Summary
Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity. rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.
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References
Braun AC (1956) The activation of two growth substance systems accompanying the conversion of normal to tumor cells in crown gall. Cancer Res 16:53–56
Braun AC (1959) A demonstration of the recovery of the crown gall tumor cell with the use of complex tumors of single cell origin. Proc Natl Acad Sci USA 45:932–938
Braun AC, Wood HN (1976) Suppression of the neoplastic state with the acquisition of specilized functions in cells, tissues, and organs of crown gall teratomas of tobacco. Proc Natl Acad Sci USA 73:496–500
Chilton M-D, Drummond HJ, Merlo DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–271
Chilton M-D, Saiki RK, Yadav N, Gordon MP, Quetier F (1980) T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci USA 77:4060–4064
De Greve H, Decraemer H, Seurinck J, Van Montagu M, Schell J (1981) The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6S3. Plasmid 6 (in press)
Drummond MH, Gordon MP, Nester EW, Chilton M-D (1977) Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours. Nature 269:535–536
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Gurley WB, Kemp JD, Albert MJ, Sutton DW, Callis J (1979) Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines. Proc Natl Acad Sci USA 76:2828–2832
Ilernalsteens JP, Van Vliet F, De Beuckeleer M, Depicker A, Engler G, Lemmers M, Holsters M, Van Montagu M, Schell J (1980) The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. Nature 287:654–656
Kemp JD (1976) Octopine as a marker for the induction of tumorous growth by Agrobacterium tumefaciens strain B6. Biochem Biophys Res Commun 69:816–822
Leemans J, Shaw Ch, De Blaere R, De Greve H, Hernalsteens JP, Van Montagu M, Schell J (1981) Intermediate Ti-vectors for site specific mutagenesis of T-DNA and for transfer of genes to plant cells. J Mol Appl Genet (in press)
Lemmers M, De Beuckeleer M, Holsters M Zambryski P, Depicker A, Hernalsteens JP, Van Montagu M, Schell J (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours. J Mol Biol 144:355–378
Martón L, Wullems GJ, Molendijk L, Schilperoort RA (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277:129–130
Murashige T, Skoog S (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–497
Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87
Otten LA, Schilperoort RA (1978) A rapid microscale method for the detection of lysopine-and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500
Sacristán MD, Melchers G (1977) Regeneration of plants from “habituated” and “Agrobacterium-transformed” single cell clones of tobacco. Mol Gen Genet 152:111–117
Schell J, Van Montagu M (1977) The Ti plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of NIF genes in plants? In: Hollaender A (ed) Genetic engineering for nitrogen fixation. Plenum Press, New York, p 159–179
Schell J, Van Montagu M, De Beuckeleer M, De Block M, Depicker A, De Wilde M, Engler G, Genetello C, Hernalsteens JP, Holsters M, Seurinck J, Silva B, Van Vliet F, Villarroel R (1979) Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proc R Soc Lond B 204:251–266
Schieder O (1978) Somatic hybrids of Datura innoxia Mill.+Datura discolor Bernh. and of Datura innoxia Mill.+Datura stramonium L. var. tatula L. I. Selection and characterization. Mol Gen Genet 162:113–119
Schilperoort RA, Klapwijk PM, Hooykaas PJJ, Koekman BP, Ooms G, Otten LABM, Wurzer-Figurelli EM, Wullems G, Rörsch A (1978) A. tumefaciens plasmids as vectors for genetic transformation of plant cells. In: Thorpe TA (ed) Frontiers of plant tissue culture, Proc. 4th Int. Congress of plant tissue and cell culture. University of Calgary, Alberta, p 85–94
Schröder J, Schröder G, Huisman H, Schilperoort RA, Schell J (1981) The mRNA for lysopine dehydrogenase in plant tumor cells is complementary to a Ti-plasmid fragment. FEBS Lett 129:166–168
Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW (1980a) Intergation and organization of Ti plasmid sequences in crown gall tumors. Cell 19:729–739
Thomashow MF, Nutter R, Postle K, Chilton M-D, Blattner FR, Powell A, Grodon MP, Nester EW (1980b) Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens. Proc Natl Acad Sci USA 77:6448–6452
Turgeon R, Wood MN, Braun AC (1976) Studies on the recovery of crown gall tumor cells. Proc Natl Acad Sci USA 73:3562–3564
Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability.. Nature 252:169–170
Willmitzer L, Otten L, Simons G, Schmalenbach W, Schröder J, Schröder G, Van Montagu M, De Vos G, Schell J (1981) Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures. Mol Gen Genet 182:255–262
Willmitzer L, De Beuckeleer M, Lemmers M, Van Montagu M, Schell J (1980) The Ti-plasmid derived T-DNA is present in the nucleus and absent from plastids of plant crown-gall cells. Nature 287:359–361
Wood HN, Binns AN, Braun AC (1978) Differential expression of oncogenicity and nopaline synthesis in intact leaves derived from crown gall teratomas of tobacco. Differentiation 11:175–180
Wullems GJ, Molendijk L, Schilperoort RA (1980) The expression of tumour markers in intraspecific somatic hybrids of normal and crown gall cells, from Nicotiana tabacum. Theor Appl Genet 56:203–208
Yadav NS, Postle K, Saiki RK, Thomashow MF, Chilton M-D (1980) T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature 287:458–461
Yang F, Montoya AL, Merlo DJ, Drummond MH, Chilton M-D, Nester EW, Gordon MP (1980) Foreign DNA sequences in crown gall teratomas and their fate during the loss of the tumorous traits. Mol Gen Genet 177:707–714
Zambryski P, Holsters M, Kruger K, Depicker A, Schell J, Van Montagu M, Goodman HM (1980) Tumor DNA structure in plant cells transformed by A. tumefaciens. Science 209:1385–1391
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Communicated by W. Arber
This paper is dedicated to Prof. Georg Melchers on the occasion of his 75th birthday, in recognition and gratitude for his relentless and enthousiastic pioneering efforts in the field of experimental plant genetics and cell biology
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Otten, L., De Greye, H., Hernalsteens, J.P. et al. Mendelian transcmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens . Molec. Gen. Genet. 183, 209–213 (1981). https://doi.org/10.1007/BF00270619
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DOI: https://doi.org/10.1007/BF00270619