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Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

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Abstract

The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. InNicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F1 monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the “discordant” segregation values observed between corresponding R1 and F1 progenies in up to 9% of the evaluated transformants. In addition, 5% of the transformants showed a phenotypic loss of resistance. In the F3 generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F4 generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved.N. plumbaginifolia exhibited more instability thanN. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.

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References

  • Assaad, F.F. and Signer, E.R. (1992) Somatic and germinal recombination of a direct repeat inArabidopsis, Genetics 132, 553–66.

    Google Scholar 

  • Cherdshewasart, W. (1991a) Total DNA extraction-alternative protocols. In Negrutiu, I. and Gharti-Chhetri, G.B., eds,A Laboratory Guide for Cellular and Molecular Plant Biology, pp. 161–6. Basel/Switzerland: Birkhauser Verlag AG.

    Google Scholar 

  • Cherdshewasart, W. (1991b) Southern blot analysis of transgenicNicotiana sp. In Negrutiu, I. and Gharti-Chhetri, G.B. eds,A Laboratory Guide for Cellular and Molecular Plant Biology, pp. 214–224. Basel/Switzerland: Birkhauser Verlag, AG.

    Google Scholar 

  • Cherdshewasart, W. (1991c) Assessing methylation of inserted DNA by restriction with isoschizomeric enzymes and inducing demethylation with 5-azacytidine. In Negrutiu, I. and Gharti-Chhetri, G.B. eds,A Laboratory Guide for Cellular and Molecular Plant Biology, pp 277–86. Basel, Switzerland: Birkhauser Verlag AG.

    Google Scholar 

  • Derr, L.K. and Strathern, J.N. (1993) A role for reverse transcripts in gene conversion.Nature 361, 170–3.

    Google Scholar 

  • Flavell, R.B. (1985) Repeated sequences and genome change. In Hohn, B. and Dennis, E.S. eds.,Genetic Flux in Plants pp. 130–56. Vienna: Springer Verlag.

    Google Scholar 

  • Gal, S., Pisan, B., Hohn, T., Grimsley, N. and Hohn, B. (1991) Genomic homologous recombinationin planta.EMBO J. 10, 157–78.

    Google Scholar 

  • Gharti-Chhetri, G.B., Cherdshewasart, W., Dewulf, J., Paszkowski, J., Jacobs, M. and Negrutiu, I. (1990) Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration, its inheritance and factors affecting these processes inNicotiana plumbaginifolia.Plant Mol. Biol. 14, 687–96.

    Google Scholar 

  • Gharti-Chhetri, G.B., Cherdshewasart, W., Dewulf, J., Jacobs M. and Negrutiu, I. (1992) PEG-mediated direct gene transfer-transient gene expression and integration pattern, genetic transmission and expression (instability of foreign genes in twoNicotiana sp.Physiol. Plant. 85, 345–451.

    Google Scholar 

  • Hain, R., Stabel, P., Czervilovsky, A.P., Steinbiss, H.-H., Herrera-Estrella, L. and Schell, J. (1985) Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts.Mol. Gen. Genet. 199, 161–8.

    Google Scholar 

  • Hilson, P., Dewulf, J., Delporte, F., Install, P., Jacquemin, J.M., Jacobs, M. and Negrutiu, I. (1990) Yeast RAS2 affects cell viability, mitotic division and transient gene expression inNicotiana species.Plant Mol. Biol. 14, 669–85.

    Google Scholar 

  • Hobbs, S.L.A., Kpodar, P. and DeLong, C.M.O. (1990) The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants.Plant Mol. Biol. 15, 851–64.

    Google Scholar 

  • Jongsma, M., Koornneef, M., Zabel, P. and Hille, J. (1987) Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences.Plant Mol. Biol. 8, 383–94.

    Google Scholar 

  • Mittelsten Scheid, O., Paszkowski, J. and Potrykus, I. (1991) Reversible inactivation of a transgene inArabidopsis thaliana.Mol. Gen. Genet. 228, 104–12.

    Google Scholar 

  • Matzke, M.A. and A.J.M. Matzke (1990) Gene interactions and epigenetic variation in transgenic plants.Developmental Genetics 11, 214–23.

    Google Scholar 

  • Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene intoPetunia results in reversible co-suppression of homologous genes in trans.Pl. Cell 2, 279–89.

    Google Scholar 

  • Negrutiu, I., Dewulf, J., Pietrzak, M., Botterman, J., Reitveld, E., Wurzer-Figurelli, E.M., Ye, D. and Jacobs, M. (1990) Hybrid genes in the analysis of transformation conditions II. Transient expression vs stable transformation — analysis of parameters influencing gene expression levels and transformation,Physiol. Plant. 79, 197–205.

    Google Scholar 

  • Negrutiu, I., Shillito, R.D., Potrykus, I., Baisini, M.G. and Sala, F. (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts.Pl. Mol. Biol. 8, 363–73.

    Google Scholar 

  • Paszkowski, J., Shillito, R.D., Saul, M.W., Mandak, V., Hohn, T., Hohn, B. and Potrykus, I., (1984) Direct gene transfer to plants.EMBO J. 3, 2717–22.

    Google Scholar 

  • Peterhans, A., Schulupmann, H., Basse, C. and Paszkowski, J. (1990) Intrachromosomal recombination in plants.EMBO J. 9, 3437–45.

    Google Scholar 

  • Pichersky, E. (1990) Nomad DNA — a model for movement and duplication of DNA sequences in plant genomes.Pl. Mol. Biol. 15, 437–48.

    Google Scholar 

  • Potrykus, I., Paszkowski, J., Saul, M.W., Negrutiu, I. and Shillito, R.D. (1987) Direct gene transfer to plants: facts and future. — In Green, C.E., Somers, D.A., Hackett, W.P. and Biesboer, D.D. eds,Plant Tissue and Cell Culture, pp. 289–302. New York: Alan R. Liss Inc.

    Google Scholar 

  • Potrykus, I., Paszkowski, J., Saul, M.W., Petruska, J. and Shillito, R.D. (1985) Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer.Mol. Gen. Genet. 199, 167–77.

    Google Scholar 

  • Schimke, R.T., Sherwood, S.W., Hill, A.B. and Johnston, R.N. (1986) Overreplication and recombination of DNA in higher eukaryotes: potential consequences and biological implications.Proc. Natl. Acad. Sci. USA. 83, 2157–61.

    Google Scholar 

  • Shillito, R.D., Saul, M.W., Müller, M. and Potrykus, I. (1985) High efficiency direct gene transfer to plants.Bio/Technology 3, 1099–103.

    Google Scholar 

  • Smith, G.P. (1976) Evolution of repeated sequences by unequal crossover.Science 191, 528–38.

    Google Scholar 

  • Swoboda, P., Hohn, B. and Gal. S. (1992) Somatic homologous recombination in plants: the recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects.Mol. Gen. Genet. (in press).

  • van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M. and Stuitje, A.R. (1990) Flavonoid genes inPetunia: addition of a limited number of gene copies may lead to a suppression of gene expression.Pl. Cell 2, 291–9.

    Google Scholar 

  • Zhu, Z., Hughes, K.W. and Huang, L. (1991) Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum.In Vitro Cell Dev. Biol. 27, 77–83.

    Google Scholar 

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Cherdshewasart, W., Gharti-Chhetri, G.B., Saul, M.W. et al. Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants. Transgenic Research 2, 307–320 (1993). https://doi.org/10.1007/BF01976171

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