Summary
Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 104 pollen grains were GUS+. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS+ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.
Similar content being viewed by others
Abbreviations
- GUS:
-
ß-glucuronidase
- CaMV:
-
Cauliflower Mosaic Virus
- MCS:
-
multicellular structure
- NPTII:
-
neomycin phosphotransferase
- PEG:
-
polyethylene glycol
- X-gluc:
-
5-bromo-4-chloro-3-indolyl glucuronide
- DAPI:
-
4,6-diamidino-2-phenylindole
- Tris:
-
Tris(hydroxymethyl)aminomethane hydrochloride
- EDTA:
-
ethylenedinitrilo tetraacetic acid, disodium salt dihydrate
References
Alwen A, Benito Moreno RM, Vicente O, Heberle-Bors E (1992) Transgenic Res 1:63–70
Benito Moreno RM, Macke F, Hauser MT, Alwen A, Heberle-Bors E (1988) In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants, Springer, Berlin Heidelberg New York, pp 137–142
Chase SS (1969) Bot Rev 35:117–167
Datta SK, Wenzel G (1987) Plant Sci 48:49–54
Duijs JG, Voorrips RE, Custers JBM (1989) Acta Bot Neerl 38:343–344
Gaillard A, Vergne P, Beckert M (1991) Plant Cell Rep 10:55–58
Garrido D, Charvat B, Benito-Moreno RM, Alwen A, Vicente O, Heberle-Bors E (1991) In: Negrutiu I, Gharti-Chhetri G (eds) A laboratory guide for cellular and molecular plant biology, Birkhäuser, Basel, pp 59–69
Heberle-Bors E, Reinert J (1980) Naturwissenschaften 67:311
Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F (1992) Plant Science 86:89–96
Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers S, Fraley R (1985) Science 227:1229–1231
Huang B, Bird S, Kemble R (1991) In Vitro Cell Dev Biol 27:28–31
Huang B (1992) In Vitro Cell Dev Biol 28:53–58
Jefferson RA (1987) Plant Mol Biol Rep 5:387–405
Klein TM, Wolf ED, Wu R, Sanford JC (1987) Nature 327:70–73
Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Bio/technology 6:559–566
Kuhlmann U, Foroughi-Wehr B, Graner A, Wenzel G (1991) Plant Breeding 107:165–168
Maniatis T, Fritsch BF, Sambrook J (1982) Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Mascarenhas JP, Hamilton DA (1992) Plant J 2:405–408
Matthews BF, Abdul-Baki AA, Saunders JA (1990) Sex Plant Reprod 3:147–151
Morrison RA, Evans DA (1988) Bio/Technology 6:684–690
Murashige T, Skoog F (1962) Physiol Plant 15:473–497
Neuhaus G, Spangenberg G, Mittelsten-Scheid O, Schweiger HG (1987) Theor Appl Genet 75:30–36
Pechan PM (1989) Plant Cell Rep 8:387–390
Plegt LM, Ven BCE, Bino RJ, Salm TPM, van Tunen AJ (1992) Plant Cell Rep 11:20–24
Siva Reddy V, Leelavathi S, Sen S (1985) Physiol Plant 63:309–314
Stöger E, Benito Moreno RM, Ylstra B, Vicente O, Heberle-Bors E (1992) Transgenic Res 1:71–78
Swanson EB, Coumans MP, Wu SC (1987) Plant Cell Rep 6:94–97
Twell D, Klein TM, Fromm ME, McCormick S (1989) Plant Physiol 91:1270–4
Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L (1990) Mol Gen Genet 220:245–50
Ziauddin A, Simion E, Kasha KJ (1990) Plant Cell Rep 9:69–72
Zumbrunn G, Schneider M, Rochaix JD (1989) Technique 1:204–16
Author information
Authors and Affiliations
Additional information
Communicated by I. Potrykus
Rights and permissions
About this article
Cite this article
Stöger, E., Fink, C., Pfosser, M. et al. Plant transformation by particle bombardment of embryogenic pollen. Plant Cell Reports 14, 273–278 (1995). https://doi.org/10.1007/BF00232027
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00232027