BY‐2 Cells: Culture and Transformation for Live Cell Imaging

Federica Brandizzi1, Sarah Irons1, Anne Kearns1, Chris Hawes1

1 Oxford Brookes University, Oxford
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.7
DOI:  10.1002/0471143030.cb0107s19
Online Posting Date:  August, 2003
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Abstract

Tobacco Bright Yellow‐2 (BY‐2) suspension cells are a widely used biological material for studying plant cell morphology and physiology. These cells are easy to transform and maintain in culture and tolerate transformation with fluorescent proteins such as the green fluorescent protein and its derivatives. These, by the addition of plant or mammalian targeting sequences, can be directed to specific subcellular locations for the study of cell dynamics in vivo.

This unit describes the production of BY‐2 cell stable transformants via an Agrobacterium based method to permit the visualisation of cellular components in vivo by epifluorescence or confocal microscopy.

     
 
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Table of Contents

  • Basic Protocol 1: Establishing a BY‐2 Suspension Culture
  • Basic Protocol 2: Routine Culture of BY‐2 Cells
  • Basic Protocol 3: Aphidicolin‐Mediated Synchronization of Cell Cultures
  • Basic Protocol 4: Stable Transformation of BY‐2 Cells Mediated by Agrobacterium for Visualization of Subcellular Organelles
  • Alternate Protocol 1: Stable Transformation of BY‐2 Cells with Two Constructs
  • Basic Protocol 5: Preparation of BY‐2 Cells for Fluorescence Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishing a BY‐2 Suspension Culture

  Materials
  • Calli of wild‐type and/or transformed BY‐2 cells (available from various laboratories) grown on solid BY‐2 medium (see recipe) in petri dish
  • 50‐ml conical flasks containing 20 ml liquid BY‐2 medium (see recipe), covered with aluminum foil and autoclaved
  • Suitable filter‐sterilized antibiotics, for culturing transformed BY‐2 cells only
  • Razor blades, sterile
  • Packet of aluminum foil squares for covering flasks, sterile
  • Shaking incubator, 25°C

Basic Protocol 2: Routine Culture of BY‐2 Cells

  Materials
  • 50‐ml conical flasks containing 20 ml liquid BY‐2 medium (see recipe), covered with aluminum foil and autoclaved
  • Suitable filter‐sterilized antibiotics, for transformed BY‐2 cells only
  • Wild‐type or transformed stationary‐phase BY‐2 cells (i.e., 7‐day‐old cultures) grown in suspension (see protocol 1)
  • Packet of aluminum foil squares for covering flasks, sterile
  • Trimmed 1‐ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile
  • Shaking incubator, 25°C

Basic Protocol 3: Aphidicolin‐Mediated Synchronization of Cell Cultures

  Materials
  • 50‐ml conical flasks containing 20 ml liquid BY‐2 medium (see recipe), covered with aluminum foil and autoclaved
  • 5 mg/ml aphidicolin (Fisher) in dimethyl sulfoxide, store up to 1 yr at 4°C
  • Suitable filter‐sterilized antibiotics, for transformed BY‐2 cells only
  • Wild‐type or transformed stationary‐phase BY‐2 cells (i.e., 7‐day‐old cultures) grown in suspension (see protocol 1)
  • Five 100‐ml beakers, covered with aluminum foil and autoclaved
  • 20‐µm nylon filter mounted on a cut plastic autoclavable 50‐ml beaker, sterile
  • Packet of aluminum foil squares for covering flasks, sterile
  • Trimmed 1‐ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile
  • Shaking incubator, 25°C
  • 1‐liter liquid waste container

Basic Protocol 4: Stable Transformation of BY‐2 Cells Mediated by Agrobacterium for Visualization of Subcellular Organelles

  Materials
  • YEB medium (see recipe) containing appropriate filter‐sterilized bacterial selection antibiotic
  • Agrobacterium tumefaciens transformed with vector containing appropriate GFP construct (e.g., strain GV3101::pMP90; Konez and Schell, ) transformed with another plasmid (e.g., pVKHI8En6, pBII21) which contains GFP and the insert of interest.
  • 3‐day‐old wild‐type BY‐2 suspension culture (see protocol 1)
  • Liquid BY‐2 medium (see recipe), sterile
  • Solid BY‐2 medium (see recipe) plates with plant selectable antibiotic, 100 µg/ml carbenicillin (see recipe), and 20 µg/ml timentin (see recipe)
  • Shaking incubator, 25°C
  • Trimmed 1‐ml pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile
  • 5‐ and 10‐cm petri dishes, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Forceps, sterile

Alternate Protocol 1: Stable Transformation of BY‐2 Cells with Two Constructs

  Materials
  • Solid BY‐2 medium (see recipe), for short‐term observation only
  • Wild‐type or transformed BY‐2 suspension culture (see protocol 1)
  • 70% (v/v) ethanol, for long‐term observation only
  • Valap: 1:1:1 (w/w/w) Vaseline/lanolin/paraffin (unit 13.1)
  • Electrical tape, cut in 0.5‐cm‐wide strips, for short‐term observation only
  • 76 × 26–mm glass slides and 51 × 20–mm O thickness coverslips, for short‐term observation only
  • Microwave oven, for short‐term observation only
  • Trimmed 200‐µl pipet tips (i.e., 4 to 5 mm cut off from narrow end), sterile
  • 76 × 51–mm glass slides with 18‐mm hole in the center and 24 × 24–mm O thickness coverslips, for long‐term observation only
  • 50°C oven, for long‐term observation only
  • Double‐sided tape, for long‐term observation only
  • Scalpel, for long‐term observation only
  • 2.5 × 2.5–cm piece of bioFOLIE film (Vivascience), for long‐term observation only
  • Sterile tissues, for long‐term observation only
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Figures

Videos

Literature Cited

   An, G. 1985. High‐efficiency transformation of cultured tobacco cells. Plant Physiol. 79:568‐570.
   Boisnard‐Lorig, C., Colon‐Carmona, A., Bauch, W., Hodge, S., Doerner, P., Bancharel, E., Dumas, C., Haseloff, J., and Berger, F. 2001. Dynamic analyses of the expression of the HISTONE::YFP fusion protein in arabidopsis show that syncytial endosperm is divided in mitotic domains. Plant Cell 13:495‐509.
   Brandizzi, F., Fricker, M., and Hawes, C. 2002. A greener world: The revolution of plant bioimaging. Nature Mol. Cell Biol. 3:520‐530.
   Criqui, M.C., Parmentier, Y., Derevier, A., Shen, W.H., Dong, A., and Genschik, P. 2000. Cell cycle‐dependent proteolysis and ectopic overexpression of cyclin B1 in tobacco BY2 cells. Plant J. 24:763‐773.
   Criqui, M.C., Weingartner, M., Capron, A., Parmentier, Y., Shen, W.H., Heberle‐Bors, E., Bogre, L., and Genschik, P. 2001. Sub‐cellular localization of GFP‐tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation. Plant J. 28:569‐581.
   David, K.M. and Perrot‐Rechenmann, C. 2001. Characterization of a tobacco Bright Yellow 2 cell line expressing the tetracycline repressor at a high level for strict regulation of transgene expression. Plant Physiol. 125:1548‐1553.
   Dixit, R. and Cyr, R.J. 2002. Spatio‐temporal relationship between nuclear‐envelope breakdown and preprophase band disapearance in cultured tobacco cells. Protoplasma 219:116‐121.
   Evans, D.E., Coleman, J., and Kearns, A. 2003. Plant Cell Culture—The Basics. BIOS Scientific Publishers, Oxford.
   Forreiter, C., Kirschner, M., and Nover, L. 1997. Stable transformation of an arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo. Plant Cell 9:2171‐2181.
   Geelen, D.N.V. and Inzé, D.G. 2001. A bright future for the Bright Yellow‐2 cell culture. Plant Physiol. 127:1375‐1379.
   Geelen, D., Leyman, B., Batoko, H., Di Sansebastiano, G.P., Moore, I., and Blatt, M.R. 2002. The abscisic acid‐related SNARE homolog NtSyr1 contributes to secretion and growth: Evidence from competition with its cytosolic domain. Plant Cell 14:387‐406.
   Granger, C.L. and Cyr, R.J. 2000. Microtubule reorganization in tobacco BY‐2 cells stably expressing GFP‐MBD. Planta 210:502‐509.
   Haseloff, J., Siemering, K.R., Prasher, D.C., and Hodge, S. 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. U.S.A. 94: 2122‐2127.
   Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H., and Mano, Y. 1978. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase‐alpha. Nature 275: 458‐460.
   Irons, S., Evans, D.E., and Brandizzi, F. 2003. The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelope in plants. J. Exp. Bot. 54:943‐950.
   Koncz, C. and Schell, J. 1986. The promoter of TL‐DNA gene 5 controls the tissue‐specific expression of chimeric genes carried by a novel type of agrobacterium binary vector. Mol. Gen. Genet. 204:383‐396.
   Koscianska, E. and Wypijewski, K. 2001. Electroporated intact BY‐2 tobacco culture cells as a model of transient expression study. Acta Biochim. Pol. 48:657‐661.
   Kost, B., Spielhofer, P., and Chua, N.H. 1998. A GFP‐mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16:393‐401.
   Kumagai, F., Yoneda, A., Tomida, T., Sano, T., Nagata, T., and Hasezawa, S. 2001. Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY‐2 cells stably expressing GFP‐tubulin: Time‐sequence observations of the reorganization of cortical microtubules in living plant cells. Plant Cell Physiol. 42:723‐732.
   Menges, M. and Murray, J.A.H. 2002. Synchronous Arabidopsis suspension cultures for analysis of cell‐cycle gene activity. Plant J. 30:203‐212.
   Nagata, T., Nemoto, Y., and Hasezawa, S. 1992. Tobacco BY‐2 cell line as the “HeLa” cell in the cell biology of higher plants. Int. Rev. Cytol. 132:1‐30.
   Nebenführ, A., Gallagher, L.A., Dunahay, T.G., Frohlick, J.A., Mazurkiewicz, A.M., Meehl, J.B., and Staehelin, L.A. 1999. Stop‐and‐go movements of plant Golgi stacks are mediated by the acto‐myosin system. Plant Physiol. 121:1127‐1141.
   Nebenführ, A., Frohlick, J.A., and Staehelin, L.A. 2000. Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells. Plant Physiol. 124:135‐151.
   Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T., and Machida, Y. 2001. The NPK1 mitogen‐activated protein kinase kinase kinase is a regulator of cell‐plate formation in plant cytokinesis. Genes Dev. 15:352‐363.
   Rose, A. and Meier, I. 2001. A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc. Natl. Acad. Sci. U.S.A. 98:15377‐15382.
   Saint‐Jore, C.M., Evins, J., Brandizzi, F., Batoko, H., Moore, I., and Hawes, C. 2002. Redistribution of membrane proteins between the Golgi apparatus and the endoplasmic reticulum in plants is reversible and not dependent on cytoskeleton networks. Plant J. 29: 661‐678.
   Shinmyo, A., Shoji, T., Bando, E., Nagaya, S., Nakai, Y., Kato, K., Sekine, M., and Yoshida, K. 1998. Metabolic engineering of cultured tobacco cells. Biotechnol. Bioeng. 58:329‐332.
   van der Fits, L., Deakin, E.A., Hoge, J.H.C., and Memelink, J. 2000. The ternary transformation system: Constitutive virG on a compatible plasmid dramatically increases Agrobacterium‐mediated plant transformation. Plant Mol. Biol. 43:495‐502.
   Yoshida, K., Kasai, T., Garcia, M.R.C., Sawada, S., Shoji, T., Shimizu, S., Yamazaki, K., Komeda, Y., and Shinmyo, A. 1995. Heat‐inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana. Appl. Microbiol. Biotechnol. 44:466‐472.
   Zuo, J., Niu, Q.W., Nishizawa, N., Wu, Y., Kost, B., and Chua, N.H. 2000. KORRIGAN, an Arabidopsis endo‐1,4‐β‐glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137‐1152.
Key References
   Geelen and Inze, 2001. See above.
  Reviews the applications of BY‐2 cells in plant cell biology studies. Includes a comparison of Arabidopsis and BY‐2 cells as model plant cells.
   Kodama and Komamine 1995. Synchronization of cell cultures of higher plants. Methods Cell Biol. 49:315‐329.
  Summarizes the techniques used in synchronization in different plant cultures including determination of cell number, mitotic index, and percentage of living cells.
   Nagata et al., 1992. See above.
  Comprehensively reviews the history, growth, characteristics, and applications of BY‐2 cells.
Internet Resources
   http://www.bch.msu.edu/pamgreen/bv2.htm
  Describes useful hints for transforming BY‐2 cells and culturing.
   http://botany1.bio.utk.edu/cellbiol/default.htm
  Illustrates GFP fluorescent BY‐2 cells, with most of the images from BY‐2 cells expressing a GmMan1‐GFP fusion protein to label Golgi stacks. Also shows combinations with other fluorochromes to simultaneously highlight other cellular components in interphase and cell division and in the presence of different test compounds.
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