The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent.

The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the omega' translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.

Synthesis of cytokinin glucuronides for the selection of transgenic plant cells.

Glucuronide derivatives of cytokinins have been synthesized for use as agents for the selection of plant cells transformed with a beta-glucuronidase (GUS) gene. In this selection system, the GUS gene functions as both a selectable, as well as a screenable gene. GUS liberates active cytokinin from inactive cytokinin glucuronides which then stimulates growth and regeneration of the transformed cells. The frequently used cytokinin N6-benzyladenine was conjugated to glucuronic acid at N-3 or at N-9 but only the former was a substrate for GUS. The glucuronide of isopentenyladenine was also made, by coupling at the N-3. This compound was readily hydrolysed by GUS.

A novel principle for selection of transgenic plant cells: positive selection.

A novel principle for selection of transgenic plant cells is presented. In contrast to traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), this selection method actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. Therefore, this selection strategy is termed 'positive selection'. TheE. coli ß-glucuronidase gene was used as selectable (as well as screenable) gene and a glucuronide derivative of the cytokinin benzyladenine as selective agent which is inactive as cytokinin but, upon hydrolysis by GUS, active cytokinin is released stimulating the transformed cells to regenerate. Selection ofAgrobacterium tumefaciens inoculated of tobacco leaf discs on benzyladenine N-3-glucuronide (7.5-15 mg/l) resulted in 1.7-2.9 fold higher transformation frequencies compared to kanamycin selection. A significant advantage of this selection procedure is the elimination of the need for herbicide and antibiotic resistance genes.

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco.

We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a beta-glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.

DNA methylation is involved in maintenance of an unusual expression pattern of an introduced gene.

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced beta-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.

Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota.

The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.

Employment of hydrolytic enzymes in the study of the level of DNA methylation.

A new method for the determination of the level of DNA methylation was established. The method involves enzymatic hydrolysis of DNA by nuclease P1 and bacterial alkaline phosphatase, and separation of the resulting deoxyribonucleosides by HPLC. By this method, DNA was hydrolysed completely to the five deoxyribonucleosides and the complete base composition was determined. Pairing bases were shown to occur in similar amounts, and analysis could be performed on as little as 1 microgram of DNA with a high degree of reproducibility. Among other enzymes hitherto used in order to hydrolyze DNA, micrococcal nuclease, phosphodiesterase II and nuclease P1 have been shown to cause deamination of deoxyadenosine, while deoxyribonuclease I, phosphodiesterase I and bacterial alkaline phosphatase have been shown to be sensitive to contamination by RNA, and to release 5-methyldeoxycytidine at a slower rate than the other four deoxyribonucleosides. Neither of these effects was seen with the new method.