Abstract
Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 μg ml-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5′ end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.
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References
Andebrhan T (1985) Studies on the epidemiology and control of witches' broom disease of cocoa in the Brazilian Amazon. In: Proceedings of the 9th International Cocoa Research Conference. (pp 395–402). Stephen Austin and Sons LTD, Hertford
An G, Watson BD, Stachel S, Gordon MP & Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J. 4: 277–284
Baker B, Coupland G, Fedoroff NV, Starlinger P & Schell J (1987) Phenotypic assay for excision of the maize controlling element Ac in tobacco. EMBO J. 6: 1547–1554
Bachmann BJ, Low KB & Taylor AL (1976) Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40: 116–167
Becker D, Kemper E, Schell J & Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol. Biol. 20: 1195–1197
Bevan M, Barnes WM & Chilton M-D (1989) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucl. Acids Res. 11: 369–385
Binns AN & Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Ann. Rev. Microbiol. 42: 575–606
Chilton WS, Tempe J, Matzke M & Chilton M-D (1984) Succinamopine: a new crown gall opine. J. Bacteriol. 157: 357–362
Christou P (1992) Genetic transformation of crop plants using microprojectile bombardment. Plant J. 2: 275–281
Couch JA & Fritz PF (1990) Isolation of DNA from plants high in polyphenolics. Plant Mol. Biol. Rep. 8: 8–12
Couch JA, Zintel HA & Fritz PF (1993) The genome of the tropical tree Theobroma cacao L. Mol. Gen. Genet. 237: 123–128
Depicker A, Herman L, Jacobs A, Schell J & Van Montagu M (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol. Gen. Genet. 201: 477–484
Dhaese P, De Greve H, Decraemer H, Schell J & Van Montagu M (1979) Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucl. Acids Res. 7: 1837–1849
Duhem K, Le Mercier N & Boxus Ph (1989) New data on the induction and the development of somatic embryos in Theobroma cacao L. Café Cacao Thé 32: 9–14
Figueira A & Janick J (1993) Development of nucellar somatic embryos of Theobroma cacao. Acta Horticulturae 336: 231–238
Figueira A, Janick J & Goldsbrough P (1992) Genome size and DNA polymorphism in Theobroma cacao. J. Amer. Soc. Hort. Sci. 117: 673–677
Gheysen G, Villarroel R & Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev. 5: 287–297
Greathouse DD, Laetsch WM & Phinney BO (1971) The shoot-growth rhythm of a tropical tree, Theobroma cacao. Amer. J. Bot. 58: 281–286
Guyon P, Chilton M-D, Petit A & Tempe J (1980) Agropine in ‘null-type’ crown gall tumors: Evidence for generality of the opine concept. Proc. Natl. Acad. Sci. USA 77: 2693–2697
Hofgen R & Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 16: 9877
Holford P & Newbury HJ (1992) The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus. Plant Cell Rep. 11: 93–96
Hood EE, Chilton WS, Chilton M-D & Fraley RT (1986a) T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants. J. Bacteriol. 168: 1283–1290
Hood EE, Gelvin SB, Melchers LS & Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2: 208–218
Hood EE, Helmer GL, Fraley RT & Chilton M-D (1986b) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168: 1291–1301
Jefferson RA, Burgess SM & Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene fusion marker. Proc. Natl. Acad. Sci. USA 83: 8447–8451
Lopez-Baez O, Bollon H, Eskes A & Petiard V (1993) Somatic embryogenesis and plant regeneration from flower parts of cocoa (Theobroma cacao L.) C. R. Acad. Sci. Paris, Sciences de la Vie/Life Sciences 316: 579–584
Lowe JM, Davey MR, Power JB & Blundy KS (1993) A study of some factors affecting Agrobacterium transformation and plant regeneration of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.) Plant Cell Tiss. Org. Cult. 33: 171–180
Maliga P, Sz.-Breznovits A & Marton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nature New Biol. 244: 29–30
Purdy LH & Dickstein ER (1989) Theobroma cacao, a host for Agrobacterium tumefaciens. Plant Disease 73: 638–639
Reiss B, Sprengel R, Will H & Schaller H (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30: 211–218
Rodriguez RL & Tait RC (1983) Recombinant DNA Techniques: An Introduction. Benjamin/Cummings, Menlo Park
Sambrook J, Fritsch EF & Maniatis J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sondahl M, Sereduk TB, Chen Z & Bellato CM (1989) Somatic embryogenesis and plant regeneration of cacao, patent no. 88/3078, Republic of South Africa
Thorold CA (1975) Diseases of Cocoa. Clarendon Press, Oxford
Wood GAR & Lass RA (1987) Cocoa, 4th edition. Longman Scientific & Technical and John Wiley & Sons, New York
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Sain, S.L., Oduro, K.K. & Furtek, D.B. Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens . Plant Cell Tiss Organ Cult 37, 243–251 (1994). https://doi.org/10.1007/BF00042337
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DOI: https://doi.org/10.1007/BF00042337