Finn T. Okkels

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.

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

SummaryA 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 β-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.

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 Ω′ 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.

Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry

Abstract A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants. Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch industry and which is already being widely utilized in specific food processes.

Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Abstract With the aim of making a system using the GUS gene as a selection gene, the effects of two toxin glucoronides were tested on transgenic tobacco cells ( Nicotiana tabacum L.) containing a β-glucuronidase (GUS) gene from Escherichia coli and on non-transgenic cells. No significant difference in toxicity was observed between transgenic and non-transgenic cells. We found that this most probably was due to the activity of an endogeneous GUS enzyme, which could be detected in all plant species tested, e.g. tobacco, sugar beet ( Beta vulgaris L.), oilseed rape ( Brassica napus L.), pea ( Pisum sativum L.), wheat ( Triticum sativum L.) and rhubarb ( Rheum rhaponticum L.) This indicates that GUS may be ubiquitous to plants contrary to earlier assmptions. The endogenous enzyme is active at pH 4–5 and the activity is eliminated without reducing the introduced GUS activity when pH is elevated. In addition the endogenous GuS can be selectively inhibited at high temperatures. Modifications according to these findings can be employed in standard GUS assays to avoid misinterpretations when the expression of tissue specific promoters is tested.

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.

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 β-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.

Effect of Extracellular Peroxidases on Growth of Carrot and Sugar Beet Suspension Cells

Summary It is generally accepted that an increase in peroxidase activity is temporally correlated to a decrease in growth rate of plant cells. The growth of embryogenic suspension cells of carrot was found to be inversely correlated to the peroxidase activity secreted into the growth medium. Due to the extracytoplasmic localization of the majority of peroxidase activity of suspension cells, horse radish peroxidase (HRP) was added to the growth medium of suspensions of embryogenic carrot cells and habituated sugar beet cells and the effect on the growth rate was measured. The added HRP activity was 10 to 200 fold higher than the physiological activity and it persisted throughout the growth period. The peroxidase activity secreted from the carrot suspension cells was not affected by the presence of added HRP activity. The added HRP activity did not reduce the growth of the suspension cells.