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Efficient transformation of Agrobacterium spp. by high voltage electroporation

Efficient transformation of Agrobacterium spp. by high voltage electroporation Downloaded from https://academic.oup.com/nar/article/17/20/8385/1248996 by DeepDyve user on 17 August 2022 Volume 17 Number 20 1989 Nucleic Acids Research Efficient transformation of Agrobaderium spp. by high voltage electroporation Shen Wen-jun and Brian G.Forde* Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ, UK Submitted September 15, 1989 Transformation of agrobacteria has hitherto been limited to a few isolates, and at best gives only 10^ transformants/vg (1). PIasaids are therefore usually introduced by conjugative transfer from Escherichia coll. We have found that efficient transformation of two strains widely used in plant genetic manipulation can be obtained by the application of a high voltage electric pulse under conditions similar to those giving high frequency transformation of E. coli (2). Since only small amounts of plasmid DMA are required, electroporation also opens up the possibility of direct cloning in Agrobacteriua without the need to use E. coli as an intermediate host. Strains; A. ttmefaciens LBA4404 (pRAL4404), a derivative of Ach5; A. rhizogenes LBA9402 (pR11855), a rifr derivative of NCPPB 1855. Preparation of cells: Agrobacterial cultures were grown at 29° for 24-30 h in 2 X YT medium to an OD^Q O °f 0.5-0.7. Cells were cooled on ice, pelleted, washed successively in 1, 0.5, 0.02 and 0.02 culture volumes of cold 1QJ (v/v) glycerol and resuspended in 0.01 volume 10X glycerol (10H-10'2 cells/ml). Aliquots were frozen in liquid N2 and could be stored at -70° for at least 6 weeks. Transformation: Frozen cells were thawed on ice and a 40 ill aliquot was transferred to a pre-cooled 0.2 cm electroporation cuvette (Bio-Rad Laboratories Ltd.) One Vl of plasmid DNA (2-10 ng) was mixed with the cell suspension on ice and an electric pulse applied immediately using a Gene Pulser1111 with Pulse Controller unit (Bio-Rad). Highest transformation efficiencies (up to 8 X 10s) were obtained at a field strength of 12.5 kV/cm, a capacitance of 25 uF and resistors of 400 or 600 ohms in parallel with the sample, giving time constants of 8-12 msec (Fig. 1). The cells were immediately transferred to 1 ml YMB or TY and shaken at 29° for 3 h. Aliquots of 10 ill or 100 ul were plated on YMB agar containing appropriate antibiotics and incubated for 3 d at 29°. Figure 1. Effect of field strength and pulse length on efficiency of transformation of A. tumefaciens. 7.5 ng of pBI121 DNA (3) was used for each electroporation and the pulse lengths were varied by selecting resistors of 100 to 1000 ohms at field strengths of 10 kV/cm ( O ) and 12.5 kV/cm ( • ) . 5 10 15 20 Similar results were obtained with A. Time constant (msec) rhizogenes. *To whom correspondence should be addressed References: (1) Hooykaas PJJ (1988) In Gelvin SB and Schilperoort RA (eds) Plant Mol Biol Manual, Kluwer Academic Publishers, Dordrecht, Section A4, pp 1-13. (2) Dower WJ et al. (1988) Nucl Acids Res 16:6127-6145. (3) Jefferson RA et al. (1987) EMBO J 6:3901-3907 ©IRL Press 8385 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Efficient transformation of Agrobacterium spp. by high voltage electroporation

Wen-jun, Shen; Forde, Brian G.
Nucleic Acids Research , Volume 17 (20) – Oct 25, 1989
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Oxford University Press
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Copyright © 2022 Oxford University Press
ISSN
0305-1048
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1362-4962
DOI
10.1093/nar/17.20.8385
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Abstract

Downloaded from https://academic.oup.com/nar/article/17/20/8385/1248996 by DeepDyve user on 17 August 2022 Volume 17 Number 20 1989 Nucleic Acids Research Efficient transformation of Agrobaderium spp. by high voltage electroporation Shen Wen-jun and Brian G.Forde* Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ, UK Submitted September 15, 1989 Transformation of agrobacteria has hitherto been limited to a few isolates, and at best gives only 10^ transformants/vg (1). PIasaids are therefore usually introduced by conjugative transfer from Escherichia coll. We have found that efficient transformation of two strains widely used in plant genetic manipulation can be obtained by the application of a high voltage electric pulse under conditions similar to those giving high frequency transformation of E. coli (2). Since only small amounts of plasmid DMA are required, electroporation also opens up the possibility of direct cloning in Agrobacteriua without the need to use E. coli as an intermediate host. Strains; A. ttmefaciens LBA4404 (pRAL4404), a derivative of Ach5; A. rhizogenes LBA9402 (pR11855), a rifr derivative of NCPPB 1855. Preparation of cells: Agrobacterial cultures were grown at 29° for 24-30 h in 2 X YT medium to an OD^Q O °f 0.5-0.7. Cells were cooled on ice, pelleted, washed successively in 1, 0.5, 0.02 and 0.02 culture volumes of cold 1QJ (v/v) glycerol and resuspended in 0.01 volume 10X glycerol (10H-10'2 cells/ml). Aliquots were frozen in liquid N2 and could be stored at -70° for at least 6 weeks. Transformation: Frozen cells were thawed on ice and a 40 ill aliquot was transferred to a pre-cooled 0.2 cm electroporation cuvette (Bio-Rad Laboratories Ltd.) One Vl of plasmid DNA (2-10 ng) was mixed with the cell suspension on ice and an electric pulse applied immediately using a Gene Pulser1111 with Pulse Controller unit (Bio-Rad). Highest transformation efficiencies (up to 8 X 10s) were obtained at a field strength of 12.5 kV/cm, a capacitance of 25 uF and resistors of 400 or 600 ohms in parallel with the sample, giving time constants of 8-12 msec (Fig. 1). The cells were immediately transferred to 1 ml YMB or TY and shaken at 29° for 3 h. Aliquots of 10 ill or 100 ul were plated on YMB agar containing appropriate antibiotics and incubated for 3 d at 29°. Figure 1. Effect of field strength and pulse length on efficiency of transformation of A. tumefaciens. 7.5 ng of pBI121 DNA (3) was used for each electroporation and the pulse lengths were varied by selecting resistors of 100 to 1000 ohms at field strengths of 10 kV/cm ( O ) and 12.5 kV/cm ( • ) . 5 10 15 20 Similar results were obtained with A. Time constant (msec) rhizogenes. *To whom correspondence should be addressed References: (1) Hooykaas PJJ (1988) In Gelvin SB and Schilperoort RA (eds) Plant Mol Biol Manual, Kluwer Academic Publishers, Dordrecht, Section A4, pp 1-13. (2) Dower WJ et al. (1988) Nucl Acids Res 16:6127-6145. (3) Jefferson RA et al. (1987) EMBO J 6:3901-3907 ©IRL Press 8385

Journal

Nucleic Acids Research – Oxford University Press

Published: Oct 25, 1989

Keywords: agrobacterium; electroporation; voltage

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