Summary
The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.
Similar content being viewed by others
Abbreviations
- AIM:
-
Agrobacterium infection medium
- CIM:
-
callus-inducing medium
- CTAB:
-
cetyltrimethylammonium bromide
- 2,4-D:
-
2,4-dichlorophenoxy-acetic acid
- GUS:
-
ß-glucuronidase
- hph:
-
hygromycin phosphotransferase
- IAA:
-
indole-3-acetic acid
- IBA:
-
indole-3-butyric acid
- 2ip:
-
N -(2-isopentenyl) adenine
- NPTII:
-
neomycin phosphotransferase II
- RIM:
-
root-inducing medium
- 35S:
-
cauliflower mosaic virus 35S promoter
- SIM:
-
shoot-inducing medium
References
Akama K and Tanifuji S (1990) Plant Mol. Biol. 15:337–346
Chang S-S, Park S-K, Nam H-G (1990) In: Abstract of the Fourth International Conference on Arabidopsis Research, Vienna, pp28
Christianson ML, Warnick DA (1983) Dev. Biol. 95:288–293
Clarke MC, Wei W, Lindsey K (1992) Plant Mol.Biol.Rep. 10:178–189
Doyle JJ, Doyle JL (1990) Focus 12:13–15
Estelle MA, Somerville CR (1986) Trends in Genetics 2:89–93
Feldmann KA, Marks MD (1986) Plant Sci. 47:63–69
Feldmann KA, Marks MD (1987) Mol. Gen. Genet. 208:1–9
Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:151–158
Hoekema A, Hirsch P, Hooykaas P, Schilperoort R (1983) Nature 303:179–180
Hood EE, Helmer GL, Fraley RT, Chilton, M-D (1986) J.Bacteriol. 168:1291–1301
Jefferson RA (1987) Plant Mol. Biol. Rep. 5:387–405
Jefferson RA, Kavanagh TA, Bevan M (1987) EMBO J. 6:3901–3907
Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464–466
Márton L, Browse J (1991) Plant Cell Rep. 10:235–239
Meyerowitz EM, Pruitt RE (1985) Science 229:1214–1218
Moloney MM, Walker JM, Sharma K (1989) Plant Cell Rep. 8:238–242
Murashige T, Skoog F (1962) Physiol. Plant 15:473–497
Ohta S, Mita S, Hattori T, Nakamura K (1990) Plant Cell Physiol. 31:805–813
Rédei GP, Koncz C, Schell J (1988) In: Gustafson JP, Appols R (eds) Chromosome structure and function. Plenum Press, New York, pp 175–200
Sangwan RS, Bourgeois Y, Sangwan-Norreel BS (1991) Mol. Gen. Gent. 230:475–485
Schmidt R, Willmitzer L (1988) Plant Cell Rep. 7:583–586
Southern EM (1975) J. Mol. Biol. 98:503–517
Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Proc. Natl. Acad. Sci. USA 85:5536–5540
Van Haute E, Joos H, Maes S, Warren G, Van Montagu M, Schell J (1983) EMBO J. 2:411–418
Author information
Authors and Affiliations
Additional information
Communicated by J. M. Widholm
Rights and permissions
About this article
Cite this article
Akama, K., Shiraishi, H., Ohta, S. et al. Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains. Plant Cell Reports 12, 7–11 (1992). https://doi.org/10.1007/BF00232413
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00232413