Abstract
An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.
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Boxus P, Quoirin M, Laine JM (1977) In: Rienert J, Bajaj YPS (eds) Applied and fundamental aspects of plant, cell, tissue and organ culture, Springer-Verlag, New York, pp 261–271
Bradford MM (1976) Anal Biochem 72:248–254
Bringhurst RS, Voth V (1981) In: Childers NF (ed) The strawberry, Horticultural publications, 3906 N.W. 31st place, Gainesville, Florida 32601, pp 156–164
Dellaporta SL, Wood J, Hicks JB (1983) Plant Mol Biol Rep 1:19–21
Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Proc Natl Acad Sci USA 77:7347–7351
Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCormick SM, Niedermeyer JG, Dean DA, Kretzmer KK, Mayer EJ, Rochester DE, Rogers SG, and Fraley RT (1987) Bio/Technology 5:807–813
Fraley RT, Rogers SG, Horsch RB (1986) CRC Crit Rev Plant Sci 4:1–46
Gamborg OL, Miller RA, Ojima K (1968) Expt Cell Res 50:151–158
Gasser CS, Fraley RT (1989) Science 244:1293–1299
Hanahan D (1983) J Mol Biol 166:557–580
Hancock JF, Scott DH (1988) Fruit Var J 24:102–108
Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Bio/Technology 6:915–922
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 303:179–180
Hoekema A, Huisman MJ, Molendijk L, Van den-Elzen PJM, Cornelissen BJC (1989) Bio/Technology 7:273–278
Horsch RB, Fry JE, Hoffmann ML, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227:1229–1231
James DJ (1987) Biotechnology and Genetic Engineering Reviews 5:33–79
James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Plant Cell Reports 7:658–661
Jefferson RA (1987) Plant Mol Biol Rep 5:387–405
Jefferson RA, Kavanagh TA, Bevan MW (1987) EMBO J 6:3901–3907
Kartha KK, Leung ML, Pahl K (1980) J Amer Soc Hort Sci 105:481–484
Koncz C, Schell J (1986) Mol Gen Genet 204:383–396
McCabe DE, Swain WF, Martinell BJ, Christou P (1988) Bio/Technology 6:923–926
McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Plant Cell Reports 5:81–84
McDonnell RE, Clark RD, Smith WA, Hinchee MA (1987) Plant Mol Biol Rep 5:380–386
McHughen A, Jordan MC (1989) Plant Cell Reports 7:611–614
Murashige T, Skoog F (1962) Physiol Plant 15:473–497
Nagata T, Okada K, Kawazu T, Takebe I (1987) Mol Gen Genet 207:242–244
Nehra NS, Chibbar RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C (1990) Plant Cell Reports 9:10–13
Nehra NS, Stushnoff C, Kartha KK (1989) J Amer Soc Hort Sci 114:1014–1018
Ooms G, Burrell MM, Karp A, Bevan MW, Hille J (1987) Theor Appl Genet 73:744–750
Pua E, Mehra-Palta A, Nagy F, Chua N (1987) Bio/Technology 5:815–817
Stiekema WJ, Heidekamp F, Louwerse JD, Verhoeven HA, Dijkhuis P (1988) Plant Cell Reports 7:47–50
Takashi A, Hirano H, Naito S, Komeda Y (1989) Plant Cell Reports 8:259–262
Terada R, Shimamoto K (1990) Mol Gen Genet 220:389–392
Visser RGF, Jacobsen E, Hesseling-Meinders A, Schans MJ, Witholt B, Feenstra WJ (1989) Plant Mol Biol 12:329–337
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Communicated by F. Constabel
NRCC No. 31491
During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).
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Nehra, N.S., Chibbar, R.N., Kartha, K.K. et al. Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Reports 9, 293–298 (1990). https://doi.org/10.1007/BF00232854
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DOI: https://doi.org/10.1007/BF00232854