cells, 246, 251 Morinda lucida, phenolic compounds, - 240 Morphogenesis in plants, see Phytohormone genes, transfer of to induce morphogenesis in plants Mosquitoes, activity of dipteran toxins towards, 134, 137 Multigene changes and chromosomal arrangements, 173-174 Muscle cells, crustacean, - Muscle preparations, - , - calcium and neurohormone, 21 resting and action potentials, 52 volume regulation, 54 Mutant T-DNA, phenotypes of soybeans transformed by, 187-192 characterization of tmr roots regenerated in vitro, 190 characterization of tumor lines incited by, 190-191 IAA levels of tumor lines, 191-192 rhizogenic nature of soybean tumor lines incited by, 188-190 Mutations, point, 173 N Naphthoquinone, 214-217 biosynthesis stages of, 217-219 Natural killer (NK) cells in fish kidney, 13-14 Nerve preparations, - , - calcium and neurohormone, 21 resting and action potentials, 52 volume regulation, 54 Neurohormone, and calcium, 21 Neuromuscular preparations, - , 52-54 Neuromuscular synapses, crustacean, 71 Neurons, crustacean, - Nicotiana alata, 208 Nicotiana plumbaginifolia, 193,196 Nicotiana silvestris, 203, 204 Nicotiana tabacum cell culture, 203, 204, 207, 235 transformed, shoot organogenesis, 193-195,196 NK, see Natural killer cells Nonphotosynthetic light, effect of on plants, 236-240 Nutrient conditions, effect on regulation of phenolic compound, 245-247 O Oncorhynchus masou virus (OMV), Onobrychis vicifolia, 234 Orconectes limosus, 59 Orconectes virilus, 70 Orchestia gammarella, 56 Organ and tissue culture of crustaceans, see Crustacean organ and cell culture techniques Organ culture from fish cells, 10-12, see also Fish cell culture 286 INDEX Oryza sativa, 183 Osmoregulation, of neuroendocrine system in crustaceans, - 5 gill, 54 gut and urinary bladder, - 5 Ovarian cells, crustacean, - Ovary, crustacean, 5 - Oxygen, use as electron acceptor in aerobic metabolism of insect cells, 270 P P element-mediated transformation of Drosophila, 105-109 usefulness, 105-107 of other insects, 107-108, 109-111 Pacifastacus leniusculus, 73, 74, 76 Packed-bed insect cell bioreactor, 274 Papilio xuthus cells, 268-269 Parsley cell homogenate, 229 Paulownia tormentosa, 235 Penaeusjaponicus, 55, 70 Penaeus monodon, 65, 78 Penaeus penicillatus, 78 Penaeus vannamei, 70 Percoll density-gradient cell separations, 68 pH, as important process parameter for large-scale insect cell production, 271-272 Perilla frutescens, cell cultures of, 203 Perylla ocymoides, cultured cells of, 241-242, 243 Petroselinum crispum, cell suspension cultures, 211, 225, 234 Petunia hybrida cells, 203 Phagocytosis in crustaceans, hemocyte in vitro studies of blood cells affecting, 62-63 and nonself recognition factors, - serum factors affecting, 63 Phaseolus aureus cell suspension cultures, 210 cultured cells, 227 tissue cultures, 203 Phaseolus vulgaris callus tissues, 207 cell suspension cultures, 222, 244, 248 cultured cells, 227, 235 Phenolic compound formation in plant cells and tissue culture conclusions, 251-252 occurrence and biosynthesis of anthraquinones, 211-214 enzymology of shikimate pathway, 203-205 flavonoids, 219-232 hydroxybenzoic acids and derivatives, 209-210 hydroxycinnamic acids and derivatives, 205-209 hydrozycouramine, 210-211 polymeric compounds lignin, 234-236 tannin, 232-234 regulation of effect of light, 236-240 effect of nutrient conditions, 245-247 effect of other factors, 247-251 effect of phytohormone and growth regulators, 240-245 role, 201-202 Phenotypes, 174 biochemical selection, 177-178 selective recovery from, 177-178 L-Phenylalanine ammonia lyase (PAL), 204 Phytohormone genes in plant morphogenesis conclusions, 197 history, 183-187 genetic approach to regeneration of recalcitrant plant species, 185-187 soybean plants, regenerating, 184-185 phenotypes of soybean transferred by mutant T-DNA, 187-188 characterization of tmr roots regenerated in vitro, 190 characterization of tumor lines incited by m u t a n t T-DNA, 190-191 IAA levels of tumor lines, 191-192 rhizogenic nature of soybean tumor lines incited by tmr mutant, 188-190 phenotypic effects of cytokinin gene fused with strong promoter promoter fusions, 192-193 287 INDEX shoot organogenesis in transformed Nicotiana and cucumber, 193-195 RNA transcripts and zeatin levels, 195-196 Photosynthetic light, effect, 236-240 Phytochrome system, 237 Phytohormone and growth regulators, effect of on phenolic compound, 240-245 Phytophthora megasperma effect of, 249, 250 elicitor from, 212, 230 Picea excelsa, 231 Picea glauca, 234 Pigment dispersing hormone (PDH), 62 Pieris brassicae, 130,131-132 Pinus elliotti, 234 Pinus resinosa, callus of, 226, 251 Pinus strobus, 235 Pisum sativum, 227 Pithomyces chartarum, 249 Pituitary gland cultures, derived from fish, 10 Plant morphogenesis, see Phytohormone genes, transfer to induce morphogenesis in plants Plaque forming cells (PFC), 14 Plodia interpunctella, 128 Plumbago zeylanica cell line, 241 young stems, 214 Polyphenol, see Phenolic compounds Populus nigra, 235 Populus tremuloides, 235 Potato callus protoplast, electrofusion, 150 Prawn cell cultures, 7, see also Fish cell culture Precursors, effect on phenolic compounds, 247-248 Primary fish cell culture, preparation, 2-3, see also Fish cell culture Procambarus clarkii, 55, 56, 71 Promoter fusions, 192-193 Protoplast samples, biological condition of, 154-155, see also Electromanipulation, for plant protoplast fusion Prunus avium, callus tissue, 220, 250 Pseudotsuga menziesii, cell suspension cultures, 220, 223, 230, 231 Q Quercus robur, 209, 233 Quercus stenophyllia, - 3 R Recombinant-DNA technologies for development of vaccine for foot and mouth disease, 162-163 Red pigment-concentrating hormone (RPCH), 62 Regeneration, plant difficulty of, 165 media and plantlet, 171-172 Rehmannia glutinosa, callus cells, 206 Retinal pigment epithelium (RPE) offish, 11 Rheum palmatum, callus tissues, 213 Rhizogenicity of tmr galls on whole soybean plants, 188-190 Ribes sanguineum, cultured cells, 220-221, 222, 223 Rice, as model cereal for advancing technology, 179-180, see also Anther culture, use of in germplasm development RNA transcript and zeatin levels, 195-196 Rosa damascena, 240 Rosa multiflora, 223 Rosette forming cells (RFC), 14 Rubia cordifolia, 213 Ruta graveolens, 210 S Saccharomyces cerevisiae, 249 S-aminoethylcysteine (S-AEC), 177 Salmon, see Fish cell culture Sarcophaga peregrima cells, 268-269 Scutellaria baicalensis, 225 Sensitive cell lines, resistance of to high levels of toxins, 129 Shear stress, formed in liquid media to protect insect cells, - Shellfish cell culture, preparation of, 6-7, see also Fish cell culture 288 INDEX Shikimate pathway of aromatic amino acid and phenolic compound synthesis, 203-205 Silybum marianum, 226 Snail, freshwater, see Fish cell culture Solanum brevidens mesophyll protoplast, electrofusion of, 150 Somatic cell fusion, 100-101 Soybean phenotypes transformed by m u t a n t T-DNA, 187-192 rhizogenic nature of tumor lines incited by tmr mutant, 188-190 regenerating, 184-185 Soybean cells, cultivated, 208 treatment with fungal elicitor, 250 Spinal cord tissue and cell culture from fish, 11 Spiny lobster cell cultures, 7, see also Fish cell culture Spodoptera exigua NVP, 266 Spodoptera frugiperda cells, 136, 266, 268 ovarian tissue, 263, 264 Spodoptera litoralis, 266 Staphylococcus aureus, 137 Stilbenes, 226-227 Streptocarpus dunnii, cell suspension cultures, 216, 218-219 Strobilanthes dyeriana, 224 Surface-sterilized exoskeletons, - dark-grown calluses, 243 Suspension systems for large-scale cell production, 272-273 T Tabebuia argentea, cell suspension culture, 216 Tannin condensed, 233 formation, 234 hydrolyzable, 232-233 Tectona grandis, 212 Temperature for crustacean cell culture, - effects on hemocyte, 63 for electrofusion, 156 for fish cell culture growth, - for insect cell growth, 271-272 shock, in mid- to late-uninucleated microspore stage, 169,171 Testicular cells, crustacean, - Testis and androgenic gland, crustacean, 56-58 Tissue and organ culture of crustaceans, see Crustacean organ and cell culture techniques Tmr tumor lines mutant, rhizogenic nature of tumor lines, 188-190 characterization of roots regenerated in vitro, 190 characterization of tumor lines incited by m u t a n t T-DNA, 190-191 Tobacco mesophyll protoplasts, electrofusion of, 149-150,154,157 diheterokaryons between carrot root and, 156,157-158 Toxins, insect, see Bacterial protein toxins, insect cell culture studies Transferred DNA (T-DNA), 185-187 Trichoplusia ni, 127, 264, 274 Trifolium pratense, callus of, 249 Triticum aestivum, 183 Trout, see Fish cell culture Tumor lines, soybean, see Phytohormone genes in plant morphogenesis U Uca pugilator, 55, 56,60 UV light, response of plant cells to, 237-238 V Variation, spontaneous, 176-177 Viral erythrocytic necrosis (VEN), Viral vectors, 101-102 Virus, fish identification, - isolation, - replication, in vitro models for studying, 8-9 289 INDEX w Wilkie's chemically defined media, 266, 267 Y Y-organ, crustacean, culturing, 59-60 regulation, 60-61 X Xenopus eggs, 94 somatic transformation, 112 X-organ sinus gland complex of crustacean, culturing, 61 Z Zea mays, 183 Zeatin levels, RNA transcript and, 195-196 Zebrafish embryos, clonal analysis, 12 ... a t progress in refining culture systems has been relatively slow in this area compared to insect cell culture A survey by Hink (1980) listed no less t h a n 71 cell lines from 17 insect species... (Thorgaard, 1 976 ; Etlinger, 1 976 , 1 977 , 1 978 ) C Cellular Physiology and Differentiation Both primary and continuous fish cell cultures have been employed as in vitro models for studying various cellular... and Teninges, D (1 979 ) J Virol 32, 593-605 Engelking, H M., and Leong, J C (1981) Virol 109, 47- 58 Etlinger, H M (1 976 ) In Vitro 12, 599-601 Etlinger, H M (1 977 ) Eur J Immunol 7, 881-8 87 Etlinger,