Summary
An attempt was made to transfer the T-DNA of Agrobacterium tumefaciens, previously introduced into plant cells, via protoplast fusion from one species into another. For the experiments two cell lines were used: firstly, a Nicotiana paniculata cell line transformed with the Agrobacterium strain B6S3. This cell line exhibits both hormone independent growth and synthesis of octopine as a result of the incorporated T-DNA from Agrobacterium. These two markers are dominant. The second cell line was the nitrate reductase deficient cnx-68 cell line of N. tabacum which contains an intracellular calcium oxalate druse. These two markers are recessive. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone free agar medium supplemented with 50 mM KClO3. This compound is toxic for cells possessing nitrate reductase activity. From about 1.1×107 cultivated protoplasts 18 cell lines survived the selection treatment. Of these seven exhibited the two dominant and the two recessive markers, whereas the others showed either only one or none of the recessive or only one of the dominant markers. In dot-blot experiments using species specific DNA clones of the donor and the recipient plant species it was confirmed that besides the T-DNA other nuclear genomic DNA of the donor species had also been transferred in various amounts. The possible consequences of these results for plant breeding programmes are discussed.
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References
Binding H (1974) Cell cluster formation by leaf protoplasts from axenic cultures of haploid Petunia hybrida L. Plant Sci Lett 2:185–188
Bohnert HJ, Crouse EJ, Schmitt JM (1982) Organization and expression of plastid genomes. In: Parthier B, Boulter D (eds) Encyclopedia of plant physiology. New series, vol. 14B: Nucleic acids and proteins in plants II. Springer-Verlag, Berlin, Heidelberg, New York, pp 476–530
Dudits D, Praznovsky T (1985) Intergeneric gene transfer by protoplast fusion and uptake of isolated chromosomes. In: Zaitlin M, Day P, Hollaender A (eds) Biotechnology in plant science: Relevance to agriculture in the eighties. Academic Press, Orlando, pp 115–127
Fluhr R, Aviv D, Edelman M, Galun E (1983) Cybrids containing mixed and sorted-out chloroplasts following interspecific somatic fusions in Nicotiana. Theor Appl Genet 65:289–294
Gupta PP, Gupta M, Schieder O (1982) Correction of nitrate reductase defect in auxotrophic plant cells through protoplastmediated intergeneric gene transfer. Mol Gen Genet 188:378–383
Gupta PP, Schieder O, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatible plants through asymmetric fusion. Mol Gen Genet 197:30–35
Hain R, Stabel P, Czernilofsky AP, Steinbiß H-H, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol Gen Genet 199:161–168
Hein T, Przewozny T, Schieder O (1983) Culture and selection of somatic hybrids using an auxotrophic cell line. Theor Appl Genet 64:119–122
Itho K, Futsuhara Y (1983) Interspecific transfer of only a part of genome by fusion between non-irradiated protoplasts of Nicotiana glauca and X-ray irradiated protoplasts of N. langsdorffii. Jpn J Genet 58:545–553
Larkin PJ, Brettel RIS, Ryan SA, Davies PA, Pallotta MA, Scowcroft WR (1985) Somaclonal variation: Impact on plant biology and breeding strategies. In: Zaitlin M, Day P, Hollaender A (eds) Biotechnology in plant science: Relevance to agriculture in the eighties. Academic Press, Orlando, pp 83–100
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127
Lörz H, Baker B, Schell J (1985) Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199:178–182
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York
Müller AG, Grafe R (1978) Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol Gen Genet 161:67–76
Müller-Gensert E, Schieder O (1985) Non-random plastid segregation in somatic hybrids of Datura innoxia with several other solanaceous species. Curr Genet 10:335–337
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Otten LABM, Schilperoort RA (1978) A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500
Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants EMBO J 3:2717–2722
Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer to cells of a graminaceous monocot. Mol Gen Genet 199:183–188
Saul MW, Potrykus I (1984) Species-specific repetitive DNA used to identify interspecific somatic hybrids. Plant Cell Rep 3:65–67
Schell J, Herrera-Estrella L, Zambryski P, de Block M, Joos H, Willmitzer L, Eckes P, Rosahl S, van Montagu M (1984) Genetic engineering of plants. In: Schell JS, Starlinger P (eds) The impact of gene transfer techniques in eukaryotic cell biology. Springer, Berlin, Heidelberg, New York, pp 73–90
Schieder O (1977) Hybridization experiments with protoplasts from chlorophyll-deficient mutants of some solanaceous species. Z Pflanzenphysiol 137:253–257
Schieder O (1984) Isolation and culture of protoplasts: Datura. In: Vasil I (ed) Cell culture and somatic cell genetics of plants. Academic Press, Orlando, pp 350–355
Somers DA, Naravanan KR, Kleinhofs A, Cooper-Bland S, Cocking EC (1986) Evidence for transfer of barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion. Mol Gen Genet 204:296–301
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Sparks RB, Dale RMK (1980) Characterization of 3H-labeled mitochondrial DNA from tobacco suspension culture cells. Mol Gen Genet 180:351–355
Steffen A, Eriksson T, Schieder O (1986) Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens generally not achievable with untransformed protoplasts. Theor Appl Genet 72:135–140
Taylor B, Powell A (1982) Isolation of plant DNA and RNA. Focus 4:4–6
Van Slogteren GMS, Hooykaas, PJJ, Schilperoort RA (1984) Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment. Plant Mol Biol 3:333–336
Zelcer A, Aviv D, Galun E (1978) Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts of normal Nicotiana sylvestris and X-ray irradiated protoplasts of male-sterile N. tabacum. Z Pflanzenphysiol 90:397–408
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Communicated by G. Melchers
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Müller-Gensert, E., Schieder, O. Interspecific T-DNA transfer through plant protoplast fusion. Mole Gen Genet 208, 235–241 (1987). https://doi.org/10.1007/BF00330448
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DOI: https://doi.org/10.1007/BF00330448