Summary
Combining stepper-motors for microscope stage movement with a specially designed software program has led to the establishment of an efficient cell tracking system. Cell immobilization, fixed reference points for calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (either beforehand or in retrospect), their locations fixed, and subsequent development of the individual cells monitored daily using computer-assisted relocation. In this way, the specific cell type capable of sustained division and regeneration has recently been identified within a low efficiency protoplast system of a recalcitrant species, sugarbeet. These totipotent cells originated from stomatal guard cells. Isolation and purification procedures were then optimized in a directed way to yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity for transient expression studies proved that GCPs were amenable to transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure yields 600 transgenic individuals per person per year. This number allows for the selection of the best plants with regard to copy number, DNA insert size, gene expression, and field performance. Prospects for future application of the cell finder system will be discussed.
Similar content being viewed by others
References
D'Halluin, K.; Bossut, M.; Bonne, E., et al. Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio/Technology 10:309–314; 1992.
Freytag, A. H.; Anand, S. C.; Rao-Ardelli, A. P., et al. An improved medium for adventitious shoot formation and callus induction inBeta vulgaris L. in vitro. Plant Cell Rep. 7:30–34; 1988.
Fry, J. E.; Barnason, A. R.; Hinchee, M. Genotype-independent transformation of sugarbeet usingAgrobacterium tunefaciens. In: Hallick, R. B., ed. Molecular biology of plant growth and development. Third International Congress of the International Society for Plant Molecular Biology. Tucson, AZ.#384; 1991.
Gleba, Y. Y.; Sytnik, K. M. Protoplast fusion. Genetic engineering in higher plants. Berlin, Germany: Springer-Verlag. 1984.
Glimelius, K.; Fahlesson, J.; Landgren, M., et al. Gene transfer via somatic hybridization in plants. Trends Biotechnol. 9:24–31; 1991.
Golds, T. J.; Babczinsky, J.; Rauscher, G., et al. Computer-controlled tracking of single cell development inNicotiana tabacum L. andHordeum vulgare L. protoplasts embedded in agarose/alginate films. J. Plant Physiol. 140:582–587; 1992.
Hagège, D.; Catania, R.; Micalef, H., et al. Nuclear shape and DNA content of fully-habituated nonorganogenic sugarbeet cells. Protoplasma 166:49–54; 1992.
Hall, R. D.; Pedersen, C.; Krens, F. A. Improvement of protoplast culture protocols forBeta vulgaris L. (sugarbeet). Plant Cell Rep. 12:339–342; 1993.
Hall, R. D.; Pedersen, C.; Krens, F. A. Regeneration of plants from protoplasts ofBeta vulgaris (sugar beet). In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry 29. Plant protoplasts and genetic engineering V. Berlin, Germany: Springer-Verlag; 1994:16–37.
Hall, R. D.; Riksen-Bruinsma, T.; Weyens, G., et al. Stomatal guard cells are totipotent. Plant Physiol. 112:889–892; 1996.
Hall, R. D.; Riksen-Bruinsma, T.; Weyens, G., et al. A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells. Nature Biotechnology 14:1133–1138; 1996.
Hall, R. D.; Riksen-Bruinsma, T.; Weyens, G., et al. Sugar beet guard cell protoplasts demonstrate a remarkable capacity for cell division enabling applications in stomatal physiology and molecular breeding. J. Exp. Bot. 48:255–263; 1997.
Hall, R. D.; Verhoeven, H. A.; Krens, F. A. Computer-assisted identification o fprotoplasts responsible for rare division events reveals guard-cell totipotency. Plant Physiol. 107:1379–1386; 1995.
Harms, C. T.; Potrykus, I. Fractionation of plant protoplast types by isoosmotic density gradient centrifugation. Theor. Appl. Genet. 53:57–63; 1978.
Helgeson, J. P. Somatic hybridization of wildSolanum species with potato: a potential source of diversity for breeders. In: Louwes, K. M.; Toussaint, H. A. J. M.: Dellaert, L. M. W., eds. Parental line breeding and selection in potato breeding. Wageningen, The Netherlands: Pudoc; 1989:87–94.
Horsch, R. B.; Fry, J. E.; Hoffmann, N. L., et al. A simple and general method for transferring genes into plants. Science 227:1229–1231; 1985.
Karp, A. Somaclonal variation as a tool for crop improvement. Euphytica 85:295–302; 1995.
Krens, F. A.; Jamar, D. The role of explant source and culture conditions on callus induction and shoot regeneration in sugarbeet (Beta vulgaris L.). J. Plant Physiol. 134:651–655; 1989.
Krens, F. A.; Jamar, D.; Rouwendal, G. J. A., et al. Transfer of cytoplasm from newBeta CMS sources to sugarbeet by asymmetric fusion. I. Shoot regenerated plants. Theor. Appl. Genet. 79:390–396; 1990.
Krens, F. A.; Molendijk, L.; Wullems, G. J., et al. In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74; 1982.
Krens, F. A.; Trifonova, A.; Keizer, L. C. P., et al. The effect of exogenously-applied phytophormones on gene transfer efficiency in sugarbeet (Beta vulgaris L.). Plant Sci. 116:97–106; 1996.
Kruse, T.; Tallman, G.; Zeiger, E. Isolation of guard cell protoplasts from mechanically prepared epidermis ofVicia faba leaves. Plant Physiol. 90:1382–1386; 1989.
Laparra, H.; Bronner, R.; Hahne, G. Amyloplasts as a possible indicator of morphogenic potential in sunflower protoplasts. Plant Sci. 122:183–192; 1997.
Nomura, K.; Komamine, A. Identification and isolation of single cells that produce somatic embryos at a high frequency in a carrot suspension culture. Plant Physiol. 79:988–991; 1985.
Pedersen, C.; Hall, R. D.; Krens, F. A. Petioles as the tissue source for isolation and culture ofBeta vulgaris andB. maritima protoplasts. Plant Sci. 95:89–97; 1993.
Pillai, K. G.; Rao, U.; Rao, I. V. R., et al. Induction of division and differentiation of somatic embryos in the leaf epidermis ofGaillardia picta. Plant Cell Rep. 10:599–603; 1992.
Roest, S.; Gilissen, L. J. W. Regeneration from protoplasts—a supplementary literature review. Acta Bot. Neerl. 42:1–23; 1993.
Sahgal, P.; Martinez, G. V.; Roberts, C., et al. Regeneration of plants from cultured guard cell protoplasts ofNicotina glauca. Plant Sci. 97:199–208; 1994.
Schweiger, H.-G.; Dirk, J.; Koop, H.-U., et al. Individual selection, culture and manipulation of higher plant cells. Theor. Appl. Genet. 73:769–783; 1987.
Shillito, R. D.; Saul, M. W.; Paszkowski, J., et al. High efficiency direct gene transfer to plants. Bio/Technology 3:1099–1103; 1985.
Toonen, M. A. J.; Hendriks, T.; Schmidt, E. D. L., et al. Description of somatic embryo-forming single cells in carrot suspension cultures employing video cell tracking. Planta 194:565–572; 1994.
Toonen, M. A. J.; de Vries, S. C. Use of video cell tracking to identify embryogenic cultured cells. In: Lindsey, K., ed. Plant tissue culture manual. Dordrecht, Netherlands: Kluwer Academic Publishers; 1997:H1–45.
Tran Thanh Van, K. M. Control of morphogenesis inin vitro cultures. Annu. Rev. Plant Physiol. 32:291–311; 1981.
Waara, S.; Glimelius, K. The potential of somatic hybridization in crop breeding. Euphytica 85:217–233; 1995.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Krens, F.A., Verhoeven, H.A., Van Tunen, A.J. et al. The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation. In Vitro Cell.Dev.Biol.-Plant 34, 81–86 (1998). https://doi.org/10.1007/BF02822769
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02822769