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Can elimination of the protein products of selectable marker genes in transgenic plants allay public anxieties?
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Can elimination of the protein products of selectable marker genes in transgenic plants allay public anxieties? The improvement of crop plants by the introduction of agronomically important genes using geneticengineering techniques is already at an advanced stage for certain species. During the transformation process, a selectable marker gene is introduced in tandem with the gene of interest to enable selection and recognition of transformed tissues. Efficient genetic-transformation systems have been developed for many plant species but, to date, most of the selectable marker genes used are based on strong constitutive promoters, for example, the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, which continues to direct expression of the selectable marker throughout the lifetime of the transgenic plant. Currently, the safety of the commonly used selectablemarker genes and their protein products is the subject o f some debate 1,2. Although there is no evidence that marker-gene protein products are toxic to humans or animals, the majority are of bacterial origin and encode antibiotic resistance, for example, neomycin phosphotransferase-II, npt-II (which confers resistance to kanamycin). Whether or not it is justified, transgenic plants expressing marker-gene products may not be accepted easily by the general public. Bryant and Leather 2 have supported proposals for the physical removal of marker genes from transgenic plants via the Cre/Lox site-specific recombination system 3. However, for vegetatively propagated crops, this system would disrupt the genetic composition of elite varieties, since sexual crosses and seed production would be necessary 1. Therefore, if regulatory agencies decided that marker genes should be removed, potato, apples, strawberries, sweet potato, cassava and many other crop plants would be much more difficult to improve using plant biotechnology. It has also been argued that, rather than © 1993, Elsevier Science Publishers Ltd (UK)
being a detrimental feature, the presence of marker genes and their products provides the agricultural community with an accurate and sensitive method for monitoring foreign genes in transgenic plants. Marker-gene expression is only required early in the plant-transformation process, during the selection of transformed plant tissue. Any expression of a marker gene in the mature plant is therefore unnecessary. One way of minimizing markergene expression and, hence, the presence of marker-gene protein products in transgenic plants, is to limit expression to the stages at which selection is applied. Recently, we demonstrated that a monocot, wound-induced promoter (AoPlK1) isolated from Asparagus officinalis showed strong, localized reportergene expression in plant-transfom> ation target cells 4,s. Agrobacteriummediated transformation of tobacco, potato and Arabidopsis using an AoP1Ll-npt-H construct resulted in the efficient selection of large numbers oftransgenic plants at a comparable frequency to that achieved using similar constructs which contained CaMV 35S-npt//marker genes 4,5. In the leaves, roots, young stems and tubers (in the case of potato) of these plants, the npt-II marker-gene protein product was present in extremely low amounts, in comparison with the levels found in CaMV 35S-npt iI plants. Thus, by using the particular expression characteristics of the AoPR1 promoter, the selectable marker gene is orfly expressed when it is required, i.e. during selection for transformed cells. The resultant mature transgenic plants express very low levels of marker-gene activity in a few specialized cell types, and due to the origin of the promoter, exhibit very localized expression upon wounding or pathogen invasion 6. Although promoter activation by minor damage inflicted during harvesting is possible, such
expression would be transient and localized to the wound site, and would therefore not significantly alter the overall, much lower levels of marker-gene protein products found in such plants. The approach described above, whilst not providing the ideal system, does offer a novel and extremely simple way o f reducing marker-gene protein products in transgenic plants, since it relies upon standard transfom~ation techniques, is a one-step process and does not require sexual crosses. In addition, although most plant tissues will contain very low levels of marker-gene product, by using the AoPR1 promoter to direct marker genes, marker-gene expression can still be induced by wounding, and monitored when required. A reduction in markergene expression in field-grown crop plants may also avoid any possible yield penalty that might be incurred as a result of the constitutive expression of transformation marker genes. Therefore, this approach counters possible objections to the presence of marker-gene protein products in transgenic plants, and may help to allay public anxieties towards them.
letter
References 1 Flavell,R. B., Dart, E., Fuchs, R. 1.. and Fraley, R. T. (1992) Bio/Tecknolo2y 10, 141-144 2 Bryant,J. and Leather, S. (1992) Trends Biotecknol. 10, 274-275 3 Dale, E, C. and Ow, D. W. (1991) Proc. Natl Acad. &i. USA 88, 10558~10562 40zcan, S., Firek, S. and Draper,J. (1993) Bio/Technology11, 218-221 5 Firek, S., Ozcan, S., Warner, S. A.J. and Draper, J. Plant3/Iol. Biol. (in press) 6 Warner, S. A. J., Scott, IL. and Draper,J. (1993) Plan@ 3, 191-201
Sebahattin (2zcan Simon Firek John Draper Department of Botany, University of Leicester, Leicester, UK LEI 7R5f. TIBTECH JUNE 1993 (VOL t 1)
Can elimination of the protein products of selectable marker genes in transgenic plants allay public anxieties? The improvement of crop plants by the introduction of agronomically important genes using geneticengineering techniques is already at an advanced stage for certain species. During the transformation process, a selectable marker gene is introduced in tandem with the gene of interest to enable selection and recognition of transformed tissues. Efficient genetic-transformation systems have been developed for many plant species but, to date, most of the selectable marker genes used are based on strong constitutive promoters, for example, the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, which continues to direct expression of the selectable marker throughout the lifetime of the transgenic plant. Currently, the safety of the commonly used selectablemarker genes and their protein products is the subject o f some debate 1,2. Although there is no evidence that marker-gene protein products are toxic to humans or animals, the majority are of bacterial origin and encode antibiotic resistance, for example, neomycin phosphotransferase-II, npt-II (which confers resistance to kanamycin). Whether or not it is justified, transgenic plants expressing marker-gene products may not be accepted easily by the general public. Bryant and Leather 2 have supported proposals for the physical removal of marker genes from transgenic plants via the Cre/Lox site-specific recombination system 3. However, for vegetatively propagated crops, this system would disrupt the genetic composition of elite varieties, since sexual crosses and seed production would be necessary 1. Therefore, if regulatory agencies decided that marker genes should be removed, potato, apples, strawberries, sweet potato, cassava and many other crop plants would be much more difficult to improve using plant biotechnology. It has also been argued that, rather than © 1993, Elsevier Science Publishers Ltd (UK)
being a detrimental feature, the presence of marker genes and their products provides the agricultural community with an accurate and sensitive method for monitoring foreign genes in transgenic plants. Marker-gene expression is only required early in the plant-transformation process, during the selection of transformed plant tissue. Any expression of a marker gene in the mature plant is therefore unnecessary. One way of minimizing markergene expression and, hence, the presence of marker-gene protein products in transgenic plants, is to limit expression to the stages at which selection is applied. Recently, we demonstrated that a monocot, wound-induced promoter (AoPlK1) isolated from Asparagus officinalis showed strong, localized reportergene expression in plant-transfom> ation target cells 4,s. Agrobacteriummediated transformation of tobacco, potato and Arabidopsis using an AoP1Ll-npt-H construct resulted in the efficient selection of large numbers oftransgenic plants at a comparable frequency to that achieved using similar constructs which contained CaMV 35S-npt//marker genes 4,5. In the leaves, roots, young stems and tubers (in the case of potato) of these plants, the npt-II marker-gene protein product was present in extremely low amounts, in comparison with the levels found in CaMV 35S-npt iI plants. Thus, by using the particular expression characteristics of the AoPR1 promoter, the selectable marker gene is orfly expressed when it is required, i.e. during selection for transformed cells. The resultant mature transgenic plants express very low levels of marker-gene activity in a few specialized cell types, and due to the origin of the promoter, exhibit very localized expression upon wounding or pathogen invasion 6. Although promoter activation by minor damage inflicted during harvesting is possible, such
expression would be transient and localized to the wound site, and would therefore not significantly alter the overall, much lower levels of marker-gene protein products found in such plants. The approach described above, whilst not providing the ideal system, does offer a novel and extremely simple way o f reducing marker-gene protein products in transgenic plants, since it relies upon standard transfom~ation techniques, is a one-step process and does not require sexual crosses. In addition, although most plant tissues will contain very low levels of marker-gene product, by using the AoPR1 promoter to direct marker genes, marker-gene expression can still be induced by wounding, and monitored when required. A reduction in markergene expression in field-grown crop plants may also avoid any possible yield penalty that might be incurred as a result of the constitutive expression of transformation marker genes. Therefore, this approach counters possible objections to the presence of marker-gene protein products in transgenic plants, and may help to allay public anxieties towards them.
letter
References 1 Flavell,R. B., Dart, E., Fuchs, R. 1.. and Fraley, R. T. (1992) Bio/Tecknolo2y 10, 141-144 2 Bryant,J. and Leather, S. (1992) Trends Biotecknol. 10, 274-275 3 Dale, E, C. and Ow, D. W. (1991) Proc. Natl Acad. &i. USA 88, 10558~10562 40zcan, S., Firek, S. and Draper,J. (1993) Bio/Technology11, 218-221 5 Firek, S., Ozcan, S., Warner, S. A.J. and Draper, J. Plant3/Iol. Biol. (in press) 6 Warner, S. A. J., Scott, IL. and Draper,J. (1993) Plan@ 3, 191-201
Sebahattin (2zcan Simon Firek John Draper Department of Botany, University of Leicester, Leicester, UK LEI 7R5f. TIBTECH JUNE 1993 (VOL t 1)