Stress inactivation of foreign genes in transgenic plants

ELSEVIER

Field Crops Research 45 (1996) 19-25

Field Crops Research

Stress inactivation of foreign genes in transgenic plants Inge Broer Lehrstuhl fiir Genetik, Fakultiit fiir Biologie, Universitiit Bielefeld, P.O. Box 100131, 33501 Bielefeld, Germany

Abstract The inactivation of endogenous plant genes during periods of stress or environmental changes has already been demonstrated. Although the expanding use of transgenic plants in scientific research has given hints for transgene inactivation caused by transinactivation, the first inactivation of transgene-encoded proteins caused by environmental changes was observed not before 1990. Transgenic Petunia hybrida plants, carrying the maize AI cDNA under the control of the CaMV35S RNA promoter causing a salmon red flower phenotype, were released to the field. Environmental factors, possibly including heat, lead to a stable loss of flower pigmentation, accompanied by the methylation of the viral promoter. In the same year, the inactivation of the phosphinothricin resistance gene in single-cell cultures of a transgenic Medicago sativa line was observed in more than 90% of the cells after a 10-day heat treatment. In transgenic tobacco, different transgenes could be inactivated by a heat treatment (37°C), which did not lead to changes in the growth of the plant. These inactivations seem to be not correlated with methylation. The heat-induced inactivation of herbicide resistance genes led to sensitivity to the herbicide during the heat treatment. The transgene activity was regained after the temperature decreased to normal cultivation conditions. To date, heat-induced transgene inactivation has not been reported in the field, but it may give rise to problems if it does occur. Therefore, strategies to analyse the causes of heat-induced transgene inactivation and to circumvent the problem have to be developed. Keywords: Environmental stress; Resistance; herbicide; Medicago sativa; Nicotiana tabacum; Petunia hybrida; Transgenic plants; Transinactivation

1. I n t r o d u c t i o n Most stress responses o f plants are accompanied by controlled activation and silencing o f endogenous plant genes. As an example, in tomato, the appearance of new proteins and the decrease in gene product and m R N A levels of other genes could be detected after wounding (Metha et al., 1991). Wounding increases polysome levels in storage and mature tissues in carrot (Lin et al., 1973), in potato (e.g., Ishizuka and Imaseki, 1989) and in pea (e.g., Davies et al., 1986). Severing roots and rhizomes o f Cardamine cordifolia once in the early growing season

produced moderate, but detectable changes in plant physiological traits (Louda and Collinge, 1992). In maize, anaerobiosis resulted in the induction o f a specific set o f genes and additionally in the repression o f normal cellular messages (Sachs et al,, 1980). In rice, five proteins possibly associated with water stress were overproduced in cells adapted to polyethylene glycol (PEG) (Borkird et al., 1991). Much is known about the activation o f genes during heat shock and the resulting regulation of gene expression. F o l l o w i n g heat shock, normal cellular messages are released from the polysomes and are replaced by heat-shock-specific m R N A (for review, see Gallie,

0378-4290/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0378-4290(95)00055-0

20

L Broer / Field Crops Research 45 (1996) 19-25

1993). Gene expression is also altered during plant defense against pathogens (e.g., Boller, 1988). These activations and inactivations are necessary for the controlled response of the plant to environmental stress factors. They are part of the normal regulation procedure in the plant. The location of genes in the plant genome is often important for their regulation. In contrast to endogenous genes, transgenes insert randomly into the plant's chromosome and are therefore exposed to influences of varying chromosomal environments. They are influenced by cis-acting factors not carried by the transgene but present at the insertion site (position effect). To date, this impact cannot be prevented and may be the cause of differing levels of gene expression in individual transformants (e.g., Peach and Velten, 1991). Since the discovery of the ability of Agrobacterium tumefaciens to transfer DNA into the plant cell, many different plant species have been transformed using this agent. Most of the plants analysed inherited the integrated genes according to the Mendelian laws (e.g., Otten et al., 1981). Nevertheless, Tepfer (1983) reported the lack of opines in some descendants of transgenic tobacco plants that contained a full length T-DNA. Later, similar effects were described by Potrykus et al. (1985), Budar et al. (1986) and Miiller et al. (1987). Because the transferred genes were still present in the plant's genome, it was assumed that the loss of transgeneencoded phenotypes in the offspring was caused by mutations or gene suppressions. Most of the authors dealing with this phenomenon named it 'gene-silencing' (for review, see Jorgensen, 1992). In several plant species, gene-silencing was correlated with methylation of CpG residues in the transgene sequence (e.g., John and Amasino, 1989). One of the possible causes of transgene suppression may be the interaction of the transferred gene with homologous sequences also transferred to or already present in the plant. Transinactivation phenomena such as allelic interactions and interactions between duplicated sequences at unlinked loci are described (for reviews, see Kooter and Mol, 1993; Matzke and Matzke, 1993). Examples and possible explanations concerning this effect, named 'co-suppression' are reviewed by Jorgensen (1992). Inactivation of transgenes accompanied by CpG methylation could also be observed in Arabidopsis plants after several gen-

erations of inbreeding (Kilby et al., 1992). These effects occurred mainly during meiosis. Therefore, it is possible to eliminate plants producing variegated offspring during the analysis necessary for breeding. This is not the case for silencing events that happen during the growth of the plant on the field or in the growth chamber. Additionally, none of the examples given combine the effect of environmental changes on gene expression and transgene silencing. Nevertheless, environmental changes are an important factor in the field and may, besides the described regulation of endogenous genes, also influence transgene expression. Few examples of transgene silencing induced by environmental changes during the normal growth phase of a plant or a suspension culture are present in the literature and reviewed in this paper.

2. Environmental factors influencing the expression of a maize A1 gene in transgenic Petunia

hybrida In 1990, the first transgenic plants (Petunia hybrida) were released in Germany (Meyer et al., 1992). The aim of the experiment was the trapping of transposable elements that have been genetically described in petunia. Their integration into a heterozygous transgene should result in a white or variegated phenotype, respectively. Because the frequency of integration of transposable elements into preselected genes should be between 10 -z and 10 -6, the release of 30 000 transgenic plants was necessary to have a chance to isolate some mutant phenotypes and analyse them on a molecular level. Transgenic P. hybrida plants carrying a single copy of a transgene, the expression of which confers a salmon red phenotype on the flowers of the originally white plant, were planted in the field. The mutation leading to the lack of colour in the mutant used for transformation was caused by blocking the conversion of dihydrokaempferol to dihydroquercetin. The transgene giving the salmon red colour was composed of a cDNA of the maize A1 gene fused to the 35SCaMV promoter. The A1 gene encodes a dihydroflavonol reductase, an enzyme of the anthocyanin pigmentation pathway. In transgenic plants, dihydrokaempferol was converted to leucopelargonidin, which was further metabolised to a

L Broer/Field Crops Research 45 (1996) 19-25

pelargonidin pigment. These pigments, normally not produced in petunia, gave the salmon red flower phenotype to the plant (Linnet al., 1990). Out of all transgenic plants produced, one homozygous line was chosen, which carried only one copy of the transgene per genome. Transgene expression was very stable compared to other transgenic lines. Hemizygous offspring were produced via mating of the stable transgenic line with the originally white mutant. In 1990, summer in Germany was unusually hot. During the growing season of the petunia plants in the field, plants showing white or variegated phenotypes and plants with only weak pigmentation were observed with varying frequencies. In several plants it could be demonstrated that these variations were accompanied by the hypermethylation of the 35S promoter directing the expression of the A1 gene. This was not the case in plants with homogeneous red flowering. Linn et al. (1990) assume that the loss in colour is not only dependent on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made. Nevertheless, environmental stress, possibly including heat, seems to have impact on the observed silencing. No transposon insertions could be detected in the experiment (Meyer et al., 1992).

3. Heat-treatment-induced

inactivation of the

pat-gene-encoded herbicide resistance in single-cell cultures of transgenic Medicago sativa The second transgene inactivation induced by environmental factors was also observed in Germany in 1990. In contrast to the petunia experiment described above, the direct correlation between heat and transgene inactivation could be demonstrated (Walter et al., 1992). The plant used in this study was a descendant of the transgenic Medicago sativa line T304, carrying a gene conferring resistance to the herbicide phosphinothricin (Pt). Pt is produced as phosphinotricylalanyl-alanin (Ptt) by Streptomyces hygroscopicus (Kondo et al., 1973) and S. viridochromogenes (Bayer et al., 1972). Pt is a structural analogue of glutamic acid, which competitively inhibits bacterial

21

and plant glutamine synthetases (Bayer et al., 1972; Lea et al., 1984). Resistance genes have been isolated from both producer strains (Thompson et al., 1987; Strauch et al., 1988) and transferred to several plant species (De Block et al., 1987). Both genes code for a phosphinothricin-N-acetyl-transferase which inactivates Pt. The amino acid sequence derived from the resistance-conferring phosphinothricin-N-acetyl transferase gene isolated from S. viridochromogenes (Wohlleben et al., 1988) was used to determine the corresponding DNA sequence, which was then adapted to the plant's codon usage (Eckes et al., 1989). The gene was transferred to M. sativa Ra-3 to give rise to the Pt-resistant M. sativa T304 (Eckes et al., 1989). T304 contains only one copy of the transgene (G. Donn, pers. commun., 1989). Transgenic, Pt-resistant Daucus carota and M. sativa suspension cultures which were maintained for a long period without selective pressure, occasionally revealed a high percentage of sensitive cells (Dr5ge et al., unpublished data). This phenomena has already been reported for endogenes genes in permanent cell lines owing to de novo methylation of CpG-island promoters that are normally methylation free. Only genes that are required for growth in culture were still active (Bird, 1992). According to Bird (1992), these methylations are culture specific. Therefore, we decided to quantify the observed loss of resistance using a single-cell system established by Walter et al. (1992). The single-cell culture was divided into two parts, both grown at 25°C for 60 days. Samples of the cultures were analysed with respect to their content of Pt-resistant and -sensitive cells. The growth of the cultures as well as their proportion of Pt-sensitive cells increased at the same rate during this period of time. Thereafter, a heat treatment of 37°C was applied for 10 days to one of the cultures in order to study the impact of the temperature on the stability of pat gene expression. Interestingly, the growth rate of the heat-treated culture was not affected and the cell generation time did not differ from the control. In contrast to this observation however, the percentage of Pt-sensitive cells in the culture changed dramatically. In comparison to the control culture where 12% of the cells lost the Pt-resistant phenotype, 95% of the cells derived from the heat-treated culture were found to be Pt-sensitive.

22

L Broer / Field Crops Research 45 (1996) 19-25

The loss of the Pt-resistant phenotype may be due to a deletion or a mutation of the pat gene. As shown by the PCR, the pat gene was present in all Pt-sensitive clones tested. The restriction sites in the synthetic pat gene were maintained in the PCR products and the sizes of smaller fragments were also identical to those of the wild type. Deletions or mutations were not visible. The Pt-sensitive phenotype may also have been caused by a reduced activity of the pat gene. Nevertheless, no difference in the resistance levels could be detected between transgenic Pt-sensitive and wild type cells. Additionally, the phosphinothricin-Nacetyl transferase activity was measured in transgenic and wild type calli, using the PAT-assay (Dr~Age et al., 1992). No acetylation of Pt was detectable in transgenic, Pt-sensitive clones. In contrast, Pt-resistant clones demonstrated high turnover rates of Pt.

4. Transgene-encoded phenotypes are strongly reduced during a heat treatment of transgenic Nicotiana tabacum plants The heat-induced inactivation of transgenes may be of importance when transgenic plants are used in agriculture. Because suspension cultures are artificial systems, the heat-induced loss of transgene expression was analysed in a plant system (Broer et al., 1995; Neumann et al., 1995). For that purpose the modified pat gene (Wohlleben et al., 1988) was transferred, together with a noSpro-nptlI-gene, to Nicotiana tabacum using Agrobacterium-mediated gene transfer (Broer et al., 1988). Sl-plants were selected that carry either one or at least two copies of the transgenes in the hemi- and homozygous state. The inactivation of transgene-related phenotypic characteristics in response to the heat treatment was determined by analysing aseptically grown plants. By means of Pt and Km selection, inactivation of the pat and nptlI resistance genes can be recognized by antibiotic-induced damage of the plants. Using this detection system, the inactivation of transgene-encoded functions after heat treatment was recognized in the transgenic plants analysed. It was important to ascertain whether the loss of the transgene-encoded protein activity was correlated with heat damage of the plant, and at which tempera-

tures these reactions in the tobacco plants could be detected. Therefore, we compared phenotypically visible heat-damage of the plant, indicated by parameters such as retarded growth and bleaching of leaves, with the loss of the transgene-related resistance. Reduction of the transgene-encoded phenotype could already be observed at 35°C, while a visible damage of the heat-treated plant was obtained in all test plants after 10 days of treatment of at least 40°C. After decreasing the culture temperature to 25°C again, transgene activity was regained. In order to test whether the plant's response was dependent on the copy number of the transgene, different molecularly well-characterised transformants carrying one or at least two copies were compared in this study. The response observed could not be correlated with the copy number of the transgene. Plants carrying only one copy of the transgenes showed the same phenotypic reaction as those with at least two copies (Broer et al., 1995; Neumann et al., 1995). The inactivation of the A1 gene in petunia carrying a CaMV promoter was correlated with methylation of the viral promoter sequence (Meyer et al., 1992), indicating that the viral origin of the promoter may have caused this phenomenon. Therefore, we employed fusion genes driven by a bacterial- or a virus-promoter. However, in this work, the different promoters gave the same results. It has been assumed, that the inactivation of transgenes might be influenced by the homo- or hemizygous state of the gene (e.g., de Carvalho et al., 1992). Therefore hemi- and homozygous plants selected from the offspring of the individual transformants were used in the test. No differences in heatresponse of the hemi- or homozygous transgenes could be observed (Broer et al., 1995; Neumann et al., 1995). In order to study heat-induced loss of transgene activity at the molecular level, enzyme activity assays were employed (Dr~Sge et al., 1992). The PATactivity was completely lost within 2 days of heat treatment and was not restored within the first 2 days following the heat treatment. Ten days later, PAT-activity was detectable again. The NPTII-protein content was dramatically reduced during the heat treatment of the eight heat-sensitive test plants, nevertheless a basic level remained even after 10 days of heat

I. Broer / Field Crops Research 45 (1996) 19-25

treatment (Broer et al., 1995; Neumann et al., 1995). In conclusion, the heat treatment leads to a nearly complete loss of the transgene-encoded protein. Neither protein activity, nor the protein itself coded by the different transgenes is detectable in the plants carrying heat-treatment-sensitive transgenes. The reversion of transgene activity after the reduction of the temperature proves the integrity of the DNA sequence. Therefore, mRNA instabilities as occur during heat shock (e.g., Gallie, 1993) or a reduced transcription of the transgenes, relying on epigenetic mechanisms, may be the cause for the low level of transgene expression. During heat treatment, the amount of transgene-specific RNA in heatstress-sensitive plants is not significantly reduced (Broer et al., 1995; Neumann et al., 1995). Accordingly, this phenomenon can not be correlated with the methylation of CpG residues in the transgene sequence and we were not able to detect changes in DNA-methylation of the transgenes in heat-treated, sensitive, plants. Neither prevents the incorporation of 5-azacytidine the inactivation of transgenes, nor are any differences detectable when DNA from heat-treated and control plants are restricted with methylation-sensitive endonucleases. The molecular mechanisms involved in this probably post-transcriptional phenomenon remain to be investigated.

5. Conclusion

Because transgenic plants are proposed as candidates to be applied in agriculture, it is important that even under unusual environmental conditions the desired phenotype is stably maintained in the crop. The instability of transgene expression in petunia could not strictly he correlated with heat. Several environmental and endogenous factors seem to influence the phenomenon. Actually, it has to be stated that heat alone did not lead to transgene suppression in petunia planted in the greenhouse. Transinactivation events have been described for homozygous descendants of the same transgenic line (Meyer et al., 1992), but this cannot explain instabilities in hemizygous plants like those released. Although the inactivated transgenes are hypermethylated like most

23

of those silenced by transinactivation, different factors have to be involved. In the other two examples, inactivation of transgene-encoded proteins was strictly correlated with heat treatment. Nevertheless, it cannot be excluded, that other factors may lead to the same reaction. Inactivation of the transgene-encoded protein activity found in M. sativa suspension cultures could also be detected in tobacco plants. In case that the selection of genes inactivated during the heat treatment period is dependent on the transgene itself, one possible signal for the identification may be the viral promoter region. Nevertheless, the Nos-promoter from the Agrobacterium T-DNA was also subject to transgene inactivation. Although the analysis of two different constructs is not a proof, that all transgenes will react on heat treatment in the same manner, the effect indicates that the transgene sequence itself may not be the main cause for inactivation. The fact that neither copy number, nor hemi- or homozygous state of the transgenes influence the degree of inactivation indicates, that heat-induced inactivation of transgene-encoded proteins is not due to co-suppression. Therefore, the mechanism of inactivation remains to be investigated. To date, it cannot be excluded that position effects may influence stability of transgene expression. This was also assumed by Pr~ls and Meyer (1992) concerning the influence of the insertion locus of the Al-gene and a nptII gene in the petunia chromosome on the stability of transgene expression. If this is the case, one may assume that there are some loci on the plant chromosome resistant to stress inactivation. Analysis of plants resistant to the inactivation of transgene-encoded functions may shed light on plant gene regulation and additionally offer possibilities to prevent transgene instabilities in the field. Inactivation of the A1 gene in transgenic petunia plants demonstrates the possible effects originating from environmental factors. Because loss of colour is a phenotypic reply that does not depend on the application of chemicals such as herbicides, the effect can easily be seen any time it occurs. Up to now, the inactivation of resistance genes in the field have not been reported in the literature. This may be due to the fact that the effect can only be seen if the herbicide is applied directly during a period of high temperature. Nevertheless, it cannot be excluded that

24

1. Broer / Field Crops Research 45 (1996) 19-25

the suppression of transgenes observed in the laboratory will occur in the field. It is therefore of great importance to verify our results in field tests. Additionally, it has to be stated that other plant species and transgenes may react differently on heat treatment.

Acknowledgements The author wishes to thank Katrin Neumann and Wolfgang Drrge-Laser (University of Bielefeld) for the presentation of data prior to publication and Katrin Neumann, Wolfgang Drrge-Laser and Alfred Piihler (University of Bielefeld) for helpful advice and collaboration.

References Bayer, E., Gugel, K.H., H~igele, K., Hagenmeier, H., Jessipow, S., Krnig, W.A. and Z~ihner, H., 1972. Stoffwechselprodukte yon Mikroorganismen. Phosphinothricin und Phosphinothricylalanyl-alanin. Helvetica. Chim. Acta, 55: 224-239. Bird, A., 1992. The essentials of DNA methylation. Cell, 70: 5-8. Boiler, T., 1988. Ethylen and the regulation of antifungual hydrolases in plants. Oxford Surv. Plant Mol. Cell. Biol., 5: 145-174. Borkird, C., Simons, C., Villarroel, R. and van Montagu, M., 1991. Gene expression associated with water stress adaption of rice cells and identification of two genes as hsp70 and ubiqitin. Physiol. Plant., 82: 449-457. Broer, 1., Arnold, W., Wohlleben, W. and Piihler, A., 1988. The phosphinothricin-N-acetyltransferase gene as a selection marker for plant genetic engineering. In: G. Galling (Editor), Proceedings of the Braunschweig Symposium on "Applied Plant Molecular Biology". Zentrale Stelle fiir Weiterbildung, TU Braunschweig, Germany, pp. 240-246. Broer, 1., Drrge-Laser, W., Vinnemeier, J., Krhne, S. and Neumann, K., 1995. Strong heat-treatment induced reduction of transgene encoded herbicide-resistance in tobacco (Nicotiana tabacum) plants. Plant Physiol., submitted. Budar, F., Thia-Toong, L., Van Montagu, M. and Hernalsteens, J.P., 1986. Agrobacterium-mediated gene transfer results mainly in transgenic plants transmitting T-DNA as a single Mendelian factor. Genetics, 114: 303-313. Davies, E., Ramaiah, K.V.A. and Abe, S., 1986. Wounding inhibits protein synthesis yet stimulates polysome formation in aged, exised pea epicotyls. Plant Cell Physiol., 27:1377-1386. De Block, M., Bottermann, J., Vandewiele, M., Dockx, J., Thoen, C., Gossele, V., Movva, N.R., Thompson, C., Van Montagu, M. and Leemans, J., 1987. Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J., 6: 2513-2518.

de Carvalho, F., Gheysen, G., Kushnir, S., van Montagu, M., inze, D. and Castresana, C., 1992. Suppression of /3-1,3-glucansase transgene expression in homozygous plants. EMBO J., 11: 2592-2602. Drrge, W., Broer, I. and Piihler, A., 1992. Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants. Planta, 187:142-151. Eckes, P , Vijtewaal, B. and Donn, G., 1989. Synthetic gene confers resistance to the broad spectrum herbicide L-Phosphinothricin in plants. J. Cell Biochem., suppl. 13 D. Gallie, D.R., 1993. Posttranscriptional regulation of gene expression in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 77-105. lshizuka, M. and Imaseki, H., 1989. Uncoupling of the formation of polysomes from transcription at the early stage of wound response in potato tuber slices. Plant Cell Physiol., 30: 229236. John, M.C. and Amasino, R.M., 1989. Extensive changes in DNA methylation patterns accompany activation of a silent T-DNA ipt gene in Agrobacterium tumefacies-transformed plant cells. Mol. Cell Biol., 9: 4298-4303. Jorgensen, R., 1992. Silencing of plant genes by homologous transgenes. Agbiotech. News Info, 4: 265-273. Kilby, N.J., Leyser, O. and Furuer, 1.J., 1992. Promoter methylation and progressive transgene inactivation in Arabidopsis. Plant Mol. Biol., 20:103-112. Kondo, Y., Shomura, T., Ogawa, Y., Tsuruoka, T., Watanabe, H., Totsukawa, K., Suzuki, T., Moriyama, C., Yoshida, J., Inouye, S. and Niida, T., 1973. Studies on a new antibiotic SF-1293. I. Isolation and physio-chemical and biological characterization of SF-1293 substance. Sci. Rep. Meiji Seika Kaisha, 13: 34-41. Kooter, J.M. and Mol, J.N.M., 1993. Trans-inactivation of gene expression in plants. Current Opinion Biotechnol., 4: 166-171. Lea, P.J., Joy, K.W., Ramos, J.L. and Guerrero, M.G., 1984. The action of the 2-amino-4-(methylphosphinyl)-butanoic acid (phosphinothricin) and its 2-oxo-derivative on the metabolism of cyanobacteria and higher plants. Phytochemistry, 23: 1-6. Lin, C.Y., Travis, R.L., Chia, L.-L.S.V. and Key, J.L., 1973. Changes in ribosomal activity during inkubation of Daucus carota root disks. Phytochemistry, 12: 2801-2807. Linn, F., Heidmann, 1., Saedler, H. and Meyer, P., 1990. Epigenetic changes in the expression of the maize A I gene in Petunia hybrida: Role of numbers of integrated gene copies and state of methylation. Mol. Gen. Genet., 222: 329-336. Louda, S.M. and Collinge, S.K., 1992. Plant resistance to insect herbivores: A field test on the environmental stress hypothesis. Ecology, 731: 153-169. Matzke, M. and Matzke, A.J.M., 1993. Genomic imprinting in plants: Parental effects and transinactivation phenomena. Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 53-76. Metha, R.A., Parsons, B.L., Metha, A.M., Nakhasi, H.L. and Mattoo, A.K., 1991. Differential protein metabolism and gene expression in tomato fruit during wounding stress. Plant Cell Physiol., 32: 1057-1065. Meyer, P., Linn, F., Heidmann, I., Meyer, Z.A.H., Niedenhof, I.

I. Broer/Field Crops Research 45 (1996) 19-25 and Saedler, H., 1992. Endogenous and environmental factors influence 35S promoter methylation of a maize AI gene construct in transgenic petunia and its colour phenotype. Mol. Gen. Genet., 231: 345-352. MUller, A.J., Mendel, R.R., Schiemann, J., Simons, C. and lnze, D., 1987. High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation. Mol. Gen. Genet., 207: 171-175. Neumann, K., Dr~ige-Laser, W., Ktihne, S., Piihler, A. and Broer, i., 1995. A heat-induced loss of transgene activity detected and analysed in several different transgenic Nicotiana tabacum lines. In: E.R. Schmidt (Editor), Biosafety: Stability of DNA, Horizontal Gene Transfer and Expression of Transgenes. Springer, Berlin, in press. Otten, L., de Greve, H., Hernalsteens, J.P., Van Montagu, M., Schieder, O., Straub, J. and Schell, J., 1981. Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Mol. Gen. Genet., 183: 209-213. Peach, C. and Velten, J., 1991. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol. Biol., 7: 49-60. Potrykus, I., Paszkowski, J., Saul, M.W., Petruska, J. and Shilito, R.D., 1985. Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer. Mol. Gen. Genet., 199: 169-177. Pr~51s, F. and Meyer, P., 1992. The methylation patterns of chro-

25

mosomal integratio regions influence gene activity of transferred DNA in Petunia hybrida. Plant J., 24: 465-475. Sachs, M.M., Freeling, M. and Okimoto, R., 1980. The anaerobic proteins of maize. Cell, 20: 761-767. Strauch, E., Wohlleben, W. and Piihler, A., 1988. Cloning of a phosphinothridin-N-acetyl transferase from Streptomyces viridochromogenes Tfa 494 and its expression in Streptomyces lividans and E. coli. Gene, 63: 65-74. Tepfer, D., 1983. The biology of genetic transformation of higher plants by Agrobacterium rhizogenes. In: A. Piihler (Editor), Molecular Genetics of the Bacteria-Plant Interaction. Springer Verlag, Berlin, pp. 248-258. Thompson, C.J., Rao Mova, N., Tizard, R., Crameri, R., Davies, J.E., Lauwereys, M. and Botterman, J., 1987. Characterization of the herbicide resistance gene bar from Streptomyces hygroscopicus. EMBO J., 6: 2519-2523. Walter, C., Broer, 1., Hillemann, D. and Piihler, A., 1992. High frequency, heat-treatment induced inactivation of a phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago satiua. Mol. Gen. Genet., 235: 189-196. Wohlleben, W., Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Piihler, A., 1988. Nucleotide sequence of the phosphinothricin-N-acetyl-transferase from Streptomyces viridochromogenes T'fi 494 and its expression in Nicotiana tabacum. Gene, 70: 25-37.